Department of Botany, Government College, Kota, Rajasthan, India.

*Corresponding author:Noopur Soni, Department of Botany, Government College, Kota, Rajasthan, India. Email Id: noopur9943@gmail.com

Copyright: © Soni N, et al. 2025. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Article Information:Submission: 26/05/2025; Accepted: 24/06/2025; Published: 28/06/2025

The genus Marsilea comprises amphibious ferns known for their remarkable adaptability to varied aquatic and semi-aquatic habitats. Numerous species of Marsilea exhibit morphological changes depending on their habitat or geographic location. This study investigates morphological and ecological variation across eight natural populations of Marsilea from distinct habitat types within Kota District, Rajasthan, India. Extensive field surveys were conducted across diverse habitats, including wetlands, canal systems, agricultural fields, and temporary water bodies, during different seasons. A total of 80 specimens, comprising aquatic, semi-aquatic, and terrestrial forms, were assessed for key morphological traits, including leaflet length and width, petiole length, sporocarp characteristics, and growth patterns. Environmental parameters, including soil type, water availability, and anthropogenic pressures, were also recorded to understand habitat influences. The findings reveal significant intra and interspecific variations in response to local ecological conditions, highlighting the phenotypic plasticity of Marsilea populations. Additionally, the study underscores the role of habitat disturbance and climatic factors in shaping the distribution and vitality of these species. The results contribute valuable insights into the adaptive strategies of Marsilea spp., and they show that Marsilea minuta is more ecologically versatile and stress-tolerant, making it more adaptable to different environmental conditions than Marsilea cf. coromandelina in the semi-arid landscape of Kota district, and provide a baseline for future conservation and ecological studies.

Keywords:Marsilea; Comparative Analysis; Morphological Variation; Habitat Ecology; Kota District; Rajasthan; Phenotypic Plasticity

The genus Marsilea, commonly known as water clovers or pepperworts, comprises small aquatic ferns belonging to the family Marsileaceae [1]. In India, several species of Marsilea are distributed across diverse habitats, with notable occurrences in the Kota District of the Hadoti Plateau, southeastern Rajasthan. Among these, Marsilea minuta, a cosmopolitan species, is frequently observed thriving in ditches and ponds throughout the year in the Kota region, whereas Marsilea cf. coromandelina has been recorded only during the monsoon season near Borabas village, growing in limited terrestrial habitats [2]. Notably, the genus is well recognized for its high degree of phenotypic plasticity, which enables individuals to modify their morphology in response to varying environmental conditions [3]. This characteristic is particularly significant in the Kota region, which experiences pronounced wet-dry seasonal cycles and supports a mosaic of aquatic, amphibious, and terrestrial microhabitats. Furthermore, previous studies have indicated that the genetic diversity of Marsilea plays a crucial role in promoting these plastic traits, thereby enhancing the species’ ability to adapt across heterogeneous environments [4]. Therefore, investigating the morphological variation of Marsilea species across such contrasting habitats provides valuable insight into their habitat-specific adaptations. In addition, the seasonal wetlands in this region harbour

ecologically sensitive and endemic flora, which are increasingly under threat due to anthropogenic land-use changes. Importantly, compared to aquatic environments, terrestrial habitats generally expose plants to more fluctuating environmental factors, further influencing morphological differentiation [5]. In this context, Baruah and Sarma have emphasized the significance of morpho-ecological variation and phenotypic plasticity in Marsilea, particularly in regions experiencing ecological disturbances or anthropogenic pressures (IUCN, 2022) [6,7]. Consequently, the Hadoti Plateau especially the Kota District emerges as an ideal landscape to study these dynamics, given its diverse aquatic and terrestrial systems, including permanent ponds, riverbanks, seasonal wetlands, and dry agricultural patches. Nevertheless, despite its ecological richness, this region remains understudied in terms of pteridophyte diversity and morphological adaptation. This is especially true for genera like Marsilea, which are highly responsive to subtle microenvironmental variables such as soil moisture, water depth, and human disturbance [8]. Hence, the present study focuses on easily quantifiable morphological traits such as leaflet length and width, petiole length, and sporocarp size in order to assess environmental influences on morphological differentiation. Ultimately, this research contributes not only to a better understanding of ecological adaptation and phenotypic plasticity in Marsilea but also to the growing need for region-specific baseline data that can inform both taxonomic resolution and conservation planning amid on-going habitat degradation and climate variability.

The study was conducted in Kota District, Rajasthan (23°45′– 25°53′ N, 75°09′–77°26′ E), located on the Hadoti plateau, Rajasthan in the Chambal River basin. Based on preliminary surveys, eight representative populations of Marsilea were selected from distinct habitat types (Table no.1). The dominant species at each site was identified: M. minutain the aquatic, Marsilea minuta hybrid in amphibiotic sites, and M. cf. coromandelina in the purely terrestrial site. Each site was geo-referenced using GPS, and dominant habitat features were recorded.

Sampling and Identifications: A total of 10 individuals were sampled per site (n = 80 in total). Plants were collected during the active growing season and analysed fresh for morphological traits. Parameters recorded included leaflet length and width, petiole length, number of sporocarps per plant, sporocarp shape, and size. Standard botanical keys, Flora of Rajasthan – Vol 1 and 2 and regional floras (Khullar, 1994; Chandra, 2000;Fraser-Jenkins, C. R. (2008) were used for preliminary identification.

Morphological measurements:Morphological analysis was conducted to evaluate species-specific and habitat-induced variations among Marsilea populations collected from aquatic, amphibious, and terrestrial habitats in Kota District, Rajasthan. In the laboratory, each specimen was carefully washed and assessed for the following key
morphological traits:
• Leaflet length (cm): Measured from the base to the tip of the leaflet using a digital Vernier calliper.
• Petiole length (cm): Distance from the rhizome node to the base of the first leaflet.
• Sporocarp size (length × breadth in cm): Measured using a calliper, focusing on mature sporocarps only.
• Sporocarp shape and surface features: Observed under a stereomicroscope for ribbing, hairiness, and structural characteristics.
• Number and position of sporocarps: The number and Position of sporocarps were visually recorded, with the petiole’s orientation and attachment points noted (horn).

The traits were chosen based on their ecological relevance and established use in previous Marsilea studies (Mangestuti et al., 2017; Sharma &Bhardwaja, 2019). [2,9]

Identification of Hybrids:The leaf of hybrid Marsilea is a compound, heteromorphic structure with four leaflets arranged in a clover-like pattern, exhibiting intermediate size, texture, and venation between M. minuta and M. cf. coromandelina, and often marked by variability in petiole length, leaflet symmetry, marginal curvature and sporocarps arrangements. This key is based solely on morphological observations, and further anatomical or molecular analysis is recommended to validate hybrid status.

Statistical analysis: Mean trait values were compared across habitat groups. One-way ANOVA tests (α = 0.05) assessed whether leaflet length, petiole length, or sporocarp size differed significantly among the three habitat categories (aquatic, amphibious, terrestrial). Descriptive statistics (mean ± standard deviation) were tabulated, and a bar chart was prepared to illustrate leaflet length differences.[4]

All measurements were conducted using standard protocols for aquatic Pteridophytes (Khullar, 2000; Pérez-Harguindeguy et al., 2013) [13,15].

A comparative analysis was conducted on Marsilea taxa, including Marsilea minuta, M. cf. coromendelina and Marsilea minuta hybrid, from 8 distinct habitats in the Kota region (Kanwas, Abheda

pond, Borabas pond, Kewal Nagar, Moru Kalan, Kolani, Borabas and Abhera Biological Park).
Morphological Assessment: Key morphological characters such as leaflet size, leaflet shape and margin, petiole length, sporocarp size, number and position of sporocarps, and sporocarp shape showed significant interspecific and intraspecific variation:
Leaflet length ranged from 0.8 ± 0.21 cm in Marsilea sp. from Borabas to 2.33 ± 0.51 cm from Kewal Nagar. Margins varied from entire (M. minuta) to crenate (Borabas and Abhera samples). Leaflet

shape ranged from obdeltoid and membranous (M. minuta) to rigid, leathery obtriangular forms (M. cf. coromendelina). It was observed that leaflet of Marsilea minuta hybrid spp. is larger than M. minuta and M. cf. coromendelina due to amphibious characteristics, more water availability and Heavy clay type of soil is favourable for Broad leaves of Marsilea spp.

Petiole length showed considerable variation, with the longest observed in M. minuta from kanwas (12.2 ± 0.32 cm) and the shortest in Marsilea cf. coromendelina from Borabas (4.2 ± 0.40 cm), indicating

different growth strategies and ecological adaptations. It was observed that Permanent water availability, low anthropogenic disturbance and Loam to sandy loam soil also called alluvial soil is favourable for Marsilea petiole length.

Sporocarp size ranged from 0.17 x 0.2 cm (M. minuta) to 0.42 x 0.21 cm (Marsilea sp. from Borabas). Marsilea hybrid spp. displayed ribbed, bean-like sporocarps, while the Borabas and Abhera samples had rigid, quadrilateral or square sporocarps with distinct ridges and hairs. The number and positioning of sporocarps also varied, with M. hybrid bearing 3–4 attached at the petiole base and the Kolani form producing clusters of 6–8 per node.

The results clearly demonstrate pronounced morphological variation in Marsilea across different habitat types, reflecting the genus’s inherent phenotypic plasticity. Specifically, aquatic and amphibious individuals exhibited significantly larger leaflets and longer petioles compared to their terrestrial counterparts. This enlargement of leaf structures in semi-aquatic conditions may serve to maximize photosynthetic capacity under higher light availability and simultaneously reduce the impact of anthropogenic disturbances. In contrast, the relatively smaller leaf form observed in submerged conditions might be an adaptive strategy to minimize water drag. These external morphological traits are consistent with the findings of Sharma et al. (2019), who reported that Marsilea specimens from flooded environments developed extensive aerenchyma and thinner stele, while those growing on land possessed denser internal tissues [2]. Although internal anatomy was not examined in the present study, the distinct external features likely result from similar physiological responses to varying hydric regimes.

Moreover, the relative stability in sporocarp size across habitats suggests that reproductive organs may exhibit lower plasticity than vegetative parts. In line with previous observations of M. minuta, all studied populations consistently produced small, mature sporocarps. This uniformity implies that reproductive development in Marsilea may be governed by a conserved genetic program, relatively unaffected by environmental variability. However, it is important to note that sporocarp viability and production rates factors that could also be influenced by habitat quality were not assessed in this study. Therefore, future research should address these parameters to gain deeper insight into the reproductive ecology of the genus.

These findings resonate with other studies on Marsilea. For instance, Mangestuti et al. (2017) [9] demonstrated that M. crenata individuals cultivated in soil differed markedly in leaflet thickness and stomatal density from those grown in water, highlighting habitat-driven morphological differentiation. Similarly, anatomical investigations conducted in Kota by Sharma et al. (2019) supported population-level variation correlated with habitat history.

Broadening the scope, similar trends have been documented across fern taxa. For example, Farrar (1974) [10] observed that ferns from open, drier habitats produced thicker fronds compared to those adapted to shaded, humid environments, illustrating phenotypic modulation of gametophytes in response to ecological pressures. Such parallels underscore that Marsilea, like other ferns, possesses a high degree of plasticity, enabling it to adjust morphologically to variations in light and moisture availability. Additionally, these observations are congruent with earlier reports by Smith et al. (2006) and Khullar (1994) [12,13], which emphasize the moisture-responsive plasticity in Marsilea morphology.

Furthermore, similar patterns of morphological flexibility have been noted by Khullar (1994) and Chandra (2000) [14], who documented extensive trait variability among Indian pteridophytes. The present study builds upon these findings by further confirming the role of habitat heterogeneity in shaping phenotypic expression. For example, wetlands such as Kewal Nagar and permanent ponds like Kolani supported robust individuals with larger leaflets and longer petioles. These habitats likely provide stable moisture availability, higher organic content, and microclimatic consistency conducive to optimal growth. In contrast, Marsilea populations inhabiting more ephemeral or disturbed environments such as Borabas roadside and Abhera Biological Park tended to exhibit stunted growth or greater morphological variability, likely resulting from stress-induced adaptations or developmental limitations.

Interestingly, transitional zones and anthropogenically impacted habitats harbored individuals exhibiting intermediate morphological traits that bridged characteristics of M. minuta and other closely related species. This phenomenon may suggest ongoing hybridization, introgression, or ecotypic divergence. Notably, this hypothesis finds support in the broader literature, including studies by Walker (1961) and Schneider et al. (2004) [18,19], which discuss the evolutionary implications of such intermediate forms in fern taxa. These patterns may reflect ongoing speciation events or rapid evolutionary responses to complex and fluctuating environmental conditions.

Looking ahead, future investigations should incorporate quantitative measurements of environmental variables such as water depth, soil moisture, and organic content to strengthen the ecological interpretation of the observed trends. Preliminary observations already indicate that aquatic sites maintained standing water throughout the year, while terrestrial sites underwent complete desiccation during summer months. Therefore, it is reasonable to infer that the observed morphological variations arise not only from plastic responses but also potentially from local adaptations to differing moisture regimes.

This study provides comprehensive evidence of significant morphological variability among Marsilea species across distinct aquatic, amphibious, and terrestrial habitats in Kota District, Rajasthan. The observed differences in key vegetative and reproductive traits, Marsilea minuta was commonly found in permanent aquatic environments and exhibited delicate, membranous fronds with longer petioles, reflecting adaptation to submerged conditions.

In contrast, M. cf. coromandelina, primarily found in terrestrial or seasonally dry habitats, showed thicker, leathery leaflets and compact growth forms, indicating adaptations to water stress and fluctuating soil moisture. Notably, hybrid populations displayed intermediate morphological traits, suggesting possible natural hybridization events and highlighting the genus’s phenotypic plasticity. Environmental factors such as soil type, water availability, and anthropogenic disturbances played a critical role in shaping the observed morphological differences. The study reveals that permanent water bodies with loamy soils promote larger vegetative structures, while temporary and disturbed sites lead to more compact growth forms and altered sporocarp features.

These findings underscore the adaptive strategies of Marsilea species in response to ecological gradients and highlight the need for further integrative taxonomic work, including molecular analyses, to clarify species boundaries and evolutionary relationships. Conservation attention is also warranted for morphologically distinct or potentially endemic populations, particularly those in ecologically vulnerable areas like Borabas, Kolani, and Abhera Biological Park, where habitat degradation poses a risk to genetic and ecological diversity. The study suggests additional molecular and physiological research to help clarify taxonomic ambiguities, evaluate adaptive responses, and inform successful biodiversity management strategies for the genus Marsilea in the Hadoti region and beyond. This will help advance species delimitation and conservation planning.

We thank the Department of Botany at the Government College, Kota for research support and access to laboratory facilities. We are grateful to our research supervisor Prof.Pratima Shrivastava, Principal, Govt. College, Kota for guidance, field assistants and for help in data collection and would like to Thank Dr. Nilima Bhardwaj madam for her support and guidance in this work.