Tree Genetics & Genomes

, 13:118 | Cite as

Genome skimming and plastid microsatellite profiling of alder trees (Alnus spp., Betulaceae): phylogenetic and phylogeographical prospects

  • Hervé Gryta
  • Céline Van de Paer
  • Sophie Manzi
  • Hélène Holota
  • Mélanie Roy
  • Guillaume Besnard
Original Article
Part of the following topical collections:
  1. Taxonomy


Alders (Alnus spp.) represent keystone species trees of riparian and mountainous habitats of the northern hemisphere. Previous genetic studies have suggested a complex intrageneric diversification with numerous events of interspecific hybridization and polyploidization. Here, we first aim to test the present taxonomical treatment of Alnus by generating phylogenetic hypotheses based on plastid and nuclear data obtained from species belonging to the three main alder subgenera (Alnus, Alnobetula, and Clethropsis). A genome-skimming strategy was used to assemble the complete plastome and the nuclear ribosomal DNA cluster of 22 Eurasian and American alder individuals. Phylogenies based on these data strongly support an early diverging subgenus Alnobetula, while members of the subgenus Clethropsis do not constitute a monophyletic clade and are embedded within the subgenus Alnus. Incongruent topologies also sustain reticulate evolution within this group. Our results thus suggest considering the subgenera Clethropsis and Alnus within the same taxonomical unit. Our second aim is to test for the utility of highly variable plastid markers (microsatellites) to investigate the phylogeographic patterns of Eurasian alder species. Fifty-two polymorphic plastid microsatellite markers were developed and tested on 33 populations of the subgenus Alnus in western Eurasia. On average, 4.3 alleles per locus were revealed in 131 individuals of Alnus glutinosa, allowing the identification of 30 chlorotypes (multiloci profiles). Strong phylogeographic signals and recurrent cytoplasmic captures between co-occurring species are revealed, demonstrating that our plastid microsatellite profiling method is suitable for tracing the post-glacial spread of maternal lineages among alder species. All these results finally support the use of nuclear genomic regions for species identification and of plastid markers for phylogeographic aspects and origin certification in genetic resource management.


Alder Alnus Chloroplast genome cpSSR Introgressive hybridization Microsatellite Nuclear ribosomal DNA Phylogeny Phylogeography Population genetics 



We thank L. Csiba and F. Forest (Jodrell Laboratory) for samples of the living collection of Royal Botanic Gardens (Kew, Richmond) and DNA extractions, R. Etienne and A. Gasc (EDB) for the help in plant genotyping, B. Grosso (EDB) for the conservation of dried specimens, and A. Roy and B. Durrington for English corrections. We deeply thank Pr. G. Nakhutsrishvili and Dr. A. Jorjadze (Tbilisi Botanical Garden and Institute of Botany), J. Bastenaire and A. Latrille (Institut Français de Georgie), L. Gogava and D. Sincu (Romanian Academy of Science), T. Manilova (Ceske Budejovice University), S. Ravera (Roma Botanical Garden), M. Dosmann (Arnold Arboretum of Harvard University), J.P. Gibson, C. Ehardt and S. Rice (University of Oklahoma), J.A. Bérubé (Université Laval), A. Bellino, D. Puntillo and L. Hugot (Conservatoire Botanique National de Corse), P. Jargeat, D. Badr, C. del Rio, and M. Gardes (EDB), for their helpful support in the field.

Author contributions

HG and GB conceived the initial project; SM, HH, and GB did the lab work; HG, MR, and GB collected plants; HG, CVDP, and GB analyzed data; HG and GB wrote the paper; and all authors commented and approved the final version of the manuscript.

Funding information

All authors are members of the Laboratoire Evolution & Diversité Biologique (EDB) part of the LABEX entitled TULIP managed by Agence Nationale de la Recherche (ANR-10-LABX-0041). We also acknowledge an Investissement d’Avenir grant of the Agence Nationale de la Recherche (CEBA: ANR-10-LABX-25-01) and an EC2CO MicrobiEn grant (ECosphère COntinentale et CÔtière, Microbiologie ENvironnementale, “Coévolution et phylogéographie des mutualismes aulne-microorganismes”). CVDP and GB were funded by the Regional Council Midi-Pyrenees (AAP 13053637, 2014-EDB-UT3-DOCT). This study also received support from the PhyloAlps project, with the help of M. Boleda and A. Iribar O. Bouchez from the Genopole in Toulouse (INRA) helped with the Illumina sequencing.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11295_2017_1204_MOESM1_ESM.docx (4.4 mb)
ESM 1 (DOCX 4524 kb)


  1. Bandelt HJ, Forster P, Röhl A (1999) Median-joining networks for inferring intraspecific phylogenies. Mol Biol Evol 16:37–48CrossRefPubMedGoogle Scholar
  2. Beatty GE, Montgomery WI, Tosh DG, Provan J (2015) Genetic provenance and best practice woodland management: a case study in native alder (Alnus glutinosa). Tree Genet Genomes 11:92CrossRefGoogle Scholar
  3. Besnard G, Bervillé A (2002) On chloroplast DNA variations in the olive (Olea europaea L.) complex: comparison of RFLP and PCR polymorphisms. Theor Appl Genet 104:1157–1163CrossRefPubMedGoogle Scholar
  4. Besnard G, Hernández P, Khadari B, Dorado G, Savolainen V (2011) Genomic profiling of plastid DNA variation in the Mediterranean olive tree. BMC Plant Biol 11:80CrossRefPubMedPubMedCentralGoogle Scholar
  5. Besnard G, Christin PA, Malé PJG, Coissac E, Ralimanana H, Vorontsova MS (2013a) Phylogenomics and taxonomy of Lecomtelleae (Poaceae), an isolated panicoid lineage from Madagascar. Ann Bot 112:1057–1066CrossRefPubMedPubMedCentralGoogle Scholar
  6. Besnard G, Khadari B, Navascués M, Fernandez-Mazuecos M, El Bakkali A, Arrigo N, Baali-Cherif D, Brunini-Bronzini de Caraffa V, Santoni S, Vargas P, Savolainen V (2013b) The complex history of the olive tree: from late quaternary diversification of Mediterranean lineages to primary domestication in the northern Levant. Proc R Soc Lond B 280:20122833CrossRefGoogle Scholar
  7. Bouille M, Senneville S, Bousquet J (2011) Discordant mtDNA and cpDNA phylogenies indicate geographic speciation and reticulation as driving factors for the diversification of the genus Picea. Tree Genet Genomes 7:469–484CrossRefGoogle Scholar
  8. Chen Z, Li J (2004) Phylogenetics and biogeography of Alnus (Betulaceae) inferred from sequences of nuclear ribosomal DNA ITS region. Int J Plant Sci 165:325–335CrossRefGoogle Scholar
  9. Corriveau JL, Coleman AW (1988) Rapid screening method to detect potential biparental inheritance of plastid DNA and results over 200 angiosperm species. Am J Bot 75:1443–1458CrossRefGoogle Scholar
  10. Cubry P, Gallagher E, O'Connor E, Kelleher CT (2015) Phylogeography and population genetics of black alder (Alnus glutinosa (L.) Gaertn.) in Ireland: putting it in a European context. Tree Genet Genomes 11:99CrossRefGoogle Scholar
  11. De Vienne DM, Giraud T, Martin OC (2007) A congruence index for testing topological similarity between trees. Bioinformatics 23:3119–3124CrossRefPubMedGoogle Scholar
  12. Dering M, Latałowa M, Boratyńska K, Kosiński P, Boratyński A (2017) Could clonality contribute to the northern survival of grey alder [Alnus incana (L.) Moench] during the last glacial maximum? Acta Soc Bot Pol 86:3523Google Scholar
  13. Douda J, Doudová J, Drašnarová A, Kuneš P, Hadincová V, Krak K, Zákravský P, Mandák B (2014) Migration patterns of subgenus Alnus in Europe since the last glacial maximum: a systematic review. PLoS One 9:e88709CrossRefPubMedPubMedCentralGoogle Scholar
  14. Excoffier L, Smouse PE, Quattro JM (1992) Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131:479–491PubMedPubMedCentralGoogle Scholar
  15. Furlow JJ (1979) The systematics of the American species of Alnus (Betulaceae). Rhodora 81:1–121Google Scholar
  16. Gamisans J (1999) La végétation de la Corse. Edisud, 2nde édition, Aix en Provence, France, 392 p Google Scholar
  17. Gavin DG, Fitzpatrick MC, Gugger PF, Heath KD, Rodríguez-Sánchez F, Dobrowski SZ, Hampe A, FS H, Ashcroft MB, Bartlein PJ, Blois JL, Carstens BC, Davis EB, de Lafontaine G, Edwards ME, Fernandez M, Henne PD, Herring EM, Holden ZA, Kong WS, Liu J, Magri D, Matzke NJ, McGlone MS, Saltré F, Stigall AL, Tsai YHE, Williams JW (2014) Climate refugia: joint inference from fossil records, species distribution models and phylogeography. New Phytol 204:37–54CrossRefPubMedGoogle Scholar
  18. Goudet J (1995) Fstat (version 1.2), a computer program to calculate F-statistics. J Hered 86:485–486CrossRefGoogle Scholar
  19. Grimm GW, Renner SS (2013) Harvesting Betulaceae sequences from GenBank to generate a new chronogram for the family. Bot J Linn Soc 172:465–477CrossRefGoogle Scholar
  20. Hamzeh M, Dayanandan S (2004) Phylogeny of Populus (Salicaceae) based on nucleotide sequences of chloroplast trnT-trnF region and nuclear rDNA. Am J Bot 91:1398–1408CrossRefPubMedGoogle Scholar
  21. Havrdová A, Douda J, Krak K, Vít P, Hadincová V, Zákravský P, Mandák B (2015) Higher genetic diversity in recolonized areas than in refugia of Alnus glutinosa triggered by continent-wide lineage admixture. Mol Ecol 24:4759–4777CrossRefPubMedGoogle Scholar
  22. Heuertz M, Carnevale S, Fineschi S, Sebastiani F, Hausman JF, Paule L, Vendramin GG (2006) Chloroplast DNA phylogeography of European ashes, Fraxinus sp (Oleaceae): roles of hybridization and life history traits. Mol Ecol 15:2131–2140CrossRefPubMedGoogle Scholar
  23. Hewitt GM (1999) Post-glacial re-colonization of European biota. Biol J Linn Soc 68:87–112CrossRefGoogle Scholar
  24. Hinsinger DD, Gaudeul M, Couloux A, Bousquet J, Frascaria-Lacoste N (2014) The phylogeography of Eurasian Fraxinus species reveals ancient transcontinental reticulation. Mol Phylogenet Evol 77:223–237CrossRefPubMedGoogle Scholar
  25. Hubert F, Grimm GW, Jousselin E, Berry V, Franc A, Kremer A (2014) Multiple nuclear genes stabilize the phylogenetic backbone of the genus Quercus. Syst Biodivers 12:405–423CrossRefGoogle Scholar
  26. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A (2012) Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647–1649CrossRefPubMedPubMedCentralGoogle Scholar
  27. Kennedy PG, Walker JKM, Bogar LM (2015) Interspecific mycorrhizal networks and non-networking hosts: exploring the ecology of the host genus Alnus. In: Horton TR (ed) Mycorrhizal networks, Ecol Stud, vol 224. Springer, Berlin, pp 227–254CrossRefGoogle Scholar
  28. King RA, Ferris C (1998) Chloroplast DNA phylogeography of Alnus glutinosa (L.) Gaertn. Mol Ecol 7:1151–1161CrossRefGoogle Scholar
  29. King RA, Ferris C (2000) Chloroplast DNA and nuclear DNA variation in the sympatric alder species, Alnus cordata (Lois.) Duby and A. glutinosa (L.) Gaertn. Biol J Linn Soc 70:147–160CrossRefGoogle Scholar
  30. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874CrossRefPubMedGoogle Scholar
  31. Kundzinsh AV (1957) Alnus glutinosa and A. incana hybrids in forests of Latvian SSR (in Russian). Latv PSR Zin Akad Vēst 2:69–74Google Scholar
  32. Lepais O, Muller SD, Ben Saad-Limam S, Benslama M, Rhazi L, Belouahem-Abed D, Daoud-Bouattour A, Gammar AM, Ghrabi-Gammar Z, Bacles CFE (2013) High genetic diversity and distinctiveness of rear-edge climate relicts maintained by ancient tetraploidisation for Alnus glutinosa. PLoS One 8:e75029CrossRefPubMedPubMedCentralGoogle Scholar
  33. Magri D, Fineschi S, Bellarosa R, Buonamici A, Sebastiani F, Schirone B, Simeone MC, Vendramin GG (2007) The distribution of Quercus suber chloroplast haplotypes matches the palaeogeographical history of the western Mediterranean. Mol Ecol 16:5259–5266CrossRefPubMedGoogle Scholar
  34. Mandák B, Havrdová A, Krak K, Hadincová V, Vít P, Zákravský P, Douda J (2016a) Recent similarity in distribution ranges does not mean a similar postglacial history: a phylogeographical study of the boreal tree species Alnus incana based on microsatellite and chloroplast DNA variation. Ney Phytol 210:1395–1407CrossRefGoogle Scholar
  35. Mandák B, Vít P, Krak K, Trávníček P, Havrdová A, Hadincová V, Zákravský P, Jarolímová V, Bacles CFE, Douda J (2016b) Flow cytometry, microsatellites and niche models reveal the origins and geographical structure of Alnus glutinosa populations in Europe. Ann Bot 117:107–120CrossRefPubMedGoogle Scholar
  36. Mensous M, Van de Paer C, Manzi S, Bouchez O, Baâli-Cherif D, Besnard G (2017) Diversity and evolution of plastomes in Saharan mimosoids: potential use for phylogenetic and population genetic studies. Tree Genet Genomes 13:48CrossRefGoogle Scholar
  37. Mingeot D, Husson C, Mertens P, Watillon B, Bertin P, Druart P (2016) Genetic diversity and genetic structure of black alder (Alnus glutinosa [L.] Gaertn) in the Belgium-Luxembourg-France cross-border area. Tree Genet Genomes 12:24CrossRefGoogle Scholar
  38. Nakhutsrishvili G, Zazanashvili N, Batsatsashvili K, Montalvo CS (2015) Colchic and Hyrcanian forests of the Caucasus: similarities, differences and conservation status. Flora Medit 25:185–192Google Scholar
  39. Navarro E, Bousquet J, Moiroud A, Munive A, Piou D, Normand P (2003) Molecular phylogeny of Alnus (Betulaceae), inferred from nuclear ribosomal DNA ITS sequences. Plant Soil 254:207–217CrossRefGoogle Scholar
  40. Navascués M, Emerson BC (2005) Chloroplast microsatellites: measures of genetic diversity and the effect of homoplasy. Mol Ecol 14:1333–1341CrossRefPubMedGoogle Scholar
  41. Olofsson JK, Bianconi M, Besnard G, Dunning LT, Lundgren MR, Holota H, Vorontsova MS, Nosil P, Osborne CP, Christin PA (2016) Genome biogeography reveals the intraspecific spread of adaptive mutations for a complex trait. Mol Ecol 25:6107–6123CrossRefPubMedGoogle Scholar
  42. Parnell J (1994) Variation and hybridisation of Alnus miller in Ireland. Watsonia 20:67–70Google Scholar
  43. Peakall R, Smouse PE (2012) GenAlEx 6.5: genetic analysis in excel. Population genetic software for teaching and research—an update. Bioinformatics 28:2537–2539CrossRefPubMedPubMedCentralGoogle Scholar
  44. Petit RJ, Excoffier L (2009) Gene flow and species delimitation. Trends Ecol Evol 24:386–393CrossRefPubMedGoogle Scholar
  45. Petit RJ, Kremer A, Wagner DB (1993) Finite island model for organelle and nuclear genes in plants. Heredity 71:930–641CrossRefGoogle Scholar
  46. Petit RJ, Brewer S, Bordacs S, Burg K, Cheddadi R, Coart E, Cottrell J, M Csaikl U, van Dam B, Deans JD, Espinel S, Fineschi S, Finkeldey R, Glaz I, Goicoechea PG, Jensen JS, König AO, Lowe AJ, Madsen SF, Mátyás G, Munro RC, Popescu F, Slade D, Tabbener H, de Vries SGM, Ziegenhagen B, de Beaulieu JL, Kremer A (2002) Identification of refugia and post-glacial colonisation routes of European white oaks based on chloroplast DNA and fossil pollen evidence. For Ecol Manag 156:49–74CrossRefGoogle Scholar
  47. Petit RJ, Aguinagalde I, Beaulieu JL, Bittkau C, Brewer S, Cheddadi R, Ennos R, Fineschi S, Grivet D, Lascoux M, Mohanty A, Müller-Stark G, Demesure-Musch B, Palmée A, Martín JP, Rendell S, Vendramin GG (2003) Glacial refugia: hotspots but not melting pots of genetic diversity. Science 300:1563–1565CrossRefPubMedGoogle Scholar
  48. Põlme S, Bahram M, Yamanaka T, Nara K, Dai YC, Grebenc T, Tedersoo L (2013) Biogeography of ectomycorrhizal fungi associated with alders (Alnus spp.) in relation to biotic and abiotic variables at the global scale. New Phytol 198:1239–1249CrossRefPubMedGoogle Scholar
  49. Põlme S, Bahram M, Kõljalg U, Tedersoo L (2014) Global biogeography of Alnus-associated Frankia actinobacteria. New Phytol 204:979–988CrossRefPubMedGoogle Scholar
  50. Powell W, Morgante M, McDevitt R, Vendramin G, Rafalski J (1995) Polymorphic simple-sequence repeat regions in chloroplast genomes: applications to the population genetics of pines. Proc Natl Acad Sci U S A 92:7759–7763CrossRefPubMedPubMedCentralGoogle Scholar
  51. Pozzi AC, Bautista-Guerrero HH, Nouioui I, Cotin-Galvan L, Pepin R, Fournier P, Menu F, Fernandez MP, Herrera-Belaroussi A (2015) In-planta sporulation phenotype: a major life history trait to understand the evolution of Alnus-infective Frankia strains. Environ Microbiol 17:3125–3138CrossRefPubMedGoogle Scholar
  52. Provan J, Powell W, Hollingsworth PM (2001) Chloroplast microsatellites: new tools for studies in plant ecology and evolution. Trends Ecol Evol 16:142–147CrossRefPubMedGoogle Scholar
  53. Quantum GIS Development Team (2016) Quantum GIS geographic information system. Open source geospatial foundation project.
  54. Rambaut A (2014) FigTree v1.4.2, a graphical viewer of phylogenetic. Trees Available from
  55. Ren BQ, Xiang XG, Chen ZD (2010) Species identification of Alnus (Betulaceae) using nrDNA and cpDNA genetic markers. Mol Ecol Resour 10:594–605CrossRefPubMedGoogle Scholar
  56. Rochet J, Moreau PA, Manzi S, Gardes M (2011) Comparative phylogenies and host specialization in the alder ectomycorrhizal fungi Alnicola, Alpova and Lactarius (Basidiomycota) in Europe. BMC Evol Biol 11:40CrossRefPubMedPubMedCentralGoogle Scholar
  57. Roy M, Rochet J, Manzi S, Jargeat P, Gryta H, Moreau PA, Gardes M (2013) What determines Alnus-associated ectomycorrhizal community diversity and specificity? A comparison of host and habitat effects at a regional scale. New Phytol 198:1228–1238CrossRefPubMedGoogle Scholar
  58. Roy M, Pozzi AC, Gareil R, Nagati M, Manzi S, Nouioui I, Sharikadze N, Jargeat P, Gryta H, Moreau PA, Fernandez MP, Gardes M (2017) Alder and the golden fleece: high diversity of Frankia and ectomycorrhizal fungi revealed from Alnus glutinosa subsp. barbata roots close to a Tertiary and glacial refugium. PeerJ 5:e347CrossRefGoogle Scholar
  59. Schuelke M (2000) An economic method for the fluorescent labelling of PCR fragments. Nat Biotechnol 18:233–234CrossRefPubMedGoogle Scholar
  60. Simeone MC, Grimm GW, Papini A, Vessella F, Cardoni S, Tordoni E, Piredda R, Franc A, Denk T (2016) Plastome data reveal multiple geographic origins of Quercus group Ilex. PeerJ 4:e1897CrossRefPubMedPubMedCentralGoogle Scholar
  61. Sollars ES, Harper AL, Kelly LJ, Ramirez-gonzalez RH, Swarbreck D, Kaithakottil G, Uauy C, Havlickova L, Zohren J, Clavijo BJ, Li Y, He Z, Fellgett A, Cooper ED, Kjær ED, Downie JA, Boshier D, Lee S, Bancroft I, Caccamo M, Buggs RJ (2017) Genome sequence and diversity of European ash trees threatened by ash dieback. Nature 541:212–216CrossRefPubMedGoogle Scholar
  62. Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30:1312–1313CrossRefPubMedPubMedCentralGoogle Scholar
  63. Steele FL (1961) Introgression of Alnus serrulata and Alnus rugosa. Rhodora 63:297–304Google Scholar
  64. Stewart JR, Lister AM, Barnes I, Dalén L (2010) Refugia revisited: individualistic responses of species in space and time. Proc R Soc Lond B 277:661–671CrossRefGoogle Scholar
  65. Straub SCK, Parks M, Weitemier K, Fishbein M, Cronn RC, Liston A (2012) Navigating the tip of the genomic iceberg: next-generation sequencing for plant systematics. Am J Bot 99:349–364CrossRefPubMedGoogle Scholar
  66. Tallantire PA (1974) The palaeohistory of the grey alder (Alnus incana (L.) Moench.) and black alder (A. glutinosa (L.) Gaertn.) in Fennoscandia. New Phytol 73:529–546CrossRefGoogle Scholar
  67. Vanden Heuvel BD (2011) Alnus. In: Kole C (ed) Wild crop relatives: genomic and breeding resources, Forest trees, chap. 1. Springer-Verlag, Berlin, pp 1–14Google Scholar
  68. Vercken E, Fontaine MC, Gladieux P, Hood ME, Jonot O, Giraud T (2010) Glacial refugia in pathogens: European genetic structure of anther smut pathogens on Silene latifolia and Silene dioica. PLoS Pathog 6:e1001229CrossRefPubMedPubMedCentralGoogle Scholar
  69. Vitelli M, Vessella F, Cardoni S, Pollegioni P, Denk T, Grimm GW, Simeone MC (2017) Phylogeographic structuring of plastome diversity in Mediterranean oaks (Quercus group ilex, Fagaceae). Tree Genet Genomes 13:3CrossRefGoogle Scholar
  70. Whittemore AT, Schaal BA (1991) Interspecific gene flow in sympatric oaks. Proc Natl Acad Sci U S A 88:2540–2544CrossRefPubMedPubMedCentralGoogle Scholar
  71. Wicaksono CY, Aguirre-Guiterrez J, Nouhra E, Pastor N, Raes N, Pacheco S, Geml J (2017) Contracting montane cloud forests: a case study of the Andean alder (Alnus acuminata) and associated fungi in the Yungas. Biotropica 49:141–152CrossRefGoogle Scholar
  72. Zedane L, Hong-Wa C, Murienne J, Jeziorski C, Baldwin BG, Besnard G (2016) Museomics illuminate the history of an extinct, paleoendemic plant lineage (Hesperelaea, Oleaceae) known from an 1875 collection from Guadalupe Island, Mexico. Biol J Linn Soc 117:44–57CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Hervé Gryta
    • 1
  • Céline Van de Paer
    • 1
  • Sophie Manzi
    • 1
  • Hélène Holota
    • 1
  • Mélanie Roy
    • 1
  • Guillaume Besnard
    • 1
  1. 1.CNRS, UPS, ENSFEA, IRDLaboratoire Evolution & Diversité BiologiqueToulouseFrance

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