Vastly underestimated species richness of Amazonian salamanders (Plethodontidae: Bolitoglossa) and implications about plethodontid diversification

We present data showing that the number of salamander species in Amazonia is vastly underestimated. We used DNA sequences of up to five genes (3 mitochondrial and 2 nuclear) of 366 specimens, 189 corresponding to 89 non-Amazonian nominal species and 177 Amazonian specimens, including types or topotypes, of eight of the nine recognized species in the region. By including representatives of all known species of Amazonian Bolitoglossa, except for one, and 73% of the currently 132 recognized species of the genus, our dataset represents the broadest sample of Bolitoglossa species, specimens, and geographic localities studied to date. We performed phylogenetic analyses using parsimony with tree-alignment and maximum likelihood (ML) with similarity alignment, with indels as binary characters. Our optimal topologies were used to delimit lineages that we assigned to nominal species and candidate new species following criteria that maximize the consilience of the current species taxonomy, monophyly, gaps in branch lengths, genetic distances, and geographic distribution. We contrasted the results of our species-delimitation protocol with those of Automated Barcode Gap Discovery (ABGD) and multi-rate Poisson Tree Processes (mPTP). Finally, we inferred the historical biogeography of South American salamanders by dating the trees and using dispersal-vicariance analysis (DIVA). Our results revealed a clade including almost all Amazonian salamanders, with a topology incompatible with just the currently recognized nine species. Following our species-delimitation criteria, we identified 44 putative species in Amazonia. Both ABGD and mPTP inferred more species than currently recognized, but their numbers (23–49) and limits vary. Our biogeographic analysis suggested a stepping-stone colonization of the Amazonian lowlands from Central America through the Chocó and the Andes, with several late dispersals from Amazonia back into the Andes. These biogeographic events are temporally concordant with an early land bridge between Central and South America (~10–15 MYA) and major landscape changes in Amazonia during the late Miocene and Pliocene, such as the drainage of the Pebas system, the establishment of the Amazon River, and the major orogeny of the northern Andes.This work was partly funded by projects CGL2014-56160-P (PI: I. De la Riva) and CGL2016-75227-P (PI: C. Vilà) of the Spanish Government. AFJ was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil (CNPq procs. 132721/2015-5l) and ProEx of Programa de Posgraduação em Zoologia – PUCRS. Research in Ecuador was conducted under permits NoMAE-DNB-CM-2015-2017 and MAE-DNB-CM-2018-0105, issued by the Ministerio del Ambiente del Ecuador. Research in Colombia was conducted under expedient No. RCI0005-00-2018 issued by Autoridad Nacional de Licencias Ambientales (ANLA). Biological material from the Museo de Biodiversidad del Perú (MUBI) is recognized by the Resolución de Dirección General N° 024–2017–SERFOR/DGGSPFFS.Peer reviewe