Protocarnivore or locomotion deterrent? Function of ‘cups’ in Dipsacus fullonum (common teasel) and by Anson Lam Ho Ching

Protocarnivore or locomotion deterrent? Function of ‘cups’ in Dipsacus fullonum (common teasel) and symbiosis with Dasyhelea bilineata (midge) larvae as possible mechanism of partial carnivory Ho Ching Anson Lam

Abstract

larvae of species Dasyhelea bilineata was also discovered in cups of D. fullonum. I then discuss and weigh the likelihood of the two hypothesis and concludes that the function of locomotion deterrent precedes protocarnivory. Then, D. bilineata specialized in breeding in D. fullonum and result in a mutualistic relation where the larvae help to break down organic matter.

Teasels have connate leaves, where sessile leaves merge at the stem, forming a cup-shape formation. These ‘cups’ can collect rain water. The function is it may be to bar insects from climbing up, or to trap organic matter and invertebrates, thus gaining nutrients. This study confirms that D. fullonum specializes in collecting rainwater, and shows positive evidence for both hypothesis. A kind of _______________________________________________________________________________________

Introduction The ‘cup’ structure has long pondered biologist and there are predominantly two theories about the function of holding water in these receptacle: 1. Preventing arthropods from climbing up the stem (below referred as locomotion deterrent hypothesis); 2. As a pitfall trap for insects, which the plant can get nutrients from when dead animals are degraded (below referred as protocarnivore hypothesis). Dipsacus has been suggested to be partially carnivorous a few times (Darwin, 1877; Christy, 1923; Simons, 1981). Protocarnivorous plants, sometimes refer as paracarnivorous (Schnell, 2002), subcarnivorous or borderline carnivorous, can trap and kill insects or other animals. However, they lack the ability to produce and secrete digestive enzymes, thus often rely internal food web to break down dead bodies for the plants’ absorption (Jolivet, 1998). However, in Jolivet’s book (1998), Dipsacus was not considered to be protocarnivorous since it is a dicot, while ‘qualified’ protocarnivorous plants are all monocots. Shaw and Shackleton(2011) attempted to provide experimental evidence that Dipsacus Fullonum

benefits from carnivory, and shows that addition of dead dipteran larvae to leaf bases caused an increase in seed set and the seed mass to biomass ratio. The reservoir may be analogous to a moat around a castle, which acts as a defense mechanism to prevent invaders from crossing. It has been suggested that these ‘invaders’ can be sap-sucking aphids, or nectar-stealing ants. A study by Beal and John (1887) suggest that it is more probable the main function of ‘cups’ is to protect the plant from crawling insects, since efficiency in trapping them was not observed, but they are effective in barring crawling insects. Pollination is mainly carried out by flying insects instead of beetles or ants. Hoverflies as pollinators are common in Cambridgeshire, England (Livio, Sarah, Lynn & Ben, 1999); while bees are most common in Ontario, Canada (William, 1984). Thus pollination can still be carried out. In this study, I set out to investigate whether there is more support for locomotion deterrent hypothesis or protocarnivore hypothesis. Adaptations in D. fullonum to increase efficiency in

collecting and retaining water are also investigated, possible mechanism for D. fullonum to break down as filling the cups with water is essential to either dead bodies and obtain nutrients. functions. Furthermore, I looked into how effective is the reservoir as a barrier, and what may be the _______________________________________________________________________________________

Method This study is divided into three parts: investigating 1. Water collecting and storing efficiency; 2. Effectiveness in barring insects; 3. Procarnivory Experiment 1A Artificial rain is created by pouring water with a water can over the plant. Then, amount of water collected is measure using a syringe. The process is repeated 5 times for each treatment. The plant was treated either intact, leaves bent downwards, auxiliary branches cut, and all leaves cut leaving behind cup structures (as shown in figure 1). Figure 1: From left to right shows a) D. fullonum untreated. b) leaves artificially bent downwards. c) branches removed. d) only cup structure is left Experiment 1B Treatments were done such that leaves were bent downwards or upwards on alternating leaves up a stem. Cups were filled full with water and surface area of the reservoirs were measured. Evaporation rate was then calculated by measuring amount of water left in each cups once every day. Experiment 2 Yellow meadow ants (Lasius flavus) are placed between nodes on the stem. The cups up or down in respect to the ants are either filled with water or empty. After 30 minutes, it was recorded how many ants have passed through the cups.

Experiment 3 Ink from a gel pen was syringed into three cups, two in one plant and one in another. After three days, it was observed whether the pigments has

been transported into leaves or vasculature in the main stem.

_______________________________________________________________________________________ It was also shown that such morphology also Results Experiment 1A: Collecting and storing water as significantly reduced the water loss by evaporation main function of cups (Kruskal-Wallis, p<0.05) (fig 3). It was shown that Dipsacus has morphological features that facilitates collecting and retaining of water in their cups. Data show that leaves pointing upwards significantly increases the volume of water collected in cups in an experiment of artificial rain (Kruskal-Wallis, p<0.01). There is around 85% drop in water collected when the leaves are forced to bend downwards, or when the leaves are removed leaving behind the cup structures (fig 1).

Figure 3: percentage loss of water in cups per hour for treated and non-treated D. fullonum. Leaves bending downwards has a significant effect in increasing water evaporation rate (Kruskal-Wallis, n=19, p<0.05)

Figure 2: total volume of water collected in cups after an artificial rain for different treated D. fullonum. Aux cut= branches cut, cut= only cup structure left, intact= untreated, petiolated= leaves artificially bent downwards. Individual plants with leaves untreated collected significantly more water than other treatments (Kruskal-Wallis, n=20, p<0.01). Experiment 1B

Experiment 2: Water as a barrier to pests Figure 4 and 5 shows that water in cups is an effective barrier to prevent yellow meadow ants from walking up the stem. When the upper cup was filled with water, none of the ants successfully swam through the water to get to higher nodes of the stem (fig 5), but 1-2 ants successfully floated through the water to get to lower nodes when the lower cup was filled (fig 4). Statistics show that whether the upper cup was filled with water or not has no significant effect on the number of ant walked through the cup to go

upwards (p=0.47, Kruskal-Wallis). However, in all treatments, only less than or equal to one ant passed through and climb up. There is a tendency that ants travel downwards instead of upwards.

actively depart from the stem into the water, but instead they fell from the stem, which then floats to the leaf side, allowing the ants to land on. Experiment 3: Ink tracing experiment Pigment did not permeate epidermis. It was not found in the leaf nor in the vasculature of main stem.

Figure 4: Number of ants successfully passed downwards across a cup in 30 minutes. X-axis legend: upper cup treatment.lower cup treatment. Filling the lower cup with water significantly decreased the number of ants that passed through (Kruskal-Wallis, n=10, p=0.01)

Figure 6: Cup of D. fullonum with ink in it had its main stem peeled into strands exposing the vasculature. No blue pigment was observed. Cup under microscope Trichome protrusions into the cup were observed.

Figure 5: Number of ants successfully passed upwards across a cup in 30 minutes. X-axis legend: upper cup treatment.lower cup treatment. Filling the upper cup with water has no significant effect on number of ants that passed through (KruskalWallis, n=10, p=0.47) About how the ants travel down, the ants did not

Figure 8: Height of D. fullonum at the hill of Juniper Hall at different distances from the tree line. D. fullonum further away have shorter heights (ANOVA, n=25, p<0.001).

Figure 7: A collection of pictures taken under a light microscope (first one in x 50, others x 100) showing the glandular hairs in the cup regions of D. fullonum indicated by the red circle.

Collecting field data Data on height and number of purplish leaves was collected on the hill? In Juniper Hall. I sampled the D. fullonum there along a distance from the treeline, and D.fullonum were clustered into three patches at 2m, 5m and 7m away from the tree line. Results show that distance has significant effect on the height of D. fullonum (ANOVA, n=25, p<0.001). Height of D. fullonum at 5m and 7m is about 50 cm lower than those at 2m. Furthermore, it shows that the interaction between distance and number of purplish leaves is significant (ANOVA, n=18, p<0.01). There are fewer purplish leaves at 2m than at 7m.

Figure 9: Shows the interaction between number of purple leaves and Observing water samples from cups Samples of water were collected from cups of D. fullonum and analysed under a microscope. Different kinds of arthropods such as hoverflies, beetles, thrips, spiders, aphids, bees, wasps, moths etc. were observed. Algae is abundant. Also, living larvae were observed in all water samples collected, which were then identified to belong to family Ceratopogonidae. Pupae were kept in a petri

dish, and adults were identified to belong to genus Dasyhelea. Only one species of larvae was ever observed. It was estimated that a well watered cup holds at least 50-100 larvae

Figure: Pupal stage of Dasyhelea as indicated by red arrow

Figure: Larvae feeding on organic matter

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Discussion Experiment 1A & 1B: The main reason why I investigated whether the upward bending of leaves influences evaporation rate or water collecting efficiency is because such morphology does not maximize light capturing for photosynthesis, as the leaves should spread out horizontally. Therefore, a selective pressure favouring upward bending of leaves might have existed, and suggests that benefits from having water-filled cups outweighs the cost of reduced photosynthetic efficiency. D. fullonum therefore, has adaptations to collect and store water. There are other possible morphological adaptations, for example the leaves are folded at an angle about the central vein, which can drain rain water to the cups. Furthermore, while the lower opposite leaves are sessile and form cups, the upper opposite leaves are perfoliate or clasping, which the water drained to the stem will drip down to the uppermost cup. This has a significance that it is better to have fewer cups filled with more water, than to have many cups filled with little water which will be evaporated more quickly. The central midrib of the leaves are also strengthened with its hollow structure and vascular bundles arranged in a curved manner. This may help maintain the cup structure and resist forces from rain droplets or wind.

Experiment 2 Results show that water is effective in preventing

insects from walking up the stem with a ‘moat’ defense line. To explain why even when the upper cup was empty there was no more than one ant successful in crossing over, it was observed that the cups were not dried completely and a thin film of water remained, which the legs of ants got stuck due to low surface tension. The ants then exhibit a behaviour which they rub their feet and antennae, possibly to get rid of water. However, the main drawback of this experiment is that yellow meadow ants are not usually found on D. fullonum. Rather, they are only proxies. Thus, the experiment does not reflect the real scenario. Nonetheless, it still shows that the ‘moats’ are effective to a certain extent. Experiment 3 Ink pigment not going through the epidermis does not mean nutrients in the water do not get absorbed by the plant. It may be because the pigment particles are too large to diffuse through, or D. fullonum may have transporters for nutrients e.g. potassium or nitrate, but just not pigments. An experiment investigating nutrient flow from dead bodies in the cups to D. fullonum has been done by Shaw (2011). It was shown that the seed mass and biomass:seed mass ratio increased when a maggot was put in the cups at regular time intervals. Shaw’s experiment can be criticized that: 1. Maggots are not usually found in D. fullonum’s cups, thus does not reflect real situation; 2. In the analysis, there is no reason why the individual plants should be classified into two size classes based on the number of leaves while the number of leaves can already be used as a factor.

Using Shaw’s raw data, I independently performed statistical analysis, also using ANOVA. However, I included all the interaction terms up to fourway(number of leaves, site, water treatment and maggot treatment). Then, I removed the terms that are not significant step-wise to produce a minimal model. Results are shown in appendix x. It was mentioned in Shaw’s discussion part (2011), that ‘surprisingly cutting each leaf base to prevent water buildup had no effect on any growth parameter’. This may be because water removal treatment took place once a week, which allowed certain amount of water to accumulate during the week, or that some water was still left after removal. Nonetheless, this raised the question whether water is needed for nutrient acquirement by the plant since internal food web such as bacteria would break down organic matter quicker with presence of water, and nutrients need a liquid medium in contact with the plant in order for diffusion or direct transport across epidermis to take place. In my analysis, the interaction term of maggot treatment:water treatment is the only term containing ‘water treatment’ that yields a significant effect on growth parameters, and that is log(biomass). As shown in figure x, in general water removal counterintuitively increased the biomass of D. fullonum in the experiment. However, as shown in figure x, water removal decreases seed mass to biomass ratio, though the effect is not statistically significant.

Showdown between locomotion deterrent and protocarnivore hypotheses Based on the results, there is support for both locomotion deterrent and protocarnivore hypothesis. A morphological structure may have more than one function, and this may be the case in D. fullonum. However this brings to the question whether the initial selective pressure for developing connate leaves at the base of the leaves is for locomotion deterrent or for carnivory. The former is more probable than the latter, since relying on internal food web to degrade corpses is not reliable and efficient, plus the plant is not adapted to absorb nutrients through the epidermis of leaves and stem under the surface of water. However, were mutations taken place that made Dipsacus able to retain water in connate sessile leaves, the effect of preventing insects from climbing up the stem is more immediate. The ability to trap insects may come as a side effect and the plant might then benefit from it from absorbing nutrients. However, Christy (1923) argues that D. fullonum is so tall and has long corolla tubes that it is not worth it for insects. Questioning adaptations in D. fullonum for carnivory As early as in 1877, Francis Darwin studied the traits D. sylvestris (synonymous to D. fullonum) which may help carnivory, with the main focus on ‘protoplasmic filaments from the glandular hairs’. Darwin wrote that: 1. The leaves are smooth and steeply inclined, and form a pair of treacherous slides leading down to a pool of water and it prevents insects from going out 2. There are no spines near the cup regions so insects can’t crawl out using them as a ladder

3. Trichomes in cup regions help absorption of nutrients 4. First year leaves are hairy, second year’s are smooth- a trait that is specialized for trapping insects Regarding point 1, the upper surface of the leaf is not smooth and steep enough and animals can still climb out, as ants did in my experiment. However, Francis Darwin has remarked that ‘I have seen a beetle struggling to get out and observed his tarsi slipping, over and over again, on the smooth stalk.’ It is unlikely that the insects trapped in the cups climbed up from the bottom, but rather reach the plant by crossing from other vegetation, since the ‘moat’ is so effective at barring insects. This would explain why insect bodies are found not only in the bottommost cup, but also on higher cups. Regarding point 2, as shown in picture x, there are spines near some of the cup regions, though many of them don’t. Regarding point 3, it may be assumed that nutrients can freely diffuse across the epidermis, and the hairs can increase the surface contact area. In order to qualify D. fullonum as a carnivorous plant, it must have a mechanism to attract prey and capture them. Christy (1923) argues that the numerous dead insects present in cups cannot be due solely to accidental causes like wind-transport, rain-wash, accidental falls. Instead, she proposed that Dipsacus exudes sweeting-tasting toxic substances which attracts these creatures to the plant and also intoxicates them. Darwin had also noticed that beetles drown in the cups more rapidly than in pure water. However, these all lack evidence as support. Nonetheless, I do agree with Christy that the putrefying smell due to bacteria and organic compost may have an effect in attracting flies. Symbiosis with Dasyhelea bilineata (midge) larvae

as possible mechanism of partial carnivory D. fullonum might be on its way to evolving as a fully carnivorous plant, as the term protocarnivory suggests. For example, ability to emit volatile sweet scent molecules to attract insects, or being able to secrete its own digestive enzymes may be evolved in the future. However, it may be also be unnecessary to evolve such metabolically costly traits. From observation, there may be a symbiotic relationship between D. fullonum and Dasyhelea bilineata (in family ceratopogonidae, or the biting midges). Speculating mutual benefits, D. fullonum provides a breeding ground for Dasyhelea and protects them in the cup structures and with spines from predators. The water stored also prevents desiccation of larvae, providing larvae a medium to swim in, and food from organic matter such as dead bodies and algae. On the other hand, the larvae helps decompose organic matter through their intestinal tracts and excrete excreta. It may contain nutrients rich in e.g. phosphorus and nitrogen, more readily absorbed by the plant. It is analogous to a human gut microbiome which bacteria help digest substances that are hard to break down by human enzymes. Evidence for such symbiotic relationship comes from the fact that a species of Dasyhelea-D. bilineata, was discovered in the cups of Dipsacus ( ). Subsequent records of identifying D. bilineata was also in Dipsacusâ&#x20AC;&#x2122;s cups (). It can be believed that the larvae observed during this study were also D. bilineata. It also suggests that D. fullonum and D. bilineata may be coevolved species but there is currently no strong support.

tree line. Furthermore, it was observed that the cups of 7m teasels are dried out, have more purplish leaves and did not have larvae, but the 2m teasels have water-filled cups and have abundant larvae. The number of purplish leaves may indicate mineral deficiency and those at 7m are less healthy. The open space teasels are directly exposed to the sun, which evaporates the water quicker than when there is shade provided from trees, and D. bilineata preferentially would not breed in dried cups. The increased height may be due to the presence of larvae, or due to closer distance to the tree line. Since the two covaries a conclusion cannot be drawn. Further studies To fully understand the evolutionary development of connate leaves, one must study the genes involves in such morphology. I suggest comparing the genomes of species in genus Dipsacus, since while many possess connate leaves, some donâ&#x20AC;&#x2122;t e.g. D. pilotus. Radioactive substances can be supplied to the cups and tracked the absorption. A study on the effect of larvae to the nutrition acquirement of D. fullonum can also be carried out. Lastly, it is time to settle the score whether D. fullonum secretes sweet-smelling molecules or that putrefying smell attracts prey. References 1998 book, Interrelationship Between Insects and Plants, Pierre Jolivet Beal, W., & St. John, C. (1887). A Study of Silphium perfoliatum and Dipsacus laciniatus in Regard to Insects. Botanical Gazette,12(11), 268-270

Field data shows evidence for D. fullonum benefitting from Dasyhelea Figure x shows that the height of D. fullonum in open space is much shorter than those close to the

Comba, Livio; Corbet, Sarah; Hunt, Lynn; Warren, Ben. 1999. Flowers, nectar and insect visits: evaluating British plant species for pollinatorfriendly gardens. Annals of Botany. 83(4): 369-383.

[73070] Judd, William W. 1984. Insects associated with flowering teasel, Dipsacus sylvestris, at Dunnville, Ontario. Proceedings of the Entomological Society of Ontario. 114: 95-98. [73061] Christy M, 1923. The common teasel as a carnivorous plant. Journal of Botany, 61:33-45.

http://publikationen.ub.unifrankfurt.de/frontdoor/index/index/docId/14019 Darwin F. On the protrusion of protoplasmic filaments from the glandular hairs on the leaves of the common teasel (Dipsacus sylvestris). Proc Roy Soc Lond. 1877;26:245â&#x20AC;&#x201C;271.

_______________________________________________________________________________________ Appendix Upper cup

Lower Cup

Passed up

Passed down

Died

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