3. Results

1. Shape: 0, elliptical; 1 ovoid

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3. Length/width ratio: 0, <2; 1, >2 4. Hilum position: 0, lateral; 1, basal.

2. Seed length: 0, <1 mm; 1, 1–2 mm, 2, >2 mm

9. Periclinal wall: 0, convex; 1, flat; 2, concave

12. Epidermis: 0, well represent; 1, degenerate

shape; SH, seed shape; and SL, seed length.

13. Endothelium: 0, present; 1, absent

11. Seed coat anatomy: 0, seed coat distinct; 1, indistinct

5. Hilum character: 0, distinctly protuberant; 1, flat to indistinctly protuberant; 2, deep

10. Periclinal wall ornamentation: 0, striate; 1, papillate/granulate; 2, smooth/folded

Character number Character code Axis 1 Axis 2 Axis 3 Axis 4 SH 0.23118 0.76272 0.26612 0.263812 SL 0.007571 0.467293 0.281203 0.31876 LWR 0.654796 0.952965 0.162676 0.4085 HP 0.10317 0.025843 0.09821 0.26307 HC 0.065757 0.91809 0.06242 0.80719 PSS 0.817606 0.046239 0.7419 0.29745 ECS 0.09684 0.15219 0.04569 0.273533 AW 0.8693 0.080012 0.101048 0.143169 PW 0.88318 0.176183 0.03262 0.129772 PWO 0.491091 0.221064 0.31801 0.04532 SCA 0.995628 0.19168 0.954453 0.561853 EP 0.382648 0.61797 0.775164 0.04003 EN 0.91233 0.07943 0.531445 0.705178

> Eigenvalue 0.325 0.176 0.123 0.115 % total variance 34.355% 18.675% 13.041% 12.213%

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

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

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

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

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 1–11. A comprehensive description of seed features by family and genus is given here.

#### 3.1. Scrophularia (Scrophulariaceae s.s)

Five species of Scrophularia were 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 didyma var. lilacina ranged from 1.01 0.81 mm to 1.49 1.25 mm, and the smallest seeds of V. rotunda var. subintegra ranged 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 reticulateverrucate, as in V. persica, V. arvensis, and V. didyma var. lilacina (Figures 2F, I and 4O), reticulatecorrugate 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 roseum had the largest seeds (4.31 1.77 mm to 5.42 2.2 mm), whereas Phtheirospermum japonica had 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

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), and S. 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).

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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), and V. 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 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), and S. 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).

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Figure 3. Scanning electron micrographs of seeds of Plantaginaceae. Veronica dahurica (A, B, C), V. rotunda var. sabinteara (D, E, F), V. pusanensis (G, H, I), V. incana (J, K, L), and V. 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).

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Figure 4. Scanning electron micrographs of seeds of Plantaginaceae. Veronica nakiana (A, B, C), V. kiusiana var. diamentica (D, E, F), V. kiusiana var. glabrifolia (G, H, I), V. undulata (J, K, L), and V. didyma var. 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 3. Scanning electron micrographs of seeds of Plantaginaceae. Veronica dahurica (A, B, C), V. rotunda var. sabinteara (D, E, F), V. pusanensis (G, H, I), V. incana (J, K, L), and V. 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).

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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).

(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.

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Figure 6. Scanning electron micrographs of seeds of Orobanchaceae. Pedicularis mandshurica (A, B, C), P. resupinata var. umbrosa (D, E, F), P. verticillata (G, H, I), P. resupinata (J, K, L), and P. resupinata for albiflora (M, N, O). Scale bars: 600 μm (A, B, D, E, G, H, J, K, M, N), 42 μm (C, F, I, L, O).

(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

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).

smooth, finely papillate, or finely folded and unevenly thickened.

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Figure 7. Scanning electron micrographs of seeds of Orobanchaceae. Melampyrum koreanum (A, B, C), M. roseum (D, E, F) M. roseum var. japonicum (G, H, I), M. roseum var. ovalifolium (J, K, L), and M. 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).

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Figure 8. Scanning electron micrographs of seeds of Orobanchaceae and Phrymaceae. Melampyrum setaceum var. nakaianum (A, B, C), Lathraea japonica (D, E, F), Phtheirospermum japonicum (G, H, I), Siphonostegia chinensis (J, K, L), and Mazus 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 7. Scanning electron micrographs of seeds of Orobanchaceae. Melampyrum koreanum (A, B, C), M. roseum (D, E, F) M. roseum var. japonicum (G, H, I), M. roseum var. ovalifolium (J, K, L), and M. 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).

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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. didyma var. lilacina, (L, M) Veronicastrum sibiricum, and (N, O) Limnophila indica. em, embryo; en, endothelium; end, endosperm; and ep, epidermis.

#### 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,

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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.

slightly concave, and finely granulate, whereas the anticlinal wall was slightly raised and finely granulate to folded.

#### 3.5. Paulownia (Paulowniaceae)

3.4. Lindernia (Linderniaceae)

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embryo; en, endothelium; end, endosperm; and ep, epidermis.

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,

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. didyma var. lilacina, (L, M) Veronicastrum sibiricum, and (N, O) Limnophila indica. em,

> 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.

Figure 11. Seed anatomy of (A, B) Lathraea japonica, (C, D) Paulownia coreana and (E, F) Mazus pumilus. Abbreviations: em, embryo; en, endothelium; end, endosperm; ep, epidermis.

#### 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 Scrophularia except in three species of Veronica (V. arvensis, V. didyma var. 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. peregrina and 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 Melampyrum taxa 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 Melampyrum species.

The seed coat was well represented in Lindernia spp., 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

3.6. Mazus (Phrymaceae)

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embryo; en, endothelium; end, endosperm; ep, epidermis.

was shallow with fine folds.

layers or degenerated layers.

3.7. Seed anatomy

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

Figure 11. Seed anatomy of (A, B) Lathraea japonica, (C, D) Paulownia coreana and (E, F) Mazus pumilus. Abbreviations: em,

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 Scrophularia except in three species of Veronica (V. arvensis, V. didyma var. 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. peregrina and 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

The seeds of the six Melampyrum taxa 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 Melampyrum species.

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

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.

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 dahurica and three Pedicularis species, 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 Melampyrum species, 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 chinensis remained isolated and positioned at the bottom of the cluster. Interestingly, the subcluster formed by three Veronica species was supported by an 80% BT value, which was higher than for any other subclusters in cluster II which had bootstrap support <50%.
