2. Materials and methods

### 2.1. Specimens

1. Introduction

200 Advances in Seed Biology

many families in Lamiales [1, 2, 5–7].

flowers, and fruit to seeds.

the Plantaginaceae increased to about 1900 species [15].

In traditional classification, Scrophulariaceae sensu lato (s.l.) are the largest family in the Lamiales. The members of the family can be distinguished from related families by bilaterally symmetrical and often tubular flowers, ovaries with axile placentation and numerous ovules, and many seeded capsular fruits. However, recent molecular studies have suggested that it is not the presence of a suite of uniquely derived characteristics that allows for the easy recognition of a member of Scrophulariaceae s.l. but, rather, the absence in Scrophulariaceae of the synapomorphies that characterize closely related families [1]. The taxonomic problem of the Scrophulariaceae s.l. is one aspect of the widespread difficulties that reflect the problems of describing natural groups within the order Lamiales. As with most large families, previous classification of the family includes many treatments that differ in their circumscription (for example, see [2]). The most influential classifications for the nineteenth century concept of Scrophulariaceae were those of in [3, 4], from which most contemporary classifications of the family are derived. A large-scale investigation of phylogenetic relationships in the Scrophulariaceae s.l. and related families using DNA sequence data has radically altered the circumscription of

Before the revolution in molecular systematics, many studies proposed that Scrophulariaceae s.l. were monophyletic and several morphologically similar groups of taxa, which are now assigned to different families, and were placed together (for example, see [8]). For the first time in [2], the study identified two clearly separated clades consisting of members of the family and suggested that the Scrophulariaceae are polyphyletic. Subsequently, a third clade was identified consisting of parasitic members of the Scrophulariaceae and Orobanchaceae [9–11]. Later on five distinct phylogenetic lineages composed mainly of taxa previously assigned to Scrophulariaceae were recognized [5]. However, the emerging classification for the plants which are traditionally assigned to Scrophulariaceae consists of at least seven groups that bear the rank of family [12–14]. Most notable, following changes in the circumscription of families and the disintegration of Scrophulariaceae s.l., were the dramatic changes in the size of some families: the Scrophulariaceae itself was more than halved in size to just over 1800 species, and

The morphological similarities and differences among the groups of taxa and their alignment in various families usually depend on the characters emphasized by different researchers. On the other hand, complications in discriminating between genera or groups of genera are usually the result of the available suite of usable characters, and thus, several comparative studies were carried out. In general, systematics uses morphological characteristics to carve diversity into its taxonomic subunits, and since the beginning of the discipline, plant systematics has frequently used morphological character ranges from roots, leaves, inflorescence,

Due to its great uniformity, seed morphology has been recognized as an important source of useful phylogenetic information. A number of angiosperm taxa have already been investigated intensively in terms of their seed morphology, in combination with phenetic or phylogenetic analyses at the genus level. In the past, the variation in seed morphology has been used More than 2500 seeds from 41 taxa and 56 accessions were investigated, corresponding to 13 genera of family Scrophulariaceae s.l. originating from seed bank and herbarium specimens at the Korea National Arboretum, Pocheon, Korea. Names of investigated species and accession numbers are presented in Table 1.

#### 2.2. Scanning electron microscopy

Sampling seeds were directly taken from seed bank (stored in <sup>18</sup> C) and thus no pretreatment was needed for scanning electron microscopy (SEM). Before SEM observation, the seed samples were rinsed with absolute ethyl alcohol and sputtered with gold coating in a KIC-IA COXEM ion-coater (COXEM Co., Ltd., Korea). SEM photographs were taken with the help of COXEM CX-100S scanning electron microscope at 20 kV at the Seed Test Laboratory of the Korea National Arboretum. Scale bars were added manually during image alignment.

#### 2.3. Light microscopy

At least 10 to 12 seeds from each species were sectioned and investigated under light microscope. Microscopic slides were prepared using conventional microtome resin method. Mature seeds were dehydrated through alcohol series (50, 70, 80, 90, 95, and 100%). The complete dehydrated seeds were transferred in alcohol/Technovit combination (3:1, 1:1, 1:3, and 100% Technovit) and then embedded in Technovit 7100 resin. Histo-blocks were prepared from each


Morphological Studies on Seeds of Scrophulariaceae s.l. and Their Systematic Significance http://dx.doi.org/10.5772/intechopen.70572 203


Table 1. List of the plant species with their voucher number included in this study.

embedded materials and then sectioned using a Leica RM2255 rotary microtome (Leica Microsystems GmbH, Germany). Serial sections of 4–6 μm thickness were cut with stainless blades, fixed onto a slide glass, and dried on electric slide warmer for about 12 h. In order to stain, dried slides were immersed in 0.1% Toluidine blue O for 60–90 s, rinsed with running water, and again dried with an electric slide warmer for more than 6 h to remove water. After complete removal of the water, slides were mounted with Entellan (Merck Co., Germany) and pressed with metal blocks for a couple days to remove air bubbles. After 2 days, the prepared slides were observed under an AXIO Imager A1 light microscope (Carl Zeiss, Germany). Photomicrographs were taken with an AxioCam MRc5 attached camera system, and seed coat measurement was carried out by using AxioVision software for Windows (release 4.7, 2008). Multiple image alignment was arranged by Photoshop CS4 for Windows 2007. None of the image-alteration facilities of Photoshop were used to modify the original images.

#### 2.4. Morphometry and data analysis

Family Genus Species Voucher number

L. crustacea (L.) F.Muell. L3029

M. roseum Maxim. L2324 M. roseum var. japonicum Franch. & Sav. L1707 M. roseum var. ovalifolium Nakai ex Beauverd L10163 M. setaceum (Maxim. ex Palib.) Nakai L8578 M. setaceum var. nakaianum (Tuyama) T.Yamaz. L3449

P. resupinata f. albiflora (Nakai) W.T.Lee L9042 P. resupinata L. 2014KNA031 P. resupinata var. umbrosa Kom. ex Nakai L10246 Pedicularis verticillata L. L8699 Phtheirospermum japonicum (Thunb.) Kanitz L9691

V. dahurica Steven L10154 V. didyma var. lilacina (H. Hara) T.Yamaz. L2006 V. incana L. L10791 V. kiusiana var. diamantiaca (Nakai) T.Yamaz. L10532 V. kiusiana var. glabrifolia (Kitag.) Kitag. L9413 V. linariifolia Pall. ex Link L7983 V. longifolia L. L9903 V. nakaiana Ohwi 2014GB024 V. peregrina L. L2695 V. persica Poir. L2702 V. pusanensis Y. Lee L11087 V. pyrethrina Nakai L10564 V. rotunda var. subintegra (Nakai) T.Yamaz. L10130 V. undulata Wall. 2014 cc56

Mazus Mazus pumilus (Burm.f.) Steenis L10128 Lathraea Lathraea japonica Miq. 2014REC006 Melampyrum Melampyrum koreanum K. J. Kim & S. M. Yun L9710

Pedicularis Pedicularis mandshurica Maxim. L10193

Siphonostegia Siphonostegia chinensis Benth. L10657

Veronica Veronica arvensis L. L3304

Veronicastrum Veronicastrum sibiricum (L.) Pennell L10658

Paulowniaceae Paulownia Paulownia coreana Uyeki 2014 REC081 Plantaginaceae Limnophila Limnophila indica (L.) Druce L10453

Linderniaceae Lindernia Lindernia procumbens (Krock.) Philcox L10041

Phrymaceae Orobanchaceae

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A total of approximately 2500 seeds were used for morphometric measurement. Digital images of whole seeds were taken with a Leica DFC420 C multifocal camera attached to a Leica MZ16 FA microscope (Leica Microsystems). The length and width of a minimum of 20 seeds from each taxon were measured from digital images using Leica LAS V3.8 software for Windows. Seed length (SL) and width were measured, length/width ratios (LWRs) were calculated, and mean values are presented. Individual seed morphological parameters and their features are given in Appendix 1.

For the analysis, 13 seed characters were treated as non-ordered and coded as unweighted consecutive numbers (Table 2 and Appendix 2). Correspondence analysis (CA) and cluster analysis (UPGMA) were performed to reveal whether the seed features allowed the grouping of taxa using PAST version 3.14 [36]. The eigenvectors and character scores of the first four axes in CA are presented in Table 3, together with the percentage of total variance. The results were presented in a two-dimensional biplot of axis 1 (in X axis) and axis 2 (Y-axis). To visualize the relationship between the different taxa based on seed features, a cluster analysis-based UPGMA tree using Euclidean distance measurement was constructed. Bootstrap support values were based on 100 replicates, and values above 50 were presented above tree branches.


Table 2. Seed characters with their coding states used in analysis.


AW, anticlinal wall; EN, endothecium; EP, epidermis; HC, hilum character; HP, vn; LWR, length/width ratio; PSS, primary surface sculpture; PW, periclinal wall; PWO, primary wall ornamentation; SCA, seed coat anatomy; ECS, epidermal cell shape; SH, seed shape; and SL, seed length.

Table 3. Eigenvectors and character scores of the first four axes of a CA of the 13 seed characters.
