{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T17:24:00Z","timestamp":1789665840857,"version":"4.0.1"},"reference-count":56,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2023,4,18]],"date-time":"2023-04-18T00:00:00Z","timestamp":1681776000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100008725","name":"National Agency of Investigation and Innovation (ANII, Uruguay)","doi-asserted-by":"publisher","award":["POS_EXT_2015_122792"],"award-info":[{"award-number":["POS_EXT_2015_122792"]}],"id":[{"id":"10.13039\/100008725","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100008725","name":"National Agency of Investigation and Innovation (ANII, Uruguay)","doi-asserted-by":"publisher","award":["T0214\/33883\/2019\/sm"],"award-info":[{"award-number":["T0214\/33883\/2019\/sm"]}],"id":[{"id":"10.13039\/100008725","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Zempelin-Stiftung-Deutsches Stiftungszentrum","award":["POS_EXT_2015_122792"],"award-info":[{"award-number":["POS_EXT_2015_122792"]}]},{"name":"Zempelin-Stiftung-Deutsches Stiftungszentrum","award":["T0214\/33883\/2019\/sm"],"award-info":[{"award-number":["T0214\/33883\/2019\/sm"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Genes"],"abstract":"<jats:p>The species delimitation of the marine bivalve species complex Aequiyoldia eightsii in South America and Antarctica is complicated by mitochondrial heteroplasmy and amplification bias in molecular barcoding. In this study, we compare different data sources (mitochondrial cytochrome c oxidase subunit I (COI) sequences; nuclear and mitochondrial SNPs). Whilst all the data suggest that populations on either side of the Drake Passage belong to different species, the picture is less clear within Antarctic populations, which harbor three distinct mitochondrial lineages (p-dist \u2248 6%) that coexist in populations and in a subset of individuals with heteroplasmy. Standard barcoding procedures lead to amplification bias favoring either haplotype unpredictably and thus overestimate the species richness with high confidence. However, nuclear SNPs show no differentiation akin to the trans-Drake comparison, suggesting that the Antarctic populations represent a single species. Their distinct haplotypes likely evolved during periods of temporary allopatry, whereas recombination eroded similar differentiation patterns in the nuclear genome after secondary contact. Our study highlights the importance of using multiple data sources and careful quality control measures to avoid bias and increase the accuracy of molecular species delimitation. We recommend an active search for mitochondrial heteroplasmy and haplotype-specific primers for amplification in DNA-barcoding studies.<\/jats:p>","DOI":"10.3390\/genes14040935","type":"journal-article","created":{"date-parts":[[2023,4,19]],"date-time":"2023-04-19T03:20:39Z","timestamp":1681874439000},"page":"935","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Mitochondrial Heteroplasmy and PCR Amplification Bias Lead to Wrong Species Delimitation with High Confidence in the South American and Antarctic Marine Bivalve Aequiyoldia eightsii Species Complex"],"prefix":"10.3390","volume":"14","author":[{"given":"Mariano","family":"Mart\u00ednez","sequence":"first","affiliation":[{"name":"Functional Ecology, Helmholtz Centre for Polar and Marine Research, Alfred Wegener Institute, Am Handelshafen 12, 27570 Bremerhaven, Germany"},{"name":"Oceanograf\u00eda y Ecolog\u00eda Marina, Instituto de Ecolog\u00eda y Ciencias Ambientales, Facultad de Ciencias, Universidad de la Rep\u00fablica, Igu\u00e1 4225, Montevideo 11400, Uruguay"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7620-0613","authenticated-orcid":false,"given":"Lars","family":"Harms","sequence":"additional","affiliation":[{"name":"Helmholtz Centre for Polar and Marine Research, Alfred Wegener Institute, Am Handelshafen 12, 27570 Bremerhaven, Germany"},{"name":"Helmholtz Institute for Functional Marine Biodiversity at the University of Oldenburg (HIFMB), Ammerl\u00e4nder Herrstrasse 231, 26129 Oldenburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Doris","family":"Abele","sequence":"additional","affiliation":[{"name":"Functional Ecology, Helmholtz Centre for Polar and Marine Research, Alfred Wegener Institute, Am Handelshafen 12, 27570 Bremerhaven, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christoph","family":"Held","sequence":"additional","affiliation":[{"name":"Functional Ecology, Helmholtz Centre for Polar and Marine Research, Alfred Wegener Institute, Am Handelshafen 12, 27570 Bremerhaven, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,4,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"313","DOI":"10.1098\/rspb.2002.2218","article-title":"Biological identifications through DNA Barcodes","volume":"270","author":"Hebert","year":"2003","journal-title":"Proc. Biol. Sci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"302","DOI":"10.3389\/fevo.2019.00302","article-title":"Review and interpretation of trends in DNA Barcoding","volume":"7","author":"DeSalle","year":"2019","journal-title":"Front. Ecol. Evol."},{"key":"ref_3","first-page":"294","article-title":"DNA Primers for amplification of mitochondrial Cytochrome c Oxidase Subunit I from diverse metazoan invertebrates","volume":"3","author":"Folmer","year":"1994","journal-title":"Mol. Mar. Biol. Biotechnol."},{"key":"ref_4","unstructured":"Steinke, D., Prosser, S.W.J., and Hebert, P.D.N. (2016). Marine Genomics, Springer."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"995","DOI":"10.1093\/molbev\/msv343","article-title":"Selection on mitochondrial variants occurs between and within individuals in an expanding invasion","volume":"33","author":"Rollins","year":"2016","journal-title":"Mol. Biol. Evol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"355","DOI":"10.1111\/j.1471-8286.2007.01678.x","article-title":"BOLD: The Barcode of Life Data System (Http:\/\/Www. Barcodinglife. Org)","volume":"7","author":"Ratnasingham","year":"2007","journal-title":"Mol. Ecol. Notes"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"806","DOI":"10.1080\/24701394.2019.1665036","article-title":"Conserved Numts mask a highly divergent mitochondrial-COI gene in a species complex of Australian stingless bees Tetragonula (Hymenoptera: Apidae)","volume":"30","author":"Zuntini","year":"2019","journal-title":"Mitochondrial DNA Part A"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"781","DOI":"10.1111\/1755-0998.12119","article-title":"Nuclear mitochondrial pseudogenes in Austinograea alayseae hydrothermal vent crabs (Crustacea: Bythograeidae): Effects on DNA Barcoding","volume":"13","author":"Kim","year":"2013","journal-title":"Mol. Ecol. Resour."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1080\/24701394.2020.1844677","article-title":"Mitochondrial heteroplasmy and pseudogenes in the Freshwater prawn, Macrobrachium amazonicum (Heller, 1862): DNA Barcoding and phylogeographic implications","volume":"32","author":"Iketani","year":"2021","journal-title":"Mitochondrial. DNA Part A"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1460","DOI":"10.1038\/s41598-018-38001-8","article-title":"Paternal leakage and mtDNA heteroplasmy in Rhipicephalus spp. Ticks","volume":"9","author":"Mastrantonio","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"375","DOI":"10.1093\/molbev\/msv224","article-title":"A Distinct mitochondrial genome with DUI-Like inheritance in the ocean quahog Arctica islandica","volume":"33","author":"Abele","year":"2016","journal-title":"Mol. Biol. Evol."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Doublet, V., Souty-Grosset, C., Bouchon, D., Cordaux, R., and Marcad\u00e9, I. (2008). A thirty million year-old inherited heteroplasmy. PLoS ONE, 3.","DOI":"10.1371\/journal.pone.0002938"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"509","DOI":"10.1093\/genetics\/118.3.509","article-title":"Length and restriction site heteroplasmy in the mitochondrial DNA of american shad (Alosa sapidissima)","volume":"118","author":"Bentzen","year":"1988","journal-title":"Genetics"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1093\/molbev\/msg025","article-title":"Paternal leakage of mitochondrial DNA in the great tit (Parus major)","volume":"20","author":"Kvist","year":"2003","journal-title":"Mol. Biol. Evol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1007\/s00294-010-0331-1","article-title":"The Occurrence of mtDNA heteroplasmy in multiple cetacean species","volume":"57","author":"Vollmer","year":"2011","journal-title":"Curr. Genet."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1016","DOI":"10.1093\/icb\/icz061","article-title":"Natural heteroplasmy and mitochondrial inheritance in bivalve molluscs","volume":"59","author":"Ghiselli","year":"2019","journal-title":"Integr. Comp. Biol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"e2760","DOI":"10.7717\/peerj.2760","article-title":"Pursuing the quest for better understanding the taxonomic distribution of the system of Doubly Uniparental Inheritance of mtDNA","volume":"4","author":"Gusman","year":"2016","journal-title":"PeerJ"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"7463","DOI":"10.1073\/pnas.91.16.7463","article-title":"An unusual type of mitochondrial DNA inheritance in the blue mussel Mytilus","volume":"91","author":"Zouros","year":"1994","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1007\/102_2018_4","article-title":"Doubly Uniparental Inheritance of mtDNA: An unappreciated defiance of a general rule","volume":"Volume 231","author":"Zouros","year":"2019","journal-title":"Cellular and Molecular Basis of Mitochondrial Inheritance"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Doucet-Beaupr\u00e9, H., Breton, S., Chapman, E.G., Blier, P.U., Bogan, A.E., Stewart, D.T., and Hoeh, W.R. (2010). Mitochondrial phylogenomics of the bivalvia (Mollusca): Searching for the origin and mitogenomic correlates of Doubly Uniparental Inheritance of mtDNA. BMC Evol. Biol., 10.","DOI":"10.1186\/1471-2148-10-50"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1111\/j.1439-0469.2007.00421.x","article-title":"A Mitochondrial phylogeography of Brachidontes variabilis (Bivalvia: Mytilidae) reveals three cryptic species","volume":"45","author":"Terranova","year":"2007","journal-title":"J. Zool. Syst. Evol. Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"413","DOI":"10.1007\/s00227-012-2099-y","article-title":"Heteroplasmy in a deep-Sea protobranch bivalve suggests an ancient origin of Doubly Uniparental Inheritance of mitochondria in bivalvia","volume":"160","author":"Boyle","year":"2012","journal-title":"Mar. Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1016\/j.ympev.2013.05.018","article-title":"Into the Deep: A phylogenetic approach to the bivalve subclass protobranchia","volume":"69","author":"Sharma","year":"2013","journal-title":"Mol. Phylogenetics Evol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1016\/j.seares.2013.12.008","article-title":"Differential adaptations between cold-stenothermal environments in the bivalve Lissarca Cf. miliaris (Philobryidae) from the Scotia Sea islands and Antarctic Peninsula","volume":"88","author":"Reed","year":"2014","journal-title":"J. Sea Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"445","DOI":"10.4067\/S0716-078X2012000400007","article-title":"Divergence time estimations and contrasting patterns of genetic diversity between antarctic and southern South America benthic invertebrates","volume":"85","author":"Poulin","year":"2012","journal-title":"Rev. Chil. Hist. Nat."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"200603","DOI":"10.1098\/rsos.200603","article-title":"Gene flow in the antarctic bivalve Aequiyoldia eightsii (Jay, 1839) suggests a role for the Antarctic Peninsula coastal current in larval dispersal","volume":"7","author":"Barnes","year":"2020","journal-title":"R. Soc. Open Sci."},{"key":"ref_27","first-page":"269","article-title":"A molecular perspective on the invasibility of the Southern Ocean benthos: The impact of hypoxia and temperature on gene expression in South American and Antarctic Aequiyoldia bivalves","volume":"14","author":"Held","year":"2023","journal-title":"Front. Physiol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1016\/j.pocean.2018.09.004","article-title":"Cryptic speciation in Southern Ocean Aequiyoldia eightsii (Jay, 1839): Mio-Pliocene trans-Drake Passage separation and diversification","volume":"174","author":"Rosenfeld","year":"2019","journal-title":"Prog. Oceanogr."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"e1800235","DOI":"10.1002\/pmic.201800235","article-title":"Connecting proteomics to Next-Generation Sequencing: Proteogenomics and its current applications in biology","volume":"19","author":"Low","year":"2019","journal-title":"Proteomics"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2310","DOI":"10.1111\/mec.13165","article-title":"SNP genotyping and population genomics from expressed sequences\u2014Current advances and future possibilities","volume":"24","author":"Pespeni","year":"2015","journal-title":"Mol. Ecol."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Garc\u00eda, G., R\u00edos, N., Guti\u00e9rrez, V., Serra, S., and Loureiro, M. (2019). Transcriptome-based SNP discovery and validation in the hybrid zone of the neotropical annual fish Genus Austrolebias. Genes, 10.","DOI":"10.3390\/genes10100789"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"5911","DOI":"10.1111\/mec.14338","article-title":"Population transcriptomic characterization of the genetic and expression variation of a candidate progenitor of Miscanthus energy Crops","volume":"26","author":"Yan","year":"2017","journal-title":"Mol. Ecol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Rogier, O., Chateigner, A., Amanzougarene, S., Lesage-Descauses, M.-C., Balzergue, S., Brunaud, V., Caius, J., Soubigou-Taconnat, L., Jorge, V., and Segura, V. (2018). Accuracy of RNAseq based SNP discovery and genotyping in Populus nigra. BMC Genom., 19.","DOI":"10.1186\/s12864-018-5239-z"},{"key":"ref_34","unstructured":"Bushnell, B. (2021, July 24). BBMap. BBMap Short Read Aligner, and Other Bioinformatic Tools. Available online: https:\/\/sourceforge.net\/projects\/bbmap\/."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"3211","DOI":"10.1093\/bioinformatics\/bts611","article-title":"SortMeRNA: Fast and accurate filtering of ribosomal RNAs in metatranscriptomic data","volume":"28","author":"Kopylova","year":"2012","journal-title":"Bioinformatics"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"644","DOI":"10.1038\/nbt.1883","article-title":"Full-length transcriptome assembly from RNA-Seq data without a reference genome","volume":"29","author":"Grabherr","year":"2011","journal-title":"Nat. Biotechnol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1038\/nmeth.1923","article-title":"Fast gapped-read alignment with Bowtie 2","volume":"9","author":"Langmead","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"2078","DOI":"10.1093\/bioinformatics\/btp352","article-title":"The sequence alignment\/map format and SAMtools","volume":"25","author":"Li","year":"2009","journal-title":"Bioinformatics"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"2156","DOI":"10.1093\/bioinformatics\/btr330","article-title":"The variant call format and VCFtools","volume":"27","author":"Danecek","year":"2011","journal-title":"Bioinformatics"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"3193","DOI":"10.1111\/mec.14792","article-title":"These aren\u2019t the loci you\u2019e looking for: Principles of effective SNP filtering for molecular ecologists","volume":"27","author":"Puritz","year":"2018","journal-title":"Mol. Ecol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"313","DOI":"10.1016\/j.ympev.2012.08.023","article-title":"MITOS: Improved de novo metazoan mitochondrial genome annotation","volume":"69","author":"Bernt","year":"2013","journal-title":"Mol. Phylogenetics Evol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"4216","DOI":"10.1111\/mec.12856","article-title":"What is genetic differentiation, and how should we measure It--GST, D, neither or both?","volume":"23","author":"Verity","year":"2014","journal-title":"Mol. Ecol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"479","DOI":"10.1093\/genetics\/131.2.479","article-title":"Analysis of Molecular Variance inferred from metric distances among DNA haplotypes: Application to human mitochondrial DNA restriction data","volume":"131","author":"Excoffier","year":"1992","journal-title":"Genetics"},{"key":"ref_44","unstructured":"RCore, T. (2016). R: A Language and Environment for Statistical Computing, R foundation for statistical computing."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1111\/1755-0998.12549","article-title":"Vcfr: A package to manipulate and visualize variant call format data in R","volume":"17","author":"Knaus","year":"2017","journal-title":"Mol. Ecol. Resour."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"3070","DOI":"10.1093\/bioinformatics\/btr521","article-title":"Adegenet 1.3-1: New tools for the analysis of genome-wide SNP data","volume":"27","author":"Jombart","year":"2011","journal-title":"Bioinformatics"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"e281","DOI":"10.7717\/peerj.281","article-title":"Poppr: An R package for genetic analysis of populations with clonal, partially clonal, and\/or sexual reproduction","volume":"2","author":"Kamvar","year":"2014","journal-title":"PeerJ"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1","DOI":"10.18637\/jss.v022.i04","article-title":"The Ade4 package: Implementing the duality diagram for ecologists","volume":"22","author":"Dray","year":"2007","journal-title":"J. Stat. Softw."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"419","DOI":"10.1093\/bioinformatics\/btp696","article-title":"Pegas: An R package for population genetics with an integrated\u2013modular approach","volume":"26","author":"Paradis","year":"2010","journal-title":"Bioinformatics"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"20190186","DOI":"10.1098\/rstb.2019.0186","article-title":"Faraway, so close. The comparative method and the potential of non-model animals in mitochondrial research","volume":"375","author":"Milani","year":"2020","journal-title":"Philos. Trans. R. Soc. Lond. B Biol. Sci."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"1624","DOI":"10.1093\/molbev\/msz117","article-title":"Sequencing disparity in the genomic era","volume":"36","author":"David","year":"2019","journal-title":"Mol. Biol. Evol."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"140424","DOI":"10.1098\/rsos.140424","article-title":"Regional differentiation and extensive hybridization between mitochondrial clades of the Southern Ocean giant sea spider Colossendeis megalonyx","volume":"2","author":"Dietz","year":"2015","journal-title":"R. Soc. Open Sci."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"3439","DOI":"10.1111\/j.1365-294X.2011.05173.x","article-title":"Evidence of hidden biodiversity, ongoing speciation and diverse patterns of genetic structure in giant antarctic amphipods","volume":"20","author":"Baird","year":"2011","journal-title":"Mol. Ecol."},{"key":"ref_54","unstructured":"Held, C. (2003). Antarctic Biology in A Global Context, Gregor Rare Books."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"203","DOI":"10.24199\/j.mmv.2014.71.16","article-title":"The identity of juvenile Polynoidae (Annelida) in the Southern Ocean revealed by DNA taxonomy, with notes on the status of Herdmanella gracilis ehlers Sensu augener","volume":"71","author":"Neal","year":"2014","journal-title":"Mem. Mus. Vic."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"2502","DOI":"10.1111\/j.1365-294X.2012.05512.x","article-title":"Comprehensive sampling reveals circumpolarity and sympatry in seven mitochondrial lineages of the Southern Ocean crinoid species Promachocrinus kerguelensis (Echinodermata)","volume":"21","author":"Hemery","year":"2012","journal-title":"Mol. Ecol."}],"container-title":["Genes"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4425\/14\/4\/935\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T19:18:16Z","timestamp":1760123896000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4425\/14\/4\/935"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,4,18]]},"references-count":56,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2023,4]]}},"alternative-id":["genes14040935"],"URL":"https:\/\/doi.org\/10.3390\/genes14040935","relation":{},"ISSN":["2073-4425"],"issn-type":[{"value":"2073-4425","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,4,18]]}}}