ARTICLE https://doi.org/10.1038/s41467-021-21757-5 OPEN Evolution of the locomotor skeleton in Anolis lizards reflects the interplay between ecological opportunity and phylogenetic inertia ✉ Nathalie Feiner 1 , Illiam S. C. Jackson 1,2, Edward L. Stanley 3 & Tobias Uller1 Anolis lizards originated in continental America but have colonized the Greater Antillean islands and recolonized the mainland, resulting in three major groups (Primary and Secondary 1234567890():,; Mainland and Greater Antillean). The adaptive radiation in the Greater Antilles has famously resulted in the repeated evolution of ecomorphs. Yet, it remains poorly understood to what extent this island radiation differs from diversification on the mainland. Here, we demonstrate that the evolutionary modularity between girdles and limbs is fundamentally different in the Greater Antillean and Primary Mainland Anolis. This is consistent with ecological opportu- nities on islands driving the adaptive radiation along distinct evolutionary trajectories. However, Greater Antillean Anolis share evolutionary modularity with the group that reco- lonized the mainland, demonstrating a persistent phylogenetic inertia. A comparison of these two groups support an increased morphological diversity and faster and more variable evolutionary rates on islands. These macroevolutionary trends of the locomotor skeleton in Anolis illustrate that ecological opportunities on islands can have lasting effects on mor- phological diversification. 1 Department of Biology, Lund University, Lund, Sweden. 2 College of Natural Sciences, University of Texas at Austin, Austin, TX, USA. 3 Department of ✉ Natural History, Florida Museum of Natural History, University of Florida, Gainesville, FL, USA. email: [email protected] NATURE COMMUNICATIONS | (2021) 12:1525 | https://doi.org/10.1038/s41467-021-21757-5 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21757-5 ineages that colonize islands often rapidly diversify along Anolis and Primary Mainland Anolis. However, the evolutionary distinct evolutionary trajectories, as famously demonstrated modularity of Greater Antillean Anolis was shared with the group L 1 2 by the Darwin Finches and Hawaiian silverswords . that recolonized the mainland, a pattern accompanied by higher Important reasons for this include that islands can harbor a morphological diversity and faster and more variable evolu- range of ecological settings, few competing species, and low tionary rates on islands. These macroevolutionary trends illus- predation3,4. In comparison, lineages that establish on the trate how morphological diversification is shaped by the interplay mainland typically face more limited opportunities to diversify between ecological opportunity and phylogenetic inertia. since ecological niches tend to be already occupied by similar organisms, and competition and predation may be severe. Results Ecological opportunities do not exist in isolation, however, and A phenotyping of 704 individuals from 271 species (including they will be realized only insofar as there are phenotypes that can four closely related non-Anolis species) allowed us to unravel exploit them5. In the short term, the capacity to adapt is limited patterns of evolutionary diversification in the locomotor skeleton by the phenotypes that can be generated from standing genetic of Anolis lizards. Specimens from museum collections were variation6,7. In the longer term, adaptive diversification depends scanned using microcomputed tomography (micro-CT). We used on the capacity of development to generate phenotypes that can – 3D geometric morphometrics to capture variation in the shape of interact with the environment in novel ways8 10. Differences in the pectoral and pelvic girdles with 18 landmarks each (Supple- development, physiology, and behavior can lead to persistent mentary Table 1)38,39, as well as univariate length measurements differences between clades in the extent to which parts of the of 15 limb elements that capture morphological variation in the organism evolve together (i.e., evolutionary modularity and fore- and hindlimbs relative to body size (Supplementary Fig. 1). integration11, reviewed in refs. 12,13). Yet, the relationship We supplemented these four blocks (pelvic girdle, pectoral girdle, between adaptive diversification and evolutionary modularity – forelimb, and hindlimb) with centroid size as a proxy for body remains poorly understood11,14 18. On the one hand, diversifi- size, resulting in a dataset comprising 124 features or traits. To cation into novel ecological opportunities may require changes in allow an inclusive analysis of the entire locomotor skeleton, we variational properties, in particular less constrained covariation – standardized (z-transformed) the 124 traits to account for the fact between parts19 22. For example, primates specialized in vertical that they are not on a commensurate scale40. This procedure clinging and leaping (e.g., lemurs and tarsiers) have weaker removes certain properties (e.g., the original trait variances) from phenotypic integration between fore- and hindlimbs compared to the dataset40,41, but the transformation allows us to infer mor- other quadruped primates23. In contrast, the extraordinary phological differences among Anolis groups across the entire diversification of beak and skull shape in Hawaiian honeycreepers locomotor skeleton, which is our primary focus. Analyses that and Darwin finches proceeded along the same evolutionary would be compromised if performed on standard normal deviates covariance as other birds, illustrating that the existing develop- (e.g., disparity analysis)40 were performed on girdles and limbs mental and functional integration of the avian head is fully separately to allow retention of original trait variances42. capable of generating extreme morphologies24. The biogeographic history of the species-rich Anolis lizards offers an outstanding opportunity to explore how ecological Greater Antillean Anolis show greater morphological disparity opportunity and developmental bias shape adaptive diversifica- than the Secondary Mainland clade. Anolis species belonging to tion. Early in the history of the Anolis genus, original mainland the Greater Antilles, Lesser Antilles, and the Primary and Sec- forms (hereafter Primary Mainland) from continental America ondary Mainland groups occupy largely overlapping regions in colonized Greater Antillean islands where they diversified into morphospace, although slight differences exist (Fig. 1b, c, and more than 100 species (Fig. 1a). Subsequently, Anolis lizards most Supplementary Fig. 2). Variation in PC1 and PC2 is associated closely related to extant Jamaican species dispersed back to with the shape of the pelvic and pectoral girdle, respectively, Central and South America (hereafter Secondary Mainland) and whereas variation in PC3 is also associated with variation in the gave rise to over 100 extant species25–27. The numerous small relative lengths of limb bones (Supplementary Table 2). Body size islands of the Lesser Antilles that typically contain only one or does not load strongly on any of the first three PCs. two species per island were colonized in two waves, one early The evolution of the locomotor skeleton is characterized by a fi = = wave from the Primary Mainland clade, and one later wave from signi cant phylogenetic signal (Kmult_total 0.571; Kmult_girdles 28 = fi the Greater Antilles (Fig. 1a). 0.586; Kmult_limbs 0.945; all P < 0.001), which justi es taking a Greater Antillean Anolis have produced neither more species formal comparative phylogenetic approach (see “Methods”). nor an overall higher diversity in gross morphology than main- Using this framework, we find that species of the Greater Antilles land anoles29–31. However, the Greater Antillean and mainland occupy a larger volume in morphospace (Fig. 1b, c and Anolis appear to differ in the functional relationship between Supplementary Fig. 2) and show higher morphological disparity ’ = = morphology (e.g., relative limb length) and aspects of the lizards (Procrustes variance (PV)girdles 0.024; PVlimbs 0.051) 32–34 = ecology (e.g., perch diameter) . In particular, the Greater than species inhabiting the Lesser Antilles (PVgirdles 0.015, = = = Antillean islands are characterized by the presence of up to six Pgirdles_GAvsLA 0.006; PVlimbs 0.011, Plimbs_GAvsLA 0.002) = ecomorphs, each adapted to a certain microhabitat that imposes and species of the Secondary Mainland clade (PVgirdles 0.017, 35–37 = = distinct functional demands on locomotion . Pgirdles_GAvsMLsec <0.001;PVlimbs 0.038, Plimbs_GAvsMLsec 0.036). In this work, we test if the adaptive radiation on the Greater In contrast, the morphological disparity of the Primary Mainland Antilles was accompanied by faster or more variable evolutionary clade is on par with the high levels attained by the Greater = = rates and increased morphological disparity of the locomotor Antillean group (PVgirdles 0.022, Pgirdles_GAvsMLpri 0.460; = = skeleton than on the mainland. Further, we test if island and PVlimbs 0.047, Plimbs_GAvsMLpri 0.707; Supplementary Tables 3 mainland diversifications have proceeded along similar and dee- and 4). Thus, the colonization of the Greater Antillean islands was ply conserved patterns of covariation in the locomotor skeleton, not accompanied by a more extensive exploration of morphospace or if the adaptive radiation on the Greater Antilles was accom- than on the mainland, but