Zhou et al. BMC Plant Biology (2019) 19:574 https://doi.org/10.1186/s12870-019-2155-6 METHODOLOGY ARTICLE Open Access Phenotypic variation of floral organs in Malus using frequency distribution functions Ting Zhou1,2, Junjun Fan1,2,3, Mingming Zhao1,2,4, Donglin Zhang3, Qianhui Li1,2, Guibin Wang1,2, Wangxiang Zhang1,2,4* and Fuliang Cao1,2* Abstract Background: Phenotypic diversity of floral organs plays an important role in plant systematic taxonomy and genetic variation studies. Previous research have focused on the direction of variation but disregarded its degree. Phenotypic variation (including directions and degrees) of 17 floral traits from wild to cultivated crabapples were explored by comparing their distributions and deviations in three different dimensions: floral organ number, size, and the shape. Results: Except for petal number, petal length / petal width, and sepal length / sepal width, the analyzed floral traits of cultivated crabapples all showed downward distributed box bodies in box plot analysis and left deviations of fitted curves in frequency distribution function analysis when compared to the wild, which revealed consistent variation directions of petaloid conversion (pistils or stamens → petals), size miniaturization (large → small), and shape narrowness (petal shape: circular → elliptic; sepal shape: triangular → lanceolate). However, only seven floral traits exhibited significant differences in box plot analysis, while all of the traits in frequency distribution function analysis were obviously offset. The variation degrees were quantitatively characterized by sizing traits > shaping traits > numbering traits and by horizontal dimensions > radial dimensions. Conclusions: Frequency distribution function analysis was more sensitive than the box plot analysis, which constructed a theoretical basis for Malus flower type breeding and would provide a new quantitative method for future evaluation of floral variation among different groups of angiosperms at large. Keywords: Malus spp., Floral variation, Direction, Degree, Frequency distribution Background are semi-double or double flowered. Additionally, germ- Crabapples (Malus spp.) are small trees and shrubs in the plasm with larger flowers are also rare, resulting in a scar- rose family, valued for their charming flowers, colorful city of double-flowered and novel-typed cultivars available small fruits (≤5 cm), and diverse growth habits. They also in today’smarket[8]. have an added advantage of wide environmental adaptabil- Flowers are one of the most ornamental features of ity, facilitating their world-wide prominence as landscape garden plants [9–11]. They display extremely high and gardens focal points [1–3]. After a long period of nat- variation in size, color, structure, and function, which ural selection and crossbreeding, Malus germplasm are the products of continuous remodeling to adapt present a high level of diversity, with a steadily increasing to different environmental conditions and pollinators number of varieties and cultivars in relation to their wild and the important foundations for germplasm innova- ancestors [4–7]. While nearly 1200 Malus taxa are re- tions [12–19]. Currently, most floral variation studies corded in Fiala’s “Flowering Crabapple” book,lessthan5% have been restricted to the anatomical examinations and genetic interpretations for their development on the basis of phylogenetics and molecular genetics, * Correspondence: [email protected]; [email protected] 1College of Forestry, Nanjing Forestry University, Nanjing 210037, China combining with the ABC (DE) and the quartet models Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhou et al. BMC Plant Biology (2019) 19:574 Page 2 of 11 [20–28]. Moreover, variation analyses mainly occurred two groups (P values were 0.2896, 0.1021, 0.4922, above the species level (at the macro level, mainly de- 0.1959, and 0.1394, respectively). termined by paleontology and comparative morph- In terms of floral organ size (Fig. 1-II), larger distri- ology methods) [29–31]. Based on phenotypes and bution ranges were shown in flower diameter, petal statistical principles, few studies were carried out with length, sepal length, sepal width, and pedicel length more intuitive estimations of floral variation below of cultivated crabapples. Among them, distribution the species level (at the micro level, mostly deter- ranges of flower diameter and petal length of culti- mined by genetics, ecology, and low-level systematics vated crabapples completely encompassed the ranges methods) [30, 31]. Chu et al. (2009) summarized the of wild ones (the proportions of transgressive individ- main phenotypic variation of Malus floral organs uals were both 5.6%). Downward distribution trends based on intuitive experience and comparative were presented in sepal length and sepal width, with morphology, which had been widely accepted by re- smaller values in cultivated crabapples than the wild; searchers [32–34]. These results, however, were rela- whereas for the pedicel length, the upward distribu- tively imprecise because of the subjectivity of the tion trend was presented together with higher values methods. Furthermore, these studies did focused on (the proportions of transgressive individuals were 9.3, the direction of variation and disregarded its degree. 15.7, and 5.6%, respectively). On the contrary, sizing Using box plot and frequency distribution function traits of petal width, claw length, pistil length, stamen analyses, phenotypic variation of floral organs from length and pedicel thickness of cultivated crabapples wild to cultivated crabapples were explored to: (1) de- were distributed across a smaller range. Distribution termine the variation rules (including directions and ranges of claw length and pistil length of cultivated degrees) governing floral changes between the two crabapples were completely encompassed by those of groups in three different dimensions: floral organ the wild. And downward distribution trends were pre- number, size, and the shape; (2) compare the effect of sented in petal width and pedicel thickness, with different analytical methods on generating the vari- smaller values in cultivated crabapples than the wild; ation rules; and (3) provide a theoretical basis for the whereas the upward distribution trend was presented inheritance and improvement of Malus germplasm. in stamen length together with higher values (the proportions of transgressive individuals were 5.6, 5.6, Results and 0.9%, respectively). Except for pedicel length (P = Box plot analysis of phenotypic variation of floral organs 0.9660), pistil length (P = 0.0567), petal length (P = between wild and cultivated crabapples 0.0783), and claw length (P = 0.4040), the other six Figure 1 shows box plots for 17 phenotypic traits sizing traits of flower diameter, petal width, sepal reflecting Malus floral organ number, size, and shape. length, sepal width, stamen length, and pedicel thick- Comparative analyses of distributions and differences ness, all showed significant differences between the between the two groups were carried out, one includ- two groups (P values were 0.0244, 0.0005, 0.0001, ing 25 wild crabapples and the other including 108 0.0001, 0.0237, and 0.0001, respectively). cultivated ones. Except for petal number, petal length In terms of floral organ shape (Fig. 1-III), petal / petal width, and sepal length / sepal width, all culti- length / petal width and sepal length / sepal width of vated crabapples’ box bodies of the other floral traits cultivated crabapples were both distributed across a showed downward distributions relative to the wild. larger range that completely encompassed the distri- Specific distributions of all the phenotypic traits were bution ranges of those in wild crabapples (the propor- as follows: tions of transgressive individuals were 8.3 and 7.4%, In terms of floral organ number (Fig. 1-I), petal num- respectively). Significant variation was presented in ber, pistil number, and petal, pistil and stamen number petal length / petal width between wild and cultivated of cultivated crabapples were distributed across a larger groups (P = 0.0030); however, differences in sepal range that completely encompassed the distributions of length / sepal width did not reach the significant level wild crabapples (the proportions of transgressive individ- (P = 0.5298). uals were 4.6, 23.1 and 6.5%, respectively). However, with regards to the stamen number and the pistil and Frequency distribution function analysis of phenotypic stamen number, smaller distribution ranges were shown variation of floral organs between wild and cultivated in the cultivated relative to the wild, although certain crabapples transgressive individuals (smaller individuals) still existed For a clearer analysis of floral variation from wild to cul- (the proportions of transgressive individuals were 2.8 tivated