Research Article |
Corresponding author: Nelson Colihueque ( ncolih@ulagos.cl ) Academic editor: George Sangster
© 2021 Nelson Colihueque, Alberto Gantz, Margarita Parraguez.
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:
Colihueque N, Gantz A, Parraguez M (2021) Revealing the biodiversity of Chilean birds through the COI barcode approach. ZooKeys 1016: 143-161. https://doi.org/10.3897/zookeys.1016.51866
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The mitochondrial cytochrome c oxidase subunit I (COI) gene is an effective molecular tool for the estimation of genetic variation and the identification of bird species. This molecular marker is used to differentiate among Chilean bird species by analyzing barcodes for 76 species (197 individuals), comprising 28 species with no previous barcode data and 48 species with sequences retrieved from the BOLD and GenBank databases. The DNA barcodes correctly identified 94.7% of the species analyzed (72 of 76 species). Mean intraspecific K2P distance was 0.3% (range 0–8.7%). Within the intraspecific divergence range, three species, Phrygilus gayi, Sephanoides sephanoides and Curaeus curaeus, showed relatively high intraspecific divergence (1.5–8.7%), possibly due to the presence of a species complex or geographic isolation of sub-populations. Mean interspecific K2P distance was 24.7% (range 1.3–43.5%). Consequently, the intraspecific K2P distance showed limited overlap with interspecific K2P distance. The mean intraspecific divergence in our study was similar to that found in temperate regions of South America (0.24%). However, it was approximately one order of magnitude lower than values reported for bird species in tropical regions of northern South America (1.8–2.13%). This result suggests that bird species from Chile show low levels of genetic structure and divergence. The small overlap between intra- and inter-specific distances implies that COI barcodes could be used as an effective tool to identify nearly all the Chilean bird species analyzed.
Aves, biodiversity, COI, genetic variation, Neotropical birds, taxonomy
Birds are among the animal groups that have been subjected to extensive DNA barcoding. Currently, DNA barcodes are publicly available for 41% of known bird species in the world, with data for nearly 4300 species from 37 of 39 recognized avian orders (
In recent years, a number of DNA barcoding studies have assessed the efficacy of using COI data to identify South American birds. Analyses of hundreds of bird species from countries in this region, such as Argentina (
The Chilean avifauna comprises of 443 species, if we only consider species that are residents or regular visitors (
Little is known about the population structure and genetic diversity of the taxa that currently compose the diversity of birds in Chile (
We obtained samples from across central and southern Chile, mainly from the Cachapoal (34°S) and Osorno, Ranco, and Valdivia provinces (40°–41°S). Samples corresponded to dead birds found along the highways which were collected between 2012 and 2019 by volunteers and the authors; date and site of collection (at least at province level) were recorded immediately. Collection sites were georeferenced using Google Earth based on locality names. Information about vouchers and collection sites is available in Suppl. material
The primer pairs BirdF1 (5’-TTCTCCAACCACAAAGACATTGGCAC-3’) and BirdR1 (5’-ACGTGGGAGATAATTCCAAATCCTG-3’), as well as BirdF1 and BirdR2(5’-ACTACATGTGAGATGATTCCGAATCCAG-3’)(
The sequences obtained were aligned and edited using GENEIOUS 4.0.2 software (Biomatters Ltd.). Base substitution saturation, a phenomenon that may decreases the amount of phylogenetic information contained in a sequence dataset, was tested based on the index of substitution saturation (ISS) (
Barcodes were analyzed for a total of 76 unique species (197 individuals), including 32 species sequenced in this study (68 individuals) and 48 species (129 individuals) from BOLD and GenBank (see Suppl. material
The COI barcode correctly identified 94.7% of the species studied (72 of 76 species). That is, these 72 species had unique DNA barcodes that did not overlap with the barcodes of any other species. Interspecific K2P distance ranged from 1.3 to 43.5% (mean 24.7%). In most cases, the ML tree, as shown in Figure
Maximum likelihood (ML) tree derived from the analysis of COI sequences for 76 bird species from Chile. The numbers at the nodes represent the percentage of bootstrap support. GenBank or BOLD accession number for each specimen are shown. Scale indicates the sequence divergence estimated from the number of nucleotide substitutions per site. The asterisk indicates the contradictory clusters found in the tree. Charadrius alexandrinus is currently known as Charadrius nivosus.
Comparisons of K2P-pairwise distances at two taxonomic levels for 76 species of birds from Chile. Intraspecific distance was calculated for species which two or more sequences were available.
Taxonomic level | Number of individuals | Number of taxa | Number of comparisons | Genetic distances (%) | |||
---|---|---|---|---|---|---|---|
Mean | SE | Minimum | Maximum | ||||
Intraspecific | 164 | 43 | 448 | 0.3 | 0.0 | 0.0 | 8.7 |
Interspecific | 197 | 76 | 2850 | 24.7 | 0.1 | 1.3 | 43.5 |
The between-species differences in COI sequences were greater than the within-species differences. Mean intraspecific K2P distance was 0.3% (range 0–8.7%), while mean interspecific K2P distance was ca. two orders of magnitude larger, at 24.7% (range 1.3–43.5%) (see Table
Intraspecific K2P genetic distances for 43 species of birds from Chile, calculated when two or more sequences were available. Bold face indicates species with high mean intraspecific divergence that overlapped the minimum interspecific distance (> 1.3%). ‡ Currently known as Charadrius nivosus.
Species or subspecies | Number of individuals | Genetic distances (%) | |||
---|---|---|---|---|---|
Mean | SE | Minimum | Maximum | ||
Anas georgica | 2 | 0.0 | 0.0 | 0.0 | 0.0 |
Aphrastura masafuerae | 2 | 0.0 | 0.0 | 0.0 | 0.0 |
Attagis gayi | 3 | 0.0 | 0.0 | 0.0 | 0.0 |
Burhinus supercialiaris | 2 | 0.0 | 0.0 | 0.0 | 0.0 |
Charadrius alexandrinus‡ | 3 | 0.0 | 0.0 | 0.0 | 0.0 |
Charadrius alticola | 2 | 0.2 | 0.2 | 0.2 | 0.2 |
Charadrius falklandicus | 2 | 0.0 | 0.0 | 0.0 | 0.0 |
Charadrius modestus | 2 | 0.2 | 0.2 | 0.2 | 0.2 |
Chloephaga rubidiceps | 2 | 0.0 | 0.0 | 0.0 | 0.0 |
Chroicocephalus maculipennis | 2 | 0.5 | 0.3 | 0.5 | 0.5 |
Curaeus curaeus | 2 | 8.7 | 1.7 | 8.7 | 8.7 |
Elaenia albiceps chilensis | 4 | 0.0 | 0.0 | 0.0 | 0.0 |
Enicognathus leptorhynchus | 4 | 0.2 | 0.2 | 0.0 | 0.5 |
Eudyptes chrysocome | 8 | 0.1 | 0.1 | 0.0 | 0.5 |
Haematopus leucopodus | 2 | 0.0 | 0.0 | 0.0 | 0.0 |
Haematopus palliatus | 2 | 0.0 | 0.0 | 0.0 | 0.0 |
Larus belcheri | 2 | 0.2 | 0.2 | 0.2 | 0.2 |
Leucophaeus modestus | 4 | 0.0 | 0.0 | 0.0 | 0.0 |
Limosa haemastica | 2 | 0.0 | 0.0 | 0.0 | 0.0 |
Milvago chimango | 5 | 0.0 | 0.0 | 0.0 | 0.0 |
Mimus thenca | 3 | 0.0 | 0.0 | 0.0 | 0.0 |
Molothrus bonariensis | 2 | 0.2 | 0.2 | 0.2 | 0.2 |
Oceanites oceanicus | 2 | 0.0 | 0.0 | 0.0 | 0.0 |
Oreopholus ruficollis | 2 | 0.0 | 0.0 | 0.0 | 0.0 |
Phegornis mitchelli | 2 | 0.0 | 0.0 | 0.0 | 0.0 |
Phrygilus alaudinus | 15 | 0.5 | 0.1 | 0.0 | 0.9 |
Phrygilus atriceps | 3 | 0.0 | 0.0 | 0.0 | 0.0 |
Phrygilus fruticeti | 7 | 0.3 | 0.1 | 0.0 | 0.5 |
Phrygilus gayi | 7 | 1.5 | 0.4 | 0.2 | 3.2 |
Phrygilus plebejus | 7 | 0.3 | 0.1 | 0.0 | 0.5 |
Phrygilus unicolor | 2 | 0.2 | 0.2 | 0.2 | 0.2 |
Recurvirostra andina | 2 | 0.0 | 0.0 | 0.0 | 0.0 |
Sephanoides sephanoides | 2 | 2.2 | 0.7 | 2.2 | 2.2 |
Spheniscus magellanicus | 2 | 0.5 | 0.3 | 0.5 | 0.5 |
Spinus barbatus | 4 | 0.1 | 0.1 | 0.0 | 0.2 |
Strix rufipes | 2 | 0.0 | 0.0 | 0.0 | 0.0 |
Theristicus melanopis | 4 | 0.1 | 0.1 | 0.0 | 0.2 |
Thinocorus orbignyianus | 6 | 0.1 | 0.1 | 0.00 | 0.2 |
Troglodytes musculus chilensis | 3 | 1.1 | 0.4 | 0.0 | 1.7 |
Turdus falcklandii | 3 | 0.3 | 0.2 | 0.2 | 0.5 |
Tyto alba | 17 | 0.1 | 0.1 | 0.0 | 0.5 |
Vanellus chilensis | 6 | 0.0 | 0.0 | 0.0 | 0.0 |
Zenaida auriculata auriculata | 4 | 0.4 | 0.2 | 0.0 | 0.7 |
Intrageneric K2P genetic distances. Genetic distances within species were excluded.
Genera | Number of taxa | Genetic distances (%) | |||
---|---|---|---|---|---|
Mean | SE | Minimum | Maximum | ||
Attagis | 2 | 4.3 | 0.0 | 4.3 | 4.3 |
Charadrius | 5 | 16.1 | 1.1 | 1.9 | 24.3 |
Haematopus | 2 | 4.3 | 0.0 | 4.3 | 4.3 |
Larus | 2 | 1.3 | 0.1 | 1.2 | 1.4 |
Leucophaeus | 2 | 3.2 | 0.0 | 3.2 | 3.2 |
Phalacrocorax | 3 | 8.5 | 2.4 | 3.8 | 11.4 |
Phrygilus | 7 | 14.3 | 0.1 | 2.4 | 19.4 |
Pygoscelis | 2 | 8.4 | 0.0 | 8.4 | 8.4 |
Spheniscus | 2 | 2.2 | 0.3 | 1.9 | 2.4 |
Vanellus | 2 | 7.7 | 0.0 | 7.7 | 7.7 |
The mean intraspecific divergence in our study (0.3%) was ca. one order of magnitude lower than values reported for bird species in tropical regions of northern South America (1.8 and 2.13%), reported by
The mean intraspecific distances recorded in this analysis of Chilean birds (0.3%, based on 43 species) are largely consistent with other reported values for birds in temperate regions of South America, particularly Argentina (0.24%) (
DNA barcoding studies of birds from temperate regions of South America have reported that a relatively small number of species show deep divergence. For instance,
The limited numbers of bird species with deep divergence in temperate regions of South America may reflect distinct a pattern of regional biodiversity in comparison with tropical avifauna. Alternatively, as noted by
A practical utility of DNA barcoding lies in the use of divergence values as a preliminary screening of taxonomic diversity, for example, to screen for within-species divergence. Future work can follow up by examining unusual cases, especially species that show deep divergence. This approach may be useful in understanding the genetic structure of Curaeus curaeus, Phrygilus gayi and Sephanoides sephanoides, as these two species showed the largest intraspecific distances among the species analyzed (above 1.3%). A possible interpretation of this result is that these distances reflect a species complex. In fact, the maximum likelihood tree indicated that P. gayi individuals formed at least 3 clusters rather than a cohesive unit, suggestive of different lineages.
The finding that the greatest intrageneric divergence was found in Charadrius (16.1%) and Phrygilus (14.3%) is noteworthy. In the case of the Phrygilus genus this divergence pattern has been interpreted in the context of a marked phylogeographic structure, which is associated with broad altitudinal and latitudinal distributions of species across the Andean mountains (
In conclusion, this study indicates that DNA barcoding with COI markers is highly accurate for identifying Chilean bird species, as the barcode sequence for nearly every species studied was markedly distinct from that of any other species. Our analysis identified significant interspecific divergence, roughly two orders of magnitude higher than the intraspecific values observed, reflecting a clear barcode gap. In addition, most of the species analyzed showed low intraspecific divergence. This pattern is consistent with data for birds from other temperate regions of southern South America but contrasts with studies on birds from tropical regions of South America, which often show deep intraspecific divergence. Thus, these data reflect the existence of different evolutionary patterns associated with specific regions within the continent. We hope that this step-by-step effort focused on obtaining and assessing the DNA barcodes of the Chilean avifauna, will be useful for increasing knowledge of national biodiversity. This approach may also facilitate the establishment of a dataset of avian barcodes from Chile, enhancing the scope of local studies.
We would like to thank Soraya Sade, Oscar González, Jaime Rau, and Cristina Parraguez for providing samples. The suggestions and constructive comments of all those who helped to improve the final version of this manuscript are gratefully acknowledged, in particular the efforts of the editor and reviewers, which greatly improved the quality and clarity of the work. We dedicate this paper to the memory of Roberto Schlatter, an outstanding researcher of the birds of Chile, who passed away in October 2016.
Figure S1. Photographs of external morphology of bird specimens collected from Chile, with lateral, dorsal, or ventral views of specimens showing plumage color and overall appearance.
Data type: image
Explanation note: Data on specimen voucher number with size bar to estimate specimen size are provided.
Tables S1
Data type: molecular data
Explanation note: Table S1. List of Chilean birds sequenced in this study for the COI marker, with voucher numbers, collection localities, and with BOLD and GenBank accession numbers. For all specimens, tissue samples from muscle were taken.
Tables S2
Data type: molecular data
Explanation note: Table S2. List of Chilean bird COI sequences obtained from BOLD and GenBank databases, with voucher numbers and collection localities.