Table 1.
Events in Leiuperinae early development.
These events, here listed by morphological structure, were used to construct summarized developmental sequences per species, thus some may be missing in some taxa.
Fig 1.
Leiuperinae embryos at tailbud stage.
Physalaemus: (A) Ph. aff. albonotatus. (B) Ph. albifrons. (C) Ph. albonotatus. (D) Ph. biligonigerus. (E) Ph. carrizorum. (F) Ph. cicada. (G) Ph. cuvieri. (H) Ph. riograndensis. (I) Ph. santafecinus. Pleurodema: (J) Pl. borellii. (K) Pl. bufoninum. (L) Pl. cordobae. (M) Pl. diplolister. (N) Pl. guayapae. (O) Pl. thaul. Pseudopaludicola: (P) Ps. falcipes. (Q) Ps. mystacalis. Scale bar 1 mm. Five species were not included because tailbud embryos were lacking in our ontogenetic series. Note the diversity of size, shape, and pigmentation, from small, kyphotic, white embryos of most Physalaemus, to large, almost straight, dark embryos in some Pleurodema.
Fig 2.
Body ciliation in leiuperine embryos.
Ventral views of specimens with fully developed ciliation (left), older embryos with ciliation highly reduced (middle), and details of ciliated cells (sampled from the abdominal region at full development; right). (A) Physalaemus aff. albonotatus, embryos at GS23 and GS25. (B) Pleurodema thaul, embryos at GS22 and GS23. (C) Pl. nebulosum clade: Pl. nebulosum, embryo at GS23, and Pl. guayapae, embryo at GS27. (D) Pseudopaludicola: Ps. falcipes, embryos at GS23 and GS25, and detail of ciliated cells of Ps. mystacalis. Scale bars 200μm (left and middle), 10μm (right). Note the rounded or oblong cells, and the differences in regression timing among Pleurodema species, early in Pl. thaul and highly delayed in Pl. guayapae.
Fig 3.
Gill development in Pseudopaludicola.
Details of gill morphology and ciliation (left), embryos with gills at full development (middle), and gills regressing (right). (A) Ps. falcipes. (B) Ps. mystacalis. Scale bars 100μm. Note the overall poor development in the genus.
Fig 4.
Gill development in Physalaemus.
Details of gill morphology and ciliation (left), embryos with gills at full development (middle), and gills regressing (right). (A) Ph. fernandezae. (B) Ph. albifrons. (C) Ph. carrizorum. (D) Ph. riograndensis. Scale bars 10μm (left), 100μm (middle and right). Note the poor and brief development of gills of Ph. fernandezae as compared with the other species in the genus.
Fig 5.
Gill development in Pleurodema.
Details of gill morphology and ciliation (left), embryos with gills at full development (middle), and gills regressing (right). (A) Pl. diplolister. (B) Pl. guayapae. (C) Pl. thaul. (D) Pl. bibroni. Scale bar 10μm (left), 100μm (middle and right). Note the comparatively poor and brief development of gills in Pl. thaul and Pl. bibroni.
Fig 6.
Diversity of adhesive glands in Leiuperinae.
Physalaemus: (A) Ph. albonotatus. (B) Ph. henselii. (C) Ph. santafecinus. (D) Pseudopaludicola falcipes. Pleurodema: (E) Pl. bibroni. (F) Pl. guayapae. Scale bars 20μm. Note the general arrangement in the insets, and the differences in size and shape of Pleurodema glands as compared with those of Physalaemus and Pseudopaludicola.
Fig 7.
Adhesive gland ontogeny in Pleurodema thaul.
(A) Initial morphogenetic field at GS17. (B) Detail of cells with microvilli at the secretory region (arrow in A). (C) Morphogenetic field split and in a V-shape configuration at GS19. (D) Detail of secretory region (arrow in C). (E) Adhesive gland at full development–GS23–, as two circular structures with central secretory tissue. (F) Regressing adhesive glands at GS24–25, with a reduced secretory region. Scale bars 100μm, excepting B and D 10μm. In the insets, besides the differences in gland morphology note the position ventral to the oral disc conserved along the ontogeny.
Fig 8.
Diversity of hatching glands in Leiuperinae.
Pseudopaludicola: (A) Ps. falcipes at GS17–18. (B) Ps. mystacalis at GS17–18. Physalaemus: (C) Ph. albifrons at GS17–18. (D) Ph. biligonigerus at GS17–18. (E) Ph. fernandezae at GS17–18. Pleurodema: (F) Pl. borellii at GS22–23. (G) Pl. bibroni at GS17–18. Scale bars 5μm. Individual secretory cells are indicated with arrows. Note the general arrangement as shaded areas in the insets, and the long microvilli in Pl. borellii.
Fig 9.
Hatching gland ontogeny in Pleurodema bufoninum.
(A) Embryo at GS19, showing gland arrangement and pigmentation in the cephalic region. Secretory cells (arrows) in embryos at (B) GS20. (C) GS21. (D) GS22. (E) GS23. (F) GS25. Scale bars 10μm. The densest arrangement take place around GS22, co-occurring with gills at full development.
Fig 10.
Scheme of oral disc trajectories in Physalaemus.
Shaded trajectories and assignation of species to oral configurations labeled C1-C5 are as modeled in Vera Candioti et al. [35]. Additional species in our work are indicated in red and their larval oral configurations and trajectories inserted on the original scheme. The diagram emphasizes on ventral and ventrolateral gaps development (VG and VLG respectively), whereas the timing of differentiation of row P3 regarding mental papillae is simplified since it can vary between species sharing the same trajectory. Micrographs show transient oral discs of (A) Ph. carrizorum, and (B,C) Ph. cicada, highlighting representatives of the new trajectories. Note the small papilla filling the ventral gap in Ph. carrizorum, and the alternative transient morphologies of Ph. cicada embryos, with or without ventral gap.
Fig 11.
Oral disc ontogeny in Pseudopaludicola mystacalis.
(A) Rows A1 and P1. (B) Rows A2, P2, and commissural papillae differentiated. (C) Labial teeth on all tooth ridges, marginal papillae at commissures and medially at the mental region. (D) Larval oral disc ending this trajectory, i.e., two lower labial ridges and persisting ventrolateral gaps (oral disc C1 sensu [29]). (E) A rare larval oral disc with a third labial tooth row formed from mental papillae/ridge. (F) A further alternative larval oral disc with two lower labial tooth rows and a wide, bufonid-like ventral gap. Scale bars 20μm. Note the highly polymorphic larval oral disc, and the intraspecific diversity in oral developmental pathways they imply.
Fig 12.
Oral disc ontogeny in Pleurodema cordobae.
(A) Rows A1, P2, and commissural papillae. (B) Rows A2, P1, and a short P3 are formed, and marginal papillae progress medially. (C) Marginal papillae with ventral gap. (D) Labial teeth emerging on A1, P1, and P2. (E) Labial teeth on A2, and marginal papillae widely spaced. (F) Detail of a labial tooth from P2. Scale bars 20μm, excepting F 2μm. Note the small, transient ventral gap co-occurring with a short P3.
Fig 13.
Oral disc ontogeny in the Pleurodema thaul clade.
Pl. bufoninum: (A) Rows A1, P2, and commissural papillae evident. (B) Lower lip more defined. (C) P1 and A2 outlined, and lower marginal papillae progressing medially; D) P3 outlined and labial teeth emerging on A1 and P2. (E) Labial teeth on all rows, and marginal papillae widely spaced. P. thaul: (F) Detail of the lower lip of a Stage 37 tadpole. Scale bars 200μm. Note the larval oral disc with the alternate papillae giving a double-like appearance to the lower lip margin.
Fig 14.
Sequence heterochronies in developmental trajectories of leiuperines.
Developmental events (N = 24; data from S3 Appendix; X-axis) are plotted according to their order in the sequence (Y-axis), as compared to the reference trajectory of Odontophrynus americanus. AG adhesive gland first visible, TB tail bud, 1G first gill pair bud, TL = BL tail length / body length = 1, 2G second gill pair bud, 1GB first gill pair branched, 2GB second gill pair branched, A1 labial tooth ridge A1, P2 labial tooth ridge P2, OB operculum at gill base, FP first marginal papillae, P1 labial tooth ridge P1, A2 labial tooth ridge A2, GFD gills at full development, OM operculum medially fused, IC first coil in digestive tract, RGC right gill covered by operculum, MP marginal papillae complete, LGC left gill covered by operculum, ES spiracle developed, HLB hind limb buds, AGA adhesive glands absent, LOD oral disc fully formed, HL26 hind limbs at GS26. Species are not individualized since main variations among genera are emphasized. Asterisks point out the event after which hatching occurs for each species. The numbered arrows indicate main heterochronic changes: 1) the accelerated full development of gills and spiracle differentiation of Pseudopaludicola; 2) a late adhesive gland regression in Pleurodema; 3 and 4) variations in the ontogeny of rows P1 and P2 that differentiate Pseudopaludicola and Physalaemus from Pleurodema; 5) the early acquisition of larval oral disc in Ph. carrizorum; and 6) the extremely early development of hind limbs in Pl. guayapae.
Fig 15.
Ancestral reconstruction of embryonic morphological characters.
Data are taken from S4 Appendix. The first four characters are recorded in embryos at tailbud stage. (A) Length. (B) Angle of dorsal curvature; the smallest angles correspond to highly kyphotic embryos. (C) Yolk relative area. (D) Pigmentation: absent, present. (E) Gill pairs: 2, 3. (F) Adhesive glands: absent, type C, type D. The black bar indicates the ancestor of Leiuperinae. Note the overall high diversity in leiuperine tailbud embryos, contrasting with comparatively conserved gill and adhesive gland morphologies.
Fig 16.
Ancestral reconstruction of oral disc features.
Data are taken from S4 Appendix. (A) Labial tooth row P3. (B) Ventrolateral gaps. (C) Ventral gap. The black bar indicates the ancestor of Leiuperinae. Ambiguity in species we studied indicates the intraspecific occurrence of at least two alternative states. Developmental data on Engystomops are lacking to ascertain whether gaps are absent or transient, so ambiguity is indicated as well. Note the overall highly diverse oral features in leiuperines, including two or three lower labial tooth rows combined with a variety of marginal papillae arrangements.
Fig 17.
Ancestral reconstruction of developmental sequences.
Data are taken from S5 Appendix. Events (N = 27): (1) AG adhesive gland first visible, (2) TB tail bud, (3) 1G first gill pair bud, (4) 2G second gill pair bud, (5) 1GB first gill pair branched, (6) 2GB second gill pair branched, (7) A1 labial tooth ridge A1, (8) P2 labial tooth ridge P2, (9) 3G third gill pair bud, (10) OB operculum at gill base, (11) TL = BL tail length / body length = 1, (12) GFD gills at full development, (13) OM operculum medially fused, (14) FP first marginal papillae, (15) P1 labial tooth ridge P1, (16) A2 labial tooth ridge A2, (17) P3 labial tooth ridge P3, (18) AGS adhesive glands separated, (19) IC first coil in the digestive tract (20) HLB hind limb buds, (21) RGC right gill covered by operculum, (22) MP marginal papillae complete, (23) LGC left gill covered by operculum, (24) LOD oral disc fully formed (25) ES spiracle developed, (26) HL26 hind limbs at GS26, (27) AGA adhesive glands absent. Accelerations (A) and decelerations (D) of events reconstructed as synapomorphies are indicated above and below branches respectively, and main shifts discussed in the text are colored according to morphological structures they describe: adhesive glands (violet), tail and hind limbs (red), gills (green), and oral disc (yellow).