Form Alternation of the Gonopod and Chela from Breeding to Non-breeding Season in Males of the Crayfish Cambaroides dauricus (Decapoda: Cambaroididae)

© 2024 Academia Sinica, Taiwan Open Access Form Alternation of the Gonopod and Chela from Breeding to Non-breeding Season in Males of the Crayfish Cambaroides dauricus (Decapoda: Cambaroididae) Alda1,* 1Independent Researcher, Fushun, Liaoning Province, 113006, China. *Correspondence: E-mail: [email protected] (Alda) Received 23 January 2024 / Accepted 10 April 2024 / Published 4 September 2024 Communicated by Benny K.K. Chan The occurrence of cyclic morphological alternation in male crayfish of the family Cambaridae following molting is widely acknowledged. However, there remains a contentious issue within the genus Cambaroides: Some previous studies have proposed that male Cambaroides exhibit a pair of noncorneous Form II gonopods during the non-breeding season, while others argue that these species lack Form II in adult males. This study examined the color and shape of the corneous tips on the gonopod to determine its form. Additionally, morphometric methods were utilized to distinguish between Form II adults and juveniles. The results of the study confirm the presence of Form II adults in Cambaroides dauricus and Cambaroides similis. The Form I gonopod is characterized by four golden-colored corneous terminal elements, whereas the Form II gonopod features blunt, non-corneous terminal elements, on which the color is the same as that of the surrounding surface. Furthermore, cyclic dimorphism on the gonopod of C. dauricus was observed seasonally, and distinct morphological differences in the chela were noted between Form I and Form II adults. Key words: Cyclic dimorphism, Cornification, Life history, East Asian freshwater crayfish, Juvenile, Adult BACKGROUND The presence of cyclic dimorphism in male crayfish of the family Cambaridae and the genus Cambaroides has been a subject of prolonged debate (Fitzpatrick 1995). Male cambarid crayfish are categorized as Form I adults, Form II adults, and Juveniles based on the morphology and functionality of the gonopod, chela and other characters (Faxon 1884; Hobbs 1945; Scudamore 1948). Form I adult male crayfish molt into Form II during the non-breeding season and revert to Form I during the subsequent molt into the breeding season (Scudamore 1948). The Form I gonopod exhibits corneous terminal elements, while the Form II gonopod features blunt and non-corneous terminal elements. The juvenile gonopod is morphologically similar to that of Form II (Hart 1953). Scudamore (1948) substantiated the idea that sex hormones may regulate the cycle of changes in male sexual form during molting. Within the family Cambaridae, the Form I gonopod was believed to signify sexual maturity or seasonal reproductive activity of males (Larson and Magoulick 2011). The concept of dimorphism of the gonopod was first introduced by Hagen (1870). Faxon (1885) hypothesized the existence of a cycle: Form I in the breeding season would molt into Form II in the non-breeding season. To date, cyclic dimorphism has only been proven in members of the family Cambaridae (Faxon 1885; Hart 1953; Shen et al. 2000; Kawai et al. 2013). Additionally, dimorphism in the chela of male crayfish has been recorded in some American cambarids (Faxon 1884). It was observed that the Form I male of the family Cambaridae Citation: Alda. 2024. Form alternation of the gonopod and chela from breeding to non-breeding season in males of the crayfish Cambaroides dauricus (Decapoda: Cambaroididae). Zool Stud 63:24. doi:10.6620/ZS.2024.63-24. Zoological Studies 63:24 (2024) doi:10.6620/ZS.2024.63-24 1 © 2024 Academia Sinica, Taiwan possesses larger hooks on ischia compared to the Form II male (Faxon 1884; Hobbs 1945). The hook on ischia also exhibits cyclic dimorphism, considered a common characteristic of Cambaroides and the family Cambaridae (Hobbs 1974). Nevertheless, several studies suggested that the conversion from Form I to Form II does not occur universally (Taylor 1985). In recent years, the cyclic dimorphism of female Cambaridae has been discovered and studied (Wetzel 2002; Hamasaki et al. 2020; Schuster et al. 2022; Kawai and Mclay 2023), whereas it has not been observed in female Cambaroides. This distinction arises from the fact that, based on Kawai et al. (1994) and my own unpublished data, adult female Cambaroides molt only once a year in their natural habitat. Genus Cambaroides has six known species. Among them, Cambaroides dauricus is one of the most widespread East Asian crayfish (Kawai et al. 2015). It is found across northeast China, Mongolia, the far east of Russia, and North Korea, extending from the Yalu River basin in the south to the Heilongjiang River basin in the north (Kawai et al. 2015). Localities of C. similis, situated farther south, encompass the Liaodong Peninsula and the Korean Peninsula (Kawai and Min 2005). Cambaroides japonicus is restricted to Hokkaido and the northeast of Honshu (Kawai et al. 2015). Cambaroides schrenckii is distributed in both China and Russia, primarily in the Ussuri River basin, the Lower Songgari River, the Lower Heilongjiang River, and Sakhalin Island (Kawai and Tudge 2008; Kawai et al. 2013 2015; Alda and Kawai 2022). While the life histories of C. japonicus and C. similis have been studied over the last three decades (Kawai et al. 1994 1995; Kawai and Saito 1999 2001; Kawai and Scholtz 2002; Jung et al. 2009), the absence of Form II in adult males of Cambaroides has been a subject of scrutiny. Kawai et al. (2015) proposed the lack of cyclic dimorphism in adult males within the genus Cambaroides, establishing a characteristic that supports the group’s classification as an independent monophyletic family Cambaroididae, beneath the superfamily Astacoidea. The study also revealed that there is no difference in the hooks on ischia of C. japonicus between the breeding and nonbreeding seasons (Kawai and Saito 1999). However, other researchers had reported that the adult male of Cambaroides exhibited an alternate form of the gonopod (Faxon 1884; Hobbs 1974). Faxon (1884) also provided a description based on just four specimens. He described Form I C. dauricus with strongly developed hooks on ischia, while Form II C. japonicus exhibited weaker ones. My earlier collection of specimens (unpublished) also provides evidence supporting the presence of Form II in adult males of C. dauricus and C. similis. To validate the occurrence of form alternation in these Cambaroides species, a comprehensive morphological study was conducted on collected samples. This investigation unequivocally confirmed the presence of such dimorphism in adult males of C. dauricus. The principal emphasis of this study lay in the meticulous examination of C. dauricus specimens, supplemented by a detailed examination of a limited number of C. similis specimens and an exceedingly sparse sampling of C. schrenckii specimens. MATERIALS AND METHODS Study area The study was conducted in the Hunehe River basin in Fushun City, Liaoning Province, China. Considering the low population density of every species of the genus Cambaroides in China, I collected samples from different sampling sites along branches of the river basin of the Hunehe River to avoid catching excessive samples at one site (Fig. 1). The total length of the Hunehe River is 415 km; the major tributaries in the Fushun City area are the Yingge River, Zhangdang River, Suksuhu River, She River, Dongzhou River, and Sarhv River. The sources of these rivers are all mountain streams; the width of the streams ranges from 0.5–10 m. The altitude of the sites ranges from 498–562 m (Jiubing Town), 355–410 m (Tangtu Town), 219–265 m (Zhangdang Town), 289–316 m (Shangjiahe Town), 544–582 m (Wandianzi Town), and 318–354 m (Qingyuan Town). Given the limited variations in altitude, latitude, longitude, and habitat across these sites (Fig. 1), it is reasonable to assume the absence of significant environmental or climatological distinctions. Sampling A total of 177 male Cambaroides dauricus specimens were collected during the study, with 172 samples preserved in 95% ethanol. The remaining 5 individuals were not subjected to ethanol fixation. Additionally, 15 male Cambaroides similis were collected in Liaoning Province, China (Table 1), and 2 male Cambaroides schrenckii were acquired from the Wutong River, Hebei Town, Hegang City, Heilongjiang Province, China. All samples were obtained between April and October, spanning the years 2017 to 2022 (Table 1). Each 1–2 km line transect involved nocturnal manual collection of visible crayfish during stream tracing. Simultaneously, a rudimentary recording of the spermatophores and spawns of females was conducted. Crayfish collection was suspended from November to March due to frozen streams and extremely low page 2 of 14Zoological Studies 63:24 (2024) © 2024 Academia Sinica, Taiwan temperatures (e.g., averaging -10.7°C during the 2020 winter to 2021 spring) in the Fushun City area. Additionally, molting does not occur in winter months (Mitchell and Smock 1991; Kawai et al. 1994). In October 2021, 17 Form I adult male C. dauricus specimens were collected from Fushun County, Fushun City, Liaoning Province, China. These specimens were kept in aquariums (1200 mm * 400 mm * 250 mm, L * W * D), with a controlled water depth of 100 mm. Plastic nets were employed to segregate every crayfish, although they shared the same water supply. The diet comprised fish, shrimp, and Elodea aquatic plants. Water temperature remained uncontrolled, varying from 0 to 25.9°C with seasonal changes, and natural light Fig. 1. Sample Site Localities: Zhangdang Town Site (a), Shangjiahe Town Site (b), Tangtu Town Site (c), Jiubing Town Site (d), Qingyuan Town Site (e), Wandianzi Town Site (f). Table 1. Details of sample collection Species Month Number Site Cambaroides dauricus Apr. 15 Zhangdang Town, Fushun County May. 18 Zhangdang Town, Fushun County Jun. 27 Wandianzi Town, Qingyuan County; Qingyuan Town, Qingyuan County; Tangtu Town, Fushun County Jul. 28 Wandianzi Town, Qingyuan County; Tangtu Town, Fushun County Aug. 26 Shangjiahe Town, Xinbin County Sept. 12 Jiubing Town, Fushun County Oct. 51 Zhangdang Town, Fushun County; Shangjiahe Town, Xinbin County Total 177 106 corneous, 71 non-corneous Cambaroides similis Jul. 7 Gushanzi Town, Haicheng City Sept. 6 Anbo Town, Pulandian District, Dalian City Oct. 2 Gushanzi Town, Haicheng City Total 15 5 corneous, 10 non-corneous page 3 of 14Zoological Studies 63:24 (2024) © 2024 Academia Sinica, Taiwan illuminated the laboratory setting. Morphometric measurements and statistical analyses One hundred and seventy two C. dauricus (101 corneous and 71 non-corneous) and fifteen C. similis (5 corneous and 10 non-corneous) samples were measured. Morphometric measurements were conducted using a vernier caliper on the following characters: POCL (postorbital carapace length), GL (gonopod length), PL (propodus length of chela), PaL (palm length of chela), PaW (palm width of chela), PaD (palm depth of chela), and DL (dactyl length of chela). Analysis focused on 172 C. dauricus samples, considering ratios such as DL/PL, PaL/PL, PaW/PL, PaD/PL, PaD/PaL, PaW/PaL, PaD/PaW, PL/POCL, DL/POCL, PaL/POCL, PaW/POCL, and PaD/POCL for chela morphometrics, and the ratio GL/POCL for gonopod morphometrics. A one-way ANOVA was conducted on these morphometric ratios, with "Form of male crayfish" as the factor, and Tukey tests for pairwise differences, to discern variations in chela and gonopod dimensions among the forms. The allometric growth equation (y = a*x^b) was applied to model the relative dimensions of these chela morphometric characters. Method of form confirmation Male crayfish were classified into three distinct forms based on the size of the chela, gonopod, carapace, and the cornification of the gonopod in the present study. These forms are delineated as Form I adults, Form II adults, and Juveniles. The determination of form is facilitated by the examination of the shape and color of cornification on the surface of gonopod terminal elements, which encompass the mesial process, centrocaudal process, centrocephalic process, and caudal process (Fig. 2). Crayfish exhibiting more sharp terminal elements with a distinctive golden color, differing from the surrounding surface of the gonopod's tip, are classified as corneous Form I adults. Conversely, crayfish lacking sharp terminal elements and displaying a color congruent with the surrounding surface of the gonopod's tip are identified as non-corneous Form II adults or Juveniles (Fig. 2). The Form II adults and Juveniles were determined through the following morphological analysis and statistical methods: Crayfish smaller than the smallest Form I adult were identified as juveniles (Kawai and Saito 1999; Jones and Eversole 2011). However, it's imperative to note that a Form II crayfish, surpassing the size of the smallest Form I, doesn’t automatically qualify as an adult. In this study, for non-corneous samples, crayfish with < 16.22 mm POCL (the POCL of the smallest Form I male C. dauricus in this study, refer to results, Fig. 5) were categorized as Juveniles, while those with > 16.22 mm POCL encompassed oversized Juveniles and Form II adults. Principal Component Analysis (PCA) was initially conducted on POCL, PL, PaL, PaW, PaD, DL, and GL (Fig. 3). Subsequently, utilizing the score of PC1 (Eigenvalue = 6.8738, Variance = 98.20%, refer to results, Fig. 3) and GL/POCL, Hierarchical Cluster Analysis (HCA) was executed (Mean, Euclidean distance). The samples that clustered together on one branch, which contained those smaller than 16.22 mm POCL, were designated as Juveniles, while the remaining samples were classified as Form II adults. RESULTS The Juveniles and Form II adults In 71 non-corneous samples of C. dauricus, with a minimum size of 16.22 mm observed in the smallest Form I POCL, it was challenging to completely distinguish between Juveniles and Form II adults (Fig. 3). Several sampling points exhibited overlap within the 95% confidence ellipse. Hierarchical cluster analysis (HCA) revealed the convergence of these non-corneous C. dauricus samples, encompassing both Form II adults and Juveniles, into three distinct branches (Fig. 4). Samples situated within the branch corresponding to POCL measurements < 16.22 mm were identified as Juveniles (n = 28). The remaining two branches represented Form II adults (n = 43). Notably, five oversized samples (POCL: 17.02, 17.18, 17.28, 17.30, and 17.68 mm) were still classified as Juveniles. Within the Form II adult category, a distinction was made between branches indicative of “moderate size” and “large size” individuals (Fig. 4). Gonopod form alternation adjusted for POCL In this study, C. dauricus (n = 172) measured 10.24–37.92 mm POCL (mean = 22.39 mm, SD = 5.52) and 2.30–14.74 mm GL (mean = 9.12 mm, SD = 2.23). C. similis (n = 15) measured 12.84–38.16 mm POCL (mean = 20.95, SD = 6.49) and 8.82–15.78 mm GL (mean = 8.38, SD = 3.07). The smallest corneous gonopods are presented at 16.22 mm POCL for C. dauricus and 20.64 mm POCL for C. similis (Fig. 5). Samples with a POCL larger than 16.22 mm (C. dauricus) and 20.64 mm (C. similis) in corneous specimens suggest Form I adults. This POCL (16.22 mm) could also be regarded as the size of the page 4 of 14Zoological Studies 63:24 (2024) © 2024 Academia Sinica, Taiwan Fig. 2. Gonopod of Cambaroides dauricus: Form I, 28.38 mm POCL, 10.60 mm GL (a); Form II, 30.18 mm POCL, 11.92 mm GL (b). Gonopod of Cambaroides similis: Form I, 20.64 mm POCL, 8.76 mm GL (c); Form II, 22.18 mm POCL, 8.98 mm GL (d). Gonopod of Cambaroides schrenckii: Form I, 27.14 mm POCL, 11.64 mm GL (e); Form II, 27.08 mm POCL, 11.48 mm GL (f). The terminal elements: mesial process (MP); central projection: centrocaudal process (CaP), centrocephalic process (CeP); caudal process (CP). Scale bars = 1 mm. page 5 of 14Zoological Studies 63:24 (2024) © 2024 Academia Sinica, Taiwan smallest adult male of C. dauricus. Non-corneous samples beyond 16.22 mm POCL (C. dauricus) and 20.64 mm POCL (C. similis), without an absence in large size (Fig. 5), indicate the presence of Form II adults. Simultaneously, a Form II adult of C. schrenckii, with a POCL of 27.08 mm, is depicted in the photograph (see Fig. 2). Form I adult C. dauricus measured 16.22–37.92 mm POCL (mean = 23.85 mm, SD = 4.73, n = 101). Form II adult C. dauricus measured 17.76–35.02 mm POCL (mean = 24.08 mm, SD = 4.38, n = 43). Juvenile C. dauricus measured 10.24–17.68 mm POCL (mean = 14.54 mm, SD = 1.96, n = 28). The annual cyclic life history of adult males and females of Cambaroides dauricus in the Hunehe River basin The percentage of Form I in total adult males remained consistently high in April, May, September, and October (Fig. 6). April (water temperature: 4.9°C) Fig. 3. The Principal Component Analysis (PCA) Biplots of noncorneous C. dauricus samples (n = 71). Employing a threshold of POCL exceeding 16.22 mm as a basis for grouping, the analysis reveals a discernible clustering pattern, where data points are predominantly segregated into two distinct clusters. Notably, a small number of points lie within the overlapping region of the 95% confidence ellipses delineating these clusters. This observation indicates that solely relying on the POCL criterion above 16.22 mm is insufficient for categorical differentiation. Specifically, these points may pertain to either the Juveniles category, characterized by POCL values below 16.22 mm, or the Form II adults group. The axes correspond to the principal components, where PC1 elucidates 98.20% of the total variance (Eigenvalue: 6.8738), while PC2 accounts for 1.05% of the variance (Eigenvalue: 0.0732). Fig. 4. Hierarchical Cluster Analysis Tree depicting 71 non-corneous samples, encompassing 43 Form II adults and 28 Juveniles. The left side of the tree displays the POCL of the Form II adults and Juveniles. Notably, Juvenile samples with a POCL larger than 16.22 mm are highlighted with bold typeface. page 6 of 14Zoological Studies 63:24 (2024) © 2024 Academia Sinica, Taiwan and May are early spring in the northeast of China, and Form I adults reached 100%. In late May, the water temperature was still very cold (7.7°C), Form I adult males had kept corneous throughout winter. Some females had spawned, while others still carried spermatophores. June was the transition period from late spring to summer (water temperature: 13.7°C), and the proportion of Form I began to decline. During this period, a substantial number of adult males molted, and most adult females brought spawns on their swimming legs. July marked the entrance into summer (water temperature: 16.6°C), and the proportion of Form I was the lowest (Fig. 6). The proportion of Form I significantly increased as the season progressed from late summer to early autumn in August. During this period, males and females prepared for mating; Form II adult males molted back to Form I, and adult females also molted. On August 23rd, 2022 (temperature: 19.2°C), I observed five females that had completed mating, carrying spermatophores on their annulus ventralis and the base between the fourth and fifth pereopods. September (temperature: 10.2°C) and October (temperatures: 9.5°C and 3.4°C) corresponded to mid-autumn to late autumn, with the proportion of Form I nearly reaching 100%. Autumn contained numerous matings as the water temperature dropped below 3.4°C. Winter, lasting from November to March of the following year, saw no molting (Mitchell and Smock 1991; Kawai et al. 1994), and the proportion of Form I remained constant. The form alternation of adult male C. dauricus aligned with the seasonal shifts between breeding and non-breeding periods. Dimorphism of the Chela A total of 172 C. dauricus samples, including 144 adult specimens, underwent thorough examination through one-way ANOVA. The results indicate that, relative to the overall body size, Form I chela exhibited greater length, width, and depth compared to Form II (refer to Table 2 and Fig. 7). However, minimal alterations were observed in the ratios between the various morphological characteristics within the chela. This suggests that while the chela vary in size between Form I and Form II, the fundamental shape of the chela remains unchanged. Further elaboration on the distinct Fig. 5. GL fitted for POCL. The upper section of the scatter plot features corneous gonopods, indicative of the Form I adults, while the lower section includes non-corneous gonopods, encompassing the Form II adults and Juveniles. The corneous gonopod’s minimum POCL is 16.22 mm for C. dauricus (a) and 20.64 mm for C. similis (b). In C. dauricus, the black line illustrates the curve of the Form I adult GL fitted for POCL (n = 101), with GL/POCL ranging from 0.3584 to 0.4932 (mean = 0.4153, SD = 0.0219). The red line represents the curve of the Form II adult GL fitted for POCL (n = 43), with GL/POCL ranging from 0.3701 to 0.4305 (mean = 0.4034, SD = 0.0150). Meanwhile, the blue line signifies the curve of the juvenile GL fitted for POCL (n = 28), featuring GL/POCL ranging from 0.2246 to 0.4324 (mean = 0.3813, SD = 0.0424). Significantly, differences in GL/POCL among the Form I, Form II, and Juvenile were observed in C. dauricus (F2, 169 = 20.653, P = 0.000), with the Form I adult displaying a longer GL than the Form II adult when adjusted for POCL (F1, 142 = 10.568, P = 0.001). page 7 of 14Zoological Studies 63:24 (2024) © 2024 Academia Sinica, Taiwan relationships is provided in the subsequent section (Table 2). After adjusting for POCL, significant differences were observed in PL among Form I, Form II, and Juvenile (F2, 169 = 50.673, P = 0.000). Specifically, the PL of Form I exceeded that of Form II (F1, 142 = 12.987, P = 0.000). Similarly, significant differences were found in PaL (F2, 169 = 50.764, P = 0.000), with Form I surpassing that of Form II (F1, 142 = 9.711, P = 0.002). The trend continued with PaW (F2, 169 = 81.610, P = 0.000), where Form I exhibited greater width than Form II (F1, 142 = 10.980, P = 0.001), and PaD (F2, 169 = 71.714, P = 0.000), where Form I had a deeper dimension compared to Form II (F1, 142 = 12.717, P = 0.000). Additionally, DL displayed significant differences (F2, 169 = 28.885, P = 0.000), with Form I surpassing Form II (F1, 142 = 14.577, P = 0.000). Upon adjusting for PL, significant differences persisted among Form I, Form II, and Juvenile for PaL (F2, 169 = 10.976, P = 0.000), PaW (F2, 169 = 16.234, P = 0.000), PaD (F2, 169 = 2.845, P = 0.000), and DL (F2, 169 = 29.576, P = 0.000). However, no significant distinctions emerged between Form I and Form II for PaL (F1, 142 = 0.143, P = 0.706), PaW (F1, 142 = 0.140, P = 0.709), PaD (F1, 142 = 1.051, P = 0.307), and DL (F1, 142 = 0.021, P = 0.886). Upon adjusting for PaL, no significant difference was observed in PaW among Form I, Form II, and Juvenile (F2, 169 = 2.759, P = 0.066). However, significant differences were noted in PaD (F2, 169 = 9.082, P = 0.000). PaW (F1, 142 = 0.448, P = 0.504) and PaD (F1, 142 = 0.828, P = 0.364) of Form I were not larger than for Form II. Upon adjusting for PaW, significant differences were detected among Form I, Form II, and Juvenile for PaD (F2, 169 = 14.369, P = 0.000), with Form I displaying greater depth than Form II (F1, 142 = 9.361, P = 0.003). Molting, form alternation, and death of adult male Cambaroides dauricus during feeding in the aquarium From October 2021 to October 2022, 17 adult male C. dauricus samples were kept in aquariums with water temperatures ranging from 0 to 25.9°C. Spring molting occurred between 18.5 and 23.0°C during the late spring and early summer, with 10 individuals successfully molting in June and July (58.82% of total samples). Mortality peaked at temperatures from 22.2 to 25.9°C, with 11 individuals (64.71% of total samples) succumbing between July 25th and August 12th. Notably, 4 individuals (23.53% of total samples and 36.36% of dead samples) died post-molting, and 7 (41.18% of total samples and 63.64% of dead samples) died without molting. Among the survivors, one individual died on August 28th, foregoing autumn molting, while 5 re-molted in September (29.41% of total samples, 83.33% of survivors). All Form I crayfish, which successfully molted in June and July, altered into Form II after spring molting. Meanwhile, Form II crayfish that successfully molted and survived in September all reverted to Form I after autumn molting. 12 individuals (70.59% of total samples) died during feeding (Table 3). No instances were observed of adult forms changing from Form II to Form II simultaneously. DISCUSSION Kawai and Saito (1999) reported the absence of the Form II gonopod in adult male C. japonicus and detected no difference in chela length between breeding and non-breeding seasons (Kawai and Saito 2001). As a result, they suggested the lack of cyclic dimorphism in C. japonicus. Relying on specimens of C. japonicus, C. similis, and C. schrenckii, previous studies have consistently shown that the gonopod of the genus Cambaroides maintains Form I consistently throughout both breeding and non-breeding seasons (Kawai and Min 2005; Kawai and Tudge 2008; Kawai et al. 2013). Nevertheless, reports of Form II adult males in the genus Cambaroides also exist. Faxon (1884) described Form II C. japonicus with smaller and non-corneous terminal elements of the gonopod, suspecting a dual form in males of Cambaroides, similar to Cambarus. Kawai (unpublished), upon inspecting Faxon’s specimens, noted their dry or ethanolic preservation for hundreds of years, causing the color of the terminal elements to Fig. 6. The annual cyclic life history of adult male and female C. dauricus in the Upper Hunehe River basin. The top half illustrates spermatophores and spawns on the female in various months. The bottom half features a histogram displaying the proportion of Form I males (n = 106) among all adult male C. dauricus (n = 149) across different months. The accompanying dot plots provide information on water temperature. page 8 of 14Zoological Studies 63:24 (2024) © 2024 Academia Sinica, Taiwan become indistinguishable. Given the presence of sharp apexes on the gonopod in all specimens, he dismissed their classification as Form II. Hart (1953) mentioned Form II C. similis and C. schrenckii and Form I C. japonicus in his examined specimens. The current study supports the occurrence of the Form II gonopod in adult male C. dauricus, C. similis, and C. schrenckii. Building on prior studies and additional evidence presented in the current study, the prevailing opinion is that cyclic dimorphism occurs in adult male C. dauricus, C. similis, and C. schrenckii, while adult male C. japonicus only exhibits Form I. Form alternation absence The alternation from Form I to Form II in males of the family Cambaridae has been generally accepted and discussed, while some investigations have shown that a complete alternation between Form I and Form II did not necessarily occur (Taylor 1985). Another pattern of dimorphism absence is observed in the family Astacidae, where the gonopod of the adult male consistently remains non-corneous with a membranaceous tip (Hagen 1870; Faxon 1885). Incidentally, Schuster et al. (2022) mentioned that many crayfishes in the southern USA do not strictly obey the rules of form alternation. Their observations in Alabama revealed instances where many crayfishes remained in Form I throughout the year. The absence of Form II in adult C. japonicus may be attributed to its annual molting cycle. Kawai et al. (1994) substantiated that the molting season of C. japonicus occurs from June to October, exhibiting a singular peak. In laboratory rearing, Kawai et al. (1995) observed that both male and female adult Cambaroides molt only once a year, suggesting an annual molting cycle for C. japonicus. C. japonicus is primarily restricted to islands, and predominantly found in Hokkaido. The limited and northern distribution range of C. japonicus localities may further inhibit the occurrence of a second molting event in Fig. 7. The association among morphometric characteristics of the chela in C. dauricus. The legend conventions remain consistent across all figures, with the solid circles representing the Form I adults, the hollow boxes denoting the Form II adults, and the hollow triangles indicating the Juveniles. Additionally, the black curves correspond to the Form I adults, the red curves to the Form II adults, and the blue curves to the Juveniles. page 9 of 14Zoological Studies 63:24 (2024)