Research Article
Research Article
Performance of intron 7 of the β-fibrinogen gene for phylogenetic analysis: An example using gladiator frogs, Boana Gray, 1825 (Anura, Hylidae, Cophomantinae)
expand article infoRuth Amanda Estupiñán§, Sávio Torres de Farias§, Evonnildo Costa Gonçalves|, Mauricio Camargo, Maria Paula Cruz Schneider|
‡ Instituto Federal da Paraíba, João Pessoa, Brazil
§ Universidade Federal da Paraíba, João Pessoa, Brazil
| Universidade Federal do Pará, Belém, Brazil
Open Access


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 (Rubinoff and Holland 2005). Frog mitochondrial DNA (mtDNA) has high sequence evolution rates and many gene arrangements, making it difficult to find conserved regions (Zhang et al. 2013). The low mutation rates in mtDNA may also limit the ability to distinguish related species (Ballard and Whitlock 2004; Nabholz et al. 2008, 2009).

Introns in nuclear protein coding genes have several properties that make them useful for phylogenetic analyses of recently evolved vertebrates (Igea et al. 2010; Schmitz et al. 2017). Because they are flanked by conserved exons, they are easily amplified by polymerase chain reaction (PCR) in a variety of taxa that provide sites for PCR primers (Prychitko and Moore 2003). Introns evolve more slowly than mtDNA (Prychitko and Moore 1997, 2000; Johnson and Clayton 2000) and more rapidly than nuclear exon sequences (Hughes and Yeager 1997; Li 1997).

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 (Zhang et al. 2013; Lourenço et al. 2015; Faivovich et al. 2021; Pereyra et al. 2021). Rapidly evolving noncoding introns are used to resolve problematic nodes at the species, genus, and family levels (Prychitko and Moore 1997; Igea et al. 2010; Folk et al. 2015), and they show more robust and congruent phylogenetic signals than exons (Chen et al. 2017).

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) (Gonçalves et al. 2007; Velo-Antón et al. 2008; Brunes et al. 2010, 2014; Prado et al. 2012; Maia-Carvalho et al. 2014; Menezes et al. 2016, 2020). These results are in contrast with previous studies related to FGBI7 in amphibians. Thus, FGBI7 is a valuable marker for assessing phylogenetic relationships at the family level and is likely suitable for phylogenetic analyses between closely related taxa that have recently diverged (Sequeira et al. 2006; Teixeira et al. 2015).

With 99 taxa, the Neotropical gladiator frogs of Boana Gray, 1825, constitute the third largest genus within Hylinae (Frost 2023). The phenotypically very similar species and lack of reliable diagnostic characters difficult the precise identity of Boana. Studies of cariology, morphology, vocalizations, and molecular characters have revealed cryptic species, new species, and changes in the classification of Boana groups (Caminer and Ron 2014, 2020; Duellman et al. 2016; Fouquet et al. 2016, 2021; Orrico et al. 2017; Ferro et al. 2018; Peloso et al. 2018; Pinheiro et al. 2019a; Lyra et al. 2020; Faivovich et al. 2021).

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.

Materials and methods

Taxonomic sampling and DNA isolation

DNA samples were obtained from captured specimens and donations from herpetological collections (Appendix 1: Table A1). Samples included taxa from most known species groups of Boana (Pinheiro et al. 2019a; Faivovich et al. 2021).

Total DNA extraction from muscle or liver tissue was performed using the SDS -proteinase K/phenol-chloroform extraction method (Sambrook and Rusell 2001). FGBI7 was sequenced on tissues from twenty-four Boana species (ingroup), three taxa of Aplastodiscus, one sample of Bokermannohyla circumdata, one sample of Nesorohyla kanaima, and one of Callimedusa tomopterna (outgroups). Primers 5’-CCATGACAATACACAACGGC-3’ and 5’-ACCACCATCCACCACCATC-3’ were designed based on the sequence of Xenopus laevis (Roberts et al. 1995). After selecting the most conserved regions of FGBI7, the NCBI Primer- BLAST tool was used to design the target-specific primers (Ye et al. 2012). The amplification protocol was based on a 25-µL solution of 0.5–2.0 µL of the DNA template, 2.5 µL of 10× PCR buffer, 0.5 µL of each primer (10 pmol/µL), 0.5–1.5 µL of MgCl2, 1 µL of the dNTPs, and 0.15 µL of Ex Taq DNA polymerase. The PCR protocol included 3 min at 94 °C, 35 (or 30) cycles of 1 min at 94 °C, 1 min at 60 °C (or 59 s and 55 °C), and 1 min at 72 °C, and a final extension at 75 °C for 5 min.

Sequencing and alignment

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 (Katoh and Standley 2013). Alignments were edited using BioEdit (Hall 1999). Exon sequences were then concatenated using Sequence Matrix 1.7.8 (Vaidya et al. 2011) and subjected to various phylogenetic analysis methods using the same parameters as for individual genes. Genes were concatenated, although some sequences within the Boana taxa were not available. Missing bases that corresponded to unsequenced data were marked with a question mark.

To compare polymorphic sites and basic sequence statistics, Boana sequences were analyzed for conserved, variable, parsimony-informative, and singleton sites using MEGA X (Kumar et al. 2018). The number of sites without missing data (Pb*) was calculated for all genes by adding the conserved sites (C-S), singleton sites (S-S), and informative parsimony sites (P-I).

Phylogenetic analysis

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 (Goloboff and Catalano 2016), and phylogenetic trees were constructed using the New Technology Search routine. Parameters selected included sectorial search, ratchet, drift, and tree fusing. A specific search was performed with an initial setting of 100 levels and run 100 times to define the minimum sequence length. Deletions were considered as a fifth base type.

Support for clades was tested using a jackknife procedure with a removal rate of 0.36, which is the most congruent value with bootstrapping (Farris et al. 1996), with absolute frequencies of 50 RAS + TBR per replicate for a total of 1,000 replicates. Consistency indices (CI), retention indices (RI), and rescaled consistency indices (CR) were calculated.

MB analysis of the evolutionary model were performed using MEGA X. For sequences with many gaps, the “use all sites” setting was selected (Tamura et al. 2011). Bayesian and Akaike information criteria were used to select the most appropriate nucleotide substitution model (Posada and Buckley 2004). The MB was run in MrBayes 3.2.7 (Ronquist et al. 2012), and sequences were considered as individual partitions for each model.

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 (Rambaut et al. 2018) and Bayesian probability theory.

The ML analysis was performed with MEGA X software (Kumar et al. 2018) using the best substitution model generated with the same program. Bootstrap support values were used to estimate clade support based on 1,000 replicates. Missing data and gaps were included in the analyses using the “use all sites” commands. Tree inference options included nearest neighbor replacement and initial tree for ML with automatic configuration (default: NJ /BioNJ); system resource use, number of threads 1.

Phylogenetic trees were compared for each marker based on their topology and monophyletic groups defined for Boana (Faivovich et al. 2005, 2021; Pinheiro et al. 2019a). Trees were edited in Inkscape 0.48.5, FigTree V 1.4.4 (Rambaut 2016), and MEGA X.

Mean evolutionary rates of ND1, CYTB, and FGBI7 nuclear genes

Mean evolutionary rates for Boana species were based on mitochondrial and informative genes such as ND1 and CYTB from GenBank (Zhang et al. 2013). For phylogenetic inference ML, the sequences of each gene were submitted to the MEGA X software. Molecular dating for each tree, including the FGBI7 obtained, was performed using the RelTime method (Tamura et al. 2018), a fast and powerful dating algorithm very similar to the results obtained by the Bayesian method (Mello et al. 2017; Mello 2018).

To establish a chronological scale for clade/lineage evolution, the divergence times established by Duellman et al. (2016) were used to calibrate the phylogenetic trees of the mitochondrial genes ND1, CYTB, and FGBI7. The following three divergence times were used for the ND1 tree: I – divergence between Aplastodiscus and Boana, at 34.2 Ma; II – divergence of Boana pulchella group from the other Boana groups, at 22.6 Ma; and III – separation between Boana pellucens and Boana rufitela, at 5.30 Ma. The FGBI7-based phylogenetic tree was calibrated with the same divergence time of 34.2 Ma. The CYTB-based phylogenetic tree was calibrated with the divergence time between Bokermannohyla and Aplastodiscus + Boana of 36.8 Ma.

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) (Tamura et al. 2018). Mean evolutionary rates were calculated based on the absolute rates of all clades of ND1, CYTB, and FGBI7. After setting the calibration conditions, the “use all sites” option was selected to include all gaps and missing data in the branch length calculation (Mello 2018).


FGBI7 DNA sequences

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.

Nuclear DNA (nDNA) contribution to the phylogeny of Boana

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 1).

Table 1.

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

Polymorphic sites

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 1). Although the P-I percentage did not differ between concatenated genes (C-genes) and FGBI7, the percentage of P-I/Pb* parsimony-informative sites was higher for FGBI7. With the exception of 28S, the frequency of A+T was higher than 50% for all genes, with FGBI7 having the highest value.

Phylogenetic hypothesis

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. 1). Analysis of the seven nDNA markers using three different phylogenetic methods led to conflicting hypotheses about the monophyly of Boana. Seven groups of Boana were more frequently classified as monophyletic by the three methods using TYR, FGBI7, and CXCR4. FGBI7 suggests monophyly of B. pellucens group, similar to CXCR4, which also supports polyphyly of B. punctata. All groups examined were polyphyletic for 28S (Table 2).

Figure 1. 

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.

Table 2.

Monophyletic species groups recovered by the three phylogenetic methods for single and concatenated gene phylogeny.

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 3).

Table 3.

Consistency and retention indices of individual and concatenated genes.

CI 0.607 0.802 0.797 0.615 0.745 0.563 0.669 0.635
RI 0.782 0.857 0.791 0.729 0.819 0.655 0.577 0.714
CR 0.475 0.687 0.630 0.448 0.610 0.369 0.386 0.453

Mean evolutionary rate

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. 2).

Figure 2. 

Molecular dating of gene trees A ND1 B CYTB C FGBI7 using the RelTime method in MEGA X.


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 (Faivovich et al. 2013, 2021; Pinheiro et al. 2019a). The phylogeny MP best agreed with these studies, showing B. punctata group as polyphyletic and the groups B. semilineata group, B. albopunctata group, B. pellucens group, B. faber group, and B. pulchella group as monophyletic.

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 Pinheiro et al. (2019a).

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 (Miklós et al. 2004; Granados et al. 2013). Indels are a valuable source of phylogenetic information that can influence the phylogenetic outcome and have less homoplasy than nucleotides (Houde et al. 2019). The phylogenetic utility of indels may vary between individual genes; therefore, the phylogenetic weight of a single indel compared to that of a nucleotide should be explored (Pasko et al. 2011). The number, size, and distribution of indels within a sequence likely reflect the complex phenomena that lead to their accumulation over an evolutionary period and the different approaches used to analyze the available data (Houde et al. 2019).

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 (Edwards et al. 2007).

However, when gene tree and species tree data support a robust and congruent hypothesis (Granados et al. 2013; Ai and Kang 2015), it is possible that species trees can be resolved using only two or three few loci. These results lead to a paradigm for combining data in phylogenomics that focuses on the uniqueness of species histories and ignores the diversity of individual gene histories.

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). Tonini et al. (2015) concluded that phylogeneticists should continue to make explicit comparisons between the results of modern and classical methods.

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 (Phillips et al. 2004).

Congruence was observed not only at the tips of trees but also at deeper inner branches. Chen et al. (2003) proposed a reliable alternative strategy in which only one bootstrap value is considered as the threshold for clade significance. In this alternative strategy, the same clade repeatedly derived from different data sets is accepted even at low bootstrap values, rather than a strongly supported clade derived from a single data set. Congruence analysis reveals different evolutionary signals in the underlying collection of genes and allows for a more conservative interpretation of phylogenomic signals (Thiergart et al. 2014). The use of FGBI7 proved to be a complementary technique for resolving the Boana phylogeny. By confirming specific clades within the Boana phylogeny, the use of integrated traits may be better suited to elucidate the history of a clade (Salichos and Rokas 2013).

The congruence between the topology resulting from the use of FGBI7 in this study and the results reported by Pinheiro et al. (2019a) and Faivovich et al. (2021) may be due to the design of a specific primer for FGBI7 analysis. Other studies have also shown satisfactory results with the use of FGBI7-specific primers for different study groups (Sequeira et al. 2006; Teixeira et al. 2015). Designing PCR primers to screen primers against a user-selected database avoids nonspecific amplification and highlights a variable sequence of the marker to establish relationships. Although the design of specific primers is time consuming (Small et al. 2004), it has significant advantages over universal primers, particularly in terms of gene amplification, sequence quality and variation, and searching for a phylogenetic signal (Cai and Ma 2016).

Intraspecific variability in body color, description of new species, and research on declining taxa of the Hylidae (AmphibiaWeb 2021; IUCN 2021) are challenges for future studies that should be addressed using integrative trait taxonomy (Hillis 2019; Pinheiro et al. 2019b). Despite the short sequence observed in the FGBI7-based analysis, this is a versatile gene that can be used to address a variety of phylogenetic and taxonomic questions. The elucidation of taxa thought to be geographically widespread that are in fact cryptic species, such as Boana (Estupiñán et al. 2016; Fouquet et al. 2016; Caminer et al. 2017; Orrico et al. 2017; Cryer et al. 2019), and the agreement with previously proposed phylogenetic hypotheses supported by the informative sites for parsimony demonstrate the high performance of FGBI7.

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 (Zheng et al. 2011; Near et al. 2012; Stöck et al. 2012). The mtDNA and nDNA evolution rates in this study were similar to those estimated by Ehl et al. (2019). These authors estimated evolution rates from E-2 to E-3 for mtDNA and from E-3 to E-4 for nDNA. However, the amplitude of evolutionary rates was lower for FGBI7 compared with mtDNA.

The divergence dates of the FGBI7 data were close to those obtained by Fouquet et al. (2021) for B. punctata group (B. cinerascens + B. punctata) (x̄ = 9.66 ± 0.76 Ma) and B. albopunctata (x̄ = 16.8 ± 0.70 Ma). Divergence times for B. fasciata were determined by Duellman et al. (2016), Feng et al. (2017), and Fouquet et al. (2021). Although Feng et al. (2017) criticized the geological dating method used by Duellman et al. (2016) and instead calibrated their dating data with fossil records, their results differed significantly from those of Fouquet et al. (2021), who used the same calibration method. Duellman et al. (2016) and Fouquet et al. (2021) found mean divergence times of 4.71 ± 1.06 Ma for B. fasciata, while those for CYTB were overestimated by 13.22 Ma.

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 (Feng et al. 2017). Using a threshold of 62 Ma and based on nDNA and mtDNA data, Duellman et al. (2016) estimated the origin of Boana to be 34 Ma, with a mean diversification rate within their groups of 19.1 ± 4.6 Ma. This estimate is close to the divergence time calculated by Fouquet et al. (2021) for B. albopunctata (17.3 Ma) and by FGBI7 in the present study.

Recent divergence times inferred from mtDNA sequences tend to overestimate times for basal clades (Maddin et al. 2012). On the other hand, estimates of divergence times for more recent nodes based on nuclear loci are inaccurate because significantly fewer mutations have accumulated between comparatively young lineages (Wilke et al. 2009). Another way to address this divergence is to compare these results with additional evidence. The considerable amplitude between the appearance of Boana is estimated to be 30 Ma and the onset of divergence of the Amazonian clade of B. albopunctata group is estimated to be 10 Ma. This limits our understanding of divergence times using only a single type of molecular marker. We propose that FGBI7 should be used for anuran clades that originated between 30 and 70 Ma, while mtDNA should be used for clades that originated between 25 and 30 Ma and diverged until recently (< 2 Ma). Thus, it is possible to construct an nDNA-based time tree for a reduced set of taxa representing all genera, reconstruct different lineage-level time trees using mtDNA data, and compare the performance of the different approaches (Ehl et al. 2019).

The use of FGBI7 in this study showed that, unlike other nuclear genes already used to generate phylogenetic hypotheses of Anura (e.g.: Wiens et al. 2010; Pyron and Wiens 2011; Duellman et al. 2016) or in the phylogenies of Boana and some of their groups (Faivovich et al. 2013, 2021; Caminer and Ron 2014; Pinheiro et al. 2019a; Lyra et al. 2020), has great potential to reveal relationships between lineages of very close clades.

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 Fouquet et al. (2021), Vasconcellos et al. (2021), and Rainha et al. (2021), contributing to future studies on Boana evolution and systematics.

Although estimating divergence times for clades is a difficult task (Mello 2018), the approach proposed in this study estimated the average evolutionary rate for Boana using two mitochondrial genes and FGBI7. Therefore, we recommend the use of FGBI7 for the analysis of clades such as Boana with temporal ranges between 30 and 70 Ma and the use of the mtDNA genes for lineages with thresholds from the origin of Boana, between 25 and 30 Ma, to recent times (< 2 Ma).


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).


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Appendix 1

Table A1.

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
Boana aguilari MT824211 MT824337 KF751464 Faivovich et al. (2013, 2021)
Boana albomarginata KF751476 OQ448590 AY844384 AY844568 AY844794 AY844218 MRT 5870: Jussara, Bahia, Brazil. Faivovich et al. (2005, 2013)
Boana albopunctata OQ448612 AY844569 AY844795 AY844041 MRT 8229, Petrolina, Goiás, Brazil. Faivovich et al. (2005)
Boana balzani MT824213 OQ448599 AY844395 AY844582 AY844806 AY844226 MNCN/ADN 5785, Camino a San Onofre, Carrasco, Cochabamba, Bolivia. Faivovich et al. (2005, 2021)
Boana benitezi KF751477 AY844396 AY844583 AY844227 Faivovich et al. (2005, 2013)
Boana bischoffi MT824219 OQ448607 AY844398 MT824343 MT824526 AF 327, Fazenda Intervales, Estado de São Paulo. Faivovich et al. (2005, 2021)
Boana boans KF751478 OQ448591 AY844588 AY844809 AY844055 AY844231 MPEG 17385, Arredores da Fazenda passo Formoso, Manicoré, Amazonas. Faivovich et al. (2005, 2013)
Boana botumirim MT824344 Faivovich et al. (2021)
Boana buriti OQ448601 MT824346 MT824484 CHUNB 30653, Brasilia, Distrito Federal. Faivovich et al. (2021)
Boana caingua KF751479 OQ448602 MT824352 AY844812 AY844057 AY844234 AF 515, Ribeirão Grande São Paulo, São Paulo. Faivovich et al. (2005, 2013, 2021)
Boana calcarata AY844235 Faivovich et al. (2005)
Boana cambui MT824356 MT824486 MT824534 Faivovich et al. (2021)
Boana cinerascens KF751480 OQ448595 AY844610 DQ283466 RAET 505, Estação Científica Ferreira Penna, Melgaço, Pará, Brazil. Faivovich et al. (2005, 2013)
Boana cipoensis MT824357 MT824487 Faivovich et al. (2021)
Boana cordobae KF751481 AY844411 MT824460 MT824516 AY844066 AY844244 Faivovich et al. (2005, 2013, 2021)
Boana crepitans KF751482 AY844601 AY844067 Faivovich et al. (2005, 2013)
Boana curupi MT824227 MT824359 MT824489 Faivovich et al. (2021)
Boana ericae AY844416 AY844605 MT824537 Faivovich et al. (2005, 2021)
Boana faber AY844607 Faivovich et al. (2005)
Boana fasciata KX200378 AY844608 Faivovich et al. (2005); Feng et al. (2017)
Boana freicanecae MT824217 MT824366 MT824538 Faivovich et al. (2021)
Boana geographica OQ448611 QCAZ 16809: Estación Científica Yasuní. PUCE, Laguna, Orellana, Ecuador. in confirmation process
Boana gladiator MT824212 MT824368 Faivovich et al. (2021)
Boana goiana MT824372 MT824491 MT824541 Faivovich et al. (2021)
Boana guentheri MT824245 OQ448608 MT824373 MT824492 AY844253 CFBH 3386: Terra de Areia, Rio Grande do Sul, Brazil. Faivovich et al. (2005, 2021)
Boana heilprini AY844613 Faivovich et al. (2005)
Boana jaguariaivensis MT824374 MT824494 Faivovich et al. (2021)
Boana joaquini KF751484 OQ448605 AY844421 MT824376 AY844256 CFBH 1068: Urubici, Santa Catarina, Brazil. Faivovich et al. (2005, 2013, 2021)
Boana lanciformis AY844619 AY844081 AY844258 Faivovich et al. 2005
Boana lemai KF751485 AY844423 AY844620 AY844082 AY844259 Faivovich et al. (2005, 2013)
Boana leptolineata MT824246 OQ448604 AY844424 AY844621 AY844839 AY844083 AY844260 CFBH 8504: São Francisco de Paula, Rio Grande do Sul, Brazil. Faivovich et al. (2005, 2021)
Boana lundii AY844623 AY844085 AY844262 Faivovich et al. (2005)
Boana marginata KF751486 AY844426 AY844624 MT824542 AY844263 Faivovich et al. (2005, 2013, 2021)
Boana marianitae OQ448610 AY844427 MT824378 AY844843 MNCN/ADN 5901: Camino a Bella Vista, Florida, Santa Cruz, Bolivia. Faivovich et al. (2005, 2021)
Boana melanopleura KF751487 OQ448600 MT824379 HM444787 MTD-TD 1146: Huancabamba, Pasco, Peru. Faivovich et al. (2013, 2021)
Boana multifasciata GQ365986 AY844436 AY844633 AY844093 AY844270 Faivovich et al. (2005, 2010)
Boana nympha KF751488 AY844661 AY844112 AY844289 Faivovich et al. (2005, 2013)
Boana pardalis AY844637 Faivovich et al. (2005)
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 Faivovich et al. (2021)
Boana polytaenia MT824241 OQ448606 AY844443 MT824429 MT824508 MT824547 CFBH 8394: Cristina, MG. Faivovich et al. (2005, 2021)
Boana pombali MT824247 MT824431 MT824511 MT824552 Faivovich et al. (2021)
Boana prasina MT824436 MT824554 Faivovich et al. (2021)
Boana pulchella OQ448609 AY844445 MT824443 MT824513 MT824557 AY844278 CHUNB 37686: Pilar do Sul, Estado de São Paulo, Brazil. Faivovich et al. (2005, 2021)
Boana punctata OQ448596 AY844645 QCAZ18185: Estación Biológica Jatun Sacha, Napo. Faivovich et al. (2005)
Boana raniceps KF751489 OQ448613 AY844646 AY844863 AY844103 MRT 6706: UHE Lajeado Tocantins. Faivovich et al. (2005, 2013)
Boana riojana MT824238 AY844447 MT824462 MT824518 AY844279 Faivovich et al. (2005, 2021)
Boana roraima KF751490 AY844448 AY844650 AY844104 AY844280 Faivovich et al. (2005, 2013)
Boana rufitela OQ448598 AY844652 AY844867 AY844105 AY844282 CHP-STRI:5114: Quebrada Guabalito, Palmarazo, Parque Nacional General de División Omar Torrijos Herrera, Provincia de Coclé. Faivovich et al. (2005)
Boana semiguttata OQ448603 AY844452 MT824466 MT824519 MT824559 AY844285 CFBH 242: Piraquara, Paraná, Brazil. Faivovich et al. (2005, 2021)
Boana semilineata KF751491 AY844453 AY844656 AY844108 AY844286 Faivovich et al. (2005, 2013)
Boana sibleszi KF751492 OQ448593 AY844455 AY844658 AY844873 AY844110 AY844288 ROM 39561: Mount Ayanganna, Guyana. Faivovich et al. (2005, 2013)
Boana stellae MT824229 MT824475 MT824567 Faivovich et al. (2021)
Boana stenocephala MT824479 MT824520 MT824563 Faivovich et al. (2021)
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. Berneck et al. (2016)
Aplastodiscus albosignatus OQ448616 AY844385 KU184114 AY844796 AY844042 AY844219 CFBH 7711: Parque Estadual Serra do Mar, Santa Virginia, São Luís do Paraitinga, São Paulo, Brazil. Faivovich et al. (2005)
Aplastodiscus eugenioi KF751465 Faivovich et al. (2013)
Aplastodiscus leucopygius KF751466 AY844261 Faivovich et al. (2005; 2013)
Aplastodiscus perviridis KF751467 AY844201 Faivovich et al. (2005, 2013)
Aplastodiscus weygoldti OQ448615 AY844467 KU184124 AY844887 KU184257 AF 68: São Paulo do Aracã, Espírito Santo, Brazil. Faivovich et al. (2005)
Bokermannohyla circumdata KF751468 OQ448619 AY844409 AY844817 AY844064 AY844242 IT-H0562, MZUSP 93551: Juquitiba, Estado de São Paulo. Berneck et al. (2016); Faivovich et al. (2005; 2013)
Callimedusa tomopterna GQ366024 OQ448618 AY844497 AY844715 AY844157 AY844328 MPEG 17368, Near Fazenda Passo Formoso, Manicoré, Amazonas, Brazil. Faivovich et al. (2010, 2005)
Callimedusa vaillanti AY844921 Faivovich et al. (2005)
Myersiohyla liliae MH251236 Pinheiro et al. (2019a)
Myersiohyla inparquesi AY844291 Faivovich et al. (2005)
Nesorohyla kanaima GQ365994 OQ448617 AY844617 MH251240 AY844079 ROM 39586: Mount Ayanganna, Guyana. Faivovich et al. (2005, 2010); Pinheiro et al. (2019a)

Supplementary material

Supplementary material 1 

Phylogenetic trees and molecular dating of gene trees

Ruth Amanda Estupiñán, Sávio Torres de Farias, Evonnildo Costa Gonçalves, Mauricio Camargo, Maria Paula Cruz Schneider

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.

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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