The Pakaraima Mountains are an ancient mountain range along the borders of Guyana, Brazil and Venezuela. The high plateau is drained by multiple river systems in all directions. Although hypotheses have been presented for the biogeographical relationships of lowland rivers, the interconnectivity of rivers on the top of the plateau is unknown. With multiple complex rivers in a small, upland area, we predicted a high level of endemism for stream fishes and complex biogeographical relationships. We explored this with the incredibly diverse pencil catfish genus Trichomycterus. Using collections from recent expeditions to the Pakaraima Mountains of Guyana, we amplified three mitochondrial (16S, COI and Cytb) and one nuclear marker (rag2). We constructed individual gene trees and a concatenated tree to determine the placement of these taxa within the Trichomycterus of the trans-Andean/Amazonian clade. Herein, we identify six endemic lineages of Trichomycterus from the highlands of the Pakaraima Mountains. Of the identified lineages, we find two species occupying multiple basins, suggesting that Pakaraima streams either maintain connectivity or had some degree of recent connectivity.

24Aim 25The Pakaraima Mountains are an ancient mountain range along the borders of Guyana, Brazil, 26 and Venezuela. The high plateau is drained by multiple river systems in all directions. Although 27 hypotheses have been presented for the biogeographic relationships of lowland rivers, the 28 interconnectivity of rivers on the top of the plateau is unknown. With multiple complex rivers in 29 a small, upland area we predicted a high level of endemism for stream fishes and complex 30 biogeographic relationships. We explore this with the incredibly diverse pencil catfish genus 31Trichomycterus. Only two species are known from the region. In this study, we 1) confirm the 32 discovery of multiple endemic Trichomycterus species in the region, 2) determine the 33 phylogenetic placement of our samples to posit biogeographical scenarios, and 3) provide 34 clarification for the identification of T. guianensis based on morphology. 35 Location 36 Pakaraima Mountains, a part of the Guiana Shield in Guyana, South America 37 Taxon 38 Pencil catfish genus Trichomycterus 39 Methods 40Using collections from recent expeditions to the Pakaraima Mountains of Guyana, we amplified 41 three mitochondrial (16S, COI, and cytb) and two nuclear markers (myh6 and RAG2). We 42 constructed individual gene trees as well as a concatenated tree to determine the placement of 43 these taxa within the Trichomycterus of the Trans-andean/Amazonian clade. 44 Results 45Our results identify six unique lineages in the highlands of Guyana. Only two species, 46Trichomycterus guianensis and T. conradi, were previously known to science. 47 Main Conclusions 48The Pakaraima Mountains of South America are a region of high endemism, as demonstrated 49 here in Trichomycterus catfishes. We find two species occupying multiple basins, suggesting that 50 Pakaraima streams either maintain or had some degree of recent connectivity. We identify six 51 endemic lineages of Trichomycterus from the highlands of the Pakaraima Mountains. The upper 52 portions of the study rivers have been connected either through surface flow or by stream 53 capture. Both processes have occurred on multiple time scales and are independent of the 54 patterns seen in the lowlands. 55