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

Taina Barbosa de Souza1, Daniela Cristina Ferreira2 , Hugmar Pains da Silva3, André Luiz Netto-Ferreira4 and Paulo Cesar Venere2

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The family Lebiasinidae includes a number of miniature and medium-sized fish species that are endemic to the Neotropical region. Pyrrhulina is the second most speciose lebiasinid genus and it is also the one with the most taxonomic uncertainties. In this context, the present study focused on the Pyrrhulina morphospecies found in a number of different drainage basins in South America to test the alternative proposals on the arrangement of the taxonomic units found within what is assumed to be a single nominal species, Pyrrhulina australis, based on a DNA Barcoding approach. The results of the analyses indicate that Pyrrhulina australis is a species complex, with intraspecific (within-group) genetic distances of up to 3.74%, well above the Optimal Threshold of 1.79% defined in the present study. The species delimitation analyses revealed a surprising level of diversification among the morphospecies evaluated, in particular, in the clade that encompasses Pyrrhulina australis (from the Paraguay River) + P. cf. rachoviana (Lower Paraná River), P. aff. australis I (Araguaia River)/II (Paraguay River)/III (Upper Paraná River)/IV(Guaporé River),and P. marilynae (Teles Pires River), which were arranged in six distinct evolutionary lineages that align with the geographical distribution of the respective drainage basins.

Keywords: Cryptic species, Freshwater fish, Molecular identification, Small fishes, Species delimitation methods.


A família Lebiasinidae é endêmica da região Neotropical, composta por espécies de peixes em miniaturas e de médio porte. O gênero Pyrrhulina é o segundo maior em número de espécies e o grupo com mais incertezas taxonômicas dentre os representantes dessa família. Diante disso, o objetivo deste estudo foi avaliar morfoespécies provenientes de diferentes bacias de drenagens, a fim de testar as diferentes propostas de reconhecimento de unidades taxonômicas dentro do que se acreditava ser a espécie nominal Pyrrhulina australis, utilizando o princípio do DNA Barcoding. Os resultados mostraram que Pyrrhulina australis é um complexo de espécies, com distância intraespecífica dentro do grupo de 3,74%, valor acima do Threshold Ótimo de 1,79% definido para este estudo. As análises de delimitação de espécies indicam uma surpreendente diversidade dentro das morfoespécies analisadas, especialmente no clado que engloba Pyrrhulina australis (rio Paraguai) + P. cf. rachoviana (baixo rio Paraná), P. aff. australis I (rio Araguaia), II (rio Paraguai), III (alto rio Paraná), IV(rio Guaporé)e P. marilynae (rio Teles Pires),que apresentou seis linhagens evolutivas que podem estar relacionadas ao padrão de distribuição pelas bacias de drenagens.

Palavras-chave: Delimitação de espécies, Espécies crípticas, Identificação molecular, Peixes de água doce, Peixes de pequeno porte.


The Lebiasinidae (Characiformes) is endemic to the Neotropical region, and is composed of approximately 75 valid species (Van der Laan, Fricke, 2023). The lebiasinids are miniature to medium-sized fish that have fusiform bodies and are known popularly as pencilfish or, in Brazil, as “pirrulinas” or “charutinhos” (Géry, 1977; Malabarba, Malabarba, 2020). Based on a molecular analysis, Melo et al. (2022) estimated that the family experienced a notable diversification event during the Cenozoic era, approximately 30 million years ago, which gave rise to the present-day lineages. The six valid genera in two subfamilies, Lebiasininae and the Pyrrhulininae, with the latter being the more speciose, comprising 47 species distributed among four genera (Weitzman, Weitzman, 2003; Netto-Ferreira et al., 2011; Van der Laan, Fricke, 2023): Nannostomus Günther, 1872(20 species), Copella Myers, 1956 (six species), Copeina Fowler, 1906(two species), and Pyrrhulina Valenciennes,1847 (19 species).

The 19 valid species (Fricke et al., 2023) of Pyrrhulina are distributed in the Amazon and Araguaia-Tocantins basins, as well as in the Paraguay-Paraná-La Plata hydrographic network, and the Laguna dos Patos and Tramandaí River systems in southern South America (Weitzman, Weitzman, 2003; Venere, Garutti, 2011; Bertaco et al., 2016; Dagosta, de Pinna, 2019). The genus houses most of the current taxonomic uncertainties within the Lebisinidae (Netto-Ferreira, Marinho, 2013), including a range of inconsistencies in the descriptions and diagnosis of the species of this group (Géry, 1977), which are difficult to distinguish on the basis of their morphological traits. This has led to the incorrect identification of many species in the past (Vieira, Netto-Ferreira, 2019).

In the first study based on genomic data, Ferreira et al. (2022) were able to determine the phylogenetic relationships among Pyrrhulina australis Eigenmann & Kennedy, 1903, P. marilynae Netto-Ferreira & Marinho, 2013, P. obermulleri Myers, 1926, P. brevis Steindachner, 1876, and P. semifasciata Steindachner, 1876, with a high level of genetic divergence among the species, which correlated with the geographic distribution of the taxa among the respective drainage basins. This was an important initial step toward understanding of the phylogenetic relationships among the species of the group, given the general lack of data available for species of Pyrrhulina. Although Pyrrhulina was under-represented in the aforementioned study, the close relationship between P. australis and P. marilynae obtained by the authors agree with the relationships proposed by Netto-Ferreira, Marinho (2013), based on morphological data, placing them within the “Pyrrhulina australis group” with P. rachoviana Myers, 1926 and P. vittata Regan, 1912. Except for P. vittata, the taxonomic limits among these species have been the subject of considerable debate by systematic biologists, and represent a major challenge for the definition of the taxonomy of the group. Pyrrhulina australis isa broadly-distributed species that is found between Argentina and French Guiana, in the Amazon (including the Araguaia-Tocantins system) and Paraguay-Paraná-La Plata basins, and adjacent coastal drainages (Weitzman, Weitzman, 2003). The species was reviewed by Zarske, Géry (2004), who synonymized P. macrolepis Ahl & Schindler, 1937 and P. rachoviana with P. australis. Netto-Ferreira, Marinho (2013) considered the synonymization of P. rachoviana to be erroneous. However, Zarske (2016) subsequentially confirmed the validity of P. rachoviana, and argued that P. marilynae would be a synonym of P. rachoviana instead. That conclusion was based on Zarske’s hypothesis that the type locality of P. rachoviana to be in the Amazon basin, contradicting Myers (1926) , who suggested the type specimens of the species as originating from Rosário, Argentina.

The precise identification of species becomes imperative and challenging, especially when dealing with the vast diversity of neotropical ichthyofauna, estimated at around 9,000 fish species (Reis et al., 2016). However, despite this extensive diversity, many natural stocks are facing local or global extinction processes before even being known to science (Manel et al., 2020). In this context, molecular tools such as DNA barcoding can provide valuable insights for the resolution of complex taxonomic questions, in particular, the delimitation of species that are difficult to diagnose based solely on their morphological traits, and this approach has been widely used in phylogenetic studies of an enormous diversity of organisms (see e.g., Hebert et al., 2003; Pereira et al., 2013; Díaz et al., 2016; Machado et al., 2017; Costa-Silva et al., 2018; Arruda et al., 2019; Ramirez et al., 2020).

Given this scenario, the present study aimed to delimitate the species of the Pyrrhulina australis group, sensu Netto-Ferreira, Marinho (2013), based on the application of DNA barcoding. In addition, the present data allowed to objectively evaluate the different taxonomic proposals for the Pyrrhulina australis group – i.e., a single species (Zarske, Géry, 2004), two species (Zarske, 2016) or three (Netto-Ferreira, Marinho, 2013) – and to test the validity of the species that make up the group proposed by Netto-Ferreira, Marinho (2013), based on the morphological evidence.

Material and methods

Study area and sample collection. The samples of the nominal species of Pyrrhulina australis, P. marilynae,and P. obermulleri were collected in the different drainage basins (Paraguay, Paraná, Araguaia-Tocantins, Tapajós, Guaporé, Madeira, and Maroni) in South America (Fig. 1). Additional sequences were obtained from GenBank and the BOLD systems database (Tab. 1). Samples of P. obermulleri, P. spilota Weitzman, 1960, and P. filamentosa Valenciennes, 1847 (Tab. 1) were included as outgroups in relation to the P. australis group. The species Nannostomus marginatus Eigenmann, 1909 (BOLD registration numbers GBMND24648-21, GBMND24649-21), was included as an outgroup to root the tree in the analysis performed with MrBayes. The species were identified based on the morphological traits (Fig. 2) described by Zarske, Géry (2004) and Netto-Ferreira, Marinho (2013) or the probable distribution of the taxa, estimated from the type locality, in the case of P. rachoviana (Weitzman, Weitzman, 2003).

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

TABLE 1 | Voucher specimen information of the Pyrrhulina specimens analyzed in the present study, including the collecting locality, geographic coordinates, and BOLD accession numbers. The sequences of the specimens marked with an asterisk were obtained from the BOLD. Brazilian states: MT = Mato Grosso; PR = Paraná; RO = Rondônia; SP = São Paulo.

Species – consensus MOTU



Number of the sampled specimens



Sequence id.

Voucher CPUFMT

BOLD systems accession number

Pyrrhulina aff. australis IV – MOTU 6

Pontes e Lacerda, MT, Brazil

Amazon (Guaporé)



















Pyrrhulina obermulleriMOTU 7

Porto Velho, RO, Brazil

Amazon (Madeira)


































Pyrrhulina marilynaeMOTU 3

Sorriso, MT, Brazil

(Teles Pires)







Pyrrhulina aff. australis IMOTU 2

Barra do Garças, MT, Brazil

Araguaia, Tocantins (Araguaia)



















Pyrrhulina australis– MOTU 1

Poconé, MT, Brazil

(Bento Gomes)















Pyrrhulina cf. rachoviana*- MOTU 1

Victoria, Argentina

Lower Paraná (Paraná)








Pyrrhulina aff. australis III*- MOTU  5

Cravinhos, SP, Brazil

 Upper Paraná (Pardo)








Pyrrhulina aff. australis III*- MOTU  5

Marilena, PR, Brazil

Upper Paraná (Paranapanema)










Pyrrhulina filamentosa*- MOTU 9

Sipaliwini, Suriname

Maroni (Paloemeu)










Pyrrhulina spilota*- MOTU 8

Puchana, Peru

Amazon (Mómon)









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 wereobtained from the BOLD systems database, and no physical specimens were collected in the field.

The voucher specimens were deposited in the ichthyological collection of the Universidade Federal do Mato Grosso, Cuiabá (CPUFMT 7757–7762), and the tissue samples are stored in the Laboratório de Genética e Citogenética Animal (LABGEN) of the Instituto de Biociências of the UFMT. The gene sequences were deposited in the BOLD systems database, under the accession numbers shown in Tab. 1.

Extraction, amplification, and sequencing of the DNA. The DNA was extracted using the saline extraction protocol of Aljanabi, Martinez (1997). The COI gene was amplified using the primers COI FISH F1 and FISH R1 described by Ward et al. (2005). The reagents and cycling conditions were the same as those described by Arruda et al. (2019).

The amplicons of the COI gene were purified and sequenced by Biotecnologia Pesquisa e Inovação (BPI, https://bpibiotecnologia.com.br). The samples were purified using a magnetic bead kit of the AMpure XP type, and sequenced with a BigDye® Terminator v. 3.1 Cycle Sequencing kit (Applied Biosystems), following the manufacturer’s protocols. The samples were sequenced automatically by capillary electrophoresis in an ABI3730xl Genetic Analyzer (Applied Biosystems).

Data analysis. The raw sequences were edited and the presence of gaps was verified in Geneious® 7.1.3 (Kearse et al., 2012) while the sequences were aligned in Mega v. 11 (Tamura et al., 2021) using the ClustalW algorithm (Thompson et al., 2003). The aligned sequences were inspected in MEGA for the identification of stop codons, pseudogenes, and deletions and insertions. The sequences were tested in DAMBE7 (Xia et al., 2003) to determine the saturation of nucleotide substitutions.

The mean intraspecific and interspecific genetic distances between the morphospecies and the consensus MOTUs were calculated in Mega v. 11 using the Kimura-2-Parameter (K2P) model (Kimura, 1980), following Hebert et al. (2004), who employed the minimum interspecific and the maximum intraspecific distances identified in Excel, using the Minimum and Maximum functions. The Molecular Operational Taxonomic Units (MOTUs) were identified in the JMotu software (Jones et al., 2011) based on the Optimum Threshold (OT). The OT was calculated in the SPIDER (SPecies IDentify and Evolutions in R, Brown et al., 2012) package using the “LocalMinima” function in the R environment v. 3.6.3 (https://www.r-project.org; R Development Core Team, 2016). The groups formed by the Assemble Species by Automatic Partitioning software (ASAP, Puillandre et al., 2021) were estimated at the site https://bioinfo.mnhn.fr/abi/public/asap/asapweb.html using the K2P substitution model, with all other parameters at the default values. The partition was selected based on the second-highest significant score that was closest to that of the OT generated by SPIDER.

The best evolutionary model (HKY+G) for the coalescence analyses was identified in JModeltest2 v. 2.1.6, implemented on the CIPRES platform (Miller et al., 2010). The Maximum Likelihood Poisson Tree Processes (PTP) analysis was based on a Bayesian ultrametric tree generated in MrBayes (version available at CIPRES), using the HKY substitution model, gamma rated, Markov Chain Monte Carlo (MCMC) of 10,000,000 generations, sump burn-in 9.001, with Nannostomus marginatus, as the outgroup.

This non-ultrametric tree was used in the analysis of the PTP model at the site https://species.h-its.org/ (Zhang et al., 2013), with the following parameters: rooted, 40,000 MCMC generations, with the outgroup omitted from the analysis. All other configurations were the default values. The Bayesian ultrametric input tree used for the Generalized Mixed Yule Coalescence (GMYC) method was constructed in BEAST2 (version available at CIPRES) with the following parameters: HKY+G model, gamma shape, relaxed molecular clock with lognormal distribution, and the birth-death speciation model, which was run three times independently, initiated using random trees, each with 50 million generations, based on the Markov Chain Monte Carlo (MCMC), with 25% of the topologies being discarded as burn-in during each run. The results of the three runs were combined in LogCombiner v. 2. Effective Sample Size (ESS > 200) was verified in Tracer v. 1.6. The three files with the “.tree” extension were combined in Treeannotator v. 1.8 (available at CIPRES), visualized in FigTree v. 1.4, and exported with a NEWICK final extension. This final tree was used in the GMYC analysis, which was run in the SPLITs (SPecies LImits by Threshold Statistics; Monaghan et al., 2009) package in the R environment v. 3.6.3, using a single threshold model.

The definition of Consensus MOTUs was established by assessing the congruence among the previously mentioned delimitation methods, specifically through the agreement between two or more methods. However, in groups where the congruence between the analyses was not observed, we opted to employ the Optimal Threshold in the formation.


A total of 46 COI sequences were obtained for analysis, including 34 sequences obtained for the present study, and 12 retrieved from the BOLD systems database. Following edition and alignment, the sequences were 628 base pairs long, of which 127 were variable sites. No insertions, deletions or stop codons were observed, which indicate that the fragments used in the analysis were of adequate quality. The Index of nucleotide substitution (Iss) also indicated the lack of base saturation, given that the observed value (R² = 0.1168) was lower than Iss.c (R² = 0.7358), which also supported the quality of the data for the species delimitation analyses.

The Bayesian Inference analysis grouped all the representatives of the Pyrrhulina australis species group in a monophyletic clade, sister to ((P. filamentosa, P. spilota) P. obermulleri)). The integrated analysis based on the different species delimitation approaches recognized six, well-supported (> 95%) monophyletic lineages within the P. australis species group (Fig. 3), with a mean intraspecific distance within the nominal species of 3.74%. These findings indicate that Pyrrhulina australis is a species complex with five independent lineages: MOTU 1 – P. australis + P. cf. rachoviana (Lower Paraná and Paraguay rivers); MOTU 2 – Pyrrhulina aff. australis I (Araguaia River); MOTU 4 – P. aff. australis II (Paraguay River); MOTU 5 – P. aff. australis III (Upper Paraná River), and MOTU 6 – Pyrrhulina aff. australis IV (Guaporé River). The other MOTUs (Fig. 3) correspond to Pyrrhulina marilynae (MOTU 3 – Teles Pires River), P. obermulleri (MOTU 7 – Madeira River), P. spilota (MOTU 8 – Mómon River), and P. filamentosa (MOTU 9 – Paloemeu River).

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.

In the genetic distance analyses, the OT approach indicated the existence of seven MOTUs, whereas the ASAP indicated nine. The OT was 0.0179 (1.79%), and grouped all the individuals identified a priori as P. australis (Paraguay and Upper Paraná basins), P. cf. rachoviana (Lower Paraná), and the P. marilynae morphospecies(Teles Pires River), except for specimen 99, which was collected from the Paraguay basin and identified as P. australis. In this analysis, MOTUs 2 (P. aff. australis I from the Araguaia River) and 6 (P. aff. australis IV from the Guaporé River) were distinct from the P. australis group. The ASAP, in turn, defined nine MOTUs, subdividing P. australis into six MOTUs: P. australis + P. cf. rachoviana (Lower Paraná and Paraguay rivers); P aff. australis I (Araguaia River); P. marilynae (Teles Pires River); P. aff. australis II (Paraguay River, individual 102), P. aff. australis III (Parapanema and Pardo rivers), and P. aff. australis IV (Guaporé River).

The results of the coalescence analyses and the PTP revealed the presence of 11 MOTUs, with high levels of support for each of the groups. Individuals 81 and 88 (P. obermulleri) were assigned to the same MOTU, separate from the other individual of this species. The analysis also divided P. australis into seven MOTUs, as in the arrangement of the ASAP, albeit with three MOTUs for the individuals representing P. aff. australis II (Paraguay River), P. aff. australis III (Upper Paraná and Pardo rivers), and P. aff. australis III (Upper Paraná and Paranapanema rivers).

The arrangement defined by the GMYC analysis based on the Maximum Likelihood model (L = 322.998) was significantly (p = 0.005) different from the null model (L0 = 314.3342). While the arrangement defined by the GMYC was very similar to the configuration of the MOTUs established by the PTP, in this analysis, the nominal species P. obermulleri forms only a single MOTU. These analyses indicate the existence of 10 distinct entities, with a confidence interval of 10–12. However, eight of these entities are groups formed by more than one sequence, while the other two – P. marilynae (MOTU 3, Teles Pires River) and P. aff. australis II (MOTU 4, Paraguay River) – are singletons.

The mean intra- and interspecific genetic distances between the nominal species and the consensus MOTUs are shown in Tab. 2. The maximum intraspecific and minimum interspecific distances between the consensus MOTUs and the nominal species are shown in Tab. 3. These results are presented graphically in the plots of the genetic distances between the nominal species (Fig. 4) and the consensus MOTUs (Fig. 5).

TABLE 2 | Mean K2P interspecific genetic distances obtained for the Pyrrhulina morphospecies or consensus MOTUs identified in the present study. The values in bold script in the diagonal are the mean within-group genetic distances (%).











1. Pyrrhulina australis





2. Pyrrhulina spilota 





3. Pyrrhulina obermulleri





4. Pyrrhulina filamentosa





Consensus MOTU










1. Pyrrhulina aff. australis IV (MOTU 6, Guaporé River)










2. Pyrrhulina spilota (MOTU 8, Mómon River)










3. Pyrrhulina obermulleri (MOTU 7, Madeira River)










4. Pyrrhulina australis + Pyrrhulina cf. rachoviana

(MOTU 1, Paraguay and Lower Paraná)










5. Pyrrhulina aff. australis II (MOTU 4, Bento Gomes River)










6. Pyrrhulina aff. australis I (MOTU 2, Araguaia River)










7. Pyrrhulina australis III (MOTU 5, Upper Paraná)










8. Pyrrhulina marilynae (MOTU 3, Teles Pires River)










9. Pyrrhulina filamentosa (MOTU 9, Paloemeu River)











TABLE 3 | Maximum intraspecific and minimum interspecific K2P genetic distances between the Pyrrhulina morphospecies and consensus MOTUs identified in the present study (%).


Maximum intraspecific distance (%)

Minimum interspecific distance (%)

Pyrrhulina australis



Pyrrhulina obermulleri



Pyrrhulina spilota 



Pyrrhulina filamentosa



Consensus MOTU



Pyrrhulina aff. australis IV (MOTU 6, Guaporé River)



Pyrrhulina spilota (MOTU 8, Mómon River)



Pyrrhulina obermulleri (MOTU 7, Madeira River)



Pyrrhulina australis + Pyrrhulina cf. rachoviana (MOTU 1, Paraguay and Lower Paraná)



Pyrrhulina aff. australis I (MOTU 2, Araguaia River)



Pyrrhulina aff. australis III (MOTU 5, Upper Paraná)



Pyrrhulina. aff. australis II (MOTU 4, Paraguai)



Pyrrhulina marilynae (MOTU 3, Teles Pires River)



Pyrrhulina filamentosa (MOTU 9, Paloemeu River)




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| 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.

The mean intraspecific genetic distance within the Pyrrhulina australis speciescomplex is 3.74%, which is higher than the threshold of 1.79% (Tab. 2), with a maximum value of 7.32% (Tab. 3). A mean intraspecific distance of 1.09% (Tab. 2) was recorded in MOTU 5 (P. aff. australis III, Upper Paraná), with a maximum distance of 1.94% (Tab. 3). Additionally, the lowest minimum interspecific distance of 0.48% (Tab. 3) was found in MOTU 4 (P. aff. australis I, Paraguay) and MOTU 5. No significant divergence was found within the other groups of morphospecies and MOTUs.

The greatest mean distance between nominal species was 15.01%, recorded between P. filamentosa and P. spilota. The lowest mean interspecific distance was 8.95%, recorded between the P. australis groupand P. spilota (Tab. 2). The lowest mean inter-MOTU genetic distance was 0.96%, observed between P. marilynae (MOTU 3) and P. aff. australis II(MOTU 4, Paraguay River; Tab. 2). The greatest interspecific distance between nominal species (10.40%) was observed in P. filamentosa, while the lowest value (7.64%) was recorded in P. australis and P. obermulleri (Tab. 3).

When the minimum interspecific and maximum intraspecific distances are compared among the morphospecies (Fig. 4), P. australis (sensu lato) is the only nominal species in quadrant II, which means that both distances are above the threshold of 1.79%, indicating the presence of cryptic species in this group. By contrast, MOTU 3 (P.marilynae, Teles Pires River) falls in quadrant III (Fig. 5), where both inter- and intraspecific distances are lower than 1.79%, which indicates that the species may have diverged recently. Similar conclusions apply to MOTUs 1 (P. australis + P. cf. rachoviana, Paraguay and Lower Paraná basins) and 2 (P. aff. australis, Paraguay River), which are also located in quadrant III (Fig. 5). Only MOTU 5 (P. aff. australis IV – Upper Paraná basin) is found in quadrant IV, which implies that the morphological identification does not correspond to the molecular identification. Five MOTUs – 2 (P. aff. australis I, Araguaia River), 6 (P. aff. australis IV, Guaporé River), 7 (P.obermulleri, Madeira River), 8 (P. spilota, Mómon River), and 9 (P. filamentosa, Paloemeu River) – are located in quadrant I (Fig. 5). This indicates that the morphological and molecular analyses produced the same arrangement, given that the highest interspecific distances were over 1.79% and the lowest intraspecific distances were less than 1.79%.


The species delimitation analyses based on genetic distance (OT and ASAP) and coalescence (PTP and GMYC), together with the Bayesian phylogenetic analysis, which has well-supported clades, indicate the existence of nine distinct molecular clades in the dataset analyzed in the present study, with some variation and divergence among the results of the different delimitation methods. The congruence between these analyses is commonly employed in species delimitation based on a single locus (e.g.,Machado et al., 2018; Ramirez et al., 2020; Nogueira et al., 2021). Multiple approaches are necessary due to the computational limitations of each method commonly used for species delimitation (Carstens et al., 2013).

The clade composed of MOTUs 1 (P. australis + P. cf. rachoviana, Lower Paraná and Paraguay basins), 2 (Pyrrhulina aff. australis I, Araguaia River), 3 (P. marilynae, Teles Pires River), 4 (P. aff. australis II, Paraguay River), 5 (P. aff. australis III, Upper Paraná River), and 6 (Pyrrhulina aff. australis IV, Guaporé River), are distributed in six monophyletic lineages, with a maximum intraspecific distance of 7.32% within the nominal species P. australis, which is thus unlikely to represent a single, monophyletic species, but rather, a species complex.

The MOTU 1, which includes P. australis (from the Paraguay River) and P. cf. rachoviana (from the Lower Paraná basin) was a consensus arrangement in all the species delimitation methods used in the present study, which further highlights the debate on the relationship between these two species. While P. rachoviana was identified as a synonym of P. australis by Zarske, Géry (2004), these authors concluded that the type locality of P. rachoviana (Rosário, Argentina), was erroneously identified by Myers (1926), whereas Zarske (2016) suggested that the species did in fact originate from the Amazon basin. However, the contestation of Zarske, Géry (2001) and Zarske (2016) is not supported by any conclusive evidence, except for an inconspicuous line extending along the whole length of the body in the original description of Myers (1926). The only way to resolve this question would be to run a molecular analysis of the P. rachoviana type specimens and identify their closest relationship with specimens from a given drainage basin. As the type specimens of P. rachoviana were not available for analysis in the present study, it was decided to follow the original species description and consider Rosário in Argentina to be the type locality, contradicting more recent studies. The results of the present study do in fact indicate that the Pyrrhulina population from Rosário is part of the same MOTU as P. australis, which is consistent with the synonymization suggested by Zarske, Géry (2004) and contradicts Netto-Ferreira, Marinho (2013).

In the case of MOTU 3 (P. marilynae, Teles Pires River), the group is well defined by the different species delimitation methods, despite the low mean genetic distance, of 0.96% (Tab. 2), in comparison with P. aff. australis II (Paraguay River). This MOTU was well differentiated from MOTU 1 (P. australis + P. cf. rachoviana, Paraguay and Lower Paraná basins), however, with a mean distance of 2.17% between the two groups (Tab. 2), which is well above the OT. This further reinforces the close proximity of P. australis and P. marilynae, as demonstrated in the genomic study of Ferreira et al. (2022) and ratified by the findings of Moraes et al. (2021), who showed that the diploid chromosome number of P. marilynae from the Amazon basin (2n = 32) is the lowest of any Pyrrhulina species, given that all the others analyzed in the present study have diploid numbers of 2n = 40–42. This difference is the result of major structural chromosomal rearrangements, reinforced by the highly dynamic configuration of the repetitive DNA of these fish (Moraes et al., 2021). These findings contradict, once again, the synonymy between P. rachoviana and P. marilynae proposed by Zarske (2016), based on the comparison of the coloration patterns of specimens from the Lower Amazon, rather than the Lower Paraná basin. Given this, in addition to the analysis of the type specimens of P. rachoviana mentioned above, it would be necessary to examine specimens from the Amazonian populations described by Zarske (2016) for the definitive resolution of these taxonomic questions.

The allocation of the specimens from the Paraguay and Lower Paraná basins (i.e., MOTU 1 – P. australis and P. cf. rachoviana) in quadrant III of Fig. 5 indicates a possible recent divergence, hybridization, or synonymy, as discussed by Hebert et al. (2004). This allocation is supported by consistent interspecific genetic distance compared to specimens of the Upper Paraná basin (i.e., MOTU 5, P. aff. australis III), confirming that MOTU 1 and MOTU 5 represent distinct lineages. By contrast, the genetic distances between these individuals and those from the Upper Paraná basin (MOTU 5) reach 2.53%, which is consistent with the conclusions of Costa-Silva et al. (2018), i.e., that the historical isolation of the fish populations of the Upper Paraná basin has been upheld, with this scenario being reflected in the significant genetic distances between the different populations. It is important to note here that, while MOTU 5 (P. aff. australis IV from the Upper Paraná-Pardo and Paranapanema rivers) is located in quadrant IV (Fig. 5), this morphospecies was the only one allocated to quadrant III in Fig. 4, which indicates that the morphological identification of the specimens contradicts the molecular analysis (Hebert et al., 2004). This MOTU also has a mean intraspecific distance of 1.09% (Tab. 2), which may be related to the fact that this group includes individuals from the same basin, but from different rivers. In the present analysis, these groups were not considered to be distinct MOTUs due to the fact that their genetic distances were below the OT, although a more comprehensive sample of the population of the Upper Paraná basin would be necessary to provide a more conclusive interpretation of the relationships among the taxa.

This same divergence pattern has been found in the small-bodied species of the family Parodontidae from the La Plata basin, with mean genetic distances of 0.3% between the populations of the Uruguay and Paraguay basins, but 6.1% between these populations and those of the Upper Paraná basin (Bellafronte et al., 2013). Using the DNA barcode, Costa-Silva et al. (2018) compared the fish faunas of the Paraguay River and Upper Paraná basin, including P. australis. In the specific case of this morphospecies, the authors did not record any significant genetic distances (K2P) between the basins, although they did conclude that it may be undergoing a process of speciation, based on the findings of the other analytical approaches employed in the study. These findings may be related to the historical context of the Upper Paraná basin, which is separated from rest of the basin by the Guaíra Falls, which formed an insurmountable barrier for almost all the local fish species. In the 1980s, the construction of the Itaipu dam at the Guaíra Falls had an enormous impact on local fish diversity, affecting both sedentary and migratory species (Agostinho et al., 2007; Díaz et al., 2016). Even so, the specimens from the Paraguay and Lower Paraná basins (MOTU 4) are not divided, given that, while they are from geographically distant areas. These areas are located within environments that are connected by the flood pulse of the Pantanal wetlands, which plays a major role in the local hydrological regime, and has a fundamental influence on the similarity of the fish faunas of the Paraguay-Paraná system (Quirós et al., 2007).

The divergence between the P. australis MOTUs may be related to the fact that small-bodied fish do not undertake major migrations, which leads to the accumulation of genetic diversity in isolated populations. This pattern of genetic divergence is typical of fish species of small size, such as Piabina argentea Reinhardt, 1867, in which Pereira et al. (2013) recorded high intraspecific genetic distances, with means of 2.0–5.6%, a finding that supported the separation of the species into at least six distinct evolutionary units. In a molecular analysis of samples of Nannostomus eques Steindachner, 1876 from the Amazon basin based on the mitochondrial D-Loop, Terencio et al. (2012) concluded that the populations were subdivided into two evolutionary units, separated by very high interspecific genetic distances, of between 5.5% and 8.3%. These findings confirm the hypothesis that the limited dispersal and geographic subdivision of the populations, which is typical of small-bodied species, facilitate the appearance of new species through geographic isolation (Terencio et al., 2012). This genetic diversity was also found in other Nannostomus species, with divergent genetic lineages being found in four of the nominal species, with a mean interspecific distance of 19% within this group, which obviously means that a threshold of 2% is inadequate for the diagnosis of this group of species (Benzaquem et al., 2015).

The results of the present study reinforce the importance of analytical tools that take the evolutionary history of the target groups into account for the interpretation of diversity patterns, with an Optimal Threshold (OT) of 1.79% being considered here. This OT value was below the standard limit of divergence established by Ward et al. (2009) for fishes, that is 2.1%, although many studies have found that an OT of less than 2% is effective for the delimitation of species (e.g., Bellafronte et al., 2013; Machado et al., 2017; Arruda et al., 2019). The mean intraspecific distance in the Pyrrhulina australis group (including P. marilynae and“P. rachoviana”) was above this threshold (3.74%), which confirms the separation of this group into six genetic lineages. Species delimitation analyses of closely-related taxa based on COI sequences have also reported thresholds lower than the standard value, reaching less than 1% in some cases (inter-MOTU distances), which have been attributed in general to the recent divergence of the taxonomic units (Pereira et al., 2013; Ramirez, Galetti, 2015; Ramirez et al., 2017).

Previous taxonomic (Ardila-Rodríguez 1994, 1999, 2000, 2001, 2002, 2004, 2008a,b, 2010; Netto-Ferreira, 2010, 2012; Netto-Ferreira et al., 2011, 2013; Netto-Ferreira, Marinho, 2013; Vieira, Netto-Ferreira, 2019) and revisionary studies (Weitzman, 1966; Weitzman, Cobb, 1975; Marinho, Menezes, 2017) with lebiasinid genera have shown the color pattern as the most relevant source of characters for recognizing species among congeners, as the aforementioned studies revealed meristic and morphometric characters to be well conserved among representatives of the family, being useful to distinguish very few species among their congeners. Likewise, within Pyrrhulina the pigmentation pattern is also and is commonly used in species diagnoses and identification keys (Zarske, Géry, 2004; Netto-Ferreira, Marinho, 2013; Marinho, Netto-Ferreira, 2014; Vieira, Netto-Ferreira, 2019). Although the external morphological examination of the representatives of the MOTUS, agreed with the characters proposed by Netto-Ferreira, Marinho (2013) for the Pyrrhulina australis species group, no conspicuous anatomical character allowed their prompt recognition, reinforcing the difficulty in obtaining unique diagnostic characters distinguishing closely related species. Instead, the color pattern variation among each MOTUs confirmed the importance of that source of characters for the systematics of the genus. The morphospecies P. aff. australis I (Araguaia River, Fig. 2A) differs from P. australis (Paraguay basin, Fig. 2E) by presenting reddish coloration on the dorsal and anal fins, as well as presenting a discrete, straight, slender stripe passing onto scales of lateral series 3 and 4, bordered by clearer stripes dorsal- and ventrally. Pyrrhulina marilynae (Teles Pires River, Fig. 2D) differs from P. australis (Paraguay River, Fig. 2E) by having a conspicuous dark stripe along the body, a characteristic pattern described for P. marilynae, and Pyrrhulina zigzag Zarske & Géry, 1997 (Zarske, Géry, 2004; Netto-Ferreira, Marinho, 2013; Vieira, Netto-Ferreira, 2019). Specimens of P. aff. australis IV (Fig. 2 D), present a dark blotch on the caudal fin that extends to the tip of the median caudal-fin rays, a pattern also observed in P. marilynae (Netto-Ferreira, Marinho, 2013) and other Pyrrhulina, not included in the present contribution, collected in the Amazon basin (Zarske, Géry, 2004). The specimens attributable to P. rachoviana (sensu Myers)and Pyrrhulina aff. australis IV also exhibit a distinct checkerboard pattern along the body (Figs. 2B–D), absent in the morphospecies from the Araguaia-Tocantins and Paraná-Paraguay basins (Figs. 2A, E). Besides the lack of the characters defining the P. australis species group (sensu Netto-Ferreira, Marinho, 2013), P. obermulleri can be distinguished from the members of that clade by the primary stripe extending posteriorly to the pectoral girdle; P. filamentosa differs from that species group by having a long, streamlined body, with the largest scale count among Pyrrhulina (25–28 scales on the lateral line series); and P. spilota can be readily recognized by the presence of a series of dark blotches on the flanks and anal-fin rays.

The MOTUs of P. obermulleri, P. spilota, and P. filamentosa were well-defined by the different species delimitation methods employed in the present study. These species are distributed allopatrically across the drainages of the Amazon and Maroni basins. P. filamentosa (Maroni basin), exhibits a high inter-specific distance of 15.01% from P. spilota (Amazon basin) and 14.48% from P. obermulleri (Madeira basin). The evolution of Neotropical ichthyofauna is closely linked to the evolutionary history of drainage basins (Reis et al., 2016), and the Maroni basin, situated in the Guiana Shield, represents an endemic zone with distinct geological formation compared to the Brazilian Shield and lowlands (Lundberg et al., 1998; Albert et al., 2011). These patterns favor allopatric speciation due to the interruption of gene flow between geographically separated populations, associated with limited dispersal capabilities of these species (Albert et al., 2011, 2020; Bernardi, 2013). Similar patterns of allopatric speciation were observed in the geographical distributions of MOTUs 1to 6 within the P. australis complex, across the Paraná-Paraguay, Guaporé, Tapajós, and Araguaia-Tocantins river basins. These basins have a complex history of geographical, and the evolution of ichthyofauna has been influenced by vicariance events between the Amazonia and Paraguay basins approximately 30 Ma (Lundberg et al., 1998), as well as events involving the dispersal of Amazonian species into the Paraguay basin during the last 10 Ma (Lundberg et al., 1998; Carvalho, Albert, 2011). These events have contributed to the sharing of fauna among the different river basins (Carvalho, Albert, 2011; Dagosta, de Pinna, 2019). However, the gene tree was not capable, on its own, of testing adequately the phylogenetic relationships among the Pyrrhulina species, it is necessary to integrate the use of multiple molecular markers and morphological data (Oliveira et al., 2011; Roxo et al., 2017; Ramirez et al., 2020), which will demand the application of a more comprehensive approach for the definitive diagnosis of the relationships among the Pyrrhulina species.

Overall, then, the results of the analyses presented here indicate an unexpected level of diversity within the Pyrrhulina australis morphospecies, which contrasts with the findings of the previous studies of the diversity of this species. The “P. australis” morphospecies (sensu lato) was subdivided into six evolutionary lineages related systematically with the geographic distribution of the populations in the different drainage basins, and were well diagnosed, forming a clade that encompasses the Pyrrhulina australis + P. cf. rachoviana, P. aff. australis I/II/III/IV, and P. marilynae morphospecies. In this context, it would be most parsimonious to conclude that the geographic subdivisions of the populations are favoring speciation processes in this genus, including the detection of a possible species complex centered on the nominal species P. australis. In the specific case of the species identified as P. australis and P. aff. australis, previous studies have shown that, while they are highly similar in morphological terms and both have the same diploid chromosome number of 2n = 40, they do present divergences in certain classes of repetitive DNA, and can be considered to be two distinct evolutionary units of P. australis (Moraes et al., 2017).

Despite the results obtained in the present study, a definitive conclusion regarding the proposed synonymy of P. australis and P. rachoviana could not be provided, as we did not have access to the sequences of the type specimens of both species. The objective recognition of the relationships of the namebearing types with the MOTUs recognized herein are a necessary step to determine which of the lineages will be described as new species, helping to further restrict the confusion on the taxonomy of the genus. Considering that the plasticity of the aforementioned morphological/coloration features allowing the recognition of the morphospecies and would permit diagnosing the undescribed species, were not examined in detail, no nomenclatural act was taken on this occasion, and will be the subject of future investigation. The integration of new molecular markers, both mitochondrial and nuclear, is also necessary to better comprehend the evolutionary relationships within the group (Oliveira et al., 2011; Ramirez et al., 2020). Even so, the present study has made important advances in the understanding of the specific limits of the genus Pyrrhulina, and provides important insights that should help to resolve the taxonomic uncertainties of this fish group.


Thanks are due to Dr. Carolina B. Machado (UFSC) for support during the analyses employed herein. Authors were financially supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq process 421733/2017–9 INAU II and INPP, 446925/2014–43 to PCV; process 130650/2019–6 to TBS and 13834/2021–0 to ALNF), Fapemat Universal (Fundação de Amparo à Pesquisa do Mato Grosso; process 0336943/2017 to DCF). FAPERGS (Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul; call 14/2022 ARD/ARC to ALNF).


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Taina Barbosa de Souza1, Daniela Cristina Ferreira2 , Hugmar Pains da Silva3, André Luiz Netto-Ferreira4 and Paulo Cesar Venere2

[1]    Programa de Pós Graduação em Ecologia e Conservação da Biodiversidade, Universidade Federal de Mato Grosso, Avenida Fernando Correa da Costa, 2367, Bairro Boa Esperança, 78060-900 Cuiabá, MT, Brazil. (TBS) taina.souzab1@gmail.com.

[2]    Laboratório de Genética e Citogenetica Animal, Instituto de Biociências, Universidade Federal de Mato Grosso, Av. Fernando Correa da Costa, 2367, Bairro Boa Esperança, 78060-900 Cuiabá, MT, Brazil. (DCF) ferreiradc@gmail.com (corresponding author), (PCV) paulo.venere@gmail.com.

[3]    Faculdade de Ciências Agrárias, Biológicas, Engenharia e Saúde, Universidade do Estado de Mato Grosso, Campus Universitário Professor Eugênio Carlos Stieler de Tangará da Serra, Avenida Inácio Bittencourt Cardoso, 6967 E, Bairro Jardim Aeroporto, 78301-532 Tangará da Serra, MT, Brazil. (HPS) painsbio@gmail.com.

[4]    Laboratório de Ictiologia, Departamento de Zoologia, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, 91501-970 Porto Alegre, RS, Brazil. (ALNF) alnferreira@gmail.com.

Authors’ Contribution

Taina Barbosa de Souza: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Visualization, Writing-original draft, Writing-review and editing.

Daniela Cristina Ferreira: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Software, Supervision, Visualization, Writing-review and editing.

Hugmar Pains da Silva: Conceptualization, Data curation, Methodology, Visualization, Writing-review and editing.

André Luiz Netto-Ferreira: Conceptualization, Data curation, Visualization, Writing-review and editing.

Paulo Cesar Venere: Conceptualization, Data curation, Funding acquisition, Resources, Supervision, Visualization, Writing-review and editing.

Ethical Statement​

The fish sampling was supported by the license number 15226–2, provided by the Instituto Chico Mendes de Conservação de Biodiversidade (ICMBio), through the Sistema de Autorização e Informação em Biodiversidade (SISBIO).

Competing Interests

The authors declare no competing interests.

How to cite this article

Souza TB, Ferreira DC, Silva HP, Netto-Ferreira AL, Venere PC. DNA Barcoding of Pyrrhulina australis (Characiformes: Lebiasinidae) reveals unexpected cryptic diversity in the group. Neotrop Ichthyol. 2023; 21(4):e230037. https://doi.org/10.1590/1982-0224-2023-0037

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Distributed under

Creative Commons CC-BY 4.0

© 2023 The Authors.

Diversity and Distributions Published by SBI

Accepted September 26, 2023 by Matt Kolmann

Submitted April 12, 2023

Epub November 13, 2023