Introduction
Palms, members of the Arecaceae family, are perennial, flowering monocotyledons in the order Arecales and are characterized by woody stems. This highly diverse family includes 181 genera and approximately 2600 species (Baker and Dransfield Reference Baker and Dransfield2016). The Arecaceae family includes several economically important species, including coconut, arecanut and palmyrah palm, which are cultivated globally and support the livelihoods of millions.
Coconut palms are abundant in tropical coastal regions and play a vital role in island ecosystems by providing food, fibres, beverages, medicine, shelter and handicrafts. For this reason, they are often called ‘Kalpavriksha’ or the ‘tree of life’. The coconut (Cocos nucifera L.) has a diploid chromosome number of 2n = 32 and is the only species of its genus (Swaminathan and Nambiar Reference Swaminathan and Nambiar1961). Coconut is classified into two main groups, tall (typica) and dwarf (nana), based on the plant habit and breeding behaviour. Tall types are cross-pollinating and thus highly heterozygous. Dwarf types, by contrast, are self-pollinating; hence, populations are more homozygous (Narayana and John Reference Narayana and John1949; Niral and Jerard Reference Niral, Jerard, Nampoothiri, Krishnakumar, Thampan and Achuthan2018).
Areca, a genus of about 76 palm species, includes Areca catechu L. (arecanut) as the only cultivated species. This allotetraploid palm (2n = 32) is widely used in Asian and Pacific cultures for chewing its dried kernel, known as betel nut. In addition, the nuts of other wild species such as Areca triandra (2n = 32) and Areca concinna (2n = 32) are also used locally as masticatories (Venkatasubban Reference Venkatasubban1945; Hu et al. Reference Hu, Huang, Hung, Hsu and Chiang2009). Areca triandra and A. concinna exhibit traits such as suckering habit, smaller nuts and resistance to Phytophthora meadii, making them valuable genetic resources for arecanut improvement (Muralikrishna et al. Reference Muralikrishna, Sharadraj, Gangaraj, Nagaraja, Karun and Rajesh2018; Rajesh et al. Reference Rajesh, Nagaraja, Sabana and Muralikrishna2024). Similarly, related genera such as Normanbya normanbyi and Actinorhytis calapparia are considered important genetic resources. The arecanut palms are broadly classified into tall, semi-tall and dwarf types, based on plant height (Balasimha and Rajagopal Reference Balasimha and Rajagopal2004).
Another significant palm species, the palmyrah palm (Borassus flabellifer L.), is a dioecious and slow-growing tree with a chromosome number of 2n = 36. Widely distributed across South and Southeast Asia, through India, Sri Lanka, Burma, to New Guinea, it serves as a vital source of palm sugar, beverages, edible fruits and wood for construction (Morton Reference Morton1988; Pipatchartlearnwong et al. Reference Pipatchartlearnwong, Juntawong, Wonnapinij, Apisitwanich and Vuttipongchaikij2019).
Globally, coconut production is 66,674 million nuts, with India leading at 20,736 million nuts annually (ICC, Reference International Coconut Community2021). Arecanut production worldwide is 26.33 lakh tonnes, of which India contributes 16.66 lakh tonnes, the highest among producing countries (Anonymous 2022). There are about 140 million palmyrah palms globally, with India accounting for 102 million, of which 51 million palms are distributed in Tamil Nadu alone (Aravindan et al. Reference Aravindan, Sharmila, Tamilnayagan, Sivalingam, Auxcila, Gomathi, Kalpana, Hazaana, Rahale and Prasanthrajan2023; Jerard et al. Reference Jerard, Sumitha, Hebbar and Patel2025). Thus, these palm crops play a vital role in the plantation sector economy at both the national and global levels.
The deoxyribonucleic acid (DNA) content of an unreplicated haploid chromosome set (n), measured in picograms (pg) or base pairs (bp), is known as the C-value or genome size of a species (Greilhuber Reference Greilhuber2005). Nuclear genome size is a fundamental parameter in species classification, offering critical insights into taxonomy and the evolutionary relationships among higher plants. It varies considerably across plant species and serves as a key resource in genetic research, advancing our understanding of genetic diversity, cytology and genomics (Jatt et al. Reference Jatt, Lee, Rayburn, Jatoi and Mirani2019). It also has a substantial impact on growth rates and cell cycles, ultimately influencing an organism’s morphology, physiology and adaptability (Gunn et al. Reference Gunn, Baudouin, Beule, Ilbert, Duperray, Crisp, Issali, Konan and Rival2015). Hence, a database of ploidy levels and DNA content is vital for any crop breeding program.
Flow cytometry (FC) is a widely adopted technique for estimating nuclear DNA content (2C) and ploidy levels in plants. Compared to traditional chromosome counting, FC is faster, more accurate, cost-effective, and allows high-throughput analysis of nuclei from various plant tissues with minimal sample preparation (Qui et al. Reference Qui, Hamernick, Ortiz and Watkins2020). As a result, it has become the method of choice for studying intraspecific and interspecific genome size variation in many plant species, including perennial plantation crops (oil palm [Srisawat et al. Reference Srisawat, Kanchanapoom, Pattanapanyasat, Srikul and Chuthammathat2005], cashew [Aliyu Reference Aliyu2012], date palm [Jatt et al. Reference Jatt, Lee, Rayburn, Jatoi and Mirani2019], coffee [Ortega et al. Reference Ortega, Ortega, Medrano and Cazares2019] and tea [Sharma et al. Reference Sharma, Kaushik and Raina2019]).
However, genome size variation among palm species, including coconut, arecanut, their wild relatives and the palmyrah palm together, has not been extensively studied using FC. Most of the available studies are confined to intraspecific variation of genome size among different accessions in a single palm species. Notably, information on the nuclear genome size of wild Areca relatives such as A. triandra, A. concinna, N. normanbyi and A. calapparia is currently unavailable. These wild taxa represent valuable reservoirs of novel alleles for traits such as disease resistance, stress tolerance and quality improvement, and thus hold significant potential for pre-breeding and genetic enhancement of palms. Similarly, genome size data for B. flabellifer are also lacking.
To bridge these knowledge gaps, the present study aims to estimate genome size variation among conserved palm germplasm accessions using FC. The specific objectives are:
(i) To characterize genome size variation across related palm genera; and (ii) to assess intra- and interspecific genome size variation among accessions of A. catechu and its wild relatives, as well as intraspecific variation among C. nucifera accessions and B. flabellifer (palmyrah) types.
The findings are expected to enhance understanding of genome size diversity, evolutionary relationships and cytogenetic differentiation among palm species, while providing baseline genomic information essential for plant genetic resource characterization, conservation, pre-breeding and whole-genome sequencing initiatives.
Materials and methods
Plant material
A total of 29 samples were analysed in this study, comprising 20 accessions of coconut (11 talls, 4 dwarfs and 5 cross combinations) (Table 1), 3 accessions of arecanut, 2 related species of A. catechu, viz., A. concinna and A. triandra, 2 related genera of A. catechu, viz., N. normanbyi and A. calapparia and 2 types of palmyrah, viz., male and female palms (Table 2). The coconut and palmyrah palm samples were sourced from the National Field Gene Bank at the Indian Council of Agricultural Research–Central Plantation Crops Research Institute (ICAR-CPCRI) in Kasaragod, Kerala, India (located at 12°18′ N latitude and 75°00′ E longitude, with an elevation of approximately 10.7 metres above mean sea level). Arecanut and its related species and genera were collected from the field gene banks at ICAR-CPCRI, Regional Station, Vittal, Karnataka, India (12°15′ N latitude and 75°25′ E longitude). All the palms were maintained under irrigated conditions, with fertilizers applied according to standard recommendations.
List of coconut accessions, their place of collection, habit and key characters
Table 1 Long description
Palm entries are compared by species or accession, growth habit, collection location, and distinguishing traits such as crown posture, suckering, flowering, and nut color and shape. Arecanut accessions vary from tall VTL11 from Indonesia with a partially drooping crown, to semi-tall VTL3 from China with early flowering and crinkled leaf tips, to dwarf VTL56 from India with short internodes, an erect crown, reduced leaf length, and small oval to round yellow to orange-red nuts. Areca concinna from Sri Lanka is dwarf and suckering with red oblong nuts, while Areca triandra from Indonesia and Mauritius is semi tall to tall, suckering, and has red to pinkish red oblong nuts. Normanbya normanbyi from Australia is semi tall and non-suckering with red pear-shaped nuts, and Actinorhytis calapparia from Saigon is tall and non-suckering with crimson red, oval, bold nuts. Palmyrah palm entries from India are both tall, with male plants described by stout inflorescences with multiple branches and small sessile staminate flowers, and female plants by fewer spikes with solitary, larger spherical flowers. Comparisons are descriptive rather than quantitative, and some traits may vary within populations beyond what is listed for each entry.
List of Areca species, wild relatives and palmyrah palm types, their place of collection, habit and key characters
Sample preparation
For the analysis, fresh, tender and disease-free leaf samples were collected from each of the identified palms. A total of six samples were analysed for each accession. The collected leaf samples were placed in individual poly covers and immediately transported to the laboratory and stored at 4°C until further use.
Nuclei were isolated using Galbraith buffer, modified with the addition of polyvinylpyrrolidone (PVP) and β-mercaptoethanol to eliminate the inhibitory effects of phenolic compounds present in the palm leaf samples on staining of nuclei with propidium iodide (PI). The leaf sample size and incubation period after sample preparation followed the protocol standardized for coconut by Ranjini et al. (Reference Ranjini, Niral and Chandran2020). The nuclei isolation buffer used in this study contained MgCl2, sodium carbonate, 3-(N-morpholino)propanesulfonic acid (MOPS) and Triton X-100 (Galbraith buffer), supplemented with 1% PVP-10 and 15 mM β-mercaptoethanol. Fresh buffers were prepared and used for sample preparation each time.
A total of 100 mg of fresh leaf tissue of each sample was chopped together with 50 mg of leaf tissues from the reference standard, Pisum sativum cv. Citrad in a Petri dish containing 1.5 mL of cold isolation buffer (Galbraith buffer) using a razor blade for approximately 90 seconds. Seeds of the reference standard were procured from the Institute of Experimental Botany, Olomouc, Czech Republic, and have a known genome size of 2n = 9.09 pg (Dolezel et al., Reference Doležel, Greilhuber and Suda2007). The nuclear suspension was filtered through a 50 µm filter to remove larger leaf debris, followed by filtration through a 20 µm nylon net filter to isolate intact nuclei. To the filtered lysate, 2.5 µL of 10 mg/mL DNase-free RNase A and PI (50 µg/mL) were added. The mixture was then incubated for 15 minutes at 4°C in darkness before analysis. RNase A was added to prevent staining of double-stranded RNA, as PI is not specific to DNA, which could otherwise result in inaccurate estimation of nuclear DNA content.
FC analysis
The relative fluorescence intensities of PI-stained nuclei were measured using a BD Accuri C6 flow cytometer (Becton Dickinson, USA) with laser excitation at 488 nm (blue laser). Prior to analysis, the instrument was calibrated using BD fluorescent calibration beads (eight-peak and six-peak beads) to ensure proper alignment. Samples were analysed at a flow rate of approximately 100 nuclei per second, with a minimum of 10,000 nuclei acquired per sample. Data were analysed using BD C6 Flow Plus software. The reliability of the results was assessed by calculating the average coefficient of variation (CV) for G1 peaks, with CV values below 3% considered reliable (Galbraith et al. Reference Galbraith, Harkins, Maddox, Ayres, Sharma and Firoozabady1983).
For the analysis of samples of A. concinna, A. triandra, N. normanbyi, A. calapparia and B. flabellifer, external standardization was employed. This was due to the fact that internal standardization using pea as a reference resulted in overlapping histogram peaks and elevated CV values. To minimize experimental error, identical sample preparation conditions were applied to both the palm samples and the external reference standard (pea). The reference standard was analysed after each sample run to ensure accuracy and consistency.
Genome size estimation
The mean fluorescence units for both the unknown samples and the reference standard were recorded based on the positions of the G0/G1 histogram peaks (non-replicating phase of the cell cycle), and nuclear DNA content was calculated following the method of Dolezel et al. (Reference Dolezel, Bartos, Voglmayr and Greilhuber2003).
\begin{align*}2C\,value\,of\,the\,sample &= \frac{{\,Peak\,value\,of\,the\,unknown\,sample}}{{Peak\,value\,of\,the\,reference\,sample}}\\
&\quad x\,Reference\,2C\,DNA\,content\end{align*}The genome size calculated was expressed in pg/2C and converted to megabase pairs (Mbp) by a conversion factor of 1pg = 978 bp (Dolezel et al. Reference Dolezel, Bartos, Voglmayr and Greilhuber2003).
Statistical analysis
The differences between the 2C DNA content among palm species were statistically tested using IBM SPSS version 2.0 software. Cluster analysis was performed using MVSP 3.22 statistical software.
Results
Interspecies variations of genome size among palm species
The 2C DNA content of seven palm species was analysed using FC, based on the relative fluorescence intensity of each sample. The mean genome size varied significantly among the species, ranging from 5.56 pg/2C in A. calapparia to 12.70 pg/2C in B. flabellifer, representing a 2.28-fold difference.
The average genome size of coconut is 5.86 pg/2C, which is slightly higher than the 2C value of the palm species A. calapparia (2C = 5.56 pg). The mean 2C DNA content reported for A. catechu is 6.46 pg, while related species showed values of 8.50 pg in A. concinna and 8.72 pg in A. triandra, representing increases of 20.50% and 22.70%, respectively. Further, among the related genera of A. catechu, 2C DNA contents of 7.40 pg and 5.56 pg were observed in N. normanbyi and A. calapparia, respectively, which are 9.50% higher and 8.90% lower than the mean genome size of A. catechu. Overall, these findings highlight substantial genome size variation among palm species.
Intraspecies variations of genome size in palm species
In coconut, genome size variation was assessed across 20 conserved germplasm accessions, including 11 tall, 4 dwarf and 5 cross combinations originating from South and Southeast Asia and the Pacific Ocean islands, representing diverse morphological traits (Table 1). The 2C DNA content ranged from 5.66 pg in the dwarf accession Chowghat Orange Dwarf (COD) to 6.16 pg in the tall accession San Ramon Tall (SNRT), with a mean value of 5.86 pg (excluding cross combinations), corresponding to an estimated haploid (1Cx) genome size of 2865.54 Mb. The extent of variation within the tall and dwarf groups was approximately 6.49% and 1.04%, respectively, indicating lower variability among dwarf accessions. Cross combinations exhibited intermediate genome sizes, ranging from 5.80 to 6.01 pg/2C (Table 3; Figure 1).
FC histograms showing nuclear DNA content of C. nucifera (coconut) accessions using P. sativum as an internal reference standard. The X-axis (FL2-A) represents fluorescence intensity proportional to DNA content, and the Y-axis (Counts) indicates the number of nuclei. Sample peaks correspond to (a) Chowghat Orange Dwarf (COD), (b) West Coast Tall (WCT) and (c) West Coast Tall × Chowghat Orange Dwarf, with the reference peak representing P. sativum cv. Citrad.
Nuclear DNA content of palm species determined by FC
Table 3 Long description
Nuclear DNA content and estimated genome size are reported for 29 palm entries using flow cytometry, with means and standard deviations for 2C DNA (pg), 1C DNA (pg), and 1C genome size in megabase pairs. Palmyrah palm shows the highest values: male at 12.70 pg per 2C (6.35 pg per 1C; 6210.30 Mbp) and female at 12.59 pg per 2C (6.24 pg per 1C; 6109.53 Mbp). Coconut accessions and hybrids are tightly grouped, ranging from 5.66 to 6.16 pg per 2C, corresponding to about 2768.85 to 3015.58 Mbp per 1C; the lowest coconut entry is COD (5.66 pg per 2C; 2768.85 Mbp) and the highest is SNRT (6.16 pg per 2C; 3015.58 Mbp). Arecanut-related entries vary more widely: Actinorhytis calapparia is lowest at 5.56 pg per 2C (2722.23 Mbp), cultivated arecanut lines are around 6.32 to 6.60 pg per 2C (about 3092.32 to 3231.54 Mbp), and other species reach 8.50 to 8.72 pg per 2C (about 4158.58 to 4264.76 Mbp). Across all entries, higher 2C DNA values align with higher 1C DNA and higher megabase-pair estimates. Letter groupings next to means indicate which entries differ statistically, and a reported critical difference at the 0.05 level is provided for each measurement column.
* Different letters indicate statistical significance.
In A. catechu, intraspecific variation was evident among the accessions studied. The 2C DNA content ranged from 6.32 pg in VTL-56 to 6.60 pg in VTL-11, with VTL-3 showing an intermediate value of 6.45 pg. This represents a 4.24% difference between the highest and lowest values, confirming the presence of intraspecific genome size variation. The average haploid (1C) genome size was estimated to be 3159.88 Mb (Table 3; Figure 2).
FC histograms showing nuclear DNA content of A. catechu (arecanut) accessions using P. sativum as an internal reference standard. The X-axis (FL2-A) represents fluorescence intensity proportional to DNA content, and the Y-axis (Counts) indicates the number of nuclei. Sample peaks correspond to (a) VTL-11, (b) VTL-3 and (c) VTL-56, with the reference peak representing P. sativum cv. Citrad.
In Palmyrah, male and female palms exhibited 2C DNA contents of 12.70 and 12.59 pg, respectively, corresponding to a difference of 1.78%. The average haploid genome size was estimated at 6159.91 Mb (Table 3; Figure 3).
FC histograms showing nuclear DNA content of selected palm species using P. sativum cv. Citrad as an external reference standard. The X-axis (FL2-A) represents fluorescence intensity proportional to DNA content, while the Y-axis (Counts) indicates the number of nuclei. Histograms correspond to (a) P. sativum (reference), (b) A. triandra, (c) A. concinna, (d) N. normanbyi, (e) A. calapparia, (f) B. flabellifer (male) and (g) B. flabellifer (female), where peak positions were used to estimate genome size relative to the external standard.
Figure 3 Long description
The image A showing a flow cytometry histogram titled by the sub-image label a. The vertical axis label is Count. The horizontal axis label is FL2-A. The horizontal axis tick labels are 0, 500,000, 1,000,000 and 1,500,000. A single tall peak is plotted near the low end of FL2-A. Text inside the plot reads M2 98.3 percent. The image B showing a flow cytometry histogram titled by the sub-image label b. The vertical axis label is Count. The horizontal axis label is FL2-A. The horizontal axis tick labels are 0, 1,000,000, 2,000,000 and 2,969,481. A single tall peak is plotted near the low end of FL2-A. Text inside the plot reads M2 99.2 percent. The image C showing a flow cytometry histogram titled by the sub-image label c. The vertical axis label is Count. The horizontal axis label is FL2-A. The horizontal axis tick labels are 0, 1,000,000, 2,000,000 and 2,973,842. A single tall peak is plotted near the low end of FL2-A. Text inside the plot reads M1 91.0 percent. The image D showing a flow cytometry histogram titled by the sub-image label d. The vertical axis label is Count. The horizontal axis label is FL2-A. The horizontal axis tick labels are 0, 1,000,000, 2,000,000 and 2,973,842. A single tall peak is plotted near the low end of FL2-A. The image E showing a flow cytometry histogram titled by the sub-image label e. The vertical axis label is Count. The horizontal axis label is FL2-A. The horizontal axis tick labels are 0, 500,000, 1,000,000 and 1,598,170. A single tall peak is plotted near the low end of FL2-A. Text inside the plot reads M2 98.6 percent. The image F showing a flow cytometry histogram titled by the sub-image label f. The vertical axis label is Count. The horizontal axis label is FL2-A. The horizontal axis tick labels are 0, 1,000,000, 2,000,000 and 3,576,224. A single tall peak is plotted near the low end of FL2-A. Text inside the plot reads M2 94.5 percent. The image G showing a flow cytometry histogram titled by the sub-image label g. The vertical axis label is Count. The horizontal axis label is FL2-A. The horizontal axis tick labels are 0, 2,000,000 and 4,397,145. A single tall peak is plotted near the low end of FL2-A. Text inside the plot reads M2 94.8 percent. Across a to g, each histogram shows one dominant peak near the low end of FL2-A. The annotated percentages are M2 98.3 percent in a, M2 99.2 percent in b, M1 91.0 percent in c, M2 98.6 percent in e, M2 94.5 percent in f and M2 94.8 percent in g.
Cluster analysis
A dendrogram was constructed using similarity coefficient values derived from Unweighted Pair Group Method with Arithmetic mean (UPGMA) analysis to evaluate clustering patterns among the studied palm species based on genome size variation in conserved genetic resources (Figure 4). The analysis resolved two major clusters, designated as Cluster A and Cluster B. Cluster A was relatively small and consisted exclusively of B. flabellifer (male and female palms).
Dendrogram showing clustering of palm species based on Euclidean distance derived from 2C nuclear DNA content estimated by FC. Species are grouped according to genome size similarity, reflecting their relative genetic relationships.
Figure 4 Long description
A dendrogram displays the clustering of various palm species based on Euclidean distance, derived from 2C nuclear DNA content. The horizontal axis represents the Euclidean distance, ranging from 9.6 to 0.0. The dendrogram is constructed using the UPGMA method. At the top, Borassus flabellifer (female and male) forms a distinct cluster. Below, Nannorrhops ritchiana and Areca triandra are grouped together. Further down, Areca catechu (with various labels such as VTL155, VTL158, VTL151) and Areca concinna are clustered. The largest cluster includes multiple Cocos nucifera entries, each labeled with different codes like RTC, TPT, COT and others, indicating different samples or varieties. Acrocomia aculeata and Actinorhytis calapparia are also included in this cluster. The dendrogram visually represents the genetic relationships and genome size similarities among the studied palm species.
Cluster B, the larger group, comprised the remaining 27 samples and was further subdivided into 2 subclusters, B1 and B2. Subcluster B1 included N. normanbyi, A. triandra and A. concinna, which exhibited similar genome sizes. Within B1, the arecanut-related species are grouped together to form a distinct subgroup (B1a).
Subcluster B2 was further divided into B2a and B2b. The accessions of A. catechu were grouped within subgroup B2a. The second subgroup, B2b, comprised all coconut accessions along with A. calapparia. In comparison to Clusters A and B1, the samples in Cluster B2 generally exhibited lower genome size values.
Within B2b, coconut accessions such as SNRT, PHOT, AGT and WCT formed a distinct group characterized by relatively higher genome sizes among the coconut samples analysed. Notably, all four dwarf coconut accessions formed a uniform and distinct subgroup, irrespective of their geographic origin, within the broader cluster of tall and cross combinations.
Discussion
Genome size data in palms remain limited, with most studies restricted to single-species or intraspecific variation. The present study provides a comparative assessment across conserved palm germplasm species, including C. nucifera, A. catechu and B. flabellifer, and underutilized taxa such as A. triandra, A. concinna, N. normanbyi and A. calapparia, thereby establishing a broader genomic framework for Arecaceae and strengthening palm genetic resource characterization.
Clear interspecific variation in genome size was observed among diploid palm species despite a conserved chromosome number within the family. The comparatively larger genome size of palmyrah relative to coconut and arecanut reflects substantial genomic diversity within Arecaceae, consistent with earlier reports of wide C-value variation despite a relatively narrow chromosome number range (2n = 26–36) across approximately 2500 species (Swaminathan and Nambiar Reference Swaminathan and Nambiar1961; Leitch et al. Reference Leitch, Beaulieu, Chase, Leitch and Fay2010; Cornejo et al. Reference Cornejo, Palomino, Mendez and Dirzo2012). Slightly higher DNA estimates reported using Feulgen microdensitometry compared to the present FC values may be attributed to methodological differences, including interference from tannins affecting staining efficiency (Greilhuber Reference Greilhuber1986; Roser et al. Reference Roser, Johnson and Hanson1997). Among Areca-related taxa, higher genome sizes observed in A. triandra, A. concinna and N. normanbyi, and the lower value in A. calapparia, indicate substantial genomic divergence from cultivated A. catechu. Although A. catechu and A. triandra share the same chromosome number (2n = 32), differences in chromosomal structure, including longer and more symmetrical chromosomes in A. triandra, may account for the observed genome size variation. This supports earlier cytological evidence (Bavappa and Raman Reference Bavappa and Raman1965; Bavappa et al. Reference Bavappa, Nair and Ratnambal1975) and reported associations between chromosome length and genome size in palms (Cornejo et al. Reference Cornejo, Palomino, Mendez and Dirzo2012). These findings highlight genome size as a complementary trait for characterizing conserved germplasm and differentiating closely related species.
In coconut, significant intraspecific variation was observed among conserved accessions, with dwarf types exhibiting lower and more uniform genome sizes and tall types showing greater variability. This pattern is consistent with their breeding systems, where cross-pollinated tall types tend to exhibit higher genetic variability compared to relatively uniform dwarf types (Narayana and John Reference Narayana and John1949; Eilam et al. Reference Eilam, Anikster, Millet, Manisterski, Assif and Feldman2007). Comparable genome size ranges have been reported in earlier studies (Neto et al. Reference Neto, Pereira, Geronimo, Azevedo, Ramos and Pereira2016; Ranjini et al. Reference Ranjini, Niral and Chandran2020), and such variation is likely associated with differences in repetitive DNA content and chromosomal organization (Raveendranath and Ninan Reference Raveendranath and Ninan1973; Bennetzen et al. Reference Bennetzen, Ma and Devos2005; Sharma et al. Reference Sharma, Kaushik and Raina2019). Analysis of coconut cross combinations further demonstrated that most hybrids exhibited intermediate genome sizes, while some showed elevated DNA content relative to parental means, suggesting possible genomic interactions or instability. Similar deviations have been reported in maize and oil palm (Rayburn et al. Reference Rayburn, Biradar, Bullock and Mcmurphy1993; Madon et al. Reference Madon, Phoon, Clyde and Mohd2008). These findings underscore the potential of genome size estimation as a rapid proxy for hybrid identification, particularly in perennial crops where conventional methods are time-consuming.
In arecanut, the observed intraspecific variation among conserved accessions is consistent with earlier reports in palms (Madon et al. Reference Madon, Phoon, Clyde and Mohd2008; Camillo et al. Reference Camillo, Leao, Alves, Formighieri, Azevedo, Nunes, Capdeville, Mattos and Souza2014; Jatt et al. Reference Jatt, Lee, Rayburn, Jatoi and Mirani2019; Kavyashree et al. Reference Kavyashree, Nagaraja, Ranjini, Niral and Rajesh2023). In palmyrah, male and female palms are morphologically indistinguishable except for their inflorescences (Pipatchartlearnwong et al. Reference Pipatchartlearnwong, Juntawong, Wonnapinij, Apisitwanich and Vuttipongchaikij2019). However, only minor differences in genome size were detected between sexes, indicating limited applicability for sex discrimination.
Cluster analysis revealed that grouping was primarily driven by genome size and species identity rather than geographic origin. Palmyrah formed a distinct cluster due to its larger genome size, while Areca species and coconut accessions formed separate clusters. The distinct clustering of A. catechu from its wild relatives aligns with earlier reports of genetic divergence between cultivated and wild taxa (Nagaraja et al. Reference Nagaraja, Ananda and Rajesh2022; Rajesh et al. Reference Rajesh, Nagaraja, Sabana and Muralikrishna2024). Similarly, the uniform clustering of dwarf coconut accessions irrespective of origin supports their narrow genetic base and probable origin from a limited number of ancestral palms (Perera et al. Reference Perera, Russell, Provan and Powell2000; Teulat et al. Reference Taulat, Aldam, Trehin, Lebrun, Barker, Arnold, Karp, Baudouin and Rognon2000; Arunachalam et al. Reference Arunachalam, Jerard, Damodaran and Ratnambal2005; Rajesh et al. Reference Rajesh, Sabana, Rachana, Rahman, Jerard and Karun2015).
The present study shows that genome size is a useful parameter for distinguishing palm species and assessing variation within conserved germplasm. The patterns observed across palm species indicate their value as a complementary tool for species identification and hybrid validation. By using a comparative approach, this study demonstrates the practical importance of genome size estimation in palms. These findings support the use of FC-based genome size analysis in routine germplasm evaluation and breeding programs.
Conclusion
This study provides the first comprehensive assessment of intra- and interspecific genome size variation in conserved palm germplasm using FC. The results confirm its effectiveness as a rapid and reliable tool for germplasm characterization and species differentiation at early developmental stages. Genome size variation observed in coconut and arecanut, along with clear divergence between cultivated A. catechu and its wild relatives, underscores its potential application in hybrid verification and pre-breeding, where conventional identification methods are time-consuming and often unreliable in palms. The baseline genome size data generated for underutilized and wild palm taxa represent a valuable resource for future genomic studies, including whole-genome sequencing, comparative analysis and molecular marker development, thereby contributing to improved conservation, utilization and genetic improvement of economically important palm species.
Acknowledgements
The authors acknowledge the Director, ICAR-CPCRI, Kasaragod, for providing the necessary research facilities.
Author contributions
T.N.R.: conceptualization, data acquisition, interpretation of data, writing of the original draft of the manuscript; V.N.: resources provided, review and editing of the manuscript; N.R.N.: sampling for analysis and review and editing of the manuscript; Y.D.: data interpretation and review and editing of the manuscript; T.N.M.: sampling for analysis and data analysis; U.K.G.: sampling for analysis. All authors contributed to the revision of the manuscript and approved the final version.
Data availability statement
All data generated during this study are included in this article.
Funding statement
This work was supported by the Indian Council of Agricultural Research–Central Plantation Crops Research Institute (ICAR-CPCRI), Kasaragod, under the Institute fund.
Competing interests
Authors have no conflict of interest with reference to interests of a financial or personal nature.
