Conservation Genetics

, Volume 13, Issue 1, pp 223–234 | Cite as

Microsatellite evidence for high clonality and limited genetic diversity in Ziziphus celata (Rhamnaceae), an endangered, self-incompatible shrub endemic to the Lake Wales Ridge, Florida, USA

  • Matthew A. GitzendannerEmail author
  • Carl W. Weekley
  • Charlotte C. Germain-Aubrey
  • Douglas E. Soltis
  • Pamela S. Soltis
Research Article


Genetic data are often crucial for designing management strategies for rare and endangered species. Ziziphus celata is an endangered sandhill shrub endemic to the Lake Wales Ridge of central Florida. This self-incompatible clonal species is known from only 14 wild populations, most of which are small (under 100 plants). Focusing on the five populations discovered in 2007, we evaluate the level of genetic diversity and identify clonal lineages within the wild populations of the species with a set of microsatellite loci. To account for somatic mutations and genotyping errors, we identified clonal lineages using a threshold cutoff for pair-wise genetic distances among samples. The microsatellites had up to 18 alleles/locus, and, consistent with outcrossing, samples were highly heterozygous (average population level H o  = 0.69). Most populations of Z. celata consist of a single clone, and the most diverse population has only 10 clones. Overall Z. celata comprises 41 multi-locus genotypes, and 30 clonal lineages. With nearly 1,000 recorded plants (595 genotyped) and only 30 clonal lineages, Ziziphus celata is highly clonal: clonal richness, R = 0.049. The pair-wise distance method facilitates identification of clonal lineages, avoiding overestimation of clonal diversity. In most cases, the samples that grouped into a lineage were one to four plants differing from a surrounding genotype by a single microsatellite repeat insertion/deletion mutation, consistent with these having arisen via somatic mutations. Our data will enable managers to incorporate extant diversity from wild populations into ex situ collections. Additionally, our research demonstrates the utility of microsatellites for conservation of imperiled species, identifying genotypes of high priority for preservation.


Clonality Conservation genetics Genetic diversity Lake Wales Ridge, Florida Rare species conservation 



The authors thank S. Anak and P. Soria for assistance in the lab; funding was provided by the US Fish and Wildlife Service, the Plant Conservation Program of the Florida Division of Forestry, and Archbold Biological Station. We thank E.S. Menges and three anonymous reviewers for helpful comments on the manuscript.


  1. Abrahamson WG, Johnson AF, Layne JN, Peroni PA (1984) Vegetation of the Archbold Biological Station, Florida: an example of the southern Lake Wales Ridge. Fla Sci 47:209–250Google Scholar
  2. Aigner PA (2004) Ecological and genetic effects on demographic processes: pollination, clonality and seed production in Dithyrea maritima. Biol Conserv 116:27–34. doi: 10.1016/S0006-3207(03)00170-8 CrossRefGoogle Scholar
  3. Arnaud-Haond S, Duarte CM, Alberto F, Serrão EA (2007) Standardizing methods to address clonality in population studies. Mol Ecol 16(24):5115–5139PubMedCrossRefGoogle Scholar
  4. Avise JC, Hamrick JL (1996) Conservation genetics: case histories from nature. Kluwer Academic Publishers, NorwellGoogle Scholar
  5. Balloux F, Lehmann L, De Meeûs T (2003) The population genetics of clonal and partially clonal diploids. Genetics 164:1635PubMedGoogle Scholar
  6. Branch LC, Clark AM, Moler PE, Bowen BW (2003) Fragmented landscapes, habitat specificity, and conservation genetics of three lizards in Florida scrub. Conserv Genet 4(2):199–212CrossRefGoogle Scholar
  7. Byers DL, Meagher TR (1992) Mate availability in small populations of plant species with homomorphic sporophytic self-incompatibility. Heredity 68:353–359CrossRefGoogle Scholar
  8. Castoe TA, Poole AW, Gu W, Jason de Koning AP, Daza JM, Smith EN, Pollock DD (2010) Rapid identification of thousands of copperhead snake (Agkistrodon contortrix) microsatellite loci from modest amounts of 454 shotgun genome sequence. Mol Ecol Res 10(2):341–347CrossRefGoogle Scholar
  9. Chistiakov DA, Hellemans B, Volckaert FA (2006) Microsatellites and their genomic distribution, evolution, function and applications: a review with special reference to fish genetics. Aquaculture 255(1–4):1–29CrossRefGoogle Scholar
  10. Christman SP, Judd WS (1990) Notes on plants endemic to Florida scrub. Fla Sci 53:52–73Google Scholar
  11. Coates F, Walsh NG, James EA (2002) Threats to the survival of the Grampians pincushion lily (Borya mirabillis, Lilliaceae)—a short-range endemic from western Victoria. Aust Syst Bot 15:477–485CrossRefGoogle Scholar
  12. Coile NC, Garland MA (2003) Notes on Florida’s endangered and threatened plants, 4th edn (PDF Version). Florida Department of Agriculture and Consumer Services, Division of Plant Industry, GainesvilleGoogle Scholar
  13. Cullings K (1992) Design and testing of a plant-specific PCR primer for ecological and evolutionary studies. Mol Ecol 1(4):233–240CrossRefGoogle Scholar
  14. DeLaney KR, Wunderlin RP, Hansen BF (1989) Re-discovery of Zizipus celata (Rhamnaceae). Sida 13:325–330Google Scholar
  15. Dobson AP, Rodrigues JP, Roberts WM, Wilcove DS (1997) Geographic distribution of endangered species in the United States. Science 275:550–553PubMedCrossRefGoogle Scholar
  16. Dolan RW, Yahr R, Menges ES, Halfhill MD (1999) Conservation implications of genetic variation in three rare species endemic to Florida rosemary scrub. Am J Bot 86(11):1556–1562PubMedCrossRefGoogle Scholar
  17. Douhovnikoff V, Dodd RS (2003) Intra-clonal variation and a similarity threshold for identification of clones: application to Salix exigua using AFLP molecular markers. Theor Appl Genet 106(7):1307–1315PubMedGoogle Scholar
  18. Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15Google Scholar
  19. Ellis MM, Weekley CW, Menges ES (2007) Evaluating stability in Ziziphus celata, a highly endangered clonal shrub endemic to Lake Wales Ridge, central Florida. Endanger Species Res 3(2):125–132CrossRefGoogle Scholar
  20. Estill JC, Cruzen MB (2001) Phytogeography of rare plant species endemic to the southeastern United States. Castanea 66:3–23Google Scholar
  21. Frankham R (2005) Genetics and extinction. Biol Conserv 126:131–140CrossRefGoogle Scholar
  22. Glémin S, Petit C, Maurice S, Mignot A (2008) Consequences of low mate availability in the rare self-incompatible species Brassica insularis. Conserv Biol 22(1):216–221PubMedCrossRefGoogle Scholar
  23. Godt MJW, Race T, Hamrick JL (1997) A population genetic analysis of Ziziphus celata, an endangered Florida shrub. J Hered 88(6):531–533Google Scholar
  24. Halkett F, Simon J-C, Balloux F (2005) Tackling the population genetics of clonal and partially clonal organisms. Trends Ecol Evol 20:194–201. doi: 10.1016/j.tree.2005.01.001 PubMedCrossRefGoogle Scholar
  25. Hoebee S, Thrall P, Young A (2008) Integrating population demography, genetics and self-incompatibility in a viability assessment of the Wee Jasper Grevillea (Grevillea iaspicula McGill., Proteaceae). Conserv Genet 9(3):515–529CrossRefGoogle Scholar
  26. Honnay O, Bossuyt B (2005) Prolonged clonal growth: escape route or route to extinction? Oikos 108(2):427–432CrossRefGoogle Scholar
  27. Honnay O, Jacquemyn H (2008) A meta-analysis of the relation between mating system, growth form and genotypic diversity in clonal plant species. Evol Ecol 22:299–312. doi: 10.1007/s10682-007-9202-8 CrossRefGoogle Scholar
  28. Judd WS, Hall DW (1984) A new species of Ziziphus (Rhamnaceae) from Florida. Rhodora 86:381–387Google Scholar
  29. Jusaitis M, Adams M (2005) Conservation implications of clonality and limited sexual reproduction in the endangered shrub Acanthocladium dockeri (Asteraceae). Aust J Bot 53:535–544CrossRefGoogle Scholar
  30. Kao T, Tsukamoto T (2004) The molecular and genetic bases of S-RNase-based self-incompatibility. Plant Cell Online 16(90001):72–83CrossRefGoogle Scholar
  31. Kijas JMH, Fowler JCS, Garbett CA, Thomas MR (1994) Enrichment of microsatellites from the citrus genome using biotinylated oligonucleotide sequences bound to streptavidin-coated magnetic particles. BioTechniques 16:657–660Google Scholar
  32. Kimpton SK, James EA, Drinnan AN (2002) Reproductive biology and genetic marker diversity in Grevillea infecunda (Proteaceae), a rare plant with no known seed production. Aust Syst Bot 15:485–492CrossRefGoogle Scholar
  33. Kwak MM, Bekker RM (2006) Ecology of plant reproduction: extinction risks and restoration perspectives or rare plant species. In: Waser NM, Ollerton J (eds) Plant–pollinator interactions: from specialization to generalization. The University of Chicago Press, Chicago, pp 362–386Google Scholar
  34. Lewis PO, Crawford DJ (1995) Pleistocene refugium endemics exhibit greater allozymic diversity than widespread congeners in the genus Polygonella (Polygonaceae). Am J Bot 82(2):141–149CrossRefGoogle Scholar
  35. McDonald DB, Hamrick JL (1996) Genetic variation in some plants of Florida Scrub. Am J Bot 83(1):21–27CrossRefGoogle Scholar
  36. McKay JK, Latta RG (2002) Adaptive population divergence: markers, QTL and traits. Trends Ecol Evol 17(6):285–291CrossRefGoogle Scholar
  37. Meirmans PG, Van Tienderen PH (2004) Genotype and genodive: two programs for the analysis of genetic diversity of asexual organisms. Mol Ecol Notes 4(4):792–794CrossRefGoogle Scholar
  38. Menges ES, Dolan RW, Yahr R, Gordon DR (2001) Comparative genetics of seven plants endemic to Florida’s Lake Wales Ridge. Castanea 66(1/2):98–114Google Scholar
  39. Menges ES, Dolan RW, Pickert R, Yahr R, Gordon DR (2010) Genetic variation in past and current landscapes: conservation implications based on six endemic Florida scrub plants. Int J Ecol 2010:12pp. doi: 10.1155/2010/503759
  40. Menges ES, Weekley CW, Hamzé SI, Pickert RL (2007) Soil preferences for listed plants on the Lake Wales Ridge in Highlands Country, Florida. Fla Sci 70:24–39Google Scholar
  41. Myers RL, Ewel JJ (1990) Ecosystems of Florida. University Press of Florida, GainesvilleGoogle Scholar
  42. Navascues M, Stoeckel S, Mariette S (2010) Genetic diversity and fitness in small populations of partially asexual, self-incompatible plants. Heredity 104:482–492PubMedCrossRefGoogle Scholar
  43. Ouborg NJ (2010) Integrating population genetics and conservation biology in the era of genomics. Biol Lett 6(1):3–6PubMedCrossRefGoogle Scholar
  44. Parks JC, Werth CR (1993) A study of spatial features of clones in a population of Bracken Fern, Pteridium aquilinum (Dennstaedtiaceae). Am J Bot 80:537–544CrossRefGoogle Scholar
  45. Peakal R, Smouse PE (2006) Genalex 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol Ecol Notes 6(1):288–295CrossRefGoogle Scholar
  46. Richards AJ (1997) Plant breeding systems, 2nd edn. Chapman & Hall, LondonGoogle Scholar
  47. Rozenfeld AF, Arnaud-Haond S, Hernández-García E, Eguíluz VM, Matías MA, Serrão E, Duarte CM (2007) Spectrum of genetic diversity and networks of clonal organisms. J R Soc Interface 4(17):1093–1102PubMedCrossRefGoogle Scholar
  48. Schnittler M, Eusemann P (2010) Consequences of genotyping errors for estimation of clonality: a case study on Populus euphratica Oliv. (Salicaceae). Evol Ecol 24:1417–1432. doi: 10.1007/s10682-010-9389-y CrossRefGoogle Scholar
  49. Schuelke M (2000) An economic method for the fluorescent labeling of PCR fragments. Nat Biotechnol 18(2):233–234PubMedCrossRefGoogle Scholar
  50. Scobie AR, Wilcock CC (2009) Limited mate availability decreases reproductive success of fragmented populations of Linnaea borealis, a rare, clonal self-incompatible plant. Ann Bot, mcp007Google Scholar
  51. Swofford DL (2003) PAUP* Phylogenetic analysis using parsimony (* and other methods), version 4. Sinauer, SunderlandGoogle Scholar
  52. Turner WR, Wilcove DS, Swain HM (2006) Assessing the effectiveness of reserve acquisition programs in protecting rare and threatened species. Conserv Biol 20:1657–1669Google Scholar
  53. US Fish and Wildlife Service (1999) Florida Ziziphus. In: Multi-species recovery plan for the threatened and endangered species of South Florida. US Fish and Wildlife Service, Atlanta, pp. 1986–1999Google Scholar
  54. US Fish and Wildlife Service (2009) Florida ziziphus (Ziziphus celata) 5-year status review: summary and evaluation, 23 pp. US Fish and Wildlife Service, AtlantaGoogle Scholar
  55. Warburton CL, James EA, Fripp YJ, Trueman SJ, Wallace HM (2000) Clonality and sexual reproductive failure in remnant populations of Santalum lanceolatum (Santalaceae). Biol Conserv 96(1):45–54CrossRefGoogle Scholar
  56. Ward DB, Austin DF, Coile NC (2003) Endangered and threatened plants of Florida, ranked in order of rarity. Castanea 68(2):160–174Google Scholar
  57. Weekley CW, Menges ES (2008) Experimental introductions of Florida Ziziphus on Florida’s Lake Wales Ridge, USA. In: Soorae PS (ed) Global re-introduction perspectives: re-introduction studies from around the Globe. IUCN/SSC Re-introduction Specialist Group, Abu Dhabi, pp 256–261.
  58. Weekley CW, Race T (2001) The breeding system of Ziziphus celata Judd and D.W. Hall (Rhamnaceae), a rare endemic plant of the Lake Wales Ridge, Florida, USA: implications for recovery. Biol Conserv 100(2):207–213CrossRefGoogle Scholar
  59. Weekley CW, Kubisiak TL, Race T (2002) Genetic impoverishment and cross-incompatibility in remnant genotypes of Ziziphus celata (Rhamnaceae), a rare shrub endemic to the Lake Wales Ridge. Florida Biodivers Conserv 11(11):2027–2046CrossRefGoogle Scholar
  60. Weekley CW, Menges ES, Pickert RL (2008). An ecological map of Florida’s Lake Wales Ridge: a new boundary delineation and an assessment of post-Columbian habitat loss. Fla Sci 71: 45–64.,etal.-2008-FlaSci-LWRboundary.pdf Google Scholar
  61. You F, Huo N, Gu Y, Luo M, Ma Y, Hane D, Lazo G, Dvorak J, Anderson O (2008) BatchPrimer3: a high throughput web application for PCR and sequencing primer design. BMC Bioinform 9:253CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Matthew A. Gitzendanner
    • 1
    • 2
    Email author
  • Carl W. Weekley
    • 3
  • Charlotte C. Germain-Aubrey
    • 1
  • Douglas E. Soltis
    • 1
  • Pamela S. Soltis
    • 2
  1. 1.Department of BiologyUniversity of FloridaGainesvilleUSA
  2. 2.Florida Museum of Natural HistoryUniversity of FloridaGainesvilleUSA
  3. 3.Archbold Biological StationLake PlacidUSA

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