Study species
Closely related native and nonnative species were used for this
experiment to control for species responses that are attributable to
phylogeny and isolate the response of plant origin (native, nonnative)
to climate change and soil biota. We chose species from Eugenia, in
the Myrtaceae family, because there are closely-related native and
nonnative species of the same genus and functional group which
co-occur in subtropical hammock habitat of Florida (Liu et al. 2007).
All three species are relatively abundant throughout Florida
(Wunderlin et al. 1996). These small tree and shrub species are found
in subtropical habitats in Florida, central and South America, and the
Caribbean. Eugenia uniflora, or Surinam cherry, is native to Brazil
and has been introduced to much of South America outside of Brazil, in
addition to Asia, Australasia-Pacific Region, Europe, and North
America (Wunderlin et al. 1996; ISSG 2016). Eugenia uniflora
associates with arbuscular mycorrhizal fungi (Zangaro et al. 2005).
Eugenia uniflora was introduced to Florida as an ornamental and for
its edible fruit prior to 1931, and has been widely planted in central
and south Florida, especially for hedges (Langeland et al. 2008).
Eugenia uniflora has a high impact on ecological communities (FLEPPC
2017), is able to invade upland habitat, and is located south of the
freeze line in Florida. Eugenia uniflora is considered Category I, as
designated by the Florida Exotic Pest Plant Council (FLEPPC 2017),
which is a species that causes large ecological damage through the
displacement of native species, changing community structures or
ecological functions, or hybridizing with natives.
Growth chamber Experiment set up
Changes in growth and germination of our three Eugenia study species
was monitored in pots placed in growth chambers, using upland hammock
soils from their current range in Florida and from their potential
climatically-induced expanded range. Central Florida is the current
northern limit of Eugenia species in Florida (Wunderlin and Hansen
2003), and so we chose a site with hammock habitats that was north and
well outside of their current range, as predicted by the poleward
expansion of species (Parmesan and Yohe 2003). Future temperature
conditions of the northern site were estimated with a Low, B1 emission
scenario; for a range of SRES emissions scenarios, and using global
climate projections from the Fourth IPCC Assessment (IPCC 2007;
Girvetz et al. 2009). Pots were placed in growth chambers where
diurnal variation in daylength and temperatures were simulated, with
the high and low daily temperatures determined by the average daily
maximum and minimum temperatures for the month of May in Jacksonville,
FL (Florida Climate Center, Center for Ocean-Atmospheric Prediction
Studies), the northernmost site from where soil was collected. The
pots experienced environmental conditions simulated for current (2010)
and future (2050) conditions, with 10 hours of light per day and 30/17
°C and 31/18 °C and day/night temperatures (Table 1a & b).
Seed Sampling
Seeds were haphazardly collected from populations located within Hugh
Taylor Birch State Park, in south Florida, for the one nonnative and
two native study species. Seeds were collected for each species at the
peak of seed production for their species. Seeds for the native
species were collected on December 17th, 2011, and seeds for the
nonnative Eugenia species were collected on April 28th, 2012. The
fruit covering from each seed was removed by hand and the seeds were
surface sterilized in 5% bleach solution for fifteen minutes, and
washed with de-ionized water, prior to planting.
Soil Sampling
Soil was collected from three hammock habitat sites within each of the
central, south, and north Florida sites. Soil biota was collected in
the form of fresh field-collected soil from one of two sources: the
current home range [Central Florida (Cape Canaveral, FL), South
Florida (Hugh Taylor Birch State Park)] or within the projected new
range [North Florida (Timucuan Ecological and Historic Preserve,
Florida)]. Soils were collected from all three Florida source regions
within one week prior of the potting date, to ensure viability of the
soil microbiota. In the south and central Florida sites, we collected
soil from hammock habitats within natural areas which were at least 20
meters from Eugenia shrubs or seedlings. In the north Florida site, we
collected soil from randomly placed transects (using random point
generator feature of ArcMap, ESRI, Redlands, CA) within hammock
habitats. Within each of these three sites, two, 10-meter transects
were laid within hammock habitat, at least 5m away from roads. Every
two meters, 10cm deep soil samples were collected and placed into a
Ziploc bag. The two, 10 m transects were parallel and at least 10 m
apart. Soil samples were combined within each site, sieved to 2 mm,
and added to the pots within one week of collection (as in, Hawkes et
al. 2011). We pooled soils within each site to provide a soil inoculum
treatment representing all possible soil microbes in that site and the
average density found within that site (Cahill et al. 2017), which is
a common treatment used to understand the effect of soil microbes on
plant germination and growth (Grman and Suding 2010; Lau and Lennon
2011; Farrer and Suding 2016), however this method can artificially
decrease variation in plant-microbe interactions (Reinhart and Rinella
2016; Rinella and Reinhart 2017; Rinella and Reinhart 2018). While
variation in plant-microbe interactions is decreased with pooling
samples, this method of soil pooling is preferable when the objective
is to understand if the average pathogen density found in each of two
regions differentially effects plant growth (Cahill et al. 2017). The
soil biota treatment is one of several treatments, where we evaluate
plant-microbial interactions in relation to those treatments.
The soil biota treatment was fresh field-collected soil from each of
the central, south, and north Florida sites. For the control
treatment, we sterilized half of these field-collected soils from the
current and new ranges. The sterile soil inoculum was autoclaved three
times, and we mixed the soil in between autoclave events, to ensure
sterilization of the soils. The soil biota and sterile control inocula
comprised 5% of the mass of the pot, to ensure sufficient inoculation
of the soil biota to the pot while also maintaining the same nutrient
conditions and soil characteristics across all treatments (as in
Reinhart and Callaway, 2004).
For each species, eight seeds were planted into a minimum of seven,
sterile replicate pots (4 x 4 x 6") filled with sterile potting
mix (MetroMix 366 sterile potting soil) and one of two soil inoculum
treatments (sterile, nonsterile), two temperature treatments 2010
(e.g. ‘current’) and 2050 (e.g. ‘future’ temperature conditions at our
northernmost site) and three site treatments (south, central, and
north), for a total of 86 pots for the nonnative species and 105 pots
each for the native species. Eight to nine replicate pots per
treatment were made for the native species (Table 1a), and seven
replicate pots were made per treatment for the nonnative species
(Table 1b), as the native species have lower germination rates,
relative to the nonnative Eugenia species (Stricker and Stiling 2013).
The potting dates were the 27th of January, 2012, for the native
species and the 9th of May, 2012, for the nonnative species, in accord
with their fruiting phenology and when the seeds were collected. After
germination, pots were kept in a growth chamber for the next 12 weeks,
to assess growth. They were watered daily with equal amounts of water,
approximately 15-20 ml. Pots were rotated daily within the growth
chamber, to control for positional effects. Care was taken to ensure
that the soil biota were not cross-contaminated between pots by using
sterile techniques. Germination was monitored weekly until after the
appearance of the first germinant, at which point monitoring occurred
daily. Daily monitoring ceased after the pots were monitored daily for
two weeks with no new germination. Two weeks after germination ceased
for each species, we selected a maximum of four seedlings to remain,
and removed all other seedlings from the pot, taking care not to
disturb the soil. Twelve weeks following initiation of germination,
the remaining plants were harvested for total above and below-ground
biomass. Shoots were cut at ground level and oven-dried separately in
paper bags at 60 °C for 2 days. The roots were carefully washed to
remove soil particles and also oven-dried at 60 °C in paper bags.
After drying, shoots and roots were weighed with a precision balance
to determine dry weight.
Literature cited
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Miller HL, eds. Intergovernmental Panel on Climate Change. Cambridge,
United Kingdom and New York, NY, USA.: Intergovernmental Panel on
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Girvetz EH, Zganjar C, Raber GT, Maurer EP, Kareiva P, Lawler JJ.
2009. Applied Climate-Change Analysis: The Climate Wizard Tool. Plos
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