DNA Barcoding Identifies Illegal Parrot Trade.

Illegal trade threatens the survival of many wild species, and molecular forensics can shed light on various questions raised during the investigation of cases of illegal trade. Among these questions is the identity of the species involved. Here we report a case of a man who was caught in a Brazilian airport trying to travel with 58 avian eggs. He claimed they were quail eggs, but authorities suspected they were from parrots. The embryos never hatched and it was not possible to identify them based on morphology. As 29% of parrot species are endangered, the identity of the species involved was important to establish a stronger criminal case. Thus, we identified the embryos' species based on the analyses of mitochondrial DNA sequences (cytochrome c oxidase subunit I gene [COI] and 16S ribosomal DNA). Embryonic COI sequences were compared with those deposited in BOLD (The Barcode of Life Data System) while their 16S sequences were compared with GenBank sequences. Clustering analysis based on neighbor-joining was also performed using parrot COI and 16S sequences deposited in BOLD and GenBank. The results, based on both genes, indicated that 57 embryos were parrots (Alipiopsitta xanthops, Ara ararauna, and the [Amazona aestiva/A. ochrocephala] complex), and 1 was an owl. This kind of data can help criminal investigations and to design species-specific anti-poaching strategies, and demonstrate how DNA sequence analysis in the identification of bird species is a powerful conservation tool.

[1]  M. Holmgren,et al.  Science on the Rise in Developing Countries , 2004, PLoS biology.

[2]  J. M. González,et al.  A fluorimetric method for the estimation of G+C mol% content in microorganisms by thermal denaturation temperature. , 2002, Environmental microbiology.

[3]  Mudjekeewis D. Santos,et al.  Species composition of by-catch from milkfish (Chanos chanos) fry fishery in selected sites in the Philippines as determined by DNA barcodes , 2014, Mitochondrial DNA. Part A, DNA mapping, sequencing, and analysis.

[4]  S. Ball,et al.  DNA barcodes for biosecurity: invasive species identification , 2005, Philosophical Transactions of the Royal Society B: Biological Sciences.

[5]  G. Amato,et al.  A molecular phylogeny of Amazona: implications for Neotropical parrot biogeography, taxonomy, and conservation. , 2004, Molecular phylogenetics and evolution.

[6]  J. Eberhard,et al.  PHYLOGENY AND BIOGEOGRAPHY OF THE AMAZONA OCHROCEPHALA (AVES: PSITTACIDAE) COMPLEX , 2004 .

[7]  M. Kimura.  A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences , 1980, Journal of Molecular Evolution.

[8]  P. Hebert,et al.  Comprehensive DNA barcode coverage of North American birds , 2007, Molecular ecology notes.

[9]  M. Nei,et al.  MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. , 2011, Molecular biology and evolution.

[10]  A. Baker,et al.  Combined nuclear and mitochondrial DNA sequences resolve generic relationships within the Cracidae (Galliformes, Aves). , 2002, Systematic biology.

[11]  E. Tavares,et al.  Phylogeny and biogeography of Yellow-headed and Blue-fronted Parrots ( Amazona ochrocephala and Amazona aestiva ) with special reference to the South American taxa , 2007 .

[12]  Jeremy R. deWaard,et al.  Biological identifications through DNA barcodes , 2003, Proceedings of the Royal Society of London. Series B: Biological Sciences.

[13]  Andrew R. Deans,et al.  Extreme diversity of tropical parasitoid wasps exposed by iterative integration of natural history, DNA barcoding, morphology, and collections , 2008, Proceedings of the National Academy of Sciences.

[14]  M. Smith,et al.  DNA BARCODING: CO1 DNA barcoding amphibians: take the chance, meet the challenge , 2008, Molecular ecology resources.

[15]  Joanne Abramson,et al.  The Large Macaws: Their Care, Breeding, and Conservation , 1996 .

[16]  Mudjekeewis D. Santos,et al.  Illegal trade of regulated and protected aquatic species in the Philippines detected by DNA barcoding , 2016, Mitochondrial DNA. Part A, DNA mapping, sequencing, and analysis.

[17]  T. Burke,et al.  Parrot Evolution and Paleogeographical Events: Mitochondrial DNA Evidence , 1998 .

[18]  Arie van der Meijden,et al.  Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians , 2005, Frontiers in Zoology.

[19]  J. Robertson,et al.  DNA detective: a review of molecular approaches to wildlife forensics , 2010, Forensic science, medicine, and pathology.

[20]  A. Baker,et al.  Phylogenetic relationships and historical biogeography of neotropical parrots (Psittaciformes: Psittacidae: Arini) inferred from mitochondrial and nuclear DNA sequences. , 2006, Systematic biology.

[21]  Antoinette Kotze,et al.  DNA barcoding as a tool for species identification in three forensic wildlife cases in South Africa. , 2011, Forensic science international.

[22]  John C. Avise.  Molecular Markers, Natural History and Evolution , 1994, Springer US.

[23]  C. Hänni,et al.  Food and forensic molecular identification: update and challenges. , 2005, Trends in biotechnology.

[24]  T. A. Hall,et al.  BIOEDIT: A USER-FRIENDLY BIOLOGICAL SEQUENCE ALIGNMENT EDITOR AND ANALYSIS PROGRAM FOR WINDOWS 95/98/ NT , 1999 .

[25]  U. Ramakrishnan,et al.  Tracing the geographic origin of traded leopard body parts in the indian subcontinent with DNA‐based assignment tests , 2015, Conservation biology : the journal of the Society for Conservation Biology.

[26]  P. Hebert,et al.  Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species , 2003, Proceedings of the Royal Society of London. Series B: Biological Sciences.

[27]  A. Baker,et al.  DNA Barcode Detects High Genetic Structure within Neotropical Bird Species , 2011, PloS one.

[28]  C. Cicero,et al.  Open access, freely available online Correspondence DNA Barcoding: Promise and Pitfalls , 2022 .

[29]  N. Collar,et al.  Threatened birds of the Americas , 1992 .

[30]  John C. Avise,et al.  Molecular Markers, Natural History, and Evolution , 1993 .

[31]  Paula Franco Moreira.  Country Profile: Brazil , 2010 .

[32]  Amanda D. Roe,et al.  Patterns of evolution of mitochondrial cytochrome c oxidase I and II DNA and implications for DNA barcoding. , 2007, Molecular phylogenetics and evolution.

[33]  M. Vences,et al.  Deciphering amphibian diversity through DNA barcoding: chances and challenges , 2005, Philosophical Transactions of the Royal Society B: Biological Sciences.

[34]  J. Felsenstein.  CONFIDENCE LIMITS ON PHYLOGENIES: AN APPROACH USING THE BOOTSTRAP , 1985, Evolution; international journal of organic evolution.

[35]  A. Vogler,et al.  DNA-based species delineation in tropical beetles using mitochondrial and nuclear markers , 2005, Philosophical Transactions of the Royal Society B: Biological Sciences.

[36]  A. Baker,et al.  Single mitochondrial gene barcodes reliably identify sister-species in diverse clades of birds , 2008, BMC Evolutionary Biology.

[37]  Katherine F. Smith,et al.  Summarizing the Evidence on the International Trade in Illegal Wildlife , 2010, EcoHealth.

[38]  L. Frézal,et al.  Four years of DNA barcoding: current advances and prospects. , 2008, Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases.

[39]  D. Tautz,et al.  Reverse taxonomy: an approach towards determining the diversity of meiobenthic organisms based on ribosomal RNA signature sequences , 2005, Philosophical Transactions of the Royal Society B: Biological Sciences.

[40]  P. Hebert,et al.  Identification of Birds through DNA Barcodes , 2004, PLoS biology.

[41]  A. Hoelzel.  Molecular genetic analysis of populations: a practical approach. , 1993 .

[42]  R. Ward,et al.  DNA barcoding Australia's fish species , 2005, Philosophical Transactions of the Royal Society B: Biological Sciences.

[43]  J. Brookfield,et al.  Single-locus and multilocus DNA fingerprinting. , 1992 .

[44]  James Haile,et al.  Egg forensics: An appraisal of DNA sequencing to assist in species identification of illegally smuggled eggs , 2011, Forensic Science International: Genetics.

[45]  D. Rubinoff.  Utility of Mitochondrial DNA Barcodes in Species Conservation , 2006, Conservation biology : the journal of the Society for Conservation Biology.

[46]  S. Palumbi,et al.  Which whales are hunted? A molecular genetic approach to monitoring whaling. , 1994, Science.

[47]  Andrew P. Martin,et al.  Barcoding bushmeat: molecular identification of Central African and South American harvested vertebrates , 2010, Conservation Genetics.

[48]  R. Ward,et al.  An analysis of nucleotide and amino acid variability in the barcode region of cytochrome c oxidase I (cox1) in fishes , 2007 .

[49]  E. Tavares,et al.  PHYLOGENETIC RELATIONSHIPS AMONG SOME NEOTROPICAL PARROT GENERA (PSITTACIDAE) BASED ON MITOCHONDRIAL SEQUENCES , 2004 .

[50]  R. Collevatti,et al.  The role of demography and climatic events in shaping the phylogeography of Amazona aestiva (Psittaciformes, Aves) and definition of management units for conservation , 2009 .

[51]  Rob Ogden,et al.  Validation of the barcoding gene COI for use in forensic genetic species identification. , 2007, Forensic science international.