Sexual dimorphism refers to well-defined differences in phenotypic and morphological traits between males and females of the same species (Fazhan et al., 2021; Roterman et al., 2025). It is widespread across the animal kingdom and also occurs in many plant species (Roterman et al., 2025). In animals, morphological sexual dimorphism often arises from sexual selection, driven by mate choice and intraspecific competitions (Fazhan et al., 2021; Hidir et al., 2021), as well as from ecological pressures that favor sex specific adaptations (Wada, 1999; Waiho et al., 2021). Beyond primary morphological traits, many crustaceans exhibit marked dimorphism in secondary sexual characteristics (Meldola, 1900). For example, males often develop larger chelipeds, which serve as weapons for defense and competition during mating, whereas females typically possess a broader abdominal pleon to facilitate egg carrying and reproductive feasibilities (Hidir et al., 2021). A well-studied case is the mud crab genus Scylla, in which these morphological differences are pronounced (Fazhan et al., 2021).

Sexual dimorphism in hydrothermal vent crabs, including members of the Bythograeidae (e.g., Bythograea thermydron, Austinograea spp.) and Kiwaidae (e.g., Kiwa hirsuta, “Yeti crab”), encompasses morphological, physiological, and behavioral differences between male and female sexes. These traits are shaped by the extreme physicochemical conditions of vent ecosystems, such as high hydrostatic pressure, low light availability, and chemically enriched fluids, and often serve reproductive, ecological, and survival functions. Despite their significance, studies on sexual dimorphism in vent-associated species remain limited. Notable examples include research on Kiwa hirsuta and Kiwa puravida (Kiwaidae) (Azofeifa-Solano et al., 2022; Roterman et al., 2025) and selected Bythograeidae species (Guinot and Hurtado, 2003). In contrast, most detailed accounts of brachyuran sexual dimorphism derive from non-vent taxa, such as the varunid crab Gaetice depressus (Takeda et al., 2024), freshwater potamids (Najafi et al., 2023), fiddler crabs (Ocypodidae) (Levinton and Weissburg, 2021), and hermit crabs (Parapaguridae) (Candiotto et al., 2023).

The grapsoid crab X. testudinatus inhabits the extreme sulfur-rich shallow water hydrothermal vent ecosystems of the western Pacific volcanic arc, with a restricted distribution from Taiwanese to Japanese islands (Ng et al., 2000; Thirunavukkarasu et al., 2025b). First they were recorded from the shallow volcanic rises off Kueishan Island, Taiwan, the species name “testudinatus” derives from the Latin term for “turtle shell,” reflecting both its carapace morphology and also habituated locality of Turtle Island of Taiwan (Jeng et al., 2004a; b; Ng et al., 2000, 2007, 2014). This species serves as a valuable model for investigating population expansion, evolutionary ecology, metabolic regulation, genetic adaptation, and meta-population dynamics in the extreme environments (Ki et al., 2009; Allen et al., 2020; Yang et al., 2022; Tseng et al., 2025; Thirunavukkarasu et al., 2025a). Hydrothermal vent ecosystems present unique physicochemical challenges, including elevated levels of temperatures, metal concentrations, toxic chemical release and fluctuating pH, which can shape sexual dimorphism by driving the evolution of sex specific structural, mechanical, and biochemical traits. X. testudinatus thrives under intense volcanic activity, toxic vent plumes, and unstable substrates, making this crab an ideal model to explore how sexual dimorphism may reflect divergent functional strategies and adaptive capacities under extreme ecological stressors.

Physicochemical stressors in the hydrothermal vent ecosystems affect exoskeletal chemical composition and structural patterns in the vent crustacean populations. For example, X. testudinatus reported varying concentrations of trace metals accumulation in its exo-skeleton and soft other tissues likes gills and muscles (Peng et al., 2011; Zeng et al., 2018) and morphological sexual dimorphism (in carapace width, chela size, and mass) has also been documented in this species (Tseng et al., 2020). These findings support the plausibility of environmental stimuli as a driving factor in structural and biochemical changes leading to sexual dimorphism. Our earlier study of sexual dimorphism in X. testudinatus (Tseng et al., 2020) was based on a limited set of morphological parameters, focusing primarily on external traits such as wet weight, carapace width, and chela length. That study did not incorporate comprehensive multivariate statistical analyses, nor did it assess exoskeletal microstructural surface topography and sex specific elemental composition, leaving the mechanistic basis of sexual dimorphism unresolved. In the present study, we address these gaps by integrating an expanded suite of morphometric variables with advanced statistical approaches, high resolution morphological observations, and detailed elemental analyses. Specifically, the application of SEM, TEM, EDX, and XPS allowed us to link sex-specific morphology with underlying structural organization and elemental differentiation. These methodological and analytical advances provide novel insights into the functional and adaptive significance of sexual dimorphism in X. testudinatus, offering a mechanistic framework that strengthens and extends our earlier findings in the context of shallow hydrothermal vent environments.

Sexual dimorphic characteristics in crustaceans can be quantified and distinguished both qualitatively and quantitatively through robust numerical and statistical analyses based on standardized morphometric approaches (Bertin et al., 2002). Such methods allow detailed differentiation between sexually dimorphic species (Asahina, 2018) and across life stages (Parvizi et al., 2017). The rigid exoskeleton and the presence of spines and other external structures in many crustacean taxa facilitate these measurements, making them suitable models for morphological and morphometric studies (Fazhan et al., 2021). Consequently, analyses of morphological and morphometric traits in crustaceans are valuable tools for taxonomic classification, as well as for understanding ecological, behavioral, and evolutionary processes (Alencar et al., 2014; Fazhan et al., 2021).

Scanning Electron Microscopy (SEM) has become a powerful imaging tool, providing high-resolution, three-dimensional images of specimen surface topography, including cuticle scale patterns, medulla structures, and surface textures (Peng et al., 2024). In studies of sexual dimorphism, SEM is widely used to identify and analyze morphological differences between males and females (Naem, 2007; Peng et al., 2024; Thirunavukkarasu et al., 2024). Transmission Electron Microscopy (TEM) complements SEM by enabling detailed investigation of cellular and subcellular structures, providing insights into reproductive and evolutionary adaptations (Ge et al., 2022; Guo et al., 2022). Sex specific differences in chemical composition and elemental content can occur at multiple biological levels, from whole tissues to cells and molecules (Peng et al., 2011; Chen et al., 2016; Zeng et al., 2018; Lee et al., 2022; Mei et al., 2022), often reflecting hormonal, genetic, metabolic, and reproductive roles. Such differences can be detected using SEM combined with Energy-Dispersive X-ray spectroscopy (EDX) and X-ray photoelectron spectroscopy (XPS). We hypothesize that sexual dimorphism in X. testudinatus has evolved through sexual selection and ecological role differentiation, whereby males develop more robust body sizes to compete for mates and defend territories, while females exhibit traits that enhance reproductive success, including stronger exoskeletons and defensive mechanisms. The present study tests this hypothesis by assessing extended morphometric traits, surface topography, internal and external cuticle structures, and elemental composition across different regions of the exoskeleton.

Vent crabs of X. testudinatus (Supplementary Figures 1A, B) were collected from the active shallow water hydrothermal vent of Kueishan Island in the western Pacific Ocean at northeastern Taiwan (Supplementary Figure 2) by SCUBA diving at the depth ranging from 18 to 20 m. As earlier described by Tseng et al. (2020), the traps (width 60.5 cm; mouth opening 43 × 21 cm) were non-selective with respect to sexes, as they rely on active movement and attraction rather than size and sex specific mechanisms. Both the male and female crabs were capable of entering the trap through the same opening, and there was no sex specific baiting nor mesh size effects. Moreover, the traps were designed based on information from other ecological studies that reported no evidence of systematic sex-biased capture. In total three crab traps were applied, and they were covered by the 333 μm sized mesh to prevent the escape of collected crabs. The crab traps were hand-picked by SCUBA divers after around 2 hours of dipping and the collected crabs were immediately transferred into new plastic troughs with native water and slowly acclimated with normal seawater collected from the nearest coastal areas of the Fulong coast, Taiwan. Some of the crabs were stored in 4% buffer formalin (BFS) for scanning electron microscopy study. For elemental and transmission electron microscopy studies the samples were washed thrice in native water and again washed with glass distilled water, stored in the freezer at -20°C and covered with sterile autoclaved plastic bags. Furthermore, live and stored specimens were transported to the laboratory and handled with care. Only adult males and females were subjected to all analyses, with Adult Carapace Width (CW) 23.10± 0.53 mm in male and 20.20 ± 0.69 mm CW in females (Tseng et al., 2020). Based on these criteria, a total of 100 crabs were selected from 583 individuals to elucidate distinct variations and to document pronounced differentiation between male and female populations.

In the shallow vent crab X. testudinatus, males were larger and heavier than females, as indicated by differences in carapace length, carapace width, and total body weight. The male population studied were in the size range of carapace width around 23.10 ± 0.53 mm; Carapace length 21.66 ± 0.47 mm and total weight 6.50 ± 0.70 g and the females were in a range of carapace width 20.20 ± 0.69 mm; Carapace length 18.69 ± 0.57 mm and total weight 4.37 ± 0.37 g. They were employed to highlight sexual dimorphism and to analyze the interrelationships among different morphometric characters, particularly in relation to total carapace width and length in the species.

Our results provide strong support for the hypothesis that the shallow-vent crab X. testudinatus exhibits clear sexual dimorphism expressed through distinct morphometric and morphological traits. Comprehensive morphometric analyses not only confirm but extend earlier findings for this species (Tseng et al., 2020). The observed dimorphism likely reflects adaptive responses to the extreme physicochemical conditions of shallow hydrothermal vents, characterized by elevated temperatures (optimum ~25°C, reaching up to 116°C), high metal concentrations (e.g. Fe, Cu, Zn), and highly variable, acidic pH (1.75 to 4.6).

Sex specific traits such as a broader female abdomen, which enhances egg brooding capacity, and more robust male chelae, likely linked to mate competition and territorial defense, are ecologically meaningful under the strong physical and chemical constraints of shallow hydrothermal vent environments. These traits likely reflect differential energy allocation strategies that maximize reproductive success in habitats characterized by fluctuating temperature, sulfide exposure, and patchy food resources. Comparable patterns of sexual dimorphism have been reported in vent-associated taxa including Bythograea spp. (Guinot and Hurtado, 2003) and Kiwa puravida (Azofeifa-Solano et al., 2022), as well as in coastal and deep-sea crabs such as Gaetice depressus (Takeda et al., 2024), Goniopsis cruentata (Da Silva et al., 2024), Callinectes sapidus (Jiménez et al., 2023), Carcinoplax mistio (Prema et al., 2024), Paromola cuvieri (Capezzuto et al., 2023), Taliepus dentatus (Barría and Antecao, 2025), and Chionoecetes opilio (Mullowney et al., 2023). The recurrence of these sex-biased morphological traits across phylogenetically and ecologically diverse crab lineages highlights their adaptive significance in facilitating reproductive efficiency, niche partitioning, and behavioral specialization in stressful marine environments, including shallow hydrothermal vent systems.

Detailed morphometric analyses of X. testudinatus revealed that the allometric growth patterns of morphological traits were sex specific, with males showing dominant morphological characteristics compared to females. Our results demonstrate that all morphometric traits, except abdominal length, showed significant sexual dimorphism and optimistic allometric growth. Notably, the lack of a significant correlation between carapace width and length with abdominal length suggests that abdominal growth in X. testudinatus may proceed independently of overall carapace size. This finding aligns with Lee et al. (2024), who reported sex specific morphometric characters and distinct allometric growth patterns in the commercially important sand crab Ovalipes punctatus from the Korean coast. In both sexes of O. punctatus, carapace width and abdominal width exhibited optimistic allometric growth, whereas orbital spine width showed negative allometric growth. Similarly, in the present study, abdominal width correlated positively and significantly with other morphometric traits, except for abdominal length. Comparable developmental stages of abdominal growth were documented in female Ovalipes catharus a congener where significant morphological changes occur during maturation at carapace widths of approximately 30 - 40 mm (Davidson and Marsden, 1987). Such allometric shifts during maturation are common among decapods and are often reflected as notable increases in the slope of allometric growth functions.

Statistical analysis revealed highly significant differences (p ≤0.01) in carapace length and width between male and female X. testudinatus, confirming pronounced sexual dimorphism. Males exhibited significantly larger carapace dimensions, likely linked to mating competition and territorial behaviors (Tseng et al., 2020; Fazhan et al., 2021). Correspondingly, males displayed larger chelae (claws) and wider carapaces, whereas females had shorter, more rounded carapaces and broader abdomens adapted for egg brooding (Tseng et al., 2020; Fazhan et al., 2021). The flattened carapace of X. testudinatus is a key adaptation to hydrothermal vents, enhancing current resistance, attachment to rocky substrates, and tolerance to extreme thermal and chemical stress, while potentially supporting symbiotic bacteria (Toyota et al., 2020; Tseng et al., 2020).

Scanning electron microscopy revealed that fine granular textures were more pronounced and more densely distributed in females than in males, particularly on the anterolateral regions of the carapace. This sexual dimorphism in surface granulation may play important roles in reproductive maturity, camouflage, mating recognition, protection, and mechanical durability (Scalici and Gibertini, 2009; Toyota et al., 2020; Fazhan et al., 2021). The carapace of X. testudinatus shows sex specific differences: females have a robust, finely structured surface for protection, metal sequestration, and brooding, while males have smoother carapaces supporting mobility and competition (Zbinden et al., 2018; Tunnicliffe, 1992). Shared traits ridges, spines, and a dome-shaped form enhance fouling resistance, thermal tolerance, and mechanical strength, consistent with adaptations seen in other vent crabs (Kiwa hirsuta, Bythograeidae) (Goffredi et al., 2008; Thatje et al., 2015; Leignel et al., 2017). The hexagonal ommatidial arrangement, similar in both sexes, maximizes field of view and motion detection in shallow, turbid vent habitats, reflecting a conserved, functionally specialized visual system (Müller et al., 2007; Vannier et al., 2016; Greco et al., 2013; Jenkins et al., 2022).

Crystallinity in crab shells reflects the degree of structural order within their bio-polymeric and mineral components, primarily chitin and calcium carbonate (Ogresta et al., 2021). A higher crystallinity index corresponds to a more organized molecular arrangement, which often translates into enhanced mechanical strength. X-ray diffraction (XRD) analyses revealed significant differences in shell crystallinity between male and female X. testudinatus (Souza et al., 2011; Ogresta et al., 2021). Female shells exhibited higher crystallinity indices, indicating a more ordered and crystalline structure, whereas male shells displayed lower crystallinity, suggesting a more amorphous, less organized matrix. These differences may be linked to distinct physiological roles and behavioral strategies. Females often require stronger, more resilient shells to defend territories, which could be supported by increased crystallinity and consequent mechanical robustness (Chen et al., 2008; Sneddon et al., 2000; Ledesma et al., 2010). Variations in shell crystallinity influence hardness, resistance to predation, and environmental adaptability, and they also bear practical significance for the extraction and industrial application of shell-derived biomaterials such as chitin, where crystallinity affects solubility and processing characteristics (Monteiro et al., 2023).

In summary, the elevated crystallinity observed in female crab shells likely reflects adaptive structural modifications aligned with their ecological and reproductive demands, while the comparatively lower crystallinity in males may correspond to alternative functional requirements. Understanding these sex specific differences enhances insight into crab shell biology and informs potential biomaterial applications (Chen et al., 2008; Souza et al., 2011; Ogresta et al., 2021).

Energy Dispersive X-ray Spectroscopy (EDX) and X-ray Photoelectron Spectroscopy (XPS) analyses of X. testudinatus shell samples revealed elemental compositions dominated by carbon (C), oxygen (O), and calcium (Ca), with minor quantities of magnesium (Mg) and phosphorus (P). The weight percentages of carbon and oxygen were approximately 35% and 40%, respectively, while calcium constituted around 20% of the shell matrix. These results align with the established biochemical composition of crab shells, primarily composed of calcium carbonate (CaCO₃) and chitin, an organic polymer rich in carbon and oxygen (Chen et al., 2008; Fabritius et al., 2009; Goldstein et al., 2017; Ismail et al., 2022). Ismail et al. (2022) similarly reported calcium, carbon, and oxygen as the major elements in crab shells, confirming the predominance of mineralized and organic phases. The detection of trace elements such as Mg and P likely reflects physiological residues or environmental incorporation (Goldstein et al., 2017; Ismail et al., 2022).

Comparative analyses between male and female X. testudinatus corroborate prior findings by Zeng et al. (2018), who observed that males accumulated higher concentrations of manganese (Mn), mercury (Hg), and potassium (K), whereas females exhibited greater boron (B) accumulation, indicating sex specific elemental uptake patterns. In contrast, female crab shells exhibited higher crystallinity indices, which may provide greater structural protection. This enhanced shell crystallinity could be an adaptive response to increased mortality risk arising from male populations, as males exhibit more aggressive and combative behaviors that lead to higher rates of cannibalism and female mortality (Chen et al., 2008). Collectively, these findings reveal nuanced sexual dimorphism in X. testudinatus encompassing both morphological and biochemical dimensions. To fully elucidate the physiological and metabolic underpinnings of these differences, further investigation into sex specific activity and metabolism is warranted. Moreover, population genetic and transcriptomic analyses will provide new insights into the morphological and functional differences within the panmictic population.

This study provides clear evidence of pronounced sexual dimorphism in X. testudinatus from the shallow hydrothermal vents of Kueishan Island, Taiwan. Integrating morphometric analyses with SEM, TEM, EDX, and XPS techniques revealed consistent sex specific differences in exoskeletal morphology, crystal orientation, and elemental composition. These traits suggest divergent functional adaptations that enhance resilience and reproductive success under extreme vent conditions.

Distinct cuticle architectures and crystallinity patterns indicated sex specific strategies for coping with thermal, chemical, and mechanical stresses. Together, these findings highlight X. testudinatus as an effective model for investigating adaptive dimorphism in extreme marine environments. By linking morphometric, structural, and elemental traits, this work advances the understanding of crustacean adaptation and informs future studies on the physiological and genetic mechanisms underlying environmental resilience.