Mycorrhizal diversity, seed germination and long-term changes in population size across nine populations of the terrestrial orchid Neottia ovata HANS JACQUEMYN, * MICHAEL WAUD, * † VINCENT S. F. T. MERCKX, ‡ BART LIEVENS † and REIN BRYS * *KU Leuven, Department of Biology, Plant Conservation and Population Biology, B-3001 Leuven, Belgium, †KU Leuven, Campus De Nayer, Department of Microbial and Molecular Systems (M 2 S), Laboratory for Process Microbial Ecology and Bioinspirational Management (PME&BIM), B-2860 Sint-Katelijne-Waver, Belgium, ‡Naturalis Biodiversity Center, Leiden University, Leiden, the Netherlands Abstract In plant species that rely on mycorrhizal symbioses for germination and seedling establishment, seedling recruitment and temporal changes in abundance can be expected to depend on fungal community composition and local environmental condi- tions. However, disentangling the precise factors that determine recruitment success in species that critically rely on mycorrhizal fungi represents a major challenge. In this study, we used seed germination experiments, 454 amplicon pyrosequencing and assessment of soil conditions to investigate the factors driving changes in local abun- dance in 28 populations of the orchid Neottia ovata. Comparison of population sizes measured in 2003 and 2013 showed that nearly 60% of the studied populations had declined in size (average growth rate across all populations: 0.01). Investigation of the mycorrhizal fungi in both the roots and soil revealed a total of 68 species of puta- tively mycorrhizal fungi, 21 of which occurred exclusively in roots, 25 that occurred solely in soil and 22 that were observed in both the soil and roots. Seed germination was limited and significantly and positively related to soil moisture content and soil pH, but not to fungal community composition. Large populations or populations with high population growth rates showed significantly higher germination than small pop- ulations or populations declining in size, but no significant relationships were found between population size or growth and mycorrhizal diversity. Overall, these results indicate that temporal changes in abundance were related to the ability of seeds to ger- minate, but at the same time they provided limited evidence that variation in fungal communities played an important role in determining population dynamics. Keywords: extinction debt, mycorrhizal fungi, orchids, population dynamics, seed germination Received 12 November 2014; revision received 24 April 2015; accepted 7 May 2015 Introduction Recruitment from seeds represents a major factor deter- mining the distribution and abundance of plant popula- tions and therefore represents a key component in the life history of plants (Clark et al. 2007). In general, two major factors determine whether and where recruitment will take place. If plants do not produce a sufficient number of viable seeds or seeds are unable to reach vacant sites, recruitment will be limited by seed avail- ability. If, on the other hand, the availability of suitable environmental conditions is limited, seedling recruit- ment will be limited due to microsite limitation (Eriks- son & Ehrl en 1992). In plant species that rely on mycorrhizal symbioses for germination and seedling establishment, the availability of suitable mycorrhizal fungi may constitute an important part of microsite lim- Correspondence: Hans Jacquemyn, Fax: +32 16321968; E-mail: hans.jacquemyn@bio.kuleuven.be © 2015 John Wiley & Sons Ltd Molecular Ecology (2015) 24, 3269–3280 doi: 10.1111/mec.13236