Appendix A. Methodological details of common garden experiments for testing effects of pathogens and herbivores on native and introduced clover species.
This includes the following: (1) Details of methods and dates, (2) Table A1: Details of sample sizes, census dates, and harvest dates, and (3) Table A2: Number of individuals of each species surviving in final sample.
Details of methods and dates
In each year, we germinated seeds and then raised the seedlings in the Bodega Marine Reserve (BMR) greenhouse until plants were established. Plants were grown in Ray Leach Conetainers (2.5 cm × 16.5 cm, Stuewe and Sons, Corvallis, Oregon, USA) (Years 13) or plastic sleeves (Year 4) in soil collected in the field site, and watered with overhead misting, until transplanting into the field site.
Year 1. In 19971998, seeds were germinated in three cohorts from 19 November14 December and grown in conetainers until transplanted into the field between 18 December1 January. Plants that died were replaced until Feb. 7. Two plants of each species were randomized in 24 blocks (N = 48). Each block was at least 5 m from every other block, and plants were arranged every 0.5 m in rows 1 m apart. Deer netting was laid on top of the blocks. Seven blocks were used for severity censuses.
Year 2. In 19981999, seeds were germinated 1519 December, grown in conetainers in the greenhouse, and transplanted into the field 6 February, replacing dead plants until 20 March. A 90-m × 105-m grid was laid out with posts every 15 m, providing 42 sites for blocks, of which 36 were used for this experiment. Each block had one plant from each species, randomized in three rows (1 m apart) of six plants (0.5 m apart). Deer netting was laid on top of the blocks. Seven blocks were used for severity censuses.
Year 3. In 19992000, seeds were planted into conetainers 1721 December and transplanted into the field 24 February. We transplanted 40 pairs of control and fungicide plants per species into one large, randomized grid; pairs were 5 m apart in each direction, and plants in a pair were 0.3 m apart. The fungicide plants were sprayed weekly with DaconilTM + Tween 80; the control plants were sprayed with distilled water + Tween 80.
Year 4. In 20002001, intact soil cores were extracted in plastic sleeves 5 cm diameter and brought into the BMR greenhouse. Seeds were planted into these soil cores 13 January and transplanted into the field 13 March into their original holes, completely randomized for both species and fungicide treatment (6 replicates of each). Because they remained in plastic sleeves, these plants were protected from competition and belowground disturbance by gophers. The fungicide plants were sprayed weekly with Dithane F45TM + the surfactant RainguardTM; the control plants were sprayed with distilled water + RainguardTM.
TABLE A1. Details of sample sizes, census dates, and harvest dates for four years of common garden experiments. Year 3 and Year 4 included fungicide (F) and control (C) treatments.
Year 1 |
Year 2 |
Year 3 |
Year 4 |
|
19971998 |
19981999 |
19992000 |
20002001 |
|
Census dates for |
9Feb, 21Feb, 7Mar, 23Mar, 4Apr, 18Apr, 9May |
5Feb, 20Feb, 7Mar, 20Mar, 3Apr, 17Apr, 1May, 15May, 22May |
At harvest |
At harvest |
Census dates for |
8Mar, 21Mar, 3Apr, 1720Apr, 18May |
21Mar, 24Apr, |
At harvest |
At harvest |
Harvest dates |
1018 May |
2124 May |
2028 May |
1114 May |
Number native species |
9 |
10 |
8 |
9 |
Number introduced species |
7 |
8 |
8 |
8 |
Number individuals |
48 |
34 |
40F + 40C |
6F + 6C |
TABLE A2. Number of individuals of each species surviving to the final sample. Year 3 and Year 4 include both fungicide and control plants in the sample size. The two invasive species are indicated in bold. A dash (–) indicates the species was not available for planting that year. Number of individuals planted per species is given in Table A1.
Origin |
Species |
Year 1 |
Year 2 |
Year 3 |
Year 4 |
Introduced |
M. arabica |
46 |
16 |
69 |
12 |
Introduced |
M. lupulina |
– |
20 |
63 |
12 |
Introduced |
M. polymorpha |
38 |
20 |
63 |
12 |
Introduced |
T. campestre |
39 |
9 |
45 |
12 |
Introduced |
T. dubium |
37 |
12 |
55 |
11 |
Introduced |
T. glomeratum |
27 |
17 |
42 |
11 |
Introduced |
T. repens |
34 |
14 |
64 |
12 |
Introduced |
T. subterraneum |
42 |
17 |
63 |
12 |
Native |
T. barbigerum |
44 |
11 |
60 |
12 |
Native |
T. bifidum |
37 |
12 |
61 |
12 |
Native |
T. fucatum |
46 |
17 |
63 |
11 |
Native |
T. gracilentum |
32 |
13 |
60 |
12 |
Native |
T. macraei |
24 |
13 |
49 |
12 |
Native |
T. microcephalum |
39 |
6 |
45 |
12 |
Native |
T. microdon |
30 |
23 |
57 |
12 |
Native |
T. variegatum |
– |
16 |
– |
– |
Native |
T. willdenovii |
35 |
17 |
– |
12 |
Native |
T. wormskjoldii |
38 |
17 |
66 |
11 |