DOI: 10.7280/d17d5h
title: Data from: Lineage and latitudinal variation in Phragmites australis tolerance to herbivory: implications for invasion success

description: Herbivores play a critical role in plant invasions either by facilitating
 or inhibiting species establishment and spread. However, relatively few
 studies with invasive plant species have focused on the role of plant
 tolerance and how it varies geographically to influence invasion success.
 We conducted a common garden study using two lineages (native and
 invasive) of the grass Phragmites australis that are prevalent in North
 American wetlands. Using 31 populations collected across a broad
 geographic range, we tested five predictions: 1) the invasive lineage is
 more tolerant to simulated folivory than the native lineage, 2) tolerance
 to herbivory decreases with increasing latitude of origin of the
 populations, 3) estimates of tolerance are correlated with putative
 tolerance traits and plasticity in those traits, 4) a tradeoff exists
 between tolerance and resistance to herbivory, and 5) tolerance has a
 fitness cost. Response to folivory varied substantially among populations
 of P. australis, ranging from intolerance to overcompensation. Our model
 selection procedure deemed lineage to be an important predictor of
 tolerance but, contrary to our prediction, the native lineage was 19% more
 tolerant to folivory than the invasive lineage. Tolerance for both
 lineages exhibited a u-shaped relationship with latitude. A
 tolerance-resistance tradeoff was evident within the invasive but not the
 native lineage. Also, tolerance was positively correlated with belowground
 biomass allocation, leaf silica concentrations, specific leaf area and
 plasticity in stem density, and negatively correlated with the relative
 growth rate of the population and plasticity in putative resistance
 traits. Lastly, although we did not detect costs of tolerance, our results
 highlight that fast growth rates can maintain high fitness in the presence
 of herbivory. Herbivory and plant defense strategies for P. australis
 lineages in North America exhibit complex biogeographic patterns that
 cause substantial heterogeneity in enemy release and biotic resistance
 and, consequently, invasion success.
description: From Croy et. al. (2020): <b>Study
 system</b> <i>Phragmites australis
 </i>is a 2-5 m tall perennial grass commonly found in wetlands,
 estuaries, salt marshes, ponds, and rivers on every continent except for
 Antarctica (Clevering and Lissner 1999). Although present in North
 American wetlands for millennia (Hansen 1978, Orson 1999), <i>P.
 australis </i>began spreading aggressively, dominating wetlands and
 negatively impacting native plant species, hydrologic regimes, nutrient
 cycles, and ecosystem function (Chambers et al. 1999, Meyerson et al.
 2009, 2010). The rapid spread is attributed to the introduction of an
 invasive Eurasian lineage (Haplotype M; <i>P. australis</i>
 <i>australis</i>) that first appeared in the herbarium record
 about 150 years ago (Chambers et al. 1999, Saltonstall 2002). Populations
 of the Eurasian lineage in North America are genotypically diverse
 (Saltonstall 2003) and despite being clonal, genotypic variation has been
 identified within patches (McCormick et al. 2010). Additional haplotypes
 have been introduced from Europe, North Africa (e.g., Lambertini
 <i>et al.</i>, 2012; Meyerson and Cronin, 2013) and Asia
 (Lambert et al. 2016), but their known distributions are localized and
 none were included in this study. Throughout North America, at least 14
 closely related native endemic haplotypes have been identified
 (Saltonstall 2002, Meadows and Saltonstall 2007, Vachon and Freeland
 2011). These native haplotypes have been given subspecies status
 (<i>P. australis</i> <i>americanus</i>) but are
 collectively referred to as the native lineage. In Europe<i> </i>where
 it is considered native, <i>P. australis</i> is host to a
 diverse assemblage of arthropod herbivores with over 170 species
 identified. In contrast, only 26 herbivore species have been reported in
 North America (Tewksbury et al. 2002). Within North America, herbivory on
 <i>P. australis</i> is primarily attributed to herbivores
 introduced from Europe, most prominently stem-galling flies in the genus
 <i>Lipara</i> (Lambert et al. 2007, Cronin et al. 2015, Allen
 et al. 2015) and the mealy plum aphid <i>Hyalopterus pruni</i>
 (Lozier et al. 2009, Cronin et al. 2015). Based on previous research,
 there is strong support for the enemy release hypothesis: the invasive
 lineage suffers substantially less herbivory from each of three major
 feeding guilds (aphids, stem-gallers and leaf chewers) in its invasive
 than native range, and in comparison to co-occurring plants of the native
 lineage in North America (Cronin et al. 2015, Allen et al. 2015, Bhattarai
 et al. 2017a, b). Because these differences in herbivore abundance or
 damage between the native and invasive lineages are also manifested under
 common garden conditions, it suggests greater resistance to herbivory by
 the invasive than native lineage of <i>P. australis</i> and
 that resistance is genetically based (Lambert and Casagrande 2007, Allen
 et al. 2017, Bhattarai et al. 2017b). Interestingly, Cronin et al. (2015)
 found that leaf damage and stem-galler incidence decreased with increasing
 latitude for the native lineage but not invasive lineage, and these
 non-parallel latitudinal gradients in herbivory resulted in stronger enemy
 release at southern than northern latitudes (Cronin et al. 2015). This
 genotypic and latitudinal variation in herbivory lends credence to a
 biogeographic investigation into the role of plant tolerance to herbivory
 in invasion success. <b>Common
 garden</b> The common
 garden design used in this experiment is detailed in Bhattarai et al.
 (2017b). Briefly, rhizomes from <i>P. australis</i> patches
 were collected from field sites throughout North America spanning 19.5° of
 latitude and 55.9° of longitude and planted in a common garden established
 at Louisiana State University, Baton Rouge, LA (30.35° N, 91.14° W) in
 2009 (Figure 1; Appendix A: Table S1). Plants were grown for at least one
 year prior to the start of the experiment to minimize the influence of
 maternal effects. In early March 2014, we potted 10 replicates for each of
 31 source populations of <i>P. australis </i>(13 native, 18
 invasive) in 7.6-liter pots. Each replicate consisted of a single rhizome
 cutting (10-15 g) potted in sand to standardize nutrients and initial
 starting conditions. Due to lower than expected propagation success, we
 added five additional replicate pots for each population in early May
 2014. We fertilized each pot with Osmocote<sup>®</sup> (58
 grams/pot of 3 month, slow-release in March followed by 58 grams/pot of 9
 month, slow-release in June of 15-9-12 NPK, The Scotts Miracle-Gro
 Company<sup>®</sup>, Marysville, Ohio) and
 Ironite<sup>®</sup> (1.7 grams/pot;
 Pennington<sup>®</sup>, Madison, Georgia) to ensure that
 resources were standardized. We repeatedly sprayed the plants with a
 non-systemic insecticide (Ortho<sup>®</sup> Malathion; The
 Scotts Miracle-Gro Company<sup>®</sup>, Marysville, Ohio) to
 prevent herbivore damage. Finally, we placed potted plants from each
 population in the same plastic pool (1.2 m diameter) filled with tap
 water. Plants subjected to different levels of folivory within a
 population were randomly distributed within pools, and populations were
 randomly distributed within the common garden.
 <b>Folivory treatment and tolerance
 </b> We implemented an
 artificial folivory treatment to simulate the effects of heavy folivory, a
 common approach in the study of tolerance to herbivory (e.g., Marquis
 1988, Tiffin and Inouye 2000, Vergés et al. 2008, Ashton and Lerdau 2008,
 Lurie et al. 2017). <i>Phragmites australis </i>lineages vary
 widely in resistance to folivory (Cronin et al. 2015, Bhattarai et al.
 2017b) and, consequently, it would have been difficult to achieve a
 standardized level of herbivory among source populations without varying
 herbivore density and/or exposure time. We assigned plants within a
 population at random to either a folivory or no folivory treatment.
 Starting in late May 2014, we clipped 40% of the leaf area, followed by a
 monthly removal of 40% of the new growth until late August. We clipped
 plants from the top-down to reflect herbivore behavior, because they
 generally remove the newest, most palatable leaves first. Although 40%
 folivory is a severe damage treatment, this level of herbivory has been
 observed in the field (Cronin et al. 2015). At the end of the growing season
 (early November in Louisiana), we harvested above- and below-ground plant
 material, which was then air-dried on benchtops in the greenhouse until
 completely dried (2 mo.), and measured with a hanging scale
 (Pesola<sup>©</sup>,Schindellegi, Switzerland; precision of ±
 0.3%). Because flowering frequencies for <i>P. australis
 </i>in the first year following propagation from small rhizome
 cuttings are quite low, and biomass encapsulates all aspects of asexual
 reproduction, we used the total dry biomass at the end of the season as
 our proxy for fitness. Moreover, we had two planting dates owing to the
 need for supplemental plants, and it was our intention to include a
 blocking effect for planting date in our statistical models. However, we
 did not have sufficient replication within the second block to calculate
 tolerance for each source population. To account for possible differences
 in final plant size between planting dates, we used least-squares means
 for plant biomass (above-, belowground, and total biomass) computed from a
 general linear model that included the main and interactive effects of
 clipping treatment and population, as well the main effect of planting
 date. From this, we obtained a planting date-independent estimate of plant
 biomass for each population in both the clipped and unclipped treatments.
 With these estimates, we calculated tolerance for each population using
 log-response ratios, where Tolerance<sub>total</sub> =
 <i>ln</i>[mean end-of-season total biomass of clipped
 plants/mean end-of-season total biomass of unclipped plants] (Hedges et
 al. 1999). A Tolerance<sub>total </sub>value of zero would
 indicate no effect of folivory on end-of-season biomass whereas a
 Tolerance<sub>total</sub> &gt; 0 would indicate
 overcompensation. Overall, the larger the value of
 Tolerance<sub>total</sub>, the greater the tolerance of that
 population. We also calculated log-response ratios for aboveground
 (Tolerance<sub>above</sub>) and belowground
 (Tolerance<sub>below</sub>) biomass separately to compare the
 relative impacts of folivory on the shoot and root biomass, respectively.
 Lastly, we note here that the above method for estimating tolerance
 necessitated that the population, not the individual pot, was our unit of
 replication in this experiment, resulting in a single estimate of
 tolerance for each population. We averaged the estimates for dependent
 variables at the population-level to avoid pseudo-replication (rhizomes
 were collected from same pool of plants for each population and thus are
 not completely independent samples).
 <b>Putative tolerance and resistance
 traits</b> In addition
 to population-level measures of tolerance, we also measured a suite of
 plant functional traits that are widely recognized as correlates of plant
 tolerance and resistance to herbivore damage. How these traits change in
 response to a folivore treatment can provide clues as to the mechanisms
 underlying plant defenses. Putative tolerance traits like relative growth
 rate (RGR), root-shoot ratio, stem density, and photosynthetic rate are
 closely associated with compensatory ability (e.g., Meyer, 1998; Strauss
 and Agrawal, 1999; Tiffin, 2000). To estimate plant RGR, we divided total
 end-of-season biomass by the number of days in the growing season. With
 the exception of our blocked design, all plants were propagated and
 harvested at the same time, and so we believe that a linear estimate of
 RGR captures variation in RGR across populations. To account for the fact
 that some plants grew for a longer period of time than others due to the
 blocked design, we accounted for the blocking effect RGR statistically as
 was done for biomass (see above). In addition to RGR, we measured stem
 density at the last census of the growing season (late August 2014) and
 calculated root mass fraction (= belowground biomass/total biomass;
 Pérez-Harguindeguy et al., 2016). High rates of
 photosynthesis are thought to be a potential mechanism of tolerance, and
 specific leaf area is a frequently used correlate for photosynthetic
 ability (Pérez-Harguindeguy et al. 2016). In August 2014, we photographed
 and subsequently collected the uppermost fully open leaf of three stems
 per pot. The leaves were dried at 50°C for 72 hours before being weighed.
 We measured leaf area using ImageJ software (Schneider et al. 2012), and
 calculated specific leaf area as the ratio of area to dry biomass for a
 leaf (mm<sup>2 </sup>mg<sup>-1</sup>). Using a
 porcelain pestle and mortar, we ground the dry leaf material into fine
 powder for leaf chemical analyses (see below).
 Foliar nitrogen
 and carbon serve important physiological and ecological functions, with
 nitrogen content linked to photosynthetic ability (Evans 1989) and both
 carbon and nitrogen content shown to influence herbivore performance
 (Agrawal 2004, Imaji and Seiwa 2010, Cronin et al. 2015). In particular,
 because nitrogen is often positively correlated with plant RGR and RGR is
 a putative tolerance trait, plants with high foliar nitrogen content are
 expected to better compensate for herbivore damage than plants with low
 nitrogen content (e.g., Vergés et al., 2008; Bagchi and Ritchie, 2011;
 Mundim et al., 2012). Therefore, we assayed dried leaf tissues for percent
 carbon (%C), percent nitrogen (%N), and C:N ratio using an elemental
 analyzer at Brown University Environmental Chemistry Facilities (http://www.brown.edu/Research/Envchem/facilities/).             In addition to foliar nutrients, we measured leaf toughness and total phenolics, two putative resistance traits known to correlate with <i>P. australis</i> herbivory (Cronin et al. 2015, Bhattarai et al. 2017b). Using a penetrometer (Itin Scale Co., Inc., Brooklyn, NY), leaf toughness (force [kg] required to push a blunt steel rod [4.8 mm in diameter] through the leaf) was measured on the uppermost fully open leaf on three randomly selected stems per plant. Total phenolics (nM/g of dried leaf tissue) were estimated using a modified version of the Folin-Ciocalteu method (Waterman and Mole 1994, Cronin et al. 2015) at the University of Rhode Island, Kingston, RI. Although never before examined in <i>P. australis</i> in the context of plant defense, we also analyzed plants for silica content (g kg<sup>-1</sup>) at the Louisiana State University AgCenter Soil Testing and Plant Analysis Lab, using methods adapted from Kraska and Breitenbeck (2010). Silica is a known herbivore defense in grasses (McNaughton et al. 1985, Massey et al. 2006, Reynolds et al. 2012).             Putative resistance traits sometimes fail to predict, or are only weakly correlated with, true resistance measured in terms of herbivore preference or performance on different plants (e.g., Fritz and Simms, 1992; Bhattarai et al., 2017b). The best test of tolerance-resistance tradeoffs is with true measures, not putative correlates of each trait. Although we did not measure true resistance in this study, we used data from Bhattarai et al. (2017b) that quantified <i>P. australis</i> resistance to a generalist leaf-chewing herbivore, <i>Spodoptera frugiperda</i> (fall armyworm; Lepidoptera: Noctuidae) on the same clonal populations used for this study and from within the same common garden. Briefly, in this bioassay, Bhattarai et al. (2017b) reared individual pre-weighed <i>S. frugiperda</i> larva on replicate plants of each source population for eight days. Afterward, larval biomass and leaf area consumed were measured and plant palatability to the herbivore (biomass conversion efficiency) was calculated (proportional change in larval fresh mass per unit area of leaf consumed; all measurements <i>ln</i>-transformed). The inverse of this metric was used as a measure of resistance. Between the two experiments, there were 20 overlapping populations that used identical genetic material for plant propagation. Finally, we note that although resistance and tolerance were measured one year apart, our work suggests that population-level resistance is strongly genetically based and relatively fixed over time (Allen et al. 2017, Bhattarai et al. 2017b). Moreover, clonal integrity was maintained in the garden over the years by preventing sexual reproduction (via removal of all panicles prior to floral maturation) and external contamination of our populations from naturally occurring seed sources was exceedingly unlikely given that the nearest source of <i>P. australis</i> was &gt; 100 km away. Thus, we feel confident that these two data sets allow for a valid assessment of a tolerance-resistance tradeoff among <i>P. australis</i> populations. Also, see the following manuscript for description on the common garden design. Allen, W. J. et al. 2017. Biogeography of a plant invasion: drivers of latitudinal variation in enemy release. - Global Ecology and Biogeography 26: 435–446. Allen, W. J. et al. 2020. Intraspecific and biogeographical variation in foliar fungal communities and pathogen damage of native and invasive Phragmites australis. - Global Ecology and Biogeography in press. Bhattarai, G. P. et al. 2017. Biogeography of a plant invasion: genetic variation and plasticity in latitudinal clines for traits related to herbivory. - Ecol Monogr 87: 57–75.