{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T14:48:02Z","timestamp":1785854882065,"version":"3.56.0"},"reference-count":49,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T00:00:00Z","timestamp":1785801600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Programa Fortalecimiento Grupos de Investigaci\u00f3n UTA","award":["4769-24"],"award-info":[{"award-number":["4769-24"]}]},{"name":"Centro de Investigaci\u00f3n y Desarrollo de Excelencia de Inter\u00e9s Nacional ANID","award":["CIN250047"],"award-info":[{"award-number":["CIN250047"]}]},{"award":["CIN250047"],"award-info":[{"award-number":["CIN250047"]}],"id":[{"id":"https:\/\/ror.org\/01297db79","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Unidad de Medio Ambiente del Consejo de Pueblos Atacame\u00f1os"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biology"],"abstract":"<jats:p>The Chilean flamingo (Phoenicopterus chilensis) is a widely distributed South American species inhabiting diverse wetland ecosystems from Ecuador and Peru south to Chile and Argentina, and east to Brazil, Uruguay, and Paraguay, ranging from sea level to high-altitude Andean salars. Despite its broad distribution, local populations are increasingly exposed to anthropogenic pressures, raising conservation concerns at regional scales. In this study, we generated mitogenomic resources and developed a molecular framework for species identification using non-invasive samples. We sequenced genomic DNA from P. chilensis and assembled its complete mitogenome, enabling the in silico evaluation of commonly used metabarcoding markers. Subsequently, we collected eight fecal samples from a feeding site in a high-Andean lagoon where P. chilensis co-occurs with Phoenicoparrus andinus and Phoenicoparrus jamesi. As a positive control, nine additional fecal samples were collected from the Zool\u00f3gico Nacional, Santiago, Chile, where only P. chilensis is maintained. DNA was extracted and initially screened via Sanger sequencing. To robustly assign taxonomic identity, we implemented a probabilistic framework combining maximum likelihood and Bayesian inference to estimate the posterior probability of species membership based on observed genetic variants. This approach revealed the presence of two genetically distinct groups not attributable to P. chilensis, indicating the coexistence and detectability of other flamingo taxa through fecal DNA. Our results demonstrate the value of integrating mitogenomic resources, metabarcoding, and statistical inference for non-invasive species identification in mixed-species systems. This framework provides a scalable tool for biodiversity monitoring and has direct implications for conservation strategies and evidence-based policy development in fragile high-Andean ecosystems.<\/jats:p>","DOI":"10.3390\/biology15151295","type":"journal-article","created":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T13:42:36Z","timestamp":1785850956000},"page":"1295","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Mitogenome-Informed Metabarcoding for Non-Invasive Identification of Sympatric Flamingos in Northern Chile"],"prefix":"10.3390","volume":"15","author":[{"given":"Stephanie","family":"Barr\u00eda-Iriarte","sequence":"first","affiliation":[{"name":"Plataforma de Monitoreo Gen\u00f3mico y Ambiental, Departamento de Qu\u00edmica, Facultad de Ciencias, Universidad de Tarapac\u00e1, Arica 1000007, Chile"},{"name":"Centro Nacional de Investigaci\u00f3n en R\u00edos, Invasiones y Sistemas (IRIS), Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Diego","family":"Soto-Jim\u00e9nez","sequence":"additional","affiliation":[{"name":"Laboratorio de Bioqu\u00edmica, Departamento de Qu\u00edmica, Facultad de Ciencias, Universidad de Tarapac\u00e1, Arica 1000007, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Flavia","family":"Salcedo-D\u00edaz","sequence":"additional","affiliation":[{"name":"Unidad de Medio Ambiente del Consejo de Pueblos Atacame\u00f1os (UMA-CPA), San Pedro de Atacama 1410000, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-0343-6113","authenticated-orcid":false,"given":"Noem\u00ed","family":"Labra-Or\u00f3stica","sequence":"additional","affiliation":[{"name":"Unidad de Medio Ambiente del Consejo de Pueblos Atacame\u00f1os (UMA-CPA), San Pedro de Atacama 1410000, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7913-7963","authenticated-orcid":false,"given":"Pablo","family":"Aguilar","sequence":"additional","affiliation":[{"name":"Laboratorio de Complejidad Microbiana y Ecolog\u00eda Funcional, Instituto Antofagasta, and Centro de Bioingenier\u00eda y Biotecnolog\u00eda (CeBiB), Universidad de Antofagasta, Antofagasta 1240000, Chile"},{"name":"Departamento de Biotecnolog\u00eda, Facultad de Ciencias del Mar y Recursos Biol\u00f3gicos, Universidad de Antofagasta, Antofagasta 1240000, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8273-9743","authenticated-orcid":false,"given":"Pablo","family":"Valladares-Fa\u00fandez","sequence":"additional","affiliation":[{"name":"Departamento de Biolog\u00eda, Facultad de Ciencias, Universidad de Tarapac\u00e1, Arica 1000007, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-1612-171X","authenticated-orcid":false,"given":"Juan P.","family":"Mu\u00f1oz","sequence":"additional","affiliation":[{"name":"Laboratorio de Bioqu\u00edmica, Departamento de Qu\u00edmica, Facultad de Ciencias, Universidad de Tarapac\u00e1, Arica 1000007, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3380-389X","authenticated-orcid":false,"given":"Claudio","family":"Quezada-Romegialli","sequence":"additional","affiliation":[{"name":"Plataforma de Monitoreo Gen\u00f3mico y Ambiental, Departamento de Qu\u00edmica, Facultad de Ciencias, Universidad de Tarapac\u00e1, Arica 1000007, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1227","DOI":"10.1007\/s10531-024-02816-x","article-title":"Still standing on one leg: A systematic review of threats, priorities, and conservation perspectives for flamingos (Phoenicopteridae)","volume":"33","author":"Delfino","year":"2024","journal-title":"Biodivers. Conserv."},{"key":"ref_2","first-page":"73","article-title":"Bill Morphology of South American Flamingos","volume":"104","author":"Mascitti","year":"2002","journal-title":"Condor Ornithol. Appl."},{"key":"ref_3","first-page":"328","article-title":"Distribution and Spatial Patterning of Flamingos in the Andean Altiplano","volume":"96","author":"Hurlbert","year":"1979","journal-title":"Auk"},{"key":"ref_4","first-page":"276","article-title":"Seasonal Distribution, Abundance, and Nesting of Puna, Andean, and Chilean Flamingos","volume":"109","author":"Caziani","year":"2007","journal-title":"Condor Ornithol. Appl."},{"key":"ref_5","first-page":"103","article-title":"Waterbird Richness in Altiplano Wetlands of Northwestern Argentina","volume":"24","author":"Caziani","year":"2001","journal-title":"Waterbirds Int. J. Waterbird Biol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"827","DOI":"10.1007\/s11273-022-09872-6","article-title":"The arid Andean plateau waterscapes and the lithium triangle: Flamingos as flagships for conservation of high-altitude wetlands under pressure from mining development","volume":"30","author":"Marconi","year":"2022","journal-title":"Wetl. Ecol. Manag."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Guti\u00e9rrez, J.S., Moore, J.N., Donnelly, J.P., Dorador, C., Navedo, J.G., and Senner, N.R. (2022). Climate change and lithium mining influence flamingo abundance in the Lithium Triangle. Proc. R. Soc. B Biol. Sci., 289.","DOI":"10.1098\/rspb.2021.2388"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"e37","DOI":"10.1017\/S0959270924000273","article-title":"A review of the ecology and conservation of the Andean Flamingo Phoenicoparrus andinus and Puna Flamingo P. jamesi in South America","volume":"34","author":"Derlindati","year":"2024","journal-title":"Bird Conserv. Int."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Torres, C.R., Ogawa, L.M., Gillingham, M.A.F., Ferrari, B., and van Tuinen, M. (2014). A multi-locus inference of the evolutionary diversification of extant flamingos (Phoenicopteridae). BMC Evol. Biol., 14.","DOI":"10.1186\/1471-2148-14-36"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"3519","DOI":"10.3109\/19401736.2015.1074198","article-title":"Complete mitochondrial genome of the American flamingo, Phoenicopterus ruber (Phoenicopteriformes, Phoenicopteridae)","volume":"27","author":"Luo","year":"2016","journal-title":"Mitochondrial DNA Part A"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"170027","DOI":"10.1038\/sdata.2017.27","article-title":"Metazoan mitochondrial gene sequence reference datasets for taxonomic assignment of environmental samples","volume":"4","author":"Machida","year":"2017","journal-title":"Sci. Data"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"4493","DOI":"10.1038\/s41598-018-22817-5","article-title":"Demonstration of the potential of environmental DNA as a tool for the detection of avian species","volume":"8","author":"Ushio","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_13","first-page":"425","article-title":"Metabarcoding reveals waterbird diet in a French Ramsar wetland: Implications for ecosystem management","volume":"9","author":"Fablet","year":"2024","journal-title":"Knowl. Manag. Aquat. Ecosyst."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"631","DOI":"10.1002\/ece3.4787","article-title":"From feces to data: A metabarcoding method for analyzing consumed and available prey in a bird-insect food web","volume":"9","author":"Vesterinen","year":"2019","journal-title":"Ecol. Evol."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Sacc\u00f2, M., Campbell, M.A., Aguilar, P., Salazar, G., Berry, T.E., Heydenrych, M.J., Lawrie, A., White, N.E., Harrod, C., and Allentoft, M.E. (2025). Metazoan diversity in Chilean hypersaline lakes unveiled by environmental DNA. Front. Ecol. Evol., 13.","DOI":"10.3389\/fevo.2025.1504666"},{"key":"ref_16","first-page":"3","article-title":"Diet and feeding selectivity of the Andean Flamingo Phoenicoparrus andinus and Chilean Flamingo Phoenicopterus chilensis in lowland wintering areas","volume":"68","author":"Polla","year":"2018","journal-title":"Wildfowl"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1186\/s40693-014-0015-1","article-title":"Diet of the Chilean flamingo Phoenicopterus chilensis (Phoenicopteriformes: Phoenicopteridae) in a coastal wetland in Chilo\u00e9, southern Chile","volume":"87","author":"Tobar","year":"2014","journal-title":"Rev. Chil. Hist. Nat."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"523","DOI":"10.4067\/S0718-19572017000300009","article-title":"Variaci\u00f3n interanual en la dieta invernal del flamenco chileno Phoenicopterus chilensis (Aves: Phoenicopteriformes) en el humedal marino de Bah\u00eda Caul\u00edn, Chilo\u00e9, sur de Chile","volume":"52","author":"Tobar","year":"2017","journal-title":"Rev. Biol. Mar. Y Oceanogr."},{"key":"ref_19","unstructured":"Andersson, R., Gebhard, C., Miguel-Escalada, I., Hoof, I., Bornholdt, J., Boyd, M., Chen, Y., Zhao, X., and Andrews, S. (FastQC: A Quality Control Tool for High Throughput Sequence Data, 2010). FastQC: A Quality Control Tool for High Throughput Sequence Data, Version 0.11."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"10","DOI":"10.14806\/ej.17.1.200","article-title":"Cutadapt removes adapter sequences from high-throughput sequencing reads","volume":"17","author":"Martin","year":"2011","journal-title":"EMBnet. J."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Morgan-Richards, M., Trewick, S.A., Bartosch-H\u00e4rlid, A., Kardailsky, O., Phillips, M.J., McLenachan, P.A., and Penny, D. (2008). Bird evolution: Testing the Metaves clade with six new mitochondrial genomes. BMC Evol. Biol., 8.","DOI":"10.1186\/1471-2148-8-20"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e72","DOI":"10.1002\/edn3.72","article-title":"Novel universal primers for metabarcoding environmental DNA surveys of marine mammals and other marine vertebrates","volume":"2","author":"Valsecchi","year":"2020","journal-title":"Environ. DNA"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1038\/nmeth.3869","article-title":"DADA2: High-resolution sample inference from Illumina amplicon data","volume":"13","author":"Callahan","year":"2016","journal-title":"Nat. Methods"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"6968","DOI":"10.1038\/s41467-022-34630-w","article-title":"Muscle5: High-accuracy alignment ensembles enable unbiased assessments of sequence homology and phylogeny","volume":"13","author":"Edgar","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Price, M.N., Dehal, P.S., and Arkin, A.P. (2010). FastTree 2\u2014Approximately maximum-likelihood trees for large alignments. PLoS ONE, 5.","DOI":"10.1371\/journal.pone.0009490"},{"key":"ref_26","unstructured":"R Core Team (2020). RA Language and Environment for Statistical Computing, R Foundation for Statistical Computing."},{"key":"ref_27","unstructured":"Posit Software (2025). RStudio: Integrated Development Environment for R, Posit Software."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1111\/zsc.12576","article-title":"Mitogenomes provide insights into the phylogeny and evolution of brittle stars (Echinodermata, Ophiuroidea)","volume":"52","author":"Sun","year":"2023","journal-title":"Zool. Scr."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"e73270","DOI":"10.1002\/ece3.73270","article-title":"Mining for Mitochondria: 68 Mitogenomes for Wrasses and Parrotfishes (F: Labridae) From Off-Target UCE Data","volume":"16","author":"Swami","year":"2026","journal-title":"Ecol. Evol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2045","DOI":"10.1111\/j.1365-294X.2012.05470.x","article-title":"Towards next-generation biodiversity assessment using DNA metabarcoding","volume":"21","author":"Taberlet","year":"2012","journal-title":"Mol. Ecol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"5872","DOI":"10.1111\/mec.14350","article-title":"Environmental DNA metabarcoding: Transforming how we survey animal and plant communities","volume":"26","author":"Deiner","year":"2017","journal-title":"Mol. Ecol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1931","DOI":"10.1111\/j.1365-294X.2011.05403.x","article-title":"Who is eating what: Diet assessment using next generation sequencing","volume":"21","author":"Pompanon","year":"2012","journal-title":"Mol. Ecol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"612","DOI":"10.1177\/1747493018778713","article-title":"Multisociety Consensus Quality Improvement Revised Consensus Statement for Endovascular Therapy of Acute Ischemic Stroke","volume":"13","author":"Sacks","year":"2018","journal-title":"Int. J. Stroke"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1985","DOI":"10.1111\/2041-210X.13276","article-title":"Non-specific amplification compromises environmental DNA metabarcoding with COI","volume":"10","author":"Collins","year":"2019","journal-title":"Methods Ecol. Evol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"6310","DOI":"10.1002\/ece3.6362","article-title":"In silico and empirical evaluation of twelve metabarcoding primer sets for insectivorous diet analyses","volume":"10","author":"Tournayre","year":"2020","journal-title":"Ecol. Evol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"819","DOI":"10.1111\/1755-0998.12355","article-title":"Universal and blocking primer mismatches limit the use of high-throughput DNA sequencing for the quantitative metabarcoding of arthropods","volume":"15","author":"Mir","year":"2015","journal-title":"Mol. Ecol. Resour."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Elbrecht, V., and Leese, F. (2015). Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass\u2014Sequence Relationships with an Innovative Metabarcoding Protocol. PLoS ONE, 10.","DOI":"10.7287\/peerj.preprints.1023v1"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1975","DOI":"10.1038\/s41598-018-20427-9","article-title":"Methylation-based enrichment facilitates low-cost, noninvasive genomic scale sequencing of populations from feces","volume":"8","author":"Chiou","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1186\/2049-2618-2-19","article-title":"Choice of bacterial DNA extraction method from fecal material influences community structure as evaluated by metagenomic analysis","volume":"2","author":"Bahl","year":"2014","journal-title":"Microbiome"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"17909","DOI":"10.1038\/s41598-022-20888-z","article-title":"Natural and after colon washing fecal samples: The two sides of the coin for investigating the human gut microbiome","volume":"12","author":"Piancone","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"e8881","DOI":"10.1002\/ece3.8881","article-title":"DNA metabarcoding quantifies the relative biomass of arthropod taxa in songbird diets: Validation with camera-recorded diets","volume":"12","author":"Verkuil","year":"2022","journal-title":"Ecol. Evol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"604","DOI":"10.1111\/1755-0998.12508","article-title":"How to limit false positives in environmental DNA and metabarcoding?","volume":"16","author":"Ficetola","year":"2016","journal-title":"Mol. Ecol. Resour."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1231","DOI":"10.1111\/1755-0998.13512","article-title":"Strategies for sample labelling and library preparation in DNA metabarcoding studies","volume":"22","author":"Bohmann","year":"2022","journal-title":"Mol. Ecol. Resour."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"945","DOI":"10.1093\/genetics\/155.2.945","article-title":"Inference of population structure using multilocus genotype data","volume":"155","author":"Pritchard","year":"2000","journal-title":"Genetics"},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Wright, E.S., and Vetsigian, K.H. (2016). Quality filtering of Illumina index reads mitigates sample cross-talk. BMC Genom., 17.","DOI":"10.1186\/s12864-016-3217-x"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1620","DOI":"10.1111\/1755-0998.13227","article-title":"Tagsteady: A metabarcoding library preparation protocol to avoid false assignment of sequences to samples","volume":"20","author":"Bohmann","year":"2020","journal-title":"Mol. Ecol. Resour."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1289","DOI":"10.1111\/1755-0998.12402","article-title":"Tag jumps illuminated\u2014Reducing sequence-to-sample misidentifications in metabarcoding studies","volume":"15","author":"Schnell","year":"2015","journal-title":"Mol. Ecol. Resour."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1186\/s13323-015-0022-2","article-title":"Maternal ancestry and population history from whole mitochondrial genomes","volume":"6","author":"Kivisild","year":"2015","journal-title":"Investig. Genet."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1525","DOI":"10.1098\/rspb.2005.3056","article-title":"Problems with mitochondrial DNA as a marker in population, phylogeographic and phylogenetic studies: The effects of inherited symbionts","volume":"272","author":"Hurst","year":"2005","journal-title":"Proc. R. Soc. B Biol. Sci."}],"container-title":["Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-7737\/15\/15\/1295\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T13:51:48Z","timestamp":1785851508000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-7737\/15\/15\/1295"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,4]]},"references-count":49,"journal-issue":{"issue":"15","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["biology15151295"],"URL":"https:\/\/doi.org\/10.3390\/biology15151295","relation":{},"ISSN":["2079-7737"],"issn-type":[{"value":"2079-7737","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,4]]}}}