Overall, our results suggest that the Polythoridae MRCA first appeared around the Eocene, however most of the diversification within the family appeared during the Miocene, Pliocene and Pleistocene epochs (Fig. 1). During these periods there were a few major geographical events occurring, including the uplift of different regions of the Andes, as well as the formation of the Pebas and Acre Systems[1, 18]. While the ancestral area for the MRCA of Polythoridae is uncertain, our biogeographical analyses suggest an origin—or at least early diversification—in the Northwestern and Northeastern Andes for most genera in Polythoridae. Chalcopteryx-Chalcothore are the only two genera with a MRCA that may have had an Amazonian origin (Fig. 2, Supplementary Figure S4).
Our comparison of the three different biogeographical scenarios (Control, P&AS, and ANDES) found no significant effect of these major geographical events on the speciation in the family Polythoridae. We found this result surprising, as a number of polythorid genera are associated exclusively with the Andes and show patterns of diversification that align with mountain building events (Fig. 2). For example, the genus Polythore contains an Andean clade which shows a concordant pattern with the North Andes uplift. Likewise, Cora s.s, also shows a similar pattern separating two clades from the Northwest and Northeast Andes (Fig. 2). [11]
Moreover, our results suggest that most of the diversification within Polythoridae is due to dispersal events rather than vicariance (Supplementary Fig. 2). However, the distribution of those vicariance events (Supplementary Fig. 2.) in the time calibrated phylogeny is mainly within groups distributed along the Andes or that have representatives in Central America, reflecting the influence of these major geographical events on diversification.
We don’t see a significant influence of major South American geological events (e.g. Pebas and Acre Systems and Andes uplift) on the diversification of this family. Changes in the climatic conditions over that period of geological time may have a stronger influence on the biogeography of the family, which our current analyses focused on geological events will not capture. However, these events may cause significant changes in other biotic and abiotic conditions of different regions that may promoted Polythoridae damselflies dispersion to new suitable habitats. Despite the fact that damselflies are generalist predators in both terrestrial and aquatic ecosystems, their distribution patterns might be affected by the vegetation and climatic barriers over evolutionary time. A recent definition of the Neotropical forest relies in the combination of the following abiotic and biotic parameters: climate, floristic composition, vegetation structure and plant physiognomy [19]. Jaramillo [18] suggests that the formation of Neotropical terrestrial communities can be divided in two major phases, Cretaceous and Cenozoic, based on the defining parameters Neotropical forests in these two geological eras. In our case the Cenozoic phase is more relevant, and it was characterized by the dominance of the flowering plants [20–23]. Particularly, during the Paleogene, fossil and palynological evidence suggests that Neotropical forests were multi-stratified, like our current forests but with two major differences[18, 22]. First, the mean annual temperature was 1.5-2 ºC higher [24–27] and the CO2 level was almost double than in extant forests[28]. Second, there was a significantly lower diversity of plants in comparison with actual forest diversity. Moreover, during the Paleogene, the Paleocene-Eocene Thermal Maximum (hereafter PETM)[29] was a warming event produced by a significant addition of carbon due to volcanism in the North Sea [28]. The PETM was a major climate change event, and fossil evidence suggests that vegetation during this period was around 30% more diverse, and that extinction rates did not change while origination rates doubled [18]. Other DNA-based phylogenies of plants and herbivorous insects show radiations around those times [30–32]. Besides the PETM there was great variation in the global temperature during the Eocene (e.g. Early Eocene Thermal Maxima) and Oligocene epochs [18, 33, 34]. During the Neogene, new biomes within the Neotropical forest flourish, like savannas, dry forest, xerophytic forest, deserts, montane forest and paramos [18]. These biomes are determined by the precipitation regimes rather than temperature [19]. However, questions in regards how climatic and vegetation can influence the diversification of these damselfly family remains untested.
We found multiple interchanges among the Amazon and Andean regions; many of them involved movement within and between the different ranges of the Andes, as well as movement into the Amazon, Guiana Shield, Venezuela Highlands, the Tumbes-Chocó-Magdalena Valley, and Central America (Supplementary Fig. 2). The different genera within Polythoridae showed an array of biogeographical patterns. The genus Polythore shows a pattern of dispersal from the Andean regions to the Amazon in some clades, as well as movement from the Northern to Central Andes (Fig. 2). On the other hand, Euthore moved from the Northwestern to Northeastern Andes and then to the Venezuelan highlands (Fig. 2). This genus is currently found at locations from 1000–2000 meters in elevation; it is plausible that they colonized new habitats that became available during mountain building events in the Andes and Venezuelan highlands. Cora s.s and Miocora diversification was more likely driven by dispersal, as they cover a range of landscape types, and have moved across the Isthmus of Panama, the only groups in Polythoridae to do so. Interestingly, the age at which both of these genera diversified matches the age suggested by recent geological studies of the Central American Seaway closure during the middle of the Miocene [2, 35]. This closure may also influence the ITCZ (Inter Tropical Convergence Zone)[36] that will have affected precipitation regimes in the Neotropical Region [37].
These damselflies are restricted to fast flowing forested streams and waterfalls in montane forests; these habitat requirements might be the limiting factor of their distribution [38–40]. However, as general predators they are not limited by the distribution of food sources such as the host plant for other phytophagous insects (e.g. Lepidoptera). They are likely good dispersers in their immature stage, moving with the flow of creeks, but distribution between watersheds is likely to be limited, which could explain the vicariance patterns observed in our results.
Our analyses suggest that Polythoridae has been diversifying through an episodic pattern (Table 2). Speciation (λ) and net diversification (λ-µ) show an overall increase through time, while relative extinction (λ⁄µ) and extinction (µ) rates seem to remain somewhat constant through their evolutionary history. However, our branch-specific model of diversification shows a significant shift for the Andean Clade within the genus Polythore which might suggest that at least for this genus the intensified Andean uplift during the Pliocene has been promoting speciation (Fig. 3). The Andean uplift produced great modifications to the landscape; one of the major changes was in the flux of the hydrographic system towards the east, producing the actual Amazon and Orinoco basins. Furthermore, when the Andes reached their modern elevation by the end of the Miocene (i.e. 5–6 Ma) [1, 41–44], it generates two new biomes: cloud (montane) forest and paramo[18, 45]. The montane forest is the key habitat for the genus Polythore, and it has been suggested that the slopes of the Andes are an engine for speciation as the increase in topographic complexity generates diversity of microenvironments [1]. While all these species have relatively similar habitat requirements, they have generally disjunct distributions within the Andes, such that any local stream normally hosts only a single Polythore species, sometimes two species. The wing color diversity characteristic of Polythore is highest within the Polythoridae–the central Andean Polythore have wings that include bands of black, orange and yellow patterns, while those of the Northeastern Andes have intense black and white patterning (except for P. concinna, which is orange). This color diversity also appears to be polymorphic within some of these clades[13, 14, 39]. Some of the color diversity could be explained by sexual selection, with local mate choices driving diverse color patterns in different regions[46, 47]. Likewise, wing color may be under other constraints, such as thermal tolerance or selection by predators [48]. Having robust phylogenetic hypotheses will allow for further exploration of the reasons behind this radiation in Polythore, where population-level analysis will be a likely next step to disentangling the complex history of these striking creatures.