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Fig. 3 in Sperm characteristics as additional evidence of close relationship between Lebiasina and Piabucina (Characiformes: Lebiasinidae: Lebiasininae)

Fig. 3. Spermatozoon of Lebiasina melanoguttata (a, b, c), L. aff. uruyensis1 (d, e, f) and L. aff. uruyensis2 (g, h, i). a, d, g: longitudinal sections. b, c, e, f, h, i: transverse sections from top to posterior region. The nucleus (n) of all species is dropshaped and slightly elongated towards the flagellar axis. The flagellum (f) lies lateral to the nucleus. The centriolar complex (p, d) and nuclear fossa (arrow) is superolateral. Note the presence of the striated rootlets (r) on opposite sides of the distal centriole (d). The proximal centriole (p) is slightly oblique relative to the distal centriole (d) (a-inset, d-inset, g-inset). The cytoplasmic canal is present (asterisk). The midpiece (pi) is short, asymmetrical, and contains the oblong mitochondria (m) and vesicles (v). a: axoneme, d: distal centriole, f: flagellum, m: mitochondria, n: nucleus, p: proximal centriole, r: striated rootlet, v: vesicle, asterisk: cytoplasmic canal, arrow: nuclear fossa. Bar = 0.5 µm.

Fig. 5 in Sperm characteristics as additional evidence of close relationship between Lebiasina and Piabucina (Characiformes: Lebiasinidae: Lebiasininae)

Fig. 5. Schematic representation of spermatozoa of Lebiasina and Piabucina. Both spermatozoa are very similar and share primarily the lateral nucleus, superolateral centriolar complex, striated rootlets, oblong mitochondria and some vesicles in the midpiece.

Fig. 4 in Sperm characteristics as additional evidence of close relationship between Lebiasina and Piabucina (Characiformes: Lebiasinidae: Lebiasininae)

Fig. 4. Spermatozoon of Piabucina boruca (a, b, c), P. elongata (d, e, f) and P. panamensis (g, h, i). a, d, g: longitudinal sections. b, c, e, f, h, i: transverse sections from top to posterior region. The nucleus (n) of all species is drop-shaped and slightly elongated towards the flagellar axis. The flagellum (f) lies lateral to the nucleus. The centriolar complex (p, d) and nuclear fossa (arrow) is superolateral. Note the presence of the striated rootlets (r) on opposite sides of the distal centriole (d). The proximal centriole (p) is slightly oblique relative to the distal centriole (d) (a-inset, d-inset, g-inset). The cytoplasmic canal is present (asterisk). The midpiece (pi) is short, asymmetrical, and contains the oblong mitochondria (m) and vesicles (v). a: axoneme, d: distal centriole, f: flagellum, m: mitochondria, n: nucleus, p: proximal centriole, r: striated rootlet, v: vesicle, asterisk: cytoplasmic canal, arrow: nuclear fossa. Bar = 0.5 µm.

Fig. 1 in Sperm characteristics as additional evidence of close relationship between Lebiasina and Piabucina (Characiformes: Lebiasinidae: Lebiasininae)

Fig. 1. Spermiogenesis process representative for Lebiasina and Piabucina. Figure subunits are longitudinal sections of spermatids corresponding to (a) Lebiasina aff. uruyensis1, (b) L. aff. uruyensis1, (c) Piabucina boruca, and (d) P. elongata. The flagellum is initially lateral to the nucleus. A slightly movement of the nucleus towards the flagellar axis is present. Consequently the centriolar complex and the nuclear fossa is superolateral. During spermiogenesis, the formation of two striated rootlets occurs on opposite sides of the distal centriole. c: cytoplasm, d: distal centriole, f: flagellum, n: nucleus, p: proximal centriole, pi: midpiece, r: striated rootlet, asterisk: cytoplasmic canal, arrow: nuclear fossa. Bar = 0.5 µm.

FIGURE 3 in DNA Barcoding of Pyrrhulina australis (Characiformes: Lebiasinidae) reveals unexpected cryptic diversity in the group

FIGURE 3 | Dendrogram of the Pyrrhulina species based on a Bayesisan Inference analysis of the COI sequences obtained in the present study. The red bars represent the consensus MOTUs, defined according to the congruity between the results of the species delimitation methods applied in the present study. The black bars represent the Molecular Operational Units (MOTUs) formed by the different species delimitation methods: Optimal Threshold (OT); Assemble Species by Automatic Partitioning (ASAP); Poisson Tree Processes (PTP) and Generalized Mixed Yule Coalescence (GMYC). Bars marked with a star represent the same MOTU under the OT analysis. The sequence codes in bold script indicate the samples obtained from the BOLD systems database.

FIGURE 2 in DNA Barcoding of Pyrrhulina australis (Characiformes: Lebiasinidae) reveals unexpected cryptic diversity in the group

FIGURE 2 | Morphospecies considered in this study: A. Pyrrhulina aff. australis I (Araguaia, modified from Venere, Garuti, 2011); B. P. obermulleri (Madeira); C. P. aff. australis IV (Guaporé); D. P. marilynae (Teles Pires) and E. P. australis (Pantanal, Paraguay basin). Pyrrhulina spilota and P. filamentosa are not shown here because the sequences were obtained from the BOLD systems database, and no physical specimens were collected in the field.

FIGURE 1 in DNA Barcoding of Pyrrhulina australis (Characiformes: Lebiasinidae) reveals unexpected cryptic diversity in the group

FIGURE 1 | Geographic distribution of the Pyrrhulina morphospecies and MOTUs throughout South American river basins.

FIGURE 5 in DNA Barcoding of Pyrrhulina australis (Characiformes: Lebiasinidae) reveals unexpected cryptic diversity in the group

FIGURE 5 | Quadrant plot showing the maximum K2P intraspecific distances and the maximum K2P interspecific in percentages for the MOTUs of Pyrrhulina identified in the present study. The lines indicate the threshold (1.79%) between the intra- and interspecific distances. The morphology of the species in quadrant I is consistent with the molecular identification. The species in quadrant II probably have cryptic forms. Species present in quadrant III are likely the result of recent divergence, hybridization or synonimization, while quadrant IV represents a lack of correspondence between the morphological and molecular identifications.

FIGURE 4 in DNA Barcoding of Pyrrhulina australis (Characiformes: Lebiasinidae) reveals unexpected cryptic diversity in the group

FIGURE 4 | Quadrant plot showing the maximum K2P intraspecific distances and the maximum K2P interspecific in percentages for the nominal Pyrrhulina species analyzed in the present study. The lines indicate the threshold (1.79%) between the intra- and interspecific distances. The morphology of the species in quadrant I is consistent with the molecular identification. The species in quadrant II probably have cryptic forms. Species present in quadrant III are likely the result of recent divergence, hybridization or synonimization, while quadrant IV represents a lack of correspondence between the morphological and molecular identifications.

FIGURE 5 in Tracking the evolutionary pathways among Brazilian Lebiasina species (Teleostei: Lebiasinidae): a chromosomal and genomic comparative investigation

FIGURE 5 | First Row: Mitotic chromosome spreads of Lebiasina minuta males after CGH— interspecific comparisons (A–D). Male-derived genomic probe of L. minuta (A); L. melanoguttata (B); L. bimaculata (C) hybridized against male metaphase plates of L. minuta (D). Second Row: Mitotic chromosome spreads of Lebiasina minuta males after CGH— intraspecific comparisons (E–H). DAPI image (E); Male-derived genomic probe of L. minuta (F); Female-derived genomic probe of L. minuta (G) hybridized against male metaphase plates of L. minuta (H). The common genomic regions of both compared karyomorphs are depicted in yellow. Scale bar = 5 µm.

FIGURE 4 in Tracking the evolutionary pathways among Brazilian Lebiasina species (Teleostei: Lebiasinidae): a chromosomal and genomic comparative investigation

FIGURE 4 | Whole chromosome painting (WCP) highlighting the first chromosome pair of Lebiasina minuta completely hybridized with the probe from the first chromosome pair of L. bimaculata.

FIGURE 6 in Tracking the evolutionary pathways among Brazilian Lebiasina species (Teleostei: Lebiasinidae): a chromosomal and genomic comparative investigation

FIGURE 6 | Representative idiograms of L. bimaculata (A); L. melanoguttata (B) and L. minuta (C) highlighting the distribution of the 18S (green) and 5S (red) rDNA sequences; (CGG)n microsatellite (blue) and C-positive heterochromatin (black): Data for L. bimaculata and L. melanoguttata are from Sassi et al. (2019).

FIGURE 3 in Tracking the evolutionary pathways among Brazilian Lebiasina species (Teleostei: Lebiasinidae): a chromosomal and genomic comparative investigation

FIGURE 3 | Metaphase chromosomes of Lebiasina minuta hybridized with microsatellite probes (A, B and C) and telomeric probes (D), using red signals. Scale bar = 5 µm.

FIGURE 2 in Tracking the evolutionary pathways among Brazilian Lebiasina species (Teleostei: Lebiasinidae): a chromosomal and genomic comparative investigation

FIGURE 2 | Male and female karyotypes of Lebiasina minuta after A. Giemsa staining, B. C-banding, and C. "double-FISH" with 5S (red) and 18S (green) rDNA probes. Scale bar = 5 µm.

FIGURE 1 in Tracking the evolutionary pathways among Brazilian Lebiasina species (Teleostei: Lebiasinidae): a chromosomal and genomic comparative investigation

FIGURE 1 | Distribution of Lebiasina species with available cytogenetic data, highlighting the Brazilian state of Pará (orange) and Ecuadorian (purple) territories A. 1. L. bimaculata, 2. L. melanoguttata (Sassi et al., 2019), and 3. L. minuta (this study). B. Highlights the position of A in South America, and C. indicates that, although close, species 2 and 3 does not share an overlapped distribution.

Fig. 1 in Lebiasina yepezi, a new Lebiasininae (Characiformes: Lebiasinidae) from the Serra Parima-Tapirapecó mountains

Fig. 1. Lebiasina yepezi: a) MNRJ 39067 holotype (152.9 mm SL), Brazil, Amazonas, rio Marari, tributary of rio Padauari, Serra Tapirapecó; b) MNRJ 38917, paratype (97.5 mm SL), same data as holotype.

Fig. 3 in Lebiasina yepezi, a new Lebiasininae (Characiformes: Lebiasinidae) from the Serra Parima-Tapirapecó mountains

Fig. 3. Anal-fin of Lebiasina yepezi evidencing the sexual dimorphism: a) Male (MNRJ 39067 - holotype) 152.9 mm SL; b) female (MNRJ 38917 - paratype) 97.5 mm SL.Arrow indicates breeding tubercles on the scales.

Fig. 2 in Lebiasina yepezi, a new Lebiasininae (Characiformes: Lebiasinidae) from the Serra Parima-Tapirapecó mountains

Fig. 2. Anterior portion of the skull in dorsal view of: a) Lebiasina astrigata (MEPN 4418, 94.5 mm SL); b) Lebiasina yepezi. (MZUSP 81128, 42.8 mm SL), showing the usual T-shaped mesethmoid of Lebiasininae and the absence of lateral projections, respectively (FRO - frontal; LAT - lateral ethmoid; MES - mesethmoid; NAS - nasal; PRE - premaxillary).

Fig. 4 in Lebiasina yepezi, a new Lebiasininae (Characiformes: Lebiasinidae) from the Serra Parima-Tapirapecó mountains

Fig. 4. Map of northern portion of South America with the distribution of Guyana Shield species of Lebiasininae: Lebiasina yepezi (stars), L. unitaeniata (diamonds), L. uruyensis (squares), L. taphorni (triangle) and L. yuruaniensis (circles). White polygons represent type-localities.