4. Discussion

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

Figure 13. Seed morphological relationship among the taxa as displaced by UPGMA cluster diagram. Numbers above

the branch represent bootstrap value.

218 Advances in Seed Biology

#### 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 Melampyrum and 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 chinensis is elliptical and beaked, that of P. coreana is winged, and seed of V. didyma var. lilacina is 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 Veronica and Pedicularis [30, 32–34, 37, 38]. Out of 15 taxa of Veronica observed in this study, V. arvensis, V. didyma var. lilacina, and V. persica were reticulate-verrucate with a centrally located tubercle in epidermal cells, V. undulata had a reticulate-corrugate seed surface, and V. peregrina had 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. peregrina as 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. didyma var. lilacina, and V. persica had the most poorly differentiated seed coat, represented by a thin epidermal layer of almost degenerated cells, whereas V. peregrina and V. undulata had the most clearly defined epidermis and endothelium among the Veronica species. 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 Pedicularis taxa, there are substantial dissimilarities in secondary ornamentation and anticlinal wall formation. The anticlinal wall of P. mandshuricais, P. verticillata, and P. resupinata var. umbrosa was highly raised, whereas that of P. resupinata and P. resupinata f. albiflora was only slightly raised. The regular-reticulate, membranous-reticulate, cristate-reticulate, and the undulate primary ornamentations have been mentioned in previous studies [30, 33, 38–40], but the reticulatetuberculate primary ornamentation found in P. resupinata f. albiflora is reported for the first time here. Nevertheless, the reticulate seed surface of Pedicularis is a common feature among genera of the families Orobanchaceae and Plantaginaceae [26, 30, 41–43]. The most usual and consistent feature observed among the five taxa of Pedicularis was a seed coat comprising a clearly defined epidermis and endothelium. On the other hand, most of the seed features were consistent within six Melampyrum taxa; variation was only observed in the gross surface sculpture with colliculate (M. roseum and M. roseum var. japonicum), rugose (M. roseum var. ovalifolium and M. setaceum), and rugose + colliculate (M. koreanum and M. setaceum var. nakaianum) surfaces.

In most cases, the seed coat comprised the epidermis and the endothelium. Nevertheless, in all Melampyrum and some Veronica species, 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 Scrophularia and Verbascum. Although they did not include any Melampyrum species, similar to our result, they indicated that some Veronica species consisted only of an epidermis, and no endothelium.

According to the CA analyses, the close affinities among the species of Scrophularia are well supported by their proximity to one another. Similarly, the proximity of Melampyrum species and of Pedicularis species is also apparent. Alternatively, in the CA plot, Veronica species 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. didyma var. 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. persica share 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

seed anatomy data, V. arvensis, V. didyma var. lilacina, and V. persica had the most poorly differentiated seed coat, represented by a thin epidermal layer of almost degenerated cells, whereas V. peregrina and V. undulata had the most clearly defined epidermis and endothelium among the Veronica species. All the species with a reticulate-striate, cristate surface had well-characterized

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 Pedicularis taxa, there are substantial dissimilarities in secondary ornamentation and anticlinal wall formation. The anticlinal wall of P. mandshuricais, P. verticillata, and P. resupinata var. umbrosa was highly raised, whereas that of P. resupinata and P. resupinata f. albiflora was only slightly raised. The regular-reticulate, membranous-reticulate, cristate-reticulate, and the undulate primary ornamentations have been mentioned in previous studies [30, 33, 38–40], but the reticulatetuberculate primary ornamentation found in P. resupinata f. albiflora is reported for the first time here. Nevertheless, the reticulate seed surface of Pedicularis is a common feature among genera of the families Orobanchaceae and Plantaginaceae [26, 30, 41–43]. The most usual and consistent feature observed among the five taxa of Pedicularis was a seed coat comprising a clearly defined epidermis and endothelium. On the other hand, most of the seed features were consistent within six Melampyrum taxa; variation was only observed in the gross surface sculpture with colliculate (M. roseum and M. roseum var. japonicum), rugose (M. roseum var. ovalifolium and M. setaceum), and rugose + colliculate (M. koreanum and M. setaceum var.

In most cases, the seed coat comprised the epidermis and the endothelium. Nevertheless, in all Melampyrum and some Veronica species, 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 Scrophularia and Verbascum. Although they did not include any Melampyrum species, similar to our result, they indicated that some Veronica species consisted

According to the CA analyses, the close affinities among the species of Scrophularia are well supported by their proximity to one another. Similarly, the proximity of Melampyrum species and of Pedicularis species is also apparent. Alternatively, in the CA plot, Veronica species 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. didyma var. 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. persica share an annual habit without rhizomes and flowers in terminal inflorescence; however, the latter species differs

epidermis but indistinct endothelium.

220 Advances in Seed Biology

nakaianum) surfaces.

only of an epidermis, and no endothelium.

from the former two by having a longer pedicel than bract [44].

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 Melampyrum species can be differentiated from each other by primary sculpture and the nature of the periclinal wall; and UPGMA tree indicated that M. setaceum remains isolated from the rest of the Melampyrum species (59% BT). Morphologically, M. setaceum differed 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, Paulownia is 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 Lindernia species are very different from each other in terms of shape, primary sculpture, and epidermal cell shape. The primary surface sculpture of L. procumbens is 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. procumbence are sessile, elliptical to oblong, and somewhat rhomboid with entire or weakly toothed margins, whereas the leaves of L. crustacea are shortly petiolate, triangular-ovate to broadly ovate, and shallowly crenate or serrate. After observing the seeds of 14 Lindernia species, [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 Siphonostagia was separated first from rest of the genera despite belonging to the same family, Plantaginaceae, with Pedicularis. Pedicularis, which is known to be hemiparasitic 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 Veronica species (V. arvensis, V. didyma var. lilacina, and V. persica) constituted a remarkable case making a separate subcluster with 80% BT, whereas the rest of Veronica species grouped either with Scrophularia/Veronicastrum or with Pedicularis/ Phtheirospermum. Many of the Veronica species combined with Veronicastrum and Scrophularia, although BT support for this subcluster was <50%. In our results, Scrophularia shared some similar seed features with Veronica and Veronicastrum; however, from a morphological point of view, the position of the Scrophularia in this subcluster is very confusing. As Veronica and Veronicastrum are characterized by perennial rhizomatous herbs (although some Veronica are annual and without rhizosomes), four corolla lobes, two stamens, and capitate stigmas. Instead, Scrophularia are characterized by perennial herbs without rhizomes, five corolla lobes, four didynamous stamens, and bifid stigmas.

As predicted, three Veronica species (V. arvensis, V. didyma var. 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 Chamaedrys and 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 Scrophularia is comparable, although later one is without cristae. The seed coat anatomy indicates that the epidermis and endothelium are informative characters. All the Scrophularia species have indistinct epidermis but distinct endothelium, whereas most of the Veronica has distinct epidermis and endothelium. On the other hand, all the Melampyrum are 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, 5–7]. 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.
