Open access peer-reviewed chapter

Morphological Studies on Seeds of Scrophulariaceae s.l. and Their Systematic Significance

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Balkrishna Ghimire, Go Eun Choi, Hayan Lee, Kweon Heo and Mi Jin Jeong

Submitted: March 14th, 2017 Reviewed: August 11th, 2017 Published: December 6th, 2017

DOI: 10.5772/intechopen.70572

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Abstract

This study employed scanning electron microscopy and light microscopy to observe seed surface micromorphology and seed coat anatomy in the Scrophulariaceae s.l. to investigate seed characters of taxonomic importance. Seeds of 41 taxa corresponding to 13 genera of the family were carefully investigated. Seeds were minute and less than or slightly larger than 1 millimeter in length except for Melampyrum and Pedicularis species. The seed shape ranged from elliptical to broad elliptical and ovoid. In the studied species the surface sculpture was predominantly reticulate-striate, regular reticulate, sometimes colliculate, and rugose, or - rarely - ribbed, as in Lindernia procumbens and Paulownia coreana. Seed coats comprised the epidermis and the endothelium. Nevertheless, in all Melampyrum and some Veronica species the seed coat was very poorly represented and only formed by a papery layer of epidermis. According to correspondence analysis (CA) and unweighted pair group method with arithmetic mean (UPGMA) based cluster analysis the close affinities among the species of Scrophularia were well supported by their proximity to one another. Similarly, the proximity of Melampyrum species and Pedicularis species cannot be denied. In contrast, Veronica species were divided into two groups in CA plots and even three in the UPGMA tree. Regardless of the limited range taxa considered we found that similarities and differences in seed morphology between different genera could help us to understand the systematic relationships involved.

Keywords

  • Scrophulariaceae s.l.
  • seed morphology
  • seed anatomy
  • scanning electron microscopy
  • light microscopy
  • surface sculpture
  • seed coat
  • systematics

1. Introduction

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 many families in Lamiales [1, 2, 57].

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 [911]. 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 [1214]. 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 Plantaginaceae increased to about 1900 species [15].

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, flowers, and fruit to seeds.

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 variously in plant systematics ranging from identification [16, 17] and taxonomic circumscription [18, 19] to phylogenetic inference [20, 21] and character-state evolution [22, 23]. Both macro- and micromorphological seed characters have been shown to be of essential systematic importance within and among the genera of traditional Scrophulariaceae, Orobanchaceae, and Plantaginaceae[2435], in which seed morphological characters have been used widely to differentiate the different taxa or to find affinities between them.

Recent studies on seed morphology of Scrophulariaceae s.l. have focused mainly on common genera like Veronica, Scrophularia, and Pedicularis. Juan et al. [30] observed fruits and seeds of Scrophulariaceae from southwest Spain, and the systematic significance of seed morphology of Veronicaand Pedicularishas been examined in some comprehensive studies [3234]. Despite the aforementioned, no comparative studies on seed anatomy or seed coat characteristics together with surface structure have been conducted on any genera of Scrophulariaceae s.l. It is necessary to make extensive investigation of the seed morphology and anatomy of Scrophulariaceae s.l. to determine whether they can be used as additional support for disintegration of genera in the family. The objectives of this study were to (1) understand the utility of seed morphology and anatomy in Scrophulariaceae s.l. systematics, (2) discuss the proximity of studied genera based on these characters, and (3) highlight the characters that can be used to describe different genera and possible variation in infrageneric classification of Veronicaon a similar basis.

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2. Materials and methods

2.1. Specimens

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.

FamilyGenusSpeciesVoucher number
LinderniaceaeLinderniaLindernia procumbens(Krock.) PhilcoxL10041
L. crustacea(L.) F.Muell.L3029
Phrymaceae
Orobanchaceae
MazusMazus pumilus(Burm.f.) SteenisL10128
LathraeaLathraea japonicaMiq.2014REC006
MelampyrumMelampyrum koreanumK. J. Kim & S. M. YunL9710
M. roseumMaxim.L2324
M. roseumvar. japonicumFranch. & Sav.L1707
M. roseumvar. ovalifoliumNakai ex BeauverdL10163
M. setaceum(Maxim. ex Palib.) NakaiL8578
M. setaceumvar. nakaianum(Tuyama) T.Yamaz.L3449
PedicularisPedicularis mandshuricaMaxim.L10193
P. resupinataf. albiflora(Nakai) W.T.LeeL9042
P. resupinataL.2014KNA031
P. resupinatavar. umbrosaKom. ex NakaiL10246
Pedicularis verticillata L.L8699
Phtheirospermum japonicum(Thunb.) KanitzL9691
SiphonostegiaSiphonostegia chinensisBenth.L10657
PaulowniaceaePaulowniaPaulownia coreanaUyeki2014 REC081
PlantaginaceaeLimnophilaLimnophila indica(L.) DruceL10453
VeronicaVeronica arvensisL.L3304
V. dahuricaStevenL10154
V. didymavar. lilacina(H. Hara) T.Yamaz.L2006
V. incanaL.L10791
V. kiusianavar. diamantiaca(Nakai) T.Yamaz.L10532
V. kiusianavar. glabrifolia(Kitag.) Kitag.L9413
V. linariifoliaPall. ex LinkL7983
V. longifoliaL.L9903
V. nakaianaOhwi2014GB024
V. peregrinaL.L2695
V. persicaPoir.L2702
V. pusanensisY. LeeL11087
V. pyrethrinaNakaiL10564
V. rotundavar. subintegra(Nakai) T.Yamaz.L10130
V. undulataWall.2014 cc56
VeronicastrumVeronicastrum sibiricum(L.) PennellL10658
LinariaLinaria japonicaMiq.L8982
Scrophulariaceae s.s.ScrophulariaScrophularia buergerianaMiq.L8488
S. grayanaMaxim. ex Kom.L10574
S. kakudensisFranch.L9643
S. koraiensisNakaiL9617
S. takesimensisNakaiL12516

Table 1.

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

2.2. Scanning electron microscopy

Sampling seeds were directly taken from seed bank (stored in −18°C) and thus no pre-treatment 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 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

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 Xaxis) 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.

 1. Shape: 0, elliptical; 1 ovoid
 2. Seed length: 0, <1 mm; 1, 1–2 mm, 2, >2 mm
 3. Length/width ratio: 0, <2; 1, >2
 4. Hilum position: 0, lateral; 1, basal.
 5. Hilum character: 0, distinctly protuberant; 1, flat to indistinctly protuberant; 2, deep
 6. Primary surface sculpture: 0, reticulate; 1, colliculate; 2, others
 7. Epidermal cell shape: 0, rectangular; 1, polygonal; 2, irregular
 8. Anticlinal wall: 0, shallow to indistinctly raised; 1, distinctly raised
 9. Periclinal wall: 0, convex; 1, flat; 2, concave
10. Periclinal wall ornamentation: 0, striate; 1, papillate/granulate; 2, smooth/folded
11. Seed coat anatomy: 0, seed coat distinct; 1, indistinct
12. Epidermis: 0, well represent; 1, degenerate
13. Endothelium: 0, present; 1, absent

Table 2.

Seed characters with their coding states used in analysis.

Character numberCharacter codeAxis 1Axis 2Axis 3Axis 4
1SH−0.231180.76272−0.266120.263812
2SL0.0075710.4672930.281203−0.31876
3LWR0.6547960.9529650.162676−0.4085
4HP0.103170.025843−0.098210.26307
5HC0.0657570.91809−0.06242−0.80719
6PSS0.8176060.046239−0.74190.29745
7ECS−0.09684−0.15219−0.045690.273533
8AW0.86930.0800120.1010480.143169
9PW0.883180.176183−0.032620.129772
10PWO0.4910910.221064−0.31801−0.04532
11SCA0.995628−0.191680.9544530.561853
12EP0.382648−0.617970.775164−0.04003
13EN0.91233−0.079430.5314450.705178
Eigenvalue0.3250.1760.1230.115
% total variance34.355%18.675%13.041%12.213%

Table 3.

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

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.

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3. Results

A total of 41 taxa from Scrophulariaceae s.l. were studied, belonging to six families: Scrophulariaceae sensu stricto (s.s.; 1 genus, 5 species), Plantaginaceae (4 genera, 14 species, 4 varieties), Orobanchaceae (5 genera, 9 species, 4 varieties, 1 forma), Linderniaceae (1 genus, 2 species), Phrymaceae (1 species), and Paulowniaceae (1 species). Selected scanning electron micrographs and light micrographs of seeds are presented in Figures 111. A comprehensive description of seed features by family and genus is given here.

Figure 1.

Scanning electron micrographs of seeds of Scrophulariaceae s.l.Scrophularia kakudensis(A, B, C),S. koraiensis(D, E, F),S. takesimensis(G, H, I),S. buergeriana(J, K, L), andS. grayana(M, N, O). Scale bars: 300 μm (A, B, D, E, G, H, J, K, M, N), 30 μm (C, O), and 20 μm (F, I, L).

Figure 2.

Scanning electron micrographs of seeds of Plantaginaceae.Veronica peregrina(A, B, C),V. arvensis(D, E, F),V. persica(G, H, I),V. longifolia(J, K, L), andV. linariifolia(M, N, O). Scale bars: 300 μm (D, E, G, H, J, K, M, N), 150 μm (A, B), and 20 μm (C, F, I, L, O).

Figure 3.

Scanning electron micrographs of seeds of Plantaginaceae.Veronica dahurica(A, B, C),V. rotundavar.sabinteara(D, E, F),V. pusanensis(G, H, I),V. incana(J, K, L), andV. pyrethrina(M, N, O). Scale bars: 300 μm (A, B, D, E, G, H, J, K), 150 μm (M, N), and 20 μm (C, F, I, L, O).

Figure 4.

Scanning electron micrographs of seeds of Plantaginaceae.Veronica nakiana(A, B, C),V. kiusianavar.diamentica(D, E, F),V. kiusianavar.glabrifolia(G, H, I),V. undulata(J, K, L), andV. didymavar.lilacina(M, N, O). Scale bars: 300 μm (A, B, D, E, G, H, M, N), 150 μm (J, K), and 20 μm (C, F, I, L, O).

Figure 5.

Scanning electron micrographs of seeds of Plantaginaceae, Linderniaceae, and Paulowniaceae.Limnophila indica(A, B),Linaria japonica(C, D),Veronicastrum sibiricum(E, F),Lindernia crustacea(G, H),Lindernia procumbens(I, J),Paulownia coreana(K, L). Scale bars: 1000 μm (K), 600 μm (C), 150 μm (A, G), 100 μm (L), 42 μm (F), 30 μm (B, D, H), and 15 μm (J).

Figure 6.

Scanning electron micrographs of seeds of Orobanchaceae.Pedicularis mandshurica(A, B, C),P. resupinatavar.umbrosa(D, E, F),P. verticillata(G, H, I),P. resupinata(J, K, L), andP. resupinataforalbiflora(M, N, O). Scale bars: 600 μm (A, B, D, E, G, H, J, K, M, N), 42 μm (C, F, I, L, O).

Figure 7.

Scanning electron micrographs of seeds of Orobanchaceae.Melampyrum koreanum(A, B, C),M. roseum(D, E, F)M. roseumvar.japonicum(G, H, I),M. roseumvar.ovalifolium(J, K, L), andM. setaceum(M, N, O). Scale bars: 1000 μm (A, D, G, J, M), 100 μm (B, E, H, K, N), and 42 μm (C, F, I, L, O).

Figure 8.

Scanning electron micrographs of seeds of Orobanchaceae and Phrymaceae.Melampyrum setaceumvar.nakaianum(A, B, C),Lathraea japonica(D, E, F),Phtheirospermum japonicum(G, H, I),Siphonostegia chinensis(J, K, L), andMazus pumilus(M, N, O). Scale bars: 1000 μm (A, B), 600 μm (D), 200 μm (E, J, K), 300 μm (G, H), 100 μm and 42 μm (C, F, I, L, M, NM), and 20 μm (O).

Figure 9.

Seed anatomy of (A, B)Scrophularia buergeriana, (C)S. koraiensis, (D, E)Veronica peregrina, (F)V. undulata, (G, H)V. incana, (I)V. pusanensis, (J, K)V. didymavar.lilacina, (L, M)Veronicastrum sibiricum, and (N, O)Limnophila indica. em, embryo; en, endothelium; end, endosperm; and ep, epidermis.

Figure 10.

Seed anatomy of (A)Linaria japonica, (B, C)Lindernia procumbens, (D, E)Phtheirospermum japonicum, (F, G)Pedicularis mandshurica,(H, I)P. resupinata,(J, K)Melampyrum roseum, and (L)Siphonostegia chinensis. Abbreviations: em, embryo; en, endothelium; end, endosperm; ep, epidermis.

Figure 11.

Seed anatomy of (A, B)Lathraea japonica, (C, D)Paulownia coreanaand (E, F)Mazus pumilus. Abbreviations: em, embryo; en, endothelium; end, endosperm; ep, epidermis.

3.1. Scrophularia(Scrophulariaceae s.s)

Five species of Scrophulariawere investigated in this study (Figure 1A–O). Seeds were minute with a small of variation in size within studied species; they were ovoid to broadly elliptical in shape and usually black or sometimes brown in color (Appendix 1, online supplementary resource; Figure 1A, B, D, E, G, H, J, K, M, N). The hilum was terminally positioned and slightly protruding. In all species, gross surface sculpture was typical reticulate-striate, and epidermal cells were polygonal or elongated in one direction (Figure 1C, F, I, L, O). The periclinal wall (PW) of the testal cell was slightly concave with parallel striation, and the anticlinal wall (AW) was highly raised, straight to sinuous, or wavy and unevenly thickened.

3.2. Limnophila, Linaria, Veronica, and Veronicastrum(Plantaginaceae)

Altogether, 18 taxa from 4 genera of Plantaginaceae, including 11 species and 4 varieties of the genus Veronica, were investigated. Seeds were minute: the largest seeds of Veronica didymavar. lilacinaranged from 1.01 × 0.81 mm to 1.49 × 1.25 mm, and the smallest seeds of V. rotundavar. subintegraranged from 0.44 × 0.32 mm to 0.81 × 0.49 mm. Seed were pale yellow or dark brown to black in color, and seed shape (SS) ranged from ovoid, broad ovoid, to sub-spherical, and mostly they were flat or plano-convex and bifacial (Figures 2A–O, 3A–O, 4A–O, and 5A–F). The surface sculpture was predominantly reticulate-striate, cristate, and sometimes reticulate-verrucate, as in V. persica, V. arvensis, and V. didymavar. lilacina(Figures 2F, I and 4O), reticulate-corrugate as in Veronica undulata(Figure 4L), or typical reticulate as in V. peregrina(Figure 2C). Epidermal cell shape was polygonal, elongated, and isodiametric or rarely irregular as in V. arvensis(Figure 2F). The periclinal wall of the surface cell was flat to slightly concave and striate but rarely folded, whereas the anticlinal wall was slightly to highly raised, straight to wavy, folded, and sometimes of uneven thickness.

3.3. Pedicularis, Melampyrum, Lathraea, Phtheirospermum, and Siphonostegia(Orobanchaceae)

A total of 14 taxa belonging to 5 genera (6 taxa from Melampyrum, 5 taxa from Pedicularis, and 1 species from each three genera) of Orobanchaceae were investigated. The seeds were larger than the taxa of Scrophulariaceae s.s. and Plantaginaceae. Melampyrum roseumhad the largest seeds (4.31 × 1.77 mm to 5.42 × 2.2 mm), whereas Phtheirospermum japonicahad the smallest (1.04 × 0.49 mm to 1.89 × 0.84 mm; Figures 6A–O, 7A–O, and 8A–L). Mostly seeds were elliptical and cylindrical (Melampyrum), ellipsoidal, navicular (Pedicularis), ovoid to subglobose (Lathraea japonica), ovoid (Phtheirospermum japonica), or elliptical beaked (Siphonostegia chinensis). The surface sculpture was highly variable, being rugose, colliculate, reticulate, scalariform, or papillate. The testa dermal cell shape was predominantly irregular; in some species, it was rectangular to elongated or polygonal-isodiametric. The testa periclinal wall was concave, flat concave, or slightly convex, with either smooth to finely folded or sessile papillae. The anticlinal walls were mostly raised but sometimes shallow and sometimes deep. The raised wall was smooth, finely papillate, or finely folded and unevenly thickened.

3.4. Lindernia(Linderniaceae)

Two species were included and seeds were ovoid or ovoid to oblong (Figure 5G–J). The surface was ribbed, ridged, or rugose pitted. The epidermal cell shape was rectangular, elongated (L. procumbens), or irregular (Lindernia crustacea). The testal periclinal wall was flat, slightly concave, and finely granulate, whereas the anticlinal wall was slightly raised and finely granulate to folded.

3.5. Paulownia(Paulowniaceae)

An endemic species, P. coreana, was investigated. The seeds were small, fluffy winged, and pale yellow to white in color (Figure 5K, L). The testa surface was ribbed, and epidermal cells were polygonal-isodiametric in shape. The testa periclinal wall was flat to slightly concave, and its surface was smooth, whereas the anticlinal wall was raised, straight to sinuous, and unevenly thickened.

3.6. Mazus(Phrymaceae)

Only Mazus pumilus was included in this study, and seeds were small ovoid or ellipsoidal in shape (Figure 8M–O). The surface sculpture was colliculate with rectangular to polygonal epidermal cells. The periclinal wall was convex with fine folds, whereas the anticlinal wall was shallow with fine folds.

3.7. Seed anatomy

The seed coat in Scrophulariaceae s.s. was somehow distinct, thin, and fairly comparable in all studied species. The epidermis was represented by a layer of degenerated cells followed by one or two endothelium layers (Figure 9A–C). In most places, endothelium cells were degenerated.

In 18 taxa of Plantaginaceae, the seed coat was distinct and more well characterized than in Scrophulariaexcept in three species of Veronica(V. arvensis, V. didymavar. lilacina, and V. persica), in which the seed coat was unclear and degenerated (Figures 9D–O and 10A). The epidermis was well represented in all species except in the aforementioned species, and it was noticeable in V. peregrinaand Veronicastrum sibericum(Figure 9L, M). In all species, the epidermis was followed by one or two endothelium layers which were represented by either distinct cells in layers or degenerated layers.

The seeds of the six Melampyrumtaxa were easily distinguished from rest of the Orobanchaceae species. They had largest seeds among the studied genera and a very thin seed coat in transverse section (Figures 10D–L and 11A, B). The epidermis was well characterized, and endothelium was present in eight taxa of the family, excluding all Melampyrumspecies.

The seed coat was well represented in Linderniaspp., P. coreana, and M. pumilus, although the epidermal cells were more clearly noticeable in the former rather than the latter two (Figures 10B, C and 11C–F). In M. pumilus, the epidermis was characterized by a degenerated layer of cells. The endothelium was present but poorly developed in all species.

3.8. Data analyses

The relationships among the taxa for the 13 seed characteristics were analyzed using correspondence analysis (CA) and cluster analyses (Figures 12 and 13). In CA, the first four axes explained 78.869% of the total variance of the analyzed data (Table 3). Axis 1 described 34.355% of the variance based on the values of primary surface sculpture (PSS), anticlinal wall (AW), periclinal wall (PW), periclinal wall ornamentation (PWO), seed coat anatomy (SCA), and endothecium (EN) (Table 3). Axis 2 explained 18.675% of the data variability, of which seed shape (SH), seed length (SL), seed length/width ratio (LWR), and hilum character (HC) were the significant variables for the ordination of the species. Correspondingly, axis 3 and axis 4 were explained by 13.041 and 12.213% of the data variability, respectively. CA biplots revealed a cluster of taxa corresponding with primary surface sculpture, periclinal wall ornamentation, and seed coat anatomy (Figure 12). When the distribution of seed surface characters on the CA biplots was observed, most of the taxa with a reticulate surface were grouped on the negative side of axis 2, whereas the taxa with a colliculate and other types of surface were grouped on the opposite side. Likewise, all the taxa with striate periclinal walls were grouped on the negative side of axis 2, whereas taxa with papillate/granulate and smooth/folded walls, except Veronica dahuricaand three Pedicularisspecies, were distributed on the positive side of axis 2. The cluster-based UPGMA tree revealed two main clusters [supported by a 100% bootstrap (BT) value]: cluster I included Melampyrum, Mazus, Lathraea, Paulownia, Limnophila, Linaria, and Lindernia,whereas cluster II comprised species of Pedicularis, Veronica, Phtheirospermum, Veronicastrum, Scrophularia, and Siphonostegia(Figure 13). In cluster I, there were two subclusters: the first one, which was supported by an 84% BT value, included Melampyrumspecies, and the second was formed by the remaining taxa of cluster I with bootstrap support of less than 50%. Correspondingly, in cluster II, there were three separate subclusters: Pedicularis/Phtheirospermum/Veronica, Veronica/Veronicastrum/Scrophularia, and three species of Veronica. Siphonostegia chinensisremained isolated and positioned at the bottom of the cluster. Interestingly, the subcluster formed by three Veronicaspecies was supported by an 80% BT value, which was higher than for any other subclusters in cluster II which had bootstrap support <50%.

Figure 12.

Correspondence analysis (CA) biplot of 13 seed characters sampled for 41 taxa of Scrophulariaceae s.l. Samples of different genera are represented by different symbols. AW, anticlinal wall; EN, endothecium; EP, epidermis; HC, hilum character; HP, hilum position; 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; SL, seed length.

Figure 13.

Seed morphological relationship among the taxa as displaced by UPGMA cluster diagram. Numbers above the branch represent bootstrap value.

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4. Discussion

4.1. Variations in seed morphology

This study demonstrated the high diversity of seed morphology in Scrophulariaceae s.l. in terms of seed shape, hilum character, primary ornamentation, epidermal cell characters, and seed coat anatomy. Variations are mainly found in seed primary sculpture, surface cell shape, and periclinal wall ornamentation. Seeds are minute, and most of the taxa are less or slightly larger than 1 millimeter in length except Melampyrumand Pedicularis. Within each taxon, seed size is variable; however, overall not much variation was found among the different species. That is why size is not very useful for the description of a particular taxon. Seed shape is also very heterogeneous, even with the same species. Mostly in studied species, the seed shape ranged from elliptical, broad elliptical, to ovoid. Elliptical seeds were cylindrical, navicular, flattened, or plano-convex. Seed of Siphonostegia chinensisis elliptical and beaked, that of P. coreanais winged, and seed of V. didymavar. lilacinais cupular. In general, however, in Scrophulariaceae s.l., seed shape related directly to its relative position in the fruit [30].

As far as surface sculpture and ornamentation are concerned, we found quite similar patterns in several species of same genera, particularly in Veronica, Pedicularis, Scrophularia, and Melampyrum. Our result agrees with previous studies regarding Veronicaand Pedicularis[30, 3234, 37, 38]. Out of 15 taxa of Veronicaobserved in this study, V. arvensis, V. didymavar. lilacina, and V. persicawere reticulate-verrucate with a centrally located tubercle in epidermal cells, V. undulatahad a reticulate-corrugate seed surface, and V. peregrinahad a typical reticulate surface, whereas the rest of the species had a reticulate-striate surface with a cristate wall. In terms of the systematic significance of seed morphology in Veronica, Muñoz-Centeno et al. [32] described eight types of surface pattern in 123 species, and our samples represent four of them, although reticulate-striate is the most dominant pattern. Our results disagree with those of previous study [32] in terms of the surface pattern of V. peregrinaas these previous authors mentioned a reticulate-corrugate surface pattern; in contrast, in our samples we observed a typical reticulate pattern. As regards seed anatomy data, V. arvensis, V. didymavar. lilacina, and V. persicahad the most poorly differentiated seed coat, represented by a thin epidermal layer of almost degenerated cells, whereas V. peregrinaand V. undulatahad the most clearly defined epidermis and endothelium among the Veronicaspecies. All the species with a reticulate-striate, cristate surface had well-characterized epidermis but indistinct endothelium.

Regarding Pedicularis, the results of our study agree with those of earlier results (for example, see [33]), that the regular-reticulate surface pattern is common among the studied taxa. Although gross primary sculpture looks like a reticulate pattern in all five Pedicularistaxa, there are substantial dissimilarities in secondary ornamentation and anticlinal wall formation. The anticlinal wall of P. mandshuricais, P. verticillata, and P. resupinatavar. umbrosawas highly raised, whereas that of P. resupinataand P. resupinataf. albiflorawas only slightly raised. The regular-reticulate, membranous-reticulate, cristate-reticulate, and the undulate primary ornamentations have been mentioned in previous studies [30, 33, 3840], but the reticulate-tuberculate primary ornamentation found in P. resupinataf. albiflorais reported for the first time here. Nevertheless, the reticulate seed surface of Pedicularisis a common feature among genera of the families Orobanchaceae and Plantaginaceae [26, 30, 4143]. The most usual and consistent feature observed among the five taxa of Pediculariswas a seed coat comprising a clearly defined epidermis and endothelium. On the other hand, most of the seed features were consistent within six Melampyrumtaxa; variation was only observed in the gross surface sculpture with colliculate (M. roseumand M. roseumvar. japonicum), rugose (M. roseumvar. ovalifoliumand M. setaceum), and rugose + colliculate (M. koreanumand M. setaceumvar. nakaianum) surfaces.

In most cases, the seed coat comprised the epidermis and the endothelium. Nevertheless, in all Melampyrumand some Veronicaspecies, the seed coat was very poorly represented and consisted only of a papery layer of epidermis. In this regard, our results agree with the findings of Juan et al. [30] who described the seed coat of Scrophulariaceae as being composed of the epidermis and endothelium; the latter is a useful character with which to distinguish the seeds of certain genera, particularly Scrophulariaand Verbascum. Although they did not include any Melampyrumspecies, similar to our result, they indicated that some Veronicaspecies consisted only of an epidermis, and no endothelium.

According to the CA analyses, the close affinities among the species of Scrophulariaare well supported by their proximity to one another. Similarly, the proximity of Melampyrumspecies and of Pedicularisspecies is also apparent. Alternatively, in the CA plot, Veronicaspecies are divided into two clusters. One is characterized by a concave periclinal wall, striate wall ornamentation, and a distinct seed coat, whereas the other, comprising only four species (V. arvensis, V. persica, V. didymavar. lilacina, and V. peregrina), has a convex periclinal wall and either papillate or smooth folded wall ornamentation. In addition, these four species differ in gross surface pattern as they have reticulate-verrucate or typical reticulate (V. peregrina) ornamentation instead of a reticulate-striate, cristate surface in the rest of the species (except V. undulata). Morphologically, V. arvensis, V. peregrina, and V. persicashare an annual habit without rhizomes and flowers in terminal inflorescence; however, the latter species differs from the former two by having a longer pedicel than bract [44].

4.2. Systematic implications of seed characters

In the UPGMA tree based on the seed morphological and anatomical characters, two major clusters were obtained, of them cluster II was supported by a 100% BT value (Figure 13). Cluster I, which included Lindernia, Linaria, Limnophila, Paulownia, Lathraea, Mazus, and Melampyrum, was a highly heterogeneous group of plants in life form and nature, ranging from annual or perennial herbs to trees, erect or prostrate, and creeping or submerged amphibious forms. Within cluster I, two subclusters were distinguished: the first one, supported by an 84% BT value, contained only the genus Melampyrum, whereas the remaining genera formed a separate subcluster. Six Melampyrumspecies can be differentiated from each other by primary sculpture and the nature of the periclinal wall; and UPGMA tree indicated that M. setaceumremains isolated from the rest of the Melampyrumspecies (59% BT). Morphologically, M. setaceumdiffered from other five species by linear to linear-lanceolate leaves and lanceolate bracts, whereas the rest of the species had lanceolate leaves and ovate to ovate lanceolate bracts. In another subcluster of cluster I, two other subclusters contained Lindernia(51% BT) and the remaining five genera (Linaria, Limnophila, Paulownia, Lathraea, and Mazus), forming a very heterogeneous group containing members from four families. Out of the five genera, Paulowniais a deciduous or evergreen tree, and Limnophila, an annual or perennial herb, usually grows in marshy areas with erect, prostrate, or creeping stems.

Despite being clustered in the same clade, seeds of two Linderniaspecies are very different from each other in terms of shape, primary sculpture, and epidermal cell shape. The primary surface sculpture of L. procumbensis ribbed with rectangular/elongated surface cells, whereas that of L. crustacea is rugose and pitted with irregular cells. These two species also differ in their leaf morphologies as the leaves of L. procumbenceare sessile, elliptical to oblong, and somewhat rhomboid with entire or weakly toothed margins, whereas the leaves of L. crustaceaare shortly petiolate, triangular-ovate to broadly ovate, and shallowly crenate or serrate. After observing the seeds of 14 Linderniaspecies, [45] classified the genus with ribbed and unribbed seeds and also indicated that this character shows good correspondence with subdivision of the genus explained in [46].

In cluster II, the Siphonostagiawas separated first from rest of the genera despite belonging to the same family, Plantaginaceae, with Pedicularis. Pedicularis, which is known to be hemi-parasitic like Melampyrum, formed the topmost subcluster in the tree (77% BT) and remained quite far from Melampyrum. Apart from their parasitic nature, these two genera share some morphological features including leafy bracts, campanulate calyces, didynamous stamens, and capitate stigmas. Yet again, three Veronicaspecies (V. arvensis, V. didymavar. lilacina, and V. persica) constituted a remarkable case making a separate subcluster with 80% BT, whereas the rest of Veronicaspecies grouped either with Scrophularia/Veronicastrumor with Pedicularis/Phtheirospermum. Many of the Veronicaspecies combined with Veronicastrumand Scrophularia,although BT support for this subcluster was <50%. In our results, Scrophulariashared some similar seed features with Veronicaand Veronicastrum; however, from a morphological point of view, the position of the Scrophulariain this subcluster is very confusing. As Veronicaand Veronicastrumare characterized by perennial rhizomatous herbs (although some Veronicaare annual and without rhizosomes), four corolla lobes, two stamens, and capitate stigmas. Instead, Scrophulariaare characterized by perennial herbs without rhizomes, five corolla lobes, four didynamous stamens, and bifid stigmas.

As predicted, three Veronicaspecies (V. arvensis, V. didymavar. lilacina, and V. persica) form an isolated group. Based on the infrageneric classification as done in [47], V. arvensis (with haploid chromosome, n = 8) belongs to subgenus Chamaedrysand V. didyma var. lilacina(as V. polita) and V. persica, both having base chromosome n = 7, belong to Pocilla. V. linariifolia, V. longifolia, V. dahurica, and V. incana, all have the chromosome number n = 17, and the reticulate-striate, cristate group belongs to Pseudolysimachium, whereas V. peregrina(n = 9) belongs to subgenus Beccabunga.The present results confirmed what was reported in previous studies on Veronica(for example, [32, 34]), that seed morphological data can be employed to evaluate the relationships of taxa within the genus and that seed characters can also provide additional support to infrageneric discrimination in Veronica.

The SEM investigations reveal that the reticulate surface pattern as a primary sculpture is a confusing case. The pattern is found in the members of Orobanchaceae, Plantaginaceae, and Scrophulariaceae, although quite variations are observed in secondary striation and anticlinal wall pattern. The current study likewise concurs that there is no even single seed features representing all investigated species. Nonetheless, few seed characters somehow nicely characterized the group of particular taxa, for instance, seeds of Orobanchaceae are either elliptical cylindrical or ovoid in shape and of Plantaginaceae are elliptical flattened or ovoid flattened. Correspondingly, reticulate-striate surface sculpture of Veronica, Veronicastrum,and Scrophulariais comparable, although later one is without cristae. The seed coat anatomy indicates that the epidermis and endothelium are informative characters. All the Scrophulariaspecies have indistinct epidermis but distinct endothelium, whereas most of the Veronicahas distinct epidermis and endothelium. On the other hand, all the Melampyrumare without both layers, but other Plantaginaceae have well-distinct epidermis and endothelium.

The concept of Scrophulariaceae s.l. has changed considerably since the application of molecular approaches in plant systematics. Studies have shown that the traditional Scrophulariaceae are an unusual assemblage of plants distributed throughout the phylogenetic tree of Lamiales [1, 2, 57]. Despite the limited number of taxa investigated, the present seed morphological analysis highlights the high heterogeneity existing within the studied taxa of Scrophulariaceae s.l., which may be observed at a higher taxonomic rank than genus. In particular, primary surface sculpture, anticlinal and periclinal walls of epidermal cell, epidermal cell shape, and seed coat layers are the important seed features observed in this study, and these features can be used to distinguish different groups of taxa in the family. The results of this study therefore suggest that seed coat micromorphology and anatomy have significant taxonomic importance.

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Acknowledgments

This study was supported financially by the project ‘Studies on the Establishment of SeedBank base for the Asian network’ at the Korea National Arboretum (KNA), Pocheon, Korea.

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Appendix

Plant nameShapeAverage size (mm)Length/width ratioHilumPrimary surface sculpture
Lindernia procumbensElliptical, oblong0.35 × 0.162.11Basal, distinctly protuberantRibbed
L. crustaceaOvoid, sub globose0.38 × 0.31.26Basal, distinctly protuberantRugose, pitted
Mazus pumilusOvoid, elliptical0.42 × 0.261.36Basal, distinctly protuberantColliculate
Lathraea japonicaOvoid2.03 × 1.691.2Lateral, distinctly protuberantRugose
Melampyrum koreanumElliptical, cylindrical4.73 × 1.972.43Lateral, flat to indistinctly protuberantRugose/poorly colliculate
M. roseumElliptical, cylindrical4.96 × 1.982.51Lateral, flat to indistinctly protuberantColliculate
M. roseumvar. japonicumElliptical, cylindrical3.79 × 1.652.32Lateral, flat to indistinctly protuberantColliculate
M. roseumvar. ovalifoliumElliptical, cylindrical4.35 × 1.742.51Lateral, flat to indistinctly protuberantRugose
M. setaceumElliptical, cylindrical4.15 × 1.552.7Lateral, flat to indistinctly protuberantRugose
M. setaceumvar. nakaianumElliptical, cylindrical4.97 × 1.92.62Lateral, flat to indistinctly protuberantRugose/poorly colliculate
Pedicularis mandshuricaElliptical, navicular2.99 × 1.272.38Basal, distinctly protuberantReticulate
P. resupinataf. albifloraElliptical, navicular2.83 × 1.272.25Basal, distinctly protuberantReticulate, tuberculate
P. resupinataElliptical2.69 × 1.322.05Basal, distinctly protuberantReticulate
P. resupinatavar. umbrosaElliptical2.62 × 1.082.49Basal, distinctly protuberantReticulate
P. verticillataElliptical, navicular2.27 × 1.161.95Basal, distinctly protuberantReticulate
Phtheirospermum japonicumOvoid1.19 × 0.731.64Basal, distinctly protuberantReticulate
Siphonostegia chinensisElliptical, beaked0.92 × 0.462.05Basal, deepReticulate
Paulownia coreanaElliptical, winged1.58 × 0.742.13Basal, distinctly protuberantRibbed
Limnophila indicaOvoid, elliptical0.95 × 0.461.73Basal, distinctly protuberantRugose
Veronica arvensisOvoid, flattened0.96 × 0.641.51Basal, distinctly protuberantReticulate-verrucate/Rugose
V. dahuricaBroad elliptical, flattened1.02 × 0.891.16Lateral, distinctly protuberantReticulate-striate, cristate
V. didymavar. lilacinaOvoid, cupular1.27 × 11.28Lateral, distinctly protuberantReticulate-verrucate
V. incanaOvoid, flattened0.71 × 0.491.46Basal, distinctly protuberantReticulate-striate, cristate
V. kiusianavar. diamantiacaBroad elliptical, flattened1.17 × 1.011.36Lateral, distinctly protuberantReticulate-striate, cristate
V. kiusianavar. glabrifoliaBroad elliptical, flattened1.17 × 1.011.16Lateral, distinctly protuberantReticulate-striate, cristate
V. linariifoliaBroad elliptical, flattened1.19 × 0.851.4Basal, distinctly protuberantReticulate-striate, cristate
V. longifoliaBroad elliptical, flattened1.19 × 0.991.54Lateral, distinctly protuberantReticulate-striate, cristate
V. nakaianaBroad ovoid, flattened0.94 × 0.741.28Basal, distinctly protuberantReticulate-striate, cristate
V. peregrinaElliptical, flattened0.71 × 0.411.73Basal, distinctly protuberantFaintly reticulate
V. persicaOvoid, flattened1.17 × 0.791.49Basal, distinctly protuberantReticulate-verrucate
V. pusanensisBroad elliptical, flattened1.01 × 0.791.29Lateral, distinctly protuberantReticulate-striate, cristate
V. pyrethrinaBroad elliptical, flattened0.95 × 0.781.23Lateral, distinctly protuberantReticulate-striate, cristate
V. rotundavar. subintegraBroad elliptical, flattened0.99 × 0.771.29Lateral, distinctly protuberantReticulate-striate, cristate
V. undulataBroad ovoid, plano-convex0.62 × 0.401.56Lateral, distinctly protuberantReticulate-corrugate
Veronicastrum sibiricumOvoid, plano-convex0.73 × 0.461.61Lateral, distinctly protuberantReticulate-striate, cristate
Linaria japonicaOvoid, flattened2.03 × 1.681.68Basal, deepColliculate
Scrophularia buergerianaOvoid10.3 × 0.711.47Basal, deepReticulate-striate
S. grayanaOvoid1.08 × 0.761.45Basal, deepReticulate-striate
S. kakudensis.Ovoid1.13 × 0.731.56Basal, deepReticulate-striate
S. koraiensisOvoid1.2 × 0.741.64Basal, deepReticulate-striate
S. takesimensisOvoid1.01 × 0.61.72Basal, deepReticulate-striate

Epidermal cell shapePericlinal wallAnticlinal wallSeed coatEpidermisEndothelium
Rectangular, elongatedConcave, finely granulateSlightly raised, straight, finely granulatedDistinctWell representedAbsent
IrregularConcave, finely granulateSlightly raised, foldedDistinctWell representedAbsent
Rectangular, PolygonalConvex, finely foldedShallow, finely foldedDistinctWell representedPresent
IrregularConcave, smooth to foldedDistinctly raised, straight to wavy, finely folded, unevenly thickenedDistinctWell representedPresent
IrregularConvex, smooth to finely foldedShallow or slightly raisedIndistinctDegeneratedAbsent
IrregularConvex, smooth to finely foldedShallowIndistinctDegeneratedAbsent
IrregularConvex or pitted, smooth to finely foldedShallow, smooth, unevenly thickenedIndistinctDegeneratedAbsent
IrregularConvex or pitted, smooth to finely foldedSlightly raised, smooth, unevenly thickenedIndistinctDegeneratedAbsent
IrregularConcave or pitted, smooth to finely foldedSlightly raised, smooth, unevenly thickenedIndistinctDegeneratedAbsent
IrregularConvex, smooth to finely foldedShallowIndistinctDegeneratedAbsent
Rectangular, elongatedFlat, wartyDistinctly raised, straight, smooth to warty, unevenly thickenedDistinctWell representedPresent
Rectangular, elongatedFlat with sessile tubercleSlightly raised, straightDistinctWell representedPresent
Rectangular, elongatedFlat with sessile micro papillaeSlightly raised, straight,DistinctWell representedPresent
Rectangular, elongatedFlat with sessile micro papillaeDistinctly raised, straight, smooth or finely folded, unevenly thickenedDistinctWell representedPresent
Rectangular, elongatedFlat, full of micro papillaeDistinctly raised, straight, smooth or finely folded, unevenly thickenedDistinctWell representedPresent
Polygonal, isodiametricFlat, smoothDistinctly raised, straight to sinuous, unevenly thickenedDistinctWell representedPresent
Polygonal, isodiametricFlat, smooth to finely foldedDistinctly raised, straight to sinuous, unevenly thickenedDistinctDegeneratePresent
Polygonal, isodiametricFlat to slightly concave, smoothDistinctly raised, straight to sinuous, unevenly thickenedDistinctWell representedPresent
IrregularSlightly convex, wartyShallowDistinctWell representedPresent
Polygonal, elongated/isodiametricConvex with central tubercle, smoothDistinctly raised, straight to wavy, smoothIndistinctDegeneratedPresent
Polygonal, elongated/isodiametricConcave, striateDistinctly raised, straight to wavy, smoothDistinctWell representedPresent
Polygonal, isodiametricConvex with central tubercle, smoothDistinctly raised, straight to wavy, smoothIndistinctDegeneratedAbsent
Polygonal, elongated/isodiametricConcave, striateDistinctly raised, straight to wavy, foldedDistinctWell representedPresent
Polygonal, elongated/isodiametricConcave, striateDistinctly raised, straight to wavy, foldedDistinctWell representedPresent
Polygonal, elongated/isodiametricConcave, striateDistinctly raised, straight to wavy, foldedDistinctWell representedPresent
Polygonal, elongated/isodiametricConcave, striateDistinctly raised, straight to wavy, foldedDistinctWell representedPresent
Polygonal, elongated/isodiametricConcave, striateDistinctly raised, straight to wavy, folded, unevenly thickenedDistinctWell representedPresent
Polygonal, elongatedConcave, striateDistinctly raised, straight to wavy, folded, unevenly thickenedDistinctWell representedPresent
Polygonal, elongated/isodiametricFlat, smooth/foldedSlightly raised, folded, unevenly thickenedDistinctWell representedPresent
Polygonal, isodiametricConvex with central tubercle, smoothSlightly raised, folded, unevenly thickenedIndistinctDegeneratedAbsent
Polygonal, elongated/isodiametricConcave, striateDistinctly raised, straight to wavy, folded, unevenly thickenedDistinctWell representedPresent
Polygonal, elongatedConcave, striateDistinctly raised, straight to wavy, folded, unevenly thickenedDistinctWell representedPresent
Polygonal, elongatedConcave, striateDistinctly raised, straight to wavy, folded, unevenly thickenedDistinctWell representedPresent
Polygonal, isodiametricConcave, striateDistinctly raised, folded, unevenly thickenedDistinctWell representedPresent
Polygonal, elongatedConcave, striateDistinctly raised, straight to wavy, folded, unevenly thickenedDistinctWell representedPresent
Polygonal, isodiametricConvex, finely foldedShallow, finely folded, straight to wavyDistinctWell representedPresent
Polygonal, elongatedConcave, striateDistinctly raised, straight to sinuous, unevenly thickenedDistinctDegeneratedPresent
Polygonal, elongatedConcave, striateDistinctly raised, straight to sinuous, unevenly thickenedDistinctDegeneratedPresent
Polygonal, elongatedConcave, striateDistinctly raised, straight to sinuous, unevenly thickenedDistinctDegeneratedPresent
Polygonal, elongatedConcave, striateDistinctly raised, straight to sinuous, unevenly thickenedDistinctDegeneratedPresent
Polygonal, elongatedConcave, striateDistinctly raised, straight to sinuous, unevenly thickenedDistinctDegeneratedPresent

Appendix 1.

Seed characters studied for Scrophulariaceae s.l.

Plant name12345678910111213
Lindernia procumbens0011020021001
L. crustacea1001022021001
Mazus pumilus1001010, 1002000
Lathraea japonica1100022102000
Melampyrum koreanum021112, 12002111
M. roseum0211112002111
M. roseumvar. japonicum0211112002111
M. roseumvar. ovalifolium0211122002111
M. setaceum0211122022111
M. setaceumvar. nakaianum021112, 12002111
Pedicularis mandshurica0211000111000
P. resupinataf. albiflora021100, 20011000
P. resupinata0211000011000
P. resupinatavar. umbrosa0211000111000
P. verticillata0201000111000
Phtheirospermum japonicum1101001112000
Siphonostegia chinensis0001201112000
Paulownia coreana0111021112000
Limnophila indica1001222001000
V. arvensis110100, 21101111
V. dahurica0100001120000
V. didymavar. lilacina1100001101111
V. incana1001001120000
V. kiusianavar. diamantiaca0100001120000
V. kiusianavar. glabrifolia0100001120000
V. linariifolia0101001120000
V. longifolia0100001120000
V. nakaiana1001001120000
V. peregrina0101001012000
V. persica1001001001111
V. pusanensis0100001120000
V. pyrethrina0000001120000
V. rotundavar. subintegra0000001120000
V. undulata1100001122000
Veronicastrum sibiricum1000001120000
Linaria japonica1101211002000
Scrophularia buergeriana1101201120010
S. grayana1101201120010
S. kakudensis1101201120010
S. koraiensis1101201120010
S. takesimensis1101201120010

Appendix 2.

Data matrix of the character states used for analysis.

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Written By

Balkrishna Ghimire, Go Eun Choi, Hayan Lee, Kweon Heo and Mi Jin Jeong

Submitted: March 14th, 2017 Reviewed: August 11th, 2017 Published: December 6th, 2017