Article Fine- Responses of Populus tomentosa Forests to Stand Density

Huijuan Bo 1, Chunyan Wen 1, Lianjun Song 2, Yatao Yue 1 and Lishui Nie 1,*

1 College of Forestry, Beijing Forestry University, Beijing 100083, China; [email protected] (H.B.); [email protected] (C.W.); [email protected] (Y.Y.) 2 Huayang Forest Tree Nursery, Xingtai 054700, Hebei, China; [email protected] * Correspondence: [email protected]; Tel.: +86-136-0113-146

 Received: 8 July 2018; Accepted: 7 September 2018; Published: 13 September 2018 

Abstract: Stand density directly affects the distribution of ecological factors such as light, heat, and water in forest communities and changes the diversity and structure of undergrowth species, thereby affecting soil health. Fine can provide water and nutrients to plants rapidly in the fierce competition of soil resources, so as to get rid of environmental factors. This study examined the fine-root responses of the Populus tomentosa clone S86 to three stand densities (plant × row spacing: 2 × 2 m, 4 × 3 m, 4 × 5 m). We measured the biomass, morphology, and nitrogen content of lower- (1–3 order) and higher-order (>3 order) fine roots, and analyzed soil chemical properties in 10–30 cm. The soil from the density (2 × 2 m) stands showed lower soil organic matter content, available nitrogen, available phosphorous, and available potassium than others. Obviously, lower and higher-order fine roots were different: biomass of the >3 order accounted for 77–87% of the total biomass, 1–3-order fine-root diameter around 0.28–0.38 mm, while >3-order fine root were 1.28–1.69 mm; the length of 1–3-order fine root was longer than the >3 order, and root length density, specific root length, and nutrient content between the 1–3 and >3 orders were different. At 2 × 2 m, 1–3-order fine-root biomass was the highest, 132.5 g/m3, and the 1–3-order fine-root length, diameter, surface, root length density was also the highest; at the same time, the 1–3-order fine-root total nitrogen and organic matter content was also the highest, while the >3 order was highest under 4 × 3 m or 4 × 5 m. The findings of this study show that stand density affected the available nutrient content of the soil. When soil resources were poor, the biomass, morphology, and chemical content of fine roots were adjusted to increase the nutrient absorption rate, particularly in the lower-order roots.

Keywords: fine-root order; fine-root nutrient; fine-root morphology; soil nutrient

1. Introduction Nutrients are essential to tree growth and development [1]. Fine roots play an important role in the cycling of water, nutrients, and carbon in terrestrial ecosystems [2]. Trees increase nutrient absorption from soil by increasing the number of fine roots [3,4], typically considered to be those that are 0–1 or 0–2 mm in diameter [2,5,6]. However, the exact mechanism of nutrient uptake, including how fine-root positioning in the soil influences function, is a neglected area of study [7]. Previous research suggests that individual roots should be divided into different orders based on distance away from the plant; thus, distal roots are first-order, originating from second-order (more proximal) roots [2,8], and so on. This categorization is useful for an examination of linkages between fine-root structure and function. For example, specific root length and root nitrogen concentration decrease with increasing root order, suggesting that physiology is relevant to root nitrogen [3,9]. Lower, first-, and second-order roots primarily absorb soil nutrients and water, while third- to fifth-order fine-root transport and store these resources [10–12]; hereafter, fine-root categories are referred to using Arabic numerals.

Forests 2018, 9, 562; doi:10.3390/f9090562 www.mdpi.com/journal/forests Forests 2018, 9, 562 2 of 14

For instance, a dissection of fine roots from Phellodendron amurense revealed that 1–3-order (first- to third-order) roots exhibited physiological characteristics related to absorption, whereas 4–5-order (fourth- and fifth-order) roots contained continuous phellem tissue indicative of functions related to conduction [13,14]. Both genetic [8,15,16] and environmental (e.g., soil characteristics) factors [17–20] influence fine-root behavior. Fine-root architecture, morphology, and chemistry can shift to maximize uptake in response to temporal and spatial resource fluctuations [21,22]. Under poor soil conditions, for example, fine-root biomass increased over a larger area [20,23] or decreased [24,25]. Furthermore, some studies have reported increases in specific root length, presumably to assimilate more nutrients [26–30], although other observations of reduced [31] or unchanged [24] indicate the need for more detailed research [32]. Regardless, root length is especially important for absorbing relatively immobile nutrients [33]. The correlation was not significant for diameter with either mass or length response to fertilization [34]. While trees can adjust phenotype in response to environmental change [22], the fine-root phenotype appears to be more plastic. Some studies have demonstrated variation in fine-root biomass, morphology, and architecture under differing stand densities, which is a major environmental factor in forest communities [35,36]. While the mechanisms for these morphological changes to fine roots are unclear, we know that stand density directly affects the distribution of light, heat, moisture, and other ecological factors in forest communities, resulting in changes to undergrowth species diversity and structure [27,37,38]. Moreover, soil nutrient content is directly proportional to density [39,40]. Thus, all of these variables may interact to influence fine-root plasticity. When strategically integrated into agroecosystems, fast-growing hybrid poplar plantations are a major source of biomass, wood/pulp production, and ecosystem services (e.g., phytoremediation, habitat creation, soil stabilization, hydrological control, and disturbance regulation) [41–44]. Populus tomentosa is ideal for research on fine-root responses to stand density. These trees have horizontal slanting roots [45] with a high proportion of fine roots distributed in the topsoil, and soil nutrient content is an important influencing factor for fine-root distribution [1]. Therefore, we studied P. tomentosa under three stand densities. We obtained samples to measure the biomass, morphology, and nutrient content of fine roots, and examined how these variables respond to changing soil resources. We hypothesized that (1) different densities alter available soil nutrients in soil depths of 10–30 cm, (2) fine roots adjust their biomass, morphology, and chemistry in response to different soil nutrients, (3) low-order fine roots are more sensitive to soil nutrient levels than high-order roots, and (4) low- and high-order roots differ significantly in biomass, morphology, and nutrient content.

2. Materials and Methods

2.1. Study Site The study took place in the Huayang Forest Tree Nursery, located in the heart of Huabei Great Plains (36◦500–37◦470 N, 113◦520–115◦490 E), a flat, alluvial region that experiences a warm-temperate, continental, semi-arid monsoon climate. Elevation ranges from 30 to 50 m. Monthly mean temperatures are −2.5 ◦C in January and 27.0 ◦C in July. Average total annual precipitation is approximately 497.7 mm, with 70% of rainfall occurring between July and September. Annual sunshine duration is estimated to be 2575 h, and there are approximately 198 frost-free days, these meteorological conditions come from 50 years (1960–2010) data of Weixian meteorological stations. As a key plantation for hybrid poplar breeding and large-scale timber production designated by the National Forestry Bureau of China, 26.7 ha of poplar stands were established in spring 2007. These stands began as dormant 2-year-old bare-rooted stem cutting, originating from stem cuttings from four P. tomentosa hybrid clones: P. tomentosa 1316, P. tomentosa B331, P. tomentosa BT17, and P. tomentosa S86 ( P. tomentosa × P. bolleana, P. alba × P. glandulosa). The stands contained seven density levels (2 × 2 m,♀ 2 × 3 m, 3 × 3 m, 4 × 3 m,♂ 4 × 4 m, 4 × 5 m, and 4 × 6 m). The average stem Forests 2018, 9, 562 3 of 14 height and base diameter of cuttings were 5.8 m and 6.1 cm, respectively. The experimental soil had a sandy loam texture. Table1 shows basic physical and chemical properties of the soil [ 46]. The data was from the average values of four layers (0–20 cm, 20–40 cm, 40–60 cm, and 60–100 cm), that we have dug 500 pits in the experimental site in 2007, and each pit has four layers. Table2 shows the mean stand properties under three densities.

Table 1. Soil physical and chemical properties at the experimental site.

Total Available Available Bulk Total Field Organic Matter N P K pH Density Porosity Capacity g/kg g/kg mg/kg mg/kg g/cm3 %% 8.6 ± 0.13 0.6 ± 0.09 8.1 ± 0.11 90 ± 0.08 8.6 ± 0.12 1.4 ± 0.11 47 ± 0.09 26 ± 0.12 Note: Total N represent total nitrogen; Available P represents available phosphorous, and Available K represents available potassium. The data was from the average values of four layers (0–20 cm, 20–40 cm, 40–60 cm, and 60–100 cm). We dug 500 pits at the experimental site in 2007, and each pit had four layers.

Table 2. Mean stand properties under three densities.

Stand Density T1 T2 T3 Basal area (m2) 132 288 360 Mean DBH (cm) 12.4 ± 0.31 18.1 ± 0.24 16.4 ± 0.13 Mean Dominant Height (m) 14.6 ± 0.25 16.6 ± 0.12 17.6 ± 0.35 Note: The mean DBH was measured with a diameter at breast height of 1.3 m, with an accuracy of 0.01 cm; the height was measured with an altimeter, with an accuracy of 0.1 m. DBH represents diameter at breast height. The data was measured in July 2016.

2.2. Experimental Design The experimental design was a single factor completely randomized design. The factor was P. tomentosa S86 planting density, with three levels (line × row: 2 × 2 m [T1], 4 × 3 m [T2], and 4 × 5 m [T3]; respective areas: 132 m2 (20 plants), 288 m2 (14 plants), and 360 m2 (10 plants). Every level contained three replicates, resulting in nine plots. Each plot was longer on the north–south side and wider on the east–west side, ensuring a more even distribution of light per tree. Tree row direction is along the north–south axis. Two rows of trees were planted for protection at a density of 4 × 3 m.

2.3. Data Collection Annual growth stages in P. tomentosa can be divided into six phases: germination, spring vegetative, spring capped, summer vegetative, overwintering preparation, and dormancy. Maximum root growth occurs during the summer vegetative phase [47]. After examining the distribution of roots, we found fine roots distributed more at a 10–30 cm level, and we then selected five standard trees from each plot in July 2016. A shovel was used to remove a 20 × 20 cm soil block (sampling at each tree spacing and distance direction) at approximately 80 cm distance from each selected tree, the block was separated into 10–30 cm segments, yielding 90 samples. These root-containing segments were carefully extracted and immediately placed on a white bag to remove soil and loose organic matter. Within several minutes to a few hours, samples were transported to the lab and frozen. Ninety soil samples (without roots) were collected using the quartet method and bagged.

2.4. Fine Root Analysis Soil particles and fine roots were washed with gauze and dipped in cooled deionized water. Roots were categorized into different orders. The first group contained 1–3-order (0.28–0.38 mm) roots and higher orders (>3; 1.28–1.69 mm) were placed in a second group; the former group contained more subcategories because the first-order fine roots of P. tomentosa are very complex. Samples from both groups were scanned to obtain images for analysis of root diameter, length, surface area, and volume Forests 2018, 9, x FOR PEER REVIEW 4 of 14

contained more subcategories because the first-order fine roots of P. tomentosa are very complex. ForestsSamples2018 ,from9, 562 both groups were scanned to obtain images for analysis of root diameter, length,4 of 14 surface area, and volume in WinRHIZO (Pro2005c) (Shijiazhuang, Heibei, China). Roots were then wrapped in filter paper, heated for 10–30 min at 85 °C, and dried at 65–70 °C to a constant weight. in WinRHIZOThe fine root (Pro2005c) were ground (Shijiazhuang, and then Heibei, were sieved China). (2 Rootsmm) in were preparation then wrapped for nutrient in filter analysis paper, heated for 10–30 min at 85 ◦C, and dried at 65–70 ◦C to a constant weight. (total nitrogen, total phosphorus, and total potassium) using H2SO4–H2O2 digestion and then N (total nitrogen),The fine P (total root phosphorus), were ground K and (total then potassium were sieved) concentrations (2 mm) in preparation were measured for nutrient using a analysisKjeldahl (totalnitrogen nitrogen, meter total (Hangzhou, phosphorus, Zhejiang, and total China) potassium), a vanadium using H2SO molybdenum4–H2O2 digestion yellow and thencolorimetric N (total nitrogen),method, and P (total a flame phosphorus), photometric K method, (total potassium) respectively concentrations [48]; C (organic were carbon measured) concentration using a Kjeldahls were nitrogenmeasured meter using (Hangzhou, a Carlo Erba Zhejiang, NA 1500 China), NC elemental a vanadium analyzer molybdenum (Elantech, yellow Lakewood, colorimetric NJ, USA). method, and a flame photometric method, respectively [48]; C (organic carbon) concentrations were measured using2.5. Soil a CarloAnalysis Erba NA 1500 NC elemental analyzer (Elantech, Lakewood, NJ, USA).

2.5. SoilSoil Analysis samples were air-dried, ground, and then sieved (2 mm) for measuring available N (an alkali solution diffusion method), P (an NaHCO3 extraction method), and K (an NH4OAc extraction Soil samples were air-dried, ground, and then sieved (2 mm) for measuring available N (an alkali method). Soil organic matter was sieved (0.25 mm) and measured using a heat of dilution method solution diffusion method), P (an NaHCO extraction method), and K (an NH OAc extraction [48]. Hydrolysis nitrogen is abbreviated by 3available N, available phosphorous is4 abbreviated by method). Soil organic matter was sieved (0.25 mm) and measured using a heat of dilution method [48]. available P, available potassium is abbreviated by available K, and soil organic carbon is abbreviated Hydrolysis nitrogen is abbreviated by available N, available phosphorous is abbreviated by available by SOC. P, available potassium is abbreviated by available K, and soil organic carbon is abbreviated by SOC.

2.6.Data2.6.Data AnalysesAnalyses Fine-rootFine-root morphologicalmorphological variablesvariables collectedcollected includedincluded specificspecific rootrootlength length(SRL, (SRL, m/g),m/g), root length 3 density (RLD,(RLD, m/mm/m3),), and and root root C:N C:N,, calculated as follows: SRL = fine fine-root-root length/fine-rootlength/fine-root dry dry mass mass,, RLD == fine-rootfine-root length/volume of of soil soil block block,, and and C:N C:N = = fine fine-root-root C C content/fine content/fine-root-root N N content content.. MeansMeans andand standard standard deviations deviation ofs variablesof variables per plotper wereplot calculatedwere calculated in Excel in 2010. Excel Three 2010. one-way Three ANOVAsone-way ANOVAs (in SPSS 20.0, (in SPSS Shanghai, 20.0, China)Shanghai, were China used) towere determine used to (1) determine significant (1) differences significant between differences the twobetween fine-root the two groups fine in-root soil groups nutrient in content, soil nutrient (2) significant content, differences(2) significant across differences differing across stand densitiesdiffering instand total densities fine-root in biomass, total fine morphology,-root biomass, and morphology, nutrient content, and nutrient and (3) significantcontent, and index (3) differencessignificant betweenindex difference the two fine-roots between groups. the two Duncan’s fine-root multiple groups comparison. Duncan’s tests multiple were employed comparison to separate tests were the meansemployed when to theseparate main the effect means of the when ANOVA the main was significant.effect of the ANOVA was significant.

3. Results

3.1. Differences in Soil Available NutrientsNutrients Soil organic matter varied varied from from 6.33 6.33 ±± 0.230.23 g/kg g/kg to 9.90 to 9.90 ± 0.37± 0.37 g/kg g/kg and available and available N varied N varied from from23.9 ± 23.9 0.61± mg/g0.61 to mg/g 32.65 to ± 32.650.52 mg/g± 0.52; both mg/g; exhibited both exhibited the following the following density-related density-related pattern: T2 pattern: > T3 > T2T1 >(Figure T3 > T1 1a,(Figureb). Available1a,b). Available P and K P peaked and K peakedat T3 (Fig at T3ure (Figure 1 c,d).1 c,d).The ANOVA The ANOVA results results indicated indicated that thatdensity density exerted exerted a significant a significant effect effect on soil on soilavailable available nutrients, nutrients, except except available available P. P.

12 35 ( a ) a a ( b )

10 30 b

b c 8 25 c

6 Available N(mg/g) 20 Organic matter Organic matter (g/kg)

4 15

2 10 Figure 1. Cont.

Forests 2018, 9, x FOR PEER REVIEW 5 of 14 ForestsForests2018 2018, 9,, 562 9, x FOR PEER REVIEW 5 of5 14of 14

20 140 20 140 ( d ) ( c ) ( d ) ( c ) a a b a b a a a 15 110 15 110 b b

Available P (mg/g) c Available K (mg/g) Available P (mg/g) 10 80 c

10 Available K (mg/g) 80

5 50 5 50 T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3 Figure 1. Soil effective nutrient content in three stand densities of Populus tomentosa S86. Data are the FigureFigure 1. Soil1. Soil effective effective nutrient nutrient content content in in three three stand stand densities densities ofof PopulusPopulus tomentosa S86. Data Data are are the soil mean effective nutrient content ± standard deviation. Letters represent differences between thesoil soil mean mean effective nutrientnutrient contentcontent± ± standardstandard deviation. deviation. Letters Letters represent represent differences differences between between densities (α = 0.05). T1 represents the density of 2 × 2 m, T2 represents the density of 4 × 3 m, T3 densitiesdensities (α (α= 0.05).= 0.05). T1 T1 represents represents the the density density of of 2 2× × 22 m,m, T2T2 representsrepresents thethe density of 4 × 33 m, m, T3 represents the density 4 × 5 m (line × row spacing). And (a) describes the contents of soil organic T3represents represents the the density density 4 4 ×× 5 5m m (line (line × ×rowrow spacing). spacing). And And (a) (describea–d) describes the contents the contents of soil of organic soil matter; (b) describes alkali-hydrolyzed nitrogen; (c) describes available phosphorus; (d) describes organicmatter; matter, (b) describe alkali-hydrolyzeds alkali-hydrolyzed nitrogen, availablenitrogen; phosphorus (c) describe ands available available phosphorus; potassium under (d) describe three s available potassium under three planting densities. plantingavailable densities, potassium respectively. under three planting densities.

3.2. Responses of Fine-RootFine-Root Biomass 3.2. Responses of Fine-Root Biomass Fine-rootFine-root biomass biomass significantly significantly differed differed across across the the three three density density levels levels and and between between the two the tworoot Fine-root biomass significantly differed across the three density levels and between the two root rootorder order groups groups (1–3-order (1–3-order and >3 and-order >3-order roots) roots).. Total and Total >3 and-order >3-order group biomass group biomass peaked peakedat T2 (Fig ature T2 order groups (1–3-order and >3-order roots). Total and >3-order group biomass peaked at T2 (Figure (Figure2), which2), was which significantly was significantly higher than higher at thanother at density other densitylevels, and levels, soil and available soil available nutrients nutrients (organic 2), which was significantly higher than at other density levels, and soil available nutrients (organic (organicmatter and matter N) andwere N) higher were higher at this at density this density (Figure (Figure 1). 1Interestingly,). Interestingly, the the biomass biomass of of the 1–3-order1–3-order matter and N) were higher at this density (Figure 1). Interestingly, the biomass of the 1–3-order group increasedincreased significantlysignificantly with increasing density.density. BiomassBiomass ofof thethe >3-order>3-order group accounted for group increased significantly with increasing density. Biomass of the >3-order group accounted for 77–87%77–87% of the totaltotal biomassbiomass (Figure(Figure3 3).). Thus,Thus, P. tomentosa finefine roots exhibit differential growth at 77–87% of the total biomass (Figure 3). Thus, P. tomentosa fine roots exhibit differential growth at different standstand densities.densities. different stand densities.

800 a 1-3 group 800 a 1-3 group ＞ ) Aa 3 group 3 ＞ ) Aa 3 group 3 b total 600 total 600 b c Ab c Ab Ac 400 Ac 400 root biomass (g/m biomass root root biomass (g/m biomass root Fine -

200Fine - 200 Ba Ba Bb Bc Bb Bc

0 0 T1 T2 T3 T1 T2 T3 Figure 2. Fine root biomass in three stand densities of Populus tomentosa S86. Figure 2. Fine root biomass in three stand densities of Populus tomentosa S86. Figure 2. Fine root biomass in three stand densities of Populus tomentosa S86.

Forests 2018, 9, 562 6 of 14 ForestsForests 20182018,, 99,, xx FORFOR PEERPEER REVIEWREVIEW 66 ofof 1414

1-31-3 groupgroup ＞＞33 groupgroup 100%100%

50%50% root biomass root biomass ratio ) (% root biomass root biomass ratio ) (% Fine - Fine -

0%0% T1T1 T2T2 T3T3

FigureFigure 3.3.3. Fine-root FineFine--rootroot biomass biomassbiomass ratio ratioratio in threeinin threethree stand standstand densities densitiesdensities of Populus ofof PopulusPopulus tomentosa tomentosatomentosaS86. Data S86.S86. are DataData the fine-root areare thethe meanfinefine--rootroot biomass meanmean± biomassbiomassstandard ±± deviation. standardstandard Capitaldeviation.deviation. letters CapitalCapital represent lettersletters significant representrepresent differences significantsignificant between differencesdifferences the 1–3-orderbetweenbetween thethe group 11––33--order andorder the groupgroup >3-order andand thethe group >3>3--orderorder under groupgroup the same underunder density thethe samesame (α = densitydensity 0.05); lower(α(α == 0.05);0.05); letters llowerower represent lettersletters differencesrepresentrepresent differencesdifferences in the same inin thethe group samesame between groupgroup betweenbetween different differentdifferent densities. densities.densities. T1 represents T1T1 representsrepresents the density thethe densitydensity of 2 ×ofof 22 ×× m, 22 T2m,m, representsT2T2 representsrepresents the density thethe densitydensity of 4 × ofof3 m,44 ×× T3 33 representsm,m, T3T3 representsrepresents the density thethe 4densitydensity× 5 m 4 (line4 ×× 55× mmrow (line(line spacing); ×× rowrow spacing)spacing) different;; groupsdifferentdifferent were groupsgroups the 1–3-orderwerewere thethe 11 group––33--orderorder and groupgroup >3-order andand group. >3>3--orderorder groupgroup..

3.3.3.3. Differences Differences inin Fine-RootFineFine--RootRoot MorphologyMorphology Fine-rootFineFine--rootroot morphology morphologymorphology differed differeddiffered across acrossacross density densitydensity levels, levels,levels, with numerous withwith numerousnumerous 1–3-order 11–– fine33--orderorder roots fine branchingfine rootsroots andbranchingbranching proliferating andand proliferatingproliferating at T1. No such atat T1.T1. complexity NoNo suchsuch complexitycomplexity occurred at occurredoccurred T2 and T3. atat T2T2 andand T3.T3. WeWe observedobservedobserved thatthat fine-rootfinefine--rootroot length,length,length, surfacesurface area,area, RLD,RLD, andand SRLSRL werewere significantlysignificantlysignificantly higherhigher inin thethe 1–3-order11––33--orderorder groupgroup thanthanthan ininin thethethe >3-order>3>3--orderorder groupgroup (Figure(Fig(Figureure4 4).).4). In InIn contrast, contrast,contrast, fine-root finefine--rootroot diameter diameterdiameter and andand volume volumevolume werewere significantlysignificantlysignificantly higherhigher inin thethe >3-order>3>3--orderorder groupgroup thanthan inin thethe 1–3-order11––33--orderorder group.groupgroup.. FinerFiner diameter,diameter, longerlonger rootroot length,length, andand greatergreater SRLSRL allall indicateindicate strongerstronger absorptionabsorption capacity, capacity, suggestingsuggesting thatthat thethe 1–3-order11––33--orderorder rootsroots cancan bebe logicallylogically groupedgroupedgrouped together.together.together. TotalTotal rootrootroot length,length,length, surfacesurfacesurface area,area, andand RLDRLDRLD ofofof allallall finefinefine roots,roots,roots, 1–3-order11––33--orderorder roots,roots,roots, andandand >3-order>3>3--orderorder rootsroots werewere highesthighest atat T1T1 density,density, whilewhile totaltotal diameter,diameter, volume,volume, andand SRL SRL were were highest highest under under T3 T3 (Fig (Figure(Figureure 4 4).4).). However,However, SRL SRL waswas lowestlowest lowest underunder under T2,T2, T2, possiblypossibly possibly becausebecause because ofof ofincreasedincreased increased soilsoil soil resourcesresources resources andand and biomassbiomass biomass atat thatthat at thatdensity.density. density. WithinWithin thethe 1–3-order11––33--orderorder group,group, thethe greatestgreatest fine-rootfinefine--rootroot length,length, diameter,diameter, surfacesurface area,area, andand RLDRLD werewere observedobserved atat T1;T1; inin thethe >3-order>3>3--orderorder group, group, the the greatestgreatest fine-rootfinefine--rootroot length,length, surfacesurface area,area, andand volumevolume werewere observedobserved atat T2T2 (Figure(Fig(Figureure4 4).4).). These TheseThese results resultsresults suggest suggestsuggest that thatthat the thethe >3-order >3>3--orderorder group groupgroup responded responderespondedd less lessless strongly stronglystrongly to toto densitydensity variationvariation thanthan thethe 1–3-order11––33--orderorder group.group.group.

3535 ((aa)) 22 ((bb)) aa 1-31-3 groupgroup aa 30 30 ＞＞33 groupgroup aa bb aa bb 1.51.5 2525 totaltotal bb 2020 bb bb bb 11 b bb b 1515 Diamter(mm) Diamter(mm) Root length (cm) Root length (cm) a 1010 a aa 0.50.5 bb bb bb bb 55

0 0 0 0

Figure 4. Cont.

ForestsForests2018 ,20189, 562, 9, x FOR PEER REVIEW 7 of7 14 of 14

3.5 (c) 0.15 a a (d) b a 3.0 a 0.12

) a 2 ) 2.5 3 a 0.09 a 2.0 b a c a

b (cm Volume 0.06

Surface (cm area 1.5 a a b b 0.03 1.0 b

0.5 0

40 a (e) 40 (f) a a b b 30 30 b b b

20 20 SRL (cm/g) RLD (m/m3) a 10 10 a a a a c a a a a 0 0 T1 T2 T3 T1 T2 T3 FigureFig 4.ureFine-root 4. Fine- morphologyroot morphology for two for groups two ofgroups roots andof roots all roots and combined all roots incombined three stand in densitiesthree stand of Populusdensities tomentosa of PopulusS86. tomentosa Data are theS86. fine-root Data are morphology the fine-root± morphologystandard deviation. ± standard Letters deviation. represent Letters differencesrepresent between differences densities between (α = 0.05).densities T1 represents (α = 0.05). the T1 densityrepresents of 2 the× 2 density m, T2 represents of 2 × 2 m, the T2 density represents of 4 the× 3 density m, T3 represents of 4 × 3 m, the T3 density represents 4 × 5 the m (linedensity× row 4 × spacing),5 m (line different× row spacing) groups, weredifferent the 1–3-ordergroups were groupthe and 1–3- theorder >3-order group and group. the The>3-order root group. length, The surface, root length, and volume surface per, and 0.008 volume m3. SRLper 0.008 represent m3. SRL specificrepresent root length, specific m/g; root RLDlength, represent m/g; RLD root represent length density, root length m/m density,3.(a–f) describem/m3. Under the root three length, planting diameter,densities surface,, (a) describe volume,s RLD the root and SRLlength under; (b) threedescribe plantings diameter densities.; (c) describes surface; (d) describes volume; (e) describes RLD; (f) describes SRL. 3.4. Differences in Fine-Root Nutrient Content 3.4In. theDifferences >3-order in roots, Fine-Root organic Nutrient C (464.27–534.93 Content g/kg) and total K (3.87–3.97 g/kg) were higher than in the 1–3-orderIn the >3 roots.-order However, roots, organic the latter C (464.27 group– had534.93 significantly g/kg) and highertotal K total(3.87 N–3.97 (25.29–31.98 g/kg) were g/kg) higher andthan total in P the (2.30–2.53 1–3-order g/kg) roots. than However, the former the group.latter group The C:Nhad ratiosignificantly was significantly higher total higher N (25.29 in the–31.98 >3-orderg/kg) rootsand total than P in (2.30 the– 1–3-order2.53 g/kg) group, than the possibly former due group. to the The nitrogen C:N ratio of the was 1–3-order significantly group higher was in higherthe (Figure>3-order5). roots than in the 1–3-order group, possibly due to the nitrogen of the 1–3-order group wasGenerally, higher ( acrossFigure all5). fine roots (total, 1–3 order, and >3 order), T1 density resulted in higher nutrientGenerally, content (except across K), all but fine there roots was (total, no difference 1–3 order, between and >3 T2 order and T3), T1 (Figure density5). Mean resulted organic in higher C in allnutrient fine roots con rangedtent (except from 438.83K), but to there 495.33 was g/kg, no difference increasing between as density T2 increased.and T3 (Figure Average 5). Mean N content organic rangedC in from all fine 23.59 roots to 26.02 ranged g/kg; from T1 438.83 fine roots to 495.33 had significantly g/kg, increasing higher as N density than roots increased. under the Average other N twocontent density ranged levels. from Moreover, 23.59 to the 26.02 total g/kg; fine-root T1 fine N roots of 1–3-order had significant increasedly higher as density N than increased, roots under but the N inother >3-order two density roots showed levels. theMoreover, opposite the response. total fine Although-root N of soil1–3- organicorder increased matter was as density relatively increased, low underbut T1 N (Figurein >3-order5), fine-root roots showed organic the C wasopposite highest response. at this density. Although This soil pattern organic may matter be the was result relatively of fiercelow competition under T1 ( forFigure limited 5), resources,fine-root organic leading C to was more highest C being atinvested this density. into fineThis roots. pattern may be the resultTotal of P contentfierce competition in fine roots for (total) limited varied resources, from 2.13 leading to 2.33 to more g/kg. C Total being P invested from all fineinto rootsfine roots. was significantlyTotal higher P content under in T3fine than roots under (total) the varied other twofrom densities. 2.13 to 2.33 Total g/kg. fine-root Total KP rangedfrom all from fine 3.80roots to was 3.83significantly g/kg, and density higher hadunder no T3 significant than under effect the on other this variable.two densities. Total fine-root K ranged from 3.80 toThe 3.83 C:N g/kg, ratio and density in all finehad no roots significant ranged effect from on 18.05 this tovariable 19.08. and exhibited the following density-relatedThe C:N pattern: ratio in T1 all (19.08) fine >roots T3(18.60) ranged > T2(18.05)from 18.05 (Figure to 19.085). However,and exhibited density the did following not density-related pattern: T1 (19.08) > T3(18.60) > T2(18.05) (Figure 5). However, density did not

ForestsForests 20182018, 9, ,x9 FOR, 562 PEER REVIEW 8 of8 14 of 14 significantly affect total fine-root C:N. When fine roots were examined by group, we observed that thesignificantly 1–3-order roots affect had total significant fine-rootly C:N. lower When C:N fine under roots T2, were whereas examined >3-order by group, roots we had observed significantly that the higher1–3-order C:N under roots hadT1. significantly lower C:N under T2, whereas >3-order roots had significantly higher C:N under T1.

600 35 (a) (b) total a a 1-3 550 ＞3 a 30 500 b b b a a b b 450 b 25 b b

Total nitrogen(g/kg) Total a b Organic carbon (g/kg) carbon Organic 400 b b ab 20 350

3.0 (c) 4.2 (d)

a b 2.5 (g/kg) 4.0 a a b a a b a b a a b potassium a 2.0 b 3.8 a a a Total Total phosphorus(g/kg) Total

1.5 3.6

30 (e) a 25 b b a nitrogen ratio 20 a a

a a - Carbon b 15

10 T1 T2 T3

FigureFigure 5. Total 5. Total and and group group fine fine-root-root nutrients nutrients in in three three stand stand densities densities of Populus tomentosatomentosa S86.S86. DataData are arethe the fine-rootfine-root meanmean nutrientnutrient content ± standardstandard deviation. deviation. Letters Letters represent represent significant significant differences differences betweenbetween densities densities (α (=α 0.05).= 0.05). T1 T1represents represents the the density density of of2 × 2 2× m,2 m,T2 T2represents represents the the density density of of4 ×4 3× m,3 m, T3 T3represents represents the the density density 4 × 4 5× m5 m(line (line × row× row spacing). spacing). And And (a) ( adescribe–e) describes the thecontents contents of total of total nitrogen nitrogen,; (b)organic describe carbon,s the contents total phosphorus, of organic total carbon potassium; (c) describe and carbon-nitrogens the contents of ratio total under phosphorus; three planting (d) describedensities,s the respectively; contents of differenttotal potassium; groups were:(e) describe 1–3-orders carbon roots-nitrogen and >3-order ratio roots. under three planting 4.densities, Discussion respectively and Conclusions; different groups were: 1–3-order roots and >3-order roots.

4. Discussion4.1. Defining and Fine Conclusions Roots

4.1. DefiningPopulus Fine tomentosa Roots has relatively more fibrous roots than other trees (such as Platycladus orientalis, Fraxinus mandshurica, Larix olszensis, Cunninghamia lanceolata)[35,49,50], necessitating a division into thePopulus 1–3-order tomentosa and >3-order has relatively groups formore analysis. fibrous Here,roots wethan demonstrated other trees that(such the as two Platycladus groups are orientalisdifferent, Fraxinus from their mandshurica morphology, Larix and olszensis nutrient, content.Cunninghamia Previous lanceolata research) [35,49,50] indicated, thatnecessitating first-, second-, a divisionand third-order into the 1 fine–3-order roots and do not>3- differorder ingroups diameter for analysis. or length, Here, but SRL we changeddemonstrated significantly that the between two groupsthird- are and different fourth-order from their fine rootsmorphology [51]. Additionally, and nutrient an content. anatomical Previous analysis research of fine indicated roots (≤ 2that mm) firstrevealed-, second that-, and they third can be-order split fine into roots at least do two not groups: differ non-lignifiedin diameter or absorption length, but roots SRL (1–3 changed mm) and significantly between third- and fourth-order fine roots [51]. Additionally, an anatomical analysis of

Forests 2018, 9, 562 9 of 14 more lignified roots with no absorption capacity (>4 mm) [11,52]. At the same time, they suggested that the fine root should be split into two distinct classes or pools: absorptive fine roots and transport fine roots [2]. Together, these results suggest that existing methods of categorizing fine roots have ecological validity. Fine root categories also differ strongly in their response to soil resource availability [8]. At a density of 2 × 2 m, the 1–3-order group would show more adaptation in their morphology (Figure5) and nutrient content than the >3-order group (Figure5). The mechanism of this plasticity may be an increase in the number of cortical cells for the 1–3-order group, which, when affected by the external environment, would show obvious changes [53–55]. Similarly, research on sugar maple roots has shown that roots <0.5 mm in diameter have more N [9] and a higher respiration rate (by 2.4–3.4 times) than thicker roots [9].

4.2. Morphological Characteristics of 1–3-Order and >3-Order Fine Roots Variation in morphology was noted across the two fine-root groups; biomass, diameter, and volume were lower in the 1–3-order group, but root length, surface area, SRL, and RLD were higher than the >3-order fine roots. Likewise, previous research has demonstrated that, as root order increases, root length, diameter, and biomass also increase, while SRL decreases [56–58]. In contrast, a separate study demonstrated that fine-root biomass did not increase with root order [59]. While these studies differ in terms of the tree species examined and the exact response of root morphological characteristics [56], all of them indicate that the majority of fine-root biomass was in senior branch roots. Variations between studies may also be due to methodological difficulties in isolating fine roots from soil without damage, increasing the likelihood of estimation errors. Our study also showed that low-order fine roots had significantly higher total N than higher-order roots. Similarly, first-order roots in Populus balsamifera had the highest N content, a characteristic closely related to respiration rate [9]. This outcome is possibly attributable to the large amount of secondary tissue in the fourth- and fifth-orders roots; these tissues generally have lower physiological activity and do not require high N investment [11]. However, the 1–3-order group contained significantly less organic matter than the >3-order group—in line with previous results [60]—a pattern that may be due to the shorter life span and rapid turnover of lower-order roots [61]. Finally, the >3-order roots had significantly higher C:N than the 1–3-order roots, probably as a result of the low N content. The value of the C:N ratio indicates that, when organic matter input is low (i.e., ~1 g), low-order roots require more maintenance from the tree.

4.3. Variation in Fine Root Traits under Differing Stand Density In this study, three different soil environments were constructed by varying stand density, and this variation significantly affected soil available nutrients (Figure1). The effects of stand density on fine-root biomass have been extensively studied, but the results are inconsistent. Various studies have found increasing, decreasing, or nonlinear trends in fine-root biomass as stand density increases [35,62–64]; this inconsistency may be a factor of density-related changes to soil nutrient content. Notably, biomass in the 1–3-order group decreased with decreasing density, possibly because soil resources are relatively abundant at lower densities (4 × 3 m), so more absorptive roots were not necessary for nutrient uptake. Furthermore, the 1–3-order group had greater surface area and volume at high densities (2 × 2 m, where soil resources were lower), suggesting that fine roots stretch as far as possible to increase contact with soil under low nutrient conditions, thereby elevating resource absorption [61]. However, an experiment using Drew nutrient solution sand cultures showed that, under high ammonium, nitrate, and phosphorus concentrations, total root length increased significantly, as did first- and second-order root growth [32]. These opposing findings may be attributable to the fact that a nutrient concentration gradient was not directly measured in this study; instead, we primarily Forests 2018, 9, 562 10 of 14 examined the effects of density, which influences nutrient content and affects competition between fine roots, an aspect that the previous study did not consider. Specific root length is the ratio of fine-root length and biomass; thus, it is closely related to physiological activity. Specifically, absorption capacity in fine roots is higher with greater SRL [65], an adaptive response that allows the tree to invest less in biomass, but absorb more nutrients. In this study, SRL was higher under densities of 2 × 2 m and 4 × 5 m, possibly because of poorer soil resources that require fine-root extension to increase nutrient absorption at relatively low cost. We also found that when SOC, available N, available P, and available K were high (i.e., in the soil of the 4 × 3 m stand), only total P content of the fine roots increased. Other studies on wheat and grass also showed that fine-root N content did not increase, even under N-rich conditions [66]. An experimental study similarly demonstrated that increasing N in soil did not increase N content in fine roots [9]. These results may be due to the fact that fine-root nutrient content has a threshold value that is not representative of soil nutrient content. Instead, aboveground parts may contain the remaining nutrients. Forest can affect the distribution of carbon and the rate of nutrient absorption [67,68]. Although it has been reported that Populus tomentosa and ectomycorrhizal (sclerodermataceae) form the mycorrhizae, which significantly increase the root biomass of poplar [69], but also improve the host tree’s general conditions and stress tolerance [70]. However, the current study was carried out in an artificial forest and there was no mycorrhizal formation. Despite the strengths of this study, there are some notable limitations. First, variations in soil moisture and temperature may influence fine-root distribution, but examining these factors was beyond the current scope [19,71–73]. Second, forest mycorrhiza can affect C distribution and nutrient absorption rates; indeed, a previous study found that mycorrhizae significantly increased P. tomentosa root biomass. As this work was performed in artificial forests, however, mycorrhizal effects were not examined. Thus, further research accounting for soil moisture, temperature, and mycorrhizal presence is recommended to validate fine-root characteristics in response to soil resource variation under differing stand densities. Plants exhibit high phenotypic plasticity in response to environmental factors. Although the aboveground parts can exhibit differences in size, growth rate, and dry matter distribution even within the same plant [12], variation in tree density can affect growth and biomass partitioning [74,75]. Hyatt et al. found that increased density resulted in higher ratios of stem to leaf biomass in Abution [76]. While Al Afast et al. did not observe significant correlations between fine-root traits and above-ground biomass in Populus [77]. This study showed that soil nutrient content is lowest at high stand density (2 × 2 m), causing fine roots to change drastically in terms of biomass, morphology, and nutrient content. The fine-root biomass of 4 × 3 m increased by over 30% at 2 × 2 m, and the fine-root biomass of 4 × 5 m was 35%, 146.28% less than at 4 × 3 m and 2 × 2 m. The stand volume of 4 × 3 m (86.80 m3/hm2) decreased by over 28% at 2 × 2 m (120.60 m3/hm2), and the fine-root biomass of 4 × 5 m (64.75 m3/hm2) was 25%, 46% less than at 4 × 3 m and 2 × 2 m. In the future, we will carry out the effect of density on aboveground biomass, so as to compare with the underground fine roots. This study suggests that the management of dense forests should include fertilization or thinning to improve soil nutrient content and therefore forest productivity.

Author Contributions: Additional support was provided by the Huayang Forest Tree Nursery. L.N., L.S., and H.B. conceived and designed the experiments; H.B., L.S., and Y.Y. performed the experiments; H.B., C.W., and Y.Y. collected samples and analyzed the data; H.B. wrote the paper. Funding: This research received no external funding. Acknowledgments: This work was supported by the Key Technologies R&D Program of China (2015BAD09B02). Conflicts of Interest: The authors declare no conflict of interest. Forests 2018, 9, 562 11 of 14

References

1. Nan, D.; Xi, B.Y.; Jeremiah, R.P. Root biomass distribution of triploid Populus tomentosa under wide-and narrow-roe spacing planting scheme and its responses soil nutrients. Chin. J. Plant Ecol. 2013, 10, 961–971. 2. McCormack, M.L.; Dickie, I.; Eissenstat, D.M.; Fahey, T.; Fernandez, C.; Guo, D.; Helmisaari, H.-S.; Hobbie, E.; Iversen, C.; Jackson, R.; et al. Redefining fine roots improves understanding of belowground contributions to terrestrial biosphere processes. New Phytol. 2015, 207, 505–518. [CrossRef][PubMed] 3. Bilbrough, C.J.; Caldwell, M.M. The effects of shading and N status on root proliferation in nutrient patches by the perennial grass Agropyron desertorum in the field. Oecologia 1995, 103, 10–16. [CrossRef][PubMed] 4. Eissenatat, D.M.; Caldwell, M.M. Seasonal timing of root growth in favorable microsites. Ecology 1988, 69, 870–873. [CrossRef] 5. Majdi, H.; Pregitzer, K.; Moren, A.S. Measuring fine root turnover in forest ecosystems. Plant Soil 2005, 276, 1–8. [CrossRef] 6. Jagodzi´nski,A.M.; Kałucka, I. Fine root biomass and morphology in an age-sequence of post-agricultural Pinus sylvestris L. stands. Dendrobiology 2011, 66, 71–84. 7. Vogt, K.A.; Vogt, D.J.; Palmetto, P.A. Review of root dynamics in forest ecosystem grouped by climate, climatic forest type and species. Plant Soil 1996, 187, 159–219. [CrossRef] 8. Pregizter, K.S.; De Forest, J.L.; Burton, A.J. Fine root architecture of nine North American trees. Ecol. Monogr. 2002, 72, 293–309. 9. Pregitzer, K.S.; Laskowski, M.J.; Burton, A.J. Variation in sugar maple root respiration with root diameter and soil depth. Tree Physiol. 1998, 18, 665–670. [CrossRef][PubMed] 10. Guo, D.L.; Xia, M.X.; Wei, X. Anatomical traits associated with absorption and mycorrhizal colonization are linked to root branch order in twenty-three Chinese temperate tree species. New Phytol. 2008, 18, 673–683. [CrossRef][PubMed] 11. Hishi, T. Heterogeneity of individual roots within the fine root architecture: Causal links between physiological and ecosystem functions. J. For. Res. 2007, 12, 126–133. [CrossRef] 12. Zadworny, M.; Eissenstat, D. Contrasting the morphology, anatomy and fungal colonization of new pioneer and fibrous roots. New Phytol. 2011, 190, 213–221. [CrossRef][PubMed] 13. Zadworny, M.; McCormack, M.L.; Rawlik, K.; Jagodzi´nski,A.M. Seasonal variation in chemistry, but not morphology, in roots of Quercus robur growing in different soil types. Tree Physiol. 2015, 35, 644–652. [CrossRef][PubMed] 14. Wei, X.; Lin, Y.; Chen, H.B. Anatomical and functional heterogeneity among different root orders of phellodendron amurense. J. Plant Ecol. 2008, 6, 1238–1247. 15. Espeleta, J.F.; West, J.B.; Donovan, L.A. Tree species fine-root demography parallels habitat specialization across a sand hill soil resource gradient. Ecology 2009, 90, 1773–1787. [CrossRef][PubMed] 16. King, J.; Pregitzer, K.; Zak, D.; Holmes, W. Fine root chemistry and decomposition in model communities of

nort—Temperate tree species show little response to elevated atmospheric CO2 and varying soil resource availability. Oecologia 2005, 146, 318–328. [CrossRef][PubMed] 17. Finer, L.; Helmisaari, H.S.; Lohmus, K. Variation in fine root biomass of three European tree species: Beech (Fagus sylvatica L.), Norway spruce (Picea abies L. Karst.), and Scots pine (Pinus sylvestris L.). Plant Biosyst. 2017, 141, 394–405. [CrossRef] 18. Hodge, A. The plastic plant: Root responses to heterogeneous supplies of nutrients. New Phytol. 2004, 162, 9–24. [CrossRef] 19. Schenk, H.J. Vertical vegetation structure below ground: Scaling from root to globe. Prog. Bot. 2005, 66, 341–373. [CrossRef] 20. Yuan, Z.Y.; Chen, H.Y. Fine root biomass, production, turnover rates, and nutrient contents in Boreal Forest ecosystems in relation to species, climate, fertility, and stand age: Literature review and meta-analyses. Crit. Rev. Plant Sci. 2010, 29, 204–221. [CrossRef] 21. Bardgett, R.D.; Mommer, L.; Vries, F.T. Going underground: Root traits as drivers of ecosystem process. Trends Ecol. Evol. 2014, 29, 229–238. [CrossRef][PubMed] 22. Monique, W.; Frank, J.; Eric, J.W. Fine-root trait plasticity of beech (Fagus sylvatica) and spruce (Picea abies) forests on two contrasting soils. Plant Soil 2017, 415, 175–188. Forests 2018, 9, 562 12 of 14

23. Hertel, D.; Strecker, T.; Muelle-Haubold, H.; Leuschner, C. Fine root biomass and dynamics in beech forests across a precipitation gradient-is optimal resource partitioning theory applicable to water—Limited mature trees? J. Ecol. 2013, 29, 204–221. [CrossRef] 24. Brunner, I.; Herzog, C.; Dawes, M.A. How tree roots respond to drought. Front Plant Sci. 2015, 6, 547. [CrossRef][PubMed] 25. Leuschner, C.; Hertel, D. Fine root biomass of temperate forests in relation to soil acidity and fertility, climate, age and species. Prog. Bot. 2003, 64, 405–438. 26. Cui, M.; Caldwell, M.M. Nitrate and phosphate uptake by Agropyron desertorum and Artemisia tridentate from soil patches with balanced and unbalanced nitrate and phosphate supply. New Phytol. 1998, 139, 267–272. [CrossRef] 27. Jackson, R.B.; Manwaring, J.H.; Caldwell, M.M. Rapid physiological adjustment of roots to localized soil enrichment. Nature 1990, 344, 58–60. [CrossRef][PubMed] 28. Jackson, R.B.; Caldwell, M.M. Kinetic responses of Pseudoroegneria roots to localized soil enrichment. Plant Soil 1991, 138, 231–238. [CrossRef] 29. Ryel, R.J.; Caldwell, M.M.; Manwaring, J.H. Temporal dynamics of soil spatial heterogenity in sage-brush-wheatgrass steppe during a growing season. Plant Soil 1996, 184, 299–309. [CrossRef] 30. Ryel, R.J.; Caldwell, M.M. Nutrient acquisition from soils with patchy nutrient distributions as assessed with simulation models. Ecology 1998, 79, 2735–2744. [CrossRef] 31. Ostonen, I.; Puttsepp, U.; Biel, C. Specific root length as an indicator of environmental change. Plant Biosyst. 2007, 141, 426–442. [CrossRef] 32. Drew, M.C. Comparison of the effects of a localized supply of phosphate, nitrate, ammonium and potassium on the growth of the seminal root system, and the shoot, in barely. New Phytol. 1975, 75, 479–490. [CrossRef] 33. Eissenstat, D.M. Costs and benefits of constructing roots of small diameter. J. Plant Nutr. 2008, 15, 763–782. [CrossRef] 34. Eissenstat, D.M.; Kucharski, J.M.; Zadworny, M.; Adams, T.S.; Koide, R.T. Linking root traits to nutrient foraging in arbuscular mycorrhizal trees in a temperate forest. New Phytol. 2015, 208, 114–124. [CrossRef] [PubMed] 35. Xu, C.Y.; Zhang, H.; Jia, Z.K. Effects of stand density and site types on root characteristics of Platycladus orientalis plantations in Beijing moutains area. J. Beijing For. Univ. 2007, 29, 95–99. 36. Wang, Z.H.; Li, R.X.; Hao, J.P. Effects of thinning on fine-root morphology in Chinese fir plantations. J. Northeast For. Univ. 2011, 6, 13–19. 37. Shao, Y.N.; Liu, Y.K.; Li, Y.H. Soil nutrient characteristics in Larix olgensis plantation with different stand densities. J. Cent. South Univ. For. Technol. 2017, 37, 27–31. 38. Tian, X.H.; Sun, R.X.; Li, J.; Zhao, C.H.; Yang, M.S. Effect of stand density on growth of Populus × euramericana ‘Neva’ plantations. Sci. Silvae Sin. 2011, 3, 184–188. 39. Zhao, X.M.; Wang, H.Y.; Sun, X.Y. Effects of different clones and growth density of triploid Populus tomentosa on nutrient concentrations in leaf. Hebei J. For. Orchard Res. 2007, 2, 117–121. 40. Yang, Y.; Liu, B.R.; Song, N.P. Effects of Artificial Caragana shrub density on spatial distribution of soil nutrients in desert steppe. Acta Prataculturae Sin. 2014, 5, 107–115. 41. FAO. Improving Lives with Poplars and Willows; FAO: Rome, Italy, 2002. 42. Isebrands, J.G.; Aronsson, P.; Ceulemans, M.C.; Coleman, M.; Dimitriou, N.D.; Doty, S.; Gardiner, E.; Heinsoo, K.; Johnson, J.D.; Koo, Y.B.; et al. Environmental applications of poplars and willows. In Poplars and Willows: Trees for Society and the Environment; Isebrands, J.G., Richardson, J., Eds.; CABI and FAO: Rome, Italy, 2014; pp. 258–336. 43. Perry, C.H.; Miller, R.C.; Brooks, K.N. Impacts of short-rotation hybrid poplar plantations on regional water yield. For. Ecol. Manag. 2011, 143, 143–151. [CrossRef] 44. Updegraff, K.; Baughman, M.J.; Taff, S.J. Environmental benefits of cropland conversion to hybrid poplar: Economic and policy considerations. Biomass Bioenerg. 2004, 27, 411–428. [CrossRef] 45. Wang, W.Q.; Jia, Y.B.; Xu, L.M. Study on the root distribution of Populus tomentosa. J. Agric. Univ. Hebei 1997, 1, 24–29. 46. Ren, Z.X. The Study of Water-Nitrogen Coupling Effect on Different Populous Tomentosa Clones Plantations. Master’s Thesis, Beijing Forestry University, Beijing, China, 2002. Forests 2018, 9, 562 13 of 14

47. Zhu, Z.T.; Zhang, Z.Y. Study on the Method of Rapid Propagation of Superior Trees of Populus tomentosa. J. Beijing For. Univ. 1986, 4, 1–17. 48. Bao, S.D. Soil Agro-Chemistrical Analysis; China Agriculture Press: Beijing, China, 2007. 49. Wang, Q.C. Fine Roots Responses of Manchunrian Ash and Korean Larch to Soil Nutrients Heterogeneity. Ph.D. Thesis, College of Forestry, Northeast Forestry University, Harbin, China, 2004. 50. Wang, Z.H.; Li, R.X.; Hao, J.P.; Guan, Q.W. Effects of thinning on fine-root morphology in Cunninghamia Lanceolata plantations. J. Northeast For. Univ. 2011, 39, 13–15. 51. Zhou, L.M. Root Identification and Varitation in Architecture of Fine Roots of Subtropical Tree Species in Southern China. Master’s Thesis, Central South University of Forestry & Technology, Changsha, China, 2015. 52. Xu, Y.; Gu, J.C.; Dong, X.Y. Fine root morphology, anatomy and tissue nitrogen and carbon contents of the first five orders in four tropical hardwood species in Hainan Island, China. Chin. J. Plant Ecol. 2011, 9, 955–964. [CrossRef] 53. Brundrett, M.C. Coevolution of roots and of land plants. New Phytol. 2002, 154, 275–304. [CrossRef] 54. Soukup, A.; Mala, J.; Hrubcova, M. Differences in anatomical structure and lignin content of roots of pedunculate oak and wild cherry-tree plantlets during acclimation. Biol. Plant. 2004, 48, 481–489. [CrossRef] 55. Wells, C.E.; Eissenstat, D.M. Beyond the roots of young seedlings: The influence of age and order on fine root physiology. J. Plant Growth Regul. 2003, 21, 324–334. [CrossRef] 56. Xiong, D.C.; Huang, J.X.; Yang, Z.J. Fine root architecture and morphology among different branch orders of six subtropical tree species. Acta Ecol. Sin. 2012, 6, 1888–1897. [CrossRef] 57. Xiong, D.C.; Huang, J.X.; Yang, Z.J. Fine root morphology and respiration among different branch orders in two plantations of young and old Cunninghamia lanceolata. J. Subtrop. Resour. Environ. 2014, 2, 45–51. 58. Shi, W.; Wang, Z.Q.; Liu, J.L. Fine root morphology of twenty hardwood species in maoershan natural secondary forest in northeastern China. J. Plant Ecol. 2008, 6, 1217–1226. 59. Li, R.X.; Ling, N.; Guan, Q.W. Effects of stand ages on carbon storage, fine-root morphology and biomass in Platycladus orientalis plantation. J. Nanjing For. Univ. 2013, 2, 21–27. 60. Xu, T.; Wang, H.T.; Wang, Y.P. Morphological and anatomical traits of poplar fine roots in successive rotation plantations. Sci. Silvae Sin. 2015, 1, 119–126. 61. Mei, L. Fine Root Distribution and Seasonal Patterns of Manchurian Ash Plantation in Relation to Soil Nutrients Avaialability. Master’s Thesis, Northeast Forestry University, Heilongjiang, China, 2004. 62. Kerr, G. Effects of spacing on the early growth of planted Fraxinus excelsior L. Can. J. For. Res. 2003, 33, 1196–1207. [CrossRef] 63. Xie, L.Z.; Li, J.N.; Wang, S.Z. Influence of stem density in Fraxinus chinensis plantation. J. Northeast For. Univ. 2014, 9, 1–5. 64. Tian, Y.M.; Wang, Q.C. Influence of planting density on biomass accumulation and root growth of Manchurian ash (Fraxinus mandshurica) in 10-year-old plantation stands. Sci. Silvae Sin. 2011, 7, 102–107. 65. Nan, H.W.; Liu, Q.; Xiao, Q.Y. Root foraging ability of spruce (Oicea asperata) in two growth stages. Chin. J. Appl. Environ. Biol. 2015, 21, 547–554. 66. Drew, M.C.; Saker, L.R. Nutrient supply and growth of the seminal root system in barely. l. The effect of nitrate concentration on the growth of axes and laterals. J. Exp. Bot. 1973, 24, 1189–1202. [CrossRef] 67. Allen, M.F. Mycorrhizal Functioning: An Integrative Plant-Fungal Process; Chapman and Hall: New York, NY,USA, 1992. 68. Smith, S.E.; Read, D.J. Mycorrhizal Symbiosis, 2nd ed.; Academic Press: San Diego, CA, USA, 1997. 69. Sun, J.J.; Sun, Y.Z. Interactions between tree roots and soil microorganisms in . World For. Res. 2015, 2, 14–18. 70. Szuba, A. of Populus. For. Ecol. Manag. 2015, 347, 156–169. [CrossRef] 71. Achat, D.; Bakker, M.; Trichet, P. Rooting patterns and fine root biomass of Pinus pinaster assessed by trench wall and core methods. J. For. Res. 2008, 13, 165–175. [CrossRef] 72. Ceccon, C.; Panzacchi, P.; Scandellari, F.; Prandi, L.; Ventura, M.; Russo, B.; Millard, P.; Tagliavini, M. Spatial and temporal effects of soil temperature and moisture and the relation to fine root density on root and soil respiration in a mature apple orchard. Plant Soil 2011, 342, 195–206. [CrossRef] 73. Yan, X.L.; Dai, T.F.; Jia, L.M. Responses of fine-root morphology and vertical distribution of Populus × euramericana ‘Guariento’ to the coupled effect of water and nitrogen. Chin. J. Plant Ecol. 2015, 08, 825–837. Forests 2018, 9, 562 14 of 14

74. Donnelly, L.; Jagodzi´nski, A.M.; Grant, O.M.; O’Reilly, C. Above- and below-ground biomass partitioning and fine-root morphology in juvenile Sitka spruce clones in monoclonal and polyclonal mixtures. For. Ecol. Manag. 2016, 373, 17–25. [CrossRef] 75. Jagodzi´nski,A.M.; Oleksyn, J. Ecological consequences of silviculture at variable stand densities. II. Biomass production and allocation, nutrient retention. Sylwan 2009, 153, 147–157. 76. Hyatt, L.A.; Cahill, J.F.; Casper, B.B. Above-ground competition does not alter biomass allocated to roots in Abutilon theophrasti. New Phytol. 1998, 140, 231–238. 77. Al Afas, N.; Marron, N.; Zavalloni, C.; Ceulemans, R. Growth and production of a short-rotation coppice culture of poplar: Fine root characteristics of five poplar clones. Biol. Bioenerg. 2008, 32, 494–502. [CrossRef]

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).