DISCUSSION:
Patterns of population-level variation in embryonic responses to maternal provisioning and environmental factors have the potential to inform how the developmental environment contributes to evolutionary change. We observed that, generally, the influence of maternal provisioning on hatchling traits did not vary across populations; however, incubation temperature exerted population-specific effects on both morphological and metabolic traits. This may be explained by a constrained relationship between egg mass and hatch mass (Deeming and Birchard, 2007), which is expected to be maximized as hatchling mass is often an important component of survival and fitness (Ronget et al., 2018; Stearns, 2000). Thus, selection instead tends to act on aspects of maternal allocation, such as egg size and number, to best match population-specific conditions (Angilletta et al., 2004; Sinervo, 1990). On the other hand, responses to incubation temperature may be in part the result of differences in natural nest temperatures across populations, which has been shown in several species (Du et al., 2019), including the alligator (Bock et al., 2020). Such differences likely select for embryonic responses to temperature that match population-specific conditions. Our results suggest that plastic responses to incubation temperature, but not maternal provisions, are a source of interpopulation trait variation and may be more likely to be modified by selection.
The four populations examined in this study encompassed a large proportion of the alligator’s latitudinal range, with two populations from the northern extent and two populations from the southern extent. While not statistically significant, we observed a trend for smaller egg masses at the northern populations relative to the southern populations. In crocodilians, egg mass scales with maternal body size (Larriera et al., 2004), and differences in maternal size might underlie population differences observed here. In mammals, animals from high latitudes tend to be larger than those from low latitudes in a pattern known as Bergmann’s rule (Blackburn et al., 1999), and while this seems to hold in turtles and birds, it does not in other reptiles (Ashton, 2002; Ashton and Feldman, 2003) and has not been examined in crocodilians. Nonetheless, larger egg sizes at southern populations might suggest the opposite pattern. Interestingly, however, allometric relationships between maternal size and egg mass can be altered by environmental conditions, such as salinity (Murray et al., 2013). Whether differences in egg size observed here are the result of differences in maternal size across populations (maximum size or age at reproduction) or population-specific allometric relationships is unknown and an interesting area of future research.
While we expected to find responses to incubation temperature consistent with latitudinal differences between our population pairs, only a few traits showed such patterns. Namely, incubation duration was more strongly influenced by incubation temperature at the northern populations relative to the southern populations. Latitudinal differences in incubation duration have been shown in several species and generally follow one of two patterns: co-gradient variation, in which cooler populations development more slowly relative to warmer populations and counter-gradient variation, in which cooler populations development more quickly than warmer populations (Conover and Schultz, 1995; Pettersen, 2020). Our results show embryos from northern populations develop slightly slower at cooler temperatures and faster at warmer temperatures compared to southern populations. While differences within temperatures were not significant, they followed patterns of both co-gradient variation (at 29.5°C) and counter-gradient variation (at 33.5°C). Similar results have been shown in Asian pond turtles (Mauremys mutica ; Zhao et al., 2015) and may suggest that the mechanisms responsible for variation in incubation duration across populations may be temperature specific. On the other hand, increased plasticity in developmental rate at the northern populations may be driven by more variable thermal environments, which have been associated with increased levels of physiological plasticity (Seebacher et al., 2015). Additional experiments incorporating more incubation treatments and populations are needed to more completely discern how the relationship between temperature and developmental rate differs across populations as well as the underlying mechanisms responsible. Interestingly, we also observed that southern populations tended to allocate more resources towards fat body mass than northern populations at both incubation temperatures. The role of the fat body in alligators is not known, and further work examining its function, including how fat body size/mass early in life might impact survival and later life fitness, is needed to more fully appreciate the potential consequences of these patterns.
Apart from latitudinal trends, there were several differences in the influence of incubation temperature between population pairs, specifically between YK and other populations. In alligators, animals at 33.5°C are generally larger in mass than those at 29.5°C (Bock et al., 2021), which was upheld across all populations. However, at YK, the reduction of hatchling mass at 29.5°C appeared particularly pronounced and appeared to drive additional phenotypic differences. Hatchling mass relative to egg mass reflects the efficiency by which maternal resources are converted into hatchling tissue and is likely a product of the energetic cost of embryonic development (Pettersen et al., 2019). The reduction in mass at YK at 29.5°C relative to the other sites suggests that development at 29.5°C at YK was particularly inefficient. Interestingly, however, animals at 29.5°C at YK tended to have residual yolk reserves that were larger or equivalent to other populations after controlling for mass. This may suggest that alligator embryos preferentially allocate resources towards residual yolk mass at the cost of reduction in overall size under sub-optimal conditions, which has also been shown in other reptiles (Murphy et al., 2020; Radder et al., 2004).
The lack of latitudinal trends in several of the morphological and metabolic traits examined here suggests that latitude may not be the best or only microclimatic proxy within which to understand variation in responses to the developmental environment, particularly incubation temperature. A similar lack of latitudinal patterns in response to incubation temperature was shown across several populations of painted turtles (Chrysemys picta ), another TSD species (Bodensteiner et al., 2019). These results may be driven by too broad a resolution of the relationship between temperature and latitude, making it impossible to discern subtle population differences, or other population-specific microclimatic variables that put selective pressure on thermal reaction norms. For instance, of the four populations examined, YK is the only coastal site, surrounded by brackish water, which may impose unique selective pressures on embryos and breeding females, resulting in differences in response to incubation temperature (e.g., Hudak and Dybdahl 2023). Additionally, other maternal effects, such as yolk composition and deposition of hormones and anthropogenic contaminants, may, in addition to temperature, influence phenotype (Bae et al., 2021; Du et al., 2010b; Groothuis et al., 2005), but were not considered here. Further, since our design focused on incubation temperatures that produce 100% males or females, population variation in response to incubation temperature may have been driven by sex differences that would not be explained by latitude. While previous work has shown that phenotypic differences between incubation temperatures are the result of temperature and not sex (Bock et al., 2023, preprint), whether sex differences exist across populations irrespective of temperature is not known. Future work examining the latter and the role of additional aspects of the developmental environment as potential drivers of variable responses to temperature across populations and the consistency of such effects across years will be particularly informative.