João Pedro (JP) Fontanelle is a postdoc at the Institute of Forestry and Conservation at the University of Toronto in Canada. He is an evolutionary biologist interested in how spatial and temporal eco-evolutionary dynamics affect micro- and macroevolution. Here, JP shares his recent work on how stingrays invaded the freshwaters and diversified in South American basins.

João Pedro (JP) Fontenelle getting ready to fish in the Amazon.
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Institute. University of Toronto, Institute of Forestry and Conservation
Academic life stage. Postdoc
Major research themes. How changes in environmental properties and connectivity affect the evolution of organisms at micro and macroevolutionary scales, their biogeography, and diversification patterns.

The unique sunset of the Amazon river basin.
Current study system. The Neotropical freshwater stingrays (subfamily Potamotrygoninae) is the only extant lineage of elasmobranchs that is exclusive to freshwater environments. They are morphologically diverse, presenting beautiful dorsal color patterns, making them very famous in the aquarium trade. It is really interesting that this subfamily achieved a high diversity and a broad distribution across almost all South American basins in a relatively short period of evolutionary time (25-20 my). That is especially cool when we think they are a marine-derived lineage, meaning their closest related lineage is found in marine environments.
Recent JBI paper. Fontenelle, J. P. Marques, F. P. L., Kolmann, M. A, Lovejoy, N. R. (2021). Biogeography of the Neotropical freshwater stingrays (Myliobatiformes: Potamotrygoninae) reveals effects of continent scale paleogeographic change and drainage evolution. Journal of Biogeography, 48(6), 1406-1419 https://doi.org/10.1111/jbi.14086

JP (left) and Jonas Batista (Instituto Mamirauá) getting ready to fish in the Amazon.
Motivation behind this paper. It started during my MSc back at the University of São Paulo in Brazil. I was working on the taxonomy of a species-complex of these stingrays (Fontenelle et al. 2017), and I started to notice the correlation between stingray species and their distribution patterns. To try to understand the processes that led to the diversification of these stingrays, I started reading more about the evolution of the group, and consequently the biogeography of Neotropical freshwater fishes, especially Lovejoy et al. (1998), Albert et al. (2006) and Albert and Reis (2011). It was fascinating to see the intimate relationship between fish groups’ diversity, South American geography, and the abiotic properties of the environment, such as water chemistry. Trying to understand a bit more about how environmental and landscape characteristics affect the evolution of freshwater lineages was the biggest inspiration for my PhD. For the stingrays, it was widely accepted that their lineage had invaded freshwater habitats through the Caribbean. However, there were still questions about the age of this event and how the group had colonized the rest of the continent. Fortunately, we were able to compile more than two decades of samples collected all over South America, which allowed us to investigate biogeographical patterns for the whole group at a continental scale.
Key methodologies. We first needed a phylogeny (relationship hypothesis tree) with a good taxonomic representation of the freshwater stingrays. We compiled the most taxonomic rich phylogeny to date, with molecular data from more than 350 specimens, accounting for over 90% of the species and sampling most of the group’s distribution. We then combined geological data and fossil evidence to calibrate key nodes in this phylogeny to produce an age hypothesis for the diversification of the group. Then, based on the literature on biogeographical areas for Neotropical freshwater fishes, we cross-referenced the distribution of our specimens to selected biogeographical areas of relevance. Finally, we reconstructed ancestral ranges over the phylogeny using six different biogeographical models and then tested their fit to our data. These models apply different parameters, such as dispersal, range expansion, vicariance, and extinction, to interpret the differences observed in reconstructed distribution patterns. Our dataset allowed us to identify changes in river connectivity over evolutionary time and how paleogeographical events were fundamental in the dispersal and diversification of the group.

Gentle trawling the bottom of the Rio Tapajós.
Unexpected challenges. One big challenge was how to interpret the ages and the reconstructed ancestral ranges for many nodes of the tree over such a large timescale and geographic area. We had to rely on geography and geology dense papers to identify and properly interpret the biogeographical patterns. These papers helped us narrow down and interpret major events for the biogeography of the stingrays and other fishes and aquatic groups with similar distributions. This led to another series of challenges regarding contrasting biogeographical patterns among different fish groups. By exploring the literature on different fish lineages, we were able to identify evidence for the same paleogeographical event affecting the fish community, but with different outcomes depending on the group. That is, the same paleogeographical event can influence the evolution of distinct groups in different ways, and this can be attributed to differences in their biology and evolutionary history.
Major results. We provided biogeographic evidence for major changes in the paleogeography of South America across 30 million years, especially for the Amazon and associated river basins, which can be used by many other biogeography studies. We presented evidence of how changes in the connectivity between and within river basins are very important to the diversification patterns of aquatic groups and how these changes may affect different freshwater groups differently. From a more fish-focused perspective, we have highlighted the importance of marine incursions on strict freshwater dominated regions for the adaptation and diversification of marine-derived lineages (MDLs) and the importance of the Pebas Mega Wetlands acting as a facilitating route for the dispersal of the stingray lineage and possibly other MDLs, from fishes like drums and pufferfishes, for example, to mammals, molluscs and even plants. It was really nice to interpret the biogeographic patterns and explain the colonization of the South American continent by this interesting group of fishes.

Retrieving longlines at dawn, after the stingrays feed time.
Next steps for this research. By extrapolating the results from this research, we are investigating macroevolutionary patterns involving biogeography, ecology, diversification, and phenotypic changes related to changes in environment and distribution. We are also working on how paleogeographic changes in the South American landscape have influenced the evolution of other fish groups and the signals in their phylogeny. Finally, we are exploring the relationships of these stingrays at a population level to study population dynamics and their relationship to taxonomic and phylogenetic diversity.
If you could study any organism on Earth, what would it be? The easy answer for that is fishes! They are fascinating solely by the fact that what we call ‘fishes’ covers more than half of the vertebrate diversity, and are found in a crazy variety of sizes, shapes and biologies. As one of my undergraduate professors used to say: “there is probably a fish example for that”. It is really hard to choose one group of fish, though. I’m particularly interested in rapidly evolving and diverse groups, and their relationship to changes in habitat; however that still accounts for a bunch of them!

A juvenile stingray in a basin for transportation (right); an adult stingray and its beautiful color pattern, fresh on the boat (left).
Anything else to add? This manuscript is the first published chapter of my PhD thesis. That adds an extra layer of satisfaction as this research is also one of the main reasons I pursued my PhD. I’ve been questioned before about how long it took us to get this manuscript in good shape and out. Still, all the years of intense work have been a real reminder of how difficult it is to work with a diverse group in a diverse area, especially when the evolution of the group is still not very well understood and the taxonomy is very complicated (which is the case of the Neotropical freshwater stingrays). It is great to provide a very important contribution to the study of these fish and the Neotropical region and its biogeography. It has been challenging from start to finish, but so worth it! We have learned a lot.