METHODS
Site description
The ULE system offers a natural template for understanding the effect of habitat heterogeneity on the spread of E. canadensis (i.e. testing invasion hypothesis 3 ), being composed of a large (surface area 3450 ha), generally shallow (mean water depth 2.3 m) central lake (ULE), fed by the River Erne, which is linked by winter floods and various channels and tributaries, to a network of over 50 small (<40 ha), shallow (<5 m) satellite lakes (loughs; Fig. 1). The system has been subjected to eutrophication since the 1950s (Battarbee 1986; Zhou 2000) with current annual concentrations of total phosphorus (TP) and total nitrogen (TN) ranging between 29-383 μg/L and 0.22-2.25 mg/L respectively. E. canadensis is thought to have colonized the ULE system in the 1880s (Simpson 1984). Records from the Botanical Society of Britain and Ireland (BSBI) and more recent macrophyte monitoring programmes indicate presence of E. canadensis in ULE and its satellite lakes from the 1950s (Table S1). The zebra mussel (Dreissena polymorpha ) also invaded this system in the 1990s, resulting in strong phytoplankton biomass reductions and increases in water transparency in the ULE (Minchin et al. 2003).
Macrophyte sampling and diversity predictors
Macrophytes were sampled in four basins within the central ULE and in 13 satellite lakes representing a gradient of nutrient-enrichment, zebra mussel occurrence and hydrological connectivity to the ULE (Fig. 1). The lakes were grouped into three categories according to Salgado et al. (2019) as: Group 1 included the ULE and lakes directly connected to it through the River Erne (Castle, and Derrykerrib) or via tributaries (Doo and Mill). These lakes are diverse (macrophyte richness= 17.4 ± 2.7), meso-eutrophic (TP= 55.1 ±11.3 μg/L), and have clear waters (secchi depth= 222 ±47 cm) and high occurrence of zebra mussels. Group 2 lakes are connected to ULE by flows through intermediate lakes and associated tributaries (Killymackan, Cornabrass and Kilturk). The lakes are diverse (macrophyte richness= 18.7 ±4.6), eutrophic (TP = 136 ±54.4 μg/L; secchi depth= 182.3 ±55 cm) and zebra mussels occur sparsely. Group 3 lakes (Head, Digh, Derryhowlaght and Gole) are more isolated than Group 2 lakes due to intervening small hills, woodlands and roads (Figure 1). These lakes are the least diverse (macrophyte richness= 9 ±3.5) and most eutrophic (TP = 176.8 ± 89.3 μg/L) and turbid (secchi depth= 113.3 ± 79.3; zebra mussels rarely occur).
Sampling was undertaken in 1m2 units, approximating to the plant neighbourhood scale (i.e. where individual native plants may compete with E. canadensis ). The lake volume infested by macrophytes (PVI) method of Canfield & Jones (1984) was used to characterize the distributions and abundances of native macrophytes (including charophytes, bryophytes and vascular plants) and of E. canadensis . Macrophytes were surveyed during the summers of 2008-09 at individual points from a boat by zig-zagging across the entire lake using grapnel sampling and visual observations with a bathyscope. At each sampling point we recorded latitude/longitude, water depth, average plant height, and species cover (%). PVI was calculated at each point as: (macrophyte cover x average height of macrophyte)/water depth. For comparisons with previous monitoring data (Table S1) and to assess E. canadensis abundance patterns at the lake scale, percentage of occurrence of E. canadensis at each lake was also calculated by dividing the number of sampling points at which E. canadensis was observed by the total number of sampling points within the lake X 100.
The number of macrophyte sampling points varied according to lake size. A minimum of 30 points was sampled for the smaller (< 10 ha) lakes (Loughs Doo, Gole, Digh, Gole and Derryhowlaght) and between 60-80 points for the remaining larger (12-30 ha) satellite lakes (Fig 1). The ULE was sampled across four separate lake zones (~30 ha): Crom State (54.161731, -7.436668), Lisnaskea Rd.–Trannish (54.225036, -7.475074), Derrylea Rd.–Trannish (54.218312, -7.469581), and Smith Strands–Trannish (54.204659, -7.480395). A total of 20 sampling points per ULE zone were surveyed and a total of 540 sampling points across all the study lakes were covered. While our sampling approach missed some macrophyte species known to occur in individual lakes, it nevertheless provides a useful representation of variation in macrophyte distributions and abundances for the majority of species at the plant neighbourhood scale (Salgado et al. 2018b).
Abiotic data
Our previous studies of the ULE system demonstrate that macrophyte communities are structured by lake water transparency, which is negatively related to nutrient concentrations (TP and TN) and positively correlated with zebra mussel occurrence (Salgado et al. 2018a, Salgado et al. 2019). Accordingly, we used water clarity as a predictor of environmental stress variation across the macrophyte sampling points. An index of water clarity for each sampling point was defined as: lake secchi depth measured at the deepest point of each lake/water depth at the sampling point.
Plant macrofossil data
Previously published plant macrofossil abundance data derived from dated sediment cores were used to represent macrophyte community changes over the last c. 120 years (Salgado et al. 2018a,b; Salgado et al. 2019a). Cores were taken from Castle Lough and the Trannish area of ULE (lake Group 1 ), from Cornabrass and Killymackan (Group 2 ), and from Gole and Head (Group 3 ). The plant-macrofossil data are available from the Dryad Digital Repository:https://doi.org/10.5061/dryad.3jj548d.
E. canadensis remains preserve poorly in lake sediments (Davies 1985) and so we inferred its temporal abundance from a recent macrophyte study in the ULE system (Salgado et al. 2019) and from available historical monitoring data (Table S1). Salgado et al. (2019) showed that macrophyte assemblages now found in the ULE or closely-connected lakes (e.g. Castle and Derrykerrib) are similar to those characterised in sediment cores prior to eutrophication (i.e. pre 1950s). However, macrophytes currently found in the more isolated eutrophic sites (e.g. Gole and Head) resembled those characteristics of sediment cores post eutrophication (i.e. post-1960). Accordingly, we used the current E. canadensis occurrence data in lakes close to the ULE as a surrogate of E. canadensis abundances prior to 1950 and the E. canadensis occurrence data for the isolated lakes to infer E. canadensis abundances during the onset of eutrophication from 1960-1980. More recent (post-1980) E. canadensis abundance data were obtained from macrophyte surveys conducted in 1988, 2006 and 2009 (Table S1). To standardise these various sources of E. canadensis occurrence into a single comparable abundance measurement, we assigned the data to the following abundance categories: 5 (100%–80% occupancy of sampled points); 4 (79%–60%); 3 (59%–40%); 2 (39%–20%); 1 (19%–1%); 0 (0%).
Statistical analysis