Most of us are accustomed to thinking about sexual dimorphism in animals.  Male lions have manes, and male deer have antlers and generally larger bodies than female deer.  In many species, male birds have more complex sings and more colorful plumage. Perhaps less familiar is that female insects are generally larger than males of the same species.  But many of us are unaware that sexual dimorphism exists in some plant species as well.

As a child, Kaoru Tsuji spent considerable time watching insects on plants.  Later, as an undergraduate at Kyoto University in Japan, she noticed that larvae of a particular geometrid moth only visited male Eurya japonica plants, but not females. This led to her graduate work on how plant sexes affect herbivorous insects, and later, more broadly, on how plant sexual dimorphism affects other species in the community.

Kaoru Tsuji gazes at female Eurya emarginata plant. Credit: Noriyo Tsuji.

At the 2014 Ecological Society of America meetings, Tsuji heard Tadashi Fukami talk about microbial communities in flower nectar, and realized that she could learn to apply Fukami’s techniques to the microbial communities living within Eurya flowers.  After working three months in Tadashi’s lab, Tsuji was now ready to explore whether two plant species, Eurya japonica and Eurya emarginata, host different communities of bacteria and fungi in the flowers of male and female plants.

Male and female flowers of the two study species visited by pollinators.  These photos are not to scale; in actuality the male flower is substantially larger.  You can get a sense of this by noting that the same insect pollinator, the fly Stomorhina obsoleta, is pictured in figure a and near the top left of figure b.

For both species, male flowers tend to be larger, while female flowers tend to have sweeter nectar. Higher sugar levels will increase the chemical stress experienced by microbial organisms living in the nectar. Because the inside of a microbial cell has a lower sugar concentration (and thus a higher water concentration) than the sugar rich nectar environment, water tends to leave the microbial cell, leading to severe dehydration. Thus Tsuji and Fukami expected to find lower microbial abundance in female flower nectar.

Complicating this situation, animal visitors, such as bees and flies, also influence the microbial community in at least two ways.  First, many nectar-colonizing microbes depend on animals to disperse them to new flowers. Second, the interaction of nectar production, water evaporation and consumption by bees and flies can change the concentration of sugar in the nectar.  If there are few (or no) animals drinking the nectar, water will evaporate, sugar will remain, and the nectar will become more and more concentrated (sweeter) as more nectar is secreted over time.  But if nectar gets consumed, the new secretions will simply replace the old nectar, and sugar levels should be relatively constant. Thus flowers without animal visitors should impose more chemical stress on microorganisms by virtue of being sweeter.

The researchers sampled nectar from 1736 flowers, and grew the nectar microbes on agar plates supplied with nutrients that would support either bacterial or fungal growth.  In addition, the researchers also placed small-mesh bags over a subset of these flowers (before they opened), to reduce animal visitation.  After five days they counted the number of colonies formed, to estimate microbial abundance. Unfortunately, microbes were rarely found in E. japonica, so most of the data are for E. emarginata flowers only.

Female flowers of Eurya emarginata visited by a fly, Stomorhina obsoleta. Agar plates showing isolated colonies of nectar-colonizing microbes are superimposed. Left and right plates have yeast and bacterial colonies, respecitevly, both isolated from E. emarginata nectar. Credit: Kaoru Tsuji and Yuichiro Kanzaki.

First, as expected, female flowers had higher nectar sugar levels than did male flowers (the Brix value measures sucrose concentration).  In addition, putting a fine mesh bag over the buds substantially increased sugar levels in nectar from flowers of both sexes.

Sucrose (Brix) concentration of exposed and bagged E. emarginata flowers of both sexes.  For box plots, the dark horizontal bar is the median value, while the box encloses the 25th and 75th percentile.

The proportion of flowers in which fungi and bacteria were detected was much greater in male flowers than in female flowers.  In male flowers only, bagging the flowers decreased fungal frequency but not bacteria frequency.

The proportion of exposed and bagged E. emarginata flowers whose nectar, when cultured in the appropriate medium, generated fungal colonies (top graph) and bacterial colonies (bottom graph).

The researchers used colony forming units (CFUs) – the number of viable colonies on the agar plate – as their measure of bacterial abundance.

Abundance of fungi (top) and bacteria (bottom) cultured in agar plates, that were swabbed with nectar derived from exposed and bagged E. emarginata flowers of both sexes. Note that the y-axis is log₁₀ CFUs, so an increase from 3 to 4 (for example) is actually a tenfold increase in number of CFUs.

As expected, fungi were less abundant in female flower nectar than in male flower nectar.  In addition, bagging the flowers substantially reduced fungal abundance. Bacteria were also less abundant in female flower nectar than in male flower nectar.  Surprisingly, bagging the flowers substantially increased bacterial abundance, despite the increased chemical stress and decreased visitation by animal visitors.

Why did bacterial abundance increase when flowers were bagged?  The researchers hypothesize that reduced fungal dispersal from bagging caused competitive release of bacteria from the fungi.  Presumably the fungi and bacteria compete for essential resources (such as amino acids) in the nectar.  Because the bags reduce fungal abundance, there are fewer fungi to out-compete the bacteria, leading to an increase in bacterial abundance.

The researchers used DNA analysis to characterize which microbial species were found in female vs. male flowers.  They discovered major differences in species composition between the sexes.  Taken together with the data on frequency and abundance, it is clear that sexual dimorphism in these plants influences microbial communities in significant ways.  Tsuji and Fukami suggest that sexual dimorphism in many species may have profound community-wide consequences that researchers are only beginning to understand and uncover.

note: the paper that describes this research is from the journal Ecology. The reference is Tsuji, K. and Fukami, T. (2018), Community‐wide consequences of sexual dimorphism: evidence from nectar microbes in dioecious plants. Ecology, 99: 2476-2484. doi:10.1002/ecy.2494. Thanks to the Ecological Society of America for allowing me to use figures from the paper. Copyright © 2018 by the Ecological Society of America. All rights reserved.