Abstract
European black pine (Pinus nigra J.F.Arnold) is an ecologically and economically important conifer growing across southern Europe and the Mediterranean Basin in a variety of habitats. Its morphological and ecological variabilities as well as geographic origin are the basis for subspecies level taxonomic identification and forestry practice recommendations. Yet, no true consensus exists and genomic resources are lacking for resolving the taxonomy of P. nigra and assessing its adaptive potential. To provide genetic tools for further research, we generated novel genomic resources using six de novo transcriptomic assemblies representing major biogeographic regions where the species is growing naturally. Using two closely related species, Pinus sylvestris and Pinus halepensis, as outgroups, we identified a set of 2200 nuclear, putatively orthologous, single-copy genes. In addition, we identified a set of 405,624 polymorphic SNPs for P. nigra. Based on these resources, we were able to confirm the division of P. nigra into two wide geographical population groups and to provide new insights into evolutionary dynamics of the species.
Similar content being viewed by others
References
Acosta JJ, Fahrenkrog AM, Neves LG, Resende MFR, Dervinis C, Davis JM, Holliday JA, Kirst M (2019) Exome resequencing reveals evolutionary history, genomic diversity, and targets of selection in the conifers Pinus taeda and Pinus elliottii. Genome Biol Evol 11:508–520
Afzal-Rafii Z, Dodd RS (2007) Chloroplast DNA supports a hypothesis of glacial refugia over postglacial recolonization in disjunct populations of black pine (Pinus nigra) in western Europe. Mol Ecol 16:723–736
Armenise L, Simeone MC, Piredda R, Schirone B (2012) Validation of DNA barcoding as an efficient tool for taxon identification and detection of species diversity in Italian conifers. Eur J Forest Res 131:1337–1353
Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120
Buschiazzo E, Ritland C, Bohlmann J (2012) Slow but not low: genomic comparisons reveal slower evolutionary rate and higher dN/dS in conifers compared to angiosperms. BMC Evol Biol 12:8
Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T (2009) trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25:1972–1973
Caudullo G, Welk E, San-Miguel-Ayanz J (2017) Chorological maps for the main European woody species. Data in Brief 12:662–666
Celinski K, Kijak H, Wojnicka-Półtorak A, Buczkowska-Chmielewska K, Sokołowska J, Chudzińska (2017) Effectiveness of the DNA barcoding approach for closely related conifers discrimination: a case study of the Pinus mugo complex. C R Biol 340(6–7):339–348
Çengel B, Tayanç Y, Kandemir G, Velioglu E, Alan M, Kaya Z (2012) Magnitude and efficiency of genetic diversity captured from seed stands of Pinus nigra (Arnold) subsp. pallasiana in established seed orchards and plantations. New For 43:303–317
Chifman J, Kubatko L (2014) Quartet inference from SNP data under the coalescent model. Bioinformatics 30:3317–3324
Christensen KI (1997) Pinaceae, Cupressaceae, Taxaceae, Ephedraceae, Salicaceae, Juglandaceae, Betulaceae, Fagaceae, Ulmaceae, Moraceae. In: Strid A, Tan K (eds) Flora Hellenica, vol vol.1. Koeltz Scientific Books, Königstein, pp 1–17
Cingolani P, Platts A, Wang le L, Coon M, Nguyen T, Wang L, Land SJ, Lu X, Ruden DM (2012) A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly 6:80–92
Climent J, Chambel MR, Santos del Blanco L, Martínez Valcuende L, Alía R (2013) Esclareciendo la variación adaptativa entre subespecies y procedencias de Pinus nigra Arnold. 6 Congreso Forestal Español. Sociedad Espanola de Ciencias Forestales Available at https://www.congresoforestal.es/actas/doc/6CFE/6CFE01-194.pdf
De La Torre AR, Li Z, Van de Peer Y, Ingvarsson PK (2017) Contrasting rates of molecular evolution and patterns of selection among gymnosperms and flowering plants. Mol Biology Evol 34:1363–1377
De Wit P, Pespeni MH, Ladner JT, Barshis DJ, Seneca F, Jaris H, Overgaard Therkildsen N, Morikawa M, Palumbi SR (2012) The simple fool’s guide to population genomics via RNA-seq: an introduction to high-throughput sequencing data analysis. Mol Ecol Resour 12:1058–1067
Debreczy Z, Rácz I (2011) Conifers around the world. DendroPress Ltd., Budapest
DePristo M, Banks E, Poplin R, Garimella K, Maguire J, Hartl C, Philippakis A, del Angel G, Rivas MA, Hanna M, McKenna A, Fennell T, Kernytsky A, Sivachenko A, Cibulskis K, Gabriel S, Altshuler D, Daly M (2011) A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Gen 43:491–498
Eckenwalder J (2009) Conifers of the world: the complete reference. Timber Press, Portland
Eckert AJ, Bower AD, Jermstad KD, Wegrzyn JL, Knaus B, Syring JV, Neale DB (2013) Multilocus analyses reveal little evidence for lineage-wide adaptive evolution within major clades of soft pines (Pinus subgenus Strobus). Mol Ecol 22:56358–55650
Emms DM, Kelly S (2015) OrthoFinder: solving fundamental biases in whole genome comparisons dramatically improves orthogroup inference accuracy. Genome Biol 16:157
Enescu CM, de Rigo D, Caudullo G, Mauri A, Houston Durrant T (2016) Pinus nigra in Europe: distribution, habitat, usage and threats. In: San-Miguel-Ayanz J, de Rigo D, Caudullo G, Houston Durrant T, Mauri A (eds) European atlas of forest tree species. Publ. Off. EU, Luxembourg, pp 015–138
Engström PG, Steijger T, Sipos B, Grant GR, Kahles A, The RGASP Consortium, Rätsch G, Goldman N, Hubbard TJ, Harrow J, Guigó R, Paul Bertone (2013) Systematic evaluation of spliced alignment programs for RNA-seq data. Nat Meth 10:1185–1181
Farjon A (2010) A handbook of the world’s conifers. Brill EJ, Leiden/Boston
Gernandt DS, Geada López G, Ortiz García S, Liston A (2005) Phylogeny and classification of Pinus. Taxon 54:29–42
Gernandt DS, Magallón S, López G, Flores OZ, Willyard A, Liston A (2008) Use of simultaneous analyses to guide fossil-based calibrations of Pinaceae phylogeny. Int J Plant Sci 169(8):1086–1099
Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A (2011) Full-length transcriptome assembly from RNA-seq data without a reference genome. Nat Biotechnol 29:644–652
Grivet D, Avia K, Vaattovaara A, Eckert AJ, Neale DB, Savolainen O, González-Martínez SC (2017) High rate of adaptive evolution in two widespread European pines. Mol Ecol 26:6857–6870
Gülsoy AD, Gülsoy AM, Çengel B, Kaya Z (2014) The evolutionary divergence of Pinus nigra subsp. pallasiana and its varieties based on noncoding trn regions of chloroplast genome. Turk J Bot 38:627–636
Huerta-Cepas J, Serra F, Bork P (2016) ETE 3: reconstruction, analysis and visualization of phylogenomic data. Mol Biol Evol 33(6):1635–1638
Isajev V, Fady B, Semerci H, Andonovski V (2004) EUFORGEN technical guidelines for genetic conservation and use for European black pine (Pinus nigra). International Plant Genetic Resources Institute, Rome
Katoh K, Standley D (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30(4):772–780
Matsuo M, Ito Y, Yamauchi R, Obokata J (2005) The rice nuclear genome continuously integrates, shuffles, and eliminates the chloroplast genome to cause chloroplast–nuclear DNA flux. Plant Cell 17:665–675
Mayol M, Riba M, Cavers S, Grivet D, Vincenot L, Cattonaro F, Vendramin GG, González-Martínez SG (2020) A multiscale approach to detect selection in nonmodel tree species: widespread adaptation despite population decline in Taxus baccata L. Evol Appl 13:143–160
McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA (2010) The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20:1297–1303
Müller KF, Quandt D, Müller J, Neinhuis C (2005) PhyDE ® 0.995: phylogenetic data editor. Available from http://www.phyde.de
Naydenov KD, Tremblay FM, Fenton NJ, Alexandrov A (2006) Structure of Pinus nigra Arn. populations in Bulgaria revealed by chloroplast microsatellites and terpenes analysis: provenance tests. Biochem Syst Ecol 34:562–574
Naydenov KD, Naydenov MK, Alexandrov A, Vasilevski K, Gyuleva V, Matevski V, Nikolic B, Goudiaby V, Bogunic F, Paitaridou D, Christou A, Goia I, Carcaillet C, Alcantara AE, Ture C, Gulcu S, Peruzzi L, Kamary S, Bojovic S, Hinkov G, Tsarev A (2016) Ancient split of major genetic lineages of European black pine: evidence from chloroplast DNA. Tree Genet Genomes 12(68):1–18
Pinosio S, González-Martínez BF, Cattonaro F, Grivet D, Marroni F, Lorenzo Z, Pausas JG, Verdú M, Vendramin GG (2014) First insights into the transcriptome and development of new genomic tools of a widespread circum-Mediterranean tree species, Pinus halepensis Mill. Mol Ecol Resour 14:846–856
Price MN, Dehal PS, Arkin AP (2010) FastTree 2 -- approximately maximum-likelihood trees for large alignments. PLoS One 5(3):e9490
Rabiee M, Sayyari E, Mirarab S (2019) Multi-allele species reconstruction using ASTRAL. Mol Phyl Evol 130:286–296
Rambaut A, Suchard MA, Xie D, Drummond AJ (2014) Tracer v1.6. Available from http://beast.bio.ed.ac.uk/Tracer
Ramos-Onsins SE, Windsor A, Mitchell-Olds T (2008) MANVa software: multilocus analysis of nucleotide variation. http://www.ub.edu/softevol/manva
Ronquist F, Teslenko M, van der Mark P, Ayres D, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61:539–542
Rubio-Moraga A, Candel-Perez D, Lucas-Borja ME, Tiscar PA, Viñegla B, Linares JC, Gómez-Gómez L, Ahrazem O (2012) Genetic diversity of Pinus nigra Arn. populations in southern Spain and northern Morocco revealed by inter-simple sequence repeat profiles. Int J Mol Sci 13:5645–5658
Ryberg PE, Rothwell GW, Stockey RA, Hilton J, Mapes G, Riding JB (2012) Reconsidering relationships among stem and crown group Pinaceae: oldest record of the genus Pinus from the Early Cretaceous of Yorkshire, United Kingdom. Int J Plant Sci 173(8):917–932
Saladin B, Leslie AB, Wüest RO, Litsios G, Conti E, Salamin N, Zimmermann NE (2017) Fossils matter: improved estimates of divergence times in Pinus reveal older diversification. BMC Evol Biol 17:95
Santini F, Serrano L, Kefauver SC, Abdullah-Al M, Aguilera M, Sin E, Voltas J (2019) Morpho-physiological variability of Pinus nigra populations reveals climate driven local adaptation but weak water use differentiation. Env Exp Bot 166:103828
Scotti-Saintagne C, Giovannelli G, Scotti I, Roig A, Spanu I, Vendramin GG, Guibal F, Fady B (2019) Recent, Late Pleistocene fragmentation shaped the phylogeographic structure of the European black pine (Pinus nigra Arnold). Tree Genet Genomes 15:76
Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22(21):2688–2890
Stamatakis A, Hoover P, Rougemont J (2008) A rapid bootstrap algorithm for the RAxML web servers. Syst Biol 57:758–771
Stöver BC, Müller KF (2010) TreeGraph 2: combining and visualizing evidence from different phylogenetic analyses. BMC Bioinformatics 11:7
Thiel D, Nagy L, Beierkuhnlein C, Huber G, Jentsch A, Konnert M, Kreyling J (2012) Uniform drought and warming responses in Pinus nigra provenances despite specific overall performances. For Ecol Manag 270:200–208
Vallauri DR, Aronson J, Barbero M (2002) An analysis of forest restoration 120 years after reforestation on badlands in the southwestern Alps. Restor Ecol 10:16–26
Van der Auwera GA, Carneiro M, Hartl C, Poplin R, del Angel G, Levy-Moonshine A, Jordan T, Shakir K, Roazen D, Thibault J, Banks E, Garimella K, Altshuler D, Gabriel S, DePristo M (2013) From FastQ data to high-confidence variant calls: the Genome Analysis Toolkit best practices pipeline. Curr Protoc Bioinformatics 43:11.10.1–11.10.33
Vidakovic M (1974) Genetics of European black pine (Pinus nigra Arnold). Ann Forest 6:57–86
von Raab-Straube E (2014) Gymnospermae. – In: Euro+Med Plantbase – the information resource for Euro-Mediterranean plant diversity. Published on the Internet http://ww2.bgbm.org/EuroPlusMed/. Accessed 11/12/2018
Wachowiak W, Trivedi U, Perry CS (2015) Comparative transcriptomics of a complex of four European pine species. BMC Genomics 16:234
Wang B, Mahani MK, Ng WL, Kusumi J, Phi HH, Inomata N, Wang XR, Szmidt AE (2014) Extremely low nucleotide polymorphism in Pinus krempfii Lecomte, a unique flat needle pine endemic to Vietnam. Ecol Evol 4:2228–2238
Zhang C, Rabiee M, Sayyari E, Mirarab S (2018) ASTRAL-III: polynomial time species tree reconstruction from partially resolved gene trees. BMC Bioinformatics 19(S6):153
Acknowledgments
Two anonymous reviewers and the associate editor are acknowledged for their helpful comments that improved the final version of this article. We thank M. Lingrand (INRA URFM, Avignon, France) for sample collection and preparation. Collection of material was made before the Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization to the Convention on Biological Diversity and was legally implemented by signatory countries. We thank IGAtech (Udine, Italy) for transcriptome sequencing and de novo assembly. We acknowledge Supercomputing Centre of Galicia (CESGA) as well as CSC—Finnish IT Center and the Finnish grid infrastructure (FGI) for Science for the allocation of computational resources.
Funding
Funding was provided by the project INIA-MAPAMA EG17-048 co-financed by FEADER (75%) according to EU Regulation 1305/2013. SO received funding from the Spanish Ministry of Economy and Competitiveness (MINECO) under PTA2015-10836-I contract. We received financial support from GenTree. This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no. 676876.
Author information
Authors and Affiliations
Contributions
BF and GGV conceived the study; FC and VV produced the data and assembled the reference transcriptome; SO performed transcriptomic data analyses; DG contributed to genetic analyses; SO, BF and DG wrote the manuscript with the help of GG; all authors revised and edited the text, and approved the final manuscript.
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Communicated by F. Gugerli
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Olsson, S., Grivet, D., Cattonaro, F. et al. Evolutionary relevance of lineages in the European black pine (Pinus nigra) in the transcriptomic era. Tree Genetics & Genomes 16, 30 (2020). https://doi.org/10.1007/s11295-020-1424-8
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11295-020-1424-8