Narrow mycorrhizal specialization and its effect on early development in the Mediterranean orchid Orchis italica

Abstract

The contribution of individual orchid mycorrhizal (OrM) fungi to orchid germination is poorly understood. We isolated Tulasnella OrM fungi from 11 populations representing nine Italian regions associated with the orchid Orchis italica. We performed in vitro germination experiments using both single and two OrM isolate combinations of Tulasnella and Ceratobasidium isolates found in O. italica and co-occurring orchids. We performed an in situ reciprocal translocation experiment of O. italica seeds in two populations and we experimentally planted seeds into two unoccupied sites to test whether the presence of suitable OrM may limit its distribution. In vitro, Tulasnella isolates initiated germination, but only two isolates resulted in seedling development, while seeds did not germinate in the presence of Ceratobasidium isolates. Combinations of two Tulasnella isolates had lower germination rates compared to single isolates. Reciprocal translocations revealed that seeds from the same population had higher germination rates. Protocorms developed in one unoccupied site, with the associated fungus identified as Tulasnella spp. These results suggest that Tulasnella vary in their efficacy in seed germination and O. italica has a narrow specialization on Tulasnella. Such information is important for understanding the range limits of orchids and their ecological dependency on OrM fungi.

Orchid mycorrhizal communities associated withOrchis italicaare shaped by ecological factors and geographical gradients

“…In line with the results of Calevo and Duffy (2023) it could be that there is a temporal succession of OrM fungi colonization of orchid roots. It is not clear whether OrM taxa other than Tulasnellaceae play equally important roles in the germination and subsequent establishment of O. italica as a fully photosynthetic individual (but see Balducci et al, 2024). Such partitioning of space on the orchid roots may result in both positive and negative residual co-occurrences detected here.…”

“…The abundance, as measured by the number of reads, of these key OrM fungal taxa decreased with increasing latitude and increased with increasing longitude. As one of these OTUs (tul25) may be important in the germination of O. italica (Balducci et al, 2024), it could be that the reduction in the abundance of these OTUs with latitude reflects the limit of O. italica in the Mediterranean. Orchis italica does not occur in northern Europe and it may be that these OrM fungi are absent or occur at such low abundance that it renders it impossible for O. italica to establish outside of its current range.…”

“…To infer the phylogenetic position of filtered fungal OTUs, a maximum likelihood (ML) analyses was performed in the CIPRES (Miller et al, 2011). Forty-three Tulasnellaceae sequences (OR507087 -OR507130) isolated from adult roots and protocorms of O. italica reported in Balducci et al (2024) were also included for phylogenetic reconstruction. An alignment was obtained with MAFFT v.7.490 (Katoh & Toh, 2010) with default parameters and subsequently trimmed using trimAI v1.2.59 on XSEDE (Capella-Gutiérrez et al, 2009).…”

“…Previous molecular studies describing the OrM fungal communities associated with the genus revealed a potential broad specificity, with species primarily associating with several fungal Operational Taxonomic Units (OTUs) of the Tulasnellaceae (Girlanda et al, 2011;Jacquemyn et al, 2010Jacquemyn et al, , 2011Mennicken et al, 2023;Oja et al, 2017;Shefferson et al, 2008). Isolation from adult roots of O. italica, a widespread Mediterranean species, revealed a relatively narrow specialization on members of Tulasnella for germination and early development (Balducci et al, 2024). However, our understanding of the whole fungal community in roots over large spatial scales and how they might influence the distribution of O. italica remains largely unknown.…”