DOI: 10.5061/dryad.7d7wm37vq
title: Aboveground herbivory causes belowground changes in twelve oak Quercus species: a phylogenetic analysis of root biomass and non‐structural carbohydrate storage

description: Plant ecosystem structure is understood to be a result of complex
 multitrophic interactions. Most multitrophic studies focus on plant
 aboveground adaptations to aboveground herbivore pressures, neglecting
 belowground adaptations in response to aboveground damage. Differential
 investment in root structures may allow plants to compensate for tissue
 loss or damage due to herbivores. Furthermore, phylogeny may constrain a
 plant’s ability to adapt belowground. We examined the belowground
 responses of 12 species of oak (Quercus) to varying locations and
 intensities of simulated herbivore damage. We first established that oak
 belowground traits responded to aboveground herbivory by measuring
 patterns of investment in coarse vs fine root structures and re-allocation
 of non-structural carbohydrates (NSC) to root storage. We then tested
 whether phylogeny could explain variations in investment patterns using
 phylogenetic independent contrasts. Plant adaptations to aboveground
 herbivory included allocating biomass and carbon reserves to root
 structures, depending on the location and intensity of herbivore damage.
 NSC re-allocation to root storage was observed when oak species
 experienced any type of damage, but damage to lateral tissues caused a
 greater re-allocation than apical damage or control treatments. We found
 that most belowground responses to aboveground herbivory are
 species-specific and may be adapted for environmental conditions or type
 of herbivory. Some responses to herbivore damage, such as changes in
 fine-root mass and root sugar concentrations, were phylogenetically
 constrained. Phylogenetic constraints generally occur when there is severe
 damage at the apical meristem. Plants may adapt to aboveground tissue loss
 due to varying herbivore pressures (i.e. varying location and intensity of
 damage) by differentially investing in root types and NSC re-allocation to
 root storage. Understanding linkages between and phylogenetic constraints
 of plant belowground responses to aboveground herbivory will improve our
 understanding of the ecological processes involved in multitrophic
 interactions.
description: <b>Oak phylogeny and
 experimental design</b> We used a
 well-resolved molecular phylogeny containing 146 species in the American
 oak clade (Hipp et al<i>.</i> 2018). Twelve species were
 chosen across the phylogeny to give an adequate representation of
 biogeographical and environmental diversity within the oak genus. We
 followed oak nomenclature described in the Oaks Names Database (Trehane
 2007). We excluded species where interspecific hybridization and
 introgression may occur, such as among the white oaks (<i>Q.
 alba</i> complex) and red oaks (<i>Q. rubra</i> complex)
 (Whittemore and Schaal 1991, Lexer et al<i>.</i> 2006, Hipp
 and Weber 2008, Moran et al<i>.</i> 2012). We sampled the
 following 12 species: <i>Q. alba, Q. coccinea, Q. laurifolia, Q.
 macrocarpa, Q. michauxii, Q. muehlenbergii, Q. nigra, Q. palustris, Q.
 rubra, Q. sinuata, Q. stellata </i>and <i>Q. virginiana
 </i>(Fig. S1)<i>.</i> Much research
 has been done on developing a highly resolved molecular phylogeny of
 American oak clades (Hipp et al<i>.</i> 2014, McVay et
 al<i>.</i> 2017, Hipp et al. 2018). Having a well-resolved
 phylogeny is crucial for comparative studies because of the
 non-independence between species, caused by hierarchical relationships
 (Ackerly and Donoghue 1995, Huey et al<i>.</i> 2019).
 Felsenstein (1985) suggested a method for weighting character values of
 closely related species when performing comparative analyses. Sister
 species may have similar trait values because of common descent and are
 therefore nested in a phylogenetic hierarchy. These relationships violate
 the assumption of independence of data points. Felsenstein’s (1985) method
 uses independent contrasts (calculated as the absolute difference
 (“contrasts”) between the actual values (“tips”) among individual species,
 using the square root of the variance for standardization) (also see
 Garland et al<i>.</i> 1992, Ward et al<i>.</i>
 2020). Independent contrasts create statistical independence of data
 points, determining if traits may be similar because of phylogeny
 (“exaptations” <i>sensu</i> Gould and Vrba 1982) and not
 representations of individual adaptations.    <i>Simulated herbivory
 treatments</i> Oak saplings
 from each of the 12 species were purchased from Mossy Oak Nativ Nursery in
 West Point, MS, United States. To avoid differences in responses due to
 ontogeny, all saplings were the same age (approximately 3-4 years old)
 (Gruntman and Novoplansky 2011). Saplings were planted in May 2017, in the
 same type of soil (all-purpose Pro-Mix<sup>®</sup> potting
 soil) to ensure that there were no nutritional or microbial effects from
 different soils (van der Putten et al<i>.</i> 2001, Pangesti
 et al.<i> </i>2014). Throughout the experiment, pots
 containing the saplings were weeded weekly. The saplings were kept in
 optimal conditions in a greenhouse with a permanent schedule of 12 h of
 light and 12 h of dark at a constant 22 °C. Daily watering was administered
 from an irrigation system to ensure each sapling received the same amount
 of water.<b> </b>We applied five treatments to
 simulate variations in damage location (apical vs. lateral) and intensity
 (25% vs 75% tissue removal) of herbivory. The initial height of each
 sapling was recorded before clipping treatments were applied to account
 for bias related to initial size. Each treatment was replicated five times
 for each of the twelve species for a total of 25 individuals per species,
 with each plant developing in a single pot. To avoid potential block
 effects, pot arrangements were randomly rotated weekly. Many plants
 express apical dominance, i.e. preferential growth of the apex shoot, or
 apical meristem over lateral tissues (Cline 1991; Aarssen 1995, Kebrom
 2017). Apical dominance often results in the overall suppression of
 lateral growth (Aarssen 1995). Similarly, studies have shown that plants
 overcompensate in other tissues due to removal or destruction of the
 apical meristem (e.g. Aarssen and Turkington 1987, Ward 2010). For
 example, damage to the apical meristem may cause an increase in lateral
 tissue growth (Gadd et al. 2001, Ward 2010, Perkovich and Ward 2021). We
 incorporated varying locations (i.e. apical vs. lateral tissue damage) and
 intensities (25% vs. 75% tissue removal at a given location) to analyze
 the effects of herbivory location and intensity on oak traits. The
 treatments were as follows (see also Perkovich and Ward 2021):
 Control: No vegetation removed (5 individuals/ oak
 species) (Fig. 1a). 25% apical removal: Removal of
 the dominant apical meristem and 25% apical shoot (5 individuals/species)
 (Fig. 1b). 75% apical removal: Removal of the
 dominant apical meristem and 75% apical shoot (5 individuals/species)
 (Fig. 1c). 25% lateral removal: Removal of apical
 meristems on lateral shoots and 25 % of lateral tissues (5
 individuals/species) (Fig. 1d). 75% lateral removal: Removal of
 apical meristems on lateral shoots and 75 % of lateral tissues (5
 individuals/species) (Fig. 1e).   <i>Aboveground regrowth,
 root mass and total non-structural carbohydrate
 concentrations</i> Saplings were harvested one year
 after planting and treatment application (May 2018). Aboveground tissues
 were separated from belowground tissues by cutting the trees at the root
 collar. Aboveground tissues were dried at 65 ℃ for 48 h and weighed to
 calculate the total aboveground biomass. We estimated aboveground biomass
 regrowth to determine investment in aboveground regrowth relative to root
 biomass. Measuring aboveground mass regrowth is especially difficult in
 plants as we cannot weigh the plants initially without uprooting and
 damaging root structure. As root structure was instrumental for this
 study, we therefore estimated aboveground mass regrowth as the residuals
 from a regression of final height and biomass (see Schulte-Hostedde et al.
 2005; Peig and Green 2009) for each treatment to account for differences
 in growth responses between treatments. Oak roots were harvested one year
 after planting (May 2018), and treatment application. Dead root material
 was excluded from all analyses. Root samples were dried at 65 ℃ for 48 h.
 Root samples from individual trees were classified as either <i>tap
 root</i>, <i>coarse root, </i>or <i>fine
 root</i>. For this study, we considered the tap root to be the
 largest, central root from which subsidiary root branches developed.
 Coarse roots were classified as roots with a diameter &gt; 2.0 mm,
 excluding the central tap root. Fine roots were roots that were &lt;
 2.0 mm in diameter (Pregitzer et al<i>. </i>2002, McCormack et
 al<i>.</i> 2015). The dry mass of each root classification was
 measured for each individual sapling. <i>Total root mass
 </i>was calculated as the sum of all three root classes from a
 single individual. After roots were dried and weighed,
 we took samples from coarse roots of each individual sapling and milled
 the dried material using a using a 2 mm mesh in a Wiley mill for analysis
 of non-structural carbohydrate (NSC) concentrations. For standardization,
 all NSC concentrations were measured using coarse-root material (McCormack
 et al. 2015). We used the NSC extraction protocol by Fournier (2001). In
 this protocol, sugars are first extracted using an ethanol wash. After
 several washes to remove sugars, the tissues are placed in 1% hydrochloric
 acid and set in a hot water bath at 100℃ for 1 h. After sugars and
 starches were extracted from the tissues, a phenol-sulfuric acid solvent
 was used to create a colorimetric reaction and measured using a microplate
 reader (Tomlinson et al<i>.</i> 2014).  All analyses were done
 in our laboratory to ensure the same conditions (Quentin et
 al<i>.</i> 2015; Landhäusser et al. 2018).