Tree Physiology 34, 415–425 doi:10.1093/treephys/tpu019

Research paper

Root diameter variations explained by anatomy and phylogeny of 50 tropical and temperate tree species

Jiacun Gu1,2, Yang Xu1, Xueyun Dong3, Hongfeng Wang1 and Zhengquan Wang1,4

1School of Forestry, Northeast Forestry University, Harbin 150040, China; 2Center for Ecological Research, Northeast Forestry University, Harbin 150040, China;

3School of Science, Harbin University, Harbin 150086, China; 4Corresponding author ([email protected], [email protected]) Downloaded from

Received August 7, 2013; accepted February 14, 2014; published online April 2, 2014; handling Editor Frederick Meinzer

Root diameter, a critical indicator of root physiological function, varies greatly among tree species, but the underlying mecha-

nism of this high variability is unclear. Here, we sampled 50 tree species across tropical and temperate zones in China, and http://treephys.oxfordjournals.org/ measured root morphological and anatomical traits along the first five branch orders in each species. Our objectives were (i) to reveal the relationships between root diameter, cortical thickness and stele diameter among tree species in tropical and temperate forests, and (ii) to investigate the relationship of both root morphological and anatomical traits with divergence time during species radiation. The results showed that root diameter was strongly affected by cortical thickness but less by stele diameter in both tropical and temperate species. Changes in cortical thickness explained over 90% of variation in root diameter for the first order, and ~74–87% for the second and third orders. Thicker roots displayed greater cortical thickness and more cortical cell layers than thinner roots. Phylogenetic analysis demonstrated that root diameter, cortical thickness and number of cortical cell layers significantly correlated with divergence time at the family level, showing similar variation trends in geological time. The results also suggested that trees tend to decrease their root cortical thickness rather by guest on May 26, 2014 than stele diameter during species radiation. The close linkage of variations in root morphology and anatomy to phylogeny as demonstrated by the data from the 50 tree species should provide some insights into the mechanism of root diameter ­variability among tree species.

Keywords: angiosperm species, root anatomy, root branch order, root morphology, temperate forests, tropical forests.

Introduction and Jackson 2000, Wells et al. 2002), and significantly con- tribute to carbon (C) and nutrient cycles in forest ecosystems Variability of root morphology among tree species is mani- (Norby and Jackson 2000, Joslin et al. 2006). Recent studies fested by diameter, length, specific root length (SRL) and found that root diameter, particularly that of root tips, varied tissue density, all of which play key roles in root functions over 10-fold among tree species in tropical (Chang and Guo (Eissenstat and Yanai 1997, Pregitzer et al. 2002, Comas et al. 2008) and temperate regions (Valenzuela-Estrada et al. 2008, 2012). In comparison with other root traits, diameter may be Comas and Eissenstat 2009, W. Shi, personal communication). the most important morphological feature. For example, root However, what caused such large variation is yet to be well absorptive efficiency mainly depends on diameter, and thinner identified and explained. Other root-trait studies with multiple roots with lower tissue density and higher SRL may absorb species showed that diameter was closely correlated with greater nutrients and water from the soil (Eissenstat and Yanai SRL (Comas and Eissenstat 2009), stele diameter (Guo et al. 1997, Eissenstat et al. 2000, Fitter 2002). In addition, thinner 2008) and the divergence time in evolutionary history (Comas roots also have shorter lifespans or faster turnover rates (Gill et al. 2012, Chen et al. 2013). Despite these previous studies,

© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 416 Gu et al. the primary traits governing root diameter variation among tree mentioned above, the main objectives of this study were (i) to species are still not well established. reveal the relationships between root diameter, cortical thick- The cortex and stele are the two main tissues in primary roots ness and stele diameter among tree species in tropical and (Esau 1977, Peterson et al. 1999, Lux et al. 2004), and their vari- temperate forests, and (ii) to investigate the relationship of ations will determine root diameter. However, the extent to which both root morphological traits (diameter) and anatomical traits these two traits influence diameter variation remains unclear. For with divergence time during species radiation. example, Hummel et al. (2007) reported that root diameter cor- related strongly with cortical (plus rhizodermis) cross-sectional Materials and methods area but only weakly with xylem diameter in 14 herbaceous Mediterranean species. In contrast, Guo et al. (2008) showed Study site that root diameter was closely correlated with stele diameter Two study sites were chosen. Site I was located in a tropical rather than cortex thickness in 23 Chinese temperate tree spe- forest in the Jianfengling National Natural Reserve (18°23′– cies. One possible reason for such a discrepancy may be that 18°50′N, 108°36′–109°05′E) in Hainan of southern China. This Hummel et al. (2007) selected only the first-order roots (root site has a tropical climate with mean January, July and annual tips), while Guo et al. (2008) included all of the first five order temperatures of 19.4, 27.3 and 24.5 °C, respectively. The roots. Considering that the proportion of the cortex decreases mean annual precipitation is 2651 mm, of which ~80% occurs

(or disappears) and the stele increases with ascending root between May and October. The soils are Humic Acrisol (Gong Downloaded from branch orders in woody plants (Guo et al. 2008, Valenzuela- et al. 1999) with high nutrients. Under such climate conditions, Estrada et al. 2008), we propose our first hypothesis that, for tropical seasonal rainforest dominates the mountain areas. Site those lower-order (or absorptive) roots, root diameter is mainly II was located in a temperate forest at the Maoershan research affected by cortical thickness but less by stele diameter. station (45°21′–45°25′N, 127°30′–127°34′E) of the Northeast http://treephys.oxfordjournals.org/ Root diameter variability may also be influenced by species Forestry University in Heilongjiang of northeastern China. This evolutionary history (Fitter 2002, Kenrick 2002). However, it is site has a continental temperate monsoon climate with mean very difficult to reconstruct root evolutionary history, due to the January, July and annual temperatures of −19.6, 20.9 and scarcity of root fossils in geological strata (Elick et al. 1998, 2.8 °C, respectively. The mean annual precipitation is 723 mm Raven and Edwards 2001, Kenrick 2002). Extant species might with 477 mm falling from June to August (Zhou 1994). The provide some insights in this regard (Peterson 1992). Based on soils are Hap-Boric Luvisols (Gong et al. 1999), well drained phylogenetic analyses with woody plants, Comas and Eissenstat with high organic matter. The site is dominated by the second- (2009) and Comas et al. (2012) suggested that extant angio- growth forest regenerated after the old growth of mixed Korean sperms with lineages diversifying more recently during the pine and hardwood forest was harvested over 70 years ago. by guest on May 26, 2014 Cretaceous had thinner roots with higher SRL than primitive angiosperms. Chen et al. (2013) found that the root diameter Species selection and root sampling of extant species significantly correlated with divergence time at We sampled a total of 50 species, 27 at tropical Site I and 23 the family level and decreased from 120 to 64 million years ago, at temperate Site II (see Table S1 available as Supplementary and was constant afterwards. These findings suggest that root Data at Tree Physiology Online). The 47 hardwoods and three diameter is substantially influenced by plant phylogeny, although conifers belong to 19 orders and 28 families, of which four fam- further tests are needed using fossil evidence when available ilies were common at both study sites. These species included (Comas et al. 2012). Anatomy is important for our understand- magnoliids, rosids, malvids, asterids and campanulids for ing of plant evolutionary trends (Seago and Fernando 2013), but angiosperms and pinales for gymnosperms, but did not include it remains unclear whether evolutionary patterns of root diam- basal angiosperms and monocots. The roots were sampled in eter are mirrored by anatomical traits. Thus, our second hypoth- August of 2009 for tropical species (Site I) and in late July of esis is that cortical thickness, number of cortical cell layers and 2007 for temperate species (Site II). At both sites, three indi- stele diameter have similar variation patterns to root diameter vidual trees were selected for each species, with ages ranging during species radiation in geological time. from 30 to 50 years and diameters at breast height (DBHs) In the current study, we sampled 50 tree species, 27 from from 15 to 20 cm. One root sample from each of the three tropical and 23 from temperate forests (i.e., latitude from 18° trees was taken from the top 20-cm soil layer (mineral soil to 45°) in China. These 50 tree species belong to 19 orders layer), following the same procedure as in Guo et al. (2008). and 28 families. Root morphological traits (diameter, SRL and Root branches (including more than five branch orders) were tissue density) and anatomical traits (cortical thickness, stele traced to the tree stem and cut from the main lateral woody diameter and number of cortical cell layers) along the first roots, which enabled us to identify and sort roots of target five branch orders (distal roots numbered as first order) were species. Once collected, each root sample was divided into measured for each species. By testing the two hypotheses two subsamples: one was gently washed in deionized water

Tree Physiology Volume 34, 2014 Tree root diameter variation and phylogeny 417 and immediately fixed in formalin-aceto-alcohol (FAA) solution and its nearest sister family diverged from their immediate (90 ml of 50% ethanol, 5 ml of 100% glacial acetic acid and ancestor as the divergence time for this family. Since the diver- 5 ml of 37% methanol); the other was immediately put on ice gence time estimated by Wikström et al. (2001) was given and transported to the laboratory within 4 h and frozen for dis- for the divergence node between two adjacent related genera, section and morphological analysis at a later date. rather than between families, we chose the divergence time of the earliest genus within a family to represent the divergence Root morphology and anatomy time for the family (Chen et al. 2013). We noted that the phy- In the laboratory, at least five intact root branches for each logenetic tree was constructed by species at the extremes of sample were dissected for morphological analysis under a ×10 south and north distribution in China and did not have any spe- stereomicroscope (Motic SMZ-140, Xiamen, China), following cies in the middle. However, the dataset covered species of the procedure described in Pregitzer et al. (2002) and Wang both primitive and newly divergent families at each study site. et al. (2006), with the distal non-woody roots regarded as first- order roots. For morphology, roots were separated by order, with Data analysis >1000, 500, 150, 50 and 20 roots collected for the first to fifth The means, median, minimum, maximum and coefficient of order per sample. After dissection, the roots of each subsample variation (CV) were calculated at branch order level for five were scanned by order with an EPSON EXPRESSION 10000XL root traits, i.e., diameter, SRL, tissue density, cortical thickness colored scanner (dots per inch = 400). The mean diameter, total and stele diameter, in both tropical and temperate sites. In Downloaded from length and volume for each order of roots were determined with order to test the first hypothesis, regression analysis was used the root system analyzer software (WinRhizo 2004b, Regent to determine the relationships between root diameter, cortical Instruments, Inc., Québec, Canada). Then these roots were oven thickness and stele diameter for the first three branch orders dried (65 °C) to determine constant weight (nearest 0.0001 g). among species at each site, and the relationship between cor- http://treephys.oxfordjournals.org/ The SRL for each order was calculated as the total length divided tical thickness and number of cortical cell layers. All statistical by the corresponding dry mass (ash-free mass basis). The tissue analysis mentioned above was performed using the SPSS soft- density for each order of roots was calculated as the dry mass ware (2010, V. 19.0: SPSS, Inc., Cary, NC, USA). (ash-free mass basis) divided by the corresponding total volume. Variations in first-order root morphological and anatomical For root anatomy, roots in each FAA subsample were dissected traits at the family level in geological time, and their corre- carefully by branch order. Twenty roots from each branch order lations with divergence time were determined by piecewise were stained with safranine-fast green, and then dehydrated in regression (Chen et al. 2013). Piecewise regression models 70, 85, 95 and 100% alcohol, embedded in paraffin; and slides are ‘broken-stick’ models, where two or more lines are joined of 8-μm-thick root sections were prepared with a microtome for at unknown points, called ‘breakpoints’ (Toms and Lesperance by guest on May 26, 2014 determination of anatomical characteristics (Guo et al. 2008). 2003). Breakpoints can be used as estimates of thresholds, The slides were photographed under a compound microscope and were used here to determine the thresholds of divergence (BX51; Olympus, Tokyo, Japan), with root diameter, cortical thick- time for root morphological and anatomical traits in geologi- ness, stele diameter and number of cortical cell layers being cal time (Chen et al. 2013). With this approach, we tested the measured from three cross-sections for each root segment. second hypothesis in our study. In addition, we used the phy- logenetically independent contrasts (PIC) method to test the Species phylogeny dependence of root morphological and anatomical trait varia- The phylogeny of the 50 species was constructed based on tion on divergence time without the influence of phylogeny. the instructions of the APG III system (APG 2009) and the PIC analysis was evaluated with the ‘analysis of traits’ (AOT) Angiosperm Phylogeny website (http://www.mobot.org/ module in PHYLOCOM 4.2, which can calculate the internal MOBOT/Research/APweb/welcome.html) (Stevens 2001). The node values for continuous traits (Felsenstein 1985, Webb free software PHYLOCOM 4.2 (Webb et al. 2008, http://phy- et al. 2008). We did not include gymnosperms in PIC analysis lodiversity.net/phylocom/) was used to construct the phyloge- as they evolved in a different lineage and may differ markedly netic tree (see Figure S1 available as Supplementary Data at in root anatomy from angiosperm species. Finally, we calcu- Tree Physiology Online), in which the resolved PHYLOMATIC lated the correlation coefficients between root diameter and tree (R20100701) was used as the backbone for our super- anatomical traits for 47 species with the PIC method. tree. The Branch Length Adjuster algorithm in PHYLOCOM in combination with estimated family ages (Wikström et al. 2001) Results was used to adjust all the branch lengths in our phylogenetic tree. In addition, we obtained divergence times of angiosperm Variations in root morphological and anatomical traits families from Wikström et al. (2001) based on the molecular Root traits showed great variability in statistical characteris- clock theory. We determined the geological time when a family tics between both tropical and temperate forests, in addition

Tree Physiology Online at http://www.treephys.oxfordjournals.org 418 Gu et al. to large differences among the first five orders (Table1 ). The three orders in tropical and temperate trees, but the effect average diameter was greater for tropical trees than for tem- of cortical thickness was greater (see Table S2 available as perate trees. Root diameter in first order varied nearly nine- Supplementary Data at Tree Physiology Online). Root diam- fold between the thinnest (Cratoxylum cochinchinense Lour.: eter was positively correlated with cortical thickness, which 135 ± 4 µm) and the thickest (Cryptocarya chinensis Hemsl.: explained 92 (tropical) and 93% (temperate) of the variations 1113 ± 17 µm) for tropical trees, but threefold for temperate in root diameter for first order, over 80% for second order and trees (Salix koreensis Anderss.: 148 ± 7 µm vs Phellodendron over 70% for third order (Figure 1). Stele diameter displayed a amurense Rupr.: 522 ± 19 µm). The average SRL was shorter relatively weaker correlation, and explained 47–57% (tropical) and tissue density was less for tropical trees than for temper- and 36–66% (temperate) of the variations in root diameters ate trees. The SRL varied over 80-fold between the shortest among the first three orders (Figure2 ). (C. chinensis: 3.5 ± 0.3 m g−1) and the longest (C. cochinchi- In root cross-sections, cortical thickness positively cor- nense: 288.5 ± 2.3 m g−1) for tropical trees, but sixfold for related with the number of cortical cell layers across the temperate trees (P. amurense: 35.7 ± 0.7 m g−1 vs Euonymus first three order roots in both tropical and temperate trees pauciflorus Sieb.: 203.8 ± 2.1 m g−1). (Figure 3). For example, for tropical trees, Eurya ciliata Merr. Cortical thickness and stele diameter were both greater for had a first-order root cortical thickness of 55.5 ± 2.7 µm tropical trees than for temperate trees (Table 1). In the first with three cortical cell layers (Figure 4a; root diameter three orders, the average cortical thickness for each order of 142 ± 4 µm), while Alseodaphne hainanensis Merr. had Downloaded from ranged from 146 to 153 µm for tropical species and from 53 a cortical thickness of 301.1 ± 11.1 µm with 12 cortical to 79 µm for temperate species. By contrast, stele developed cell layers (Figure 4b; root diameter of 905.7 ± 27.9 µm). in all orders of roots, and its diameter increased with ascend- Similarly, for temperate trees, Betula platyphylla Suk. had ing root order. The average stele diameter for each order var- a first-order root cortical thickness of 41.3 ± 2.9 µm http://treephys.oxfordjournals.org/ ied between 108 and 203 µm for tropical trees and between with four cortical cell layers (Figure 4c; root diameter of 71 and 195 µm for temperate trees. 157 ± 7.1 µm), while P. amurense had a cortical thickness of 177 ± 6.9 µm with 10 cortical cell layers (Figure 4d; root Effect of anatomical traits on root diameter diameter of 522 ± 18.8 µm). Overall, 54–77% of the inter- Multiple linear regression showed that both cortical thick- species variation in cortical thickness could be explained by ness and stele diameter influenced root diameters of the first the number of cortical cell layers (Figure 3).

Table 1. Descriptive statistics for five root traits of morphology and anatomy in 50 tropical and temperate tree species measured in this study. by guest on May 26, 2014 Root trait Root order Tropical Temperate

Minimum Maximum Mean Median CV Minimum Maximum Mean Median CV

Diameter 1 0.135 1.113 0.422 0.356 0.607 0.168 0.520 0.243 0.219 0.297 (mm) 2 0.156 1.218 0.475 0.408 0.581 0.164 0.566 0.265 0.240 0.345 3 0.160 1.359 0.552 0.478 0.547 0.212 0.806 0.340 0.302 0.445 4 0.162 1.623 0.678 0.614 0.504 0.293 1.530 0.539 0.431 0.568 5 0.175 1.878 0.843 0.822 0.470 0.394 3.209 0.959 0.809 0.682 SRL (m g−1) 1 3.48 288.53 73.94 50.23 0.968 33.70 203.82 99.83 95.67 0.377 2 3.24 220.79 48.09 36.48 1.011 24.30 130.22 84.55 76.17 0.345 3 2.52 163.49 28.67 20.02 1.129 12.70 67.87 45.06 45.92 0.382 4 1.63 135.47 16.74 11.41 1.489 3.77 34.21 18.05 16.27 0.489 5 1.13 26.65 6.76 5.58 0.770 1.309 18.96 6.70 5.82 0.604 Tissue density 1 0.078 0.317 0.162 0.150 0.376 0.136 0.407 0.272 0.305 0.321 (g cm−3) 2 0.100 0.390 0.197 0.191 0.331 0.136 0.363 0.263 0.255 0.261 3 0.117 0.541 0.242 0.225 0.387 0.155 0.524 0.343 0.351 0.293 4 0.085 0.551 0.254 0.246 0.366 0.220 0.554 0.391 0.419 0.239 5 0.149 0.646 0.299 0.264 0.336 0.183 0.519 0.384 0.404 0.222 Cortical 1 25.00 362.25 146.47 131.05 0.615 40.91 177.74 79.32 73.08 0.386 thickness 2 16.42 432.92 153.52 134.55 0.653 14.94 175.64 71.89 72.04 0.525 (μm) 3 0.00 415.75 146.23 126.71 0.760 0.00 206 .11 53.24 25.91 1.163 Stele diameter 1 38.97 261.05 108.38 91.03 0.528 37.45 154.55 70.69 54.53 0.440 (μm) 2 68.32 321.41 143.89 126.41 0.458 59.24 220.78 110.03 103.37 0.333 3 81.61 365.14 203.86 180.16 0.414 104.62 354.49 194.94 178.61 0.330 4 123.47 1352.87 470.11 422.06 1.804 199.05 1131. 23 425.58 411.15 1.125 5 147.52 1505.84 649.73 618.52 2.063 315.23 2567.17 758.22 721.34 1.986

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Figure 1. Correlations between root diameter and cortical thickness in the first three orders among 27 tropical and 23 temperate tree species in China.

Phylogenetic variations in root morphological and root diameter, cortical thickness and number of cortical and anatomical traits cell layers were still significant after the influence of phylog- Phylogenetic analysis showed that angiosperms of primitive eny was removed (R2 = 0.19–0.33, all P < 0.05; Figure 6). In families, such as Lauraceae and Magnoliaceae, commonly addition, when the phylogenetic influence was removed, the had thicker roots in first order (Figure 5). Anatomical results correlation between cortical thickness and root diameter was showed that those species of primitive families not only had strongest among the root morphological and anatomical traits thicker root diameters but also had greater cortical thickness studied (see Table S3 available as Supplementary Data at Tree and thicker stele, as well as a greater number of cortical cell Physiology Online). layers (Figures 4 and 5). Piecewise regression analysis dem- onstrated that both root morphological and anatomical traits Discussion at the family level may have similar trends of variation in geo- logical time with a consistent decline from the period of 120 Relations of root diameter to anatomical traits to 60 million years before present (MYBP), and level off after Root diameter plays a crucial role in root-driven ecophysiologi- ~60 MYBP (Figure 6). Root diameter, cortical thickness and cal processes in forest ecosystems. For example, thinner roots number of cortical cell layers, but not stele diameter, were sig- of trees usually have higher mycorrhizal colonization (Reinhardt nificantly correlated with divergence time R( 2 = 0.19–0.28, all and Miller 1990, Brundrett 2002), greater absorptive capac- P < 0.05; Figure 6). Phylogenetically independent contrasts ity (Wells and Eissenstat 2003, Dunbabin et al. 2004, Rewald also showed that the correlations between divergence time et al. 2012), shorter lifespan (Eissenstat and Yanai 1997, Kern

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Figure 2. Correlations between root diameter and stele diameter in the first three orders among 27 tropical and 23 temperate tree species in China. et al. 2004, Baddeley and Watson 2005) and faster decompo- non-woody roots at the species level, as found by this and other sition (Fehay and Hughes 1994, Schenk and Jackson 2002). studies (Esau 1977, Lux et al. 2004, Hummel et al. 2007, Guo Root diameter varies greatly among tree species, even at the et al. 2008). Second, root cortical thickness in woody plants same site (Guo et al. 2008, Comas and Eissenstat 2009), may affect root diameter primarily by changing the number of but the reasons are still not well understood. Our study dem- cortical cell layers, as tree species with greater root cortical onstrated that root diameter was mainly affected by cortical thickness have more cortical cell layers (Figure 3) in this study thickness and less by stele diameter in a large number (50) and others (Zadworny and Eissenstat 2011). Third, genetic of tropical and temperate tree species (Figures 1 and 2, see factors may also contribute to the variation in root diameter. Table S2 available as Supplementary Data at Tree Physiology Recent studies of Arabidopsis roots found that two proteins, Online). Even when the influence of phylogeny was removed, SHR (SHORT-ROOT) and SCR (SCARECROW), were key regu- root diameter was still strongly correlated with cortical thick- lators of root radial patterning, including both periclinal and ness (see Table S3 available as Supplementary Data at Tree anticlinal divisions in cortical development (Di Laurenzio et al. Physiology Online). These results support our first hypothesis 1996, Helariutta et al. 2000, Cui and Benfey 2009). Reports that cortical thickness in absorptive roots governs the variation on crops also showed that roots of different rice and common in root diameter among tree species. bean genotypes had different diameters and numbers of corti- Several possible reasons may help to explain why corti- cal cell layers (Lynch and van Beem 1993, Lafitte et al. 2001, cal thickness determines root diameter. First, the root cortex Burton et al. 2013). Our results suggest that the variation in accounts for a greater proportion of the cross-sectional area of root diameter of the 50 tree species studied should reflect the

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Figure 3. Correlations between root cortical thickness and number of cortical cell layers in the first three orders among 27 tropical and 23 temper- ate tree species in China. differences not only in root anatomy but also in phylogenetic importantly, our study found that all three anatomical traits lineage (see the discussion below). (root cortical thickness, stele diameter and number of cortical cell layers) had similar thresholds of divergence time to that Root morphology and anatomy during evolution of diameter, which suggests that all these traits share similar Root morphology is closely linked to phylogeny (Comas and trends of variation in geological time (Figure 6b–d). This finding Eissenstat 2009). Comas et al. (2012) suggested that such a was also supported by the significant correlation between root linkage should be an indication of the evolutionary adaptation diameter and anatomical traits when the phylogenetic influence of root traits to long-term environmental changes in geologi- was removed (see Table S3 available as Supplementary Data cal time. Previous studies with extant tree species have found at Tree Physiology Online). Thus, as predicted by our second that early-divergent species on the phylogenetic tree com- hypothesis, the variation in tree root diameter is likely to be monly had thicker roots than late-divergent species (Pregitzer associated with changes in anatomical structure during spe- et al. 2002, Comas and Eissenstat 2009, Comas et al. 2012). cies radiation in geological time. Recently, Chen et al. (2013) reported that the root diameter of Patterns of evolution of root morphological traits were likely angiosperm species at the family level significantly correlated related to global climate changes during the past 120 ­million with divergence time during the period of 120–20 MYBP. Our years, as proposed in some studies (e.g., Comas et al. 2012, estimated pattern of variation for root diameter (Figure 6a) Chen et al. 2013). From the Cretaceous to the Tertiary, was consistent with the findings ofChen et al. (2013). More angiosperm species occurred and radiated worldwide (Li and

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Figure 4. Typical anatomical structures of first-order roots inEurya ciliata (a), A. hainanensis (b), B. platyphylla (c) and P. amurense (d). CO, cortex;

EP, epidermis; EX, exodermis; HS, hyphal sheath; VC, vascular cylinder; D, diameter; CT, cortical thickness; NC, number of cortical cell layers. by guest on May 26, 2014

Johnston 2000, Crepet et al. 2004, Brodribb and Feild 2010),

while the atmospheric carbon dioxide (CO2) concentration and temperature gradually declined (Savin 1977, Kerp 2002, Retallack 2002). Such changes led to a relative decrease in precipitation and an increase in the degree of drought (Tardy et al. 1989, Beerling 1999). From the mid-Cretaceous to the late Tertiary (~120–10 MYBP), extant species showed that newly diverged angiosperm species decreased not only in root diameter (Comas et al. 2012, Chen et al. 2013, this study) but also in cortical thickness and number of cortical cell layers (this study), which may enable roots to take up soil nutrients and water more efficiently Comas( et al. 2012). Other evidence also suggests that thinner roots with a thin cortex generally have high absorptive capacity (Eissenstat and Yanai 1997, Rieger and Litvin 1999, Wells and Eissenstat 2003, Hishi 2007). On the other hand, leaf stomatal and vein densities in woody plants increased concomitantly with long-term gradual

declines in atmospheric CO2 to enhance their capacity for pho- tosynthetic CO uptake (Royer 2001, Brodribb and Feild 2010), Figure 5. Average diameter (a) and cortical thickness (b) of first- 2 order roots at the family level in phylogeny structure based on the which in turn may have induced the corresponding changes Angiosperm Phylogeny Group III classification. in root morphology and anatomy (Comas et al. 2012, Chen

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Figure 6. Relationships between first-order root traits and divergence time (MYBP) for angiosperm tree species at the family level: (a)–(d) piece- wise regression between root traits and divergence time; (e)–(h) PIC correlation between root traits and divergence time. et al. 2013). Thus, the co-evolution between leaf and root traits by cortex. In absorptive roots, the cortex is considered to be could be an important evolutionary strategy for late-diverging a ‘buffer zone’ to partially isolate the stele from environmen- terrestrial species to adapt to selective pressures associated tal stresses (Lux et al. 2004) such as drought (Kondo et al. with global environmental changes during the past 100 million 2000), heavy-metal contamination (Reinhardt and Rost 1995) years (Raven and Edwards 2001, Westoby and Wright 2006, and salinity (Enstone et al. 2003). In such cases, the stele may Comas et al. 2012). be less sensitive to environmental changes and may show rela- The results of this study showed that stele diameter was not tively small variation. Another reason is that the stele seems to significantly correlated with divergence time (Figure 6c). One have been to be highly conserved in evolution (Sachs 1993, possible reason may be that the root stele is entirely surrounded Roth and Mosbrugger 1996, Seago and Fernando 2013). For

Tree Physiology Online at http://www.treephys.oxfordjournals.org 424 Gu et al. example, the average cortical thickness of species before and Conflict of interest after 60 MYBP decreased by 34%, while stele diameter only decreased by 24% in our study (data not shown). Therefore, None declared. it seems to be a root evolutionary strategy that trees may tend to decrease their cortical thickness more strongly than stele Funding diameter during species radiation in geological time. Caution is needed in the interpretation of our results on evo- This research was supported by the National Basic Research lutionary patterns of root traits through geological time. First, Program of China (973 Program: 2012CB416906), the Natural as it was limited by the number of species (families) sampled, Science Foundation of China (31100470 and 30130160) and especially the early-divergent species, the relationship between the Program for Changjiang Scholars and Innovative Research root diameter and divergence time established by piecewise Team in University (IRT1054). regression (Figure 6) may be weak. Thus, more species rep- resenting early divergence times, such as basal angiosperms (e.g., Amborellales, Austrobaileyales and Chloranthales), should References be included in future studies, in order to improve the resolu- APG (Angiosperm Phylogeny Group) (2009) An update of the angio- tion of patterns of root-trait evolution. Second, even though our sperm phylogeny group classification for the orders and families of flowering plants: APG III. Bot J Linn Soc 161:105–121. Downloaded from results provided a clear comparison of the differences in root Baddeley J, Watson C (2005) Influences of root diameter, tree age, soil traits between typical tropical and temperate species, species depth and season on fine root survivorship in Prunus avium. Plant in the transitional zone were lacking, which may lead to phy- Soil 276:15–22. logenetic analysis overweighted by species at the extremes Beerling DJ (1999) Increased terrestrial carbon storage across the Palaeocene–Eocene boundary. Palaeogeogr Palaeoclimatol of the distribution. Future studies should sample more spe- Palaeoecol 161:395–405. http://treephys.oxfordjournals.org/ cies across widely distributed study sites with replication, and Brodribb TJ, Feild TS (2010) Leaf hydraulic evolution led a surge in under a broad range of environmental conditions. leaf photosynthetic capacity during early angiosperm diversification. Ecol Lett 3:175–183. Brundrett MC (2002) Coevolution of roots and mycorrhizas of land Conclusion plants. New Phytol 154:275–304. Burton AL, Lynch JP, Brown KM (2013) Spatial distribution and pheno- The current study demonstrates that cortical thickness in typic variation in root cortical aerenchyma of maize (Zea mays L.). absorptive roots may be the most important factor influenc- Plant Soil 367:263–274. ing diameter size in both tropical and temperate trees. Cortical Chang WJ, Guo DL (2008) Variation in root diameter among 45 com- mon tree species in temperate, subtropical and tropical forests in thickness explained over 90% of the variation in root diameter China. J Plant Ecol (Chinese Version) 32:1248–1257. by guest on May 26, 2014 for the first order, and over 80 and 70% for the second and Chen WL, Zeng H, Eissenstat DM, Guo DL (2013) Variation of first- third orders. Thicker roots generally showed greater corti- order root traits across climatic gradients and evolutionary trends in cal thickness and more cortical cell layers than thinner roots. geological time. Glob Ecol Biogeogr 22:846–856. Comas LH, Eissenstat DM (2009) Patterns in root trait variation Among extant tree species, root diameter and two anatomi- among 25 co-existing North American forest species. New Phytol cal traits (cortical thickness and number of cortical cell layers) 182:919–928. showed significant correlations with divergence time, exhibiting Comas LH, Mueller KE, Taylor LL, Midford PE, Callahan HS, Beerlingz similar trends. Our results suggest that the variation in tree root DJ (2012) Evolutionary patterns and biogeochemical significance of angiosperm root traits. Int J Plant Sci 173:584–595. morphological traits should be associated with the change in Crepet WL, Nixon K, Gandolfo D (2004) Fossil evidence and phylogeny: anatomical structure during species radiation in geological time. the age of major angiosperm clades based on mesofossil and macro- fossil evidence from Cretaceous deposits. Am J Bot 91:1666–1682. Cui H, Benfey PN (2009) Cortex proliferation: simple phenotype, com- Supplementary data plex regulatory mechanisms. Plant Signal Behav 4:551–553. Di Laurenzio L, Wysocka-Diller J, Malamy JE, Pysh L, Helariutta Y, Freshour Supplementary material is available at Tree Physiology online. G, Hahn MG, Feldmann KA, Benfey PN (1996) The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root. Cell 86:423–433. Acknowledgments Dunbabin VM, Rengel Z, Diggle AJ (2004) Simulating form and func- tion of root systems: efficiency of nitrate uptake is dependent on The authors thank Xing Wei, Sen Song, Wei Shi, Ying Liu, Jinliang root system architecture and the spatial and temporal variability of Liu and Haibo Chen for their help with field and laboratory work, nitrate supply. Funct Ecol 18:204–211. and Dr Dongmei Jin, Jinlong Zhang and Peili Fu for their help with Eissenstat DM, Yanai RD (1997) The ecology of root lifespan. Adv Ecol statistical analysis. They also thank two anonymous reviewers for Res 27:1–60. Eissenstat DM, Wells CE, Yanai RD, Whitbeck JL (2000) Building roots comments and suggestions, which improved the manuscript, and in a changing environment: implications for root longevity. New Dr Harbin Li for editing the manuscript and insightful comments. Phytol 147:33– 42.

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Elick JM, Driese SG, Mora CI (1998) Very large plant and root traces Pregitzer KS, De Forest JL, Burton AJ, Allen MF, Ruess RW, Hendrick from the early to middle Devonian: implications for early terrestrial RL (2002) Fine root architecture of nine North American trees. Ecol

ecosystems and atmospheric p(CO2). Geology 26:143–146. Monogr 72:293–309. Enstone DE, Peterson CA, Ma F (2003) Root endodermis and exoder- Raven JA, Edwards D (2001) Roots: evolutionary origins and biogeo- mis: structure, function, and responses to the environment. J Plant chemical significance. J Exp Bot 52:381–401. Growth Regul 21:335–351. Reinhardt DH, Rost TL (1995) Salinity accelerates endodermal devel- Esau K (1977) Anatomy of seed plants. 2nd edn. Wiley, New York, pp opment and induces an exodermis in cotton seedling roots. Environ 215–255. Exp Bot 35:563–574. Fehay TJ, Hughes JW (1994) Fine root dynamics in a northern hard- Reinhardt DR, Miller RM (1990) Size classes of root diameter and wood forest ecosystem, Hubbard Brook Experimental Forest, NH. J mycorrhizal fungal colonization in two temperate grassland com- Ecol 82:533–548. munities. New Phytol 116:129–136. Felsenstein J (1985) Phylogenies and the comparative method. Am Retallack GJ (2002) Carbon dioxide and climate over the past 300Myr. Nat 125:1–15. Philos Trans R Soc Lond A 360:659–673. Fitter AH (2002) Characteristics and functions of root systems. In: Rewald B, Raveh E, Gendler T, Ephrath JE, Rachmilevitch S (2012) Waisel Y, Eshel E, Kafkafi U (eds) Plant roots: the hidden half. 3rd Phenotypic plasticity and water flux rates of Citrus root orders under edn. Dekker Press, New York, USA, pp 15–32. salinity. J Exp Bot 63:2717–2727. Gill RA, Jackson RB (2000) Global patterns of root turnover for ter- Rieger M, Litvin P (1999) Root system hydraulic conductivity in spe- restrial ecosystems. New Phytol 147:13–31. cies with contrasting root anatomy. J Exp Bot 50:201–209. Gong ZT, Chen ZC, Luo GB, Zhang GL, Zhao WJ (1999) Soil reference Roth A, Mosbrugger V (1996) Numerical studies of water conduction in with Chinese soil taxonomy. Soils 31:57–63. land plants: evolution of early stele types. Paleobiology 22:411–421. Guo DL, Xia MX, Wei X, Chang WJ, Liu Y, Wang ZQ (2008) Anatomical Royer DL (2001) Stomatal density and stomatal index as indicators Downloaded from

traits associated with absorption and mycorrhizal colonization are of paleoatmospheric CO2 concentration. Rev Palaeobot Palynol linked to root branch order in twenty-three Chinese temperate tree 114:1–28. species. New Phytol 180:673–683. Sachs T (1993) A developmental perspective of the evolution of the Helariutta Y, Fukaki H, Wysocka-Diller J, Nakajima K, Jung J, Sena G, stele. Int J Plant Sci 154:225–227. Hauser M-T, Benfey PN (2000) The SHORT-ROOT gene controls Savin SM (1977) The history of the earth’s surface temperature during http://treephys.oxfordjournals.org/ radial patterning of the Arabidopsis root through radial signaling. the past 100 million years. Ann Rev Earth Planet Sci 5:319–355. Cell 101:555–567. Schenk HJ, Jackson RB (2002) The global biogeography of roots. Ecol Hishi T (2007) Heterogeneity of individual roots within the fine root Monogr 72:311–328. architecture: causal links between physiological and ecosystem Seago JL Jr, Fernando DO (2013) Anatomical aspects of angiosperm functions. J For Res 12:126–133. root evolution. Ann Bot 112:223–238. Hummel I, Vile D, Violle C, Devaux J, Ricci B, Blanchard A, Garnier E, Stevens PF (2001). Angiosperm phylogeny website. Version 12, July Roumet C (2007) Relating root structure and anatomy to whole- 2012. http://www.mobot.org/MOBOT/research/APweb/ (10 August plant functioning in 14 herbaceous Mediterranean species. New 2012, date last accessed). Phy tol 173:313 –321. Tardy Y, N’Kounkou R, Probst J-L (1989) The global water cycle and Joslin JD, Gaudinski JB, Torn MS, Riley WJ, Hanson PJ (2006) Fine-root continental erosion during Phanerozoic time (570 my). Am J Sci

turnover patterns and their relationship to root diameter and soil 289:455–483. by guest on May 26, 2014 depth in a 14C-labeled hardwood forest. New Phytol 172:523–535. Toms JD, Lesperance ML (2003) Piecewise regression: a tool for iden- Kenrick P (2002) The origin of roots. In: Waisel Y, Eshel E, Kafkafi U tifying ecological thresholds. Ecology 84:2034–2041. (eds) Plant roots: the hidden half. 3rd edn. Dekker Press, New York, Valenzuela-Estrada LR, Vera-Caraballo V, Ruth LE, Eissenstat DM USA, pp 1–14. (2008) Root anatomy, morphology, and longevity among root orders Kern CC, Friend AL, Johnson JM-F, Coleman MD (2004) Fine root in Vaccinium corymbosum (Ericaceae). Am J Bot 95:1506–1514. dynamics in a developing Populus deltoids plantation. Tree Physiol Wang ZQ, Guo DL, Wang XR, Gu JC, Mei L (2006) Fine root architec- 24:651–660. ture, morphology, and biomass of different branch orders of two Kerp JHF (2002) Comment on ‘300-million-year record of atmospheric Chinese temperate tree species. Plant Soil 288:155–171. carbon dioxide from fossil plant cuticles’. Nature 415:38. Webb CO, Ackerly DD, Kembel SW (2008) Phylocom: software for Kondo M, Aguilar A, Abe J, Morita S (2000) Anatomy of nodal roots in the analysis of phylogenetic community structure and trait evolution. tropical upland and lowland rice varieties. Plant Prod Sci 3:437–445. Bioinformatics 24:2098–2100. Lafitte HR, Champoux MC, McLaren G, O’Toole JC (2001) Rice root Wells CE, Eissenstat DM (2003) Beyond the roots of young seed- morphological traits are related to isozyme group and adaptation. lings: the influence of age and order on fine root physiology. J Plant Field Crops Res 71:57–70. Growth Regul 21:324–334. Li P, Johnston MO (2000) Heterochrony in plant evolutionary studies Wells CE, Glenn DM, Eissenstat DM (2002) Changes in the risk of through the twentieth century. Bot Rev 66:57–88. fine-root mortality with age: a case study in peach, Prunus persica Lux A, Luxova M, Abe J, Morita S (2004) Root cortex: structural and (Rosaceae). Am J Bot 89:79–87. functional variability and responses to environmental stress. Root Westoby M, Wright IJ (2006) Land-plant ecology on the basis of func- Res 13:117–131. tional traits. Trends Ecol Evol 21:261–268. Lynch J, van Beem JJ (1993) Growth and architecture of seedling roots Wikström N, Savolainen V, Chase MW (2001) Evolution of the angio- of common bean genotypes. Crop Sci 33:1253–1257. sperms: calibrating the family tree. Proc R Soc B Biol Sci 268: Norby RJ, Jackson RB (2000) Root dynamics and global change: seek- 2211–2220. ing an ecosystem perspective. New Phytol 147:3–12. Zadworny M, Eissenstat DM (2011) Contrasting the morphology, anat- Peterson CA, Enstone DE, Taylor JH (1999) Pine root structure and omy and fungal colonization of new pioneer and fibrous roots. New its potential significance for root function. Plant Soil 217:205–213. Phytol 190:213–221. Peterson RL (1992) Adaptations of root structure in relation to biotic Zhou XF (1994) Long-term research on China’s forest ecosystems. and abiotic factors. Can J Bot 70:661–675. Northeast Forestry University Press, Harbin, China.

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