ARTICLE IN PRESS Flora 204 (2009) 371–381 www.elsevier.de/flora Trichome micromorphology of Iranian Stachys (Lamiaceae) with emphasis on its systematic implication Yasaman Salmakia, Shahin Zarrea,, Ziba Jamzadb, Christian Bräuchlerc a Department of Plant Biology, School of Biology, College of Science, University of Tehran, PO Box 14155-6455 Tehran, Iran Research Institute of Forests and Rangelands, PO Box 13185-116 Tehran, Iran c LMU Munich, Department Biology I, Biodiversity Research-Systematic Botany, Laboratory Prof. Dr. G. Heubel, Menzinger Street 67, 80638 Munich, Germany b Received 24 March 2008; accepted 12 May 2008 Abstract Trichomes of 37 taxa of the genus Stachys and one species of Sideritis (S. montana) were examined using light and scanning electron microscopy. The indumentum shows considerable variability among different species, but is constant among different populations of one species, and therefore, affords valuable characters in delimitation of sections and species. The characters of taxonomic interest were presence of glandular and non-glandular trichomes, thickness of the cell walls, number of cells (unicellular or multi-cellular), presence of branched (dendroid) trichomes, presence of vermiform trichomes, orientation of trichomes in relation to the epidermal surface, curviness of trichomes, and presence of papillae on trichome surface. Two basic types of trichomes can be distinguished: glandular and nonglandular trichomes. The glandular trichomes can in turn be subdivided into subtypes: stalked, subsessile, or sessile. The stalks of the glandular trichomes can be uni- or multi-cellular. Simple unbranched and branched trichomes constitute two subtypes of non-glandular trichomes. Our data do not provide any support for separation of Sideritis from Stachys. The following evolutionary trends are suggested here for Stachys: vermiform trichomes with stellate base are primitive against vermiform trichomes with tuberculate base, long vermiform trichomes are primitive against the short simple trichomes, appressed trichomes are advanced against spreading ones, and loss of glandular trichomes is advanced against their presence. Overall, trichome micromorphology is more useful in separation of species within sections rather than characterizing large natural groups known as sections, except for few cases. r 2008 Elsevier GmbH. All rights reserved. Keywords: Lamiaceae; Stachys; Sideritis; Trichome; Micromorphology; Iran Introduction Stachys L. (Lamiaceae, Lamioideae) is a large assemblage of herbaceous annuals and perennials or sub-shrubs with an estimated 275–300 species worldwide Corresponding author. E-mail address: zarre@khayam.ut.ac.ir (S. Zarre). 0367-2530/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2008.11.001 (Bhattacharjee, 1980; Lindqvist and Albert, 2002; Mabberley, 1997; Willis and Airy-Shaw, 1973). The genus is distributed mainly in warm temperate regions of the Mediterranean and SW Asia, Southern Africa, North and South America. Iran as a center of diversity of Stachys houses about 35 species (Rechinger, 1982). These species are sometimes highly polymorphic morphologically and morphological differences are often ARTICLE IN PRESS 372 Y. Salmaki et al. / Flora 204 (2009) 371–381 obscure. The majority of species prefers alpine and subalpine habitats and grows under various ecological conditions in habitats like rocky places, mountain steppes, stream banks or sometimes in forests. The most important taxonomic treatments for Eurasian species of Stachys so far were those of Ball (1972), Bhattacharjee (1980) and Rechinger (1982), which are mainly based on morphological characters. The taxonomic value of the indumentum as well as its implication in systematics and phylogenetics are well known in Lamiaceae and related families as Verbenaceae and Scrophulariaceae (Abu-Assab and Cantino, 1987; Ascensão et al., 1999; Cantino, 1990; Metcalfe and Chalk, 1950). Trichome micromorphology has been suggested to be useful in the phylogeny reconstruction (Abu-Assab and Cantino, 1987) and systematics of Lamiaceae (Cantino, 1990) as well as some of its genera (e.g. Marin et al., 1994; Navarro, 1995; Navarro and El Oualidi, 2000; Puech, 1984). However, there are only a few works dealing with detailed analysis of trichomes in Stachys (e.g., Giuliani et al., 2008), focusing the species mostly distributed in tropical Africa (Demissew and Harley, 1992). Apart from the detailed structure, trichome morphology was used in the taxonomy of Stachys in different ways, mainly in order to separate certain sections or to delimit closely related species. Sect. Ambleia for example is characterized by the presence of dendroid or stellate trichomes (Bhattacharjee, 1982), while the presence of elongated glandular trichomes on the calyx was considered as an important character for separating S. benthamiana from its closest allies within sect. Fragilicaulis, i.e. S. kurdica and S. ballotiformis (Rechinger, 1982). However, no detailed trichome micromorphological study has been conducted on the genus indicating the possible phylogenetic importance of trichome characters in the systematics or phylogenetics of the genus. Recent molecular studies (Lindqvist and Albert, 2002), however, neither support the current infrageneric classifications (Bhattacharjee, 1980, 1982; Rechinger, 1982), nor give a delimitation of Stachys as currently circumscribed towards its closest relatives, e.g. Sideritis L., Phlomidoschema (Benth.) Vved. and Prasium L. Therefore, a detailed micromorphological study can also bring clarity about many aspects of phylogeny of the genus. In the framework of providing a revision of the genus Stachys for the Persian ‘‘Flora of Iran’’, we performed a detailed micromorphological study on this genus including pollen morphology (Salmaki et al., 2008a), nutlet sculpturing (Salmaki et al., 2008b), and trichome micromorphology. Our aims were to document trichome micromorphology of native species of Stachys in Iran, in order to evaluate the systematic significance of the resulting data and discuss them on the background of both molecular results and traditional classification. Materials and methods Trichomes of 37 taxa of the genus Stachys (35 spp. and 2 subspp.) and one species of Sideritis (Sideritis montana) were studied. As a rule, five different specimens of each taxon were analyzed. In several cases more than five specimens were considered for one species to ensure certainty about the stability of trichome characters among different specimens of one species. The herbarium material for this study was removed from specimens (Table 1) deposited mainly in the herbarium of Research Institute of Forests and Rangelands, Iran (TARI), and herbarium of Tehran University (TUH). A list of voucher specimens used in the present study including some notes on the location of the plants is given in Table 1. Trichomes were obtained from leaves and calyces and studied with a stereo- or a usual light microscope. For light microscopy, mostly, cross sections of leaf blades and petioles were made by hand using commercial razor blades. The sections were stained with safranin – fast green (Gerlach, 1977) with some modifications. The cross sections were dehydrated through an ethanol gradient and finally xylol (99.5%), and then mounted on slides using Canada balsam. They were studied using an Olympus microscope model BX50 light microscope with 400X to 1000X magnifications. For the scanning electron microscopy (SEM), small pieces of leaves and calyces were fixed on aluminum stubs using double-sided adhesive, and were coated with a thin layer (ca. 25 nm) of gold–palladium. The SEM micrographs were taken in a SEM-440I (Leo, England) at an accelerating voltage of 10–15 kV. The type of indumentum was described and classified. The general classification scheme and the terminology follow Roe (1971), Cantino (1990), as well as Navarro and El Oualidi (2000). Results The main types of the investigated trichomes and their distribution among the species studied are summarized in Table 2. Selected SEM and LM micrographs of common indumentum types are presented in Figs. 1–42. Type of indumentum shows considerable variability among different species, but was constant among different populations of one species, and therefore, affords valuable characters in delimitation of sections and species. The characters of taxonomic interest were presence of glandular and non-glandular trichomes, thickness of the ARTICLE IN PRESS Y. Salmaki et al. / Flora 204 (2009) 371–381 Table 1. 373 Collection data and ecology of Stachys specimens examined here from their trichome micromorphology. Species Section Collection data Stachys acerosa Boiss.* Aucheriana S. asterocalyx Rech.f. Fragilicaulis S. atherocalyx K. Koch Olisia S. aucheri Benth. S. balansae Boiss. & Kotschy ex Boiss. Aucheriana Eriostomum S. ballotiformis Vatke Fragilicaulis S. benthamiana Boiss.* Fragilicaulis S. byzantina K. Koch Eriostomum S. cretica L. Eriostomum S. fruticulosa M. Bieb.* Thamnostachys S. iberica M.Bieb. Olisia S. inflata Benth. S. ixodes Boiss. & Hausskn. ex Boiss.* S. kermanshahensis Rech.f.* Ambleia Aucheriana Fragilicaulis S. koelzii Rech. f.* S. kurdica Boiss. & Hohen. Aucheriana Fragilicaulis S. lavandulifolia Vahl Zietenia S. laxa Boiss. Buhse.* Ambleia S. megalodonta Hausskn. & Bornm. ex P.H. Davis S. multicaulis Benth. S. obtusicrena Boiss.* Fragilicaulis Aucheriana Ambleia S. palustris L. Stachys S. persepolitana Boiss.* S. persica S. G.Gmel. ex C. A. Mey. Olisia Eriostomum S. pilifera Benth.* Aucheriana S. pubescens Ten. Olisia S. schtschegleevii Sosn. ex Grossh. Ambleia S. setifera C. A. Mey. subsp. iranica (Rech. f.) Rech. f. S. setifera C. A. Mey. subsp. daenensis (Gandog.) Rech.f. * S. setifera C. A. Mey. subsp. setifera Setifolia Kohgiluyeh va Boyer-Ahmad: Yasuj, Barandaz. Mozaffarian, 25154 (TUH) Kohgiluyeh va Boyer Ahmad: between Behbahan and Dehdasht, Assadi and Aboohamzeh, 38701 (TARI) West Azarbayjan: Uromieh, Kay village, Zarre and Salmaki, 36530 (TUH) Fars: Dena mt., Kamarsiah, Safayian, 29 (TARI) East Azarbayjan: about 21 km to Assalem from Khalkhal, Zarre and Salmaki, 36532 (TUH) Kurdestan: Kamyaran to Varmahang, Zarre and Salmaki, 36517 (TUH) Kurdestan: Kamyaran to Varmahang, Zarre and Salmaki, 36524 (TUH) Mazandaran: Firuzkuh road, Orim village, Zarre and Salmaki, 36516 (TUH) Kermanshah: Kerend to Sar-e Pol-e Zahab and Sarmil, Zarre and Salmaki, 36507 (TUH) Zanjan: 5 km after Halab to Zanjan, Zarre and Salmaki, 36506 (TUH) Mazandaran: S of Ramsar, W Javaher deh, Runemark and Maassoumi, 20775 (TARI) Hamadan: Avaj to Razan, Zarre and Salmaki, 36505 (TUH) Fars: Shiraz, Dasht-e Arjan, Foroughi, 17486 (TARI) Kermanshah: Tout-Shami to Mare-khamoush, Zarre and Salmaki, 36504 (TUH) Lurestan: Aligudarz, Maassoumi, 75544 (TARI) Kermanshah: 18 km to Kermanshah, Zarre and Salmaki, 36513 (TUH) Mazandaran: 13 km to Dizin from Gachsar, Zarre and Salmaki, 36521 (TUH) Mazandaran: Gaduk pass near pol-e Veresk, Zarre and Salmaki, 36525 (TUH) Kurdestan: Marivan to Saghez, Zarre and Salmaki, 36503 (TUH) Hamadan: 3 km to Ganjnameh, Zarre Salmaki, 35891 (TUH) Chahar Mahal-e Bakhtiari: Sabzkuh, Zarre and Salmaki, 20074 (TUH) B3 Afyon (Turkey): Çay ilçesi, Eber Gölü, Dönmez Emir, 2949 (HUB) Fars: 15 km Firouzabad, Assadi and Sardabi, 41489 (TARI) Mazandaran: 40 km.Tunekabon to Jannat Rudbar, Zarre and Salmaki, 8466 (TUH) Chahar Mahal-e Bakhtiari: Borojen, Ganduman, Kuh-e Baraftab, Mozaffarian, 57772 (TARI) Mazandaran: Firuzkuh road, Orim village, Zarre and Salmaki, 36518 (TUH) East Azarbayjan: Kaleybar, Ghaleh Babak, Zarre and Salmaki, 17508 (TUH) Hamadan: Avaj to Razan, Zarre and Salmaki, 36523 (TUH) S. spectabilis Choisy ex DC. Eriostomum Setifolia Setifolia Kurdestan: Marivan to Saghez, Zarre and Salmaki, 36502 (TUH) Tehran: 30 km to Firuzkuh from Tehran, Zarre and Salmaki, 36514 (TUH) West Azarbayjan: Dizaj to Boz-e Sina mountains, Zarre and Salmaki, 36531 (TUH) ARTICLE IN PRESS 374 Y. Salmaki et al. / Flora 204 (2009) 371–381 Table 1. (continued ) Species Section Collection data S. subaphylla Rech. f. Ambleia S. sylvatica L. Stachys S. trinervis Aitch. & Hemsl.* Ambleia S. turcomanica Trautv. Ambleia S. veroniciformis Rech. f.* Fragilicaulis Sideritis montana L.  Gorgan: 70 km of Chaman-Bid, Assadi and Maassoumi, 21491 (TARI) Guilan: Asalem to Khalkhal, Assadi and Maassoumi, 16394 (TARI) Gorgan: Azad Shahr, near Bojnourd Wendelbo et al., 11067 (TARI) Khorassan: Bojnourd to 21 km Gifan, Assadi and Maassoumi, 50221(TARI) Lurestan: Oshtorankuh, Assadi and Mozaffarian, 37189 (TARI) Mazandaran: Firuzkuh road, Orim village, Zarre and Salmaki, 36533 (TUH) Species endemic to Iran are indicated by an asterisk. Table 2. Some characteristic features of trichomes in the examined Stachys taxa and in Sideritis montana. Density Surface Stalked glandular trichomes Sub-sessile or sessile glandular trichomes Branched trichomes Short simple trichomes Long simple trichomes Vermiform trichomes Smooth Smooth Smooth Smooth Smooth + + + + + + + + + +                + + + + + sect. S. S. S. S. S. Eriostomum byzantina persica spectabilis cretica balansae Very Very Very Very Very sect. S. S. S. Setifolia setifera subsp. iranica setifera subsp. daenensis setifera subsp. setifera Sparse Sparse Sparse Smooth Smooth Smooth + + +       + + + + + +    Sparse Sparse Smooth Smooth +  + +    + +    Sparse Sparse Very sparse Very sparse Smooth Smooth Papillate Papillate   +    +      + + + + + +       sect. Thamnostachys S. fruticulosa Sparse Papillate    +   sect. Zietenia S. lavandulifolia Dense Smooth  +    + sect. Aucheriana S. multicaulis S. pilifera Sparse Dense Smooth Smooth + + + +   +  + +   sect. Stachys S. sylvatica S. palustris sect. S. S. S. S. Olisia iberica atherocalyx pubescens persepolitana dense dense dense dense dense ARTICLE IN PRESS Y. Salmaki et al. / Flora 204 (2009) 371–381 375 Table 2. (continued ) S. S. S. S. sect. S. S. S. S. S. S. S. Density Surface Stalked glandular trichomes Sub-sessile or sessile glandular trichomes Branched trichomes Short simple trichomes Long simple trichomes Vermiform trichomes acerosa aucheri koelzii ixodes Sparse Dense Sparse Dense Smooth Smooth Smooth Smooth + + + + + + + +     +  +  + + + +     Ambelia laxa turcomanica inflata schtschegleevii trinervis obtusicrena subaphylla Dense Dense Dense Dense Sparse Dense Dense Smooth Smooth Smooth Smooth Smooth Smooth Smooth        + + + + + + + + + + + + + +                      Sparse 7Dense 7Dense Sparse Sparse Dense Sparse Sparse Papillate Papillate Papillate Papillate Smooth 7Papillate Papillate Smooth   +  + +     + + + +  +         + + + + + + + +      +  +         sect. Fragilicaulis S. kurdica S. ballotiformis S. benthamiana S. megalodonta S. veroniciformis S. kermanshahensis S. asterocalyx Sideritis montana cell walls, number of cells (unicellular or multi-cellular), presence of few-armed and branched (dendroid) trichomes, presence of vermiform trichomes, orientation of trichomes in relation to the epidermal surface (erect, subappressed or appressed), curviness of trichomes (flexuous, curved or hooked), and presence of papillae on trichome surface. Two basic types of trichomes can be distinguished: glandular and non-glandular trichomes. Based on the variations observed, glandular trichomes can in turn be subdivided into two subtypes: stalked (e.g. S. ixodes, Fig. 7; S. persica, Fig. 14), subsessile (e.g. S. pubescens, Fig. 41) or sessile (e.g. S. byzantina, Fig. 35; S. multicaulis, Fig. 8; S. persica, Fig. 15). The stalks of the glandular trichomes can be uni- (e.g. S. kermanshahensis, Fig. 20) or multi-cellular (e.g. S. ixodes, Fig. 7; S. persica, Fig. 14). Simple unbranched (e.g. S. pilifera, Fig. 10) and branched trichomes (e.g. S. laxa, Fig. 1) can be considered as two subtypes of nonglandular trichomes. Four species S. iberica, S. atherocalyx, S. persepolitana (all of sect. Olisia), and S. fruticulosa (sect. Thamnostachys, Figs. 28 and 29) do not possess any glandular trichomes. Subsessile trichomes are mostly very shortly stalked and bladder-like at heads (S. pubescens, Fig. 41). Stalked glandular trichomes show variation in the size, shape, cell number, and position of the stalk and the head. Stalked glandular trichomes can be short (up to 30 mm) with a capitate head (e.g. S. pilifera, Fig. 11), short with a clavate head (e.g. S. lavandulifolia, Fig. 30), and long (up to 150 mm) with a capitate head (e.g. Stachys setifera). Based on the size, non-glandular trichomes of the simple type are short (from 50 mm in S. persepolitana to 500 mm in S. ballotiformis), long (up to 2000 mm, e.g. in S. pilifera, Fig. 10) and vermiform or extremely long (up to 5000 mm, e.g. in S. cretica, Fig. 12). Short simple trichomes are uni- or bi-cellular (or multi-cellular). Unicellular short simple trichomes show considerable variation and can be subdivided into four groups: (1) conical in shape, appressed, composed of thick-walled cells, densely covered by micro-papillae (e.g. S. persepolitana, Fig. 25), (2) acicular in shape, erect, composed of thick-walled cells, densely covered by micro-papillae (e.g. S. asterocalyx, Fig. 16), (3) similar to the latter, but thin-walled cells, smooth on surface, tuberculate at base (e.g. Sideritis montana, Fig. 33), and (4) similar to the latter, but curved at tip (e.g. S. ballotiformis, Fig. 18). Bi- and multi-cellular short simple trichomes range from erect (e.g. S. ballotiformis, Fig. 34; S. veroniciformis, Fig. 23) to appressed (e.g. S. fruticulosa, Figs. 28 and 29). They can be smooth on surface (S. ballotiformis, Fig. 18; S. veroniciformis, Fig. 23), or papillate (e.g. S. persepolitana, Fig. 26), curved (e.g. S. ballotiformis, Fig. 17), or ARTICLE IN PRESS 376 Y. Salmaki et al. / Flora 204 (2009) 371–381 Figs. 1–15. SEM micrographs of trichomes in Stachys: (1) S. laxa, (2) S. obtusicrena, (3) S. trinervis, (4, 5) S. acerosa, (6) S. aucheri, (7) S. ixodes. (8, 9) S. multicaulis, (10, 11) S. pilifera, (12, 13) S. cretica, (14, 15) S. persica, (1, 4, 7, 14) scale bar ¼ 30 mm; (2, 3, 5, 12) scale bar ¼ 100 mm; (6, 9, 11, 13, 15) scale bar ¼ 10 mm; (10) scale bar ¼ 200 mm. straight at tip (e.g. S. persepolitana, Fig. 26; S. veroniciformis, Fig. 23), and articulated (e.g. S. megalodonta, Fig. 22), or non-articulated (e.g. S. kurdica, Fig. 21). Long simple trichomes can be inflated at their nodes (e.g. S. acerosa, Fig. 5; S. pilifera, Fig. 10), or normally articulated. In the former type the basal cells are elongated, while in the latter the basal cells are tuberculate. All members of sect. Eriostomum (Fig. 13) and S. lavandulifolia (Figs. 31 and 32) are covered by very long vermiform trichomes. In the former the basal part is stellate, but in the latter group it is 7tuberculate. Branched (dendritic) trichomes are composed of 25 arms at each node (e.g. S. inflata, Fig. 38; S. schtchegleevii, Fig. 42). Depending on the trichome length they can be composed of one (e.g. S. trinervis, Fig. 3) to several branched nodes (e.g. S. obtusicrena, Fig. 2). ARTICLE IN PRESS Y. Salmaki et al. / Flora 204 (2009) 371–381 377 Figs. 16–30. SEM micrographs of trichomes in Stachys: (16) S. asterocalyx, (17, 18) S. ballotiformis, (19) S. benthamiana, (20) S. kermanshahensis, (21) S. kurdica, (22) S. megalodonta, (23) S. veroniciformis, (24) S. atherocalyx, (25, 26) S. persepolitana, (27) S. setifera, (28, 29) S. fruticulosa, (30) S. lavandulifolia. (16–17, 21–22, 25, 27–30) scale bar ¼ 10 mm; (18–20, 23, 24, 26) scale bar ¼ 30 mm. Discussion The classification of trichomes presented here considerably enhances the importance of these characters within the genus. Our data are in accordance with previous studies in Lamiaceae confirming the usefulness of indumentum characters in taxonomic identification at different infrageneric levels (Abu-Assab and Cantino, 1994; Giuliani et al., 2008) and makes it possible to suggest systematic relationships among species and grouping them. Presence of multi-cellular branched, and also subsessile glandular trichomes with a unicellular cap have been suggested to perform synapomorphies against other character states in Lamioideae (Abu-Assab and Cantino, 1994). Glandular trichomes have taxonomic value at specific and subspecific level ARTICLE IN PRESS 378 Y. Salmaki et al. / Flora 204 (2009) 371–381 Figs. 31–42. SEM and LM micrographs of trichomes in Stachys spp. and Sideritis: (31, 32) S. lavandulifolia, (33) Sideritis montana, (34) S. ballotiformis, (35) S. byzantina, (36, 37) S. cretica, (38) S. inflata, (39) S. megalodonta, (40) S. persica, (41) S. pubesens, (42) S. schtchegleevii, (31) scale bar ¼ 200 mm; (32, 36, 38, 42) scale bar ¼ 100 mm; (33–35, 37, 41) scale bar ¼ 10 mm; (39, 40) scale bar ¼ 30 mm. and also can be considered as phylogenetically informative characters. For example, subsessile or sessile glandular trichomes are the most common type of trichomes in the family and probably represent an ancestral state (Navarro and El Oualidi, 2000). On the other hand, trichome type, distribution and cover vary widely in some genera among different sections (for example in Teucrium: Navarro and El Oualidi, 2000), providing valuable characters for comparison. It was also shown that different structures of the same individual plant may vary in indumentum in some genera of Lamiaceae (Doroszenko, 1986; Husain, 1983; Husain et al., 1990). Furthermore, investigations in some genera of Lamiaceae, such as Plectranthus, confirm the need for a better terminology on trichome characters in Lamiaceae (Ascensão et al., 1999). The taxonomy of Stachys is very difficult mainly due to great variation in macromorphological characters, particularly under different ecological conditions. Demissew and Harley (1992) suggested that the Tropical African species of Stachys can be divided into three natural groups based on trichome features and nutlet microsculpturing patterns, which are to some extent in accordance with subgeneric classification suggested by Bhattacharjee (1980). These groups also find some support from biogeographical data and ecology. Among several sources of micromorphological data surveyed by our team (Salmaki et al., 2008a, b) hair micromorphology provides the most valuable characters. Our detailed study on Iranian species of Stachys shows that several trichome characters can be of taxonomical value, but the possible phylogenetic importance of these features could not be referred here in details as the few molecular systematic studies conducted on the genus include only few species distributed in the Old World (Lindqvist and Albert, 2002). ARTICLE IN PRESS Y. Salmaki et al. / Flora 204 (2009) 371–381 Variation in trichome characters appears to have particular value, not only in classification at sectional rank in some cases, but also in separating different species of Stachys from their relatives. Sometimes these characters are useful in delimitation of formerly introduced sections. Sect. Ambleia, for example, is characterized by presence of dendroid or stellate trichomes. It seems that the presence or absence of branched trichomes in Stachys has an important infrageneric phylogenetic significance and can be considered as a synapomorphy for this group, as none of the known primitive species of the genus (see Lindqvist and Albert, 2002) represents this feature. Based on the variation mentioned above, Stachys can be divided into several groups, which are discussed below in a comparative context within the formal sections known in the genus. S. sect. Ambleia Benth. (Figs. 1–3) As mentioned above, the members of this rather isolated section are characterized by having branched trichomes (Bhattacharjee, 1980). Beside this feature, most species attributed to this section possess stipules at the base of their leaves. Among the species of this section only S. trinervis, representing the shortest trichomes in the section, shows one branching node (Fig. 3), while other species are furnished with multinodal trichomes provided by a long axis (Figs. 1 and 2). S. trinervis is also well characterized by having the largest nutlets in the section and a colliculate type of nutlet microsculpturing (Salmaki et al., 2008b). So, the branched trichomes provide not only an important synapomorphy (see above), probably supporting the monophyly of the section, but also enough variation for separating a few species from their relatives. S. sect. Aucheriana Bhattacharjee (Figs. 4–11) Almost all types of glandular trichomes are observed simultaneously on individual plants. The simple trichomes in this section are thin walled, swollen at internodes and smooth on surface (Table 2, Figs. 4–11). The species of the sect. Aucheriana macro-morphologically provide a homogeneous group characterized by spinescent leaves, calyx teeth, characteristic shoot structures as well as distinct bracts. The species of this section which is endemic to West Iran can be divided into two groups each of three species. One group, with S. ixodes, S. pilifera and S. aucheri, is characterized by very long spreading trichomes (up to 2.5 mm) beside a widely distributed rhizomatous growth habit and flexuous branches, while another group including S. acerosa, S. koelzii, and S. multicaulis is defined by shorter (up to 1 mm) and appressed trichomes, as well as cushion 379 forming habit and more rigid branches. The high variation provided by the trichomes among the species of this section can be used as effective means for separating taxa in this section. S. sect. Eriostomum (Hoffmanns. & Link) Dum. (Figs. 12–15) In this homogeneous section (Bhattacharjee, 1982), the dense and vermiform (lanate) trichomes covering the underlying glandular layer are characteristic. Although vermiform trichomes are observed in S. lavandulifolia (sect. Zietenia) too, these are stellate at base in this species, while they are more or less tuberculate in sect. Eriostomum. The species of this section are suggested to be primitive in the genus based on 5S-NTS sequences of nuclear ribosomal DNA (Lindqvist and Albert, 2002). Based on outgroups suggested for Stachys s.str. clade (Lindqvist and Albert, 2002) the occurrence of long vermiculate trichomes should be considered as a plesiomorphy in Stachys. S. sect. Fragilicaulis Bhattacharjee (Figs. 16–23) The members of this section were divided into two separate sections: subsect. Fragiles and Multibracteolatae (Bhattacharjee, 1982; Rechinger, 1982). Two species of the latter, i.e. S. kermanshahensis and S. veroniciformis, are characterized by stalked glandular trichomes, which occur also in S. benthamiana of subsect. Fragiles. Moreover, the presence of dense, long and simple trichomes in S. kermanshahensis can be used for its separation from S. veroniciformis. In subsect. Fragiles, presence of densely papillose appressed short trichomes characterizes S. asterocalyx, and the presence of short glandular trichomes separates all other members of subsect. Fragiles from S. benthamiana with stalked glandular trichomes. S. kurdica and S. ballotiformis are characterized by a double indumentum consisting of long spreading simple trichomes mixed with short subappressed simple ones. However, the long spreading trichomes are very sparse or even lacking in S. kurdica. These taxa are morphologically very similar. Based on our field experiences, it seems that the long simple trichomes can be fallen with age, and the short trichomes remain. Therefore, it is likely that S. ballotiformis is a synonym of S. kurdica (the older name), and the differences considered as important for their separation are age dependent and taxonomically not relevant. The variation of trichomes in sect. Fragicaulis is high enough to provide some evidence for separation of species, but there is no indumentum character that could ARTICLE IN PRESS 380 Y. Salmaki et al. / Flora 204 (2009) 371–381 be considered as an important synapomorphy of the section. Therefore, trichome micromorphology supports the unique systematic position of this species. S. sect. Olisia Dum. (Figs. 24–26) S. sect. Zietenia (Gled.) Benth. (Figs. 30–32) Three out of four Iranian species attributed to sect. Olisia by Bhattacharjee (1982) and Rechinger (1982) are characterized by non-glandular trichomes. S. pubescens which is obviously glandular hairy differs from other species also based on pollen morphological characters (Salmaki et al., 2008a). Based on all available data sect. Olisia in the present form seems to be a heterogeneous and probably polyphyletic one. For a comparison between this section and S. montana see below. Based on trichome micromorphology, this species is characterized by long vermiform trichomes. These trichomes are stellate at base and the longest one among the studied species. The glandular trichomes in this species are extremely short (up to 10 mm) and clavate at apex. All these features correlate this species to Betonica (not shown) and suggest a basal position for this section. S. sect. Setifolia Bhattacharjee (Fig. 27) S. setifera with its three subspecies is the only member of this monotypic section. All these subspecies possess stalked capitate glandular trichomes with a thin-walled basal cell, short subappressed simple trichomes as well as elongated thin walled ones. Similar to other gross morphological characters, trichome micromorphology does not provide any support for infraspecific classification in this species, as indumentum characters do not show any variation between three subspecies introduced under this species. Moreover, no trichome character is useful in separating S. setifera from other sections of Stachys studied here, although according to molecular studies this species clusters in a same clade as Prasium in the cladograms obtained from 5S-NTS sequences, as the most basal group of the so-called clade Stachys s.l. (Lindqvist and Albert, 2002). S. sect. Stachys Both species of this homogeneous section distributed in Iran possess glandular trichomes, which, however, are subsessile in S. palustris and stalked mixed with subsessile in S. sylvatica. Moreover, the simple trichomes are short (about 200 mm) in S. palustris, but long (up to 2000 mm) in S. sylvatica. Therefore, the type of indumentum supports the separation of the species of this section, but it is not enough valuable for characterizing the section. S. sect. Thamnostachys Kapeller (Figs. 28 and 29) S. fruticulosa is the only member of this section in Iran and is highly adapted to extremely dried and severe conditions of semi-arid areas in NW Iran. The absence of glandular trichomes is the most characteristic feature of this species. The trichomes are mostly appressed and flattened except for their cylindrical basal cell. S. montana L. (Fig. 33) Sideritis is nested within a same clade, i.e. Stachys s.str., as are most species of Stachys in the most recent molecular systematic study presented on Stachys (Lindqvist and Albert, 2002). From the point of view of trichome micromorphology no characteristic trichome feature could be reported here for S. montana. The trichomes in this species, as the representative of the genus Sideritis, are most similar to the members of S. sect. Olisia. The only difference between S. montana and S. iberica as the lectotype of sect. Olisia is the presence of glandular trichomes. Therefore, trichome micromorphology of these investigated Sideritis species would not provide strong support for separation of Sideritis from Stachys. Conclusion The data presented here show that hair micromorphology is more useful in separation of species within sections rather than characterizing large natural groups known as sections, except for few cases. Although there is only one reliable molecular systematic study on Stachys (Lindqvist and Albert, 2002) including few species distributed in Iran, this excellent work can provide strong supports for evolution of hair characters in Stachys. One important result of the above mentioned study is the relative basal position of Betonica compared with Stachys (Bhattacharjee, 1980; Lindqvist and Albert, 2002). Betonica is covered by very characteristic long vermiculate trichomes which are stellate and branched at base. Moreover, the varied types of glandular trichomes observed in S. lavandulifolia are also known in this species. If these two taxa, known as the sister groups to other Stachys species, represent the basal taxa in the group, then their indumentum types can be regarded as plesiomorphic. So, after comparison with these basal taxa, following evolutionary trends might be suggested regarding the indumentum in Stachys: (1) vermiform trichomes with stellate base are ARTICLE IN PRESS Y. Salmaki et al. / Flora 204 (2009) 371–381 primitive against vermiform trichomes with tuberculate base, (2) long vermiform trichomes are primitive against the short simple trichomes, (3) appressed trichomes are advanced against spreading trichomes, and (4) loss of glandular trichomes is advanced against their presence. Acknowledgements We are grateful to ‘‘Alexander von Humboldt Stiftung’’ for a grant to the corresponding author as well as to the Research Council, University of Tehran for partial support of this project. Research Institute of Forests and Rangelands (Tehran) supports this project in various ways, which is much appreciated. We thank M. Eshghi (Islamic Azad University, Tehran) for her assistance in electron microscopy. References Abu-Assab, M.S., Cantino, P.D., 1987. Phylogenetic implications of leaf anatomy in subtribe Melittidinae (Labiatae) and related taxa. J. Arnold Arbor. 68, 1–34. Abu-Assab, M.S., Cantino, P.D., 1994. 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