Introduction

Dead-nettle Lamium L. (Lamiaceae) is a genus comprising 25 species of herbaceous plants (Govaerts et al. 2010). Morphological diversity within this genus is relatively low. Representatives of particular species differ in details of flower structure, leaf shape or their arrangement on the stem. Part of the taxa distinguished in Lamium are difficult to identify because of the very subtle morphological features that differentiate them. This particularly applies to L. incisum and L. confertum, which are polyploidal species of hybrid origin (Jörgensen 1927; Taylor 1991; Dvořáková 2000). L. incisum is easy to confuse with L. purpureum, while L. confertum shows a very high morphological similarity to L. amplexicaule (Szafer et al. 1986; Dvořáková 2000). There are also difficulties with the delimitation of some subspecies distinguished within Lamium. For instance, one of the controversial taxa is L. garganicum subsp. laevigatum, distinguished on the basis of pubescence and the shape of the upper lip of the corolla (Arcangeli 1882; Ball 1972), but combined into one taxon with L. garganicum subsp. garganicum by some researchers (Mennema 1989; Govaerts et al. 2010). Also the systematic order within the genus Lamium is a disputable issue. On the basis of morphological features, the genus is divided into three subgenera: Lamium, Orvala and Galeobdolon (Mennema 1989). In the biggest one—subgenus Lamium, there are three sections: Amplexicaule, Lamiotypus and Lamium. The division into subgenera has been set taking into account: pubescence of anthers, shape and pubescence of calyx, shape of corolla tube and locations of floral leaves. Division of subgenus Lamium into three sections also relies on the shape of corolla tube and the presence or absence of floral leaves. Recently, this classification, determined solely on the basis of morphological features, has been undermined by phylogenetic studies (Bendiksby et al. 2011), however a new division has not yet been proposed.

Since macromorphological features are often an insufficient criterion for identification of some representatives of Lamium, and they fail to fully reflect phylogenetic relationships within the genus, we decided to search micromorphological features characteristic for the representatives of Lamium. According to the study conducted on a group of about 5,000 species of plants, a very valuable taxonomic criterion at the level of species, order, and family is the sculpturing of the seed surface (Barthlott and Voit 1979; Barthlott 1981). The usefulness of mericarp morphology, anatomy and pericarp structure in taxonomy was also proved by numerous studies carried out in Lamiaceae at various taxonomical levels (Husain et al. 1990; Demissew and Harley 1992; Ryding 1992, 1994, 1995; Marin et al. 1994, 1996; Oran 1996; Budantsev and Lobova 1997; Jamzad et al. 2000; Guerin 2005; Moon and Hong 2006; Salmaki et al. 2008; Kahraman et al. 2011; Satıl et al. 2012). For instance, Marin et al. (1994) and Ryding (1995) concluded that nutlet characters were potentially useful within Lamiaceae at the level of section, genus and species. Studies on Stachys L. showed that nutlet micromorphological characters appear to be useful in distinguishing infrageneric groups (Demissew and Harley 1992) and in the species-level taxonomy (Satıl et al. 2012). In Salvia L., one of the largest genera of Lamiaceae, it has been shown that gross morphology of nutlets and their sculpturing pattern are variable and taxonomically useful at a species level (Oran 1996; Kahraman et al. 2011). In all these studies, the analysis based on scanning electron microscopy (SEM) has been successfully applied.

The purpose of this study was to provide a detailed description of fruit micromorphology in Lamium by using SEM and to evaluate the systematic significance of such characteristics in the light of the variation between taxa of the genus. The observed variability was also analyzed in relation to the evolutionary history of this genus.

The subject of our research was fruit characteristic for the genus Lamium, i.e., schizocarp splitting apart into four mericarps (nutlets). Each of them contains one seed covered with a dry, nonsplitting, leathery pericarp. The mericarps are ovoid, flat at the apex, triangular. They possess elaiosomes, the presence of which indicates adaptation to seed dispersal by myrmecochory (Dvořáková 2000). Morphological variation of fruits is observed between species with respect to fruit size, color and the presence or absence of convex spots on their surface. Micromorphological variability of fruits in the genus Lamium has not been widely studied until now. To date this variability has been described only for the subspecies of L. galeobdolon (L.) L. (Czarna and Bednorz 2011), which by some taxonomists (e.g., Dvořáková 2000; Mirek et al. 2002; Rothmaler 2007) is excluded to the separate genus as Galeobdolon luteum Huds. Identification of L. galeobdolon is based on three main morphological features distinguishing it from other Lamium species: color of the corolla, the number and shape of lobes in the corolla’s lower lip (Sychowa 1967; Ball 1972; Szafer et al. 1986; Krawczyk et al. 2013). It is debatable whether those key features are sufficient to place this taxon in separate genera. Opinions are divided, and taxonomists take different approaches to the problem. The comparison of nutlet micromorphology of L. galeobdolon with other Lamium species will enable determining whether the fruits of this species stand out against the tested genus.

Materials and methods

The study included 19 taxa of the genus Lamium, belonging to 14 species (Table 1). in the case of L. album subsp. album, L. amplexicaule and L. purpureum, the research material was collected in the course of our own field study. Other fruits were obtained from herbarium specimens, kindly provided by domestic and foreign herbaria: BR, COA, GJO, H, L, TUR. Three to four fully developed, mature and dry nutlets were examined for each taxon after a number of specimens had been compared under a light microscope for similarity. Nutlet length and width (Table 2) were measured in 20 samples per specimen of each taxon. The terminology of nutlet micromorphology was adopted after Barthlott (1981) and Salmaki et al. (2008).

Table 1 Information about specimens used in the study; country of origin, year of collection, voucher information
Full size table
Table 2 Characteristic of the pericarp surface of the examined Lamium species
Full size table

Observations were done under Nikon SMZ1000 light microscope equipped with Nikon Digital Sight camera. Then, 3–4 Lamium fruits of each taxa were mounted on copper tables, and sputter-coated with gold in argon atmosphere (Fine coater JFC-1200, JEOL). Mericarp texture was imaged by a scanning electron microscope (JSM 5310LV, JEOL) at 15 kV and analyzed by NSS Version 3.0 (EDS Noran System 7.0, Thermo Scientific). Light and electron microscopy allowed observing the structure of fruit surface but it was not possible to determine the origin of the protuberances present in mericarps of some species, macroscopically observed as whitish spots. Suspecting that these are thickenings of the cuticular layer, we performed cuticle and cutin staining according to the methodology of Wędzony (1996), after Heslop-Harrison (1977). Dried seeds of Lamium were placed for 20 min in auramine O solution for cuticle localization (lit). The characteristic fluorescence (excitation 405 nm, emission 470–520 nm) of cuticle was analyzed using a laser scanning microscope (LSCM, SP5, Leica), with LA AF Version: 2.4.1 build 6384. We examined fruits of L. amplexicaule with numerous spots and L. flexuosum, where spots were not observed.

Results

Morphological variation of the pericarp

Evaluation of Lamium mericarps under a light microscope revealed that their surface, colored in various shades of brown, was usually spotted with whitish, convex, shiny spots. Depending on the species, spots occurred singly or in large numbers, covering almost the entire surface of the fruit. Total absence of these spots was found only in the case of two species: L. flexuosum and L. orvala. Analysis of the smooth and spotted surface of fruits, carried out using confocal microscopy and appropriate staining, revealed cutin saturated cell walls and cuticular thickening present on their surface (Fig. 1). It was found that the protrusions seen as spots emitted fluorescent light, similarly to thickened parts of the cell wall in the area of smooth surfaces. It allows concluding that the observed structures were probably cuticular platelets.

Fig. 1
figure 1

Fruit surface of Lamium amplexicaule (a, b, c) and L. flexuosm (d, e, f) observed in light microscope (a, d) and laser scanning microscope (b, c, e, f). Wax platelets visible in b and c are marked with orange circles. Parts b and e present cuticle fluorescence observed for different values of Z axis than figures c and f

Full size image

Observation of fruit surface using SEM showed that the outer layer of the pericarp of species studied was formed by approximately isodiamteric, penta- to octagonal cells, forming reticulate surface sculpturi. L. moschatum was the only species where elongated cells were observed towards the axis of the mericarp (Fig. 2). Cell boundaries were generally straight or slightly undulated (due to the thickenings on the margins of cell walls). Anticlinal cell walls in most species were slightly channelled, although in some species (e.g., L. album, L. garganicum), an outline of the cells was difficult to observe because of the rich ornamentation of the surface of the fruit. Anticlinal walls of L. flexuosum and L. orvala were the only ones that were raised. The surfaces of the outer periclinal cell walls were usually concave, and cells with a flat surface were also observed in several taxa (Table 2). The margins of the periclinal walls were raised or flat. In some species, thickenings of the cell walls were present. They were regular (e.g., L. garganicum subsp. garganicum), ribbed (e.g. L. coutinhoi) or band-like, forming a ring on the periphery of the wall (e.g., L. maculatum).

Fig. 2
figure 2

SEM micrographs of mericarp surfaces in some taxa of Lamium: L. maculatum (a, b, c), L. moschatum (d, e, f), L. orvala (g, h, i), L. purpureum (j, k, l)

Full size image

The studied species can be divided into two main groups based on the secondary sculpturing of the cell walls observed. The first group consisted of species which cell wall thickenings formed regular papillae (L. amplexicaule, L. bifidum, L. confertum, L. coutinhoi, L. gevorense, L. incisum, L. macrodon, L. maculatum, L. purpureum). The other group comprised taxa with thickenings of the cell walls in the form of irregular ridges and tubercles (L. album, L. flexuosum, L. garganicum, L. orvala). L. moschatum did not fit into any of these groups, because the surface of the mericarps was formed by cell walls covered with a thick, even layer of the cuticle, with some slight depressions present in the central part of the cell wall. Taxa of the second group, excluding L. garganicum subsp. garganicum corsicum and L. garganicum subsp. laevigatum, had additional cuticle striations. Depending on the taxon, they were located on the margin of a cell wall or in a central portion and extended radially, parallelly or irregularly to the axis of mericarp (Table 2).

Diagnostic features of subspecies

A careful analysis of sculpturing on the surface of fruits enabled defining new diagnostic features that facilitate correct identification of similar taxa. Subspecies of L. garganicum had an easily observable feature, i.e., the presence or absence of striation. In addition, individual taxa differed in the shape of the periclinal wall and the structure of thickenings present on this wall. The difference in ornamentation of the fruits was also observed in two subspecies of L. bifidum. Subspecies balcanicum had groups of cells with band-like thickened edges and low thickenings in the form of papillae as well as areas of cells densely covered with thickenings in the form of ridges (Fig. 3). In subspecies bifidum, there were no band-like thickenings on the margins of the walls. Surface of periclinal walls was covered with tubercles to a various extent, but they did not form ridges (Fig. 3). There were no micromorphological features observed in L. album that would allow identification of the distinguished subspecies.

Fig. 3
figure 3

SEM micrographs of mericarp surfaces in some taxa of Lamium: L. album subsp. album (a, b, c), L. album subsp. barbatum (d, e, f), L. amplexicaule (g, h, i), L. bifidum subsp. balcanicum (j, k, l), L. bifidum subsp. bifidum (m, n, o)

Full size image

Diagnostic features of species of hybrid origin

Comparison of sculpturing of nutlets of L. incisum and L. purpureum enabled defining traits that distinguish these two species. Papillae of different sizes were found on the surface of the pericarp cells in L. incisum that sometimes merged into larger tubercles. They were located mainly on the periphery of the external periclinal cell walls (Fig. 4). In L. purpureum, the papillae were large and evenly distributed over the entire surface of the cell wall (Fig. 2), which differentiated this species from L. incisum.

Fig. 4
figure 4

SEM micrographs of mericarp surfaces in some taxa of Lamium: L. garganicum subsp. laevigatum (a, b, c), L. garganicum subsp. striatum (d, e, f), L. gevorense (g, h, i), L. incisum (j, k, l), L. macrodon (m, n, o)

Full size image

Features that distinguish L. confertum from L. amplexicaule were also identified. In these species they were also related to the protuberances on the surface of cells. Papillae of various sizes were present in L. confertum, merging on the edges of cells into ribbed structures (Fig. 5). L. amplexicaule had specific small papillae of equal size, distributed fairly evenly but they were slightly more numerous on the margins of the cell walls (Fig. 3).

Fig. 5
figure 5

SEM micrographs of mericarp surfaces in some taxa of Lamium: L. confertum (a, b, c), L. coutinhoi (d, e, f), L. flexuosum (g, h, i), L. garganicum subsp. corsicum (j, k, l), L. garganicum subsp. garganicum (m, n, o)

Full size image

When discussing micromorphological features specific for mericarps of L. confertum, one should pay attention to their very strong resemblance to the fruits of L. coutinhoi. The structures observed on the surface of mericarps of both species were similar to each other to such a degree that the distinction of species based on these traits was, in our opinion, impossible (Fig. 5).

Discussion

Our results showed that L. flexuosum and L. orvala differed to a large extent from other Lamium species. At the same time, a comparison of the characteristics of nutlets of L. flexuosum and L. orvala to the description of fruits of L. galeobdolon (Czarna and Bednorz 2011), indicated a high similarity of these three species. Their common features included a lack of cuticular plates, a lack of cell wall thickening in the form of papillae, the presence of striations and convex anticlinal cell walls. Furthermore, the analysis of pericarp morphology revealed no significant differences between L. galeobdolon and the remaining species from the Lamium genus. Quite the contrary, there were many common features indicating the similarity of mericarps of L. galeobdolon and the other dead-nettles, in particular L. flexuosum and L. orvala. The observed similarity can be explained by the evolutionary history of the species studied. The results of phylogenetic studies showed that L. galeobdolon did not form a separate phylogenetic line in relation to the genus Lamium and thereby confirmed that the presence of L. galeobdolon in Lamium was justified (Krawczyk et al. 2013; Krawczyk and Sawicki 2013). Molecular studies confirmed a close relationship of L. flexuosum, L. galeobdolon and L. orvala (Krawczyk et al. 2014). The infrageneric classification based on macromorphological features also emphasizes the similarity of L. galeobdolon and L. flexuosum which constitute the subgenus Galeobdolon, while L. orvala belongs to the monospecific subgenus Orvala (Mennema 1989).

The results of molecular studies were congruent with the observed morphological variability of pericarp also with respect to the status of L. garganicum subsp. laevigatum. The difference noticed in sculpturing of the fruits of this taxon compared to second subspecies of L. garganicum is an argument to consider L. garganicum subsp. laevigatum a separate taxon. This result is consistent with the analysis of variation of barcoding sequences in the genus Lamium, which also indicated the distinctiveness of this subspecies (Krawczyk et al. 2014; Krawczyk and Sawicki 2013).

Our findings showed that the analysis of fruit surface with the use of SEM allowed defining new diagnostic features for almost all studied representatives of the genus Lamium. Many of the variable nutlet characters have been shown to be diagnostic and of systematic value in other groups of Lamiaceae (Husain et al. 1990; Oran 1996; Salmaki et al. 2008). However particular features were of different taxonomic values depending on the studied group of plants. Among different nutlet characters studied, the type of sculpturing has been considered to provide the most valuable ones (Oran 1996). In Lamium, the general nutlet surface patterns described by Salmaki et al. (2008) showed rather low diagnostic value, since all the species studied within the genus were characterized by more or less reticulate sculpture. Only the thorough analysis of details of pericarp surface sculpturing provided valuable data for delimitation of dead-nettle species.

As noted by Barthlott (1981), cuticle striation is usually a valuable diagnostic feature. However the importance of this character in the identification of taxa higher than species seems to be limited. In turn, cell wall thickenings are both of diagnostic and taxonomic importance. According to Barthlott (1981), these structures most often distinguish taxa at the level of the genus and subfamily. Our results confirmed Barthlott’s observations. The type of cell wall thickening observed in the pericarp of Lamium divided the species studied into groups, which largely coincided with phylogenetic lines distinguished in the genus studied (Krawczyk and Sawicki 2013). Sculpturing of the cell walls in the form of papillae arose independently in two phylogenetic lines. One of them consists of: L. amplexicaule, L. bifidum, L. gevorense and L. incisum, while the second one comprises: L. confertum, L. coutinhoi, L. maculatum and L. purpureum. The papillae also evolved in the species L. macrodon, closely related to L. moschatum. The lack of visible cell wall thickening in L. moschatum may be the result of speciation. However, it cannot be ruled out that the cell wall thickenings are present in this species, but they are invisible due to the thick layer of cuticle on the surface of the fruit.

Conclusion

Nutlet micromorphology provides diagnostic features for almost all representatives of Lamium included in this study. One of the most valuable features, useful in distinguishing examined species, is cuticle striation, while cell wall thickenings are both of diagnostic and taxonomic importance. Although the analysis of fruit surfaces does not resolve phylogeny of the genus completely, it confirms to a large extent the picture of phylogenetic relationships derived from molecular studies. The obtained results combined with the analysis of macromorphological features and the results of phylogenetic studies provide a sound basis for creating a new taxonomical classification within the genus Lamium. However, in our opinion, a thorough reanalysis of macromorphological features is necessary to establish such a classification.