Mycorrhizal associations and reproductive isolation in three closely related Orchis species

Abstract

Background and aims: The maintenance of species boundaries in sympatric populations of closely related species requires some kind of reproductive isolation that limits gene flow among species and/or prevents the production of viable progeny. Because in orchids mycorrhizal fungi are needed for seed germination and subsequent seedling establishment, orchid-mycorrhizal associations may be involved in acting as a post-mating barrier.

Methods: We investigated the strength of post-mating barriers up to the seed germination stage acting between three closely related Orchis species (Orchis anthropophora, O. militaris and O. purpurea) and studied the role of mycorrhizal fungi in hybridization by burying seed packets of pure and hybrid seeds. After retrieval and assessment of seed germination, the fungi associating with protocorms originating from hybrid and pure seeds were determined and compared with those associating with adult individuals using DNA array technology.

Results: Whereas pre-zygotic post-mating barriers were rather weak in most crosses, post-zygotic post-mating barriers were stronger, particularly when O. purpurea was crossed with O. anthropophora. Germination trials in the field showed that seed germination percentages of hybrid seeds were in most cases lower than those originating from pure crosses. In all species pair combinations, total post-mating reproductive isolation was asymmetric. Protocorms associated with a smaller range of fungal symbionts than adult plants, but there was considerable overlap in mycorrhizal associations between protocorms and their respective parents.

Conclusions: Our results suggest that mycorrhizal associations contribute little to reproductive isolation. Pre-mating barriers are probably the main factors determining hybridization rates between the investigated species.

Fig. 1.

Flower morphology of the three investigated Orchis species. (A) Side view of the helmet, (B) front view of the lip and (C) the pollinium of O. anthropophora, O. purpurea and O. militaris.

Fig. 2.

Mycorrhizal associations identified in (A) adults and (B) protocorms obtained from different crosses between O. anthropophora (A), O. militaris (M) and O. purpurea (P). In the experimental crosses, the first letter denotes the receptor species and the second letter the donor species.

Fig. 3.

Protocorms retrieved in seed packages originating from crosses between (A) O. militaris^(♀) and O. purpurea^(♂), (B) O. purpurea^(♀) and O. anthropophora^(♂), and (C) O. anthropophora^(♀) and O. militaris^(♂).

Fig. 4.

Total reproductive isolation incorporating both pre- and post-mating barriers up to seed germination and relative contributions of reproductive barriers of reciprocal crosses between O. anthropophora, O. militaris and O. purpurea. In all crosses, the first species name denotes the receptor species and the second the donor species.