In terrestrial ecosystems, soil organisms contribute to a variety of biological and biochemical processes, which play an important role in maintaining healthy and functional ecosystems (Lavelle et al., 2006). Among soil macro-invertebrates, earthworms make up a substantial part of the soil invertebrate biomass across various terrestrial habitats and are often used as bioindicators in soil quality assessments (Barros et al., 2002; Fründ et al., 2011; Pérès et al., 2011; Pauli et al., 2011). Earthworms play a crucial role by contributing to litter decomposition, nutrient cycling, soil aeration, and the maintenance of soil structure (Edwards and Bohlen, 1996).

In natural conditions, earthworms move at an extremely slow pace of approximately 1.4–9 m/year; however, their long-distance migration is facilitated through various means, such as by the feet of birds and other animals, through the roots of displaced plants, and even through transportation of wooden logs as well as long-distance transportation of the organism for commercial purposes (Julka, 1988; Díaz Cosín et al., 2011; Tóth et al., 2020; Chen et al., 2021). While colonizing a new environment beyond their native range, earthworm species face various ecological challenges. These include changes in behavioral traits, such as dietary adaptability and physiological tolerance, as well as changes in life history characteristics like parthenogenesis, short generation times, and dispersal modes (Nouri-Aiin et al., 2022). Some studies have shown that parthenogenesis and polyploidy benefit migrating earthworm species (Edwards and Bohlen, 1996; Díaz Cosín et al., 2011).

Earthworms are generally considered hermaphrodites; cross-fertilization is the most common reproductive strategy among most earthworm species. Self-fertilization has only been observed in Eisenia andrei Bouché, 1972, wherein the worm bends itself, allowing its spermathecal pores to contact the ventral zone of its clitellum and to pass the sperm from the male pores to the spermathecae (Domínguez et al., 2003). In addition to hermaphroditism, parthenogenetic reproduction is observed in some species, most of which are polyploid (Díaz Cosín et al., 2011). Parthenogenesis is significant due to its ability to preserve polyploidy and promote the expansion of polyploid variations in new regions; a single worm can initiate a new colony (Gates, 1972). Parthenogenetic reproduction is common in the family Lumbricidae (Terhivuo and Saura, 2003; Lowe and Butt, 2008; Sosa et al., 2017), with more than 30 species being found in North America (Reynolds, 1974). Parthenogenetic morphs typically have a high reproductive capacity, produce resistant cocoons, and exhibit wide environmental or feeding tolerances, a high dispersal rate, and the ability to withstand higher parasitic burdens (Gates, 1972; Jaenike and Selander, 1979; Blakemore, 2012).

In their study, Martay and Pearce-Higgins (2020) have reported a significant decline in the bird species that rely on earthworms, due to a decline in the earthworm population. Thus, it can be concluded that the role of soil biodiversity in maintaining ecosystem health and conserving habitats for higher vertebrates is invaluable, but often gets ignored in conservation policies (Martay and Pearce-Higgins, 2020; Cui et al., 2022; Duarte et al., 2024). This oversight leads to insufficient data on the conservation status of soil organisms, raising concerns about the effectiveness of conservation areas in preserving overall ecosystem functionality.

Analyzing earthworm populations and behavior will yield valuable insights into soil conditions and the broader environment. Moreover, an understanding of the spatial distribution of earthworm species along the protected areas is crucially important as baseline information against which future changes in the protected areas can be monitored and assessed. Therefore, the primary objective of this study was to analyze the spatial distribution of earthworm species across several wildlife sanctuaries, namely, Bethuadahari Wildlife Sanctuary, Bibhutibhushan Wildlife Sanctuary, Raiganj Wildlife Sanctuary, Ballavpur Wildlife Sanctuary, and Ramnabagan Wildlife Sanctuary. Our investigation aimed to address the following questions: Does species richness vary along these wildlife sanctuaries? What is the status of native and exotic peregrine species within these protected areas? Does the natural forest support native and epigeic species? To achieve these goals, earthworm samples were collected from different wildlife sanctuaries using the TSBF (Tropical Soil Biology and Fertility) method.

The present study was part of the Zoological Survey of India’s in-house program entitled “Faunal Diversity of Wildlife Sanctuaries of West Bengal”, covering all faunal groups. Two surveys were conducted for earthworms, one in December 2021 [Ramnabagan Wildlife Sanctuary (RAWLS) and Ballavpur Wildlife Sanctuary (BAWLS)] and another in October 2023 [Bibhutibhushan Wildlife Sanctuary (BIWLS), Bethuadahari Wildlife Sanctuary (BEWLS), and Raiganj Wildlife Sanctuary, also known as Kulik Bird Sanctuary (KUWLS)]. In the West Bengal state, these sanctuaries are situated in the districts of North 24 Parganas (BIWLS), Nadia (BEWLS), North Dinajpur (KUWLS), Birbhum (BAWLS), and Purba Bardhaman (RAWLS). The Nadia district has an average yearly precipitation of 1,245 mm, while the North Dinajpur district has an average of 1,592 mm. The Birbhum district with an average yearly precipitation of 1,321 mm, the Purba Bardhaman district with an average yearly precipitation of 1,400 mm, and the North 24 Parganas district with an average yearly precipitation of 1,579 mm are the intermediate districts concerning the rainfall level. The majority of the rainy season falls between June and September, during the Southwest monsoon. According to data from the WB State Government, January is the coldest month with the lowest points of up to 10°C, while May has the greatest peak of up to 41°C. Alluvial soil is the typical soil found in these regions. These sanctuaries are situated between 45 and 66 m above mean sea level and are a part of the Gangetic biogeographical zone. The selected Wildlife Sanctuaries characterized by the tropical deciduous forest comprise the dominant tree species, Acacia auriculiformis A. Cunn. ex Benth., Anacardium occidentale Linn., Shorea robusta Gaertn., Phyllanthus emblica Linn., Terminalia bellirica (Gaertn.) Roxb., Terminalia chebula Retz., Tectona grandis L.F., Terminalia arjuna (Roxb.) Wight and Arn., Dalbergia sissoo Roxb., Bambusa tulda Roxb., Neolamarckia cadamba (Roxb.) Bosser, Limonia acidissima Linn., and Ficus racemosa Linn.

Earthworms were collected from the aforementioned five wildlife sanctuaries. Within each sanctuary, four sampling sites (each with an area of 10 × 10 m) were selected ( Supplementary Table 1 ; Supplementary Figure 1 ). At each sampling site, earthworms were collected by digging and hand sorting nine quadrants, each measuring 25 × 25 cm and up to 30 cm depth, following the TSBF method (Anderson and Ingram, 1993). In this way, a total of 20 sampling sites (5 × 4) and 180 subunits (20 × 9) were explored for quantitative and qualitative earthworm studies. The collected specimens were washed with water and preserved in 5% formalin for subsequent taxonomic identification. All pertinent morphological and anatomical characterizations of the earthworms were carried out using a Leica stereomicroscope (Model: Leica EZ4). The family level classification followed was based on the criteria set by Misirlioğlu et al. (2023). Additionally, considering the vertical distribution, size, and color of the specimens, the species were categorized into ecological groups (Bouché, 1977; Bottinelli et al., 2020). Finally, the specimens were deposited in the National Zoological Collection in the ZSI, GNC section, Kolkata. Earthworm community structure and species richness were assessed with the help of species diversity indices and a Mondrian plot using PRIMER v.7 (Clarke and Gorley, 2015), respectively. Additionally, Indicator Species Analysis (ISA) was also performed using Past version 4.13 (Hammer et al., 2001) to identify the strength of the species (p < 0.05) with the probability of association to the wildlife sanctuaries.

A total of 22 species belonging to 12 genera and 6 families, viz., Benhamiidae, Megascolecidae, Acanthodrilidae, Rhinodrilidae, Almidae, and Moniligastridae, were collected. Table 1 shows the highest number of species recorded at KUWLS (n = 15) followed by BEWLS (n = 14), BAWLS (n = 11), BIWLS (n = 10), and RAWLS (n = 8). Although KUWLS had the highest number of 15 species, the Shannon diversity index (H’ = 2.34) and Simpson’s index (D = 0.89) were the highest at BEWLS ( Table 1 ). The variance could be attributed to an uneven distribution—where a few species [Drawida nepalensis Michaelsen, 1907, Glyphidrilus gangeticus Gates, 1955 and Pontoscolex corethrurus (Müller, 1857)] dominate the ecosystem while others have significantly fewer individuals ( Table 1 and Figure 1A ). This disparity within the 15 species might contribute to the lower Shannon diversity index in KUWLS than that in BEWLS. Furthermore, the species were classified into ecological categories, viz., (1) Epigeic: Dichogaster affinis (Michaelsen, 1890), Dichogaster bolaui (Michaelsen, 1891), and Perionyx sp.; (2) Epi-endogeic: Amynthas alexandri (Beddard, 1900), Metaphire houlleti (Perrier, 1872), and Metaphire peguana (Rosa, 1890); (3) Endogeic: Lennogaster chittagongensis (Stephenson, 1917), Lennogaster yeicus (Stephenson, 1931), Octochaetona beatrix (Beddard, 1902), Octochaetona surensis (Michaelsen, 1910), Pontoscolex corethrurus (Müller, 1857), Glyphidrilus gangeticus Gates, 1955, Drawida barwelli (Beddard, 1886), Drawida calebi Gates, 1945, Drawida nepalensis Michaelsen, 1907, Dichogaster modiglianii (Rosa, 1896), Lampito mauritii Kinberg, 1867, Metaphire planata (Gates, 1926), Metaphire posthuma (Vaillant, 1868), and Polypheretima elongata (Perrier, 1872); (4) Anecic: Eutyphoeus orientalis (Beddard, 1883) and Eutyphoeus nicholsoni (Beddard, 1901). Among the identified earthworm communities, the endogeic was the most dominant (13 species), followed by the epigeic (3 species), the epi-endogeic (3 species), and the anecic (2 species). Moreover, the earthworm communities were composed of native and exotic peregrine species with the exotic species Pontoscolex corethrurus being found at most of the sites and dominating the earthworm communities at RAWLS ( Table 1 ).

The Mondrian plot ( Figure 1A ) revealed that mixed clustering might be attributed to the presence of peregrine species at each wildlife sanctuary. The exotic peregrine species Pontoscolex corethrurus and the native peregrine species Lampito mauritii and Drawida nepalensis were found in most of the study sites within the wildlife sanctuaries. Although the population of these species varied significantly among the sites, Pontoscolex corethrurus, for example, was predominantly abundant at site RA2 in RAWLS; similarly, Drawida nepalensis was predominantly abundant at KU4 in KUWLS and Lampito mauritii was predominantly abundant at BA4 in BAWLS ( Figure 1A ). It is interesting to note that the populations of other species were found to be minimal in areas where these species predominated. Furthermore, the study sites within each wildlife sanctuary showed some clustering based on the occurrence and abundance of the observed species ( Figure 1A ). Four distinct clusters were formed among the sampling sites in the wildlife sanctuaries, namely, BIWLS, KUWLS, RAWLS, and BEWLS, each with similarity scores of 63.08%, 57.32%, 56.78%, and 46.95%, respectively. Additionally, the assessment of ISA identified 9 out of 22 species as significant indicators (p < 0.05) in the selected Wildlife sanctuaries. It included three species in BEWLS, two species each in BAWLS and RAWLS, and one species each in KUWLS and BIWLS ( Figure 1B ). Except for KUWLS, all other sanctuaries harbor at least one exotic species as an indicator, namely, BIWLS, Metaphire posthuma; BEWLS, Amynthas alexandri; BAWLS, Dichogaster affinis; and RAWLS, Pontoscolex corethrurus. Interestingly, Eutyphoeus nicholsoni was found to be the only endemic species serving as an indicator at KUWLS, which also supports a greater number of species ( Figure 1B ).

In earthworm reproduction, the clitellum secretes a girdle-like protective and nutritious layer that moves toward the anterior side, collecting the ova from the ovary and sperms from spermathecae (sperm storage) and finally shedding through the anterior-most segment and forming a cocoon (Julka, 1988). A colony of the earthworm species Metaphire houlleti was observed with the parthenogenetic mode of reproduction. This species was found at 7 sites out of 20 in five different wildlife sanctuaries with a population range of 1.78–14.22 ind. m⁻². The parthenogenetic morphs were found in a single colony with a population of 14.22 ind. m⁻² at only one site near Gharial pound in BEWLS (BE2), where all were athecal morphs. In natural conditions, the formation of a cocoon mostly remains unabsorbed. Interestingly, we managed to collect specimens showing the movement of the girdle toward the anterior, indicating the formation of cocoons in parthenogenetic species. We highlighted this phenomenon in two specimens by varying the degree of movement, one at segment 8 ( Figure 2A ) and one at segment 11 ( Figure 2B ). All the parthenogenetic morphs lack spermathecae, in addition to one lacking male pores, but all have well-developed testes, seminal vesicles, and prostate glands. Moreover, the population per meter square was slightly higher (14.22 ind. m⁻²) in the parthenogenetic colony, as compared to the maximum (12.44 ind. m⁻²) at another site [Badhur tower (KU1), KUWLS] in a sexually reproducing colony.

Except for a few studies (Hale et al., 2006; Ransom, 2011; Loss et al., 2012; Craven et al., 2017), earthworms are considered an important community for the conservation of a healthy ecosystem and habitat for other vertebrates (Blouin et al., 2013; Martay and Pearce-Higgins, 2020). In this study, 22 species were documented across various wildlife sanctuaries, with species counts ranging from two to eight at each specific site, falling well within the reported range of 1–15 species (Edwards and Bohlen, 1996). The observed variability in species occurrence among these sites may be due to the factors elucidated in various studies such as environmental conditions, food availability, and specific habitat characteristics (Ekschmitt et al., 2003; Phillips et al., 2019; Tóth et al., 2020). Considering the wildlife sanctuaries, a maximum of 15 species were recorded from KUWLS; this site is also known for hosting a substantial annual bird population and has witnessed notable counts of approximately 99,383 birds, including 64,055 open bill storks, 8,969 night herons, 19,841 egrets, and 6,528 cormorants (Wildlife Sanctuary Information). Sometimes, birds and animals play an important role in the migration of earthworm species through soil adhering to their feet (Gates, 1972; Blakemore, 2009). Despite having the highest number of species at KUWLS, the values of the Shannon diversity index (H’ = 2.34) and Simpson’s index (D = 0.89) were the highest at BEWLS, which may be due to the equal distribution of earthworm species. Our findings support those of Lenka et al. (2023), who noted that an even distribution of species in an ecosystem tends to give higher Shannon diversity index values. Furthermore, according to Callaghan et al. (2019), a higher value of H’ indicates greater ecological stability and a more resilient ecosystem capable of supporting various ecological niches. Therefore, it can be concluded that the ecosystem at BEWLS is likely more suitable for the colonization of earthworm species when compared to other wildlife sanctuaries. In the present study, all the sanctuaries were characterized by the distinct indicator species. The changes in the indicator species may be reflected by the changes in the ecosystem (Siddig et al., 2016). To ensure better management strategies and conservation efforts, knowledge of both species diversity and edaphic parameters is necessary (Magray et al., 2022). Hence, it is essential to interpret the edaphic factors with respect to earthworm species diversity in wildlife sanctuaries.

The clustering of nearby study sites associated with each wildlife sanctuary might be due to the local environmental conditions, which play a significant role in the colonization and abundance of earthworm species. Previous studies contributed to the conclusion that local edaphic factors were responsible for the formation of earthworm species population patches (González et al., 2007; Demetrio et al., 2019; Singh et al., 2020; Ahmed et al., 2022). The species Lennogaster yeicus was initially described in Chaungson, Myanmar (Stephenson, 1931), and in India, it has been reported from Himachal Pradesh (Julka, 1988), Uttarakhand (Bhadauria et al., 1997), and Tripura (Chaudhuri et al., 2012; Jamatia and Chaudhuri, 2017). The report of this species in the wildlife sanctuaries is an addition to the earthworm fauna of the state of West Bengal. Overall, the presence of 54.5% native species in the protected forest supports Demetrio et al. (2023) findings, which showed that forest ecosystems support more native species than exotic ones.

Earthworms play an important role in nutrient recycling and plant growth (van Groenigen et al., 2014; Hussain et al., 2018), which is strongly influenced by their ecological categories (Huang et al., 2020). It was observed that litter decomposition substantially increased when worms from all categories were present in an ecosystem (Huang et al., 2020). Moreover, the species recorded in the present study were categorized into ecological groups, aligning with the finding that an earthworm community in an ecosystem is composed of one to two epigeic species, two to four endogeic species, and zero to two species of large anecics (Pop, 1997). In protected areas, the presence of all the ecological categories indicates greater ecological stability and a more resilient ecosystem that can support various ecological niches. Alteration of natural vegetation to pasture, cropland, and tree plantation often alters functional diversity (Decaëns and Jiménez, 2002; Smith et al., 2008). The widespread occurrence of endogeic species such as Pontoscolex corethrurus, Lampito mauritii, and Drawida nepalensis indicates their tolerance to a wide range of edaphic factors.

Parthenogenesis is considered significant due to its ability to preserve polyploidy and promote the expansion of polyploid variations in new regions, as a single worm can initiate a new colony (Gates, 1972). In the present study, a colony of parthenogenetic morphs of the species Metaphire houlleti was recorded from a site in BEWLS. Similar to the parthenogenetic morphs observed in the present study, Blakemore (2012) also reported the presence of iridescent testes and funnels in parthenogenetic morphs, whereas other studies suspected this phenomenon in parthenogenesis (Gates, 1972; Jaenike and Selander, 1979). The findings of Jaenike and Selander (1979) are supported by the current observation of Metaphire houlleti, a parthenogenetic earthworm, in the upper soil organic layer. Interestingly, we observed that this species exhibited both parthenogenetic and sexual reproduction, with the population recording the highest in the former. Our study also supports the findings that parthenogenetic morphs typically have a high reproductive capacity, contribute to rapid population growth, produce resistant cocoons, and exhibit wide environmental or feeding tolerances, a high dispersal rate, and the ability to withstand higher parasitic burdens (Gates, 1972; Jaenike and Selander, 1979; Blakemore, 2012).

Among 22 species identified from various wildlife sanctuaries of West Bengal, the number of species has varied across the sanctuaries. In comparison with the other sanctuaries, BEWLS is determined to be more appropriate for the perpetuation of earthworm communities based on the Shannon diversity index. With the exception of KUWLS, ISA has revealed exotic species as indicators across the majority of the wildlife sanctuaries. At KUWLS, it is observed that the endemic species Eutyphoeus nicholsoni might function as a possible indicator species, which also supports a greater number of species. It is also observed that the range extension of Lennogaster yeicus to the West Bengal state and the widespread occurrence of various species such as Pontoscolex corethrurus, Lampito mauritii, and Drawida nepalensis indicate their high tolerance of a wide range of edaphic factors. It is the need of the hour to assess and interpret the physical process data with respect to the diversity of earthworm species in wildlife sanctuaries to validate such indicator species. Moreover, further molecular studies are required to find out the genetic variations between parthenogenetic and sexually reproducing colonies of the earthworm species Metaphire houlleti.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The manuscript presents research on animals that do not require ethical approval for their study.

SA: Data curation, Investigation, Methodology, Resources, Writing – original draft, Writing – review & editing. JJ: Data curation, Formal analysis, Writing – review & editing. DB: Conceptualization, Supervision, Writing – review & editing. NM: Data curation, Software, Validation, Writing – original draft, Writing – review & editing.