Genetic diversity and population structure based on mitochondrial
markers
We analyzed the concatenated mitochondrial DNA sequences (COI + cyt b =
1,130 bp) of 24 individuals of T. ruficapillus belonging to the
five subspecies (Table S1). The specimens were collected in 16
localities from Peru, Bolivia, Argentina and Uruguay, covering a wide
range of the species distribution (Figure 1a). Our results showed
considerable variation within T. ruficapillus and, in particular,
the subspecies that inhabits the Atlantic Forest, T. r.
ruficapillus , clearly differed from the four Andean subspecies, with a
mean genetic differentiation between 2.6% and 3.6% (Table 1). There
was also considerable variation among the Andean subspecies, withT. r. cochabambae showing the highest genetic divergence both
with respect to T. r. ruficapillus and with the remaining Andean
subspecies (Table 1). Lower levels of differentiation were observed
between T. r. subfasciatus , T. r. marcapatae and T.
r. jaczewskii and in fact we found almost no differentiation (0.2%)
between T. r. subfasciatus and T. r. marcapatae .
Both the haplotype network (Figure 1b) and the AMOVA (Table 1) show a
marked phylogeographic structure within T. ruficapillus , with
three clearly differentiated lineages: one formed by the subspecies
present in the Atlantic Forest (T. r. ruficapillus ), another one
by the subspecies distributed in the Andes of southern Bolivia and
northwestern Argentina (T. r. cochabambae ), and the last one
containing the remaining Andean subspecies from northern Bolivia to Peru
(see Figure 1b). Consistently, ΦST values from the AMOVA
were high and significant between the subspecies of these three
different lineages (ΦST 0.7-0.83), but were not
significant between the three subspecies of the northern lineage
(T. r. subfasciatus , T. r. marcapatae and T. r.
jaczewskii ; see Table 1). The haplotype network confirmed a lack of
differentiation and shared haplotypes between T. r. subfasciatusand T. r. marcapatae , and low genetic differentiation between
these two subspecies and T. r. jaczewskii , despite its
geographically isolated and disjunct distribution.
Phylogenetic analyses and diversification dating based on
mitochondrial DNA
Bayesian and maximum parsimony (MP) reconstructions recovered T.
ruficapillus as monophyletic in relation to T. doliatus with
maximum statistical support (Figure S3). The time-calibrated ultrametric
phylogeny (Figure 1c) shows that the split between them occurred 1.65
million years ago (Mya) (95% HDP: 0.82-2.27). These reconstructions
show three clades that correspond to the lineages described above: one
includes the subspecies from the Atlantic Forest (T. r.
ruficapillus ), another one is formed by T. r. cochabambae and
the third one by T. r. subfasciatus , T. r. jaczewskii andT. r. marcapatae (see Figure 1c and Figure S3). In the case of
the Bayesian and MP reconstructions the oldest split was between the
subspecies from the Atlantic Forest and those from the Andean forest
(Figure S3), with low values of both posterior probability and bootstrap
support between the two Andean clades (0.63 and 63%, respectively).
However, the time-calibrated ultrametric phylogeny indicated that the
first split within the species, which occurred in the Pleistocene
approximately 1.29 Mya (95% HPD: 0.71-2.10 Mya), was between the
southernmost Andean subspecies (T. r. cochabambae ) and the rest
of the subspecies (Figure 1c). This analysis suggested a more recent
divergence between the subspecies from the Atlantic Forest (T. r.
ruficapillus ) and the clade that includes the rest of the Andean
subspecies (T. r. subfasciatus , T. r. jaczewskii andT. r. marcapatae ), approximately 0.97 Mya (95% HPD: 0.52-1.57
Mya). This split, however, also had low support (posterior probability:
0.58; Figure 1c).