The idea that development influence evolution and the search for universal developmental rules that could explain the diversity of life forms traces its origins back to the 19th century, when early embryologists were comparing embryos, trying to find links between them. One of the well-known, albeit incorrect, hypotheses from this period is the Haeckel recapitulation theory, which postulates that "ontogeny recapitulates phylogeny.". While the idea that development can influence the evolution shadowed after this, it resurfaced in the mid-20th century due to the contributions of Alan Turing (Turing 1952), Raup (Raup 1966), and, eventually, Gould in 1977 with his book Ontogeny and Phylogeny (Gould 1977). These findings reintroduced the concept that development and evolution are intertwined, suggesting that developmental constraints, akin to physical forces, could potentially shape the evolution of organisms (Smith et al. 1985; Hall 2012). In 2007, a groundbreaking paper identified one of these developmental constraints or rules in the patterning of mouse molars (Kavanagh et al. 2007). They found that the reaction/diffusion or Turing-like mechanisms that control tooth development constrain the relative sizes of mouse molars and identified a developmental rule able to predict the size of various rodent molars. When tested on other clades (Polly 2007) and even on other organs that undergo development similar to teeth (Young et al. 2015), this rule was found to be quite robust. However, certain species and organs appeared to deviate from it (Polly 2007; Carter and Worthington 2016; Roseman and Delezene 2019), raising interesting questions to explore: Could this rule be a particular case? Are there universal rules applicable to all organisms? How are these rules modified to generate morphological diversity?
To find answers, two things were needed: (1) an ecologically diverse group of organisms in which to study variation (2) across all stages of development and not solely in adults.
Noctilionoids bats, the group of bats we used in our paper, meets these requirements. This super-family has evolved almost all possible mammalian diets, some species specializing on fruit, nectar, pollen, fishes, insects, vertebrates or even blood (Figure 1). In tandem with the adaptation to various diets, these bats have evolved a various number of tooth shape and number in only 30-ish million years (Freeman 1998; Dumont et al. 2012; Fleming et al. 2020). On the contrary to other models, the ability to obtain specimens from field captures and the richness of museum collections (Figure 2) make possible to study the developmental mechanisms that shape this variation for all tooth classes across all developmental stages in various ecological contexts.
We first approached this by testing if premolars and molars proportions follow the ones predicted by the original rule. We realized that it was not the case, and more interestingly, that molars and premolars seemed to diverge from the rule in two different ways, suggesting that they develop and evolve independently. So, we then wondered: how to demonstrate this independence? Are these rules completely different in bats and between tooth classes? Or are there other factors (morphology, development, other developmental mechanisms) that can modify the resulting phenotypes?
Among others, one morphological trait seemed to stand out, the face/jaw length. Indeed, not only these bats have different teeth, but they also have different faces which is generally linked with their adaptation to various diets. For example, the nectar bats have an elongated face that allow them to reach the center of the flowers, and the fruits bats have a shorter face associated with a strong bite force to crush fruit pulp (Figure 3; believe me, you can feel it when they bite to escape our careful hands!). Indeed, we found a correlation between jaw length and tooth number and size in Noctilionoids and decided to investigate the downstream developmental mechanisms that could explain how the development of the jaw, teeth and developmental rules interact to explain this diversity. We combined multiple techniques and approaches. First, we reconstructed tooth development from bud to final stage using contrasted µCT scans to identify if premolars and molar development is independent and how and which of them are lost in multiple species of bats. We found that premolars and molars develop independently, in opposite directions. We also found that tooth losses occurred very differently in these two tooth classes as jaw shorten during evolution, supporting the idea of separate mechanisms for their development.
This latter observation suggested that jaw growth variation (making the jaw long or short through the modulation of growth during development) could have an impact. To verify this, we calculated the the growth rate between the different species on µCTscans and visualized the differences in cell division rate using two makers of cell division. We found that bats with elongated jaws exhibit more cell divisions compared to those with regular or shorter jaws, when teeth are forming successively. This finding support the idea that variations in jaw growth play a role in regulating both the size and sequential emergence of teeth, likely through the modulation of rules or Turing mechanisms governing their development. To confirm this intuition, we modeled the sequential emergence of premolars and molars by classical reaction/diffusion (or Turing) mechanisms in which we introduced variations in growth rates to simulate the observed patterns in bats, successfully reproducing the phenotypes observed within this family.
There are three major conclusions from our work.
1 - We found that two tooth classes, premolars and molars, develop and evolve independently from one another by two different Turing-like rules in bats, and probably other mammals. This important result bring a new piece of evidence regarding the developmental and evolutionary differences between tooth classes - a major mammalian innovation - that remain relatively obscure and limited in their taxonomic scope.
2 - The original developmental rule is not necessarily wrong – teeth and other developmental appendages develop through Turing-like mechanisms – but the equation and timing may vary between species and organs depending on other developmental parameters. Here we show how the interaction of multiple developmental constrains can generate some variation during evolution.
3 - Finally, our work demonstrates how new morphologies are reached by modulating the interaction between multiple developmental constraints during the burst of diversity that accompanies adaptive radiations. While the idea that growth rate variation is important for Turing mechanisms is not novel (Kondo 2002; Kondo and Miura 2010), our work proposes that it can facilitated the apparition of new phenotypes in teeth and potentially other ectodermal appendages.
In our future research, we will use this powerful model system to try to understand what genomic variation trigger these differences and extend these finding to other clades. As we like to say in the lab, "Bats are like Darwin finches but weirder". In this case, it's once more completely true and it helps us to better understand how morphological traits diversify during the colonization of new ecological niches, both thanks to and against developmental constraints.
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