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Intra-individual ITS polymorphism and hybridization in Pulmonaria obscura Dumort. and Pulmonaria angustifolia L. (Boraginaceae)

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Abstract

Internal transcribed spacer (ITS) is used as a molecular marker in most phylogenetical analyses in Boraginaceae, an unplaced group in the current angiosperm phylogeny. However, there is no knowledge on intra-individual polymorphism of ITS in Boraginaceae. Difficulties in PCR and sequencing of ITS in Pulmonaria species mentioned in the literature may be seen as indirect evidence of intra-individual polymorphism. This study aims to detect intra-individual polymorphism of ITS1–5.8S–ITS2 in rare species Pulmonaria angustifolia L. and common species Pulmonaria obscura Dumort. Cloning of ITS sequences of P. angustifolia, P. obscura and putative hybrid specimens from mixed population showed intra-individual polymorphism of ITS1–5.8S–ITS2 in all specimens, whereas most of them also contained pseudogenic sequences. Intra-individual sequence divergence of ITS1 and ITS2 spacers was similar in the P. angustifolia and mixed population and significantly higher than in P. obscura. P. angustifolia specimens had nucleotide polymorphisms characteristic to both P. angustifolia and P. obscura and part of P. angustifolia sequences clustered into mostly P. obscura clade on the neighbour-joining trees based on ITS1 sequence and ITS2 sequence and structure data. These results suggest a hybrid origin of all P. angustifolia populations studied. Thus, outbreeding depression is a likely explanation for the decline in populations near the northern border of distribution area of rare species P. angustifolia. Hybrid origin of the whole P. angustifolia is also probable; the central population from Poland showed a similar pattern of polymorphism as distribution edge populations.

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Acknowledgments

We thank Marcin Nobis, Joanna Zalewska-Gałosz, Vija Kreile, Egita Zviedre and Jaan Liira for their help with the fieldwork. We are indebted to Matthias Wolf for advice in structure analysis, thanks go to Meelis Pärtel, Tatjana Oja and two anonymous reviewers for valuable comments on the manuscript. We also are grateful to Robert Szava-Kovats for proofreading. This study was supported by the European Regional Development Fund (Center of Excellence FIBIR), the “Environmental protection and technology R&D programme (KESTA)” and by the Ministry of Education and Research (IUT20-29 and IUT20-31).

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  1. Institute of Ecology and Earth Sciences, University of Tartu, Lai 40, 51005, Tartu, Estonia

    Ene Kook, Kersti Püssa, Rein Kalamees, Ülle Reier & Silvia Pihu

  2. Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010, Tartu, Estonia

    Eve Vedler

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  1. Ene Kook
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Appendices

Appendix 1

See Table 4.

Table 4 Number of observed ITS types (n = 118) among Pulmonaria specimens studied
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Appendix 2

Table 5.

Table 5 Boraginaceae sequences from GenBank and sequence-structure information from ITS2 database used in neighbour-joining analyses
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Kook, E., Vedler, E., Püssa, K. et al. Intra-individual ITS polymorphism and hybridization in Pulmonaria obscura Dumort. and Pulmonaria angustifolia L. (Boraginaceae). Plant Syst Evol 301, 893–910 (2015). https://doi.org/10.1007/s00606-014-1123-8

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