16/16 Finally, thanks to @lifesciencestudios.bsky.social for the watercolors! I want to say finally that this type of interdisciplinary work is not possible at all without federal funding. Please #standupforscience

15/16 Thanks, as always, to my coauthors, including co-first author Maxime Policarpo, my advisor @tjnear.bsky.social and Richard Harrington, Eva Hoffman, Maya Stokes, and Didier Casane! Thanks as well to the Yale Peabody Museum, Yale EEB and TPG TPG for support!

14/16 The ages of the cave-related vision gene loss logically postdate the caves, so the ages we get are the minimum ages of the subterranean ecosystems! This suggests that some of these cave systems are up to 15 million years old! These systems have been here since the Miocene!

13/16 But we can go further! What about the caves themselves? The ages of eastern North America's iconic karst caves, such as Mammoth Cave, are actually really hard to date using conventional methods. Cavefishes fill the gap! (Photo: NPS)

12/16 Well, most anyways. Some, like species in the genera Amblyopsis and Typhlichthys, actually have pseudogene ages that predate their divergence from other species in each genus! Thus, these genomic markers of cave adaptation came before-these fishes speciated underground!

11/16 As it turns out, the ages we get from examining this pseudogene clock are younger than the divergence times of cavefish lineages from one another! So, they experienced cave-associated degenerative evolution AFTER diverging from other fishes, and thus independently!

10/16 To get to the bottom of this, we used a trick. By estimating generation times since a gene was pseudogenized, and then multiplying that by years per generation, we can estimate how many millions of years ago a cavefish, well, became a cavefish!

9/16 The problem is that, for single species diverging from a surface-dwelling ancestor, cave colonization and adaptation could have happened anywhere along the branch on the tree leading to the living cave dweller. This is because its closest surface relatives might be extinct!

8/16 We noticed that the number of pseudogenes might be associated with the age of cave lineages themselves. For example, Troglichthys rosae, the first obligate cave-dweller to diverge, pseudogenized 25 vision and light related genes ancestrally!

7/16 What's more, no loss of function mutations appear ancestral to lineages including multiple obligate cave-dwelling amblyopsid genera, nor are none present in Forbesichthys! This indicates independent cave colonization and subsequent gene function loss. But there's more!

6/16 Different sets of vision-related genes, including several involved in human disease, lost functionality in different lineages of obligate cave-dwelling amblyopsids! Not only that, but the loss of function mutations themselves are not identical among lineages!

5/16 Although the similarities of obligate cave-dwelling amblyopsids are not just skin deep (they have all elongated their skulls and bodies, deossified their circumorbital series, and reduced or lost pelvic fins), comparisons of their genomes show these fishes are convergent!

4/16 A clue is that eyed, pigmented species in Forbesichthys are deeply nested within blind, obligate cave dwelling amblyopsids, suggesting multiple origins. By using genomes, personal examination of key fossils, and CT scans, we built the most comprehensive tree yet to do so!

3/16 Most amblyopsids have degenerated their eyes and lost external pigment to become obligate cave-dwellers. Yet, the different ways in which the eyes of different species have degenerated has led to a 125-year debate about how many times these fishes invaded caves! (Photo: NPS)

2/16 Amblyopsids are one of North America's most obscure and most interesting vertebrate lineages. Endemic to eastern North America, they are part of a species-poor lineage that also includes pirate and trout perches and originated 80 million years ago!