Dioecious species and arbuscular mycorrhizal symbioses

Abstract

Sex-specific interactions with herbivores and pollinators have been observed in female and male plants of dioecious species. However, only a limited number of studies have revised sex-specific patterns in mycorrhizal symbiosis. To test whether female and male plants of Antennaria dioica differ in their relationship with arbuscular mycorrhizal (AM) fungi, we examined the temporal and spatial variation in AM fungi in female, male and non-reproductive A. dioica plants in three natural populations in Finland during flowering and after seed production. Our results are consistent with previous studies both under greenhouse and field conditions with the same species showing differences in AM colonization between the sexes linked with allocation to reproduction. Taken together, the results indicate that there is a sex-specific interaction between A. dioica and AM fungi. Overall, females have a greater investment in AM fungi, likely to enhance their uptake of soil nutrients and support the reproduction by seed.

In dioecious speciesthe different resource demands between the sexes^1,2 can lead to secondary sexual dimorphism in the incidence and strength of biotic interactions such as herbivory, pollination and mycorrhizas.³ Approximately 74% of angiosperm species grow in symbiosis with arbuscular mycorrhizal (AM) fungi in their roots.⁴ This symbiosis has been interpreted as mutualistic because there is a bidirectional transfer of nutrients between host plant and fungal partners, usually improving the performance of both symbionts. However, the outcome of the relationship may vary along a continuum from mutualism to parasitism depending on biotic and abiotic factors,^5-7 the symbiont identity,⁷ and the AM colonization frequency in the roots.⁸ Typically, plants are colonized by multiple fungal species⁹ and the same fungus can simultaneously colonize different plants. Given that plants and their mycorrhizal symbionts can detect and preferentially reward the most beneficial partner,¹⁰ and that the sexes in dioecious species usually differ in resource needs and allocation patterns,^1,2 it is logical to predict that the sexes in dioecious plants could differ in the cost-benefit of this symbiosis.^3,11 When differences between the sexes are detected in relation to AM frequency in roots, females are sometimes reported to possess higher AM frequency and to receive more benefits than males.^3,11 Moreover, if the differences in root AM fungal colonization are linked to the different resource demands of the sexes, we should expect a change in the colonization frequency due to resource allocation patterns toreproduction during the growing season.

Previous studies with the dioecious perennial herb Antennaria dioica under field conditions show that the sexes do not differ in the frequency of colonization by AM fungal structures (hyphae, vesicles and arbuscules) inside their roots.^12,13 In these studies root samples were collected only in August, after resource allocation to reproduction has taken place. Therefore, we recently evaluated temporal and spatial variation in AM fungal colonization in roots of A. dioicain three natural populations in central Finland.¹⁴ To confirm whether or not the AM colonization frequency differs between the sexes and whether the potential differences are linked to resource allocation to reproduction, we collected roots from plants with different reproductive status (non-reproductive plants and flowering female and male individuals) before and after resource allocation to reproduction took place (i.e., during flowering period in June and after seed production period in August).

In the present work we report the seasonal response by AM fungal structures in A. dioica. Colonization by hyphae and arbuscules differed among reproductive status and populations, and the colonization by AM hyphae, vesicles and arbuscules decreased after seed production (Table 1; Fig. 1). In terms of the frequency of arbuscules, there were significant interactions between reproductive status and population (F_4,109 = 2.9, p = 0.022), reproductive status and period (F_2,109 = 4.0, p = 0.020) and population and period (F_2,109 = 1.1, p = 0.009). Overall, females had higher frequency of arbuscules, and marginally (p = 0.071) higher frequency of hyphae than male and non-reproductive plants during flowering (June), while the frequency of vesicles did not differ between sexual status. After seed production (August) females, males and non-reproductive plants did not differ in the frequency of AM colonization, which is consistent with the previous studies of Varga and Kytöviita.^12,13 However, the authors observed that the plants that did produce flowers tended to have higher mycorrhizal frequency than plants that did not produce flowers regardless of sex (Fig. 1, Varga and Kytöviita, pers. communication). The same trend was observed in plants growing under greenhouse conditions (Fig. 1, Varga and Kytöviita, pers. communication). In all studies, the amount of AM fungal structures in roots seems lower in non-flowering plants compared with flowering ones (Fig. 1), suggesting a link between resource allocation to reproduction and AM fungal frequency, and that plants can regulate the symbiosis to match the costs and benefits of these interactions. The differences in responses observed among populations and the interactions in AM frequency between the sexual status, the populations and the period suggest a complex relationship among soil fertility, plant sex and AM fungi.

Table 1. Root colonization by AM fungi in Antennaria dioica.

	Hyphae (%)	Vesicles (%)	Arbuscules (%)
Reproductive status
Female	32.7 ± 3.3a	13.6 ± 2.7a	5.2 ± 1.6a
Male	26.8 ± 2.2ab	12.6 ± 1.8a	2.5 ± 0.6b
Non-reproductive	24.9 ± 2.0b	12.4 ± 1.5a	1.9 ± 0.5b
Populations
Population 1	32.7 ± 2.6a	13.3 ± 1.8a	4.1 ± 1.6a
Population 2	28.4 ± 2.0a	13.7 ± 1.9a	3.4 ± 0.7a
Population 3	20.2 ± 2.3b	10.9 ± 2.2a	0.9 ± 0.3b
Period
Flowering	32.3 ± 2.3a	17.5 ± 1.9a	3.8 ± 0.9a
After seed production	23.1 ± 1.4b	8.1 ± 0.9b	2.2 ± 0.4b

Mean ± SE root colonization by different AM fungal structures in roots of Antennaria dioica in reference to their reproductive status and spatial (populations) and temporal (period) variation. Different letters indicate means that are significantly different (p < 0.05) when testing for differences between reproductive status, populations and period. Bold letters indicate marginally significant differences (p < 0.10).

Figure 1. Percentage of root colonized by (A) hyphae, (B) vesicles and (C) arbuscules in Antennaria dioica plants in four different studies. The different studies are indicated by roman numbers. The studies I¹⁵ and II¹⁶ were conducted under greenhouse conditions, while the studies III¹³ and IV¹⁴ were conducted in the field. The sexes (F, females and M, males) are plotted separately except in the study II where only F are shown because only one M produced flowers. In the studies I, II and III the root samples were taken only once in a set of plants that produced flowers and in another set that did not produce flowers, while in the study IV the root samples were taken twice (in June when plants were flowering and in August after seed production). Values are means ± SE. Sample sizes for the flowering and non-flowering plants were 13 F + 6 M and 22 F + 13M; 6 F and 4 F; 61 F + 59 M and 11 F + 11 M; and 25 F + 31 M and 25 F + 31 M in study I, II, III and IV, respectively.

In addition, previous studies under greenhouse confirm that the interaction with AM fungi and A. dioica is sex-specific:^15,16 females and males of A. dioica differed in their response to the same AM fungal species (Glomuscla roideum). Females gain greater AM benefit than males when grown without stress conditions, while under stress conditions (drought or low pH) the mycorrhizal benefit is lower and similar in both sexes.^15,16 These studies suggest that the sexes may provide different amount of photosynthates to the fungus and that they also differ in the benefit derived from the symbiont. However, in natural ecosystems the interaction between AM fungi and plants is more complex,^3,11 the plants are colonized by several AM fungal species and the colonization can be species-specific.⁹ The AM fungal species that A. dioica associates with under natural conditions are unknown. In this paper, we also report AM fungal species associated to the rhizosphere of A. dioica in the three populations in central Finland. The sites are the same where we have reported AM frequency in A. dioica roots.¹⁴ In short, A. dioica rhizosphere soil samples were collected, AM spores were extracted and identified based on morphological characters under a light microscope at 100× and 400× magnification. We identified 13 different AM morphotypes associated to the rhizosphere of A. dioica (Fig. 2). Five morphotypes belonged to the genus Glomus, five belonged to the genus Acaulospora, one morphotype belonging to the genus Claroideoglomus, one morphotype belonging to the genus Funneliformis and one morphotype belonging to the genus Scutellospora were found. This is consistent with a previous study where the AM fungal genera colonizing the roots of A. dioica were characterized with molecular methods: 14 sequence types belonging to genera Glomus and Acaulospora were identified inside the roots ofA. dioicaplants collected in central Sweden.⁹ Even though the sex of the Swedish plants was not specified, the authors also analyzed seasonal dynamics in community composition and reported the highest diversity of sequence types in May and June,⁹ exactly when we also observed the highest percentage of colonization by arbuscules, and the plants had flowers.¹⁴ In autumn the diversity of sequence types decreased⁹ in line with our finding of declining AM colonization frequency toward the end of the growing season (Fig. 1).¹⁴

Figure 2. AM spores associated to rhizosphere of Antennaria dioica. (A) Claroideoglomus claroideum, (B) Glomus hoi, (C) Funneliformis mosseae, (D) Acaulospora scrobiculata like, (E) Scutellospora calospora, (F) Glomus sp1, (G) Glomus sp2, (H) Glomus sp3, (I) Acaulospora sp1, (J) Acaulospora sp2, (K) Acaulospora sp3, (L) Acaulospora sp4, (M) Glomus sp4. The genera Acaulospora and Scutellospora were placed in PVLG with Melzer’s reagent. Photos were taken with bright field microscopy and the scale is 50 µm. All photos were taken by Mauritz Vestberg.

Overall, the studies with A. dioca indicate that the symbiosis with AM fungi is sex-specific and these differences seem to be related to the different resource demands between sexes. Females have a greater investment in AM fungi and obtain a higher benefit of this interaction compared with male plants, presumably to enhance their uptake of soil nutrients and support the more expensive female function, the production of seeds. Studies involving the interactions between AM fungi and sexually dimorphic plants have increased in recent years.^3,11 However, our knowledge in this topic is still in its infancy. Future investigations should evaluate whether sex-specific interactions between AM fungi and plants are widespread in nature. More experimental ecological studies evaluating the seasonal dynamics of AM fungal communities both in the roots and in the soil are needed. In addition, little is known about how AM symbiosis can be influenced by other biotic interactions³and abiotic factors.^15,16 Finally, the possibility that AM fungi play an important role in the evolution of sexual systems as have been proposed for herbivores and pollinators,^17,18 deserves more attention.

Glossary

Abbreviations:

AM

arbuscular mycorrhizal

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.