Introduction
Polyploidy, the presence of multiple chromosome sets, plays an essential role in the evolution, adaptation and diversification of plant species. In the genus Morus (mulberry), a key crop in moriculture, polyploidy contributes to remarkable cytotype diversity, ranging from 2n = 14 to 2n = 308 (He et al. Reference He, Zhang, Qi, Zhao, Tao, Yang, Lee, Wang, Cai, Li, Lu, Liao, Luo, He, Tan, Xu, Li, Zhao, Jia, Fu, Zeng, Gao, Ma, Liang and Xiang2013; Kruthika et al. Reference Kruthika, Rukmangada and Naik2023; Mondal et al. Reference Mondal, Antony, Gnanesh, Thanavendan, Ravikumar, Sreenivasa, Doss and Vijayan2023a). Several attempts have been made to establish a link between phenotypic characteristics and ploidy level (Gnanesh et al. Reference Gnanesh, Raju, Arunakumar, Manojkumar, Pradeep, Bhavya, Sowbhagya, Burji, Mogili and Sivaprasad2023; Kruthika et al. Reference Kruthika, Rukmangada and Naik2023; Mondal et al. Reference Mondal, Dubey, Ramesha, Vijayan, Naik and Moorthy2025); however, no study has yet demonstrated the effect of ploidy on plant function in mulberry trees.
Ploidy level is acknowledged to influence elements including cell size, development rate, photosynthesis and plant architecture; nonetheless, recent findings suggest that higher ploidy levels can slow cell division, potentially affecting biomass accumulation and plant vigour (Doyle and Coate, Reference Doyle and Coate2019; Šmarda et al. Reference Šmarda, Veselý, Šmerda, Bureš, Knápek and Chytrá2016). However, the specific force of polyploidy on the fitness of tree species remains little understood (Ræbild et al. Reference Ræbild, Anamthawat-Jónsson, Egertsdotter, Immanen, Jensen, Koutouleas, Martens, Nieminen, Olofsson, Röper and Salojärvi2024), which represents a significant ecophysiological aspect. Additionally, for tree species, an open field experiment is a prerequisite to estimate a consistent outcome; this adaptation is at the discretion of the plant breeder (Mondal et al. Reference Mondal, Kumar and Gnanesh2023b). In this context, our study specifically aims to investigate the increase in ploidy effects in mulberry tree plants, with the objective of assessing how variation across different ploidy levels can be utilized for breeding, yield improvement and conservation.
Materials and methods
Study area and experimental conditions
About four-year-old clonally propagated eight mulberry trees of accessible cytotypes, including diploid (2x = 28) Morus indica L., triploid (3x = 42) Morus alba L. and tetraploid (4x = 56) Morus laevigata Wall. synonyms of Morus macroura (4x = 56), hexaploid (6x = 84) hexaploid Morus tiliaefolia Makino. synonym of Morus cathayana Hemsl. and Morus serrata Roxb., as well as 22-ploid Morus nigra L. (22x = 308), were selected for phenotypic observations in the field (open environment) condition (Fig. 1). All plants were maintained at the field genebank of CSB-Central Sericultural Germplasm Resources Centre (CSGRC), Hosur, Tamil Nadu, India (12.7198° N, 77.8107° E). Plants are maintained as per the SOP guideline of CSGRC, and the mean temperature, rainfall and humidity of the period of plant growth are 23.89 ± 2.12°C, 65.82 ± 17.22°C and 58.54 ± 85.86 mm, respectively. Due to poor growth response from pruning, especially at higher ploidy levels, we have considered three uniform plants from each cytotype that are well synchronized in growth for further study. To capture the maximum expressivity of parameters at the early stage of development under well-moistened conditions, observations were recorded from the fully spread fourth-positioned leaf of a one-month-old branch. To examine the influence of different cytotypes on traits, morphological, anatomical, biomass metrics and physiological gas exchange parameters were recorded. Three technical replicates with three biological replicates for each parameter were considered.
Morphological and cytogenetic series of mulberry cytotypes. Left to right: diploid (2x = 28), triploid (3x = 42), tetraploid (4x = 56), hexaploid (6x = 84) and 22-ploid (22x = 308). Top to bottom: plant habit; representative mature single leaves (adaxial view; scale bar: 2 cm); stomatal impressions showing guard-cell enlargement and frequency (scale bar: 10 µm). Metaphase plate with scale bar: 5 µm for diploid and triploid, where for the remaining cytotypes the scale bar is 10 µm.
Figure 1 Long description
The image presents a morphological and cytogenetic series of mulberry cytotypes. From left to right, it includes diploid (2n equals 28), triploid (2n equals 42), tetraploid (2n equals 56), hexaploid (2n equals 84) and 22-ploid (2n equals 308). Each cytotype is shown in four rows: the first row displays the plant habit, the second row shows representative mature single leaves in adaxial view, the third row illustrates stomatal impressions with guard-cell enlargement and frequency and the fourth row presents metaphase plates. The scale bars for stomatal impressions are 10 micrometers and for metaphase plates, they are 5 micrometers for diploid and triploid and 10 micrometers for the remaining cytotypes.
Chromosome study
To depict the apparent variation of chromosome number, a metaphase chromosome study was carried out using Mondal et al. (Reference Mondal, Antony, Gnanesh, Thanavendan, Ravikumar, Sreenivasa, Doss and Vijayan2023a).
Morphological characterization
Morphological parameters, including leaf length (LL), leaf width (LW), petiole length (PL) and leaf area (LA), were studied. LA was calculated using ImageJ (https://imagej.net/ij/download.html) software by processing the captured images.
Anatomical characterization
Anatomical studies were performed using a CILIKA microscope (https://www.medprimetech.com) equipped with an iPad attachment for live observation and image capture. The stomatal parameters of the leaf were studied by removing the peels from the lower surfaces. Observations focused on stomatal length (SL), stomatal width (SW), guard cell volume (GCV), stomatal frequency (SF) and chloroplast number per stomatal guard cell (CS). To calculate the GCV, the following formula was used: 10−3 × 1/4 × π × SL ×(1/2 × SW)2 (pl).
Leaf biomass assessment
Leaf fresh weight (FW) was measured immediately using an analytical balance, and images were captured for area estimation. After imaging, leaves were air-dried at room temperature until thoroughly dry, and their dry weight (DW) was recorded. For the estimation of leaf mass per unit area (LMA), the ratio of DW to LA was recorded.
Leaf gas exchange study
Portable photosynthesis systems (LI-6400XT, LI-COR, Inc., Lincoln, NE, USA) were used to measure photosynthetic rate (Pn), stomatal conductance (gs), intercellular CO₂ (Ci), transpiration rate (E), and intrinsic water use efficiency (iWUE = Pn/E) from 10:00 AM to 12:00 PM.
Statistical analysis
The descriptive statistics were calculated using Microsoft Excel 2021. Multiple comparisons using one-way ANOVA followed by Tukey Honestly Significant Difference (HSD) (ScatterPlotBar v.0.7.0; https://scatterplot.bar/index.html) were carried out. Further multivariate principal component analysis (PCA) and correlation analysis were carried out using SRPlot (https://www.bioinformatics.com.cn/en; Tang et al. Reference Tang, Chen, Huang, Zhang, Zeng, Zhang, Wu and Wang2023). The plasticity index (PI) was estimated to represent the ratio of the raw trait values from different polyploid (3x, 4x, 6x, and 22x) to diploid (2x). To depict the expressivity and plasticity of the studied parameter across the cytotypes, heatmap analysis was performed using TBtools (Chen et al. Reference Chen, Chen, Zhang, Thomas, Frank, He and Xia2020).
Results
Mulberry exhibits an extraordinary cytogenetic spectrum – spanning diploid (2n = 2x = 28) to 22-ploid (2n = 22x = 308) – yet the mechanisms by which polyploidization occurs remain unknown. Additionally, the influence of such cytotypes on the trait expressivity and plasticity has not yet been studied. Therefore, in the present study, a field experiment was undertaken to investigate the expressivity and plasticity of cytotype-specific parameters, particularly those associated with cell division, cell size and biomass metrics.
The aerial vegetative part, shoot/branch structure, leaf shape and size, stomatal structure and metaphase plates correspond to the cytotypes presented in Fig. 1. Following data standardization, the mean values were subjected to multivariate analysis and visualized through a PCA and heatmap chart (Fig. 2a–c; Table S1). The substantial variation of the studied parameters was detected following the Tukey HSD test (Fig. S1–3). The significant (p < 0.05) correlation of studied traits was also depicted across cytotypes (Fig. 2b). The multivariant PCA represents a considerable difference with a cumulative variation of 79.7% across the cytotypes, which is contributed by component 1 (PC1: 46.3%) and component 2 (PC2: 33.4%), respectively (Fig. 2a). In Fig. 2a, tetraploids (4x) have the most advantageous cytotype, as shown by the positive correlation of attributes like LL, LW and LA. Triploid (3x) showed a positive correlation with PL and photosynthetic rate (Pn). This, in turn, PCA reveals a vigorous and balanced expression of polyploid advantage, specifically in tetraploids (4x), followed by triploids (3x). Conversely, the diploid (2x) exhibited a correlation with both the stomatal frequency (SF) and the iWUE. Therefore, despite the greater number of small-sized stomata, the diploid cytotype maintains a stable equilibrium between photosynthetic rate (Pn) and transpiration rate (E). In contrast, PCA also indicates the loss of growth superiority at higher ploidy levels, including hexaploid Ms (6x) and 22-ploid (22x).
Multivariate analysis using principal component analysis (PCA) and heatmap. (a) scattered distribution of cytotypes and measured parameters along PC1 and PC2, respectively. (b) Correlations among the studied parameters. Heatmaps parameterize the (c) expressivity and (d) plasticity of measured parameters across cytotypes. The blue and yellow stars (*) represent the considerable expressivity and plasticity of 4x and 22x, respectively (LL, leaf length; LW, leaf width; PL, petiole length; LA, leaf area; FW, fresh weight; DW, dry weight; LMA, leaf mass per unit area; SL, stomatal length; SW, stomatal width; SF, stomatal frequency; CS, chloroplast number per stomatal guard cell; GCV, guard cell volume; Pn, photosynthetic rate; gs, stomatal conductance; Ci, intercellular CO₂; E, transpiration rate; and iWUE, intrinsic water use efficiency).
Figure 2 Long description
The image consists of four panels related to multivariate analysis using principal component analysis and heatmaps. Panel (a) shows a scattered distribution of cytotypes and measured parameters along PC1 and PC2, with PC1 accounting for 46.3 percent and PC2 for 33.4 percent of the variation. Different cytotypes are represented by colored shapes: diploid (2x) in red, triploid (3x) in blue, tetraploid (4x) in green, hexaploid (Ms) in purple and 22-ploid (22x) in orange. Panel (b) illustrates correlations among studied parameters, with a triangular matrix showing correlation values ranging from negative 0.8 to positive 0.8. Panels (c) and (d) are heatmaps parameterizing expressivity and plasticity of measured parameters across cytotypes. Panel (c) highlights expressivity with blue stars indicating significant expressivity for tetraploids (4x), while panel (d) highlights plasticity with yellow stars indicating significant plasticity for 22-ploid (22x). Parameters include leaf length, leaf width, petiole length, leaf area, fresh weight, dry weight, leaf mass per unit area, stomatal length, stomatal width, stomatal frequency, chloroplast number per stomatal guard cell, guard cell volume, photosynthetic rate, stomatal conductance, intercellular carbon dioxide, transpiration rate and intrinsic water use efficiency.
Most interestingly, a distinguishable variation was observed in cytotypes belonging to hexaploid (6x). For instance, cytotype 6x (Mt) is positively correlated with fresh (FW) and dry leaf weight (DW); however, LMA is more closely associated with cytotype 6x (Ms). Moreover, the significant effect of enlarged cell, cellular stoichiometry balance, and biomass was observed in 22-ploid, as evident from significant association with SL, SW, GCV, chloroplast number per stomata (CS), and LMA. Parameters including intercellular CO₂ (Ci) and stomatal conductance (gs) showed a higher association with 22-ploid (22x), followed by tetraploid (4x) (Fig. S2). Overall, the leaf gas exchange study suggests that none of the parameters showed consistency with an increased level of ploidy. Further, the estimated PI confers that (Fig. 2d) only cell size (but not number) associated parameters, including SL, SW, and GCV, consistent with 22-ploid. On the other hand, parameters that are influenced by elongation as a cumulative contribution of both cell number and size significantly influenced LL, LW, LA, etc. in tetraploid (4x).
Discussion
To delineate six cytotypes of the genus Morus in an open environment, we have analyzed functional characteristics influenced both directly and indirectly by the nucleotypic effect. Measured expressivity of parameters is presented in heatmaps (Fig. 2c), which illustrates that beyond tetraploid, key parameters showed downsizing with increasing ploidy levels, particularly in higher ploidy. However, there are no standardized metrics available within the community to evaluate/compare the polyploids of cross-species. Therefore, we assessed the pairwise ratio between polyploid cytotypes (3x, 4x, 6x, and 22x) and close relatives, diploid (2x), using the PI (Fig. 2d). It seems that the index ratio illustrates the apparent effects of cytotypes by masking the various evolutionary histories and adaptations at the species level (Šmarda et al. Reference Šmarda, Klem, Knápek, Veselá, Veselá, Holub, Kuchař, Šilerová, Horová and Bureš2023). The estimated PI confirmed that the lower limit of cell size and rate-limited attributes influenced the loss of growth superiority of the higher ploidy level. One possible interpretation of the observed consistency or trend appears to be phylogeny independence, which could be regarded as an actual consequence of polyploidy.
Higher polyploids employed a higher gs (Fig. 2c), likely to meet physiological needs like increasing nutrient uptake, especially of nitrogen (N), which results from enlarged root cells being unable to transport or absorb nutrients efficiently due to their smaller surface-to-volume ratio when water is not a limiting factor (Šmarda et al. Reference Šmarda, Klem, Knápek, Veselá, Veselá, Holub, Kuchař, Šilerová, Horová and Bureš2023). Specifically in higher polyploids, the genome size correlated with the cell size/volume, which reduces surface-to-volume ratios and enlarged stomata enhance gs, but the net effect is an inverse relationship between genome size and maximum photosynthetic rate across vascular plants (Roddy et al. Reference Roddy, Théroux-Rancourt, Abbo, Benedetti, Brodersen, Castro, Castro, Gilbride, Jensen, Jiang and Perkins2020).
Overall, the phenotyping survey shows that trait responses to increasing chromosome sets are nonlinear, especially parameters that are influenced by cell division, cell size as well and biomass. Therefore, mulberry polyploid cytotypes are involved in the loss of growth superiority alongside the genome downsizing (Kruthika et al. Reference Kruthika, Rukmangada and Naik2023). Which in turn relays that the rate-limited attributes are the determining factor for loss of growth superiority in higher polyploid cytotypes, especially hexaploid and 22-ploid. Such decoupling echoes the ‘large-genome constraint’ hypothesis, which predicts that expanded DNA content slows S-phase and mitosis, thereby imposing a rate-limiting ceiling on growth (Knight et al. Reference Knight, Molinari and Petrov2005; Van’t Hof and Sparrow, Reference Van’t Hof and Sparrow1963). Recent findings further indicate that bigger genomes restrict trait flexibility (Bhadra et al. Reference Bhadra, Šmarda, Bureš and Horová2023; Knight et al. Reference Knight, Molinari and Petrov2005), yielding diminished returns beyond the tetraploid level. Furthermore, guard-cell volume and chloroplast number scaled more proportionally with higher polyploidy, fixed a lower limit of size and consumed higher N per stomatal cell, because biogenesis of chloroplasts in the active stage of growth utilized 70–80% of N of total leaf N (Makino and Osmond Reference Makino and Osmond1991). Earlier studies suggest that chloroplast counts rise with ploidy level and appear to maintain the stoichiometric balance between nuclear- and chloroplast-encoded genes throughout polyploidization (De Smet et al. Reference De Smet, Adams, Vandepoele, Van Montagu, Maere and Van de Peer2013). Notably, overrepresented transcripts of polyploid cytotypes also showed significant association with the nucleic acid metabolism and transposition of transposable elements (TEs; Mondal et al. Reference Mondal, Dubey, Ramesha, Vijayan, Naik and Moorthy2025). All of which possibly suggests that the functioning of large genomes is likely expensive, which may account for the scarcity of young 22-ploid M. nigra trees in the temperate and Mediterranean climate regions (Kılınçer et al. Reference Kılınçer, Khanyile, Gürcan, Şimşek, Uzun and Nikbakht-Dehkordi2024).
Collectively, the lower limit of cell size and rate-limited attributes showed a significant effect of the enlarged genome in mulberry. Hence, the current study points out that benchmarking of ploidy level for selection of parental material is crucial for enhancing cellular function and developmental efficiency, thereby maximizing leaf yield for future sustainability of sericulture. It is crucial to determine in the future if the observed ‘tetraploid advantage’ or ‘22-ploid rate-limited departed growth superiority’ is due to ploidy alone or is influenced by species-specific genetic backgrounds. This can be done using a dual analytical framework that compares ploidy within species and between species. The existing varied trait expression comprehended through ex situ field surveys, additional laboratory experiments and high-throughput sequencing data is also required to complement our knowledge, especially at higher ploidy levels.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S1479262125100439.
Acknowledgements
RM thanks the Central Silk Board (CSB), Bangalore, Ministry of Textiles (MoT), Govt. of India, for the financial support, Grant ID: PIG06010SIC (Phase II). STA and SA expressed thanks to CSB, Bangalore, India, for the JRF and SRF fellowships, respectively.
Author contributions
STA data collection, analysis, visualization, original draft preparation and final editing of the manuscript; SA cytological study; RM conceived the idea, designed and executed research, prepared the original draft and final editing of the manuscript; VNN and SMM provided resources and final editing of the manuscript. All authors read and approved the manuscript.
Funding statement
The work supported by the grant ID: PIG06010SIC (Phase II), Central Silk Board (CSB), Bangalore, Ministry of Textiles (MoT), Govt. of India.
Competing interests
The authors declare that they have no known financial and non-financial competing interests.
