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Hypoxia Improves Germination of the Problematic Invader Garlic Mustard (Alliaria petiolata) of North American Forests.

INTRODUCTION

Garlic mustard [Alliaria petiolata (M. Bieb.) Cava & Grande] belongs to the family Brassicaceae and is native to Europe. It is a biennial plant producing leaves the first year (Cavers et al., 1979). In Denmark seeds germinate in March and April, and the plant overwinters as a basal rosette that remains green during the winter. In the second spring, plants flourish in April and May, set seeds in July, and senesce by mid-late August (Yasin and Andreasen, 2015). Alliaria petiolata is a shade-tolerant understory herb (Rodgers et al., 2008) and is known to exhibit shade avoidance syndromes. It has emerged from western Eurasia and now ranges as far north as Sweden and as far west as England (Cavers et al., 1979). It was introduced into North America in the mid-1800s (Rodgers et al., 2008) and spread rapidly through the eastern U.S.A. and adjacent Canada (Nuzzo, 1999), becoming a problematic invasive plant species in the eastern North American forests (Davis and Cipollini, 2014). Alliaria petiolata is recorded as a "noxious weed" in six of the 34 U.S.A. states where it is now found (Nuzzo, 2000).

In many terrestrial forests ecosystems, leaf litter makes up a significant source of unavailable nutrients added to the soil (Klotzbucher et al., 2012). Leaf litter may affect germination and inhibit early seedling establishment of the flora by several mechanisms. Leaf litter may cover seeds present on the soil surface; shade reduces the thermal amplitude in the soil and makes up a physical barrier for seed germination. It may affect seed germination biochemically by releasing secondary compounds when it decays (Xiong and Nilsson, 1999). Sometimes leaf litter reduces [O.sub.2] concentration on the forest ground. Decaying leaf litter releases many kinds of volatile compounds and gasses like methane (C[H.sub.4]); ethylene ([C.sub.2][H.sub.4]); nitrous oxide ([N.sub.2]O); and carbon dioxide (C[O.sub.2]) (Ruiz-Valdiviezo et al., 2010), which reduces [O.sub.2] concentration on the forest floor. Dense leaf litter layers also act as a physical barrier to [O.sub.2] gas exchange from the atmosphere into the soil and therefore reduces [O.sub.2] concentration in the soil. This reduction in [O.sub.2] concentration may limit die germination of some species and favor others. Leaf litter depth may play a significant role in the invasion of overgrowing Lonicera maacki and A. petiolata (Bartuszevige et al., 2007). Bartuszevige et al. (2007) studied the effect of leaf litter on the establishment, growth, and survival of A. petiolata in a deciduous forest. They found the survival of established seedlings was substantially better in plots with leaf litter than in plot where they had removed the litter.

Elevated C[O.sub.2] may reduce [O.sub.2] concentration and facilitates exotic plant invasion (Bradley et al., 2010). Invasive species like A. petiolata favor high C[O.sub.2] levels in understories, particularly at night time, where the amount of atmospheric [O.sub.2] concentration is sometimes less underneath the canopy. Some invasive species have the ability to germinate and grow faster at low [O.sub.2] levels than other native species (Manea and Leishman, 2011). Anderson and Cipollini (2013) observed A. petiolata increased growth as a response to increasing C[O.sub.2] levels.

Alliaria petiolata dominates relatives by creating a situation of reduced oxygen in its surrounding root zone. There are several factors involved in the success of A. petiolata invasion which directly or indirectly reduces the oxygen concentration (Fig. 1).

The objective of this study was to investigate whether reduced [O.sub.2] concentrations inhibited or stimulated the germination and early growth of A. petiolata. We tested the hypothesis low [O.sub.2] concentration improves the germination of A. petiolata.

Methods

PLANT MATERIAL

Alliaria petiolata plants were collected and reaped at maturity from overshadowed and moist soil near Albertslund golf club, at the roadside of Snubbekorsvej, close to Hoejbakkegaard Alle, Taastrup, Denmark (Taastrup, Denmark, 55[degrees]38'N, 12[degrees]17'E). We threshed the plants and separated the seeds from siliques by using a WINTERSTEIGER I.D 350 thresher (Amelia Earhart Drive, Salt Lake City, U.S.A.). Seeds were cleaned with a WESTRUP LA-LS seed cleaner (WESTRUP, DK-4200 Slagelse, Denmark) and stored at 5C. Seeds moisture percentage was estimated at 9 % with a grain moisture meter MILE 55 (Model: W1910/FLFM, No.1, 3rd Shangdi, Flaidian District Beijing, China).

EXPERIMENTAL LAVOUT

Two similar experiments displaced in time were performed using a complete randomized design with two replications. According to ISTA's procedures for seed testing, less than 400 seeds can be tested. In such cases at least 100 seeds must be tested in replicates of 25 or 50 (ISTA, 2011). Experiment two was performed immediately after completion of experiment one. In each experiment fifty seeds of A. petiolata per replication were placed on moist filter paper in germination boxes (ISTA, 2011). The boxes were placed randomly in air tight glass containers at 15 C. Seeds were germinated at 16 h photoperiod in a growth incubator Termaks KB 8000L (Termaks AS, Nino lab, Koge, Denmark). It contained light tubes on both sides and provided a light intensity of 12,000 Lux. Sixty ml of water was added to each germination box, which was sufficient for each 14 d germination bioassay. Seeds were assumed germinated when either radicle tissue or the cotyledons protruded beyond 2 mm of the seed coat. Germination was counted daily following a seedling evaluation protocol (AOSA, 1990) until no more seeds germinated. In total 1000 seeds were tested [5 ([O.sub.2] treatment levels) x 50 (seeds) x 2 (replicates) x 2 (experiments)].

OXYGEN CONCENTRATIONS

We obtained five oxygen concentrations (20.9, 15, 10, 5, and 2.5%) by mixing [N.sub.2] gas with [O.sub.2] in air tight glass containers (50 cm long, 30 cm wide, and 20 cm high). We injected the [N.sub.2] gas from a liquid [N.sub.2] cylinder into the containers. A Gasman Personal Gas Monitor (Crowcon Detection Instruments Ltd., Rotterdam, The Netherlands) was placed inside each container to monitor and keep the [O.sub.2] concentrations in container constant during the experiments. We investigated the relationship between germination response and oxygen concentrations.

STATISTICAL ANALYSIS

Both experiments were analyzed separately. Statistical analyses were performed using the open-source program R version 3.2.5 (http://www.R-project.org). The seed germination was modelled using a cumulative distribution function of the standard log-logistic distribution using the package drc (Ritz and Streibig, 2005; eq. 1).

F(t) = d/1+ exp[b{log(t) - log([t.sub.50])}] (1)

where F(t) is the fraction of seeds germinated at time t. The upper limit parameter (d) denotes the proportion of seeds that germinated during the experiment out of the total number of seeds. The parameter b is proportional to the slope of F at time t equal to the parameter [t.sub.50] where 50% of the total seeds germinated during the experimental period. The parameter [t.sub.50] has the same unit as the time scale in the experiment. The estimation and model checking procedures are based on treating the data as event times, that is, to record the time it lakes for germination (the event of interest) to occur as described by Ritz et at. (2013).

RESULTS

Germination percentage (d) and rate ([t.sub.50]) of A. petiolata seeds were affected by a change in [O.sub.2] concentrations (Figs. 2, 3). The d was substantially increased when the [O.sub.2] level dropped from 20.9 % to 15 % in both experiments (Fig. 2). The highest germination was found at hypoxia [O.sub.2] concentration, with maximum germination percentage and rate (Exp. 1: d = 0.57; t50= 3.91 and Exp. 2: r d = 0.42; [t.sub.50] = 3.33) (Fig. 2; Table 1). The second highest germination and rate was observed at 10 % [O.sub.2] (Exp. 1: d = 0.48; [t.sub.50] = 4.29 and Exp. 2: [t.sub.50] = 0.26; [t.sub.50] = 3.69) (Table 1), followed by 20.9 % [O.sub.2] (Exp. 1: d = 0.30; [t.sub.50] = 2.98 and Exp. 2: d = 0.27; [t.sub.50] = 4.87) (Table 1). However, a reduction in the [O.sub.2] concentration below 5% reduced d to 0.11 and increased [t.sub.50], to 4.13 in Exp. 1 and reduced d to 0.06 and increased [t.sub.50], to 6.87 in Exp. 2 (Table 1). The seeds were very sensitive to 2.5 % [O.sub.2], and germination fraction declined drastically (Fig. 2; Table 1). In Exp. 1, 10 % [O.sub.2] concentration increased germination more than 20.9 % [O.sub.2], while in Exp. 2, 10% [O.sub.2] concentration resulted in about the same germination fraction as 20.9 % [O.sub.2] (Fig. 2). The [t.sub.50] was less in days in Exp. 2 at hypoxia, compared to 20.9 % [O.sub.2], however, this relationship was opposite in Exp. 1 (Fig. 3).

DISCUSSION

Our experiments showed A. petiolata germinated well at hypoxia. This trait may enable it to adapt to [O.sub.2] deficient soils (compressed, compact, water logged, and soils with hard surfaces). We also observed the ground was moist and compressed where we collected the plants, but we were not able to measure tile [O.sub.2] level. Its ability to germinate at low [O.sub.2] concentrations may be due to a decreasing respiration per volume tissue and an increased [O.sub.2] uptake, resulting in a long-term morphological adaptation as seen from other plant species at low [O.sub.2] concentrations (Geigenberger, 2003; Yasin and Andreasen, 2016).

Seeds are living organisms, and the characteristics of seed populations change over time in their germination ability, vigor, and the number of viable and dead seeds. The effect of the [O.sub.2] concentration may also depend on the quality of the seeds (e.g. seed age, physical injuries, seed diseases, production conditions, and harvest stage) and climate conditions. We cannot expect the same germination response under field condition. Testing under field conditions is usually unsatisfactory, as the results cannot be repeated with reliability (ISTA, 2011). Therefore, it makes good sense to do such experiments in laboratories. We controlled and maintained a constant [O.sub.2] and [N.sub.2] ratio to isolate the effect of [O.sub.2]. Our aim was to test whether we could reproduce our result from the first experiment when we repeated it later. We succeeded although the seed population physiologically changed.

In our experiments we assumed the eventual toxic effect of increased C[O.sub.2] level, as a result of seeds respiration, could be ignored due to the large volume of the glass containers. However, we did not consider our estimate to be realistic comparable to field conditions, where the effect of the reduced [O.sub.2] concentration might be caused by increasing C[O.sub.2] levels. Leaf litter depth and decomposition, roots, and microbial respiration increases the C[O.sub.2] concentration and may also directly affect seeds germination in the forest.

There was variation in germination between the two experiments (Fig. 2). The reason could be the genetic variation or may be due to seed dormancy of A. petiolata. Seeds of A. petiolata can develop physiological dormancy and often do exhibited poor germination (Sosnoskie and Cardina, 2009; Yasin and Andreasen, 2015). We did not want to affect the dormancy of the seeds but kept them as they were in order to mimic seeds in nature. Use of heavy machinery and regular traffic imposed soil compaction, poor gasses exchange, lowed the [O.sub.2] concentration in capillaries, and therefore promoted hypoxia in the soil. These factors may favor germination and growth of A. petiolata. Sometimes leaf litter covers the seeds present on the ground. Decaying leaf litter releases many kinds of volatile compounds (plant secondary metabolites) and toxic gasses which may reduce [O.sub.2] concentration and hinder germination of seeds present on the forest floor. Often an increase in leaf litter depth results in reduced gas exchange from the atmosphere to the soil and therefore reduces the [O.sub.2] available for the germinating seeds. Consequently, leaf litter could play a role in the invasion of A. petiolata by limiting the germination of other species. However, reduced [O.sub.2] concentration also favors seed germination of plant species like overgrowing L. maackii (Bartuszevige el al., 2007).

In experiment one, [t.sub.50] was less at hypoxia compare to 20.9 % [O.sub.2], but it was opposite in experiment two (Fig. 3). However, the estimates are based on a few observations around [t.sub.50] making the estimates rather weak (Fig. 2; Table 1). Seeds were very sensitive to 5 % and 2.5 % [O.sub.2], and the germination declined severely.

The d and [t.sub.50] of A. peliolata were improved al hypoxia (Table 1). Anderson and Cipollini (2013) showed elevated C[O.sub.2] concentration resulted in stronger growth response of A. peliolata in the start, but later it down-regulated the photosynthesis rate. Hypoxia might enable A. peliolata to overcome seed dormancy and improved germination. This effect may contribute to explaining why A. peliolata often occurs and competes well against other plant species in shady understories where the [O.sub.2] concentration is rather low, particularly during nights. Our results also indicate hypoxia enabled A. petiolate to adapt to compressed and compact soils. The hard surfaces may be due to reduced [O.sub.2] concentrations and poor gas exchange in these soils. This hypothesis needs to be confirmed by measuring the [O.sub.2] concentration of the soil in places where the species is well-established. Yearly fecundity is an important demographic function for A. peliolata (Davis el al., 2006). Species with a small seed production become extinct rapidly and are dominated by more fecund species like A. peliolata (Biswas and Wagner, 2015; Pardini el al, 2009).

Further research should also examine whether hypoxia increases the adult size and fecundity of A. peliolata. Additional research is required to reveal which physiological mechanisms are involved at the molecular and cell level, promoting germination of A. peliolata at reduced [O.sub.2] concentrations.

Acknowledgments.--We thank the University of Copenhagen, Denmark for providing research support and facilities for the experiments and the University of Sargodha, Pakistan for awarded the Faculty Development Program (FDP) scholarship for doctorate study to Muhammad Yasin.

LITERATURE CITED

Anderson, L. J. and D. Cipollini. 2013. Gas exchange, growth, and defense responses of invasive AUiaria peliolata (Brassicaceae) and native Gettrn vemum (Rosaceae) to elevated atmospheric C[O.sub.2] and warm spring temperatures. Am. J. Bot., 100:1544-1554.

Association of Official Seed Analysis (AOSA). 1990. Rules for testing seeds. J. Seed Technol, 12:1-112. Bartuszevige, A. M., R. L. Hrenko, and D. L. Corchov. 2007. Effects of leaf litter on establishment, growth and survival of invasive plant seedlings in a deciduous forest. Am. Midi. Nat., 158:472-477.

Biswas, S. and H. Wagner. 2015. Spatial structure in invasive Alliaria peliolata reflects restricted seed dispersal. Biol. Invasions, 17:3211-3223.

Bradley, B. A., D. M. Blumenthal, D. S. Wilcove, and L. H. Ziska. 2010. Predicting plant invasions in an era of global change. Trends Ecol. EvoL, 25:310-318.

Cavers, P. B., M. I. Heagy, and R. F. Kokron. 1979. The biology of Canadian weeds.: 35. Alliaria peliolata (M. Bieb.) Cavara and Grande. Can.J. Plant Sci., 59:217-229.

Davis, A. S., D. A. Landis, V. Nuzzo, B. Blossey, E. Gerber, and H. L. Hinz. 2006. Demographic models inform selection of biocontrol agents for garlic mustard (Alliaria peliolata). Ecol. Appl., 16:2399-2410.

Davis, S. .and D. Cipollini. 2014. Do mothers always know best? Oviposition mistakes and resulting larval failure of Pieris virginiensis on Alliaria peliolata, a novel, toxic host. Biol. Invasions, 16:1941-1950. Geigenberger, P. 2003. Response of plant metabolism to too little oxygen. Cun. Opin. Plant Biol, 6:247-256.

Ista. 2011. International rules for seed testing, Germination tests. International Seed Testing Association, Basserdorf, Switzerland. Pp 5-15.

Klotzbi Cher, T., K. Kaiser, C. Stepper, E. Van Loon, P. Gerstberger, and K. Kalbitz. 2012. Long-term litter input manipulation effects on production and properties of dissolved organic matter in the forest floor of a Norway spruce stand. Plant Soil, 355:407-416.

Manea, A. and M. R. Leishman. 2011. Competitive interactions between native and invasive exotic plant species are altered under elevated carbon dioxide. Oecologia, 165:735-744.

Nuzzo, V. 1999. Invasion pattern of herb garlic mustard (Alliaria peliolata) in high quality forests. Biol. Invasions, 1:169-179.

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Unpublished report. The Nature Conservancy, Arlington, Virginia: Nature Conservancy. Pardini, E. A., J. M. Drake, J. M. Chase, and T. M. Knight. 2009. Complex population dynamics and control of the invasive biennial Alliaria petiolata (garlic mustard). Ecol. Appl., 19:387-397.

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MUHAMMAD YASIN, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Hojbakkegard Alle 13, DK-2630 Taastrup, Denmark; Department of Agronomy, University College of Agriculture, University of Sargodha, PK-40100 Sargodha, Pakistan; Seed Conservation Department, Royal Botanic Gardens, Kew, Wakehurst Place, West Sussex RH17 6TN, United Kingdom , and CHRISTIAN ANDREASEN, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Hojbakkegard Alle 13, DK-2630 Taastrup, Denmark Submitted 16 June 2016; Accepted 15 September 2017

Caption: Fig. 1.--Conceptual diagram showing factors of importance for the success of the invasion of garlic mustard caused by reduced oxygen concentration in the soil

Caption: Fig. 2.--Germination over lime (days) of garlic mustard at five (20.9, 15, 10, 5, and 2.5 % [O.sub.2]) oxygen Concentrations

Caption: Fig. 3.--Relationship between oxygen concentrations and time (days) to attain 50% of the seeds germinated, out of the total germinated seeds during the experimental period ([t.sub.50]) of garlic mustard
Table 1.--Estimated parameters and standard error (se) of the
germination curves (model 1)

                  (e): [t.sub.50] (Days)

% [O.sub.2]       Exp. 1          Exp. 2

20.9            2.98 (0.15)     4.87 (0.58)
15              3.91 (0.17)     3.33 (0.21)
10              4.29 (0.27)     3.69 (0.44)
5               4.13 (0.65)     6.87 (1.64)
2.5             8.74 (1.16)        - (-)

                  (d): Maximum germination

% [O.sub.2]       Exp. 1          Exp. 2

20.9            0.30 (0.04)     0.27 (0.04)
15              0.57 (0.04)     0.42 (0.04)
10              0.48 (0.05)     0.26 (0.04)
5               0.11 (0.03)     0.06 (0.02)
2.5             0.03 (0.01)        - (-)

                 (-b): Slope at inflection

% [O.sub.2]       Exp. 1          Exp. 2

20.9            6.79 (1.19)     3.23 (0.65)
15              5.20 (0.63)     4.23 (0.60)
10              4.00 (0.56)     3.05 (0.59)
5               3.59 (1.08)     3.81 (1.76)
2.5             8.39 (5.17)        - (-)
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Title Annotation:Notes and Discussion Piece
Author:Yasin, Muhammad
Publication:The American Midland Naturalist
Article Type:Report
Date:Jan 1, 2018
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