- Email: [email protected]
Effects of initial propagule size and water depth on Butomus umbellatus L. growth and vegetative propagation
Accepted Manuscript Title: Effects of initial propagule size and water depth on Butomus umbellatus L. growth and vegetative propagation Authors: Christian Carter, John D. Madsen, Gary N. Ervin PII: DOI: Reference:
S0304-3770(17)30395-9 https://doi.org/10.1016/j.aquabot.2018.06.003 AQBOT 3040
To appear in:
Aquatic Botany
Received date: Revised date: Accepted date:
4-12-2017 15-6-2018 23-6-2018
Please cite this article as: Carter C, Madsen JD, Ervin GN, Effects of initial propagule size and water depth on Butomus umbellatus L. growth and vegetative propagation, Aquatic Botany (2018), https://doi.org/10.1016/j.aquabot.2018.06.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effects of initial propagule size and water depth on Butomus umbellatus L. growth and vegetative propagation
Christian Carter a, 1, John D. Madsen b, 2, and Gary N. Ervin a
SC RI PT
Affiliations: a
Department of Biological Sciences, Mississippi State University, MS 39762, USA
b
Geosystems Research Institute, Mississippi State University, MS 39762, USA
Corresponding author: Gary N. Ervin
U
Department of Biological Sciences, Mississippi State University, MS 39762, USA
N
[email protected]
A
Present addresses: PO Box 4242, Rio Rico, AZ 85648
2
US Department of Agriculture, Agricultural Research Service, Exotic and Invasive Research
M
1
D
Unit, Davis, California 95616
TE
Title: Effects of initial propagule size and water depth on Butomus umbellatus L. growth and vegetative propagation
EP
Authors: Christian Carter, John D. Madsen, and Gary N. Ervin Highlights • • • •
Vegetative regrowth occurred from all rhizome fragment sizes (3, 6, 9, and 12 cm). Total plant biomass decreased approximately 70 % along the 0 - 132 cm depth gradient. Shoot:root ratios increased almost five-fold along a depth gradient from 0 - 132 cm. Rhizome bud number and mass declined at deeper depths, along with total plant mass.
CC
• • • •
A
Declarations of interest: none
1
Abstract Over the last century, flowering rush (Butomus umbellatus L.: Butomaceae) has escaped its native Eurasian range and has become a problematic species in parts of North America. As an aquatic invasive species, flowering rush has degraded wetlands in introduced areas and has interfered
SC RI PT
with human water usage. Although experimental work has been published regarding the
reproductive biology of the species, we found few manipulative experiments aimed at testing
ecological attributes of this species in the literature. The research reported here demonstrates that flowering rush is capable of aggressive clonal growth and propagation, whether from vegetative rhizome buds or from fragments of rhizomes themselves. This species was capable of growth
U
along a depth gradient from zero to 132 cm and showed an ability to adjust biomass allocation
N
along that gradient; however, biomass and asexual propagule production declined at depths
A
beyond approximately 50 cm. The combination of regrowth from relatively small propagules,
M
ability to tolerate depths of greater than one meter, and plasticity of biomass allocation along habitat gradients make this species a potential threat in many aquatic and wetland habitats in its
D
introduced range. However, certain aspects of its biomass allocation strategies may enhance
TE
chemical control for plants growing at greater depths or those regenerating from rhizome buds, both of which tend to have greater S:R biomass ratios.
EP
Keywords: asexual propagule; clonal propagation; depth gradient; invasive species; rhizomes;
A
CC
rhizome buds
2
1. Introduction Butomus umbellatus L. (Butomaceae; commonly known as “flowering rush”) is an invasive freshwater macrophyte capable of creating extensive monotypic stands, with the potential for significant negative ecosystem impacts in its invaded range. Butomus umbellatus is
SC RI PT
considered an invasive pest species in North America (Anderson et al. 1974, Kliber and Eckert 2005, Bailey and Preston 2011), where its invasion has been suggested to cause negative
ecosystem impacts such as obstruction of water delivery, degradation of recreational waters, and reduction of biodiversity in native wetlands (Countryman 1970, Boutwell 1990, Marko et al. 2015, Madsen et al. 2016a, b).
U
The first recorded account of B. umbellatus in North America occurred in 1905 near
N
Montreal, along the St. Lawrence River (Core 1941, Countryman 1970). This species has since
A
spread throughout much of the Great Lakes region, with isolated pockets throughout waterways
M
in southern Canada and across the northern U.S. (Kliber and Eckert 2005, Gunderson et al. 2016). Escape from water gardens appears to have been the most important process leading to its
D
introduction and continued spread; for example, as early as 1897, B. umbellatus was advertised
TE
and sold by North American nursery companies (Les and Mehrhoff 1999). Despite its current designation as an invasive species, a recent survey of Ontario garden centers found that one third
EP
of those businesses included B. umbellatus in their inventories (Funnell et al. 2009), and a group in Minnesota found little difficulty having the plant delivered into the state, despite its prohibited
CC
status (Maki and Galatowitsch 2004). This species can exist on a wide variety of substrates (Roberts 1972, Hroudová 1989,
A
Gunderson et al. 2016), can grow terrestrially along shores or at depths where the plant is completely submerged, and may exhibit different shoot morphology in each condition (Countryman 1970, Sarbu et al. 2009). Field surveys have found flowering rush at depths out to 4.88 m (Madsen et al. 2016b). However, at depths of around 1.5 m, plants rarely produce any 3
emergent leaves, and biomass production appears to decline at depths greater than 1.2 m (Madsen et al. 2016b). When completely submerged, the leaves are thin and ribbon-like (Boutwell 1990), and plants do not flower (Hroudová et al. 1996). However, when emerged, the leaves become rigid, the outer cell walls and cuticle thicken, stomata increase in number by 15 %, and flowering
SC RI PT
may be observed (Sarbu et al. 2009). The ability to exist along a broad water depth gradient may enhance the invasibility of B. umbellatus, as this increases access to niche space and resources
that other plant species cannot access. Furthermore, the ability of B. umbellatus to form dense stands to depths of 1.2 m or more may result in severe degradation of littoral zone habitats of invaded lakes (Madsen et al. 2016b).
U
Butomus umbellatus has two known cytotypes; a diploid and a triploid, both of which are
N
considered invasive outside their native range (Kliber and Eckert 2005, Bailey and Preston 2011).
A
Both cytotypes can be acquired commercially (Kliber and Eckert 2005, Bailey and Preston 2011),
M
but North American horticultural stock is believed to be mainly triploid (Lui et al. 2005). The diploid can self-fertilize and almost always produces an inflorescence, while triploids cannot self-
D
fertilize and are said to very rarely flower (Krahulcová and Jarolímová 1993, Kliber and Eckert
TE
2005). Diploid plants do form seeds, but this is thought to rarely serve as a method of propagation (Hroudová and Zákravský 1993a, Lui et al. 2005); thus, both cytotypes reproduce
EP
primarily vegetatively. The triploid form, on which this work focuses (Lui et al. 2005, Marko et al. 2015), has been found to have a more branching rhizome (which may lead to more rapid
CC
clonal spread), produce more above and below ground biomass, and is considered to be more
A
resistant to eutrophication than the diploid cytotype (Hroudová and Zákravský 1993a, b). Although vegetative buds form on both the rhizome and in inflorescences (Hroudová and
Zákravský 1993b, authors’ personal observations), rhizome buds constitute the vast majority of vegetative buds produced by triploid B. umbellatus (Hroudová and Zákravský 1993b, authors’ personal observations). Fragmentation of the rhizome itself also can yield new clonal ramets. 4
This can occur by mechanical disturbance of the rhizome, or autofragmentation in older plants (Hroudová 1989). While the mechanisms of flowering rush vegetative reproduction are well understood, little is known of how the size of a vegetative propagule affects growth and survivorship of the resulting daughter clone. Understanding such details in the biology and
SC RI PT
ecology of this species thus is an important component to management of freshwater habitats in this species’ invaded range.
The present work was intended to provide basic life history information concerning the invasive triploid cytotype of B. umbellatus. Observational field surveys of Madsen and
colleagues (2016b) examined biomass allocation, plant height, and rhizome bud production, and
U
they demonstrated that biomass, plant height, and density typically increased out to water depths
N
of approximately 0.7 to 1.2 m, then declined in deeper waters. Rhizome bud production and
A
belowground biomass generally were found to be negatively correlated with water depth from
M
zero to 3 m (Madsen et al. 2016b). We experimentally evaluated viability and biomass production of B. umbellatus vegetative propagules of varying sizes and assessed specifically the
D
relationship between water depth and biomass production in this species. We hypothesized that
TE
survival and biomass production both would be positively influenced by the size of initial vegetative propagules and that, in accordance with field observations, biomass production would
EP
decline with increasing water depth.
CC
2. Experimental
2.1. General methods
A
These experiments were performed within the Aquatic Plant Research Facility at R.R.
Foil Plant Science Research Center, Mississippi State University. All plant materials used in these experiments came from stock tanks housed at the mesocosm facility. The materials were
5
collected originally from Detroit Lakes, Minnesota, are of the triploid cytotype, and are clones of a single genet (Marko et al. 2015). This study consisted of two 12 week experiments, run simultaneously beginning on 29 May 2013. One examined the effects of initial propagule size on growth and production of
SC RI PT
rhizome buds by B. umbellatus, the other examined the effects of water depth on growth and
rhizome bud production. All plants were grown individually in 4 L plastic containers, which were placed in mesocosm tanks as described below, with water provided from an irrigation
reservoir adjacent to the mesocosm facility (see Wersal and Madsen 2011). To facilitate recovery of belowground structures at the end of the experiments, rhizome fragments for this work were
U
planted into commercially purchased masonry sand, amended with Osmocote® 18-16-12,
N
corresponding to 2 g fertilizer L-1 soil. The sand was capped with pea gravel to minimize
A
suspension of finer grained materials and/or fertilizer into the water column within tanks.
M
Previous work has shown that B. umbellatus is tolerant of a wide variety of sediment composition, including ability to establish in bare soil, new alluvial sand deposits, and in coarse,
D
unconsolidated substrates (Hroudová 1989, Gunderson et al. 2016). Thus, our substrate
TE
conditions were similar to those in which this species is known to establish, while also facilitating
EP
recovery of belowground structures for enumeration and biomass determination. All mesocosm tanks were kept under 30% shade cloth to reduce heat and direct sunlight
CC
on the plants (as in Wersal and Madsen 2011). Average water temperatures during 06 June 2013 to 15 August 2013 ranged from 26.2 °C to 28.3 °C (minimum 24.0 °C – 27.4 °C; maximum 28.1
A
°C – 29.2 °C). Insecticide (cyfluthrin 0.75% liquid) was applied as necessary to prevent biomass loss to herbivory, primarily from Lepidoptera larvae. 2.2. Propagule size experiment
6
For the propagule size experiment, pots containing individual buds or plant fragments were placed in 378.5 L (100 gallon) Rubbermaid® commercial stock tanks (135 cm × 79 cm × 64 cm tall). Propagules of four sizes (a single rhizome bud, which averaged approx. 1 cm in length, or a three-, six-, or nine cm rhizome fragment, with any associated axillary branches) were
SC RI PT
planted into individual containers. Containers were placed eight per stock tank with two pots of each fragment size randomly assigned to positions within each of twelve tanks. Each tank then
was assigned randomly to one of four time durations (three, six, nine, or twelve weeks), resulting in 96 pots total, with six replicates of each treatment combination (fragment size × time).
Throughout the course of this experiment, water depth was maintained at approximately 40 cm
U
above the soil surface.
N
At each time point, plants in the four tanks were harvested, separated into root, shoot,
A
rhizome, and rhizome buds, and then dried at 100 °C until no change in mass was observed in a
M
24 hour period. Only propagules producing shoots were considered for analysis. Shoot:root
and rhizome bud biomass.
D
ratios were calculated as the ratio of shoot (above-ground) material to the sum of root, rhizome,
TE
2.3. Depth gradient experiment
This experiment was conducted in twenty-eight 1900 L tanks (137 cm diameter, 157 cm
EP
tall). Two B. umbellatus rhizome segments of approximately 10 cm length were placed into each of 280 pots. Ten such pots were placed in each of the 28 tanks. Tanks were arranged 4 × 7 with
CC
the rows aligned east to west. Tanks were randomly assigned to one of seven depth treatments: 0 cm, 22 cm, 44, cm, 66 cm, 88 cm, 110 cm, and 132 cm of water above the surface of soil within
A
the planting containers. The tanks were wrapped in black visqueen to prevent light from penetrating the sides of tanks. Water was added as needed to replace loss due to evaporation and maintain the desired water depth above soil. Plants were harvested at the end of the experiment, and all biomass collected was divided into shoot, root, rhizome, and rhizome buds. The material 7
then was placed in a drying oven at 70 °C until no mass change was observed in a 24 hour period. Additionally, we examined rhizome bud production (number of buds produced) in this experiment, as a further evaluation of the impact of water depth on vegetative propagation. As in the propagule experiment, shoot:root ratios were calculated as the ratio of shoot (above-ground)
SC RI PT
material to the sum of root, rhizome, and rhizome biomass.
Light intensity was recorded eight times during 10 June to 10 August 2013, from the
water’s surface and at 22 cm intervals until reaching the soil level of the pots. Light intensity also was measured above the tanks at the time of each depth reading, and this value was used to
standardize light intensity across depths, owing to changes in cloud cover and sun position among
U
individual light intensity readings.
N
2.4. Statistical analyses
A
Data were analyzed using Minitab v17.2.1 (Minitab, Inc.). The one exception was the
M
Jonckheere-Terpstra test of propagule survival, which was conducted in R. Data were examined
D
for fit to model assumptions and transformed where necessary. Mass data from the propagule size experiment were natural log transformed prior to analyses to meet the normality assumption,
TE
owing to exponential growth of the plants over time. With one exception, analyses were conducted as general linear models, and post-hoc analyses conducted with Bonferroni
EP
corrections; all analyses evaluated statistical significance at α = 0.05. Numbers of buds produced
CC
per plant in the depth gradient experiment was examined by Poisson regression, to account for the
A
nature of those count-based data.
Percent survival in both studies was calculated as:
𝑁𝑁𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 −𝑁𝑁𝑛𝑛𝑛𝑛 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔 ℎ , where Ninitial is 𝑁𝑁𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖
the number of individuals initially planted per treatment combination, and Nno growth is the number of individuals exhibiting no growth at the termination of a given replicate. A Jonckheere8
Terpstra test was used to determine whether initial propagule size affected survival. Only one of the 280 pots in the depth gradient experiment failed to yield any plant growth at the termination of the study; therefore, no analysis was conducted on survival for that experiment. 3. Results
SC RI PT
3.1. Propagule size experiment
Final biomass generally was greater when plants started from larger propagules (Figure 1). Total biomass increased significantly with both time and propagule size, fitting an
exponential growth model in all propagule sizes (R2 = 0.86; F(time) 3,41 = 54.2; F(propagule size) 3,41 =
59.1; P < 0.001; Figure 1A). Final biomass was lowest in plants grown from rhizome buds only
U
(mean = 1.9 g per pot), and plants grown from 3 cm rhizome fragments were significantly smaller
N
(12.6 g per pot) than those from 6 or 9 cm fragments (40-60 g per pot; Bonferroni post-hoc
A
comparisons).
M
Similarly, biomass of rhizome buds increased significantly over time, and with increasing initial propagule size (R2 = 0.74; F(time) 2,24 = 34.2; F(propagule size) 3,24 = 12.0; P < 0.001; Figure 1B).
D
In the case of rhizome bud biomass, no plants produced rhizome buds at three weeks of growth,
TE
and plants arising from rhizome buds themselves failed to produce any new buds until twelve weeks of growth (Fig. 1B). In fact, only two plants arising from rhizome buds were found to
EP
have produced buds at the termination of the experiment (mean = 0.02 g rhizome bud mass per pot). Plants arising from 9 cm rhizome segments also produced significantly greater biomass of
CC
new rhizome buds (6.2 g) than did plants arising from 3 cm segments (2.4 g; Bonferroni post-hoc
A
comparisons).
The ratio of above- to belowground biomass (S:R ratio) changed over time for all initial
plant propagule sizes (R2 = 0.67; F3,41 = 11.5; P < 0.001; Figure 1C). However, the S:R ratio for plants arising from buds increased over time, whereas that for plants arising from rhizome
9
segments declined over time, across all initial rhizome segment sizes (propagule size × time interaction F9,41 = 5.8; P < 0.001). Despite the observed difference in biomass production among initial propagule size categories, plant survival demonstrated no significant correlation with initial propagule size
SC RI PT
(Jonckheere-Terpstra P = 0.76; also examined by Kruskal-Wallis analysis with P = 0.71).
Survival ranged from 54 % to 67 % of ramets used in this experiment, regardless of initial propagule size. 3.2. Depth gradient experiment
Percent light transmission declined exponentially with depth (F1,46 = 319.4; P < 0.001;
U
Figure 2), and this was accompanied by a per-plant total biomass and total rhizome bud biomass
N
decline with increasing depth (F(total) 6,21 = 25.4; F(buds) 6,21 = 12.5; P < 0.001; Figure 3A&B). In
A
particular, biomass produced was significantly lower at depths greater than one meter, in
M
comparison with depths less than a half meter for both of these measures. We also found that the shoot:root ratio (S:R; calculated as the ratio of shoot biomass to the sum of root, rhizome, and
D
rhizome bud biomass) increased significantly with depth (F6,21 = 13.8; P < 0.001; Figure 3C),
TE
again with plants grown at depths greater than one meter having significantly higher S:R ratio than plants grown at depths shallower than 50 cm.
EP
In examining the production of asexual rhizome buds, we found a significant decline in the number of rhizome buds produced per plant with an increase in depth (Poisson regression, R2
CC
= 0.72; F6,21 = 71.4; P < 0.001; Figure 4A). We also found that the mass per rhizome bud differed among depths (F6,21 = 3.9; P = 0.009; Figure 4B), and that rhizome buds produced by plants
A
grown at 110 cm depth were significantly smaller than those produced by plants grown at 22 cm or 44 cm depth.
10
Only one pot of the 280 pots in this experiment contained no surviving plants at the end of the 12 week experiment period; therefore, no survival analysis was performed for this experiment. 4. Discussion
SC RI PT
Notable findings of the present work are that belowground asexual propagules of B.
umbellatus (both rhizome fragments and rhizome buds) are viable at various sizes, from a single rhizome bud of approximately 1 cm length to rhizome fragments of ~10 cm length. Rhizome
fragments were found capable of establishing new ramets at depths from 0 to 132 cm water depth, virtually all of which survived our 12-week study. Although these asexual propagules established
U
successfully at all water depths, those that established in the deepest water depths produced
N
approximately 20% as much biomass as those in the shallowest depths and tended to have a much
A
greater allocation of resources to aboveground tissues.
M
Several other aquatic plants exhibiting a rhizomatous, graminoid growth form also have been shown to decrease their biomass production as plants are exposed to greater water depths
D
(e.g., Liefers and Shay 1981, Coops et al. 1996, Vretare et al. 2001). A comparison among
TE
Phalaris arundinacea, Phragmites australis, Scirpus maritimus, and Scirpus lacustris showed that each of these species exhibited peak biomass at approximately 0.5 m depth or shallower,
EP
although the optimal water depth varied among species (Coops et al. 1996). Each of those species also exhibited an increased S:R ratio with increasing depth, as we found with B.
CC
umbellatus.
Grace (1989) compared growth of Typha latifolia with that of Typha domingensis to
A
determine growth and biomass allocation patterns of these species across a depth gradient. He found that T. domingensis, which is more tolerant of deeper water habitats, exhibited a relatively consistent S:R ratio across depths, whereas T. latifolia shifted more biomass to production of aboveground tissues as depth increased. It was noted in that paper, however, that although many 11
emergent species have been shown to shift phenotypes with water depth, this may be accomplished via differing strategies among species. Typha domingensis, for example, maintained relatively consistent S:R ratios, but reduced the number of shoots produced at increasing depth, while increasing the mass of each individual shoot.
SC RI PT
Vretare et al. (2001) also found increased S:R ratios in P. australis at increasing water depth and suggested, as did Grace (1989) above, that this likely serves to maintain a positive
carbon balance for plants receiving reduced light and CO2 at greater water depth. Vretare et al. (2001) further speculated that plants growing at depths causing reduced allocation to
belowground structures may also face increased risk of uprooting by wind or wave effects in
U
natural systems. In highly invasive aquatic species, such as B. umbellatus, this potential for
N
uprooting may be of benefit (to the plant), rather than detriment, as the increased production of
A
buoyant aboveground tissues could assist individual ramets in dispersing to new sites following
M
such a disturbance. This, however, does pose a difficulty for those who are interested in managing populations of this species, as it suggests that stands growing in deeper depths may be
D
subject to serve as sources of propagules for invasion of other areas.
TE
Because B. umbellatus is a species that is prone to invade waterbodies in its novel range, we wished to know which propagule types may be more efficient at establishing new stands. We
EP
therefore investigated the two most relevant propagule types of B. umbellatus in its invasive range: rhizome fragments and rhizome buds. These two belowground clonal structures differ in
CC
their capacities to fulfill the functions of resource acquisition & storage, dispersal, demographic increase, plant anchorage, and protection of the developing clonal propagule (sensu Grace 1993).
A
Concomitant with this, they differed in their rates of biomass accumulation and subsequent vegetative propagation, as well as in resource allocation toward subsequent ramets. Despite rhizome buds and fragments having similar initial S:R ratios, ramets establishing from rhizome buds showed a marked increase in S:R over time, while those establishing from rhizome 12
fragments showed a slight decline in S:R (Figure 1C). These differences very likely will be useful in management of this species, by prioritizing efforts towards reducing rhizome bud production or preventing rhizome buds from developing sufficient aboveground tissues to support ramet establishment (Madsen et al. 2016a, b).
SC RI PT
As an example of the potential impact of targeting rhizome buds as a management
strategy, Marko et al. (2015) found that B. umbellatus produced approximately 400 rhizome buds per m2, with the production per unit biomass being relatively consistent among sites and across
seasons at 0.50 ± 0.04 bud g-1 of rhizome biomass. In our depth gradient experiment, we found a mean of 0.54 ± 0.04 bud g-1 of rhizome biomass, averaged across all depths. Interestingly,
U
Madsen et al. (2016a) showed that reductions in the number of rhizome buds produced was a
N
more dependable indicator of herbicide effectiveness than was overall rhizome biomass. In that
A
study, herbicide treatment (two growing season treatments of 4.2 kg diquat dibromide ha-1)
M
resulted in an 80-90 % reduction in rhizome bud biomass during in situ herbicide trials. As with our work here, Madsen et al. (2016b) also found that B. umbellatus biomass
D
production peaked at just under 1 m water depth, and declined afterward, out to a maximum
TE
observed depth of just over 3 m. Gunderson et al. (2016) found similar patterns with depth in the Niagara River (New York, USA), where B. umbellatus growth form switched from emergent to
EP
submersed at depths greater than approximately 60 cm. Owing to the reduced biomass produced by plants at greater depths and their need to produce greater amounts of aboveground biomass to
CC
reach the water’s surface (Figure 3C), those plants also may be more susceptible to chemical management. This shift in biomass allocation not only provides greater leaf area per total
A
biomass (which may increase herbicide uptake), but we also found it to be correlated with an overall lower total plant biomass and lower rhizome bud biomass at greater depths (Figure 3). This reduced biomass could result in a reduced ability to quickly recover or replace tissues damaged by management activities. 13
Acknowledgements Gray Turnage provided important technical and scientific support in execution of this work. Dr. Lisa Wallace, Tyler Schartel, and Cory Shoemaker provided conceptual suggestions in the development and interpretation of these studies, and their contributions are appreciated, as are
SC RI PT
the helpful comments provided by two anonymous reviews and Dr. E. Gross. Numerous student workers assisted in these experiments; thanks are owed to: Lee Bryant, Cody Blackwell, Parker Davis, Julie Gower, Trey Higginbotham, Trey Jackson, Olivia Osaji, John Perren, and Logan
White. Partial funding for this work was provided by Pelican River Watershed District (Detroit
Lakes, Minnesota, USA). Mention of trade names or commercial products in this publication is
U
solely for the purpose of providing specific information and does not imply recommendation or
N
endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and
A
CC
EP
TE
D
M
A
employer.
14
References Anderson, L. C., C. D. Zeis, and S. F. Alam. 1974. Phytogeography and possible origins of Butomus in North America. B. Torrey Bot. Club 101:292-296.
umbellatus in Britain and Ireland. New J. Bot. 1:28-32.
SC RI PT
Bailey, J. P., and C. D. Preston. 2011. Spatial separation of diploid and triploid Butomus
Boutwell, J. E. 1990. Flowering-rush: a plant worth watching. Aquatics 12:8-11.
Coops, H., F. W. B. ven den Brink, and G. van der Velde. 1996. Growth and morphological
responses of four helophyte species in an experimental water-depth gradient. Aquatic Botany 54:11-24.
U
Core, E. L. 1941. Butomus umbellatus in America. Ohio J. Sci. 41:79-85.
N
Countryman, W. D. 1970. The history, spread and present distribution of some immigrant aquatic
15
A
weeds in New England. Hyacinth Contr. J. 8:50-2.
M
Funnell, E., M. Heaton, F. MacDonald, and B. Brownson. 2009. The aquarium and horticultural industry as a pathway for the introduction of aquatic invasive species—outreach initiatives
TE
D
within the Great Lakes basin. Biodiversity 10:104-112.
Grace, J. B. 1989. Effects of water depth on Typha latifolia and Typha domingensis.
EP
Amer. J. Bot. 76:762-768.
Grace, J. B. 1993. The adaptive significance of clonal reproduction in angiosperms: an aquatic
CC
perspective. Aquatic Botany 44: 59-180.
Gunderson, M. D., K. L. Kapuscinski, D. P. Crane, and J. M. Farrell. 2016. Habitats colonized by
A
non-native flowering rush Butomus umbellatus (Linnaeus, 1753) in the Niagara River, USA. Aquatic Invasions 11:369-380.
Hroudová, Z. 1989. Growth of Butomus umbellatus at a stable water level. Folia Geobot. Phytotax. 24:371-385.
Hroudová, Z., A. Krahulcová, P. Zákravský, and V. Jarolímová. 1996. The biology of Butomus umbellatus in shallow waters with fluctuating water level. Pages 27-30 in J. M. Caffrey, P. R. F. Barrett, K. J. Murphy, and P. M. Wade, editors. Management and Ecology of Freshwater Plants. Springer Netherlands.
SC RI PT
Hroudová, Z., and P. Zákravský. 1993a. Ecology of two cytotypes of Butomus umbellatus III. Distribution and habitat differentiation in the Czech and Slovak Republics. Folia Geobot. Phytotx. 28:425-435.
Hroudová, Z., and P. Zákravský. 1993b. Ecology of two cytotypes of Butomus umbellatus II. Reproduction, growth and biomass production. Folia Geobot. Phytotx. 28:413-424.
U
Kliber, A., and C. G. Eckert. 2005. Interaction between founder effect and selection during
N
biological invasion in an aquatic plant. Evolution 59:1900-1913.
Karyology and breeding behaviour. Folia Geobot. Phytotx. 28:385-411.
M
16
A
Krahulcová, A., and V. Jarolímová. 1993. Ecology of two cytotypes of Butomus umbellatus I.
Liefers, V. J. and J. M. Shay. 1981. The effects of water level on the growth and reproduction of
D
Scirpus maritimus var. paludosus. Can. J. Bot. 59:118-121.
TE
Les, D. H., and L. J. Mehrhoff. 1999. Introduction of nonindigenous aquatic vascular plants in southern New England: A Historical Perspective. Biol. Invasions 1:281-300.
EP
Lui, K., F. L. Thompson, and C. G. Eckert. 2005. Causes and consequences of extreme variation in reproductive strategy and vegetative growth among invasive populations of a clonal
CC
aquatic plant, Butomus umbellatus L. (Butomaceae). Biol. Invasions 7:427-444.
A
Madsen, J.D., B. Sartain, G. Turnage, and M. Marko. 2016a. Management of flowering rush in the Detroit Lakes, Minnesota. J. Aquat. Plant Manage. 54:61-67.
Madsen, J.D., R.M. Wersal, and M.D. Marko. 2016b. Distribution and biomass allocation in relation to depth of flowering rush (Butomus umbellatus) in the Detroit Lakes, Minnesota. Invas. Plant Sci. Manage. 9:161-170.
Maki, K., and S. Galatowitsch. 2004. Movement of invasive aquatic plants into Minnesota (USA)
SC RI PT
through horticultural trade. Biol. Conserv. 118:389-396. Marko, M.D., J.D. Madsen, R.A. Smith, B. Sartain, and C.L. Olson. 2015. Ecology and
phenology of flowering rush in the Detroit Lakes Chain of Lakes, Minnesota. J. Aquat. Plant Manage. 53:54-63.
Mitchell, D. S. 1974. Aquatic vegetation and its use and control, edited by D. S. Mitchell. Paris,
U
Unesco, 1974.
N
Pieterse, A. H., and K. J. Murphy. 1990. Aquatic weeds : the ecology and management of
[England] ; New York : Oxford University Press, 1990.
M
17
A
nuisance aquatic vegetation, edited by Arnold H. Pieterse and Kevin J. Murphy. Oxford
Roberts, M. 1972. Butomus umbellatus in the Mississippi watershed. Castanea 37:83-85.
D
Sarbu, A., D. Smarandache, A. Paraschiv, and D. Mihai. 2009. Butomus umbellatus morpho-
TE
structural considerations on adaptive plasticity. Scientific Annals of “Alexandru Ioan Cuza” University of Iasi, New Series, Section II a. Vegetal Biology.
EP
Vretare, V., S. E. B. Weisner, J. A. Strand, and W. Granéli. 2001. Phenotypic plasticity in Phragmites australis as a functional response to water depth. Aquatic Botany 69:127-145.
CC
Wersal R. M. and J. D. Madsen. 2011. Comparative effects of water level variations on growth
A
characteristics of Myriophyllum aquaticum. Weed Res. 51:386-393.
Figure 1. Effects of initial propagule size on (A) total plant mass per pot, (B), per-pot biomass of
A
CC
EP
TE
D
M
18
A
N
U
SC RI PT
rhizome buds, and (C) shoot:root ratio, at 3, 6, 9, and 12 weeks of growth.
Figure 2. (A) Mean, maximum, and minimum light intensity (µmol m-2 s-1) measured at the soil surface in each tank on 10 June 2013, during mid-day. (B) Percent light transmission at the soil surface among the seven water depths examined. Data at each depth are corrected for above-
SC RI PT
water light intensities; 0 cm depth readings were taken in each of the 24 tanks just below surface
and also corrected for above-surface light levels. Percent transmission fit an exponential decline with depth (R2 = 0.87; P < 0.001). 1600
(A)
U
1200
N
800
0 0
22
44
66
M
19
A
400
88
110
132
D
Depth (cm)
80
CC
20
EP
60
40
(B)
TE
100
0
A
0
22
44
66
88
Depth (cm)
110
132
Figure 3. Effects of water depth on (A) total plant mass, (B), biomass of rhizome buds, and (C) shoot:root ratio. Means with the same letter did not differ significantly in Bonferroni post-hoc
A
CC
EP
TE
D
M
20
A
N
U
SC RI PT
comparisons; error bars represent means ±1SE.
Figure 4. Effects of water depth on (A) number of rhizome buds produced per plant and (B)
SC RI PT
biomass per rhizome bud. Means with the same letter in panel B did not differ significantly in
A
CC
EP
TE
D
M
21
A
N
U
Bonferroni post-hoc comparisons; error bars represent means ±1SE.
S0304-3770(17)30395-9 https://doi.org/10.1016/j.aquabot.2018.06.003 AQBOT 3040
To appear in:
Aquatic Botany
Received date: Revised date: Accepted date:
4-12-2017 15-6-2018 23-6-2018
Please cite this article as: Carter C, Madsen JD, Ervin GN, Effects of initial propagule size and water depth on Butomus umbellatus L. growth and vegetative propagation, Aquatic Botany (2018), https://doi.org/10.1016/j.aquabot.2018.06.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effects of initial propagule size and water depth on Butomus umbellatus L. growth and vegetative propagation
Christian Carter a, 1, John D. Madsen b, 2, and Gary N. Ervin a
SC RI PT
Affiliations: a
Department of Biological Sciences, Mississippi State University, MS 39762, USA
b
Geosystems Research Institute, Mississippi State University, MS 39762, USA
Corresponding author: Gary N. Ervin
U
Department of Biological Sciences, Mississippi State University, MS 39762, USA
N
[email protected]
A
Present addresses: PO Box 4242, Rio Rico, AZ 85648
2
US Department of Agriculture, Agricultural Research Service, Exotic and Invasive Research
M
1
D
Unit, Davis, California 95616
TE
Title: Effects of initial propagule size and water depth on Butomus umbellatus L. growth and vegetative propagation
EP
Authors: Christian Carter, John D. Madsen, and Gary N. Ervin Highlights • • • •
Vegetative regrowth occurred from all rhizome fragment sizes (3, 6, 9, and 12 cm). Total plant biomass decreased approximately 70 % along the 0 - 132 cm depth gradient. Shoot:root ratios increased almost five-fold along a depth gradient from 0 - 132 cm. Rhizome bud number and mass declined at deeper depths, along with total plant mass.
CC
• • • •
A
Declarations of interest: none
1
Abstract Over the last century, flowering rush (Butomus umbellatus L.: Butomaceae) has escaped its native Eurasian range and has become a problematic species in parts of North America. As an aquatic invasive species, flowering rush has degraded wetlands in introduced areas and has interfered
SC RI PT
with human water usage. Although experimental work has been published regarding the
reproductive biology of the species, we found few manipulative experiments aimed at testing
ecological attributes of this species in the literature. The research reported here demonstrates that flowering rush is capable of aggressive clonal growth and propagation, whether from vegetative rhizome buds or from fragments of rhizomes themselves. This species was capable of growth
U
along a depth gradient from zero to 132 cm and showed an ability to adjust biomass allocation
N
along that gradient; however, biomass and asexual propagule production declined at depths
A
beyond approximately 50 cm. The combination of regrowth from relatively small propagules,
M
ability to tolerate depths of greater than one meter, and plasticity of biomass allocation along habitat gradients make this species a potential threat in many aquatic and wetland habitats in its
D
introduced range. However, certain aspects of its biomass allocation strategies may enhance
TE
chemical control for plants growing at greater depths or those regenerating from rhizome buds, both of which tend to have greater S:R biomass ratios.
EP
Keywords: asexual propagule; clonal propagation; depth gradient; invasive species; rhizomes;
A
CC
rhizome buds
2
1. Introduction Butomus umbellatus L. (Butomaceae; commonly known as “flowering rush”) is an invasive freshwater macrophyte capable of creating extensive monotypic stands, with the potential for significant negative ecosystem impacts in its invaded range. Butomus umbellatus is
SC RI PT
considered an invasive pest species in North America (Anderson et al. 1974, Kliber and Eckert 2005, Bailey and Preston 2011), where its invasion has been suggested to cause negative
ecosystem impacts such as obstruction of water delivery, degradation of recreational waters, and reduction of biodiversity in native wetlands (Countryman 1970, Boutwell 1990, Marko et al. 2015, Madsen et al. 2016a, b).
U
The first recorded account of B. umbellatus in North America occurred in 1905 near
N
Montreal, along the St. Lawrence River (Core 1941, Countryman 1970). This species has since
A
spread throughout much of the Great Lakes region, with isolated pockets throughout waterways
M
in southern Canada and across the northern U.S. (Kliber and Eckert 2005, Gunderson et al. 2016). Escape from water gardens appears to have been the most important process leading to its
D
introduction and continued spread; for example, as early as 1897, B. umbellatus was advertised
TE
and sold by North American nursery companies (Les and Mehrhoff 1999). Despite its current designation as an invasive species, a recent survey of Ontario garden centers found that one third
EP
of those businesses included B. umbellatus in their inventories (Funnell et al. 2009), and a group in Minnesota found little difficulty having the plant delivered into the state, despite its prohibited
CC
status (Maki and Galatowitsch 2004). This species can exist on a wide variety of substrates (Roberts 1972, Hroudová 1989,
A
Gunderson et al. 2016), can grow terrestrially along shores or at depths where the plant is completely submerged, and may exhibit different shoot morphology in each condition (Countryman 1970, Sarbu et al. 2009). Field surveys have found flowering rush at depths out to 4.88 m (Madsen et al. 2016b). However, at depths of around 1.5 m, plants rarely produce any 3
emergent leaves, and biomass production appears to decline at depths greater than 1.2 m (Madsen et al. 2016b). When completely submerged, the leaves are thin and ribbon-like (Boutwell 1990), and plants do not flower (Hroudová et al. 1996). However, when emerged, the leaves become rigid, the outer cell walls and cuticle thicken, stomata increase in number by 15 %, and flowering
SC RI PT
may be observed (Sarbu et al. 2009). The ability to exist along a broad water depth gradient may enhance the invasibility of B. umbellatus, as this increases access to niche space and resources
that other plant species cannot access. Furthermore, the ability of B. umbellatus to form dense stands to depths of 1.2 m or more may result in severe degradation of littoral zone habitats of invaded lakes (Madsen et al. 2016b).
U
Butomus umbellatus has two known cytotypes; a diploid and a triploid, both of which are
N
considered invasive outside their native range (Kliber and Eckert 2005, Bailey and Preston 2011).
A
Both cytotypes can be acquired commercially (Kliber and Eckert 2005, Bailey and Preston 2011),
M
but North American horticultural stock is believed to be mainly triploid (Lui et al. 2005). The diploid can self-fertilize and almost always produces an inflorescence, while triploids cannot self-
D
fertilize and are said to very rarely flower (Krahulcová and Jarolímová 1993, Kliber and Eckert
TE
2005). Diploid plants do form seeds, but this is thought to rarely serve as a method of propagation (Hroudová and Zákravský 1993a, Lui et al. 2005); thus, both cytotypes reproduce
EP
primarily vegetatively. The triploid form, on which this work focuses (Lui et al. 2005, Marko et al. 2015), has been found to have a more branching rhizome (which may lead to more rapid
CC
clonal spread), produce more above and below ground biomass, and is considered to be more
A
resistant to eutrophication than the diploid cytotype (Hroudová and Zákravský 1993a, b). Although vegetative buds form on both the rhizome and in inflorescences (Hroudová and
Zákravský 1993b, authors’ personal observations), rhizome buds constitute the vast majority of vegetative buds produced by triploid B. umbellatus (Hroudová and Zákravský 1993b, authors’ personal observations). Fragmentation of the rhizome itself also can yield new clonal ramets. 4
This can occur by mechanical disturbance of the rhizome, or autofragmentation in older plants (Hroudová 1989). While the mechanisms of flowering rush vegetative reproduction are well understood, little is known of how the size of a vegetative propagule affects growth and survivorship of the resulting daughter clone. Understanding such details in the biology and
SC RI PT
ecology of this species thus is an important component to management of freshwater habitats in this species’ invaded range.
The present work was intended to provide basic life history information concerning the invasive triploid cytotype of B. umbellatus. Observational field surveys of Madsen and
colleagues (2016b) examined biomass allocation, plant height, and rhizome bud production, and
U
they demonstrated that biomass, plant height, and density typically increased out to water depths
N
of approximately 0.7 to 1.2 m, then declined in deeper waters. Rhizome bud production and
A
belowground biomass generally were found to be negatively correlated with water depth from
M
zero to 3 m (Madsen et al. 2016b). We experimentally evaluated viability and biomass production of B. umbellatus vegetative propagules of varying sizes and assessed specifically the
D
relationship between water depth and biomass production in this species. We hypothesized that
TE
survival and biomass production both would be positively influenced by the size of initial vegetative propagules and that, in accordance with field observations, biomass production would
EP
decline with increasing water depth.
CC
2. Experimental
2.1. General methods
A
These experiments were performed within the Aquatic Plant Research Facility at R.R.
Foil Plant Science Research Center, Mississippi State University. All plant materials used in these experiments came from stock tanks housed at the mesocosm facility. The materials were
5
collected originally from Detroit Lakes, Minnesota, are of the triploid cytotype, and are clones of a single genet (Marko et al. 2015). This study consisted of two 12 week experiments, run simultaneously beginning on 29 May 2013. One examined the effects of initial propagule size on growth and production of
SC RI PT
rhizome buds by B. umbellatus, the other examined the effects of water depth on growth and
rhizome bud production. All plants were grown individually in 4 L plastic containers, which were placed in mesocosm tanks as described below, with water provided from an irrigation
reservoir adjacent to the mesocosm facility (see Wersal and Madsen 2011). To facilitate recovery of belowground structures at the end of the experiments, rhizome fragments for this work were
U
planted into commercially purchased masonry sand, amended with Osmocote® 18-16-12,
N
corresponding to 2 g fertilizer L-1 soil. The sand was capped with pea gravel to minimize
A
suspension of finer grained materials and/or fertilizer into the water column within tanks.
M
Previous work has shown that B. umbellatus is tolerant of a wide variety of sediment composition, including ability to establish in bare soil, new alluvial sand deposits, and in coarse,
D
unconsolidated substrates (Hroudová 1989, Gunderson et al. 2016). Thus, our substrate
TE
conditions were similar to those in which this species is known to establish, while also facilitating
EP
recovery of belowground structures for enumeration and biomass determination. All mesocosm tanks were kept under 30% shade cloth to reduce heat and direct sunlight
CC
on the plants (as in Wersal and Madsen 2011). Average water temperatures during 06 June 2013 to 15 August 2013 ranged from 26.2 °C to 28.3 °C (minimum 24.0 °C – 27.4 °C; maximum 28.1
A
°C – 29.2 °C). Insecticide (cyfluthrin 0.75% liquid) was applied as necessary to prevent biomass loss to herbivory, primarily from Lepidoptera larvae. 2.2. Propagule size experiment
6
For the propagule size experiment, pots containing individual buds or plant fragments were placed in 378.5 L (100 gallon) Rubbermaid® commercial stock tanks (135 cm × 79 cm × 64 cm tall). Propagules of four sizes (a single rhizome bud, which averaged approx. 1 cm in length, or a three-, six-, or nine cm rhizome fragment, with any associated axillary branches) were
SC RI PT
planted into individual containers. Containers were placed eight per stock tank with two pots of each fragment size randomly assigned to positions within each of twelve tanks. Each tank then
was assigned randomly to one of four time durations (three, six, nine, or twelve weeks), resulting in 96 pots total, with six replicates of each treatment combination (fragment size × time).
Throughout the course of this experiment, water depth was maintained at approximately 40 cm
U
above the soil surface.
N
At each time point, plants in the four tanks were harvested, separated into root, shoot,
A
rhizome, and rhizome buds, and then dried at 100 °C until no change in mass was observed in a
M
24 hour period. Only propagules producing shoots were considered for analysis. Shoot:root
and rhizome bud biomass.
D
ratios were calculated as the ratio of shoot (above-ground) material to the sum of root, rhizome,
TE
2.3. Depth gradient experiment
This experiment was conducted in twenty-eight 1900 L tanks (137 cm diameter, 157 cm
EP
tall). Two B. umbellatus rhizome segments of approximately 10 cm length were placed into each of 280 pots. Ten such pots were placed in each of the 28 tanks. Tanks were arranged 4 × 7 with
CC
the rows aligned east to west. Tanks were randomly assigned to one of seven depth treatments: 0 cm, 22 cm, 44, cm, 66 cm, 88 cm, 110 cm, and 132 cm of water above the surface of soil within
A
the planting containers. The tanks were wrapped in black visqueen to prevent light from penetrating the sides of tanks. Water was added as needed to replace loss due to evaporation and maintain the desired water depth above soil. Plants were harvested at the end of the experiment, and all biomass collected was divided into shoot, root, rhizome, and rhizome buds. The material 7
then was placed in a drying oven at 70 °C until no mass change was observed in a 24 hour period. Additionally, we examined rhizome bud production (number of buds produced) in this experiment, as a further evaluation of the impact of water depth on vegetative propagation. As in the propagule experiment, shoot:root ratios were calculated as the ratio of shoot (above-ground)
SC RI PT
material to the sum of root, rhizome, and rhizome biomass.
Light intensity was recorded eight times during 10 June to 10 August 2013, from the
water’s surface and at 22 cm intervals until reaching the soil level of the pots. Light intensity also was measured above the tanks at the time of each depth reading, and this value was used to
standardize light intensity across depths, owing to changes in cloud cover and sun position among
U
individual light intensity readings.
N
2.4. Statistical analyses
A
Data were analyzed using Minitab v17.2.1 (Minitab, Inc.). The one exception was the
M
Jonckheere-Terpstra test of propagule survival, which was conducted in R. Data were examined
D
for fit to model assumptions and transformed where necessary. Mass data from the propagule size experiment were natural log transformed prior to analyses to meet the normality assumption,
TE
owing to exponential growth of the plants over time. With one exception, analyses were conducted as general linear models, and post-hoc analyses conducted with Bonferroni
EP
corrections; all analyses evaluated statistical significance at α = 0.05. Numbers of buds produced
CC
per plant in the depth gradient experiment was examined by Poisson regression, to account for the
A
nature of those count-based data.
Percent survival in both studies was calculated as:
𝑁𝑁𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 −𝑁𝑁𝑛𝑛𝑛𝑛 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔 ℎ , where Ninitial is 𝑁𝑁𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖
the number of individuals initially planted per treatment combination, and Nno growth is the number of individuals exhibiting no growth at the termination of a given replicate. A Jonckheere8
Terpstra test was used to determine whether initial propagule size affected survival. Only one of the 280 pots in the depth gradient experiment failed to yield any plant growth at the termination of the study; therefore, no analysis was conducted on survival for that experiment. 3. Results
SC RI PT
3.1. Propagule size experiment
Final biomass generally was greater when plants started from larger propagules (Figure 1). Total biomass increased significantly with both time and propagule size, fitting an
exponential growth model in all propagule sizes (R2 = 0.86; F(time) 3,41 = 54.2; F(propagule size) 3,41 =
59.1; P < 0.001; Figure 1A). Final biomass was lowest in plants grown from rhizome buds only
U
(mean = 1.9 g per pot), and plants grown from 3 cm rhizome fragments were significantly smaller
N
(12.6 g per pot) than those from 6 or 9 cm fragments (40-60 g per pot; Bonferroni post-hoc
A
comparisons).
M
Similarly, biomass of rhizome buds increased significantly over time, and with increasing initial propagule size (R2 = 0.74; F(time) 2,24 = 34.2; F(propagule size) 3,24 = 12.0; P < 0.001; Figure 1B).
D
In the case of rhizome bud biomass, no plants produced rhizome buds at three weeks of growth,
TE
and plants arising from rhizome buds themselves failed to produce any new buds until twelve weeks of growth (Fig. 1B). In fact, only two plants arising from rhizome buds were found to
EP
have produced buds at the termination of the experiment (mean = 0.02 g rhizome bud mass per pot). Plants arising from 9 cm rhizome segments also produced significantly greater biomass of
CC
new rhizome buds (6.2 g) than did plants arising from 3 cm segments (2.4 g; Bonferroni post-hoc
A
comparisons).
The ratio of above- to belowground biomass (S:R ratio) changed over time for all initial
plant propagule sizes (R2 = 0.67; F3,41 = 11.5; P < 0.001; Figure 1C). However, the S:R ratio for plants arising from buds increased over time, whereas that for plants arising from rhizome
9
segments declined over time, across all initial rhizome segment sizes (propagule size × time interaction F9,41 = 5.8; P < 0.001). Despite the observed difference in biomass production among initial propagule size categories, plant survival demonstrated no significant correlation with initial propagule size
SC RI PT
(Jonckheere-Terpstra P = 0.76; also examined by Kruskal-Wallis analysis with P = 0.71).
Survival ranged from 54 % to 67 % of ramets used in this experiment, regardless of initial propagule size. 3.2. Depth gradient experiment
Percent light transmission declined exponentially with depth (F1,46 = 319.4; P < 0.001;
U
Figure 2), and this was accompanied by a per-plant total biomass and total rhizome bud biomass
N
decline with increasing depth (F(total) 6,21 = 25.4; F(buds) 6,21 = 12.5; P < 0.001; Figure 3A&B). In
A
particular, biomass produced was significantly lower at depths greater than one meter, in
M
comparison with depths less than a half meter for both of these measures. We also found that the shoot:root ratio (S:R; calculated as the ratio of shoot biomass to the sum of root, rhizome, and
D
rhizome bud biomass) increased significantly with depth (F6,21 = 13.8; P < 0.001; Figure 3C),
TE
again with plants grown at depths greater than one meter having significantly higher S:R ratio than plants grown at depths shallower than 50 cm.
EP
In examining the production of asexual rhizome buds, we found a significant decline in the number of rhizome buds produced per plant with an increase in depth (Poisson regression, R2
CC
= 0.72; F6,21 = 71.4; P < 0.001; Figure 4A). We also found that the mass per rhizome bud differed among depths (F6,21 = 3.9; P = 0.009; Figure 4B), and that rhizome buds produced by plants
A
grown at 110 cm depth were significantly smaller than those produced by plants grown at 22 cm or 44 cm depth.
10
Only one pot of the 280 pots in this experiment contained no surviving plants at the end of the 12 week experiment period; therefore, no survival analysis was performed for this experiment. 4. Discussion
SC RI PT
Notable findings of the present work are that belowground asexual propagules of B.
umbellatus (both rhizome fragments and rhizome buds) are viable at various sizes, from a single rhizome bud of approximately 1 cm length to rhizome fragments of ~10 cm length. Rhizome
fragments were found capable of establishing new ramets at depths from 0 to 132 cm water depth, virtually all of which survived our 12-week study. Although these asexual propagules established
U
successfully at all water depths, those that established in the deepest water depths produced
N
approximately 20% as much biomass as those in the shallowest depths and tended to have a much
A
greater allocation of resources to aboveground tissues.
M
Several other aquatic plants exhibiting a rhizomatous, graminoid growth form also have been shown to decrease their biomass production as plants are exposed to greater water depths
D
(e.g., Liefers and Shay 1981, Coops et al. 1996, Vretare et al. 2001). A comparison among
TE
Phalaris arundinacea, Phragmites australis, Scirpus maritimus, and Scirpus lacustris showed that each of these species exhibited peak biomass at approximately 0.5 m depth or shallower,
EP
although the optimal water depth varied among species (Coops et al. 1996). Each of those species also exhibited an increased S:R ratio with increasing depth, as we found with B.
CC
umbellatus.
Grace (1989) compared growth of Typha latifolia with that of Typha domingensis to
A
determine growth and biomass allocation patterns of these species across a depth gradient. He found that T. domingensis, which is more tolerant of deeper water habitats, exhibited a relatively consistent S:R ratio across depths, whereas T. latifolia shifted more biomass to production of aboveground tissues as depth increased. It was noted in that paper, however, that although many 11
emergent species have been shown to shift phenotypes with water depth, this may be accomplished via differing strategies among species. Typha domingensis, for example, maintained relatively consistent S:R ratios, but reduced the number of shoots produced at increasing depth, while increasing the mass of each individual shoot.
SC RI PT
Vretare et al. (2001) also found increased S:R ratios in P. australis at increasing water depth and suggested, as did Grace (1989) above, that this likely serves to maintain a positive
carbon balance for plants receiving reduced light and CO2 at greater water depth. Vretare et al. (2001) further speculated that plants growing at depths causing reduced allocation to
belowground structures may also face increased risk of uprooting by wind or wave effects in
U
natural systems. In highly invasive aquatic species, such as B. umbellatus, this potential for
N
uprooting may be of benefit (to the plant), rather than detriment, as the increased production of
A
buoyant aboveground tissues could assist individual ramets in dispersing to new sites following
M
such a disturbance. This, however, does pose a difficulty for those who are interested in managing populations of this species, as it suggests that stands growing in deeper depths may be
D
subject to serve as sources of propagules for invasion of other areas.
TE
Because B. umbellatus is a species that is prone to invade waterbodies in its novel range, we wished to know which propagule types may be more efficient at establishing new stands. We
EP
therefore investigated the two most relevant propagule types of B. umbellatus in its invasive range: rhizome fragments and rhizome buds. These two belowground clonal structures differ in
CC
their capacities to fulfill the functions of resource acquisition & storage, dispersal, demographic increase, plant anchorage, and protection of the developing clonal propagule (sensu Grace 1993).
A
Concomitant with this, they differed in their rates of biomass accumulation and subsequent vegetative propagation, as well as in resource allocation toward subsequent ramets. Despite rhizome buds and fragments having similar initial S:R ratios, ramets establishing from rhizome buds showed a marked increase in S:R over time, while those establishing from rhizome 12
fragments showed a slight decline in S:R (Figure 1C). These differences very likely will be useful in management of this species, by prioritizing efforts towards reducing rhizome bud production or preventing rhizome buds from developing sufficient aboveground tissues to support ramet establishment (Madsen et al. 2016a, b).
SC RI PT
As an example of the potential impact of targeting rhizome buds as a management
strategy, Marko et al. (2015) found that B. umbellatus produced approximately 400 rhizome buds per m2, with the production per unit biomass being relatively consistent among sites and across
seasons at 0.50 ± 0.04 bud g-1 of rhizome biomass. In our depth gradient experiment, we found a mean of 0.54 ± 0.04 bud g-1 of rhizome biomass, averaged across all depths. Interestingly,
U
Madsen et al. (2016a) showed that reductions in the number of rhizome buds produced was a
N
more dependable indicator of herbicide effectiveness than was overall rhizome biomass. In that
A
study, herbicide treatment (two growing season treatments of 4.2 kg diquat dibromide ha-1)
M
resulted in an 80-90 % reduction in rhizome bud biomass during in situ herbicide trials. As with our work here, Madsen et al. (2016b) also found that B. umbellatus biomass
D
production peaked at just under 1 m water depth, and declined afterward, out to a maximum
TE
observed depth of just over 3 m. Gunderson et al. (2016) found similar patterns with depth in the Niagara River (New York, USA), where B. umbellatus growth form switched from emergent to
EP
submersed at depths greater than approximately 60 cm. Owing to the reduced biomass produced by plants at greater depths and their need to produce greater amounts of aboveground biomass to
CC
reach the water’s surface (Figure 3C), those plants also may be more susceptible to chemical management. This shift in biomass allocation not only provides greater leaf area per total
A
biomass (which may increase herbicide uptake), but we also found it to be correlated with an overall lower total plant biomass and lower rhizome bud biomass at greater depths (Figure 3). This reduced biomass could result in a reduced ability to quickly recover or replace tissues damaged by management activities. 13
Acknowledgements Gray Turnage provided important technical and scientific support in execution of this work. Dr. Lisa Wallace, Tyler Schartel, and Cory Shoemaker provided conceptual suggestions in the development and interpretation of these studies, and their contributions are appreciated, as are
SC RI PT
the helpful comments provided by two anonymous reviews and Dr. E. Gross. Numerous student workers assisted in these experiments; thanks are owed to: Lee Bryant, Cody Blackwell, Parker Davis, Julie Gower, Trey Higginbotham, Trey Jackson, Olivia Osaji, John Perren, and Logan
White. Partial funding for this work was provided by Pelican River Watershed District (Detroit
Lakes, Minnesota, USA). Mention of trade names or commercial products in this publication is
U
solely for the purpose of providing specific information and does not imply recommendation or
N
endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and
A
CC
EP
TE
D
M
A
employer.
14
References Anderson, L. C., C. D. Zeis, and S. F. Alam. 1974. Phytogeography and possible origins of Butomus in North America. B. Torrey Bot. Club 101:292-296.
umbellatus in Britain and Ireland. New J. Bot. 1:28-32.
SC RI PT
Bailey, J. P., and C. D. Preston. 2011. Spatial separation of diploid and triploid Butomus
Boutwell, J. E. 1990. Flowering-rush: a plant worth watching. Aquatics 12:8-11.
Coops, H., F. W. B. ven den Brink, and G. van der Velde. 1996. Growth and morphological
responses of four helophyte species in an experimental water-depth gradient. Aquatic Botany 54:11-24.
U
Core, E. L. 1941. Butomus umbellatus in America. Ohio J. Sci. 41:79-85.
N
Countryman, W. D. 1970. The history, spread and present distribution of some immigrant aquatic
15
A
weeds in New England. Hyacinth Contr. J. 8:50-2.
M
Funnell, E., M. Heaton, F. MacDonald, and B. Brownson. 2009. The aquarium and horticultural industry as a pathway for the introduction of aquatic invasive species—outreach initiatives
TE
D
within the Great Lakes basin. Biodiversity 10:104-112.
Grace, J. B. 1989. Effects of water depth on Typha latifolia and Typha domingensis.
EP
Amer. J. Bot. 76:762-768.
Grace, J. B. 1993. The adaptive significance of clonal reproduction in angiosperms: an aquatic
CC
perspective. Aquatic Botany 44: 59-180.
Gunderson, M. D., K. L. Kapuscinski, D. P. Crane, and J. M. Farrell. 2016. Habitats colonized by
A
non-native flowering rush Butomus umbellatus (Linnaeus, 1753) in the Niagara River, USA. Aquatic Invasions 11:369-380.
Hroudová, Z. 1989. Growth of Butomus umbellatus at a stable water level. Folia Geobot. Phytotax. 24:371-385.
Hroudová, Z., A. Krahulcová, P. Zákravský, and V. Jarolímová. 1996. The biology of Butomus umbellatus in shallow waters with fluctuating water level. Pages 27-30 in J. M. Caffrey, P. R. F. Barrett, K. J. Murphy, and P. M. Wade, editors. Management and Ecology of Freshwater Plants. Springer Netherlands.
SC RI PT
Hroudová, Z., and P. Zákravský. 1993a. Ecology of two cytotypes of Butomus umbellatus III. Distribution and habitat differentiation in the Czech and Slovak Republics. Folia Geobot. Phytotx. 28:425-435.
Hroudová, Z., and P. Zákravský. 1993b. Ecology of two cytotypes of Butomus umbellatus II. Reproduction, growth and biomass production. Folia Geobot. Phytotx. 28:413-424.
U
Kliber, A., and C. G. Eckert. 2005. Interaction between founder effect and selection during
N
biological invasion in an aquatic plant. Evolution 59:1900-1913.
Karyology and breeding behaviour. Folia Geobot. Phytotx. 28:385-411.
M
16
A
Krahulcová, A., and V. Jarolímová. 1993. Ecology of two cytotypes of Butomus umbellatus I.
Liefers, V. J. and J. M. Shay. 1981. The effects of water level on the growth and reproduction of
D
Scirpus maritimus var. paludosus. Can. J. Bot. 59:118-121.
TE
Les, D. H., and L. J. Mehrhoff. 1999. Introduction of nonindigenous aquatic vascular plants in southern New England: A Historical Perspective. Biol. Invasions 1:281-300.
EP
Lui, K., F. L. Thompson, and C. G. Eckert. 2005. Causes and consequences of extreme variation in reproductive strategy and vegetative growth among invasive populations of a clonal
CC
aquatic plant, Butomus umbellatus L. (Butomaceae). Biol. Invasions 7:427-444.
A
Madsen, J.D., B. Sartain, G. Turnage, and M. Marko. 2016a. Management of flowering rush in the Detroit Lakes, Minnesota. J. Aquat. Plant Manage. 54:61-67.
Madsen, J.D., R.M. Wersal, and M.D. Marko. 2016b. Distribution and biomass allocation in relation to depth of flowering rush (Butomus umbellatus) in the Detroit Lakes, Minnesota. Invas. Plant Sci. Manage. 9:161-170.
Maki, K., and S. Galatowitsch. 2004. Movement of invasive aquatic plants into Minnesota (USA)
SC RI PT
through horticultural trade. Biol. Conserv. 118:389-396. Marko, M.D., J.D. Madsen, R.A. Smith, B. Sartain, and C.L. Olson. 2015. Ecology and
phenology of flowering rush in the Detroit Lakes Chain of Lakes, Minnesota. J. Aquat. Plant Manage. 53:54-63.
Mitchell, D. S. 1974. Aquatic vegetation and its use and control, edited by D. S. Mitchell. Paris,
U
Unesco, 1974.
N
Pieterse, A. H., and K. J. Murphy. 1990. Aquatic weeds : the ecology and management of
[England] ; New York : Oxford University Press, 1990.
M
17
A
nuisance aquatic vegetation, edited by Arnold H. Pieterse and Kevin J. Murphy. Oxford
Roberts, M. 1972. Butomus umbellatus in the Mississippi watershed. Castanea 37:83-85.
D
Sarbu, A., D. Smarandache, A. Paraschiv, and D. Mihai. 2009. Butomus umbellatus morpho-
TE
structural considerations on adaptive plasticity. Scientific Annals of “Alexandru Ioan Cuza” University of Iasi, New Series, Section II a. Vegetal Biology.
EP
Vretare, V., S. E. B. Weisner, J. A. Strand, and W. Granéli. 2001. Phenotypic plasticity in Phragmites australis as a functional response to water depth. Aquatic Botany 69:127-145.
CC
Wersal R. M. and J. D. Madsen. 2011. Comparative effects of water level variations on growth
A
characteristics of Myriophyllum aquaticum. Weed Res. 51:386-393.
Figure 1. Effects of initial propagule size on (A) total plant mass per pot, (B), per-pot biomass of
A
CC
EP
TE
D
M
18
A
N
U
SC RI PT
rhizome buds, and (C) shoot:root ratio, at 3, 6, 9, and 12 weeks of growth.
Figure 2. (A) Mean, maximum, and minimum light intensity (µmol m-2 s-1) measured at the soil surface in each tank on 10 June 2013, during mid-day. (B) Percent light transmission at the soil surface among the seven water depths examined. Data at each depth are corrected for above-
SC RI PT
water light intensities; 0 cm depth readings were taken in each of the 24 tanks just below surface
and also corrected for above-surface light levels. Percent transmission fit an exponential decline with depth (R2 = 0.87; P < 0.001). 1600
(A)
U
1200
N
800
0 0
22
44
66
M
19
A
400
88
110
132
D
Depth (cm)
80
CC
20
EP
60
40
(B)
TE
100
0
A
0
22
44
66
88
Depth (cm)
110
132
Figure 3. Effects of water depth on (A) total plant mass, (B), biomass of rhizome buds, and (C) shoot:root ratio. Means with the same letter did not differ significantly in Bonferroni post-hoc
A
CC
EP
TE
D
M
20
A
N
U
SC RI PT
comparisons; error bars represent means ±1SE.
Figure 4. Effects of water depth on (A) number of rhizome buds produced per plant and (B)
SC RI PT
biomass per rhizome bud. Means with the same letter in panel B did not differ significantly in
A
CC
EP
TE
D
M
21
A
N
U
Bonferroni post-hoc comparisons; error bars represent means ±1SE.