Significance The formation of the Isthmus of Panama, which linked North and South America, is key to understanding the biodiversity, oceanography, atmosphere, and climate in the region. Despite its importance across multiple disciplines, the timing of formation and emergence of the Isthmus and the biological patterns it created have been controversial. Here, we analyze molecular and fossil data, including terrestrial and marine organisms, to show that biotic migrations across the Isthmus of Panama began several million years earlier than commonly assumed. An earlier evolution of the Isthmus has broad implications for the mechanisms driving global climate (e.g., Pleistocene glaciations, thermohaline circulation) as well as the rich biodiversity of the Americas.

Abstract The linking of North and South America by the Isthmus of Panama had major impacts on global climate, oceanic and atmospheric currents, and biodiversity, yet the timing of this critical event remains contentious. The Isthmus is traditionally understood to have fully closed by ca. 3.5 million years ago (Ma), and this date has been used as a benchmark for oceanographic, climatic, and evolutionary research, but recent evidence suggests a more complex geological formation. Here, we analyze both molecular and fossil data to evaluate the tempo of biotic exchange across the Americas in light of geological evidence. We demonstrate significant waves of dispersal of terrestrial organisms at approximately ca. 20 and 6 Ma and corresponding events separating marine organisms in the Atlantic and Pacific oceans at ca. 23 and 7 Ma. The direction of dispersal and their rates were symmetrical until the last ca. 6 Ma, when northern migration of South American lineages increased significantly. Variability among taxa in their timing of dispersal or vicariance across the Isthmus is not explained by the ecological factors tested in these analyses, including biome type, dispersal ability, and elevation preference. Migration was therefore not generally regulated by intrinsic traits but more likely reflects the presence of emergent terrain several millions of years earlier than commonly assumed. These results indicate that the dramatic biotic turnover associated with the Great American Biotic Interchange was a long and complex process that began as early as the Oligocene–Miocene transition.

The Isthmus of Panama is the narrow strip of land that connects North and South America and divides the Atlantic and Pacific oceans. The emergence of the Isthmus initiated one of the largest episodes of biological migration between previously disconnected landmasses, the Great American Biotic Interchange (GABI) (1⇓⇓⇓–5), one of the best natural experiments on invasive species. The closure of the Central American Seaway (CAS, the oceanic pathway along the tectonic boundary between South America and the Panama Block) and rise of the Isthmus have been linked to the onset of both thermohaline oceanic circulation and northern hemisphere glaciation (6⇓–8). Despite its broad importance, the formation of the Isthmus and its impact on the rich biodiversity of the Americas remains contentious (9). Therefore, a better understanding of when the formation of the Isthmus of Panama occurred has important implications in several scientific fields across multiple disciplines.

The timing of Isthmus formation has been assessed through different proxies. Previous studies have long suggested full closure by 3.5 Ma (7, 8, 10⇓⇓⇓⇓⇓–16). More recent geological work has suggested a longer and more complex formation, where the initial collision between South America and the Panama Block occurred between 25 and 23 Ma (17). By 20 Ma the Panama Block is suggested to have been connected to North America (18⇓⇓–21) and the width of the CAS to be 200 km (19, 20). Full closure of the CAS occurred by 10 Ma, ending the exchange of deep and intermediate waters between the Caribbean and the Pacific (11, 19, 20, 22⇓–24). However, the exchange of shallow waters between these oceans likely continued along pathways other than the CAS for many millions of years (7, 8, 10⇓⇓⇓⇓⇓–16, 25).

Over the past two decades, hundreds of studies have assumed the Isthmus of Panama to have closed at 3.5 Ma (SI Appendix), causing the separation of widespread marine populations into distinct Pacific and Caribbean groups (vicariance) and the first possible dispersals between North and South America (with the exception of stochastic long-distance dispersals). Here, we address the following questions: Given the complexities and the recent evidence of a much older geological history of the Isthmus of Panama, is 3.5 Ma an adequate age for those events? Were the suggested water corridors across the Isthmus—even if shallow and narrow—indeed effective barriers against both dispersal of terrestrial organisms and conduits of marine ones until a full closure at 3.5 Ma? We address these questions using comprehensive biological data from living and fossil organisms, where the assumption is that any well-developed terrestrial corridor would lead to both more frequent biotic dispersal between North and South America as well as a division of widespread marine organisms into distinct Caribbean and Pacific lineages (26). Biological data provide a powerful tool for this purpose compared with geological evidence, which cannot inform on the subtle differences between exposed land and shallow waters that can be crucial to the dispersal of organisms.

Recent attempts to synthesize dispersal patterns across the Isthmus of Panama are based on dated phylogenies (27⇓–29) that have included data that used a 3.5 Ma Isthmus closure as a calibration point (e.g., in calculating mutation rate). These assumptions lead to circularity if the goal is to examine timing of dispersal or vicariance across the Isthmus. Our molecular analyses differ from previous studies both qualitatively (by the elimination of the timing of the emergence of the Isthmus of Panama and the contrast between patterns in marine and terrestrial taxa) and quantitatively (735% more data points than a previous cross-taxonomic analysis) (27) and serve as a comparison with a comprehensive fossil dataset.

Another crucial aspect of the formation of the Isthmus of Panama and its impact on biological diversity entails the direction of terrestrial dispersal (e.g., north to south) and the ecology of dispersing organisms. In his seminal work on the GABI, Simpson (1) used the mammal fossil record to suggest that North American taxa had a competitive advantage over the South American fauna in that more North American taxa dispersed, survived, and diversified in southern ranges. Recent studies have suggested that ecological barriers to dispersal, such as dry savanna-like environments (30) or reduced rain forest cover (31), also prevented tropical South American taxa from migrating successfully to the north.

To explicitly address the timing of the formation of the Isthmus of Panama and its effect on geographical distributions of biota, we develop a Migration Rates Through Time (MRTT) model. Using this approach, in which migration is defined as dispersal and vicariance events collectively, we conduct an analysis of over 400 data points (SI Appendix, 1.2) of molecular divergence dates conferring dispersal events between North and South America or vicariance between the eastern Pacific and the Caribbean Sea. We compare results from extant data with over 23,000 records of American fossil mammals. Our results corroborate recent geological evidence that rather than a Pliocene, time-limited, single event, the formation of the Isthmus of Panama and the GABI were long and complex processes that began as early as the Oligocene–Miocene transition. We also show incongruent patterns in the direction of migration when comparing the molecular and fossil data, possibly reflecting differential diversification rates following dispersal. None of the ecological factors measured in the analyses can explain the statistical differences in the timing of migration across the Isthmus region among different taxa, and together with the MRTT results, this suggests that rather than intrinsic (biological) traits, extrinsic factors such as the presence of emergent land likely drove these patterns.

Discussion The patterns inferred from the migration analyses of both molecular and fossil data (Figs. 1 and 2) are strikingly consistent and robust to uncertainties associated with age estimations (see confidence intervals in Figs. 1 and 2 and sensitivity analysis in SI Appendix, Fig. S1). Our results support an initial collision of the Panama Block and South America at 25–23 Ma with development of extensive terrestrial (although not necessarily fully connected) landscapes in Panama by 20 Ma, at least some 17 Ma earlier than generally assumed. Our results further imply that over the past 10 Ma there has been substantial dispersal and vicariance across the Isthmus and that pulses of dispersal of terrestrial organisms occurred over at least three periods during the last 30 Ma. The underlying causes for these discrete migration pulses remain elusive but may be associated with landscape formation, volcanism, climate change, and/or sea level fluctuations (25, 35). North American taxa may have been preadapted to dispersal in that many of the successful lineages had migrated from Europe and Asia across northern land bridges, before their movement into South America. Despite this, our results on the North-to-South asymmetry detected in the fossil record contrast with the cross-taxonomic results from molecular phylogenies, where migrations in either direction were not found to be significantly different until ca. 6 Ma, when the migration rate from South to North America exceeds that in the opposite direction (Fig. 1B and SI Appendix, Table S1). In summary, can we assume that “no vicariant date [3.5 Ma] is better dated than the Isthmus” (26)? Our results indicate that we cannot. We show that the GABI occurred over a much longer time period than previously proposed and comprised several distinct migrational pulses. Marine and terrestrial clades exhibit similar dispersal/vicariance pulses, occurring between ca. 23–20 Ma and 8–6 Ma. An earlier connection with North America, together with evidence of a southern connection with Antarctica until 30 Ma (36), challenges the long-standing idea of South America being an island continent that evolved in “splendid isolation” (1). These realizations impact our understanding of the temporal evolution and assembly of the American biota and urge a reevaluation of how the formation and emergence of the Isthmus of Panama impacted biological exchange, oceanic currents, atmospheric circulation, and global climate change.

Materials and Methods Phylogenetic Data. We performed an analysis of available molecular phylogenies with broad representation across taxonomic groups and habitats. We excluded studies that incorporated any assumption of the timing of the closure of the Isthmus (calibrations in dated phylogenies that were directly or indirectly derived from this event). We included phylogenetic studies with at least one unambiguous instance of dispersal across the Isthmus of Panama, for instance an ancestor with a North American distribution that had a descendant lineage in South America, as well as marine studies of sister species between the Caribbean and Pacific oceans. The dataset included 424 dated dispersal or vicariance events across the Isthmus of Panama (collectively referred to here as migration events). The data were compiled from 169 dated molecular phylogenies from across the tree of life and published in 29 peer-reviewed journals (SI Appendix, Tables S8 and S9, respectively). For each biogeographic event, we also recorded mean crown age, the lower and upper confidence intervals of ages, and direction of dispersal, as well as the taxon’s altitudinal range, dispersal capability, and biome of occurrence. MRTT Analyses. Biogeographic events with associated age uncertainties were analyzed in a maximum likelihood framework to estimate rates of migration (SI Appendix, Table S10) and their variation through time. Although, ideally, such migration rates should be estimated on a per-capita basis (as for other macroevolutionary rates), their estimation in this context would be affected by unobserved extinct lineages and further biases would likely arise from combining data from multiple independent phylogenies. We therefore modeled migrations as random events resulting from a stochastic Poisson process, with a rate parameter that describes the expected waiting time between successive events. As a mean to standardize the migration rates across datasets, we rescaled the estimated absolute Poisson rates by the number of families considered in each analysis. Thus, the migration rates shown in the MRTT plots indicate the expected number of migration events per Ma per family. We emphasize that this standardization is not intended as a correction for temporal biases within each dataset but rather as a tool to facilitate comparisons across different analyses. To account for deviations from a constant rate (homogeneous) process, we tested different nonhomogeneous Poisson processes with time-varying rates, including a model with exponentially increasing rates, which might capture potential biases owed to the exponential increase of lineages in extant-taxa phylogenies. To allow for temporal changes in the migration rates, we implemented a nonhomogenous Poisson process, in which rate shifts can occur through time. Our maximum likelihood algorithm involved (i) assessing the best-fitting number of rate shifts by a stepwise AICc procedure, (ii) the optimization of their temporal placement, and (iii) the estimation of the migration rates between shifts (SI Appendix, 1.7). We used simulations to assess the most appropriate AICc thresholds, thus minimizing the risk of false positives in our analyses (SI Appendix, 1.6). We generated and analyzed 1,000 datasets resampled from the uncertainty intervals for migration dates to generate the MRTT plots and calculate 95% confidence intervals. In addition to jointly analyzing the full molecular dataset, we repeated the analyses on 14 subsets based on different criteria considering geographical, ecological, and taxonomic aspects. These tests allowed us to investigate the differential patterns of migrations linked to direction of migration and environment (terrestrial or marine) and to compare different taxonomic groups and their dispersal ability (Fig. 1B and SI Appendix, Figs. S2–S4 and 1.8). Additionally, we compared the AIC scores of two migration models with a model containing a single rate shift fixed at 3.5 Ma and a model containing two shifts in the Miocene, at 25 Ma (initial collision of the Panama block and South America) and 10 Ma (closure of the CAS) (SI Appendix, 1.8 and Table 1). GLMM Analyses. We used a GLMM approach on the molecular datasets of terrestrial/freshwater and marine taxa to test for intrinsic (biological, ecological) factors driving the differential timing of dispersal or vicariance. Ecological and biological variables (biome type, dispersal ability, dispersal direction, and elevation preference as derived from the literature and consultation with specialists) were treated as fixed effects, and we included a phylogenetic correction by treating taxonomic rank as a nested random effect (SI Appendix, Tables S4–S7). Because the molecular dates in the dataset were calibrated using a range of approaches (e.g., fossils, molecular clocks), we included calibration type as an additional variable in the model to account for any potential bias associated with how the phylogenies were time-calibrated. Fossil Data. We compiled fossil occurrence data from all available publications of South American mammalian fossils and reviewed the Cenozoic American mammal fossil record in the Paleobiology Database (https://paleobiodb.org/#/), synthesizing data from across the entire American continent. The vetted dataset comprised 23,090 fossil records from 112 families and 3,589 species. Based on first and last appearances of each species in the fossil record, we plotted diversity trajectories through time, by counting the number of species within 1 Ma bins. To explicitly incorporate dating uncertainty of fossils, we randomized the age of each fossil 1,000 times within the temporal boundaries of the geological formation in which they were found (Fig. 2B and SI Appendix, 1.4). We classified each fossil occurrence as North or South American if the taxon or its ancestor was in either North or South America before 10 Ma, following Carillo et al. (37). We then plotted the amount of immigrant species as a proportion of the total diversity in each continent within 1 Ma time bins (Fig. 2C).

Acknowledgments We thank Fernando Alda, Eldridge Bermingham, Juan David Carillo, Anthony Coates, David Dilcher, John Klicka, Harilaos Lessios, Camilo Montes, Andrés Mora, Geovanni Romero, Pierre Sepulchre, and The Panama Canal Project–Partnerships in International Research and Education participants for discussions. We are grateful to the Paleobiology Database team for reviewing and databasing the fossil data used in this study. Data analyses were run at the high-performance computing center Vital-IT of the Swiss Institute of Bioinformatics (Lausanne, Switzerland). Financial support was provided by a postdoctoral research fellowship from the Smithsonian Institution (to C.D.B.); the Wenner-Gren and Carl Tryggers foundations (D.S.); the Autoridad del Canal de Panamá, the Mark Tupper Smithsonian Fellowship, Ricardo Perez S.A., National Science Foundation Grants EAR 0824299 and OISE/EAR/DRL 0966884, and the National Geographic Society (to C.J.); National Science Foundation Grants DEB 0916695 and DEB 1354149 (to P.C.); and Swedish Research Council Grant B0569601 and European Research Council Grant 331024 under European Union’s Seventh Framework Programme FP/2007-2013 (to A.A.).

Footnotes Author contributions: C.D.B., C.J., and A.A. designed research; C.D.B., D.S., B.T.S., and P.C. analyzed data; and C.D.B. and A.A. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. R.H.R. is a Guest Editor invited by the Editorial Board.

Data deposition: Raw data for phylogenies and fossil data have been deposited in Dryad, www.datadryad.org (10.5061/dryad.6m653).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1423853112/-/DCSupplemental.