Table 1.
Summary of taxon sampling and best-fitting models for combined and individual genes, taxa, and characters.
Table 2.
Congruence of key diagnostic characters for morphology, biogeography and genetic distances between species resolved by the phylogeny (12S + 16S + COI).
Fig 1.
Phylogeny and distribution of the Pristimantis acuminatus group in the Amazon Basin.
(A) Optimal maximum likelihood tree (log likelihood = -6762.95) inferred from a partitioned analysis of 1997 aligned sites of the 12S, 16S and COI (by codon position) mtDNA genes, showing the phylogenetic relationships among 33 specimens identified as P. acuminatus sensu lato and P. tantanti from the Amazon basin. Clade A = Pristimantis limoncochensis sp. nov., clade B = P. omeviridis sp. nov., clade C = P. acuminatus sensu stricto, clade D = P. enigmaticus sp. nov., and clade E = P. tantanti. Stars denote clades with Bayesian posterior probability values1; numbers below clades represent non-parametric bootstrap support values. (B) Areas of distribution for species in the complex. Dotted circles = Localities of collection from specimens used for the phylogenetic analyses; Polygons = occurrence areas drawn as minimum convex polygons for each clade based on specimens reviewed in collections (S2 Table). Colors of clades in the phylogenetic tree correspond to colors of polygons and dotted circles on the map.
Fig 2.
Coalescent species reconstruction in *BEAST.
(A) Species tree chronogram with posterior probabilities, (B) DensiTree visualization of all estimated gene trees, and (C) DensiTree visualization of possible consensus trees for the even loci. Colors of lineages recovered by the species-tree (A) correspond to colors of polygons and doted circles in the geography of Fig 1B.
Table 3.
Descriptive morphometric statistics for species of the Pristimantis acuminatus complex.
Fig 3.
Discriminant analyses of morphological and environmental variables.
Projections of morphometrics for (A) males, (B) females and (C) environmental datasets by discriminant analyses for the Pristimantis acuminatus complex. Dots represents specimens in A-B and localities in C. Colors represents the clades recovered by the phylogenetic analysis (Fig 1), whereas the black squares represent their centroids. Note that the holotype (MCZ A19951) and paratype (MCZ A19949) of Pristimantis acuminatus are assigned to different species (Clades C and D, respectively) by the discriminant analysis (A). The 93% of the males and 81% of the females were morphological successfully assigned to species, whereas the 88% of the localities were successfully classified in the environmental space.
Table 4.
Results of successful classification in morphological-space (males/females) and environmental-space (localities), with percentage in parentheses, assigned to each clade by discriminant analysis.
Table 5.
Descriptive statistics for ecological niche models (ENMs) and their spatial representations for species within the Pristimantis acuminatus complex.
Fig 4.
Ecological niche models (ENM) and niche overlap between species within the Pristimantis acuminatus complex.
The yellow regions indicate niche overlap in pairwise comparisons of each species; D = values of Schoener's D index for niche overlap. The Minimum Training Presence (MTP) threshold and a Partial-ROC dataset was used to validate the models; over-predicted areas in the extreme west Amazonia are not shown. The star represents the type-locality of Pristimantis acuminatus (MCZ 19951) at Canelos, Pastaza Province, Ecuador.
Table 6.
Niche overlap (Schoener's D index) and Identity Test for clades within Pristimantis acuminatus complex.
Fig 5.
Holotype of Pristimantis acuminatus (MCZ 19951, A: lateral view of head, B: dorsum, C: venter).
This specimen was collected in Canelos, Pastaza province, Ecuador. Note the shagreened dorsum and absence of tympanum. Photographs by the President and Fellows of Harvard College, Museum of Comparative Zoology at Harvard University.
Table 7.
Comparisons of Pristimantis enigmaticus sp. nov., P. limoncochensis sp. nov., and P. omeviridis sp. nov. with other species from the Amazonian lowlands having a dorsal green coloration.
Fig 6.
Schematic working protocol for an integrative systematics.
Increasing black color intensity represents increasing certainty about species status in the Pristimantis acuminatus complex [48]. Colors in species represent clades shown in the phylogeny and geographic ranges in the Fig 1.
Fig 7.
Views of the body (dorsum and venter), head, hand, and foot of the holotypes of (A) Pristimantis limoncochensis, QCAZ 37277, (B) Pristimantis omeviridis, QCAZ 55392, and (C) Pristimantis enigmaticus, QCAZ 40935. Arrows indicate the absence (Pristimantis limoncochensis) or presence of tympanum (Pristimantis omeviridis and Pristimantis enigmaticus), and differences in ulnar and tarsal tubercles on arms and legs. Tags and background color have been digitally removed. Only the dorsum and venter are shown at scale. Photographs by H. M. Ortega-Andrade.
Fig 8.
Live specimens of the new species (A: Pristimantis limoncochensis sp. nov., B-C: Pristimantis omeviridis sp. nov., D-E: Pristimantis enigmaticus sp. nov.).
(A) Paratype female, QCAZ 52987; (B) holotype female, QCAZ 55392; (C) paratype male, QCAZ 55391; (D-E) holotype female, QCAZ 40935. Photographs by H. M. Ortega-Andrade (A) and S. Ron (B-E).
Fig 9.
Living specimens of the Pristimantis acuminatus complex and their relatives in the Amazon Basin.
(A) Pristimantis acuminatus, QCAZ 53263, (B) Pristimantis tantanti, CORBIDI 12987, (C-D) night and daylight color variation in Pristimantis limoncochensis sp. nov., QCAZ 52987, (E) amplectant pair of Pristimantis omeviridis sp. nov., holotype female QCAZ 55392 and paratype male QCAZ 55391, (F) Pristimantis padiali, specimen not collected, (G-H) night and daylight color variation in Pristimantis enigmaticus, specimen not collected. Photographs of (B) by V. Durán, (E) by Santiago Ron, (F) by Omar Rojas; all other photographs by H. M. Ortega-Andrade.
Fig 10.
Variation in tympanum condition.
(A) Tympanum covered by muscle, overlying skin not differentiated (Pristimantis limoncochensis sp. nov., QCAZ 52987); (B) tympanic annulus and membrane distinct, not covered by muscle (Pristimantis enigmaticus sp. nov., QCAZ 31184), overlying skin is differentiated (not shown in photo). Photographs by H. M. Ortega-Andrade.