*1.1. Isla Malpelo*

Isla Malpelo (3.5 km2, Figure 2a) is a remnant volcanic plug (hardened magma within the vent of a volcano) and is estimated to be 15–17 million years old [26], representing an older stage in the life of a volcanic island. A younger Malpelo was considerably larger and most likely vegetated [27], but now the floral communities of Malpelo's steep slopes are sparse, which likely contributes to a species-poor ecosystem [28–31]. No terrestrial vertebrates are found on Malpelo except for three endemic species of lizards: an anole (*Dactyloa agassizi*), a galliwasp (*Diploglossus millepunctatus*), and a gecko (*Phyllodactylus transversalis*). Without sufficient primary producers to anchor the trophic pyramid of Isla Malpelo's ecosystem, all three lizards are dependent upon the colonies of a unique keystone species, the Nazca Booby (*Sula granti*), which seasonally nests on the island [32]. Lizards and crabs (*Geocarcinus malpilensis*) compete inter- and intra-specifically for booby fecal matter, unprotected eggs, and dropped or regurgitated fish. *Dactyloa agassizi* is the most abundant lizard species on the island with an estimated population of 140,000–206,000 [33]. Its natural history and behavior are unique among anoles, in that individual males do not defend territories or display aggression toward one other [34], (pers. obs. KEN and MMW). This may be due to a lack of importance of territory, given the stochastic nature of nutrient availability and the lack of habitat heterogeneity. They are also very curious and unafraid of humans, readily approaching and investigating them, presumably in search of food [34], (pers. obs. KEN and MMW). *Dactyloa agassizi* belongs to a clade of anoles (*Dactyloa*, *sensu* Nicholson et al. [21]) ranging from northern Costa Rica to central South America [35–37]. A recent study examined the divergence of *D. agassizi* from mainland *D. insignis*, finding this split to predate the origin of Isla Malpelo [24]. *Dactyloa insignis* is one of the northernmost-ranging *Dactyloa* species, so the mainland ancestor of *D. agassizi* may have originated in northern Costa Rica.

## *1.2. Isla Cocos*

Isla Cocos (24 km2, Figure 2b) also has a single endemic anole, *Norops townsendi*. The environment of Isla Cocos is dramatically different from Malpelo [38], (pers. obs. KEN and MMW), being a highly vegetated and lush tropical island with cloud forest around its highest peak. Similar to Isla Malpelo, Cocos is volcanic in origin, although its origin is estimated to be much younger at 1.9–2.4 Mya [39]. Isla Cocos likely represents how Malpelo might have appeared earlier in its geologic life, while Cocos will likely more closely resemble Malpelo over time. *Norops townsendi* is found throughout the island from sea level to its peak (Cerro Yglesias, 575 m) and is very abundant [38], (pers. obs. KEN and MMW). The species is most often observed on the trunks of the trees with no obvious preference for tree species on which to perch but has been observed very high up in the canopy, as well as occasionally capturing food items on the ground (pers. obs. KEN and MMW). These observations sugges<sup>t</sup> *N. townsendi* to be an ecological generalist, although we did not collect sufficient data to specifically address the question of its trophic ecology. *Norops townsendi* was previously thought to be morphologically allied to *N. polylepis* from Costa Rica [40], but phylogenetic analyses have since suggested it is allied closer with *N. poecilopus*, a semi-aquatic anole from Panama [21,41–43], but see [20].

## *1.3. Isla Gorgona*

Isla Gorgona (13.3 km2**,** Figure 2c) is only 35 km from the coast of Colombia and, like Isla Cocos, is a small tropical island that is separated from the mainland by a deep marine channel (~270 m; [44]). The island falls entirely within Gorgona National Natural Park which covers ~600 km<sup>2</sup> [45]. It has a complex geologic history perhaps dating back to 90 Mya [46–49]. While Isla Gorgona formed separately from the mainland, there is evidence to sugges<sup>t</sup> multiple connections and subsequent separations from the mainland [50]. However, the timing of the connections is unclear. Most recently, Gorgona was connected to mainland South America from 17,000–11,000 years ago during the Pleistocene

glaciation [51,52], which may explain some of the biotic similarities between the island and mainland communities [53,54]. The dynamic geologic history sets the stage for di fferent components of Isla Gorgona's biotic community to have originated via di fferent modes: dispersal or vicariance. However, without more refined dates of the connection history we cannot test hypotheses regarding these alternatives, but we can provide interpretations of divergence dates estimated from our data.

Five anole species inhabit the island, representing a diverse range of morphological and ecological modalities as well as phylogenetic a ffinities (Figure 4). *Dactyloa gorgonae* and *Norops medemi* are endemic to Isla Gorgona, while *D. chocorum*, *D. princeps*, and *N. parvauritus* have populations in mainland South America in addition to the island. The two species of *Norops* (*N. parvauritus* and *N. medemi*) di ffer greatly from each other in ecomorphology in that *Norops medemi* is smaller and found low on the trunks of trees and is darkly colored and patterned, similar to the tree trunks on which they are found. On the other hand, one of the largest members of *Norops*, the brilliant green *N. parvauritus* has a robust body with slender limbs. This species often goes unobserved as it frequents the forest canopy and is ecologically similar to the *Dactyloa* species. This species was previously recognized as a subspecies of the widespread *N. biporcatus* (ranging from Mexico to central South America) until it was recently elevated to species status [55]. In addition to examining the timing of *N. parvauritus* on Isla Gorgona, we also used this opportunity to evaluate this proposed taxonomic change. Armstead et al. [55] had limited genetic sampling of both *N. biporcatus* (*N* = 5) and *N. parvauritus* (*N* = 3), with no individuals from Isla Gorgona, the type locality for *Norops biporcatus parvauritus*. Herein we address both deficiencies by presenting a broad genetic data across the range including from Isla Gorgona. While Armstead et al. (2017) also presented a morphological analysis to support the species elevation, past work in mainland anoles has shown that delineating species using morphology without range-wide genetic sampling can lead to the violation of a monophyletic species concept (e.g., *N. quaggulus*, [43,56]).

The other three species are in the genus *Dactyloa* (*D. chocorum*, *D. gorgonae*, and *D. princeps*) and are all highly arboreal, but di ffer in size, coloration, and perch preferences (pers. obs., this study). *Dactyloa gorgonae* is unique among all anoles in that it is bright blue in color, smaller, and slimmer than the other two *Dactyloa*, and believed to be most closely related to a mainland species (*D. chloris*), to which it is morphologically similar except in coloration. Locals on Isla Gorgona believe that *D. gorgonae* is only found within the small inhabited area on the island, where it is observed in the mostly open branches of trees, but that assertion is likely due to frequency of observations and insu fficient sampling e ffort elsewhere on the island. The forest on the island is dense, and their blue coloration is quite cryptic within the foliage of the trees (pers. obs. KEN and MMW). *Dactyloa princeps* was observed frequently on the trunks of trees while *D. chocorum* was observed rarely (only once during our visit). Both *D. princeps* and *D. chocorum* have populations on the mainland, but no studies have examined the morphological di fferences among island populations.

Understanding the biogeographic history of these Pacific anoles is a crucial initial step in setting the stage for future ecological and evolutionary studies of these species. Investigating the timing and route(s) of colonization for each island, combined with knowledge of each island's likely environment at the time of colonization, will establish the historical context for the unique extant communities. We investigated the biogeography of these Pacific Island anoles using molecular data to address the timing of divergence from mainland ancestors and to evaluate the route of colonization of each island by each of the seven species. This information was used to compare divergence of island lineages to geologic models for each island and to investigate hypotheses of overwater dispersal to each island. Islas Cocos and Malpelo are both volcanic in origin and could only have been colonized via over water dispersal, but we sought to investigate the timing and route of potential pathways. We also investigated the timing of divergence from mainland populations for all five species on Isla Gorgona and compared them to evaluate if their colonization was temporally proximate.

**Figure 4.** *Cont.*

**Figure 4.** Bayesian reconstruction of *Dactyloa* and *Norops* anoles dated using a combination of fossil calibrations and a mtDNA mutation rate. All lineages of Pacific Island anoles are colored, and posterior values are present on all nodes with <0.95 probability. (**A**) *Norops* clades pertinent to this study. (**B**) *Dactyloa* clades pertinent to this study.

Finally, we collected observations and ecomorphological measurements for comparing to those recorded for Caribbean anoles to examine the relationship in ecomorphological patterns between solitary anoles. Sexual size dimorphism is evident in many solitary species of birds [57,58], mammals [59], and lizards [22,60,61]. Solitary anoles in the Caribbean typically display sexual size dimorphism, presumably to reduce intraspecific competition [22], a trend that likewise holds in mainland populations with species-poor anole communities [62]. Given that sexual size dimorphism has not been fully evaluated in Pacific species, we seek to determine if *D. agassizi* and *N. townsendi* also adhere to this pattern.
