Genetic data confirm critical status of the reintroduced Dinaric population of Eurasian lynx

Abstract

Eurasian lynx (Lynx lynx) reintroduction to the Dinaric Mountains is considered one of the most successful reintroductions of a large predator. Six reintroduced animals founded the population, which rapidly expanded from Slovenia, through Croatia, and all the way to Bosnia and Herzegovina. However, a decrease of the population size has been observed during the last 10–15 years. Considering that possible inbreeding depression would be additive to threats like poaching, traffic mortality and prey base depletion, another extinction of this species from the Dinaric Mountains is a real possibility. We analyzed 204 samples collected between 1979 and 2010 using twenty microsatellite loci and 900-bp mitochondrial DNA control region sequence to evaluate conservation genetics aspects of this endangered population. Both markers confirmed low genetic variability of the Dinaric lynx population, and considerable effective inbreeding (0.3) compared to the source Carpathian population. Our analysis of effective population size and microsatellite variability supported field observations of decreasing population number. As a natural recolonization is a very remote possibility, we recommend population augmentation from a large source population.

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References

  1. Adams JR, Waits LP (2007) An efficient method for screening faecal DNA genotypes and detecting new individuals and hybrids in the red wolf (Canis rufus) experimental population area. Conserv Genet 8:123–131

    Article  CAS  Google Scholar 

  2. Barker JSF (2011) Effective population size of natural populations of Drosophila buzzatii, with a comparative evaluation of nine methods of estimation. Mol Ecol 20:4452–4471

    PubMed  Article  CAS  Google Scholar 

  3. Bensch S, Andrén H, Hansson B, Pedersen HC, Sand H, Sejberg D, Wabakken P, Åkesson M, Liberg O (2006) Selection for heterozygosity gives hope to a wild population of inbred wolves. PLoS One 1:e72. doi:10.1371/journal.pone.0000072

    PubMed  Article  Google Scholar 

  4. Breitenmoser U, Breitenmoser-Wursten C, Capt S (1998) Re-introduction and present status of lynx (Lynx lynx) in Switzerland. Hystrix 10:17–30

    Google Scholar 

  5. Breitenmoser-Würsten C, Obexer-Ruff G (2003) Population and conservation genetics of two re-introduced lynx (Lynx lynx) populations in Switzerland: a molecular evaluation 25 years after translocation. Progress report, KORA Bericht, Bern

  6. Broquet T, Petit E (2004) Quantifying genotyping errors in noninvasive population genetics. Mol Ecol 13:3601–3608

    PubMed  Article  CAS  Google Scholar 

  7. Butler JM, David VA, O’Brien SJ, Menotti-Raymond M (2002) The MeowPlex: a new DNA test using tetranucleotide STR markers for the domestic cat. Profiles DNA 5:7–10

    Google Scholar 

  8. Carmichael LE, Clark W, Strobeck C (2000) Development and characterization of microsatellite loci from lynx (Lynx canadiensis), and their use in other felids. Mol Ecol 9:2197–2199

    Google Scholar 

  9. Charlesworth B (2009) Fundamental concepts in genetics: effective population size and patterns of molecular evolution and variation. Nat Rev Genet 10:195–205

    PubMed  Article  CAS  Google Scholar 

  10. Chybicki IJ, Burczyk J (2009) Simultaneous estimation of null alleles and inbreeding coefficients. J Hered 100:106–113

    PubMed  Article  CAS  Google Scholar 

  11. Clark JD, Huber D, Servheen C (2002) Bear reintroductions: lessons and challenges: invited paper. Ursus 13:335–345

    Google Scholar 

  12. Čop J (1987) Propagation pattern of re-introduced population of lynx (Lynx lynx L) in Yugoslavia (1973 Slovenia—Kocevsko) and its impact on the ungulate community. In: Atti del convegno Reintroduzione dei predatori nele aree protette. Torino Italy, pp 83–91

  13. Čop J, Frković A (1998) The reintroduction of the lynx in Slovenia and its present status in Slovenia and Croatia. Hystrix 10:65–76

    Google Scholar 

  14. Crow JF, Kimura M (1970) An introduction to population genetics theory. Harper and Row, New York

    Google Scholar 

  15. Davoli F, Schmidt K, Kowalczyk R, Randi E (2012) Hair snaring and molecular genetic identification for reconstructing the spatial structure of Eurasian lynx populations. Mammal Biol. doi:10.1016/j.mambio.2012.06.003

    Google Scholar 

  16. Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the software structure: a simulation study. Mol Ecol 14:2611–2620

    PubMed  Article  CAS  Google Scholar 

  17. Excoffier L, Laval G, Schneider S (2005) Arlequin ver. 3.0: an integrated software package for population genetics data analysis. Evol Bioinform Online 1:47–50

    CAS  Google Scholar 

  18. Frankham R (1998) Inbreeding and extinction: island populations. Conserv Biol 12:665–675

    Article  Google Scholar 

  19. Frankham R (2009) Genetic considerations in reintroduction programmes for top-order, terrestrial predators. In: Hayward MW, Somers M (eds) Reintroduction of top-order predators. Blackwell, Oxford, pp 371–382

    Google Scholar 

  20. Frankham R, Ballou JD, Briscoe DA (2002) Introduction to conservation genetics. Cambridge University Press, Cambridge

    Google Scholar 

  21. Franklin IR, Frankham R (1998) How large must populations be to retain evolutionary potential? Animal Conserv 1:69–70

    Article  Google Scholar 

  22. Frković A (2001) Ris (Lynx lynx L.) u Hrvatskoj—naseljavanje, odlov i brojnost (1974–2000). Šumarski list 11–12:625–634

    Google Scholar 

  23. Gillespie JH (2004) Population genetics, a concise guide, 2nd edn. The Johns Hopkins University Press, Baltimore

    Google Scholar 

  24. Gomerčić T, Gužvica G, Đuras Gomerčić M, Frković A, Pavlović D, Kusak J, Sindičić M, Huber Đ (2009) Variation in teeth number, teeth and skull disorders in Eurasian lynx, Lynx lynx from Croatia. Folia Zool 58:57–65

    Google Scholar 

  25. Gomerčić T, Sindičić M, Đuras Gomerčić M, Gužvica G, Frković A, Pavlović D, Kusak J, Galov A, Huber Đ (2010) Cranial morphometry of the Eurasian lynx (Lynx lynx L.) from Croatia. Vet Arch 80:393–410

    Google Scholar 

  26. Goudet J (1995) FSTAT (vers. 1.2): a computer program to calculate F-statistics. J Hered 86:485–486

    Google Scholar 

  27. Goudet J (2001) FSTAT, a program to estimate and test gene diversities and fixation indices version 2.9.3. http://www.unil.ch/izea/softwares/fstat.html Updated from Goudet (1995)

  28. Gugolz D, Bernasconi MV, Breitenmoser-Würsten C, Wandeler P (2008) Historical DNA reveals the phylogenetic position of the extinct Alpine lynx. J Zool (Lond) 275:201–208

    Article  Google Scholar 

  29. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/97/NT. Nucleic Acids Symp Ser 41:95–98

    CAS  Google Scholar 

  30. Hellborg L, Walker CW, Rueness EK, Stacy JE, Kojola I, Valdmann H, Vila C, Zimmermann B, Jakobsen KS, Ellegren H (2002) Differentiation and levels of genetic variation in northern European lynx (Lynx lynx) populations revealed by microsatellites and mitochondrial DNA analysis. Conserv Genet 3:97–111

    Article  CAS  Google Scholar 

  31. Hill WG (1981) Estimation of effective population size from data on linkage disequilibrium. Genet Res 38:209–216

    Article  Google Scholar 

  32. Holm S (1979) A simple sequentially rejective multiple test procedure. Scand J Stat 6:65–70

    Google Scholar 

  33. Hundertmark KJ, Van Daele LJ (2010) Founder effect and bottleneck in an introduced, insular population of elk. Conserv Genet 11:139–147

    Article  Google Scholar 

  34. Johnson WE, Onorato DP, Roelke ME, Land ED, Cunningham M, Belden RC, Mcbride R, Jansen D, Lotz M, Shindle D, Howard J, Wildt DE, Penfold LM, Hostetler JA, Oli MK, O’Brien SJ (2010) Genetic restoration of the Florida panther. Science 24:1641–1645

    Article  Google Scholar 

  35. Keller LF, Waller DM (2002) Inbreeding effects in wild populations. Trends Ecol Evol 17:230–241

    Article  Google Scholar 

  36. Keller LF, Biebach I, Ewing SR, Hoeck PEA (2012) The genetics of reintroductions: inbreeding and genetic drift. In: Ewen JG, Armstrong DP, Parker KA, Seddon PJ (eds) Reintroduction biology: integrating science and management. Blackwell, Oxford, pp 360–394

    Google Scholar 

  37. Koritnik M (1974) Še nekaj o risu. Lovec 67:198–199

    Google Scholar 

  38. Kos F (1928) Ris (Lynx lynx) na ozemlju etnografske Slovenije. Glasnik muzejskega društva za Slovenijo 1:57–72

    Google Scholar 

  39. Koubek P, Červený J (1996) A synopsis of lynxes trapped in Slovakia and re-introduced to certain countries in Europe. Acta Sc Nat Brno 30:42–43

    Google Scholar 

  40. Lande R (1995) Mutation and conservation. Conserv Biol 9:782–791

    Article  Google Scholar 

  41. Lopez JV, Cevario S, O’Brien SJ (1996) Complete nucleotide sequences of the domestic cat (Felis catus) mitochondrial genome and a transposed mtDNA tandem repeat (Numt) in the nuclear genome. Genomics 33:229–246

    PubMed  Article  CAS  Google Scholar 

  42. Luikart G, Ryman N, Tallmon D, Schwartz M, Allendorf F (2010) Estimation of census and effective population sizes: the increasing usefulness of DNA-based approaches. Conserv Genet 11:355–373

    Article  CAS  Google Scholar 

  43. Lynch M, Conery J, Bürger R (1995) Mutation accumulation and the extinction of small populations. Am Nat 146:489–518

    Article  Google Scholar 

  44. MacDonald DW (2009) Lessons learnt and plans laid: seven awkward questions for the future of reintroductions. In: Hayward MW, Somers M (eds) Reintroduction of top-order predators. Blackwell, Oxford, pp 371–387

    Google Scholar 

  45. Menotti-Raymond M, David VA, Lyons LA, Schäffer AA, Tomlin JF, Hutton MK, O'Brien SJ (1999) A genetic linkage map of microsatellites in the domestic cat (Felis catus). Genomics 57:9–23

    Google Scholar 

  46. Meyer A, Kocher TD, Basasibwaki P, Wilson AC (1990) Monophyletic origin of Lake Victoria cichlid fishes suggested by mitochondrial DNA sequences. Nature 347:550–553

    PubMed  Article  CAS  Google Scholar 

  47. Miller C, Joyce P, Waits LP (2002) Assessing allelic dropout and genotype reliability using maximum likelihood. Genetics 160:357–366

    PubMed  Google Scholar 

  48. Milligan BG (2003) Maximum-likelihood estimation of relatedness. Genetics 163:1153–1167

    PubMed  Google Scholar 

  49. Miquel C, Bellemain E, Poillot C, Bessiére J, Durand A, Taberlet P (2006) Quality indexes to assess the reliability of genotypes in studies using noninvasive sampling and multiple-tube approach. Mol Ecol Notes 6:985–988

    Article  Google Scholar 

  50. Mirić D (1978) Ausrottungsgeschichte des Luchses auf der Balkanhalbinsel. In: Wotischkowsky U (ed) Der Luchs: Erhaltung und Wiedereinburgerung in Europa. Bernhard, Mammendorf, pp 19–24

    Google Scholar 

  51. Paetkau D, Strobeck C (1994) Microsatellite analysis of genetic variation in black bear populations. Mol Ecol 3:489–495

    PubMed  Article  CAS  Google Scholar 

  52. Peakall R, Smouse PE (2006) GENALEX 6: genetic analysis in excel. Population genetic software for teaching and research. Mol Ecol Notes 6:288–295

    Article  Google Scholar 

  53. Phillipsen IC, Funk WC, Hoffman EA, Monsen KJ, Blouin MS (2011) Comparative analyses of effective population size within and among species: ranid frogs as a case study. Evolution 65:2927–2945

    PubMed  Article  Google Scholar 

  54. Polanc P, Sindičić M, Jelenčič M, Gomerčić T, Kos I, Huber Đ (2011) Genotyping success of historical Eurasian lynx (Lynx lynx L.) samples. Mol Ecol Resour 12:293–298

    PubMed  Article  Google Scholar 

  55. Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155:945–959

    PubMed  CAS  Google Scholar 

  56. Ratkiewicz M, Matosiuk M, Kowalczyk R, Konopiski MK, Okarma H, Ozolins J, Männil P, Ornicans A, Schmidt K (2012) High levels of population differentiation in Eurasian lynx at the edge of the species’ western range in Europe revealed by mitochondrial DNA analyses. Anim Conserv. doi:10.1111/j.1469-1795.2012.00556.x

    Google Scholar 

  57. Reed DH, Lowe E, Briscoe DA, Frankham R (2003) Inbreeding and extinction: effects of rate of inbreeding. Conserv Genet 4:405–410

    Article  CAS  Google Scholar 

  58. Robinson JD, Moyer GR (2012) Linkage disequilibrium and effective population size when generations overlap. Evol Appl. doi:10.1111/j.1752-4571.2012.00289.x

    PubMed  Google Scholar 

  59. Rueness EK, Jorde PE, Hellborg L, Stenseth NC, Ellegren H, Jakobsen KS (2003) Cryptic population structure in a large, mobile mammalian predator: the Scandinavian lynx. Mol Ecol 12:2623–2633

    PubMed  Article  CAS  Google Scholar 

  60. Schmidt KR, Kowalczyk J, Ozolins P, Männi L, Fickel J (2009) Genetic structure of the Eurasian lynx population in north–eastern Poland and the Baltic states. Conserv Genet 10:497–501

    Article  Google Scholar 

  61. Schmidt K, Ratkiewicz M, Konopinski MK (2011) The importance of genetic variability and population differentiation in the Eurasian lynx Lynx lynx for conservation, in the context of habitat and climate change. Mammal Rev 41:112–124

    Article  Google Scholar 

  62. Sindičić M, Sinanović N, Majić Skrbinšek A, Huber Đ, Kunovac S, Kos I (2010) Legal status and management of the Dinaric lynx population. Veterinaria 58:229–238

    Google Scholar 

  63. Sindičić M, Gomerčić T, Galov A, Polanc P, Huber Đ, Slavica A (2012) Repetitive sequences in Eurasian lynx (Lynx lynx L.) mitochondrial DNA control region. Mitochondrial DNA 23:201–207

    PubMed  Article  Google Scholar 

  64. Skrbinšek T, Jelenčič M, Waits L, Kos I, Jerina K, Trontelj P (2012) Monitoring the effective population size of a brown bear (Ursus arctos) population using new single-sample approaches. Mol Ecol 21:862–875

    PubMed  Article  Google Scholar 

  65. Spong G, Hellborg L (2002) A near: extinction event in lynx: do microsatellite data tell the tale? Conserv Ecol 6:15

    Google Scholar 

  66. Taberlet P, Griffin S, Goossens B, Questiau S, Manceau V, Escaravage N, Waits LP, Bouvet J (1996) Reliable genotyping of samples with very low DNA quantities using PCR. Nucleic Acids Res 24:3189–3194

    PubMed  Article  CAS  Google Scholar 

  67. Tallmon DA, Koyuk A, Luikart G, Beaumont MA (2008) Onesamp: a program to estimate effective population size using approximate Bayesian computation. Mol Ecol Resour 8:299–301

    PubMed  Article  Google Scholar 

  68. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    PubMed  Article  CAS  Google Scholar 

  69. Valiére N (2002) GIMLET: a computer program for analysing genetic individual identification data. Mol Ecol Notes 2:377–379

    Google Scholar 

  70. Van Oosterhout C, Weetman D, Hutchinson WF (2006) Estimation and adjustment of microsatellite null alleles in nonequilibrium populations. Mol Ecol Notes 6:255–256

    Article  Google Scholar 

  71. von Arx M, Breitenmoser-Würsten C, Zimmermann F, Breitenmoser U (2004) Status and conservation of the Eurasian lynx (Lynx lynx) in 2001. KORA Bericht no. 19, Muri

  72. Waits LP, Luikart G, Taberlet P (2001) Estimating the probability of identity among genotypes in natural populations: cautions and guidelines. Mol Ecol 10:249–256

    PubMed  Article  CAS  Google Scholar 

  73. Wang J (2007) Triadic IBD coefficients and applications to estimating pairwise relatedness. Genet Res 89:135–153

    PubMed  Article  CAS  Google Scholar 

  74. Wang J (2011) Coancestry: a program for simulating, estimating and analysing relatedness and inbreeding coefficients. Mol Ecol 11:141–145

    Article  Google Scholar 

  75. Waples RS (2006) A bias correction for estimates of effective population size based on linkage disequilibrium at unlinked gene loci. Conserv Genet 7:167–184

    Article  Google Scholar 

  76. Waples RS, Do C (2008) ldne: a program for estimating effective population size from data on linkage disequilibrium. Mol Ecol Resour 8:753–756

    PubMed  Article  Google Scholar 

  77. Waples RS, Do C (2010) Linkage disequilibrium estimates of contemporary N e using highly variable genetic markers: a largely untapped resource for applied conservation and evolution. Evol Appl 3:244–262

    Article  Google Scholar 

  78. Ward RH, Frazier BL, Dew-Jager K, Paabo S (1991) Extensive mitochondrial diversity within a single Amerindian tribe. Proc Natl Acad Sci USA 88:8720–8724

    PubMed  Article  CAS  Google Scholar 

  79. Williamson JE, Huebinger RM, Sommer JA, Louis EE Jr, Barber RC (2002) Development and cross-species amplification of 18 microsatellite markers in the Sumatran tiger (Panthera tigris sumatrae). Mol Ecol Notes 2:110–112

    Google Scholar 

  80. Wu X, Zheng T, Jiang Z, Wei L (2007) The mitochondrial genome structure of the clouded leopard (Neofelis nebulosa). Genome 50:252–257

    PubMed  Article  CAS  Google Scholar 

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Acknowledgments

We are grateful to Vedran Slijepčević, Miha Krofel, Josip Tomaić, Josip Kusak, Franc Kljun, Marko Jonozovič, Hubert Potočnik and Ivan Kos for their help with obtaining the samples. Carlos Fernandes gave us valuable comments for data analysis. This research was in part supported by the Interreg IIIA project “Transboundary cooperation in management, conservation and research of the Dinaric lynx population”. Further, it was supported by the Slovenian Research Agency Project L1-6484 and co-funded by the Environmental Agency of the Republic of Slovenia, the Ministry of Agriculture of the Republic of Slovenia and the Institute of the Republic of Slovenia for Nature Conservation. Also we are grateful for financial support provided by Croatian State Institute for Nature Protection, Croatian Environmental protection and energy efficiency fund, and Carlsberg Croatia.

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Correspondence to Tomislav Gomerčić.

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Sindičić, M., Polanc, P., Gomerčić, T. et al. Genetic data confirm critical status of the reintroduced Dinaric population of Eurasian lynx. Conserv Genet 14, 1009–1018 (2013). https://doi.org/10.1007/s10592-013-0491-x

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Keywords

  • Lynx lynx
  • Microsatellite
  • Control region
  • Inbreeding
  • Effective population size