**Variation in Photosynthetic Traits and Correlation with Growth in Teak (***Tectona grandis* **Linn.) Clones**

**Guihua Huang 1,\*, Kunnan Liang 1, Zaizhi Zhou 1, Guang Yang <sup>1</sup> and Enarth Maviton Muralidharan <sup>2</sup>**


Received: 24 July 2018; Accepted: 10 September 2018; Published: 10 January 2019

**Abstract:** In order to interpret the patterns of genetic variation of photosynthesis and the relationships with growth traits within gene resources of teak (*Tectona grandis* Linn.), gas exchange, and chlorophyll fluorescence parameters, growth traits of plants in nursery and field trials were measured for 20 teak clones originated from different countries. The results show that there was abundant genetic variation in gas exchange, chlorophyll fluorescence, and growth among the teak clones. The measured traits were found to have generally high heritability (h2) except for intercellular concentration of carbon dioxide (CO2) (*C*i). The net photosynthetic rate (*P*n), seedling height, and individual volume of wood were significantly correlated with each other, and seedling height was significantly correlated with plant height in field trials, suggesting that *P*<sup>n</sup> and seedling height can be useful in teak breeding. Teak clones 7029, 71-5, 7219, 7412, and 7122, and provenances 3070, 3074, and 3071 had higher photosynthetic rates, and can be regarded as a key resource in teak improvement programs. This work provides useful information for teak breeding and germplasm resource management.

**Keywords:** gas exchange; chlorophyll fluorescence; growth trait; genetic variation; early selection

#### **1. Introduction**

Teak (*Tectona grandis* Linn.) is naturally distributed in India, Thailand, Myanmar, and Laos [1,2]. Its desirable hardwood properties, fine grain, and durability have made teak the luxury timber for furniture making, carving, and building around the world [3,4]. Due to its economical importance, teak has been introduced widely in the tropical regions since the 19th century, especially in Asia, Africa, Central America, and South America [5].

As one of the most valuable wood species in international markets, teak plantations have developed rapidly in the recent decade. Developing high productivity and uniform clones that can be used for plantations in different regions has become an important objective of teak breeding. Information on variation of photosynthetic parameters and their relationship with growth traits help us understand underlying processes and responses, and will be useful in tree improvement programs. During the growth process of plants, organic compounds are generated by photosynthesis, and gradually accumulate in trunks. The photosynthetic characteristics are the main measurable indicators of plant growth rates [6]. Numerous studies on breeding for high photosynthetic ability in crops have been conducted, to improve the yield [7–9], but studies on forest trees are limited [10–12]. Chu et al. 2010 [10] studied gas exchange and chlorophyll fluorescence parameters, as well as their relationship with the growth of *Populus nigra*, and found that the species originating in Serbia, southern and east Europe can be regarded as a resource with high light-use efficiency for future breeding. Teak has broad leaves and prefers warmth and sunlight, and developing clones with

high productivity and uniformity by evaluating photosynthetic characteristics can be an important goal in teak breeding. In the past, teak breeding was mainly focused on the analysis of growth indices in field experiments [13,14], and studies on photosynthetic physiology of teak are limited to those on photosynthetic responses of a single clone to simulated acid rain stress [15], photosynthetic physiological characteristics under different disturbance intensities among teak plants [16], and diurnal and seasonal photosynthetic characteristics in teak clones [17]. However, studies on teak germplasm or clones, which systematically estimate photosynthetic characteristics and correlation with growth, have not been reported.

The purpose of this study was (1) to investigate the genetic variation of photosynthetic parameters and growth traits of teak clones, (2) to reveal the correlation, if any, between photosynthetic characteristics and growth traits within the gene resources of teak, and (3) to evaluate and select superior teak resources possessing high photosynthetic efficiency for breeding.

#### **2. Materials and Methods**

#### *2.1. Materials*

A total of 20 widely cultivated teak clones propagated through tissue culture were investigated in this study. The 20 clones were selected from international provenance trial planted at Jianfeng, Hainan, China, by the Research Institute of Tropical Forestry of Chinese Academy of Forestry (RITF-CAF). A complete list of accessions with descriptions and origins is given in Table 1. Among these accessions, 10 were clones originating from India, 9 were from Myanmar sources and 1 from Nigeria.


**Table 1.** Information of 20 commercial teak clones investigated in the study.

#### *2.2. Experimental Design and Growth Parameter Measurement*

The young in vitro plantlets of teak clones were transplanted to a sterilized sand bed in the greenhouse at the Research Institute of Tropical Forestry, Chinese Academy of Forestry (RITF-CAF), in Guangzhou (113◦18 E, 23◦06 N). One month later, healthy and uniform seedlings (Ramets derived

from each clone) about 6 cm in height were transplanted into plastic pots filled with a mixture of lateritic red soil, black peat, vermiculite, and perlite (2:2:1:1, v/v/v/v)—one seedling per pot. A completely randomized block design was used in this nursery experiment with 5 seedlings in one row per plot, 6 repeats in total with 40 cm × 40 cm pot space. Seedling height and collar diameter of all seedlings in the nursery were measured at the age of one year.

Field trial was carried out at Dingan in Hainan Island (110◦19 E, 19◦39 N) and a completely randomized block design was used with 6 plants in one row per plot, 6 repeats in total, with 2.5 m × 4 m space. Plant height (H) and diameter at breast height (DBH) of each plant in field trial were measured at the age of four years.

#### *2.3. Physiological Parameter Measurement*

Three seedlings in the nursery were randomly selected for each clone, 1 seedling per plot, 3 repeats in total, in a completely randomized block design (to make sure the test was random for all 60 selected seedlings) and 3 functional leaves per seedling exposed to sunlight were chosen for study. The gas exchange parameters including net photosynthetic rate (*P*n), stomatal conductance (*G*s), intercellular CO2 concentration (*C*i), and transpiration rate (*T*r) were measured on sunny days between 8:45 and 11:30 in August 2013 with a Li-6400 portable photosynthetic apparatus (LI-COR Co. Lincoln, NE, USA) at the nursery of RITF-CAF, in Guangzhou. A leaf chamber automatic light (800 <sup>μ</sup>mol·m−2·s<sup>−</sup>1) was used when testing, with CO2 concentration 380 ± <sup>15</sup> <sup>μ</sup>mol·mol<sup>−</sup>1, temperature of the leaf chamber 30–38 ◦C, and a relative humidity 58%–68% recorded by the photosynthetic apparatus under natural conditions. Three stable values were recorded for each leaf. Chlorophyll fluorescence characteristics were measured at the same time using the German PAM-2500 Walz portable fluorescence spectrometer, the saturation pulse intensity was 4500 mol·m−2·s<sup>−</sup>1, and actinic intensity was 1000 mol·m−2·s−1. The actual quantum yield PSII (Yield), non-photochemical quenching (NPQ) and maximum photochemical efficiency of PSII (*F*v/*F*m) were also measured [18,19]. The calculation formula for Yield is Yield = (*F*m' − *F*t)/*F*m', where *F*m' is referred to maximum fluorescence under light adaptation, and *F*<sup>t</sup> denotes real fluorescence at any given time. The formula of NPQ = *F*m/*F*m' − 1; (*F*v/*F*m)=(*F*<sup>m</sup> − *F*o)/*F*m, and *F*o, *F*m, and *F*<sup>v</sup> refer to dark-adapted initial fluorescence, maximum fluorescence, and variable fluorescence, respectively. Before testing, 20 min shading treatment was carried out with a blade holder to ensure selected leaves had dark adaptation for a long enough period of time.

#### *2.4. Data Analysis*

Water use efficiency (*WUE*) was calculated by the formula *WUE* = *P*n/*T*r [20], and the coefficient of variation was calculated by the formula C = S/X, where S is the standard deviation, and X is the overall average value of each index. Clone heritability was calculated with the formula h2 = 1 − 1/*<sup>F</sup>* [21], where F is test statistic of clones in variance analysis. Individual volume of wood was calculated by the formula V = 0.4787D2H, where D is DBH, and H is plant height of field trial [22]. Variance and Duncan's multiple comparison analyses were conducted for each parameter, and correlation analyses (using Pearson's product-moment correlations) between photosynthetic parameters, water use efficiency, and growth index, were performed using SAS software (version 8.1).

#### **3. Results**

#### *3.1. Gas Exchange, Chlorophyll Fluorescence, and Growth Traits of Different Teak Clones*

Variance analysis of gas exchange, chlorophyll fluorescence, and growth parameters among teak clones are shown in Table 2. There is a significant difference in the photosynthetic parameters, water use efficiency, and growth index but not for intercellular CO2 concentration (*C*i). In addition, apart from *C*<sup>i</sup> (h2 = 0.145) and NPQ (h2 = 0.168), other parameters had high heritability (h2 = 0.670–0.903), with actual quantum yield PSII (Yield) having the highest heritability (h2 = 0.903), suggesting a strong genetic influence on the function, and that it is less affected by environment.

**Category Parameter** *F p* **Heritability (h2) Variation Coefficient** Gas exchange *P*n 5.46 <0.0001 \*\*\* 0.817 0.401 *G*s 4.53 <0.0001 \*\*\* 0.779 0.474 *C*<sup>i</sup> 1.17 0.3213 ns 0.145 0.111 *T*r 3.38 0.0004 \*\*\* 0.704 0.349 Chlorophyll fluorescence NPQ 5.93 <0.0001 \*\*\* 0.168 0.447 Yield 10.32 <0.0001 \*\*\* 0.903 0.294 *F*v/*F*m 3.03 0.0011 \*\* 0.670 0.028 Water use efficiency *WUE* 10.32 <0.0001 \*\*\* 0.903 0.474 Seedling growth at 1 year Seedling height 3.39 <0.0001 \*\*\* 0.705 0.116 Collar diameter 3.29 <0.0001 \*\*\* 0.696 0.102 Field growth at 4 years H 7.88 <0.0001 \*\*\* 0.873 0.092 DBH 6.74 <0.0001 \*\*\* 0.852 0.167 Individual volume at 4 years V 7.24 <0.0001 \*\*\* 0.863 0.327

**Table 2.** Variance analysis (ANOVA) of gas exchange, chlorophyll fluorescence, water use efficiency, and growth parameters among teak clones.

Note: *P*n: net photosynthetic rate, *G*s: stomatal conductance, *C*i: intercellular CO2 concentration, *T*r: transpiration rate, NPQ: non-photochemical quenching, Yield: the actual quantum yield PSII, *F*v/*F*m: maximum photochemical efficiency of PSII, *WUE*: water use efficiency, H: height of field growth at 4 years, DBH: diameter at breast height of field growth at 4 years, V: individual volume at 4 years. \*\* indicate highly significant difference at *p* < 0.01 level of probability, \*\*\* more highly significant difference at *p* < 0.001 level of probability, and ns no significance.

Duncan's multiple comparison analysis of photosynthetic and growth traits are listed in Tables 3–5. The ranges of the main parameters, such as *<sup>P</sup>*<sup>n</sup> and *<sup>F</sup>*v/*F*m, were 4.45 ± 1.62–14.47 ± 0.32 <sup>μ</sup>mol·m−2·s<sup>−</sup>1, 0.67 ± 0.02–0.75 ± 0.01, respectively. Water use efficiency (*WUE*) was between 1.02 ± 0.36 and 6.38 ± 1.25. Apart from the maximum photochemical efficiency of PSII *F*v/*F*<sup>m</sup> (0.028), the variation coefficients of other parameters (0.092–0.474) were great, suggesting that the teak genotypes possessed extensive variation in these traits. Results indicated that there are suitable germplasm resources for breeding of teak for high photosynthetic efficiency. Teak clones 7029, 71-5, 7219, 7412, and 7122 were selected as clones with high net photosynthetic rate based on the results.


**Table 3.** Values of gas exchange parameters among teak clones from different countries.

Note: *P*n: net photosynthetic rate, *G*s: stomatal conductance, *C*i: intercellular CO2 concentration, *T*r: transpiration rate. Values followed by the different letter of each group were significantly different at *p* < 0.05 level of probability.


**Table 4.** Values of chlorophyll fluorescence parameters and *WUE* among teak clones.

Note: NPQ: non-photochemical quenching, Yield: the actual quantum yield PSII, *F*v/*F*m: maximum photochemical efficiency of PSII, *WUE*: water use efficiency. Values followed by the different letter of each group were significantly different at *p* < 0.05 level of probability.

**Table 5.** Values of growth traits among teak clones from different countries.


Note: H: height of field growth at 4 years, DBH: diameter at breast height of field growth at 4 years. Values followed by the different letter of each group were significantly different at *p* < 0.05 level of probability.

#### *3.2. Characteristics of Gas Exchange and Chlorophyll Fluorescence of Teak Resources from Different Regions*

As shown in Table 6, net photosynthetic rate (*P*n), stomatal conductance (*G*s), transpiration rate (*T*r), and non-photochemical quenching (NPQ), and actual quantum yield (Yield) of PSII were significantly different among teak provenances.


**Table 6.** Variance analysis of photosynthetic parameters among teak clones from different provenances.

Note: *P*n: net photosynthetic rate, *G*s: stomatal conductance, *C*i: intercellular CO2 concentration, *T*r: transpiration rate, NPQ: non-photochemical quenching, Yield: the actual quantum yield PSII, *F*v/*F*m: maximum photochemical efficiency of PSII. \*\* indicate highly significant difference at *p* < 0.01 level of probability, \*\*\* more highly significant difference at *p* < 0.001 level of probability, and ns no significance.

Among the teak provenances (Table 7), 3070, 3074, and 3071 had higher *P*n, 3074 had higher *G*<sup>s</sup> and *T*r, while 20001 and 3074 had a higher *C*<sup>i</sup> value. While 8204, 3078, and 3072 showed high NPQ value (Table 8), 3074, 3071, and 3070 had higher Yield and *F*v/*F*<sup>m</sup> values. These results suggest that different teak provenances have different photosynthetic physiological characteristics. Provenances 3070, 3074, and 3071 can be considered to have high photosynthetic rates.


**Table 7.** Values of gas exchange parameters among teak clones from different provenances.

Note: *P*n: net photosynthetic rate, *G*s: stomatal conductance, *C*i: intercellular CO2 concentration, *T*r: transpiration rate. Values followed by the different letter of each group were significantly different at *p* < 0.05 level of probability.

**Table 8.** Values of chlorophyll fluorescence parameters among teak clones from different provenances.


Note: NPQ: non-photochemical quenching, Yield: the actual quantum yield PSII, *F*v/*F*m: maximum photochemical efficiency of PSII. Values followed by the different letter of each group were significantly different at *p* < 0.05 level of probability.

#### *3.3. Correlations between Photosynthetic and Growth Traits*

Correlation analyses (Table 9) of teak clone parameters showed that *P*<sup>n</sup> had significant positive correlation with *G*s, *T*r, *F*v/*F*m, seedling height and individual volume, respectively. *P*<sup>n</sup> values can therefore be regarded as a critical parameter in teak breeding, indicating potential for faster growth.


**9.**Correlationanalysisphotosyntheticcharacteristics,growthtraits,andecologicalfactorsofteakclones.

diameter of seedling growth at nursery at 1 year, H: height of field growth at 4 years, DBH: diameter at breast height of field growth at 4 years, V: individual volume at 4 years. \*\*

indicate highly significant difference at *p* < 0.01 level of probability, \* significant difference at *p* < 0.05 level of probability.

In addition, seedling height was positively correlated with collar diameter, plant height (H), and individual volume. *WUE* was significantly negatively correlated with *T*r, suggesting that teak clones with high transpiration could show low *WUE*; *G*s was positively correlated with *T*r, indicating that high stomatal conductance contributed to higher transpiration; *T*r was positively correlated with the actual quantum yield of PSII, suggesting that the higher the transpiration rate, higher the actual quantum yield of PSII would be.

#### **4. Discussion**

Plant growth and yield depend largely on photosynthesis [23,24]. Plant photosynthesis is not only affected by environmental factors, but also affected by plant genetic characteristics. It is the complex process of interaction between plant genetic and environmental factors that influences photosynthetic activity [25]. To date, ecophysiological studies on photosynthesis in forest trees were those that examined the effects of stress on photosynthetic physiology [26–29], and the photosynthetic responses to light intensity [30] and CO2 concentration [31]. The present study chiefly focused on systematically measuring photosynthetic gas exchange and chlorophyll fluorescence parameters, correlating the photosynthetic characteristics with growth, and providing a means of rapid evaluation of teak germplasm, for introduction, utilization, and improvement of teak resources in future breeding programs.

Our study showed that teak clones had high variation and high heritability (h2) for many growth and physiological traits. The results were generally consistent with the findings reported for *Populus trichocarpa* by McKown [32]. The gas exchange, chlorophyll fluorescence, and growth parameters of teak clones were highly controlled by genetic factors, especially for the actual quantum yield (Yield) of PSII. Therefore, such a parameter has high practical significance and can be effectively used for improving the efficiency of teak breeding. However, it is worth emphasizing that intercellular CO2 concentration (*C*i) and non-photochemical quenching (NPQ) were greatly influenced by the environment.

Further analysis showed that teak clones and resources from different regions vary in their photosynthetic characteristics. In this study, teak clones 7029, 71-5, 7219, 7412, 7122, and provenances 3070, 3074, 3071, which had higher *P*n, can be regarded as the key resource in future breeding and management programs. However, more teak clones from different provenances and countries need to be included in this kind of study in the future. Huang et al., 2016 [33] had suggested, after SSR molecular marker testing, that the Nigerian provenance 3078, investigated in this paper, may have originated from India. The present studies, that reveal their similar photosynthetic characteristics, further corroborates this conclusion.

The significantly positive correlation that the net photosynthetic rate has with seedling height, individual volume, *F*v/*F*m, *G*s, and *T*r, is an interesting finding of this study. In addition, seedling height was significantly and positively correlated with plant height and individual volume. Both results indicate that teak clones of high *P*<sup>n</sup> and high seedling height result in fast-growing clones. However, it is known that photosynthetic processes are influenced by environmental conditions such as light, temperature, water, and nutrients [25]. Photosynthetic rate is not the only limiting factor for growth [34]. These factors may affect growth differently for different clones, resulting in no significant relationship between *P*<sup>n</sup> and plant height or DBH of field growth at 4 years, the result being similar to previous reports [12,35].

Correlation analysis also revealed that water use efficiency was significantly but negatively correlated with *T*r, suggesting that teak clone *WUE* may decrease when transpiration rate is high in daytime. Such results were consistent with the study by Huang et al., 2016 [17], in that diurnal variation possessed a double peaked curve, with a "midday depression" phenomenon in summer, when strong sunshine often accompanied by high temperature produces excessive transpiration, followed by decline of water use efficiency. There was no significant correlation between seedling collar diameter and other parameters, except for seedling height, consistent with the results of the study on photosynthesis and growth of *Populus nigra* [10]. At the same time, the coefficient of genetic

variation of *C*<sup>i</sup> and *F*v/*F*<sup>m</sup> were lower than other photosynthetic indices in the present study, similar to photosynthetic characteristics of the clones [10]. The variation coefficients of *F*v/*F*<sup>m</sup> were small in this study (0.028) and in *Populus nigra* clones (0.024) [10]. This may be due to CO2 concentration, leaf temperature, and relative humidity fluctuating significantly under natural conditions, reducing the *F*v/*F*m compared to conditions where they remain constant [25].

Farquhar et al., 1982 [36] concluded that photosynthetic rate was controlled by stomatal factors when *P*n, *C*i, and *G*<sup>s</sup> increased or decreased at the same time. In this study, correlation analysis indicates that there was significant positive correlation between *P*n and *G*s, a positive but not significant correlation between *P*<sup>n</sup> and *C*i, *G*s, and *C*i, suggesting that the photosynthetic rate of teak was mostly controlled by stomatal factors. Plant dynamic photosynthesis was affected by many environmental factors such as light intensity, CO2 concentration, leaf temperature, and relative humidity. Fluctuating environments would have a large impact on photosynthesis. Plants have a highly responsive regulatory system to make rapid photosynthetic responses to fluctuating environments, and a number of photoprotective mechanisms allow plants to maintain photosynthesis under stressful fluctuating environments [25].

For further research, the following points need to be considered in the future studies on teak. Firstly, it is desirable that more clones from different provenances be included in this kind of study in order to analyze variation among teak resources of different provenances more efficiently. Secondly, the differences in *P*<sup>n</sup> among teak clones in this study was greater than that seen in *Populus nigra* [10] and *Populus deltoides* clones [11]. It is to be ascertained whether such a difference was caused by inherent differences in photosynthetic characteristics between the tree species, or if is due to other reasons. Thirdly, further evaluation of differences in leaf area between teak clones is needed since tree growth is restricted not only by photosynthetic efficiency, but also by photosynthetic leaf area [37,38]. Lastly, we found that photosynthetic rates of teak plants in the field trial measured at the age of 2 years were higher than that of the potted seedlings and, therefore, correlation analysis among photosynthetic parameters, photosynthetic leaf area, and growth traits in field trials need to be executed in future teak breeding programs.

#### **5. Conclusions**

Our findings have at least three important implications. First, photosynthetic parameters other than intercellular CO2 concentration (*C*i) are highly controlled by genetic factors. In addition, photosynthetic parameters and growth traits in different clones revealed abundant genetic variation. Second, the net photosynthetic rate (*P*n), seedling height, and individual volume of wood significantly correlated between each other, and seedling height was significantly correlated with plant height in field trials, suggesting *P*n and seedling height can help us in teak breeding. Third, teak clones 7029, 71-5, 7219, 7412, and 7122, and provenances 3070, 3074, and 3071, revealed to have higher photosynthetic rate, can be regarded as key resources for future breeding and germplasm resource management.

**Author Contributions:** G.H. designed and supervised implementation of the studies, supervised the statistical analyses, constructed the tables, wrote the manuscript, and crafted the final version. K.L. and Z.Z. carried out the statistical analyses and wrote the first draft of the manuscript. G.Y. supervised and carried out all technical aspects. E.M.M. participated in writing and editing the manuscript.

**Funding:** This work was supported by the ["Fundamental Research Funds for the Central Non-profit Research Institution of CAF" (grant number:No. CAFYBB2017ZA001-7)] and [the National Key Research and Development Program of China "Research Project on Teak Cultivation Techniques" (grant number: 2016YFD0600602)].

**Acknowledgments:** The authors are grateful to Bingshan Zeng and Zhenfei Qiu for providing the materials used in this study. We would like to thank anonymous reviewers for their valuable comments.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 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/).

### *Article* **Screening of Applicable SSR Molecular Markers Linked to Creeping Trait in Crape Myrtle**

### **Tangchun Zheng 1,2,3,4,5, Bo Qin 1,2,3,4,5,6, Suzhen Li 1,2,3,4,5,6, Ming Cai 1,2,3,4,5, Huitang Pan 1,2,3,4,5, Jia Wang 1,2,3,4,5, Tangren Cheng 1,2,3,4,5 and Qixiang Zhang 1,2,3,4,5,6,\***


Received: 26 April 2019; Accepted: 17 May 2019; Published: 18 May 2019

**Abstract:** Creeping plants have unique ornamental value because they have more branches and flowers and the creeping trait is rare in crape myrtle (*Lagerstroemia indica* L.). In this study, the first filial generation (F1) population was derived from *Lagerstroemia fauriei* Koehne (standard) and *L. indica* "Creole" (creeping) and the backcross1 (BC1) population was derived from the backcross of F1 individual S82 (creeping) and *L. fauriei*. The segregation of the creeping trait was analyzed for 174 seedlings of the BC1 population to examine the linkage relationship between simple sequence repeat (SSR) molecular markers and the creeping trait. Creeping genes were screened using bulked segregant analysis combined with 322 SSR primers, which were detected with good polymorphism. The results show that two SSR markers (S364 and LYS12) were detected, with genetic distances of 23.49 centimorgan (cM) and 25.86 cM from the loci controlling the plant opening angle trait and the branching angle trait, respectively. The accuracy rate for phenotypic verification using S364 and LYS12 was 76.51% and 74.14%, respectively. Our results provide basic information for the molecular marker-assisted selective breeding and cloning of the creeping gene to improve architecture diversity in the breeding of crape myrtle.

**Keywords:** *Lagerstroemia* species; simple sequence repeat markers; bulked segregant analysis; creeping trait; plant architecture

#### **1. Introduction**

Plant architecture is the result of long-term evolution and natural selection, which involve complex regulatory processes based on genetic and environmental interactions [1]. According to the different growth angles and branching directions, plant architecture can be roughly classified as standard, weeping, pillar, upright, brachytic, and dwarf [2]. Plant architecture traits have important application value for the improvement of crop production; for example, the cultivation of dwarf or semi-dwarf horticultural or agronomic crops improves yield and production efficiency because mechanized management is more conducive. The discovery and utilization of dwarf genes in rice, which greatly promoted grain production, led to the first "green revolution" in agriculture, demonstrating the importance of genetic resources [3,4]. The genetic regulation of plant architecture traits in woody plants is more complex than in herbaceous plants and plant architecture traits are susceptible to external environmental conditions [5]. Many studies have been performed on plant architecture for extreme plant types of trees, including pillar and weeping peach [6], dwarf and compact peach (*Prunus persica* L.) [7], weeping *Prunus mume* [8], weeping *Cercis chinensis* [9], and columnar and weeping apple (*Malus* × *domestica*) [10,11].

Molecular marker technology can reflect the differences between DNA sequences for different species and is widely used in plant genetic diversity analysis, genetic map construction, map-based cloning, and marker-assisted selective breeding [12–14]. Individual hybrids with target traits can be selected at the seedling stage using molecular marker-assisted selection, which reduces resource waste and breeding cost and speeds up the breeding process [15]. Bulked segregant analysis (BSA) is derived from near-isogenic line analysis, which was reported for the first time in 1991 [16]. SSR markers are widely used in relationship analysis, genetic diversity analysis, mapping quantitative trait locus (QTL), and so on. Four expressed sequence tag (EST)-SSR marker loci closely linked to the dwarf trait in pear were found by BSA technology. In *Actinidia chinensis* Planch, an EST-SSR marker was screened and the genetic distance between the marker and dwarf gene was 8.8 cM [17]. Three selected EST-SSR markers were used to determine genetic structure in 29 cultivars and were used for fruit color selection in *Prunus salicina* breeding [18]. In addition, DNA markers associated with the dwarf trait from *Brassica napus* [19], *Prunus persica* [20], and *Avena sativa* [21] have been studied.

*Lagerstroemia* L. (crape myrtle) belongs to the Lythraceae family, which includes at least 50 species of deciduous or evergreen shrubs or trees native to Southeast Asia [22]. China is located in the center of the worldwide *Lagerstroemia* distribution and origin [23]. Crape myrtle was first cultivated as an ornamental species in China approximately 1800 years ago [24]. Species in this genus are highly valued in landscaping for their graceful plant architecture, long-lasting flowering period, and colorful flowers during the summer [25]. When exposed to natural conditions, crape myrtle is a diploid plant (2n = 2x = 48) [26]. Crape myrtle is susceptible to powdery mildew; therefore, the initial purpose for hybrid breeding by interspecific hybridization between *L. indica* and *L. fauriei* was for disease resistance and then a series of excellent varieties with various plant architecture types and colorful flowers was selected from the resistant hybrid offspring ('Pocomoke") [27–29]. *Lagerstroemia* species are self-compatible and easily produce interspecific hybrids with related species. To further improve the ornamental value of hybrids with a larger flower diameter, *Lagerstroemia speciosa* and *L. indica* were used for interspecific hybridization and hybrid seedlings with traits of the parents were obtained; however, only plant height and plant width had the characteristics of the hybrids and no variation in flower color or flower size was observed [30]. Recently, research in America, Japan, and China has focused on breeding new cultivars with unique plant architecture, especially dwarf or potted crape myrtle. A few dwarf crape myrtle cultivars with many branches and large flowers were bred through intraspecific hybridization (*L. indica*) with the aim of generating dwarf plant architecture [31–35].

The molecular study of *Lagerstroemia* species has led to the development of a series of genomic SSR markers [26,36–40]. Two single nucleotide polymorphism (SNP) markers (M16337 and M38412) that are highly correlated with internode length and one SNP marker (M25207) that is highly correlated with primary lateral branch height were validated in the F1 population of *L. indica* [41]. In addition, an SSR marker linked to the dwarf gene, with a genetic distance of 23.33 cM, was screened from the *L. fauriei* × *L. indica* "Pocomoke" F1 population [35]. Currently, less is known about the plant architecture of the crape myrtle, especially the creeping trait. To analyze the linkage relation between SSR molecular markers and creeping trait, the segregation of the creeping trait was analyzed by the BSA method using the BC1 population with 174 hybrids and 322 SSR primers. Our results provide an important technical and theoretical basis for plant architecture molecular marker-assisted selective breeding for the *Lagerstroemia* species.

#### **2. Materials and Methods**

#### *2.1. Plant Materials*

The F1 population was derived from *L. fauriei* (♀, standard) and *L. indica* "Creole" (♂, creeping). To analyze the linkage relation between SSR markers and creeping trait, the BC1 segregation population was derived from a backcross of the F1 creeping individual S82 (♀) × *L. fauriei* (♂). The F1 female parent was a tree (>3 m) with standard branching and the male parent was a dwarf plant (0.3–0.5 m) with creeping branching. The F1 individual S82 had the same phenotype as *L. indica* "Creole". The linkage relationship between SSR markers was analyzed for 174 individuals of the BC1 population. All of the materials were planted in a breeding nursery at the National Engineering Research Center for Floriculture (Beijing) (40◦02' N, 115◦50' E) (Figure 1).

**Figure 1.** Plant materials and method of measurement used in this study. (**a**) *L. fauriei*; (**b**) *L. indica* "Creole"; (**c**) S82 the creeping individual of F1; (**d**,**e**) creeping and standard offsprings in BC1 population (in summer); (**f**) creeping and standard offsprings in BC1 population (in autumn); (**g**,**h**) methods of measuring branch angle and plant canopy angle. θ<sup>1</sup> and θ<sup>2</sup> are the angles of inclination of the widest position of the canopy from vertical orientation on both sides. Plant canopy angle is the sum of θ<sup>1</sup> and θ2.

#### *2.2. Phenotypic Measurement and Data Analysis*

Phenotypic traits related to plant architecture, i.e., plant height (PH), plant width (PW), plant canopy angle (PCA), branching angle of the main branch (BA), the number of main branches (NMB), and branching height (BH), were measured for 174 individuals from the S82 (♀) × *L. fauriei* (♂) population at the end of the growing season. In addition, leaf length (LL) and leaf width (LW) were measured during the peak of the growing season. PH and BH were measured at the highest point, and PW was measured at horizontal direction by ruler. LL and LW were measured with an electronic vernier caliper. Each trait was measured more than three times. NMB was directly visualized. The measurements of the above phenotypic traits were described by Ye et al. [35,37].

The branching angle of the main branch corresponds to the angle between the main branch and the horizontal direction. In [42], it is reported that the angle between the horizontal direction and the line connecting the basal branch and apical branch (angle α in Figure 1g) is a more reliable parameter than branch growing angle (angle β in Figure 1g). Moreover, this more reliable angle is more closely related to plant architecture and measures the branching angle of the main branch by the measurement of angle α. PCA and BA were measured by a huge protractor suspended at the origin. The plant canopy angle (θ = θ<sup>1</sup> + θ2, Figure 1h) was measured according to Thakur et al. [43].

Genetic variation analyses were performed on phenotype traits of BC1 population using SPSS 22.0 software (SPSS, Chicago, IL, USA). Statistical parameters included mean, maximum, minimum, variance, standard deviation, skewness, peakness, and coefficient of variation and plot the frequency distribution. The Pearson correlation coefficient between traits was calculated by the Correlations module in SPSS software and significant difference tests were performed at different levels.

#### *2.3. DNA Extraction and Detection*

Genomic DNA was extracted from fresh young leaves using the Fast DNA kit (Tiangen Biotech, Beijing, China) following the manufacturer's protocol. DNA quality and concentration were measured by 1% agarose gel electrophoresis with Gel Red [44].

#### *2.4. Construction of Near-Isogenic Pools*

The construction of near-isogenic pools was based on the methods reported by Michelmore et al. [16]. Five extremely standard individuals and five extremely creeping individuals (as described in Figure 1f) were randomly selected from 174 individuals in BC1 population and their genomic DNA was mixed to construct standard and creeping gene pools, respectively. The standard gene pool was denoted BZ, whereas the creeping gene pool was denoted BP.

#### *2.5. Screening SSR Markers Linked to Creeping Strains*

DNA from eight BC1 individuals was selected randomly to detect the polymorphism of the new SSR primers. Based on all the highly polymorphic primers in crape myrtle, two near-isogenic pools (BZ, BP) and three standard and three creeping individuals were then randomly selected and screened by SSRs. The 322 pairs of primers used in the experiment were developed using the transcriptome data from a previous study [45] (Table S1).

PCR products were detected by non-denaturing polyacrylamide gel electrophoresis. If the product strips with polymorphisms in two pools were similar for six plants, it was initially concluded that it may relate to plant architecture of crape myrtle. These primers were detected in parents and segregating populations and were correlated with phenotypic data and genetic distance (cM) between the SSR markers. The genes related to plant architecture were identified by Kosambi's method [46]. Furthermore, the validity of the screened polymorphic marker was verified by varieties in a germplasm resource nursery.

#### **3. Results**

#### *3.1. Segregation Analysis of Phenotypic Variation in BC1 Populations*

Eight statistical parameters were calculated from eight phenotypic characters (PH, PW, PCA, BA, NMB, BH, LL, and LW), and the frequency distribution histogram was constructed using SPSS 22.0 and Excel (Figure 2). In the S82 × *L. fauriei* population, the coefficients of genetic variation were between 20.09% and 35.49%, except for the BH genetic variation coefficient. The highest degree of variation was observed for PH followed by NMB, whereas the smallest variation was observed for PCA. The variability in the measured traits among individuals was greater than 10%, indicating significant genetic variation in these traits (Table 1).

**Figure 2.** Distribution of eight phenotypic traits of 174 individuals in S82 × *L. fauriei* population. (**a**) Plant height (PH); (**b**) plant width (PW); (**c**) plant canopy angle (PCA); (**d**) branching angle of the main branch (BA); (**e**) the number of main branches (NMB); (**f**) branching height (BH); (**g**) leaf length (LL); (**h**) leaf width (LW).


**Table 1.** Descriptive statistics of phenotypic characters in S82 × *L. fauriei* population.

Note: PH: plant height; PW: plant width; PCA: plant canopy angle; BA: branching angle of the main branch; NMB: the number of main branches; BH: branching height; LL: leaf length; LW: leaf width. The phenotype units are only used for the mean, standard deviation, maximum, and minimum.

#### *3.2. Correlation Analysis of Population Phenotypic Traits*

The correlation between the eight phenotypic characters was analyzed by SPSS (Table 2). PCA showed a significant positive correlation with NMB and a significant negative correlation with PH and BA. BA showed a significant positive correlation with PH and a significant negative correlation with PCA and NMB.

**Table 2.** Correlation analysis of phenotypic characters in S82 × *L. fauriei* population.


Note: PH: plant height; PW: plant width; PCA: plant canopy angle; BA: branching angle of the main branch; NMB: the number of main branches; BH: branching height; LL: leaf length; LW: leaf width. \*\* means highly significant at the 0.01 level; \* means significant at the 0.05 level.

#### *3.3. Detection of Near-Isogenic Pools, Parents, and BC1 Segregation Populations by SSR Markers*

High polymorphism was detected for 322 pairs of SSR primers for eight BC1 individuals (selected randomly from BC1). The 322 pairs of primers with high polymorphism were screened using the standard gene pool BZ and creeping gene pool BP as templates. Finally, four pairs of SSR primers with polymorphic strips were screened in two gene pools (Figure 3).

Primers that are polymorphic in both gene pools were further amplified in the parents and other hybrids to determine strip type. Of the above four primers, only S364 and LYS12 were able to amplify strips based on the phenotype of the parents (Figure S1, Table 3) and 174 individuals of the segregating population (Figure S2 and Figure S3).

**Figure 3.** Electrophoresis of four primers' amplification products in parents and gene pools. (**a**) Electrophoretic results of Q111 primers; (**b**) electrophoretic results of S364 primers; (**c**) electrophoretic results of LYS12 primers; (**d**) electrophoretic results of LYS13 primers. BZ: Standard creeping gene pool; BP: Creeping gene pool; Z1, Z2, and Z3: Three standard individuals; P1, P2, and P3: Three creeping individuals; M: DNA marker.


**Table 3.** Phenotypes and amplified strips of parents and near isogenic pools.

Note: Z: Standard individual; P: creeping individual; a: Single strip in primer S364; b: Double strip in primer S364; m: Single strip in primer LYS12; n: Double strip in primer LYS12.

S82 P a n *L. indica* 'Creole' P a n

#### *3.4. Linkage Analysis of Markers and Phenotypes*

From the continuous observation of the BC1 population, we found that PH, PW, NMB, and BH changed over time, whereas the ratio between the plant width and plant height (PW/PH), PCA, and BA maintained a relatively stable state. The statistical results of the phenotypes of the BC1 population and parents were listed in Table S2. A histogram of the frequency distribution for three traits (PW/PH, PCA, and BA) of the parents for the BC1 population was drawn using Excel software (Figure S4). Based on the phenotypic data for parents and hybrids, the plant architecture of crape myrtle was classified into

six classes using PW/PH, PCA, and BA. Artificially, PW/PH less than 1.5 were classified as G-type and all others were H-type. PCA was classified as J-type or K-type depending on whether values were less than 90◦. Similarly, BA was classified as U-type or V-type depending on whether values were greater than 60◦ (Table 4). It can be seen that types G, J, and U correspond to plants with smaller PW/PH, smaller PCA, and larger BA, respectively; namely, plant architecture will be creeping and flat. Conversely, H-, K-, and V-types correspond to those with larger PW/PH, larger PCA, and smaller BA, which means that the plant architecture is more vertical. The segregation ratio of traits in the hybrids of the population implies that the upright type has a dominant effect on the creeping type. It is preliminarily considered that the upright/creeping type of *Lagerstroemia* species is controlled by both the major gene and the minor genes.


**Table 4.** Classification of the phenotype of 174 individuals in S82 × *L. fauriei* population.

Note: PH, plant height; PW, plant width; PCA, plant canopy angle; BA, branching angle of the main branch.

According to phenotypic grading standards, phenotypic statistics, and the strip type of the parents and two gene pools, the phenotypes and strips of 174 individuals were comparatively analyzed. The results showed that phenotypes G, J, and U correspond to the strip type "m" for primer S364 and strip type "b" for primer LYS12. Phenotypes H, K, and V correspond to strip type "a" for primer S364 and strip type "n" for primer LYS12 (Table S3). Statistical analysis showed that primer S364 correlated with PCA (J-type and K-type) and primer LYS12 correlated with BA (U-type and V-type).

Among the 174 tested individuals, eight individuals did not amplify the objective strips in primer S364, whereas all individuals amplified objective strips in primer LYS12. There are 127 and 129 strips that conformed to the phenotype in primers S364 and LYS12, respectively (Table 5). Based on statistical results, the genetic distance between the S364 molecular marker and the gene that controls PCA was approximately 23.49 cM. The genetic distance between the LYS12 molecular marker and the gene that controls BA was approximately 25.86 cM. Two SSR molecular markers were verified in the BC1 population and the accuracy rate of phenotypic verification using S364 and LYS12 was 76.51% and 74.14%, respectively (Table 6).

**Table 5.** Statistics of two primer strip types in BC1 plants.


Note: "a" and "b" mean single strip and double strip in primer S364, respectively; "m" and "n" mean single strip and double strip in primer LYS12, respectively; "–" means no strip.


**Table 6.** Number of phenotypes and strip type of BC1 plants.

Two markers, S364 and LYS12, were further identified in 20 *Lagerstroemia* species or cultivars (16 were upright trees or shrubs and 4 were low shrubs with creeping or flat branches) and the strip types were recorded. As shown in Table 7, the comparison of plant architecture types and two marker strip types showed that 15 of 20 cultivars were identified by the S364 marker and the accuracy rate of phenotypic identification was 75%. Eighteen cultivars were identified with LYS12 marker bands and the accuracy rate of phenotypic identification was 90%. These results indicate that the two markers were able to accurately identify phenotypic traits in crape myrtle cultivars.

**Table 7.** Identification of two markers in 20 *Lagerstroemia* stocks with diverse plant architectures.


#### **4. Discussion**

Crape myrtle is popular and widely used in gardens because of its long flowering time and rich and colorful flowers. It can be planted alone or in clusters, used for garden flower belts, or cut flowers can be displayed in vases after pruning and pinning. The lack of varieties with diverse plant architecture types, flowers with a pleasant fragrance, and early-flowering features limits the further application of crape myrtle. Plant architecture traits (e.g., dwarf, weeping, creeping, columnar, and branching angle) have attracted much attention; these have potentially important application value for the plant architecture improvement of horticultural crops. Dwarf crape myrtles are becoming increasingly popular for use as potted plants and indoor flowers [47]. However, dwarfing and the arborization of trees are complex quantitative traits, which have greater non-additive effects on genetic performance and heritability. The columnar trait is controlled by a single gene (*Co*); however, other modification genes that may also play a role have been confirmed in apple [11]. Incomplete dominant columnar traits (pillar) also exist in peach, with a small branching angle and vertical growth of branches, but no obvious genetic segregation rule was observed for dwarf and compact traits [7].

In this study, the number of upright and creeping individuals for the F1 of *L. fauriei* and *L. indica* "Creole" was 140 and 52, respectively, and the ratio of the two plant types was 2.69/1. In the BC1 population of S82 × *L. fauriei*, the ratio of upright (138) to creeping (36) individuals was 3.83/1. However, in the BC1 population of S82 × *L. indica* "Creole", the number of upright and creeping individuals was 42 and 150, respectively, with a ratio of 1/3.57 (Table S4). In the F1 and S82 × *L. fauriei* populations, the number of upright individuals was much larger than that of creeping individuals because all hybrids were derived from crosses between upright and creeping parents. Although both parents (S82 × *L. indica* "Creole") were creeping types, upright individuals still appeared in their hybrids (Table S4). The comprehensive analysis of the segregation of two plant architecture types in three genetic populations cannot determine the number of genes controlling the creeping trait. However, the analysis shows that the upright type has a dominant effect on the creeping type. The upright/creeping plant type of crape myrtle is speculated to be controlled by a major gene and also regulated by minor genes. Our recent research results show that the dwarf trait of crape myrtle is also likely controlled by a major gene and modified by minor genes based on the phenotypic data in the F1 population of *L. fauriei* (♀) × *L. indica* "Pocomoke" (♂) [35]. Correlation analysis between phenotypic traits is helpful to advance the selection of plant architecture in crape myrtle seedlings, which can shorten the breeding time and enhance breeding efficiency. A significant positive correlation was observed between PH and PW. PCA is verified by individual traits in the BC1 population, which is similar to the correlation obtained for the F1 population generated from *L. caudate* (♀) and *L. indica* "Xiang Xueyun" (♂) [36]. Eleven quantitative characters for 192 individuals in the F1 population from a cross between *L. fauriei* (♀) and *L. indica* "Creole" (♂) were measured and analyzed; the results showed that the diversity index for plant architecture was 1.05 times greater than 1, which means that there is great potential for genetic improvement of the plant architecture [48]. In addition, most quantitative characters were significantly correlated with plant architecture and branching pattern. The results of heredity for some traits in the F1 population of *L. speciosa* and *L. indica* showed that the coefficient of variation in the F1 generation was 14.58–40.16%, which indicates significant variation [49].

Traditional breeding methods have the disadvantages of a long cycle, heavy workload, and low effectiveness for improving tree architecture. By using molecular marker-assisted selection, healthy individual plants with target traits can be selected at the seedling stage, which reduces the waste of resources, reduces the cost of breeding, and speeds up the breeding process [15]. BSA overcomes the constraints of near-isogenic lines and saves time and effort; it is widely used in marker development and gene mapping [16]. Because BSA does not require a large population, it is very popular in the study of the plant architecture traits of woody plants, such as apple (vertical traits) and peach (columnar and vertical traits) and in the development of genes and markers for peach weeping traits [2,10,50]. In ground-cover chrysanthemum, a random amplified polymorphic DNA (RAPD) marker A-10555 linked to the creeping trait was 7.96 cM from the loci controlling creeping/standard traits [51]. In crape myrtle, an SSR marker was identified and linked to the dwarf gene with a distance of 23.33 cM between the loci and dwarf gene [35]. In this study, two SSR markers (S364 and LYS12) closely linked to creeping traits in crape myrtle were obtained according to the principle of BSA. One marker was linked to PCA, with a genetic distance of 23.49 cM, and the other was linked to BA, with a genetic distance of 25.86 cM. The two markers were verified in the BC1 population, parents, species, and varieties with high accuracy, which suggests that it is feasible to use these two markers to perform molecular marker-assisted breeding of a creeping plant type for crape myrtle. Previous research indicates that the polymorphic loci assayed within 15 cm of the target locus can be identified; loci are detected with decreasing frequency as genetic distance increases [16]. Therefore, the difference between two near-isogenic pools cannot be determined with the genetic distance of 23.49 cM and 25.86 cM from creeping genes. In addition, BSA has limitations in locating quantitative traits, which can only detect large-effect QTLs and require samples with large phenotypic differences when near-isogenic pools are constructed. In the future, we can use multi-generational hybridization and backcrossing to obtain populations with more significant phenotypic segregation to construct pools and screen markers. Additionally, the number of species-specific molecular markers is a critical factor that influenced the results of this experiment. To achieve an ideal effect in the marker-assisted selection of the creeping

trait, we will develop more species-specific molecular markers to enhance the coverage area of the crape myrtle genome.

#### **5. Conclusions**

Crape myrtle is a widely used horticultural plant with important ornamental value, but it lacks creeping varieties. This study is the first to map creeping genes in *Lagerstroemia* species. In a BC1 population derived from *L. fauriei* Koehne (standard) and *L. indica* "Creole" (creeping), 174 individuals were employed to screen molecular markers linked to the creeping trait of crape myrtle among 322 SSR primers with good polymorphism using BSA and SSR technologies. Two SSR markers (S364 and LYS12), which were 23.49 cM and 25.86 cM from the loci controlling plant opening angle trait and branching angle trait, were detected and further verified in the population, parents, species, and varieties with more than 74% accuracy, respectively. Our study will lay the foundation for the QTL mapping and marker-assisted selection breeding for creeping architecture of crape myrtle.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4907/10/5/429/s1. Figure S1: Electrophoretic results of S364 primers in BC1 individuals; Figure S2: Electrophoretic results of LYS12 primers in BC1 individuals; Figure S3: Electrophoresis of S364 and LYS12 amplification products in parents. (**a**) Electrophoretic results of S364 primers; (**b**) Electrophoretic results of LYS12 primers; Figure S4: Distribution of three traits of BC1 population. P1: *L. fauriei*, P2: *L. indica* "Creole", P3: S82 individual, Table S1: Sequence of 322 pairs of primers used in the study; Table S2: Phenotypic characters of parents and BC1 plants; Table S3: Phenotype and strip type of 174 individuals in BC1 population; Table S4: Classification of phenotype of 174 individuals in F1 population of *L. fauriei* and *L. indica* "Creole".

**Author Contributions:** Conceptualization, T.Z.; data curation, T.Z., B.Q., and H.P.; formal analysis, T.Z., B.Q., and S.L.; funding acquisition, Q.Z.; investigation, B.Q., M.C., and T.C.; methodology, B.Q., S.L., and M.C.; project administration, T.Z. and Q.Z.; resources, M.C., H.P., J.W., and T.C.; software, B.Q., S.L., H.P., and J.W.; writing—original draft, T.Z. and B.Q.; writing—review and editing, T.Z. and Q.Z.

**Funding:** This work was supported by the program for Science and Technology of Beijing (No. Z181100002418006) and Special Fund for Beijing Common Construction Project.

**Acknowledgments:** We are thankful to American Journal Experts (AJE) for suggesting professional native English speaker for our manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest and this research is carried on the absence of any financial or commercial relationships that could be interpreted to a potential conflict of interest.

#### **Abbreviations**


#### **References**


© 2019 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/).

## **Morphological Characteristics and Allometric Relationships of Shoot in Two Undergrowth Plants:** *Polygonatum odoratum* **and** *Polygonatum multiflorum*

**Mirela Tulik 1,\*, Jerzy Karczewski 2, Natalia Szeliga 3, Joanna Jura-Morawiec <sup>4</sup> and Ingeborga Jarzyna <sup>5</sup>**


Received: 16 November 2018; Accepted: 17 December 2018; Published: 19 December 2018

**Abstract:** The main purpose of this investigation was to describe the spatial arrangement of shoot tissues, as seen in transverse section, and allometric relationships in two contrasting species of *Polygonatum* i.e., *Polygonatum odoratum* which commonly grows in mixed pine-oak forest with shoots rectangular in shape, and *Polygonatum multiflorum* found in oak-hornbeam forest with cylindrical shoots. The mass and length of the aerial shoots of each individual plant were measured. The shoot regions of each plant were then categorized as basal (b), central (c) or apical (a). Transverse sections of these shoot regions were subsequently cut, and the following parameters were measured: (1) Diameter of shoots, (2) thickness of the outer and inner zones of parenchyma and (3) thickness of the sclerenchyma zone. Additional allometric relationships between the various measurements were computed and determined as Pearson's correlation coefficients (*r*). Both species of *Polygonatum* differed significantly with respect to the length, diameter and thickness of the outer zone of parenchyma. Shoots of *P. multiflorum* were taller but narrower than those of *P. odoratum,* which had a significantly wider zone of outer parenchyma. Allometric relationships were stronger for *P. multiflorum,* and for both species, they were generally stronger in the basal part of the shoot. We conclude that in *P. multiflorum,* the strong correlation between the diameter and length of the shoot seems to be important to the growth in shaded environments.

**Keywords:** allometry; anatomy; *Polygonatum odoratum*; *Polygonatum multiflorum*; shape; shoot

#### **1. Introduction**

Spatial organization of the plant body is defined as plant architecture subject to genetic and environmental control [1]. It is widely believed that the purpose of the mechanical integrity of the shoot is to ensure the reproductive success and survival of the plant [2]. Such mechanical integrity, and the peripheral location (or peripheral concentration in the case of monocots) of vascular bundles and mechanical supporting tissues such as collenchyma and sclerenchyma also enable the plant to resist and respond to physical forces such as wind and are exacerbated by the weight of the plant itself [3]. Crook et al. [4] demonstrated that plants occupying different habitats are able to modify their structural investment so as to maintain a "constant factor of safety" against mechanical failure.

As a consequence, plants that grow in exposed sites differ from those occupying more sheltered environments in terms of their morphology and anatomy [5].

In a forest, light is one of the main factors affecting species diversity and coexistence of plants [6,7]. It influences the abundance [8] and composition [9] of species in the understory. The plants growing on the forest floor obtain only 0.5%–5% of incident light [10]; in tropical rainforests less than 1% [11] and less than 2%–5% in moderately humid deciduous forests [12].

We examined two species of *Polygonatum*, growing on the forest floor namely *Polygonatum odoratum* (Mill.) Druce and *Polygonatum multiflorum* (L.) All, both members of the monocotyledonous family Asparagaceae (APG III 2009). These differ from each other in several ways, including the number of flowers located in leaf axils, the size and shape of the shoot, and the habitats that they occupy. One of the most distinct differences between their habitats is the amount of light reaching the forest floor [13]. *P. odoratum* is native to Europe, Asia, and Northern Africa (http://e-monocot.org). It prefers semi-shade and a moderately exposed habitat, where it grows to a height of ~65 cm. Its white, tubular flowers (one, rarely two per axil) hang singly from the underside of the shoot, which is square to rectangular in transverse section. By contrast, *P. multiflorum* can grow to a height of 80 cm in deciduous European or Asian forests (beech, oak and hornbeam). Its tubular flowers are also white (usually 3–5 per leaf axils) and also hang from beneath the shoot which, unlike that of *P. odoratum*, is cylindrical in shape and round in transverse section.

Since mechanical design, whether it be at the cellular level or at the level of whole-plant architecture, appears to be fundamental to survival, the main purpose of this investigation was to describe the spatial arrangement of shoot tissues, as seen in transverse section, and allometric relationships in two contrasting species of *Polygonatum*.

#### **2. Materials and Methods**

Shoots of the two species used for our study (*P. multiflorum* and *P. odoratum)* were collected in the late spring of 2015 and 2017. Fifteen flowering individuals of each species were collected for each of the two years of our study. Individuals of *P. odoratum* were found in mixed pine-oak forest *Querco roboris*-*Pinetum* (W. Mat.1981) J. Mat. 1988) whereas *P. multiflorum* grew in sub-continental oak-hornbeam forest (*Tilio-Carpinetum* Tracz. 1962). Plants of each species were cut at ground level, protected from desiccation, and brought to the laboratory, where they were subjected to investigations.

Shoot length (L) was subsequently measured and three shoot regions determined for each, namely: Basal (b), located close to the base of the shoot; central (c), located in the middle part of the shoot; and apical (a), located near the apex (Figure 1).

Hand-cut transverse sections were then obtained from each of these shoot regions for both species. The protocol using carrot and cork, as recommended by Gärtner and Schweingruber [14], was adopted for preparing sections using a sliding microtome (Microm HM 440, GMI Inc, Ramsey, MN, USA). For the detection of lignified cell walls, sections were stained with Alcian blue and Safranin [15] and some sections were also observed under UV (ultraviolet) light. Photomicrographs were achieved with the aid of an Olympus system consisting of a BX61 motorized microscope (Olympus, Tokyo, Japan) and Cell P image analysis software (version 3.4) coupled to a Color View digital camera (Olympus Soft Imaging System GmbH, M ˝unster, Germeny). Based on images taken from each region of the shoot for every individual, the following parameters were measured: (1) diameter of shoot (D); (2) thickness of mechanical tissue zone (sclerenchyma zone, Ws); (3) thickness of parenchyma tissue located externally to the mechanical tissue (hereafter referred to as outer parenchyma zone (Wp)); and (4) thickness of inner parenchyma zone (Wpi, calculated and expressed as the difference between the diameter and the sum of the thicknesses of outer parenchyma and sclerenchyma zones, as follows: Wpi = D − (Wp + Ws)). Measurements were performed at two points on the circumference of each section and the average measurements calculated. The accuracy of these measurements was ±0.01 mm.

**Figure 1.** Location of three regions along the length of *Polygonatum* shoot categorized as basal (b), central (c) and apical (a). Region (b) was 0.5 cm above ground level.

In addition, we determined the mass (M) of the entire aerial part of individual plants collected in 2017 and measured shoot length (L). Accuracy of the measurements was ±0.01 g and ±0.1 mm, respectively. The allometric relationships between all measurements were characterized by allometric scaling laws of the form Y = cMb (c—constant, b—allometric scaling coefficient) and determined as Pearson's correlation coefficients (*r*) on data transformed to a natural logarithm. A similar method was used by Niklas [5], Weiner and Thomas [16], and Poorter et al. [17].

Significance of difference between means was assessed by Student's *t*-test. Analyses were computed on Statistica software, version 13.

#### **3. Results**

#### *3.1. Morpho-Anatomical Analysis of Polygonatum Shoots*

Shoot anatomy of both investigated species demonstrated significant similarity, despite differences in the cross-sectional shape of the shoots (rectangular in *P*. *odoratum* and round in *P*. *multiflorum*; Figure 2a, b). The shoots of both species were comprised of epidermis, ground tissue and collateral vascular bundles. The ground tissue was represented by the outer parenchyma, sclerenchyma and inner parenchyma (core). The collateral, closed vascular bundles, were irregularly scattered throughout the ground tissue (Figure 2c–e). Towards the outer parenchyma, the bundles were smaller in size, whereas those near the centre of the shoot were larger. The vascular bundles, especially those located peripherally, were enclosed within a sclerotic sheath comprised of layers of sclerenchyma fibres.

**Figure 2.** (**a**,**b**) Diagram showing shape of *Polygonatum* shoots as seen in transverse section; rectangular in *P. odoratum* (**a**) and circular in *P. multiflorum* (**b**). The sclerenchyma zone is marked black; the outer/inner parenchyma zone white. Vascular tissue is represented by scattered collateral bundles. (**c**–**e**) Photomicrographs of transverse sections of *P. odoratum* (**c**,**d**) and *P. multiflorum* (**e**) shoots. Scale bar = 200 μm. (**c**)—section viewed under UV light. Fluorescence of lignified cell walls of sclerenchyma cells is marked with arrow. p—outer parenchyma zone; s—sclerenchyma zone; pi—inner parenchyma zone; cvb—collateral vascular bundle.

No significant difference were observed in the thickness of the sclerenchyma zone between the species at basal level. Nevertheless, plants varied significantly with respect to the thickness of the outer zone of parenchyma, regardless of region (Table 1).


**Table 1.** Values of Student's *t*-test for the mean thickness of the outer parenchyma zone for the three regions (basal, central, and apical) along the shoots of *P. odoratum* (P. o) and *P. multiflorum* (P. m).

Means are expressed in mm, *n* = 15.

In the basal region, the mean thickness of the outer parenchyma zone of *P. odoratum* reached 0.19 mm, as compared with 0.10 mm in *P. multiflorum* (Figure 3).

**Figure 3.** Thickness of outer parenchyma zone at basal (b), central (c) and apical (a) regions along shoot of both species.

For both species, the width of the outer parenchyma zone diminished along its length, although for *P. multiflorum*, the mean value at the central region was almost identical to that for the apical region. The mean thickness of the sclerenchyma zone in the basal region was similar: 0.055 mm for *P. odoratum* and 0.053 mm for *P. multiflorum*. In both species, the diameter of shoot and thickness of the sclerenchyma zone diminished on approaching the apical region, but we observed very significant differences between values (Figure 4).

**Figure 4.** Thickness of sclerenchyma zone at basal (b), central (c) and apical (a) regions along shoot of both species. Significant differences are for central (*p* <0.0001) and apical (*p* <0.0000001) regions.

Moreover, the species differed significantly with respect to the radius/diameter, thickness of the outer parenchyma zone and sclerenchyma zone relative to the overall length of their shoots (Table 2).

**Table 2.** Values of Student's *t*-test for the mean radius and thickness of the outer parenchyma zone and sclerenchyma zone for the entire length of the shoot of tested plants.


P. o—P. odoratum; P. m—P. multiflorum. Means are expressed in mm, *n* = 45.

#### *3.2. Allometric Relationships*

Correlations between diameter (D) and length (L) of the shoot for each of the two species and for all three regions of the shoot (basal, central and apical) were very significant (Table 3, Figure 5). For *P. multiflorum*, values for *r* were greater than for *P. odoratum*. For both species, values for *r* were greater for the basal region than for the apical region of the shoot. Individuals of *P. multiflorum* had longer shoots, although these had smaller diameters.

**Table 3.** Values of Pearson's correlation coefficients (*r*) between diameter (D) and length (L) of the shoot for the two species *P. odoratum* and *P. multiflorum* based on three regions of the shoot (basal, central and apical).


Data transformed to natural logarithm. Asterisks denote the statistical significance of Pearson's correlation coefficient: \*\*\* *p* <0.001, \*\* *p* <0.01, \* *p* <0.05, *n* = 15.

**Figure 5.** *Cont.*

**Figure 5.** Relationships between diameter and length for all three regions of the shoot for the two species *P. odoratum* and *P. multiflorum.*

A relationship also existed between mass (M) and length (L) of the shoot (data ln-transformed). For both species, coefficients of correlation were similar: for *P. multiflorum r* = 0.6473 and for *P. odoratum r* = 0.6757, *p* <0.01 (Figure 6). Individuals of *P. multiflorum* were generally larger, with longer shoots and greater mass.

**Figure 6.** Relationships between mass and length of the shoot for the two species *P. odoratum* and *P. multiflorum.*

We also observed correlations between the thickness of the sclerenchyma zone (Ws) and that of the outer parenchyma zone (Wp) vs. shoot diameter (D), as well as shoot length (L), in both species (Table 4). Correlations were generally stronger for the basal part of the shoot than for the apical region, but this relationship was generally not very strong.


**Table 4.** Values of Pearson's correlation coefficients (*r*) between the thickness of the sclerenchyma zone (Ws) and the thickness of the outer parenchyma cylinder (Wp) vs. shoot diameter (D) and shoot length (L) for *P. odoratum* and *P. multiflorum* based on three regions of the shoot (basal, central and apical).

Data transformed to natural logarithm. Asterisks denote the statistical significance of Pearson's correlation coefficient: \*\*\* *p* <0.001, \*\* *p* <0.01, \* *p* <0.05, NS—non significant, *n* = 15.

#### **4. Discussion**

The organization and spatial distribution of tissues in the shoot of two species of *Polygonatum* may support the thesis that these plants are adapted to be resistant to mechanical failure, since they are composed of a variety of cells whose walls (made of cellulose and lignin) differ in elasticity [5,18]. Comparison of the shoot structure of *Polygonatum* with the anatomy of grasses reveals a universal solution to certain structural problems (in particular those relating to mechanical support) common to both groups of plants. Fundamentally, the most common type of aerial shoot anatomy in monocots is based on the presence of a peripherally situated cylinder of sclerenchyma fibres. Hypodermal sclerenchyma is also very common in grass. This tissue is dead at maturity, with thick, lignified walls enclosing an empty lumen [5,19]. The distribution of this tissue provides increased support and rigidity and helps the plant withstand forces, such as wind. Centralized arrangement of mechanical

tissues is also observed, especially in roots, as well as the shoots of submerged, aquatic angiosperms, both of which are subject to pulling strains [20]. A sclerotic sheath (a sheath of sclerenchymatous fibres surrounding the vascular bundles) may comprise the main stiffening element in many other monocots, such as palms [21]. It is stiffer by far than the surrounding parenchymatous ground tissue in which the vascular bundles are embedded [22–24]. It is also worth noting that the shoot of grasses is usually hollow e.g., the internodes of cereals. In other words, air occurs at the 'core' of grass shoots and this is the lightest and least expensive arrangement, both in terms of material and energy expenditure. Moreover, it permits longitudinal gaseous transport and is of particular value in certain marginal plants, where like the aerenchyma of true hydrophytes, it may allow the aeration of submerged organs. The arrangement of mechanical tissue, both peripherally and around each vascular bundle is considered optimal and in the case of grasses, provides excellent protection from mechanical failure (e.g., wilting), since the weight of plant per unit of shoot volume is greatly reduced. In describing the morphological and anatomical adaptation of grass shoots to mechanical stress, Frey [25] compared it with a fundamental rule of eastern fighting, namely, "flex to win". In contrast to grasses, the shoot core of *Polygonatum* is solid and parenchymatous, consisting of thin-walled parenchyma cells. Thin-walled tissues tend to possess lower elastic moduli than thick-walled tissues [5]. Nevertheless, they become increasingly important when the shoot is thick and the amount of sclerenchyma present small. Parenchyma may provide mechanical support hydrostatically by means of cell turgidity [5].

As previously mentioned, in order to be resistant to mechanical support, the shoot must be able to bend, but not break, and therefore it is necessary that the most rigid tissue is peripherally located. As a result, some sort of compromise must be reached between the distribution of mechanical and photosynthetic tissue. The question then arises: How do both species of *Polygonatum* resolve this anatomical conflict between the requirement for support and that for photosynthesis? One possible explanation in the case of *P. multiflorum* is that the turgor pressure of the inner parenchyma cells on the inside of the cylinder of sclerenchyma reinforces the shoot. As a result, these plants may grow taller and become more slender so as to absorb more solar energy for photosynthesis and other light-dependent processes. In the case of *P. odoratum*, the outer zone of photosynthetic cortical parenchyma is wider and therefore the mechanically resistant sclerenchyma is less peripheral in its distribution. As a consequence, the shoots of this species are shorter and have a greater diameter, allowing the plant to grow at greater light intensities and owing to the wider zone of chlorophyllous cortical parenchyma (outer parenchyma), photosynthesis is enhanced. It would also appear that in the case of *Polygonatum* the compromise between mechanical support and photosynthesis is subject to the environment, especially since light conditions have a particularly strong impact both on plant size and form [26,27].

The behaviour of *P. multiflorum* may also be compared with the growth strategy of sapling trees. Young and quickly growing trees produce juvenile wood external to the pith [28]. The properties of juvenile wood are different from those of mature wood and the former is thought to be mechanically more resistant. Cells of juvenile wood tend to be shorter and have thinner walls. Therefore, juvenile wood is less dense than mature wood [29]. Young trees have slender stems that are more flexible than those of older trees, may grow rapidly towards the canopy and bend without breaking. It seems that the shoots of *P. multiflorum*, which are taller and have a smaller diameter, are better adapted to compete for light than those of *P. odoratum* (despite the fact that their mechanical tissue is represented by sclerenchyma and/or parenchyma cells, and that the pattern of biomass partitioning is intermediate to that typical of herbaceous plants and trees).

Allometric relations have frequently been reported for trees (in order to estimate their total biomass or root mass—parameters that are difficult to measure), but are less often reported for herbaceous plants, which are believed to be a result of natural selection processes and adaptive evolutionary changes [30–32]. Weiner and Thomas [16] suggested that the ability of plants to adapt to the environment modifies allometric relationships, especially in annual plants, and may depend on the prevailing growing conditions [33,34]. Jarzyna [35] investigated allometric relationships for competing and non-competing plants, discovering that those for competing plants were stronger. It is likely that in both *P. odoratum* and *P. multiflorum*, amongst others, the availability of light may be a crucial factor for explaining allometric relationships. These relationships are stronger in *P. multiflorum*, probably because this species grows in poorer light conditions (oak-hornbeam forest). In moderately humid deciduous forests, the percentage of sunlight reaching through the tree foliage to the herb and moss layer on the forest floor is less than 2%–5% [12]. Moreover, light reaches the forest floor mainly in the dormant season, while in the summer time, leaves of the canopy trees block the passage of light to the ground. Based on differences in tree crown morphology and the stand structure occupied by both investigated species of *Polygonatum,* it could be assumed that in a dense forest stand occupied by *P. mutliflorum,* less light penetrates through the canopy. Thus, with limited light availability in the understory, the taller *P*. *multiflorum* might invest more energy into its aboveground biomass i.e., its supporting shoot.

#### **5. Conclusions**

Our paper contributes towards a better understanding of the relationship that exists between plant structure and the environment, based on two contrasting species of *Polygonatum* (*P. odoratum* and *P. multiflorum*) growing on the forest floor that differ from each other in the shape of the shoot, as viewed in transverse section. We conclude that the spatial distribution of tissues in the shoots of the investigated species is typical of that found in self-supporting herbaceous plants. In *P. multiflorum,* the strong correlation between shoot diameter and length seems to be important to growth in shaded habitats.

**Author Contributions:** Conceptualization, M.T. and N.S.; Investigation and Methodology, M.T., N.S. and J.J.M.; Software, J.K. and I.J.; Writing—Review and Editing, M.T., I.J., J.J.M.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare that there is no conflict of interest.

#### **References**


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