Research Article |
Corresponding author: Ruth Amanda Estupiñán ( ruthamanda.estupinan@gmail.com ) Academic editor: Uri García-Vázquez
© 2023 Ruth Amanda Estupiñán, Sávio Torres de Farias, Evonnildo Costa Gonçalves, Mauricio Camargo, Maria Paula Cruz Schneider.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Estupiñán RA, Torres de Farias S, Gonçalves EC, Camargo M, Cruz Schneider MP (2023) Performance of intron 7 of the β-fibrinogen gene for phylogenetic analysis: An example using gladiator frogs, Boana Gray, 1825 (Anura, Hylidae, Cophomantinae). ZooKeys 1149: 145-169. https://doi.org/10.3897/zookeys.1149.85627
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Boana, the third largest genus of Hylinae, has cryptic morphological species. The potential applicability of b-fibrinogen intron 7 – FGBI7 is explored to propose a robust phylogeny of Boana. The phylogenetic potential of FGBI7 was evaluated using maximum parsimony, MrBayes, and maximum likelihood analysis. Comparison of polymorphic sites and topologies obtained with concatenated analysis of FGBI7 and other nuclear genes (CXCR4, CXCR4, RHO, SIAH1, TYR, and 28S) allowed evaluation of the phylogenetic signal of FGBI7. Mean evolutionary rates were calculated using the sequences of the mitochondrial genes ND1 and CYTB available for Boana in GenBank. Dating of Boana and some of its groups was performed using the RelTime method with secondary calibration. FGBI7 analysis revealed high values at informative sites for parsimony. The absolute values of the mean evolutionary rate were higher for mitochondrial genes than for FGBI7. Dating of congruent Boana groups for ND1, CYTB, and FGBI7 revealed closer values between mitochondrial genes and slightly different values from those of FGBI7. Divergence times of basal groups tended to be overestimated when mtDNA was used and were more accurate when nDNA was used. Although there is evidence of phylogenetic potential arising from concatenation of specific genes, FGBI7 provides well-resolved independent gene trees. These results lead to a paradigm for linking data in phylogenomics that focuses on the uniqueness of species histories and ignores the multiplicities of individual gene histories.
Anura evolutionary rate, divergence time, gladiator frogs, indels, nuclear DNA, nucleotide substitution rate, phylogenetic hypothesis, polymorphic sites
Using only one type of trait, such as mitochondrial DNA (mtDNA), to detect phylogenetic relationships can lead to noise (
Introns in nuclear protein coding genes have several properties that make them useful for phylogenetic analyses of recently evolved vertebrates (
To obtain data on robust phylogenetic and temporal divergence in phylogeographic studies of frogs, nuclear intron data have often been used in conjunction with mtDNA data (
The small size of aligned base pairs (bp) and low genetic variability (variable site dataset) of FGBI7 resulted in few informative traits and discordance between mtDNA and nuclear DNA (nDNA) (
With 99 taxa, the Neotropical gladiator frogs of Boana Gray, 1825, constitute the third largest genus within Hylinae (
Two questions prompted us to conduct this study: 1) Is FGBI7 a phylogenetic signal for Boana with more robust topologies than other nuclear genes? 2) Does FGBI7 contribute to explaining the phylogeny of Boana? To answer these questions, we reconstructed the evolutionary history of Boana using several molecular markers, including FGBI7.
DNA samples were obtained from captured specimens and donations from herpetological collections (Appendix
Total DNA extraction from muscle or liver tissue was performed using the SDS -proteinase K/phenol-chloroform extraction method (
PCR products were sequenced using a MegaBACE automated DNA sequencer (GE Healthcare) and the DYEnamic ET dye terminator kit (GE Healthcare) according to the manufacturer’s instructions. Each sample was sequenced with both forward and reverse primers to confirm the observed mutations.
After searching available data in GenBank, we compared the phylogenetic signal of FGBI7 with that of C-X-C motif chemokine receptor 4 (CXCR4), single exon of recombination activating gene 1 (CXCR4), exon 1 of Rhodopsin (RHO), seven-in-absentia homolog 1 (SIAH1), exon 1 of Tyrosinase (TYR), and 28S ribosomal rDNA.
Sequence alignments were made using MAFFT version 7 (
To compare polymorphic sites and basic sequence statistics, Boana sequences were analyzed for conserved, variable, parsimony-informative, and singleton sites using MEGA X (
Each set of sequences for each marker was analyzed using maximum parsimony (MP), Bayesian analysis (MB), and maximum likelihood (ML). MP was performed in the TNT Willi Hennig Society Edition (
Support for clades was tested using a jackknife procedure with a removal rate of 0.36, which is the most congruent value with bootstrapping (
MB analysis of the evolutionary model were performed using MEGA X. For sequences with many gaps, the “use all sites” setting was selected (
One run consisted of two repeated Monte Carlo Markov chains. The run was based on considering four chains, and the default settings for the state frequency priors (statefreqpr) were set as fixed (equal) and the substitution rate priors (ratepr) were set as variable. The other priors were set to default settings, and 85 million generations were performed (with a burn-in fraction of 0.25). Stabilization of the resulting parameters was assessed using Tracer version 1.7 (
The ML analysis was performed with MEGA X software (
Phylogenetic trees were compared for each marker based on their topology and monophyletic groups defined for Boana (
Mean evolutionary rates for Boana species were based on mitochondrial and informative genes such as ND1 and CYTB from GenBank (
To establish a chronological scale for clade/lineage evolution, the divergence times established by
Divergence times were calibrated with a normal distribution and 95% confidence interval. Relative evolution rate values for each node were obtained using RelTime-Rate. Absolute evolution rates were obtained by dividing the relative rates by the scaling factor (ratio of absolute times/relative times) (
The average length of the FGBI7 sequences examined was 478 base pairs. The sequences contained both single and multiple insertions and deletions. FGBI7 sequences of 710 bp were recorded for Boana albomarginata, Boana albopunctata, Boana lanciformis, and Boana raniceps. Alignment of long (710bp) and short sequences (478 bp) revealed short and larger deletions (230–438 positions). However, polymorphism was detected when comparing the long and short sequences.
In this study, new FGBI7 sequences were generated for 24 Boana taxa. For comparison of singleton and parsimony informative sites, available sequences for 11 nuclear genes and two mitochondrial genes were retrieved from GenBank. The low number of available sequences for c-myc2, c-myc3, H3a, KIAA1239, and POMC for a large number of Boana taxa prevented their inclusion in the phylogenetic analysis of the group. CXCR4, CXCR4, RHO, SIAH1, TYR, and 28S were used for the phylogenetic evaluation of Boana (Table
Comparative polymorphic sites and basic sequence statistics in Boana nDNA.
CXCR4 | FGBI7 | RAG1 | RHO | SIAH1 | TYR | 28S | C-genes | C-genes (1) | ND1 | Cyt b | |
---|---|---|---|---|---|---|---|---|---|---|---|
S | 30 | 24 | 19 | 51 | 26 | 29 | 26 | 58 | 50 | 58 | 53 |
Pb | 676 | 478 | 428 | 316 | 397 | 532 | 823 | 3650 | 1686 | 941 | 385 |
Pb* | 675 | 466 | 428 | 316 | 397 | 532 | 786 | 3606 | 1673 | 941 | 385 |
C-S | 497 | 286 | 368 | 254 | 358 | 387 | 691 | 2817 | 1153 | 445 | 198 |
S-S(%) | 70(39) | 85(47) | 35(58) | 27(44) | 18(46) | 53(37) | 53(56) | 358(46) | 220(42) | 42(8) | 20(11) |
P-I(%) | 108(61) | 95(53) | 25(42) | 35(56) | 21(54) | 92(63) | 42(44) | 424 (54) | 300(58) | 454(92) | 167(89) |
PIS(%) (100*P-I/Pb*) | 16 | 20.39 | 5.84 | 11.08 | 5.29 | 17.29 | 5.34 | 11.76 | 17.93 | 99.51 | 99.56 |
AT (%) | 50.4 | 60.4 | 55.8 | 54.5 | 51.3 | 51.9 | 42.5 | 51.3 | 53.2 | 59.5 | 59.3 |
CG (%) | 49.6 | 39.6 | 44.2 | 45.5 | 48.7 | 48.1 | 57.5 | 48.7 | 46.8 | 40.5 | 40.7 |
Informative singleton and parsimony sites comprised between 36% and 63% of all sites. The percentage of singleton sites was generally high for all genes. The percentage of parsimony-informative sites relative to the total number of sites, excluding missing data-Pb* for each of the compared genes, showed that the data based on FGBI7, TYR, and CXCR4 gave more sensitive and highly informative performance (Table
The support values of several nodes were low, ranging from 0 to 50 in all 21 trees generated by the three phylogenetic methods applied (MP, MB, and ML) (Fig.
Phylogenetic trees corresponding to the studied markers provided more sensitive and highly informative performance (A CXCR4 B FGBI7 C TYR), and the methods used–MP, MB, and ML, corresponding to the first, second, and third trees for each marker, respectively). For Jackknife support values from the MP method, and bootstrap support values for the ML method, values below 50% were not presented.
Monophyletic species groups recovered by the three phylogenetic methods for single and concatenated gene phylogeny.
CXCR4 | FGBI7 | CXCR4 | RHO | SIAH1 | TYR | 28S | C–gene | |
---|---|---|---|---|---|---|---|---|
Boana | MP | MP, MB | * | * | MP, MB, ML | MP, MB, ML | * | * |
B. albopunctata group | MP, MB, ML | MP, MB, ML | – | * | * | MP, MB, ML | * | * |
B. benitezi group | – | – | * | * | – | MP, MB, ML | * | MP |
B. faber group | MP, MB, ML | – | – | MP, ML | – | MP, MB, ML | * | MP, MB, ML |
B. pellucens group | – | MP, MB, ML | – | – | – | – | – | MP, MB, ML |
B. pulchella group | MP, MB, ML | MP, MB, ML | MP | * | MP, MB, ML | MP, MB, ML | * | |
B. punctata group | * | * | – | * | – | – | * | * |
B. semilineata group | MP, MB, ML | MP, MB, ML | – | MP | – | MP, MB, ML | * | MP, MB; ML |
The MP and consistency indices for all nDNAs analyzed were > 0.5. The CI and CR indices showed a similar trend for all nDNA, indicating a lower degree of homoplasies with an increase in their values (Table
ND1 and CYTB gene sequence data available for Boana in GenBank were obtained for 58 and 53 species, respectively. The nucleotide substitution model GTR+G+I was run with MEGA X to generate the phylogenies. The T92+G model was chosen for the phylogenetic analysis of FGBI7. The absolute values of the mean evolutionary rates for ND1, CYTB, and FGBI7 were 1.235198E-2 ± 3.61903E-3, coefficient of variation – CV = 29%; 1.2796789E-2 ± 4.6661189E-3, CV = 36.4%; and 1.920083E-3 ± 1.07878E-3; CV = 56% replacement/site/million years, respectively.
Comparison of dating results between congruent Boana groups for ND1, CYTB, and FGBI7 showed divergence among the three genes, with some values being most similar among mitochondrial genes. However, the results diverged to a lesser extent from those obtained for the nuclear gene FGBI7. The dating results for the B. pulchella group revealed divergence times of 14.28 Ma (ND1), 15.22 Ma (CYTB), and 10 Ma (FGBI7). In addition, the B. punctata group (B. cinerascens and B. punctata) showed dating results of 11.44 Ma (ND1), 15.13 Ma (CYTB), and 9.12 Ma (FGBI7), and the B. albopunctata group showed divergence times of 21.94 Ma (ND1), 13.50 Ma (CYTB), and 16.32 Ma (FGBI7) (Fig.
While we explored the potential applicability of FGBI7 in reconstructing the phylogeny of Boana clades, our goal was to include a growing number of informative sites for future analyses, to contribute to the understanding of phylogenetic signal, and to investigate the robustness of a combination of mitochondrial and nuclear data.
Despite the short sequence (478 bp) observed in the present study, the FGBI7-based analyses were highly consistent with the previously proposed phylogenetic hypothesis based on the concatenation of mitochondrial and nuclear genes previously proposed for Boana groups (
Similar groupings were also observed among species. Some support values, such as those of B. semilineata group, B. pellucens group, and B. pulchella group, were very close to those determined by
The response of the CI and CR indices obtained by the MP analysis showed a lower degree of homoplasy for FGBI7. Therefore, a higher degree of parsimony compared with the congruent hypothesis generated considering the TYR and CXCR4 genes and using different analysis methods supports the use of FGBI7 in phylogenetic analysis of Boana.
The observed variation between the CR indices obtained for the analyzed genes can be attributed to the phylogenetic signal of indels (
The lowest proportion of parsimony-informative sites identified for C-genes is due to the noise of concatenation with 28S and RHO. After the data for these genes were removed from the analysis (C-genes (1)), the information signal increased, although it remained lower than that of FGBI7. Typically, it is believed that informativeness about species history is maximized by allowing concatenation of multiple independent loci to obtain a hypothesis congruent with the species tree. However, concatenation of sequence data can bias the phylogeny if the number of gene trees that match the species tree is small. In these cases, species tree approaches can provide better-resolved phylogenies when a large number of loci are used (
However, when gene tree and species tree data support a robust and congruent hypothesis (
The concatenated C-gene and C-gene (1) phylogenies in MP yielded very similar clades, but the C-gene tree is not supported by bootstrap values; the C-gene (1) tree showed some high support values. The FGBI7 tree, on the other hand, showed a larger number of bootstrap values compared to the C-gene trees (1).
The low reliability values obtained for multiple bootstrap and jackknife nodes and single gene trees, such as the concatenated alignment of the seven genes, indicate low robustness of the estimated topology. The low bootstrap values could be due to the small sample size and the generation of bias by signals generated by a few genes. Bootstrap values and similar support values increase with increasing numbers of sites sampled (
Congruence was observed not only at the tips of trees but also at deeper inner branches.
The congruence between the topology resulting from the use of FGBI7 in this study and the results reported by
Intraspecific variability in body color, description of new species, and research on declining taxa of the Hylidae (
This study also showed that FGBI7 for Boana has lower mean evolutionary rates than mitochondrial genes (ND1 and CYTB). The substitution rates in this study are consistent with previous reports in which nuclear genes typically had lower substitution rates than mitochondrial genes (
The divergence dates of the FGBI7 data were close to those obtained by
Assuming that the divergence threshold for Neobatrachus from Gondwana is 145 Ma and that for Hylidae is 70 Ma, and based on nDNA data and calibration of the fossil record, the origin of Boana is estimated to be 25 Ma (
Recent divergence times inferred from mtDNA sequences tend to overestimate times for basal clades (
The use of FGBI7 in this study showed that, unlike other nuclear genes already used to generate phylogenetic hypotheses of Anura (e.g.:
The topology, total informative sites, and parsimony sites of FGBI7, in combination with mitochondrial genes, allow the clarification of new lineages already proposed by other authors such as
Although estimating divergence times for clades is a difficult task (
We extend our gratitude to researchers for sample donations, to Maria Silvanira R. Barbosa for her assistance with laboratory procedures, to Daniel Fernandes Vilar Cardoso from CIMIR-RE at the Federal Institute of Paraiba for providing appropriate hardware for analysis, and to Beatriz Mello from Genetics Department – Federal University of Rio de Janeiro, for her assistance in the calculation of mean evolutionary rates and molecular dating using the RelTime method in MEGA X. We would also like to thank the Brazilian Environment Ministry, which financed the MMA/PROBIO-Brazil project “Diversity of vertebrates on the upper Marmelos River (BX 044)” and CAPES for providing a graduate RAET stipend. Collection licenses: 301 062/2003-CGFAU/LIC issued by the Brazilian Institute for the Environment and Renewable 302 Natural Resources (IBAMA).
Voucher information, localities, and GenBank accession numbers for the sequences analyzed for this study.
Species | CXCR4 | Intron 7 | RAG-1 | RHO | SIAH1 | Tyr | 28S | Voucher and source literature of sequences |
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Boana aguilari | MT824211 | MT824337 | KF751464 |
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Boana albomarginata | KF751476 | OQ448590 | AY844384 | AY844568 | AY844794 | AY844218 |
MRT 5870: Jussara, Bahia, Brazil. |
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Boana albopunctata | OQ448612 | AY844569 | AY844795 | AY844041 |
MRT 8229, Petrolina, Goiás, Brazil. |
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Boana balzani | MT824213 | OQ448599 | AY844395 | AY844582 | AY844806 | AY844226 |
MNCN/ADN 5785, Camino a San Onofre, Carrasco, Cochabamba, Bolivia. |
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Boana benitezi | KF751477 | AY844396 | AY844583 | AY844227 |
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Boana bischoffi | MT824219 | OQ448607 | AY844398 | MT824343 | MT824526 |
AF 327, Fazenda Intervales, Estado de São Paulo. |
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Boana boans | KF751478 | OQ448591 | AY844588 | AY844809 | AY844055 | AY844231 |
MPEG 17385, Arredores da Fazenda passo Formoso, Manicoré, Amazonas. |
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Boana botumirim | MT824344 |
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Boana buriti | OQ448601 | MT824346 | MT824484 |
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Boana caingua | KF751479 | OQ448602 | MT824352 | AY844812 | AY844057 | AY844234 |
AF 515, Ribeirão Grande São Paulo, São Paulo. |
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Boana calcarata | AY844235 |
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Boana cambui | MT824356 | MT824486 | MT824534 |
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Boana cinerascens | KF751480 | OQ448595 | AY844610 | DQ283466 |
RAET 505, Estação Científica Ferreira Penna, Melgaço, Pará, Brazil. |
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Boana cipoensis | MT824357 | MT824487 |
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Boana cordobae | KF751481 | AY844411 | MT824460 | MT824516 | AY844066 | AY844244 |
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Boana crepitans | KF751482 | AY844601 | AY844067 |
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Boana curupi | MT824227 | MT824359 | MT824489 |
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Boana ericae | AY844416 | AY844605 | MT824537 |
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Boana faber | AY844607 |
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Boana fasciata | KX200378 | AY844608 |
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Boana freicanecae | MT824217 | MT824366 | MT824538 |
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Boana geographica | OQ448611 | QCAZ 16809: Estación Científica Yasuní. PUCE, Laguna, Orellana, Ecuador. in confirmation process | ||||||
Boana gladiator | MT824212 | MT824368 |
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Boana goiana | MT824372 | MT824491 | MT824541 |
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Boana guentheri | MT824245 | OQ448608 | MT824373 | MT824492 | AY844253 |
CFBH 3386: Terra de Areia, Rio Grande do Sul, Brazil. |
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Boana heilprini | AY844613 |
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Boana jaguariaivensis | MT824374 | MT824494 |
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Boana joaquini | KF751484 | OQ448605 | AY844421 | MT824376 | AY844256 |
CFBH 1068: Urubici, Santa Catarina, Brazil. |
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Boana lanciformis | AY844619 | AY844081 | AY844258 |
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Boana lemai | KF751485 | AY844423 | AY844620 | AY844082 | AY844259 |
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Boana leptolineata | MT824246 | OQ448604 | AY844424 | AY844621 | AY844839 | AY844083 | AY844260 |
CFBH 8504: São Francisco de Paula, Rio Grande do Sul, Brazil. |
Boana lundii | AY844623 | AY844085 | AY844262 |
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Boana marginata | KF751486 | AY844426 | AY844624 | MT824542 | AY844263 |
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Boana marianitae | OQ448610 | AY844427 | MT824378 | AY844843 |
MNCN/ADN 5901: Camino a Bella Vista, Florida, Santa Cruz, Bolivia. |
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Boana melanopleura | KF751487 | OQ448600 | MT824379 | HM444787 |
MTD-TD 1146: Huancabamba, Pasco, Peru. |
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Boana multifasciata | GQ365986 | AY844436 | AY844633 | AY844093 | AY844270 |
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Boana nympha | KF751488 | AY844661 | AY844112 | AY844289 |
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Boana pardalis | AY844637 |
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Boana pellucens | OQ448597 | QCAZ 15354: Via Toachi-Chiriboga. Poza junto a carretera cerca del Rio Orito, Ecuador. | ||||||
Boana picturata | OQ448594 | QCAZ 15549:3 Km from Durango, em el cruce de la vá San Lorenzo e outra carretera X, Esmereldas, Ecuador. | ||||||
Boana poaju | MT824380 | MT824495 |
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Boana polytaenia | MT824241 | OQ448606 | AY844443 | MT824429 | MT824508 | MT824547 |
CFBH 8394: Cristina, MG. |
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Boana pombali | MT824247 | MT824431 | MT824511 | MT824552 |
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Boana prasina | MT824436 | MT824554 |
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Boana pulchella | OQ448609 | AY844445 | MT824443 | MT824513 | MT824557 | AY844278 |
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Boana punctata | OQ448596 | AY844645 | QCAZ18185: Estación Biológica Jatun Sacha, Napo. |
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Boana raniceps | KF751489 | OQ448613 | AY844646 | AY844863 | AY844103 |
MRT 6706: UHE Lajeado Tocantins. |
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Boana riojana | MT824238 | AY844447 | MT824462 | MT824518 | AY844279 |
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Boana roraima | KF751490 | AY844448 | AY844650 | AY844104 | AY844280 |
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Boana rufitela | OQ448598 | AY844652 | AY844867 | AY844105 | AY844282 | CHP-STRI:5114: Quebrada Guabalito, Palmarazo, Parque Nacional General de División Omar Torrijos Herrera, Provincia de Coclé. |
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Boana semiguttata | OQ448603 | AY844452 | MT824466 | MT824519 | MT824559 | AY844285 |
CFBH 242: Piraquara, Paraná, Brazil. |
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Boana semilineata | KF751491 | AY844453 | AY844656 | AY844108 | AY844286 |
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Boana sibleszi | KF751492 | OQ448593 | AY844455 | AY844658 | AY844873 | AY844110 | AY844288 |
ROM 39561: Mount Ayanganna, Guyana. |
Boana stellae | MT824229 | MT824475 | MT824567 |
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Boana stenocephala | MT824479 | MT824520 | MT824563 |
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Boana wavrini | OQ448592 | RAET 502: Estação Científica Ferreira Penna, Melgaço, Pará, Brazil | ||||||
Aplastodiscus albofrenatus | OQ448614 | KU184083 | KU184111 | KU184149 | KU184246 |
AF 101: Rio de Janeiro, Rio de Janeiro, Brazil. |
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Aplastodiscus albosignatus | OQ448616 | AY844385 | KU184114 | AY844796 | AY844042 | AY844219 |
CFBH 7711: Parque Estadual Serra do Mar, Santa Virginia, São Luís do Paraitinga, São Paulo, Brazil. |
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Aplastodiscus eugenioi | KF751465 |
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Aplastodiscus leucopygius | KF751466 | AY844261 |
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Aplastodiscus perviridis | KF751467 | AY844201 |
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Aplastodiscus weygoldti | OQ448615 | AY844467 | KU184124 | AY844887 | KU184257 |
AF 68: São Paulo do Aracã, Espírito Santo, Brazil. |
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Bokermannohyla circumdata | KF751468 | OQ448619 | AY844409 | AY844817 | AY844064 | AY844242 | IT-H0562, MZUSP 93551: Juquitiba, Estado de São Paulo. |
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Callimedusa tomopterna | GQ366024 | OQ448618 | AY844497 | AY844715 | AY844157 | AY844328 |
MPEG 17368, Near Fazenda Passo Formoso, Manicoré, Amazonas, Brazil. |
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Callimedusa vaillanti | AY844921 |
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Myersiohyla liliae | MH251236 |
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Myersiohyla inparquesi | AY844291 |
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Nesorohyla kanaima | GQ365994 | OQ448617 | AY844617 | MH251240 | AY844079 |
ROM 39586: Mount Ayanganna, Guyana. |
Phylogenetic trees and molecular dating of gene trees
Data type: pdf file
Explanation note: Phylogenetic trees corresponding to the studied markers (a. CXCR4, b. FGBI7, c. RAG-1, d. RHO, e. TYRf.SIAH1, g. 28SandC-genes), and the methods used – MP, MB, and ML, corresponding to the 1st, 2nd, and 3rd trees for each marker, respectively). For Jackknife support values from the MP method, and bootstrap support values for the ML method, values below 50% were not presented. Molecular dating of gene trees. a. ND1. b. CYTB, and c. FGBI7using the RelTimemethod in MEGA X.