fine root morphology is phylogenetically structured, but nitrogen is

Fine root morphology is phylogenetically structured,but nitrogen is related to the plant economics spectrumin temperate treesOscar J. Valverde-Barrantes*,1, Kurt A. Smemo1,2 and Christopher B. Blackwood1

1Department of Biological Sciences, Kent State University, Kent, OH 44242, USA; and 2The Holden Arboretum, 9500Sperry Rd, Kirtland, OH 44094, USA

Summary

1. Plant functional traits have revealed trade-offs related to life-history adaptations, geographi-

cal distributions, and ecosystem processes. Fine roots are essential in plant resource acquisition

and play an important role in soil carbon cycling. Nonetheless, root trait variation is still

poorly quantified and rarely related to the rest of the plant.

2. We examined chemical and morphological traits of 34 temperate arbuscular mycorrhizal

tree species, representing three main angiosperm clades (super-orders asterid, magnoliid and

rosid). We tested to what extent fine root chemical and morphological traits were correlated

similarly to the leaf economical spectrum (LES) or were structured by ancestral affiliations

among species.

3. Root traits did not display the same trade-offs as leaves (e.g. specific root length was not cor-

related with root N, whereas specific leaf area was correlated with leaf N). Moreover, 75% of

below-ground traits were phylogenetically structured according to Pagel’s k and Abouheif’s

Cmean autocorrelation tests, as opposed to 28% of above-ground traits. Magnoliids showed

thicker, less branched roots than asterids or rosids, but rosid roots exhibited lower N and higher

non-acid-hydrolysable (e.g. lignin) content than other species. In contrast, leaf traits did not dif-

fer significantly among super-orders. At the whole-tree level, chemical traits such as nitrogen tis-

sue content and lignin content were correlated between above and below-ground organs.

4. The distribution of root traits in woody temperate trees was better explained by shared

ancestry than by the nutrient content and structural trade-offs expected by the LES hypothesis.

Root chemistry and morphology differed substantially among species belonging to different

super-orders, suggesting deep divergences in resource acquisition strategies among major

angiosperm groups. Although we found partial support for the idea of whole-plant integration

based on corresponding nitrogen content across all organs (i.e. a plant economics spectrum),

our study stresses phylogenetic affiliation as the primary driver of root trait distributions

among angiosperms, a pattern that could be easily overlooked based solely on above-ground

observations.

Key-words: angiosperm evolution, arbuscular mycorrhizal trees, fine root traits, phylogenetic

trait conservatism, plant economics spectrum, root nitrogen content, specific leaf area, specific

root length

Introduction

Plant functional traits can be defined as morphological,

physiological or phenological attributes that influence the

fitness of individuals in their ecosystems (Reich et al. 2003;

Violle et al. 2007). Comparisons of functional traits have

revealed important axes of variation across species that

reflect fundamental biophysical trade-offs (Reich et al.

2003; Westoby & Wright 2006). The best known example

is the ‘leaf economic spectrum’ (LES), which predicts a

tight correlation between physiological, chemical and mor-

phological traits in leaves, ranging from leaves with high

photosynthetic rates, specific leaf area (SLA) and nitrogen

(N) but short life span, to leaves with lower metabolic*Correspondence author. E-mails: [email protected]; os.valver

[email protected]

© 2014 The Authors. Functional Ecology © 2014 British Ecological Society

Functional Ecology 2015, 29, 796–807 doi: 10.1111/1365-2435.12384

activity and N content but higher tissue protection and

longer life span (Reich et al. 1998; Wright et al. 2005). Life

history and adaptations to environmental conditions are

often considered the main drivers of the LES (Ackerley &

Cornwell 2007; Engelbrecht et al. 2007; Kraft, Valencia &

Ackerly 2008). Phylogenetic relationships, in contrast, are

rarely mentioned as a factor explaining LES syndromes,

particularly among broadleaf species. Milla & Reich

(2011), for instance, found little effect of phylogenetic

structure on the distribution of leaf traits among 57 pairs

of vicariant species growing at different altitudes in a tem-

perate broadleaf forest. Instead, leaf trait syndromes clo-

sely followed thermal regimes, indicating that ecological

filtering was far more important than shared ancestry in

explaining leaf trait differences of related species (Ackerly

& Reich 1999; Reich et al. 2003; Whitman & Aarssen

2010; but see Peppe et al. 2011).

Fine roots (the 2–3 most distal links in a root system,

Hishi 2007) exhibit wide interspecific variation in morpho-

logical and chemical traits in temperate woody plants

(Guo et al. 2008; Comas & Eissenstat 2009; Holdaway

et al. 2011). It has been suggested that such trait diversity

might be driven by trade-offs associated with stress toler-

ance, nutrient uptake rates and life-history strategies

(Grime et al. 1997; Comas & Eissenstat 2004; McCormack

et al. 2012). Because leaves and fine roots are both short-

lived organs specialized for resource capture, it is often

assumed that root trait variation is driven by compromises

between carbon investment and resource capture, similar

to those expected from the LES hypothesis. For example,

fine roots must be highly metabolically active to intercept

and take up water and nutrients from soil, requiring

investment in N to maintain transport systems, enzyme

activity and mycorrhizal symbioses (Reich et al. 2008).

Thus, correlation between N content and root respiration

rate may reflect a relationship between root N and meta-

bolic activity similar to that described for foliar organs

(Reich, Walters & Ellsworth 1997). Among woody temper-

ate trees, species classified as fast growers have shown

higher specific root length (SRL) and smaller diameter,

higher root N concentration, faster P uptake and higher

respiration rates than congeneric species with slower

growth rates (Reich et al. 1998; Comas, Bouma & Eissen-

stat 2002; Comas & Eissenstat 2004). Thus, it is possible

that fine root functional traits reflect similar trade-offs to

those predicted by the LES hypothesis (Reich et al. 2003;

Craine 2005).

An extension of the LES hypothesis states that the

adoption of an ecological strategy reflected by leaf traits

should also structure trait variation for all other organs

to maximize competitive ability (Grime et al. 1997; Reich

2014). This hypothesized integration of functional traits

across plant organs has gained recent support. Freschet

et al. (2010), for example, showed strong integration

among tissue types (leaf, stem and root) across 40 species

of subarctic flora, including 16 woody species. Integration

among leaf and root functional traits has also been

found in several grass or herb-dominated communities

(Craine et al. 2002; Birouste et al. 2011; Kembel & Cahill

2011; but see Tjoelker et al. 2005). However, studies

focused on woody plants seem to show lower integration

between leaf and root traits than non-woody plants. For

instance, Fortunel, Fine & Baraloto (2012) reported poor

correlation between foliar and coarse root traits in tropi-

cal trees. Hobbie et al. (2010) also reported no corre-

spondence between leaf and root chemical traits for 11

European tree species. Similarly, Withington et al. (2006),

Espeleta, West & Donovan (2009) and McCormack et al.

(2012) found poor correspondence between morphologi-

cal root traits, root life span and leaf life span among

temperate tree species grown in common gardens, con-

tradicting the idea of strong below- and above-ground

trait integration.

One potential explanation for the lack of clear inte-

gration of traits among roots and leaves in woody

plants is suggested by previous studies, which show a

stronger trait phylogenetic conservatism in roots (Baylis

1975; St. John 1980; Comas & Eissenstat 2009; Chen

et al. 2013) than is typically found in foliar tissues

(Wright et al. 2004). It has been hypothesized that this

trait segregation among plant lineages arose from the

unique association between roots and mycorrhizal fungi

(Brundrett 2002). Symbiotic associations with arbuscular

mycorrhizal (AM) fungi represent the ancestral state and

the most common association in terrestrial plants, cur-

rently present in >75% of angiosperms (Brundrett 2009).

AM fungi depend completely on root cortical tissue for

carbon and energy acquisition. Thus, Baylis (1975) pro-

posed that thick, non-branched, slow-growing roots were

the ancestral root type in basal angiosperms because

they are optimal for AM fungal colonization, which was

essential for the success of ancestral angiosperm groups

with little ability to obtain nutrient on their own. Subse-

quent adaptation of more recent angiosperm groups to

drier and colder conditions may have selected finer,

more branched root systems, thereby facilitating soil

exploration and nutrient absorption with less symbiotic

aid (St. John 1980; Brundrett 2002; Comas et al. 2012).

Although Baylis’ hypothesis is frequently mentioned to

explain root trait variation among angiosperm species

(Pregitzer et al. 2002; Comas & Eissenstat 2009; Hold-

away et al. 2011), few studies have addressed the ques-

tion using comparative analyses (Chen et al. 2013; Kong

et al. 2014), and little is known about how phylogenetic

conservatism in root traits may correspond with their

foliar counterparts.

In this study, we examined above- and below-ground

traits of temperate tree species representing three major

phylogenetic groups of woody angiosperms. We sampled

trees forming AM symbioses, allowing us to control for

confounding morphological and chemical modifications

associated with the switch to alternative mycorrhizal

groups. Our objectives focused on two questions: (i) Are

fine root traits more phylogenetically conserved than

© 2014 The Authors. Functional Ecology © 2014 British Ecological Society, Functional Ecology, 29, 796–807

Phylogenetic structure of woody root traits 797

corresponding above-ground traits? (ii) Even if traits are

phylogenetically conserved, does the integration of fine

root traits follow similar trade-offs as those expected based

on the LES hypothesis? For the first question, we hypothe-

sized that root traits would be more similar among closely

related species (Hypothesis 1.1). We expected that basal

angiosperms (i.e. the magnoliid clade) would be divergent

chemically and morphologically from more recently

derived groups (asterid and rosid clades). Moreover, we

hypothesized that phylogeny would be a more important

factor explaining below-ground than above-ground trait

variation (Hypothesis 1.2). For the second question, we

compared two possible but not mutually exclusive hypoth-

eses explaining the coordination between root morphologi-

cal and chemical functional traits of woody angiosperms.

If fine root functional traits follow trade-offs similar to

those observed in foliar tissues (Hypothesis 2.1, LES

hypothesis), we would expect significant correlations

between root morphological and chemical traits. Finally, if

resource capture strategies are integrated at the whole-

plant level (Hypothesis 2.2, Freschet et al. 2010), species

should show a full integration of above- and below-ground

trait syndromes commonly associated with life-history

strategy (Reich et al. 1998, 2003). Therefore, root chemical

and morphological traits should be correlated with above-

ground traits across species.

Materials and methods

STUDY SITE AND SAMPLE COLLECT ION

We sampled a set of 34 woody angiosperm species, plus two gym-

nosperms used as an out-group, replicated in two separate living

tree collections (Table S1, Supporting information, Fig. 1): The

Holden Arboretum, Ohio (40°570N and 82°280W) and Boone

County Arboretum, Kentucky (38°570N and 84°430W). The Hol-

den Arboretum (c. 300 m elevation) receives an average of 116 cm

Fig. 1. Phylogenetic relationship and trait value distribution for 36 woody species grown in common gardens in the Midwestern USA.

The traits displayed are (A) specific root length (SRL) for first-order roots; (B) ratio of non-acid-hydrolysable to nitrogen contents (NAH:

N) ratio for first-order roots; (C) above-ground branch tissue density and (D) specific leaf area (SLA). Symbol size indicates relative trait

values for each species, with smaller symbols closer to the mean value; black symbols represent values above the mean and white symbols

are below the mean. Branch lengths were standardized to have the same length; open circles represent ancestral nodes; filled circles sam-

pled species. The gymnosperms Ginkgo biloba and Thuja occidentalis were used as out-group.


798 O. J. Valverde-Barrantes et al.

precipitation per year, with an average annual temperature of

8 °C. The soils at Holden were formerly under mixed mesophytic

and beech-maple forest on gently sloped (2–6% slope) fine-silty,

mixed, active, mesic Aeric Fragiaqualfs from the Platea series.

Boone County Arboretum (c. 300 m elevation) receives 105–112 cm precipitation annually, with an average temperature of

11 °C. The site is located mostly on gently sloped fine-silty Aquic

Fragiudalfs in the Rossmoyne series, which, prior to clearing and

conversion to agriculture, was covered with deciduous hardwood

forest species typical of the oak-hickory region.

Our sampling design aimed to have a comparative representa-

tion of three main angiosperm lineages (i.e. magnoliids, asterids

and rosids) for temperate trees species forming only AM associa-

tions (Brundrett, Murase & Kendrick 1990; Wang & Qiu 2006).

Life-history description, phylogenetic relationships and details of

tree assembling are detailed in the Table S1. Above- and below-

ground samples were collected during mid-summer (July–August)

of 2010 and 2011. Samples from each species were taken from four

healthy adult trees (two individuals from each site) relatively iso-

lated from other woody plants. Root samples were obtained by

extracting two large soil cores (10 cm diameter 9 15 cm deep

PVC pipes) within 2 m of the main stem of each individual tree,

avoiding large (>5 cm diameter) roots. Samples were placed in air-

tight bags and stored at 4 °C. Additionally, roots exposed and left

in the soil were traced to the main stem and a small segment

(c. 10 cm long) that included 3–4 most distal root orders was

extracted, preserved in 45/5/50 water–acetic acid–formalin mixture

and used as a reference. In the laboratory, roots were separated

from soil by soaking in water for 12 h, gently washing with deion-

ized water and then comparing washed roots to the reference sam-

ple before further analysis.

Above-ground tissue was collected from the same individuals

that roots were taken from. The most distal branch sections that

included fully expanded sunlit leaves and first-year shoots were

cut at the junction with the basal branch. Leaves and twigs (long

shoots of first-year growth tissue with leaves still attached to the

stem) were detached from older branches immediately after collec-

tion. Leaves, twigs and branches were sealed individually in air-

tight polyethylene bags and maintained at 4 °C until fresh weight

and area could be measured (usually within 2 days; Wilson,

Thompson & Hodgson 1999). Leaf vouchers of each species were

collected and deposited in the Kent State University Herbarium

(accession numbers KS65950–KS65980).

MORPHOLOGICAL , ARCHITECTURAL AND CHEMICAL

TRA ITS

For root morphological analyses, we used five randomly selected

5- to 10-cm-long root systems comprised of the three most distal

root orders, which has been considered the portion of root sys-

tems analogous to leaf tissues (Xia, Guo & Pregitzer 2010). Root

systems were labelled according to Strahler’s stream ordering sys-

tem with the most distal roots labelled as the first order (Fitter

et al. 1991). Root image analysis of entire root systems was per-

formed using WINRHIZO software (Instrument Regent, Qu�ebec

City, QC, Canada) following Valverde-Barrantes et al. (2013) to

quantify specific root tip abundance (tips g�1 DRM), fractal

dimension and average link length (cm). Additional root systems

from each tree were dissected into three orders until c. 2 g of fresh

weight tissue was obtained from each root order. Roots from each

order (kept hydrated during dissection) were scanned, dried for

24–48 h at 65 °C and weighed. Image analysis and mass values

from each root order were combined to estimate average diameter,

SRL (m g�1 dry root mass, DRM), specific root surface area

(SRA, cm2 g�1 DRM) and root tissue density (RTD,

g DRM cm�3) for each root order from each sampled tree. For

chemical analysis, a subsample of each root set that was separated

by order was ground using a GenoGrinder (Spex SamplePrep,

Metuchen, NJ, USA) at 500 rpm for 1 min. Samples with exces-

sive fibrous material were frozen with liquid nitrogen and

ground manually with a mortar and pestle. All samples were

oven-dried again at 65 °C for 24–48 h. Proximate tissue compo-

sition was analysed in duplicates with a series of extractions as

described by Ryan, Melillo & Ricca (1990). Briefly, polar and

non-polar extractives were removed by consecutive extractions

with methanol and dichloromethane, respectively. The residue

from the polar/non-polar extractions was then separated into

acid-hydrolysable (AH; representing crystalline carbon polymers

like cellulose) and non-acid-hydrolysable (NAH; representing lig-

nin-like amorphous polymers) compounds using a two-stage

sequence of sulphuric acid digestions (Cusack et al. 2009).

Finally, all fractions were corrected for ash content after inciner-

ation of the NAH fraction at 500 °C for 4 h. Total %C and %

N were determined on duplicate 0�2–0�5 mg dried tissue samples

using an elemental analyser (Costech Analytical Model 4010,

Valencia, CA, USA).

For leaf area estimates, a minimum of five fully expanded leaves

including petioles were scanned on a flatbed scanner (Cornelissen

et al. 2003). Leaf dry matter content and SLA (cm2 g�1) were esti-

mated following Wilson, Thompson & Hodgson (1999). Leaf thick-

ness was estimated in situ measuring 10 freshly collected leaves

from each individual sampled at The Holden Arboretum. Each leaf

was measured at three points (tip, medium and base points, in the

middle of three randomly chosen leaflets in the case of compound

leaves) with an electronic thickness gauge (Eagle Technology,

Mequon, WI, USA, precision 0�01 mm) avoiding major veins or

irregular areas. Twigs and branches (including bark) were cut into

3–5 segments 2–5 cm long and scanned together, weighed, dried at

80 °C for 3 days and reweighed (Swenson and Enquist 2008). Den-

sity values for twigs and branches were calculated using volume

estimates from WinRhizo software. Additionally, we determined

fresh area per g for twigs (specific twig area, STwigA) and branches

(specific branch area, SBranchA) to have a common measured vari-

able for all tissues (analogous to SRA for roots and SLA for

leaves). Chemical analysis of above-ground tissues followed the

same procedure applied to root material.

DATA ANALYS IS

Testing for trait divergence among phylogenetic groups(Hypothesis 1.1)

We focused our analysis on differences between phylogenetic

groups at the super-order level. Firstly, we performed a linear

mixed-model ANOVA for each measured trait at the individual

level, using the REML criterion for model optimization (Bates,

Meechler & Bolker 2013). Fixed factors included super-order

(magnoliid, asterid, and rosid), site (Holden or Boone County

Arboretum), and tissue position (root order for below-ground

traits, and leaf, twig or branch for above-ground traits). Species

nested in clades was designated a random factor to account for

the non-independence of individuals of the same species. A post

hoc Tukey HSD test was performed to determine significant dif-

ferences among super-order groups. We also tested for differences

among super-orders in a multivariate context. Briefly, we created

chemical and morphological matrices for each root order and

above-ground tissue type. Then we performed a phylogenetic

principal component analysis (pPCA, Revell 2009) to find major

axes of trait variation in each tissue type. Finally, we tested for

differences among super-orders at the multivariate level by per-

forming a redundancy analysis on the first two pPCA axis of

each matrix (RDA, ter Braak 1986), using as factor matrix a cat-

egorical vector describing species at the super-order level (Jom-

bart et al. 2010).



Comparing phylogenetic conservatism between above-and below-ground traits (Hypothesis 1.2)

We tested the hypothesis of higher phylogenetic conservatism in

root traits compared with other organs by performing Abouheif’s

Cmean autocorrelation and Pagel’s k tests. Abouheif’s Cmean

analyses the autocorrelation of traits due to species topological

positions in the tree without including branch length in the calcu-

lations (Abouheif 1999; Thuiller et al. 2011). Pagel’s k coefficient

reflects the phylogenetic dependence of observed trait data with

respect to a pure Brownian model of evolution (Pagel 1999).

Both indices are therefore the most appropriate for phylogenetic

trees with standardized branch lengths (M€unkem€uller et al.

2012). Significance was estimated by comparing observed phylo-

genetic signal values to 999 permutations shuffling values at the

tips of the tree. Values significantly higher than zero indicate that

a trait is more similar among closely related taxa than expected

at random (phylogenetic signal sensu Jombart & Dray 2010).

To test whether below-ground trait variation is more often

structured by phylogeny than above-ground traits, we calculated

the difference between the number of traits showing significant A-

bouheif’s Cmean or Pagel’s k values and tested the significance

using permutation analysis assuming that equal number of traits

significantly structured by phylogeny but randomly distributed

among organs.

Correlations between morphology and chemistry withinplant tissues (Hypothesis 2.1)

We tested the LES hypothesis by calculating Pearson’s correla-

tions between species mean SRL and root C : N, and between

SLA and leaf C : N. Correlations were also calculated using phy-

logenetically independent contrasts to account for phylogenetic

non-independence of species (Felsenstein 1985). We also used a

coinertia analysis to test for trait correlations in an explicitly mul-

tivariate context. Coinertia analysis was performed on the infor-

mative morphological and chemical pPCA axes selected for

Hypothesis 1.1. This procedure finds a new set of ordination axes

that maximizes the coinertia, or sum of squared covariances,

between two data matrices, without being constrained by colinear-

ity or numbers of variables in the data matrices (Dray, Chessel &

Thioulouse 2003). We tested whether the observed coinertia value

was higher than expected by chance by performing a Monte Carlo

simulation test using 999 random permutations of the rows in

each matrix (Dol�edec & Chessel 1994).

Axes of variation integrating above- and below-groundtraits (Hypothesis 2.2)

To test for integrated variation in leaf and root traits, we first cal-

culated Pearson’s correlations, with and without phylogenetically

independent contrasts, between SRL and SLA and between root

and leaf C : N. Finally, we determined the coinertia between

above- and below-ground traits. pPCA axes included in the analy-

sis were projected in a multivariate ordination to look for domi-

nant axes of variation. To test the robustness of the correlation

among trait axes, we repeated the coinertia analysis comparing

above- and below-ground matrices before and after accounting for

phylogenetic effects using PIC scores as corrected values of all

pPCA axes (Baraloto et al. 2010).

All statistical analyses were performed in the R 2.12 statistical

platform (R Development Core Team, 2012) using the packages

lme4 (Bates, Meechler & Bolker 2013), picante (Kembel et al.

2010), ape (Paradis, Claude & Strimmer 2004), phytools (Revell

2012), adephylo (Jombart & Dray 2010), ade4 (Dray & Dufour

2004) and vegan (Oksanen et al. 2008). Trait variables were

log-transformed when necessary before statistical analysis to

account for heteroscedasticity and non-normal distribution of

residual error.

Results

TEST ING FOR TRA IT D IVERGENCE AMONG

PHYLOGENET IC GROUPS (HYPOTHESIS 1 .1 )

Similar above- and below-ground traits (e.g. per cent N)

showed similar ranges of variation among species

(Table S2). For the univariate linear mixed-model ANO-

VAs, the factors that significantly affected traits varied

among tissues (Table S3). Differences across tissues (root

orders or twigs vs. leaves) were the most important fac-

tor for most measured traits. Super-order was a signifi-

cant factor explaining most below-ground but not above-

ground trait variation, even after controlling for site and

root order, indicating a strong phylogenetic effect on

below-ground trait variation (Fig. 1, Hypothesis 1.1). Site

differences seemed to influence root chemical traits such

as polar and non-polar compounds and nitrogen content,

with Boone samples showing higher root N and polar

compound concentration but lower non-polar content

than Holden samples (Table S3). Above-ground, polar,

non-polar and AH content was also affected by site. In

a few cases, we detected an interaction between site and

super-order (Table S3). However, the interaction did not

alter the overall trait patterns observed among super-

orders.

Similarities among phylogenetic groups varied depend-

ing on the traits compared, as shown by the RDA ordina-

tion plots (Fig. 2). As expected from Baylis’ observations,

the morphology of magnoliid roots was significantly differ-

ent from asterid or rosid species (RDA analysis P < 0�005for first-order roots, Fig. 2a). Magnoliid roots were thicker

and less branched when compared with most root systems

of more derived angiosperm taxa. However, this observa-

tion did not hold for chemical traits. In this case, magno-

liid and asterid roots were on average different than rosid

roots (RDA analysis P < 0�005 for first-order roots,

Fig. 2c). Rosid species exhibited consistently lower N con-

tent and higher NAH : N ratio than other angiosperms

(Fig. 1, Table S3). A similar test for foliar traits showed

no significant differences among super-orders for either

morphological or chemical traits (P = 0�11 and 0�09,respectively, Fig. 2b,d).

COMPARING PHYLOGENET IC CONSERVAT ISM

BETWEEN ABOVE- AND BELOW-GROUND TRA ITS

(HYPOTHES IS 1 .2 )

The importance of phylogeny explaining trait variation

between above- and below-ground structures was con-

firmed by the phylogenetic signal analysis (Hypothesis

1.2). Both Abouheif’s and Pagel’s phylogenetic signal

tests were consistent with the conserved nature of root



traits across phylogenetic super-orders (72% and 56% of

the root traits studied showed significant phylogenetic

signal according to Abouheif’s Cmean and Pagel’s k,respectively, Table S4). The proportion of above-ground

traits with significant phylogenetic signal was lower

(28% and 33% of traits were significant according to

Abouheif’s Cmean and Pagel’s k, respectively). This lower

frequency of phylogenetic structure was significantly

different according to a permutation test (P = 0�0001 for

both Abouheif’s Cmean and Pagel’s k tests, Tables 1

and S4).

CORRELAT ION BETWEEN CHEMICAL AND

MORPHOLOGICAL TRA ITS WITH IN T ISSUES

(HYPOTHES IS 2 .1 )

The hypothesis that roots display a correlation between

chemical and morphological traits similar to that expected

from the LES hypothesis was not supported (Hypothesis

2.1). Increases in SRL, for instance, were not related to

C : N values (r = 0�24, P = 0�15, Fig. 3a), even though we

found the expected negative relationship in leaves for the

same species (r = �0�74, P < 0�005, Fig. 3b). At the multi-

variate level, we found a weak correlation between root

chemical and morphological pPCA trait axes (coinertia r

ranging from 0�17 to 0�34, Tables 2 and S5). Moreover,

coinertia values decreased after phylogenetic correction

(36% on average, Table 2), suggesting that part of the cor-

relation was explained by coupled root morphological and

chemical trait evolution. Similarly, twigs and branches

showed low coinertia between morphology and chemical

pPCA trait axes, which also decreased after phylogenetic

correction (Table 2). In contrast, leaf trait matrices showed

higher coinertia values (r = 0�40, P = 0�001) with no effects

of phylogeny on the relationship (Table 2).

CORRELAT IONS BETWEEN MORPHOLOGY AND

CHEMISTRY WITH IN PLANT T ISSUES (HYPOTHESIS 2 . 1 )

Specific root length was not related to SLA (r = �0�01,P = 0�97, Fig. 4a). However, the C : N ratio between

leaves and roots was closely related (r = 0�67, P < 0�001,

Table 1. Comparison of the level of phylogenetic structuring of 39

below-ground and 36 above-ground tissue traits measured in 34

species of angiosperms. Percentage of traits showing significant

phylogenetic signal (Abouheif’s Cmean index) in leaves, twigs and

branches and three orders of fine roots (N = 12 traits per tissue),

as well as the entire root system (all below-ground estimations

additionally included specific root tip abundance, fractal dimen-

sion and link length). Individual trait analyses shown in Tables S2

and S3

Trait Abouhief Cmean Pagel k

Leaf 25�00 33�33Twig 23�08 33�33Branch 36�36 33�33All above-ground 27�78 33�33First-order root 75�00 58�33Second-order root 66�67 50�00Third-order root 63�64 50�00All below-ground 71�79 56�41

Asterid

Magnoliid

Rosid Asterid

Magnoliid

Rosid

pPCA1 (98·4%)

Super-order F = 13·3, P < 0·001

(a)

Asterid Magnoliid Rosid


pPCA1 (77·09%)

(b)



Super-order F = 12·2, P < 0·001

pPCA1 (44·28%)

pPC

A2

(22·

16%

)

(d)

Super-order F = 1·7, P = 0·09

Asterid Magnoliid

Rosid

Asterid Magnoliid

Rosid

pPCA1 (82·9%)

pPC

A2

(16·

2%)

Super-order F = 2·3, P = 0·11

(c)

LeavesFirst-order roots

pPC

A2

(1·3

%)

Mor

phol

ogy

pPC

A2

(9·8

4%)

Che

mis

try

Fig 2. Ordination of first-order fine root

and leaf morphological and chemical func-

tional traits. For all panels, axis values rep-

resent the first two phylogenetic principal

component analysis (pPCA) axes, and the

amount of variation explained by each axis

(horizontal axis representing the pPCA axis

that explained the largest amount of varia-

tion). Labels represent the centroid values

for each super-order (magnoliids, asterids

and rosids). P-values were obtained from

redundancy analysis (RDA) testing differ-

ences in traits among super-orders. pPCA

loadings for traits are shown in Table S4.



Fig. 4b). Displaying all informative pPCA axes in multi-

variate space revealed two orthogonal axes between mor-

phological and chemical functional traits (Hypothesis 2.2,

Fig. 5). The first axis of variation seemed highly associated

with root and leaf N content, with leaf morphology (SLA)

as the only morphological trait aligned with the N axis. In

contrast, other morphological traits aligned across a sec-

ond axis, depicting a weak coordination between root and

stem morphological traits, but still largely independent

from the N axis. Moreover, trait correlations were influ-

enced by relatedness among species. Coinertia values

between above- and below-ground traits decreased from

r = 0�38 (P = 0�001) to 0�14 (P = 0�04) after the incorpora-

tion of phylogenetic contrasts to the pPCA axes, suggest-

ing that about c. 20% of the covariation in chemical and

morphological traits can be explained by the co-evolution

between above- and below-ground resource acquisition

organs (Fig. 5).

C : Nroot1

10 20 30 40

SRL r

oot1

(m g

–1)

0

20

40

60

80

100

120

r = –0·24, P = 0·15PIC r = –0·06, P = 0·7

(a)

C : Nleaf

10 20 30 40

SLA

(cm

2 g–1

)

0

200

400

600

800

1000

AsteridsRosidsMagnoliidsGymnosperms

(b) r = –0·74, P < 0·0001PIC r = –0·81, P < 0·0001

Fig. 3. Correlation between morphological and chemical traits for

leaves and first-order roots. (a) Specific root length (SRL) and

C : N in first-order roots; and (b) specific leaf area (SLA) and

C : N in leaves. Coefficients correspond with uncorrected Pear-

son’s correlation (r) and phylogenetic independent contrast corre-

lation values (PIC r).

Table 2. Coinertia coefficients comparing the coordination

between morphological and chemical traits within different organs

of 34 woody angiosperm species grown in common gardens in the

Midwestern USA. Coinertia coefficients represent the sum of

squared variances between matrices representing morphological

and chemical traits for each organ before (Uncorrected) and after

phylogenetic correction (Phylo-corrected)

Uncorrected Phylo-corrected

Coinertia P-value Coinertia P-value

Leaf 0�40 0�001 0�40 0�001Twig 0�18 0�01 0�05 0�20Branch 0�04 0�63 0�02 0�53All above-ground 0�29 0�005 0�24 0�003First-order root 0�17 0�03 0�04 0�67Second-order root 0�32 0�001 0�23 0�03Third-order root 0�34 0�001 0�26 0�001All below-ground 0�22 0�009 0�14 0�04

C : Nleaf

10 20 30 40

C :

Nro

ot1

10

20

30

40

AsteridsRosidsMagnoliidsGymnosperms

(b) r = 0·67, P < 0·0001PIC r = 0·56, P < 0·0001

SLA (cm2 g–1)

200 400 600 800 1000

SR

L roo

t1 (m

g–1

)

0

20

40

60

80

100

120(a) r = –0·01, P = 0·97

PIC r = 0·02, P = 0·9

Fig. 4. Comparison of leaves and first-order roots. (a) Morpho-

logical traits specific root length (SRL) and specific leaf area

(SLA); (b) C : N ratio for leaves and first-order roots. Correlation

coefficients correspond with uncorrected Pearson’s correlation (r)

and phylogenetic independent contrast correlation values (PIC r).



Discussion

EVOLUT IONARY FORCES EXPLA IN ING ROOT TRA IT

SYNDROMES

Our results support Baylis’ original hypothesis of a consis-

tent difference between root morphologies of basal and

more derived angiosperms (Baylis 1975), which is also con-

sistent with data reported elsewhere (Holdaway et al.

2011; Chen et al. 2013; Gu et al. 2014; Kong et al. 2014).

However, to our knowledge, this is the first time that, in

addition to root morphology, root chemical make-up (par-

ticularly root NAH : N ratio) has been reported to be

structured by phylogenetic history within angiosperm lin-

eages. Moreover, the significant interaction between phylo-

genetic clade and root orders suggests that the degree of

trait differentiation from distal to basal roots is also phylo-

genetically structured. Therefore, the evolutionary process

shaping functional traits in tree roots affects not only

individual root links but also the integration of root orders

at the entire root system level.

To explain hypothesized phylogenetic patterns in root

morphologies, Baylis (1975) suggested mycorrhizal depen-

dency as an important factor promoting morphological

conservatism among angiosperm root systems. This view is

consistent with theoretical evolutionary models indicating

high stability and phylogenetic conservatism in mycorrhi-

zal associations due to the complex genetic signalling

between microbial symbionts and plant roots (Kiers & van

der Heijden 2006; Markmann & Parniske 2008; Davison

et al. 2011; Kiers et al. 2011). Recent empirical studies

have also related root morphology, particularly root diam-

eter and cortex area, to the level of mycorrhizal coloniza-

tion in woody plants (Guo et al. 2008; Gu et al. 2014;

Kong et al. 2014). However, it is important to highlight

that this trend between root diameter and mycorrhizal

dependency could be limited to AM associations. Ectomy-

corrhizal associations, for instance, do not depend as heav-

ily on cortical cells for habitat because ectomycorrhizal

species create their own mantle around the root tip, thus

being less influenced by interspecific variation in root

diameter for colonization and development (Kong et al.

2014).

Another recent hypothesis is that root trait diversity in

angiosperms appeared as an adaptation to the emergence

of colder and drier climate during the Late Cretaceous

(Fletcher et al. 2008; Comas et al. 2012). According to this

hypothesis, more modern angiosperm lineages, as well as

several gymnosperms, increased lignification and reduced

xylem vessel size as they expanded into more xeric and

colder areas, becoming dominant in areas constrained by

water availability (Pittermann et al. 2012). It is thus possi-

ble that concomitant increases in SRL and tissue lignifica-

tion and decreases in diameter were a consequence of

selection for more efficient root systems in drier and colder

environments than the wet tropical sites where angio-

sperms possibly originated (Feild & Arens 2007; Feild,

Chatelet & Brodribb 2009). This view also suggests that

Coinertia axis 1 (30·9%)–1·5 –1·0 –0·5 0·0 0·5 1·0 1·5

Coi

nert

ia a

xis 2

(6·2

%)

–1·0

–0·8

–0·6

–0·4

–0·2

0·0

0·2

0·4

0·6

SLA

Twig D

STwigA

Branch D

SBranchALeaf N

Twig N

Branch N

Branch AH

SRL1

SRL2

SRL3

RTD3Root N1

Root N2Root N3

Above-ground chemical traitsBelow-ground chemical traitsAbove-ground morphological traitsBelow-ground morphological traits

Coinertia = 0·38, P < 0·0001

Phylo-coinertia = 0·14, P = 0·04

Fig. 5. Coinertia ordination of above-ground and below-ground chemical and morphological functional traits of leaves, twigs, branches

and three orders of roots. Traits shown are informative pPCA axes, and names represent the individual trait with the highest loading value

for that pPCA axis (see Table S4). Coinertia and phylo-coinertia r values represent the level of coinertia between above- and below-

ground matrices before and after performing phylogenetic contrast on pPCA axis. Both coinertia tests were significant (P = 0�0001 and

0�04 for coinertia and phylo-coinertia, respectively). For the morphological traits, SRL = specific root length for root orders 1, 2 and 3;

RTD3 = root tissue density for third-order roots; SLA = specific leaf area; SBranchA = specific branch area; STwigA = specific twig area;

D_Branch = branch diameter; and D_Twig = twig diameter. For chemical traits, N represents tissue nitrogen for each tissue;

Branch_AH = branch acid-hydrolysable content.



increases in root surface area and tissue lignification were

important adaptations to cope with low N availability,

slow decomposition rates and rough climatic conditions

common to temperate forests (Ostonen et al. 2007).

Highly branched and finer structure of roots is typically

associated with ectomycorrhizal or ericoid mycorrhizal

roots (Brundrett 2002), which developed within the rosids

and asterids, respectively. However, our data demonstrate

the presence of these root trait syndromes in AM plants,

the ancestral mycorrhizal relationship, within these lin-

eages. This supports the view that these root trait syn-

dromes were acquired by AM plants before the switch to

new fungal partners. Furthermore, fossil records indicate

that basal rosid families (i.e. Altingiaceae, Cercidiphylla-

ceae and Hamamelidaceae) have been historically distrib-

uted in temperate or high-altitude tropical forests (Wolfe

1997; Zhou, Crepet & Nixon 2001; Qi et al. 2012). Because

these basal rosid families are highly dominated by AM

associations (Wang & Qiu 2006), root traits such as

reduced diameter, high lignification and reduced link

length were likely acquired by an ancestor as a set of adap-

tations to colonize temperate areas (Wang et al. 2009).

Further analysis of root chemistry, morphology and physi-

ology in species from divergent evolutionary clades across

latitudinal gradients may help to elucidate the evolutionary

steps associated with the acquisition of root trait syn-

dromes (Guo et al. 2008), adaptive advantages under dif-

ferent ecological constraints (Gu et al. 2014) and

evolutionary processes leading to alternative mycorrhizal

partnerships.

TRA IT INTEGRAT ION AND EVOLUT ION IN ANGIOSPERM

ROOTS

Strong correlations among leaf functional traits such as

SLA and N content have been consistent across species

differing in growth form and biome distribution (Reich,

Walters & Ellsworth 1997; Wright et al. 2005). This strik-

ing global pattern suggests fundamental biophysical trade-

offs between the maximization of carbon fixation and the

minimization in water losses that limit the possible pheno-

typic combinations in foliar traits that can be successful

under natural selection (Boyce et al. 2009; Beerling &

Franks 2010; Donovan et al. 2011). A recent review by

Cornwell et al. (2014) also showed substantial variation in

leaf traits among closely related species, whereas consis-

tency between phylogeny and leaf trait values was limited

to a few highly specialized families adapted to extreme

environments. Thus, leaf traits in angiosperms seem less

constrained by ancestry and more influenced by environ-

mental conditions and life-history strategies.

Because LES has such significant influence on the under-

standing of functional trait variation (Wright et al. 2004;

McGill et al. 2006), the spectrum separating ‘fast-’ and

‘slow-growing’ species has been repeatedly applied to func-

tional trait variation in other organs, particularly fine roots

(Tjoelker et al. 2005; Kembel & Cahill 2011). Our results,

however, do not support this assumed similarity between

roots and leaves. We did not find significant correlations

between root chemical and morphological traits, suggest-

ing that morphological and chemical traits are decoupled

at the root system level. In fact, we found some species

with root trait combinations that seem contradictory to

patterns expected from the LES. For instance, some rosid

species with high SRL showed relatively high C : N ratio,

whereas magnoliids, which showed the lowest SRL, had

relatively lower C : N (Fig. 3). From the same individual

trees, we did find the expected correlations among leaf

traits, implying that the below-ground results are not an

artefact of biased sampling or lack of trait variation across

species.

In agreement with other studies involving woody plants

(Ishida et al. 2008; Baraloto et al. 2010; Fortunel, Fine &

Baraloto 2012; Chen et al. 2013), we found limited sup-

port for the idea that all tree organs are coordinated in a

whole-plant resource use strategy. The decoupling of root

chemical and morphological traits suggests that roots dis-

play a broader array of possible trait combinations than

foliar tissues for maximizing functional gains and mini-

mizing construction and maintenance costs (Donovan

et al. 2011; i.e. broader ‘phenotypic space’ sensu Pigliucci

2007). As explained above, the acquisition of functional

trait syndromes in fine roots arose independently among

plant lineages as adaptations to improve soil exploration,

deal with contrasting climatic limitations and different

mycorrhizal communities. As roots are exposed to a more

complex abiotic and biotic environment than leaves, and

the possibilities to maximize function seem broader than

those of foliar tissues, it is possible that species with simi-

lar leaf traits could present significantly different root sys-

tems.

However, this does not mean that organs are completely

independent from each other. The low but significant cor-

relation between morphological traits of roots and

branches may indicate common selective forces (e.g. simi-

lar thermal and hydraulic stresses experienced by roots

and stems), which could lead to the integration of a similar

set of functional traits. The fact that some traits, such as

tissue density, are structured phylogenetically in both roots

and stems (Swenson & Enquist 2007; Mart�ınez-Cabrera

et al. 2009; Poorter et al. 2010), could also indicate that

the ontological process involved in the construction of

these tissues is shared. However, the fundamentally differ-

ent functions of fine roots and stems, and the additional

anatomical adaptations associated with mycorrhizal com-

munities in fine roots, may limit the extent to which these

tissues can be coordinated.

In contrast, the strong correlation in N content across

plant tissues highlights the possibility of a coordinated

trait axis representing ecological strategies in plants. Nitro-

gen levels in plant tissues are usually associated with pro-

tein content, respiration and overall metabolic activity

(Evans & Seemann 1989; Lambers, Robinson & Ribas-

Carbo 2005). A previous metaanalysis by Reich et al.



(2008) proposed a common relationship between N con-

tent and respiration rates for all plant organs. In grasses

and forbs, the full integration of fine root and leaf N con-

tent is well-established (Craine et al. 2002; Tjoelker et al.

2005), but relatively few studies have evaluated similar

integrations in woody plants (Reich et al. 1998; Wright &

Westoby 1999; Laughlin et al. 2010). Our results support

the hypothesis that N content reflects inherent physiologi-

cal and life-history trade-offs across ecological guilds that

are consistent across the entire plant (Wright et al. 2004),

and not strongly structured by phylogenetic background

(Reich et al. 2008). Future studies linking root N content

with physiology and particularly nutrient acquisition rates

in woody plants are an important follow-up to this study.

In summary, this work highlights the relevance that phy-

logenetic patterns may have in the understanding of fine

root trait patterns at the interspecific level (Mueller et al.

2010; Kembel & Cahill 2011). However, it is important to

note that we have a relatively small sample of temperate

woody angiosperms that do not provide a complete picture

of angiosperm root evolution. Expanding this study to

include subtropical and tropical species and under-repre-

sented phylogenetic groups could help to elucidate the

scope of our conclusions (Donaghue 2008). Moreover, this

study was intentionally designed to minimize the influence

of environmental factors on root traits. Soil conditions can

influence the link between above- and below-ground func-

tional traits at the community level (Zangaro et al. 2007;

Holdaway et al. 2011), and both foliar and root traits in

trees have been reported to vary independently as a way to

adapt to variations in nutrient availability (Comas & Eis-

senstat 2004; Espeleta, West & Donovan 2009; Freschet

et al. 2013). Nonetheless, considering that root trait varia-

tions due to plasticity are small compared to observed var-

iation across species (Chen et al. 2013; Valverde-Barrantes

et al. 2013), we argue that the patterns found in this are

key to understanding root trait variation within and across

woody plant communities. Such information could be use-

ful in our current understanding below-ground ecological

processes and also assessing the ecological role of fine

roots over geological time-scales (Donaghue 2008; Crisp

et al. 2009; Pittermann et al. 2012).

Acknowledgements

The authors would like to thank Scott Kelsey, Amber Horning, Suhana

Chattopadhyay, Eugene Ryee, Kristine Nissel, Benjamin Villareal, Haren

Bonepudi, Josh Lucas, Mariana Romero, Jean Carlo Valverde, Mike Fulp

and Carlynn Fulp for their assistance in the field and processing samples.

Special thanks to Ethan Johnson from The Holden Arboretum and Kristo-

pher Stone and Josh Selm from Boone County Arboretum for their advice in

selecting tree individuals, and to Charlotte Hewins from The Holden Arbo-

retum for sample processing help. We also thank Andrea Case, Jean Burns

and Kevin Mueller for commenting on earlier drafts of the manuscript. This

study was supported by grants from the U.S. National Science Foundation

(DEB-0918240, DEB-0918878) and Department of Energy (DE-SC000433),

start-up funds provided Kent State University, an Art and Margaret Herrick

Research Grant, a David and Susan Jarzen Scholarship, The Holden Arbo-

retum Trust and The Corning Institute for Education and Research. No con-

flicts of interest were declared upon the publication of this manuscript.

Data accessibility

Data used in this manuscript are deposited in the online data repository

Dryad doi: 10.5061/dryad.53mc6.

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Received 18 April 2014; accepted 23 October 2014

Handling Editor: Natalia Norden

Supporting Information

Additional Supporting information may be found in the online

version of this article:

Table S1. Life-history traits of 36 temperate tree species used for

the description of above- and below-ground traits in this study.

Table S2. Summary description of morphological and chemical

traits for three root orders, leaves, twigs and branches of all 36

species studied.

Table S3. Analysis of variance comparing phylogenetic groups

(Super-order) and tissue differences for all traits measured in 34

angiosperm tree species in two different common gardens.

Table S4. Phylogenetic signal values for functional traits in 34

woody angiosperm species.

Table S5. Loadings for phylogenetic principal component analysis

(pPCA) on morphological or chemical traits for three orders of

roots, leaves, twigs and branches of 34 species of woody angio-

sperms.



fine root morphology is phylogenetically structured, but nitrogen is

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