Research Article |
Corresponding author: Ruth A. Estupiñán ( ruthamanda.estupinan@gmail.com ) Academic editor: Franco Andreone
© 2016 Ruth A. Estupiñán, Stephen F. Ferrari, Evonnildo. C. Gonçalves, Maria Silvanira R. Barbosa, Marcelo Vallinoto, 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, Ferrari SF, Gonçalves EC, Barbosa MSR, Vallinoto M, Schneider MPC (2016) Evaluating the diversity of Neotropical anurans using DNA barcodes. ZooKeys 637: 89-106. https://doi.org/10.3897/zookeys.637.8637
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This study tested the effectiveness of COI barcodes for the discrimination of anuran species from the Amazon basin and other Neotropical regions. Barcodes were determined for a total of 59 species, with a further 58 species being included from GenBank. In most cases, distinguishing species using the barcodes was straightforward. Each species had a distinct COI barcode or codes, with intraspecific distances ranging from 0% to 9.9%. However, relatively high intraspecific divergence (11.4–19.4%) was observed in some species, such as Ranitomeya ventrimaculata, Craugastor fitzingeri, Hypsiboas leptolineatus, Scinax fuscomarginatus and Leptodactylus knudseni, which may reflect errors of identification or the presence of a species complex. Intraspecific distances recorded in species for which samples were obtained from GenBank (Engystomops pustulosus, Atelopus varius, Craugastor podiciferus, and Dendropsophus labialis) were greater than those between many pairs of species. Interspecific distances ranged between 11–39%. Overall, the clear differences observed between most intra- and inter-specific distances indicate that the COI barcode is an effective tool for the identification of Neotropical species in most of the cases analyzed in the present study.
Amazon basin, amphibians, COI, DNA barcoding, identification, taxonomy
Many amphibian groups are morphologically homogeneous and tend to lack clear diagnostic traits. This means that, while there have been a number of recent advances, the taxonomy of amphibians is poorly resolved in general (see e.g.
The increasing availability of molecular data has reinforced the conclusion that morphological evolution in amphibians is often cryptic, resulting in a revitalization of amphibian taxonomy (e.g.
Short DNA sequences from a standardized region of the genome can provide a DNA “barcode” for the identification of species (
The usefulness of COI as a DNA barcode has been evaluated in Malagasy mantellids and North American plethodontid salamanders (
Using a combination of primers, COI sequences were used to successfully identify 94% of Holarctic amphibians, and showed that the overlap between intra- and inter-specific distances was the result of hybridization, the presence of species complexes or taxonomic problems (
In this context, the present study evaluated the potential of the mitochondrial COI gene as a barcode, used in combination with other traits, for the identification of Neotropical amphibians from the Amazon basin and other regions of South America. In particular, the study compares the molecular classification of the specimens with the traditional taxonomy of the group.
In order to establish a reference site for the evaluation of a barcoding approach for Amazonian vertebrates, a field survey was conducted in the BX044 polygon in the southwestern Amazon basin, an area considered to be of the highest importance for the conservation of the biome’s biological diversity (Pronabio, 2002). The polygon covers an area of 5270 km2 and is located between latitudes 08°02'52" and 08°54'46" S, and longitudes 60°50'24" and 62°10'13"W, within the Madeira-Tapajós interfluve (Fig.
Specimens were collected in January, 2004, at 74 sites located along the Maderinha, Roosevelt, and Jatuarana rivers, and their tributaries. Specimens were collected in open and dense savanna habitats, gallery and flooded forests, rainforest, and ricefields. The specimens were euthanized with a lethal dose of lidocaine (
Following the extraction of tissue samples, the specimens collected during the present study were preserved for identification at the Goeldi Museum in Belém, Brazil, where they were confirmed by M.S.H. Hoogmoed. The accuracy of COI as a barcode for the identification of species was assessed based on the most recent classification of the amphibians (
Total DNA was extracted from either muscle or liver tissue by the SDS-proteinase K/phenol-chloroform extraction method (Sambrook and Russell 2001). A partial 680-bp fragment of the COI gene was amplified using the 5-CCTGCAGGAGGAGGAGAYCC-3´ and 5-AGTATAAGCGTCTGGGTAGTC-3´ primers (
The sequences obtained were aligned and edited by BIOEDIT v. 7.0.5.3 (
Pairwise comparisons of COI sequences were conducted for three categories: (i) individuals of the same species, (ii) individuals of the same genus (excluding those of the same species), and (iii) individuals of the same family (excluding those of the same genus). The frequency distribution of intra- and interspecific genetic distances was calculated using MEGA 5 (
The variability of the COI gene between populations of the same species was also tested using the K2P model, for which the species were selected based on the largest possible sample size (number of specimens) in GenBank (A. varius, C. podiciferus, D. labialis and E. pustulosus) and the availability of accurate information on their geographic origin. Additional species were included in this analysis (see Suppl. material
COI sequences were recovered from 75% (83/110) of the specimens analyzed. Full-length PCR products (640 bps) were amplified from all of these specimens (see Suppl. material
The COI barcode identified correctly the species of 94% of the specimens examined (93 of 109 species). The COI sequences obtained for the 36 species represented by two or more specimens were most similar to one another than to those of any other species. In addition, with a few notable exceptions, which are discussed below, the differences in COI sequences between closely-related species were higher than those within species. The mean K2P distance within species was 3.0% (Fig.
In most cases, the neighbor-joining (NJ) tree reflected a relatively reduced differentiation within species in comparison with between-species divergence (Fig.
All but five of the species collected in the present study were characterized by intraspecific divergence equal to or lower than 9.9% (Suppl. material
Range of intraspecific and between-taxon divergence values recorded in the present study.
Comparison | Percentage distance |
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Intraespecific (including GenBank sequences): Atelopus varius | 0.5–1.2 |
Craugastor podiciferus | 4.1–11.4 |
Dendropsophus labialis | 0.2–9.0 |
Engystomops pustulosus | 0.0–11.4 |
Intraspecific (only species collected during the present study) | 0.0–9.9* |
Interspecific | 11.0–39.0 |
Between genera | 15.0–31.4 |
Between families (Bufonidae, Dendrobatidae, Hylidae, Craugastoridae, Leiuperidae, Microhylidae, Aromobatidae, and Leptodactylidae) | 23.0–31.0 |
Interspecific divergence varied considerably (Table
A single mitochondrial DNA barcode, derived from the COI gene, identified correctly 93 of the 109 Neotropical amphibian species analyzed in the present study. Similar barcodes (sequences) were not observed in different species, and lower distances (generally 0.0–9.9%) were observed within species than between them. The ranges of values recorded in the present study were consistent with those recorded in previous amphibian studies (Table
Within- and between-taxon distances recorded in different groups of amphibians, based on COI sequences.
Taxon or group | Geographic region | Percentage divergence in the COI gene (mean divergence) | Reference | |
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Within species | Between species | |||
Mantellidae (frogs) | Madagascar | 10.0–18.0 (5.4) | (20.7) |
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Aneides (Climbing salamanders) | USA | > 7.8 (4.3) | (13.5) |
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Litoria fallax (two lineages) | Australia | 5.0 | 11–12 |
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Ambystoma laterale-jeffesonium complex | Canada | – | 9–14 | Smith et al. (2007) |
Scinax ruber | French Guiana | 1.3–14.3 | – |
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Rhinella gr. margaritifera | Brazil, Ecuador, Peru, French Guiana | 1.0–5.1 | – |
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Engystomops pustulosus | Mexico, Guatemala, Nicaragua, Costa Rica, Panama, Colombia, Venezuela | 0.0–11.4 | – | Data from |
Dendropsophus minutus | French Guiana, Suriname, Guyana | – | 9 (uncorrectedp) |
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Hynobiidae (Asian salamanders) | China, Korea, Russia, Iran, Afghanistan, Kazakhstan | 0.0–0.061 | 0.007–0.165 |
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Chinese amphibians | China | 0–0.101 | 0.031–0.282 |
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Eight frog families | Bolivia, Brazil, Colombia, Costa Rica, Ecuador, El Salvador, French Guiana, Guatemala, Guyana Haiti, Jamaica, Mexico, Nicaragua, Panama, Peru, Suriname Trinidad and Tobago, USA, Venezuela | 0–9.9 | 11–39 | Present study |
Lower intra- and interspecific distances have been recorded for the COI barcode in most other animal groups. In butterflies, for example, mean intraspecific distances were 0.46%, while those between species ranged from 2.97% (
The high COI divergence rates recorded in the present study were nevertheless similar to those recorded in pulmonate snails (
The neighbor-joining tree indicated that most of the species and genera analyzed in the present study form relatively cohesive units. However, the data available on Dendropsophus minutus (
The greatest intrageneric distances were recorded in Hypsiboas (18.2%), Craugastor (19.7%), and Osteopilus (20.2%). The considerable distances between some Craugastor species indicates the existence of a species complex, as indicated previously for Craugastor podiciferus by
The general polytomy observed in the present study may have been the result of the phylogenetic divergence at the family and genus levels, and the relatively reduced number of terminal taxa. This may also be reflected in the considerable variation in the bootstrap values, from 0% to 92%, found in some clades.
The amplification of the COI gene is straightforward in most vertebrates (
The results of the present study support the use of COI sequences as a DNA barcode for help the identification of Neotropical amphibian species, in particular to ensure the presence of cryptic forms. However, it will still be necessary to identify the factors determining the relatively high rates of divergence observed within the populations of some of the species analyzed in the present study. It will also be important to compile a database of sequences for different molecular markers, in order to better evaluate intra- and inter-specific patterns of variability (
We are grateful to Mariana Lúcio Lyra (UNESP) and Simoni Santos da Silva (UFPA) for their valuable suggestions on the original version of the manuscript, Laíssa de Castro Pontes (LPDNA) for help with the development of the study, and the numerous researchers from Brazil and other countries who contributed samples. 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.
Data on the specimens examined in the present study
Data type: The specimens used in this study, intraspecific distances, locality data and the GenBank association number of the submitted COI sequences. The museum number and reference of each specimen.
Explanation note: Please note that some of the sequences used in the study are incompletely referenced in the GenBank barcode database because they lack some data and we are unable to rectify this because the samples were collected too long ago (1980s or before) for the missing data to be found.