DNA Barcode Detects High Genetic Structure within Neotropical Bird Species

Citation: Tavares ES, Goncalves P, Miyaki CY, Baker AJ ( DNA Barcode Detects High Genetic Structure within Neotropical Bird Species Erika Sendra Tavares 0 Priscila Gonc alves 0 Cristina Yumi Miyaki 0 Allan J. Baker 0 Bernd Schierwater, University of Veterinary Medicine Hanover, Germany 0 1 Department of Natural History, Royal Ontario Museum , Toronto, Ontario , Canada , 2 Departamento de Gene tica e Biologia Evolutiva, Instituto de Biociencias, Universidade de Sa o Paulo , Sa o Paulo, Sa o Paulo , Brazil , 3 Department of Zoology, University of Toronto , Toronto, Ontario , Canada Background: Towards lower latitudes the number of recognized species is not only higher, but also phylogeographic subdivision within species is more pronounced. Moreover, new genetically isolated populations are often described in recent phylogenies of Neotropical birds suggesting that the number of species in the region is underestimated. Previous COI barcoding of Argentinean bird species showed more complex patterns of regional divergence in the Neotropical than in the North American avifauna. Methods and Findings: Here we analyzed 1,431 samples from 561 different species to extend the Neotropical bird barcode survey to lower latitudes, and detected even higher geographic structure within species than reported previously. About 93% (520) of the species were identified correctly from their DNA barcodes. The remaining 41 species were not monophyletic in their COI sequences because they shared barcode sequences with closely related species (N = 21) or contained very divergent clusters suggestive of putative new species embedded within the gene tree (N = 20). Deep intraspecific divergences overlapping with among-species differences were detected in 48 species, often with samples from large geographic areas and several including multiple subspecies. This strong population genetic structure often coincided with breaks between different ecoregions or areas of endemism. Conclusions: The taxonomic uncertainty associated with the high incidence of non-monophyletic species and discovery of putative species obscures studies of historical patterns of species diversification in the Neotropical region. We showed that COI barcodes are a valuable tool to indicate which taxa would benefit from more extensive taxonomic revisions with multilocus approaches. Moreover, our results support hypotheses that the megadiversity of birds in the region is associated with multiple geographic processes starting well before the Quaternary and extending to more recent geological periods. - Funding: This work was supported by funding through the Canadian Barcode of Life Network from Genome Canada through the Ontario Genomics Institute, Natural Sciences and Engineering Research Council of Canada (NSERC), and other sponsors, the ROM Governors Fund, Fundacao de Amparo a` Pesquisa do Estado de Sao Paulo (FAPESP), Coordenacao de Aperfeicoamento de Pessoal de nvel Superior (CAPES) and Conselho Nacional de Pesquisa (CNPq). The funders of this research had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. One of the striking patterns in geographic distribution of terrestrial biodiversity is the increase in species richness towards lower latitudes in several groups of organisms, including birds. The possible causes for this pattern is one of the highly debated topics in ecology and evolution, even though no definitive conclusion was yet been achieved [1,2,3,4]. The Neotropical area alone holds a third of the recognized extant bird species (about 3,300 out of 10,000) [5], with a biodiversity hotspot in the tropical forests [6]. Moreover, recent phylogenies suggest the number of species in the area is underestimated because reproductively isolated lineages are frequently described in these studies [7,8,9,10,11]. In stark contrast to bird taxonomy in temperate zones, genetic evidence for species limits in the Neotropics is often discordant with traditional taxonomy due to the high incidence of species complexes. These complexes commonly feature gradual variation in morphological and behavioural characters, masking the occurrence of similar species that can be uncovered with genetic analyses [11,12,13,14,15]. DNA barcodes based on the 59 portion of the cytochrome oxidase I gene (COI) linked with specimens vouchers and locality information provides a rapid and inexpensive method to identify species and detect provisional new species [16]. Pilot DNA barcode surveys in birds of North America, sister-species pairs, and birds of Korea were successful in either identifying recognized species of birds, and detecting some potential new species, except for a minor proportion of cases where species are very recently diverged or hybridize [17,18,19,20]. Critics questioned if the success observed in North American birds could be extrapolated to the tropics [21], where species clearly exhibit a higher level of phylogeographic subdivision [22]. However, DNA barcoding has subsequently proved to be highly successful in identifying Neotropical species of birds; all 16 species (100%) of antbirds (Thamnophilidae) that were barcoded [23] and 494 of 500 (95.8%) species of birds of Argentina [24] had distinguishable COI signatures. The screening of Argentinean birds also detected 21 species with deep intraspecific structure, and revealed more complex patterns of regional divergence in the Neotropical than in the North American avifauna [24]. Even though more species will doubtlessly be shown to share barcodes when complete coverage of species and genera is available, it is clear that large-scale sequencing of COI associated with vouchered specimens and locality information is a valuable tool in understanding genetic differentiation within and among species of birds [17,21,24,25]. In this study we increased the coverage of Neotropical bird species that have been barcoded by adding 637 samples from 431 species, with higher representation in tropical forest areas of Brazil and Guyana, but also including samples from localities ranging from Mexico to Argentina and Chile. We compared these sequences with previously published sequences of congeneric species of Neotropical birds, totaling 1,431 samples from 561 different species of birds, 296 of which were represented by multiple individuals. We showed that a high success rate in species identification (93%) with DNA barcodes can be achieved in this large sample of avian biodiversity from the mega-diverse Neotropical region similar to that obtained in broad geographic surveys in the Nearctic and Palearctic regions of the world. Additionally, a higher percentage (12%) of species had multiple deep phylogeographic splits than in previous surveys, some of which are likely reproductively isolated lineages. Species identification in Neotropical birds About 93% of the spe (...truncated)