In this study, we have described the current state of knowledge of digit ratios in amphibians and conducted a detailed analysis of the digit ratio patterns in the common toad B. bufo. The main question addressed here was whether any differences exist between males and females in the three most often investigated digit ratios, namely, 2D:3D, 2D:4D, and 3D:4D. The detected sex differences in digit ratio are quite ambiguous and do not form a consistent pattern. Significant differences between sexes were found in the following digit ratios: forelimbs, left 2D:3D (variants I and II), right 2D:3D (variant I), right 2D:4D (variant II), and left and right 3D:4D (variants I and II); hindlimbs, left 2D:3D and left 3D:4D (Tables 2, S2). However, the effect size values for these digit ratios corresponded to small or medium effects, similar to those detected in newts , whereas for comparison,  found medium or large effects in the studied anuran species. We detected no differences in hindlimb 2D:4D (Tables 2, S2). We have decided to present the full results for both variants of finger numbering, as we predict that, in future studies, other digit ratios will be used more frequently (e.g., 2D:5D, 3D:5D, and 4D:5D) . We argue that variant II (i.e., the assumption of a reduced first digit, with digits II–V present on the forelimbs) should be commonly adopted in digit ratio studies in anurans (see: Limb and finger development in anurans). It is impossible to provide a detailed discussion based on the results previously obtained by  in O. pumilio and by  in Leptodactylus frogs, as these authors analyzed only 2D:4D (excluding 2D:3D and 3D:4D); we detected no sex differences in this digit ratio when using their finger numbering scheme (referred to as variant I in this study). Nevertheless, we would like to draw attention to some general problems, which in our opinion are significant irrespective of the finger numbering system adopted. Clarification of the large discrepancies in the previously obtained results on amphibians is challenging (Table 1). First, the absence of sex differences in digit ratio may be due to the small sample sizes used in the research. Among the 12 species examined, only in the case of E. pustulosus, L. podicipinus, and B. bufo did the sample size exceed 100 individuals [5, 27] (the present study). Second, in the case of monomorphic species without clear sexual dimorphism, the lack of dimorphism in the digit ratio appears to be the expected state. This may be true of some frogs (L. fuscus and L. pakeka) and salamanders (S. salamandra) [5, 24, 28], as well as of members of other evolutionary lineages such as birds, e.g., the white stork Ciconia ciconia , or mammals, e.g., the American red squirrel Tamiasciurus hudsonicus . However, it should be remembered that the lack of differences between sexes in the digit ratio may be a byproduct, or, in a case where individuals occupy different habitats, the result of natural selection (see Introduction, as well as ). Finally, digit ratio measurements may be susceptible to artificial variation resulting from the accuracy of the methods used or the condition of the preserved individuals [26, 46, 47].  suggested that the differences in the results of the two studies on O. pumilio were related to differences in the measurement methodology adopted, i.e., hand calipers versus software analysis of digital photographs. Moreover, sample preservation may induce changes in digit ratio: in the case of New Zealand geckos Woodworthia, a change in the relative length of the phalanges was found in individuals following preservation for 1 year in 10% neutral buffered formalin . This suggests that the optimal method entails the measurement of live individuals, whereas direct comparisons of digit ratios between preserved and live specimens should be avoided. Additionally, the current state of knowledge suggests that substantial interpopulational differences in digit ratio exist. This phenomenon was detected, e.g., in humans, and has been interpreted in the context of the impact of harsh environments . As a consequence, interpopulational variability should be taken into account when designing research on digit ratio, whereas heretofore, in amphibians, only two studies have been conducted on more than one population: six populations in the case of L. podicipinus  and three in the case of B. bufo (this study). However, although our sampled populations came from two habitats that had been altered and one that was relatively uninfluenced by human activity, we found no interpopulational differences.
Limb and finger development in anurans
We conducted our study using two alternative forelimb digit numbering schemes in Anura (Fig. 1), including one differing from those used in all of the earlier studies on the subject [24,25,26,27]. Our approach was based on a thorough review of the literature on the development of limbs in amphibians, which led us to call the previously implemented numbering of the digits of the forelimbs into question (referred to as variant I in this study). A general scheme of limb development in tetrapods, containing a description of the homologies of skeletal elements and based on a morphogenetic approach, was proposed by . In general, postaxial limb development in anurans is analogous to that in amniotes . However, the homology of amphibian digits and true digits of other tetrapods remains unclear [32, 50], since, in amphibians, digits develop through the differential proliferation of cells, whereas in amniotes such as mammals and birds, massive cell death of interdigital tissue is involved in the process [32, 50, 51].  speculated that “ancestors of the modern amphibians and reptiles had cell death but the modern amphibian forms have lost it” or that the mechanism of cell necrosis during limb development was established after the amniotes had separated from the early amphibians. Additionally, limb development in Anura, which is associated with amphibian metamorphosis, occurs at a much later phylotypic stage than in amniotes . Importantly, anurans, as mentioned in the Introduction, possess only four digits on their forelimbs; according to experimental studies, the lost finger is the most preaxial finger, i.e., finger I; as a consequence, anuran forelimbs contain fingers II–V [31,32,33]. The growth of fingers is a constant developmental sequence and takes place in the sequence IV, V, III, II . In general, in all Anura, the fourth digit of the forelimb is the longest . On the hindlimbs, the digits are formed in the sequence IV, III, V, II, I . In addition to phalanges, some additional skeletal elements, such as prepollex (prepollices) and prehallux (prehallices, preaxial digit-like structures), may occur in anuran limbs, albeit with considerable interspecific variation . We conclude that our approach, assuming that the first digit was reduced on anuran forelimbs (variant II), is strongly supported by earlier studies on limb development. Thus, the results concerning forelimbs in previous studies that adopted digit numeration according to  should be viewed with extreme caution [24,25,26,27].
Directional asymmetry of the digit ratio and other issues
In our dataset, asymmetry was detected for most digit ratios on the forelimbs, except for 2D:3D in variant I (corresponding to 3D:4D in variant II) (Table 3). We found significant differences in digit ratios between body sides, but the calculated effect sizes were small regardless of the finger-numbering variant used, with g values clearly less than 0.5 (Table 3). In previous studies, asymmetry in digit ratio was also found on the forelimbs of C. bransfordii and O. pumilio and on the hindlimbs of O. pumilio . Asymmetry in digit ratio is also present in other systematic groups: tailed amphibians , birds , and humans . The direction of asymmetry differed between the variants of digit numbering employed. In variant I, the digit ratio showed right-biased asymmetry; in variant II, the opposite was true (Table 3). Another paper  suggested that right digit ratios are more closely correlated than left digit ratios with sex-dependent traits because the former are more susceptible to sex steroids. It is worth noting that in Anura, the process of forelimb emergence during metamorphosis varies between sides of the body. In our opinion, this may be linked with some differences in limb development, including digit ratio patterns. In anurans, one of the forelimbs growing during metamorphosis (Gosner stages 41 to 42) emerges through the spiraculum, and the second forelimb perforates the skin. In the case of B. bufo, it is the right forelimb that emerges first, regardless of the left-side position of the spiraculum . Unfortunately, such detailed information on amphibian larval development is available for only a few species. Finally, the varying trajectories of forelimb development in anuran amphibians open up interesting possibilities for further research on digit ratios. Similarly, an interesting possibility is the inclusion of variation in locomotion modes in asymmetry research: toads and dendrobatids (e.g., O. pumilio) mainly move asymmetrically (walking, or, less frequently, jumping), whereas the other tested species are characterized by symmetrical locomotion (jumping and swimming).
Some studies indicate the phenotypic plasticity of amphibian limbs in various habitats. For example, B. bufo males from areas of intensive farmland were heavier and, importantly, were less symmetrical (in both hind- and forelimbs) than individuals from less disturbed sites, perhaps due to increased environmental stress during larval and/or postmetamorphic development . To the best of our knowledge, variability in digit lengths and digit ratios has not been analyzed in relation to habitat quality. Our results, despite the large sample, do not fill this gap, as we found no differences between the study sites. The main limitation of our data in this context was our use of only three sampling sites (two sites under strong human pressure and one in a forested area, thus resembling the species’ original habitat much more closely). As a consequence, being aware of the limitations described above, we make no attempt to provide a more profound explanation of the detected directional asymmetry. In future research, it would be advisable to use other methods (e.g., fluctuating asymmetry) and to sample more sites covering a wider area.
The final issue is that lateralization exhibits a relationship with digit ratio . Generally, hand preference in humans is a correlate of sensitivity to testosterone in the developing fetus , but  suggests that gene-based mechanisms mediate the effects of hand preference on digit ratios. In B. bufo, right-handedness was detected in 59% of individuals based on the snout-wiping test . However, in the green toad Bufotes viridis, the opposite trend was detected, whereas in R. marina, as well as in true frogs, no dominance in forelimb use was observed (reviewed in ). Therefore, even for relatively closely related species such as bufonids, no compatibility is present in this trait. Moreover,  found no correlation between forelimb use preferences and the previously mentioned sequence of forelimb emergence in some anuran species (e.g., the common spadefoot toad Pelobates fuscus and the common frog Rana temporaria), whereas such a relationship has been detected in B. bufo. In our opinion, the observed asymmetry in anurans is due to asymmetrical development and/or, possibly, to variation in locomotion modes rather than being a derivative of lateralization. However, further research on the topic is required.
Relationship between digit ratio and age
Using the second dataset (with individuals from site 2, the anthropogenically altered habitat), in which we determined individual age using skeletochronology, we found significant differences in right-forelimb 2D:4D; the relationship that we found was related to the age and size of the individual. In terms of age differences, older individuals appear to be characterized by a lower 2D:4D on the right forelimb (Fig. 2, panel: b). Interestingly, individuals at the age of 6 years exhibited both lower SDISVL values and higher SDIdigit ratio values than younger individuals (excluding those 5 years old; Fig. 3, panels: c, d). This can be interpreted as differences driven by environmental conditions during the tadpole stage or by selection during adulthood. Two scenarios may be considered in the interpretation of such results from wild populations. In the first scenario, individuals with advantageous hormonal milieus have a greater chance of survival in adulthood, which explains their overrepresentation in the older age cohorts. Notably,  showed that digit ratios are indicators of expected fitness and that early environmental effects coded in 2D:4D are of long-term relevance to reproductive success in the collared flycatcher Ficedula albicollis. In the second scenario, each age cohort exhibits specific traits resulting from the conditions in which they developed as tadpoles.
Generally, the digit ratio is sensitive to changes in the hormonal milieu (see experimental studies on the subject: [6, 8, 62]; thus, it may be used as a feature indicating environmental contamination with endocrine-disrupting substances. Amphibians are considered useful indicator organisms, especially for endocrine disruption [63, 64]. Therefore, their susceptibility to endocrine-disrupting substances during development appears certain; this applies to B. bufo as well [65,66,67]. As a consequence, digit ratio in anurans may be used as a bioindicator of some hormonal disorders, as suggested by . This assumption was later confirmed experimentally with the use of testosterone added to water with developing tadpoles . However, given the high level of sensitivity of amphibians to stress factors, it can be surmised that endocrine-disrupting substances are not the only factor capable of shaping the digit ratio pattern. An array of environmental factors, including variation in diet , competition [69, 70], the presence of predators [71, 72], diseases, and parasites, may also affect the growth trajectories and survival of metamorphs, potentially influencing adult fitness [73, 74]. Selection in juvenile amphibians is very strong, resulting in low overall survival rates at this stage; however, in temperate climates, conditions may vary from year to year, leading to different selective pressures between age cohorts. Depending on the stability of local habitat conditions, environmental factors may act with varying degrees of severity between seasons, e.g., in the form of water level fluctuations, drought, abnormally high temperatures, or varying pollution levels. As a consequence, each age cohort may show varied responses to stressors, depending on their severity and relevant interactions during development at the tadpole stage. These shifts in conditions may in turn be reflected in detectable differences in the digit ratio.
Thus, in our opinion, individuals of the same age should be used in future studies on digit ratios in amphibians in order to exclude differences between cohorts resulting from differences in developmental conditions at the time when limbs were formed. Accordingly, we recommend conducting further research under laboratory conditions, as obtaining a sufficient number of specimens of a particular age in natural conditions is expensive and time-consuming and necessitates injuring animals in the case of skeletochronology.