Vastly underestimated radiation 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.