We conducted the field research in summer 2003 at the Otter Point Creek National Estuarine Research Reserve, Abingdon, Maryland, USA (39°27′N, 76°16′W). This water body in the upper Chesapeake Bay contains 106 ha of tidal freshwater habitat. The maximum depth is 1·5 m, with a mean tidal range of 0·3 m. The Otter Point Creek Reserve is of particular interest because the presence of the monoecious strain of H. verticillata was first documented here in the summer of 2002 (J. Bortz, unpublished data). Besides H. verticillata, the submersed aquatic plant community at the study site supported Ceratophyllum demersum L., Elodea canadensis Michx., Heteranthera dubia (Jacq.) MacM., Myriophyllum spicatum L., Najas sp., Potamogeton crispus L., P. perfoliatus L., P. pusillus L., Stuckenia pectinata (L.) Böerner, Vallisneria americana and Zannichellia palustris L. Myriophyllum spicatum was the dominant species at the site in 2002, and H. verticillata has been the dominant species since 2002.
Vallisneria americana is used frequently in Chesapeake Bay to restore submersed aquatic plant beds because it was a dominant species in freshwater portions of the Bay before European settlement of the region (Brush & Hilgartner 2000) and can be propagated easily. For these reasons and because V. americana is a native macrophyte that poses little conflict with recreational interests, we decided that V. americana would represent the native freshwater submersed macrophyte community of the region.
In both field and greenhouse experiments, we created an unplanted control treatment (Bare), a treatment planted with V. americana (Vallisneria) and a treatment planted with plastic plants resembling V. americana (Plastic), to distinguish between potential mechanisms of competition and facilitation. Specifically, we predicted that if species are interacting through nutrient exploitation, H. verticillata colonization would be lower in the Vallisneria treatment than the other two treatments, effectively lending support for the pre-emption hypothesis. If so, we expected nutrient levels to be lower in the Vallisneria treatment than the Bare and Plastic treatments. Furthermore, we predicted that, if V. americana either physically inhibits or facilitates H. verticillata colonization, H. verticillata colonization would be lower (lending support for the pre-emption hypothesis) or higher (lending support for the facilitation hypothesis), respectively, in the Plastic treatment compared with the Bare treatment. Logically, the null hypothesis of no effect would be supported if no inhibitory or facilitative effects were detected. Finally, we predicted that colonization of H. verticillata into existing patches of V. americana would be greater in areas of higher H. verticillata biomass in the field or in greenhouse mesocosms with higher fragment introduction (lending support for the propagule pressure hypothesis).
We used polypropylene strapping (1·3 cm wide × 0·04 cm thick) to create artificial plants that mimicked the morphology of V. americana such that the ability to trap propagules and to affect the light environment would be equal. To test for equal effects on light, we measured light levels under the canopies of live and plastic plants (see the greenhouse methods). We did not test for equal propagule trapping ability because the test would have required detailed flume studies. Each individual plastic plant was 45 cm tall and had eight leaves. We then fastened 17 of these plants into each of 30 plastic trays measuring 34 × 24 × 9 cm. Sixty empty trays and 30 trays with the plastic plants were filled to within 1 cm of the top of the tray with substrate that was an equal mix of commercially available topsoil and sand. The final 1 cm was then filled with sand to reduce the loss of substrate to the water column. On 14 April 2003, 30 of the bare trays were seeded with V. americana. All trays were then placed in the greenhouse and submersed in 20 cm dechlorinated domestic water. Because of poor germination, we planted 17 live V. americana plants in the previously seeded trays on 23 May 2003. Resulting V. americana densities were 209 plants m−2, which was similar to reported natural densities of this species (Korschgen & Green 1988). All trays (30 with live V. americana, 30 with plastic plants and 30 unplanted) were then maintained in the greenhouse at the University of Maryland Center for Environmental Science's Appalachian Laboratory for 35 days, to permit the V. americana to root sufficiently before placement in the field.
Experimental sites at the Otter Point Creek field location were chosen based on the H. verticillata cover estimates of the previous year (2002). We selected two separate areas at Otter Point Creek that supported high (> 40% cover) to low (< 40% cover) densities of H. verticillata. In each of the two areas, we placed one experimental site in high cover, one in low cover and one midway between the high- and low-density sites, to create a biomass gradient associated with differences in propagule flux across the gradient. Measurement of propagule flux at the different sites was attempted with little success using methods described in Rybicki et al. (2001); thus existing site H. verticillata biomass, which readily produces propagules through fragmentation, was used as a surrogate for propagule pressure. All of the trays were transported to the field location on 28 June 2003 and immediately placed in the experiment sites. Five replicates of each Bare, Plastic and Vallisneria treatment were randomly placed in each site. Sediments at the site were soft enough to allow the trays to be pushed down until the top of the tray was flush with the natural sediment. Trays were placed approximately 2 m from one another and anchored in place with PVC pipe run through the centre of each tray.
To evaluate whether experimental units were indeed placed in high, medium and low densities of existing H. verticillata, we collected all above-ground biomass of each submersed aquatic macrophytes species in four randomly placed 1-m2 quadrats at each of the six experimental sites on 17 September 2003. The biomass was not rinsed because we perceived the biomass of attached detritus and epiphytic growth to be negligible. We later dried all biomass at 60 °C for 24 h and weighed the material.
We used a portable water current meter (Model 201D; Marsh-McBirney, Frederick, MD) to measure current velocity at each of the six experimental sites over a 24-h period between 25 and 26 July 2003, to test for equal current velocities between sites at different times during the tidal cycle. Velocities were measured at 10 cm above the sediment surface and just below the water surface. We also recorded depths with a PVC depth rod at each of the sites on the hour during this period.
To identify any nutrient-related mechanisms affecting the colonization success of H. verticillata, we analysed soil pore water for total nitrogen and total phosphorus in 10 randomly selected trays per treatment. We permanently installed mini-tension lysimeters (Daiki Corp., Tokyo, Japan), placed horizontally in the soil approximately 5 cm beneath the soil surface, and collected approximately 15 mL water for analysis during two sampling periods. The first soil pore water was collected on 11 July 2003. Because of time constraints imposed by Hurricane Isabel, the final soil pore water collection did not occur in the field. Instead, on 17 September 2003, all trays were collected, sealed in plastic bags and transported intact to the laboratory. Soil pore water was collected the next day from the same trays as before using the previously installed lysimeters. We stored all water samples frozen at –19 °C for 4 months until analysis. Each water sample was digested, autoclaved and analysed for total nitrogen (TN) and total phosphorous (TP) on a Lachat QuikChem Automated Flow Injection Analysis System (APHA 1998). TN and TP were analysed to account for potential shifts in inorganic and organic nutrient pools during storage.
The content of each tray was sifted to separate the substrate from the plant material. Biomass was separated into tubers, turions and above-ground biomass for H. verticillata and total biomass for V. americana. All vegetative material was dried for 24 h at 60 °C and then weighed.
We observed in the field that pumpkinseed sunfish Lepomis gibbosus L. were nesting in several of the trays and consequently removing some amount of substrate in these trays. When the trays were collected, marks had been left on the sides of the trays from algal growth, indicating how much substrate had been removed. Based on these marks, we quantified the disturbance in each tray as a percentage of the substrate that had been removed.
To control environmental conditions and regulate propagule input, we replicated the field experiment in greenhouse mesocosms (26 cm diameter × 36 cm height) using 16 unplanted mesocosms as controls (Bare), 16 mesocosms planted with V. americana (Vallisneria) and 16 mesocosms planted with the plastic plants (Plastic). Substrate was added to a depth of 9 cm. Thirteen plastic plants (36 cm tall) were added to the Plastic treatment and 13 live V. americana plants to the Vallisneria treatment such that the density of plastic and live plants was identical in the greenhouse and field experiments.
We began the experiment on 23 May 2003. On 19 August 2003, 10-cm long H. verticillata fragments with 10–20 nodes fragment−1, collected from Little Seneca Lake, Burdette, Maryland, were added to each of the treatments at densities of 0, 1, 5 or 10 fragments mesocosm−1. The dry mass of each fragment, measured on additional fragments of the same length, was 9·8 ± 0·1 mg (mean ± SE), which equated to 0, 0·2, 0·9 and 1·8 g m−2 introduced to 0, 1, 5 and 10 fragment treatments, respectively. We collected soil pore water from five randomly selected containers per treatment on 26 August 2003, using the same methodology as the field experiment. Water column nutrients from the same containers were sampled on 10 November 2003, rather than soil pore water, because H. verticillata was interacting more with the environment in the water column as a result of poor rooting in the substrate in Vallisneria treatments. We measured light availability under the canopies of plastic and live V. americana using a Model LI-1400 light meter (Li-Cor, Lincoln, NE), to test for similarity in effects on light availability. Biomass was sifted and separated into tubers, turions and above-ground biomass for H. verticillata and total biomass for V. americana. All samples were dried for 24 h at 60 °C and then weighed.
One-way anova or a non-parametric equivalent was used to test for differences among treatments in the field and greenhouse experiments. Because data often could not be transformed to meet the assumptions of normality, we used Spearman correlation analysis to test for correlations among sunfish disturbance, H. verticillata tuber biomass and total V. americana biomass in field trays. Spearman correlations also tested for a relationship between rooted H. verticillata biomass and overall biomass production. We used an ancova to assess the effects of site–treatment interactions on the dependent variable H. verticillata tuber biomass based on the categorical variable treatment and the continuous covariate pre-existing H. verticillata biomass or disturbance by sunfish.
All biomass measurements were recorded as 100% dry matter. We used SAS software (SAS Institute 2001) for all statistical tests. Unless noted otherwise, we evaluated statistical significance at P < 0·05 and we report values as means ± SE.