Conservation Genetics

, Volume 16, Issue 3, pp 717–727 | Cite as

Detecting natural hybridization between two vulnerable Andean pupfishes (Orestias agassizii and O. luteus) representative of the Altiplano endemic fisheries

  • Yareli Esquer-GarrigosEmail author
  • Bernard Hugueny
  • Carla Ibañez
  • Claudia Zepita
  • Kellie Koerner
  • Josie Lambourdière
  • Arnaud Couloux
  • Philippe Gaubert
Research Article


The detection of hybridization among freshwater fish species is of main concern for conservation programs and fish farming. We assessed the incidence of natural hybridization between two vulnerable species of Andean pupfishes (Cyprinodontidae; Orestias agassizii and O. luteus) that represent an important component of local fisheries. We combined mitochondrial and nuclear DNA sequencing, microsatellites genotyping and morphometrics to characterize hybridization patterns between the two species in lakes Titicaca and Uru Uru (N = 175). The clustering analysis of 10 microsatellite loci together with heterozygosity distribution at seven species-specific diagnostic sites in rhodopsin (nuclear DNA) proved to be a robust diagnostic tool to detect F1 and potential backcross hybrids. For the first time on a genetic basis, we confirmed the incidence of natural hybridization between the two Andean pupfishes, at frequency rates reaching almost 10 %. The morphological intermediacy criterion (relative to parental species) did not apply in hybrids, since the latter (i) deviated through their larger caudal peduncle height, and (ii) had greater, overlapping ranges in their morphometric ratios. Although viable F1 and F2 hybrids between O. agassizii and O. luteus had been reported under controlled conditions, the ecological and/or demographic factors facilitating interspecific gene flow in the wild remain speculative. We recommend that regional fish farming and restocking programs targeting O. agassizii and O. luteus call on the genetic diagnosis of hybrids to avoid the potentially deleterious impacts of releasing hybrid populations in the wild.


Andean pupfishes Conservation genetics Hybridization Morphology Orestias agassizii Orestias luteus 



We gratefully acknowledge Alexander Flores, Kelvin Herbas and Ramón Catari for field assistance (UMSA), Claude Ferrara (Direction des Collections, MNHN) for taking photographs of Orestias, and Thierry Oberdorff (UMR BOREA, MNHN) for early stage project management. This research was funded by the program “Action initiative 2008: Diversité des poisons-n°45/Radiation adaptative des Orestias dans les lacs andins”, from the Département Ressources Vivantes-Institut de Recherche pour le Développement, and Agence Nationale de la Recherche (Project “FISHLOSS-ANR-09-PEXT-008). This publication was made possible through support provided by the Institut de Recherche pour le Développement-Département Soutien et Formation to YEG. We thank three anonymous reviewers for their useful comments on an earlier version of this draft. This is publication ISEM 2014-225.

Supplementary material

10592_2015_695_MOESM1_ESM.pdf (10 mb)
Supplementary material 1 (PDF 10190 kb)


  1. Abbott R et al (2013) Hybridization and speciation. J Evol Biol 26:229–246CrossRefPubMedGoogle Scholar
  2. Anderson EC, Thompson EA (2002) A model-based method for identifying species hybrids using multilocus genetic data. Genetics 160:1217–1229PubMedCentralPubMedGoogle Scholar
  3. Aspiazu WI (2002) Hibridación artificial entre carachi (Orestias agassii) y punku (Orestias luteus) “killifish” del Lago Titicaca. In. Facultad de Agronomía, Universidad Mayor de San Andrés, Tésis de Licenciatura, La Paz, p 62Google Scholar
  4. Bustamante E, Treviño H (1980) Descripción de las pesquerías en el Lago Titicaca 1975–1979. Instituto Mar, Puno, p 73Google Scholar
  5. Campton DE (1987) Natural hybridization and introgression in fishes: methods of detection and genetic interpretations. In: Ryman N, Utter F (eds) Population genetics and fishery management. University of Washington Press, Seatle, pp 161–192Google Scholar
  6. Carson EW, Dowling TE (2006) Influence of hydrogeographic history and hybridization on the distribution of genetic variation in the pupfishes Cyprinodon atrotus and C. bifasciatus. Mol Ecol 15:667–679CrossRefPubMedGoogle Scholar
  7. Carson EW, Maza-Benignos M, Lourdes Lozano-Vilano M, Vela-Valladares L, Banda-Villanueva I, Turner TF (2014) Conservation genetic assessment of the critically endangered Julimes pupfish, Cyprinodon julimes. Conserv Genet 15:483–488CrossRefGoogle Scholar
  8. Chatfield C, Collins AJ (1980) Introduction to multivariate analysis. Chapman and Hall, London 246 pCrossRefGoogle Scholar
  9. Collins RA (2012) Investigating interspecific hybridisation in ornamental fishes. Figshare. doi: 10.6084/m9.figshare.96149
  10. Dąbrowski MJ, Pilot M, Kruczyk M, Żmihorski M, Umer HM, Gliwicz J (2014) Reliability assessment of null allele detection: inconsistencies between and within different methods. Mol Ecol Resour 14:361–373CrossRefPubMedGoogle Scholar
  11. Dray S, Dufour AB, Chessel D (2007) The ade4 package-II: two-table and K-table methods. R News 7:47–52Google Scholar
  12. Echelle AA, Carson EW, Echelle AF, Van Den Bussche RA, Dowling TE, Meyer A (2005) Historical biogeography of the New World pupfish genus Cyprinodon (Teleostei: Cyprinodontidae). Copeia 2:320–339CrossRefGoogle Scholar
  13. Esquer-Garrigos Y, Lambourdiere J, Ibañez C, Gaubert P (2011) Characterization of ten polymorphic microsatellite loci in the Andean pupfish Orestias agassizii, with cross-amplification in the sympatric O. luteus. Conserv Genet Resour 3:17–19CrossRefGoogle Scholar
  14. Esquer-Garrigos Y et al (2013) Non-invasive ancient DNA protocol for fluid-preserved specimens and phylogenetic systematics of the genus Orestias (Teleostei: Cyprinodontidae). Zootaxa 3640:373–394CrossRefGoogle Scholar
  15. Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the software structure: a simulation study. Mol Ecol 14:2611–2620CrossRefPubMedGoogle Scholar
  16. Excoffier L, Heckel G (2006) Computer programs for population genetics data analysis: a survival guide. Nature Rev Genet 7:745–758CrossRefPubMedGoogle Scholar
  17. Falk TM, Teugels GG, Abban EK, Villwock W, Renwrantz L (2003) Phylogeographic patterns in populations of the black-chinned tilapia complex (Teleostei, Cichlidae) from coastal areas in West Africa: support for the refuge zone theory. Mol Phylogenet Evol 27:81–92CrossRefPubMedGoogle Scholar
  18. Felsestein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791CrossRefGoogle Scholar
  19. Ferguson MM, Danzmann RG (1987) Deviation from morphological intermediacy in interstrain hybrids of rainbow trout, Salmo gairdneri. Environ Biol Fishes 18:249–256CrossRefGoogle Scholar
  20. Genovart M (2008) Natural hybridization and conservation. Biodivers Conserv 18:1435–1439CrossRefGoogle Scholar
  21. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  22. Hubbs CL (1955) Hybridization between fish species in nature. Syst Zool 4:1–20CrossRefGoogle Scholar
  23. Kalinowski ST (2005) HP-RARE 1.0: a computer program for performing rarefaction on measures of allelic richness. Mol Ecol Notes 5:187–189CrossRefGoogle Scholar
  24. Kundzewicz ZW et al (2008) The implications of projected climate change for freshwater resources and their management. Hydrol Sci J 53:3–10CrossRefGoogle Scholar
  25. Lauzanne L (1982) Les Orestias (Pisces, Cyprinodontidae) du petit lac Titicaca. Rev d’Hydrobiol Trop 15:39–70Google Scholar
  26. Lauzanne L (1992) Native species. The Orestias. In: Dejoux C, Iltis A (eds) Lake Titicaca: a synthesis of limnological knowledge. Kluwer Academic Publishers, Dordrecht, pp 405–419CrossRefGoogle Scholar
  27. Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451–1452CrossRefPubMedGoogle Scholar
  28. Lüssen A, Falk T, Villwock W (2003) Phylogenetic patterns in populations of Chilean species of the genus Orestias (Teleostei: Cyprinodontidae): results of mitochondrial DNA analysis. Mol Biol Evol 29:151–160Google Scholar
  29. Machordom A, Doadrio I (2001) Evidence of a Cenozoic Betic-Kabilian connection based on freshwater fish phylogeography (Luciobarbus, Cyprinidae). Mol Phylogenet Evol 18:252–263CrossRefPubMedGoogle Scholar
  30. Maldonado E, Hubert N, Sagnes P, De Mérona B (2009) Morphology-diet relationships in four killifishes (Teleostei, Cyprinodontidae, Orestias) from Lake Titicaca. J Fish Biol 74:502–520CrossRefPubMedGoogle Scholar
  31. Mallet J (2005) Hybridization as an invasion of the genome. Trends Ecol Evol 20:229–237CrossRefPubMedGoogle Scholar
  32. Müller R (1993) Critical remarks on the revision of the genus Orestias (Pisces Cyprinodontidae) by Parenti (1984). Zool Jahrb Abt fuer Anat und Ontog der Tiere 123:31–58Google Scholar
  33. Neff NA, Smith GR (1979) Multivariate analysis of hybrid fishes. Syst Zool 28:176–196CrossRefGoogle Scholar
  34. Nielsen EE, Bach LA, Kotlicki P (2006) Hybridlab (version 1.0): a program for generating simulated hybrids from population samples. Mol Ecol Notes 6:971–973CrossRefGoogle Scholar
  35. Northcote TG (1992) Eutrophication and pollution problems. In: Dejoux C, Iltis A (eds) Lake Titicaca: a synthesis of limnological knowledge. Kluwer Academic Publishers, Dordrecht, pp 551–559CrossRefGoogle Scholar
  36. Orlove BS, Levieil DP, Treviño H (1992) Social and economic aspects of the fisheries. In: Dejoux C, Iltis A (eds) Lake Titicaca: a synthesis of limnological knowledge. Kluwer Academic Publishers, Dordrecht, pp 500–504Google Scholar
  37. Parenti LR (1984) A taxonomic revision of the Andean killifish genus Orestias (Cyprinodontiformes, Cyprinodontidae). Bull Am Mus Nat Hist 178:107–214Google Scholar
  38. Peakall R, Smouse PE (2006) GenAlEx 6: genetic analysis in excel. Population genetic software for teaching and research. Mol Ecol Notes 6:288–295CrossRefGoogle Scholar
  39. Peakall R, Smouse PE (2012) GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research—an update. Bioinformatics 28:2537–2539CrossRefPubMedCentralPubMedGoogle Scholar
  40. Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155:945–959PubMedCentralPubMedGoogle Scholar
  41. Raymond M, Rousset F (1995) GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism. J Hered 86:248–249Google Scholar
  42. Rhymer JM, Simberloff D (1996) Extinction by hybridization and introgression. Annu Rev Ecol Evol Syst 27:83–109CrossRefGoogle Scholar
  43. Rice WR (1989) Analyzing tables of statistical tests. Evolution 43:223–225CrossRefGoogle Scholar
  44. Sanz N, Araguas RM, Fernandez R, Vera M, Garcia-Marin JL (2009) Efficiency of markers and methods for detecting hybrids and introgression in stocked populations. Conserv Genet 10:225–236CrossRefGoogle Scholar
  45. Schwenk K, Brede N, Streit B (2008) Introduction. Extent, processes and evolutionary impact of interspecific hybridization in animals. Philos Trans R Soc Lond B Biol Sci 363:2805–2811CrossRefPubMedCentralPubMedGoogle Scholar
  46. Scribner KT, Page KS, Bartron ML (2001) Hybridization in freshwater fishes: a review of case studies and cytonuclear methods of biological inference. Rev Fish Biol Fish 10:293–323CrossRefGoogle Scholar
  47. Stephens M, Donelly P (2003) A comparison of Bayesian methods for haplotype reconstruction. Am J Hum Genet 73:1162–1169CrossRefPubMedCentralPubMedGoogle Scholar
  48. Stephens M, Smith NJ, Donelly P (2001) A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 68:978–989CrossRefPubMedCentralPubMedGoogle Scholar
  49. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutinary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739CrossRefPubMedCentralPubMedGoogle Scholar
  50. Tchernavin VV (1944) A revision of the subfamily Orestiinae. Proc Zool Soc Lond 114:140–233CrossRefGoogle Scholar
  51. Team RDC (2008) R: A language and environment for statistical computing. R Foundation for Statistical Computing, ViennaGoogle Scholar
  52. Thielsch A, Völker E, Kraus RHS, Schwenk K (2012) Discrimination of hybrid classes using cross-species amplification of microsatellite loci: methodological challenges and solutions in Daphnia. Mol Ecol Resour 12:697–705CrossRefPubMedGoogle Scholar
  53. Tobler M, Carson EW (2010) Environmental variation, hybridization, and phenotypic diversification in Cautro Ciénegas pupfishes. J Evol Biol 23:1475–1489CrossRefPubMedGoogle Scholar
  54. Treviño H, Torres Caleron J, Roncal Gutierrez M (1992) The fishery potential. In: Dejoux C, Iltis A (eds) Lake Titicaca: a synthesis of limnological knowledge. Kluwer Academic Publishers, Dordrecht, pp 539–549CrossRefGoogle Scholar
  55. Turner BJ, Duvernell DD, Bunt TM, Barton MG (2008) Reproductive isolation among endemic pupfishes (Cyprinodon) on San Salvador Island, Bahamas: microsatellite evidence. Biol J Linn Soc 95:566–582CrossRefGoogle Scholar
  56. Vähä JP, Primmer CR (2006) Efficiency of model-based Bayesian methods for detecting hybrid individuals under different hybridization scenarios and with different numbers of loci. Mol Ecol 15:63–72CrossRefPubMedGoogle Scholar
  57. Van Damme PA, Carvajal-Vallejos F, Sarmiento J, Barrera S, Osinaga K, Miranda-Chumacero G (2009) Peces. Libro rojo de la fauna silvestre de vertebrados de Bolivia. Ministerio de Medio Ambiente y Agua, La Paz, pp 25–90Google Scholar
  58. Van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P (2004) MICRO-CHECKER: software for identifying and correcting genotyping errors in microsatellite data. Mol Ecol Notes 4:535–538CrossRefGoogle Scholar
  59. Vellard J (1992) Former lake fisheries and fish fauna of the lake. In: Dejoux C, Iltis A (eds) Lake Titicaca: a synthesis of limnological knowledge. Kluwer Academic Publishers, Dordrecht, pp 495–499Google Scholar
  60. Vila I, Pardo R, Scott S (2007) Freshwater fishes of the Altiplano. Aquat Ecosyst Health 10:201–211CrossRefGoogle Scholar
  61. Villwock W (1986) Speciation and adaptive radiation in the Andean Orestias fishes. In: Vuilleumier F, Monasterio M (eds) High altitude tropical biogeography. Oxford University Press, Oxford, pp 387–403Google Scholar
  62. Villwock W, Sienknecht U (1995) Intraspezifische variabilität im genus Orestias Valenciennes, 1839 (Teleostei: Cyprinodontidae) und zum problem der artidentität. Mitteilungen aus dem Hamburgischen Zoologischen Museum und Institut 92:381–398Google Scholar
  63. Villwock W, Sienknecht U (1996) Contribution to the knowledge and history of Chilean fishes. The cyprinodontids of the genus Orestias Val. 1839 (Teleostei: Cyprinodontidae) of the Chilean Altiplano. Medio Ambiene 13:119–126Google Scholar
  64. Vogel LS, Johnson SG (2008) Estimation of hybridization and introgression frequency in toads (genus: Bufo) using DNA sequence variation at mitochondrial and nuclear loci. J Herpetol 42:61–75CrossRefGoogle Scholar
  65. Wirtz P (1999) Mother species-father species: unidirectional hybridization in animals with female choice. Anim Behav 58:1–12CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Yareli Esquer-Garrigos
    • 1
    Email author
  • Bernard Hugueny
    • 1
  • Carla Ibañez
    • 1
    • 2
  • Claudia Zepita
    • 2
  • Kellie Koerner
    • 3
  • Josie Lambourdière
    • 4
  • Arnaud Couloux
    • 5
  • Philippe Gaubert
    • 1
    • 6
  1. 1.UMR BOREA, Département Milieux et Peuplements Aquatiques, MNHN-CNRS 7208-IRD 207-UPMCMuséum National d’Histoire NaturelleParisFrance
  2. 2.Instituto de Ecología-Unidad de LimnologíaUniversidad Mayor de San AndrésLa PazBolivia
  3. 3.LafayetteUSA
  4. 4.Département Systématique et Evolution, SSM-IFR 101Muséum National d’Histoire NaturelleParisFrance
  5. 5.GenoscopeCentre National de SéquençageEvry CedexFrance
  6. 6.Institut des Sciences de l’Evolution de Montpellier (ISEM) - UM2-CNRS-IRDUniversité de MontpellierMontpellier Cedex 05France

Personalised recommendations