Scholarly Journal of Agricultural Science Vol. 4(2), pp. 97-102 February, 2014 Available online at http:// www.scholarly-journals.com/SJAS

ISSN 2276-7118 © 2014 Scholarly-Journals

Full Length Research Paper

Nutlet micromorphology in selected species of Salvia (Lamiaceae) in Iran

Fahimeh Salimpour¹*, Fariba Sharifnia², Mahdie Ebrahymiyan³

1,2,3Department of Biology, Faculty of Biological Sciences, North Tehran Branch, Islamic Azad

University, Tehran, Iran P.O. Box: 19585-936.

Accepted 16 October, 2013

The nutlet morphology of 12 Salvia L. (Lamiaceae, Mentheae) species was examined using scanning electron microscopy. The morphology of nutlets exhibited variation in size, shape, color, attachment scar and surface sculpturing. Nutlet size ranged between 1.5 and 2.7 mm in length and between 1.0 and 2.4 mm in width. Observed shapes included spherical, ovoid and ovoid-trigonous. Three types of ornamentation were identified: reticulate, foveolate and psilate. Our results from nutlet data do not support the correlation between nutlet sculpture and stamen type in these Salvia species. Key words: Salvia, Lamiaceae, SEM, nutlet, Iran.

INTRODUCTION The genus Salvia L. (Mentheae, Lamiaceae) represents enormous and cosmopolitan assemblage of nearly 1000 species displaying a remarkable range of variation (Walker et al., 2004). Salvia has undergone marked species radiations in three regions of the world: Central and South America (500 spp.); Asia/Mediterranean (250 spp.) and East Asia (90spp.) (Walker et al., 2004). Anatolia is major center for Salvia L. where 50.6% of the 87 species are endemic (Vural and Adigüzel, 1995). Fifty-eight species of this genus are found in Iran of which 17 are endemic (Hedge, 1968; Mozzafarian, 1996). Salvia and its fruits have medicinal and cultural importannce. The fruit of Lamiaceae is typically a schizocarp consisting of indehiscent locules which separate to form fruitlets (Harley et al., 2004). The entire body may split into indehiscent fruitlets that should properly be referred to as mericarps or nutlets (Özkan et al., 2009). Ryding (2001); Hye-kyoung and Hong (2006) and Nejad Habibvash et al.(2007) in a series of papers have made detailed studies on pericarp structure in Lamiaceae. According to Harley et al. (2004), Mentheae can be divided inti three subtribes, Salviinae, Nepetinae and Mentheae. Subtribe Salviinae is recognized as a monophyletic lineage, but its largest genus Salvia proved to be para or polyphyletic (Walker et al., 2004; Walker and Sytsma, 2007). Salvia is distinguished from other 72 genera in the tribe Mentheae *Corresponding author. E-mail: drsalimpour@gmail.com.

by having the two posterior stamens aborted and the connective separating the thecae of the two expressed stamens significantly elongated. Based on this structure three main stamen types were identified (Walker and Sytsma, 2007; Hedge, 1982); type A, with a reduced theca borne on the lower connective arm; type B, lower connective arm with variously shaped connective tissue blocking access to nectar and type C, lower connective arm reduced, connective and filament not articulated. Özkan et al. (2009), suggested that nutlet character combinations are correlated with stamen type, for each taxon in Salvia based on selected species from Turkey. For example stamen with type A has spherical- foveate mericarps or taxa with stamen type B has spherical-reticulate mericarps (Özkan et al., 2009). In this paper we present results of investigation of the micro-macro morphological characters in some Salvia species and answer the question whether there is any correlation between nutlet and stamen types and whether these taxa can be distinguished by nutlet features. MATERIALS AND METHODS

The specimens belonging to 12 Salvia species were collected from several localities in Iran. Voucher specimens are deposited in herbarium of Islamic Azad University, North Tehran Branch (Table 1). Identification of species was performed using Flora Iranica (Hedge, 1982), Flora of USSR (Pobedimovea, 1978) and Flora of

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Table 1. Materials and collecting data for micro-morphological studies on nutlets in selected species of Salvia

Species Habitat

Salvia atropatana Bunge Qazvin: Alamut, 2300 m, Ebrahimian 14034 Salvia hydrangea DC. Kordestan: Changealmas, 2000m, Mansori 14008 Salvia hypoleuca Benth. Qazvin: Alamut, 2200 m, Ebrahimian 14006 Salvia limbata C. A. Mey. Hamedan: Malayer, 2360 m, Ebrahimian 14021 Salvia macrosiphon Boiss. Semnan: Semnan, 1350 m, Ebrahimian 14067 Salvia multicaulis Vahl Azerbaijan: Sabalan, 1500 m, Ebrahimian 14065 Salvia nemorosa L. Tehran: Damavand, 1300 m, Ebrahimian 14057 Salvia reuterana Boiss. Tehran: Darake, 1950 m, Ebrahimian 14051 Salvia sclarea L. Tehran: Damavand, 1700 m, Ebrahimian 14072 Salvia verticillata L. Mazandaran: Chalous to Anarak, 1600 m, Ebrahimian 14041 Salvia virgata L. Qazvin: Alamut, 2100 m, Ebrahimian 14019 Salvia xanthocheila Boiss. ex Benth. Mazandaran: Ghachsar, 2900 m, Ebrahimian 14033

Table 2. Qualitative characters of nutlet in Salvia species

Species shape color ornamentation dark stripes attachment scar shape

S. atropatana ovoid black brown foveolate present triangular S. hydrangena ovoid pale brown reticulate-foveolate absent orbicular S. hypoleuca ovoid brown reticulate present triangular S. limbata Ovoid-trigonous brown foveolate present orbicular S. macrociphon spherical black brown foveolate present triangular S. multicaulis ovoid black brown foveolate absent orbicular S. nemorosa Ovoid-trigonous brown reticulate present triangular S. reuterana ovoid pale brown psilate present orbicular S. sclarea ovoid pale brown psilate present orbicular S. verticillata spherical pale brown foveolate present orbicular S. virgata ovoid brown reticulate present orbicular S. xanthocheila Ovoid-trigonous black brown reticulate present triangular

Turkey (Davis, 1969). Nutlets were first observed with a stereomicroscope to ensure that they were of normal size and maturity. For nutlet length and width, five samples of each taxon were taken and measured. For SEM observation, dried mature nutlets of five individuals for each species were examined using a SEM microscope model XL30 (Philips). Nutlets were directly mounted on aluminum stubs using double-sided adhesive and were sputter coated with a thin layer of gold. Coated nutlets were examined and SEM micrographs were captured at 200× and 500×. Qualitative characters such as shape and color of nutlet, the shape of abscission scar and ornamentation of nutlets were examined (Table 2). Mean ± standard deviation of quantitative characters was measured by SPSS software ver.9 (Table 3). Terminology for description of morphological characteristics of the mericarps follows Stearn (1978) and Bojnansky and Fragasova (2007). RESULTS The results of nutlet morphology are presented in Table 2,3. Mericarps of the examined species are brown to black. Their length varies from 1.53 mm to 2.64 mm and their width from 1.00 to 2.39 mm. They are ovoid,

spherical or ovoid-trigonous. The surface around attachment scar is usually smooth. The color of nutlets is first greenish and turns to brownish or black at maturity. Sometimes an irregular pattern of darker stripes can be recognized under the stereomicroscope. Under SEM, the nutlets show three types of surface sculpturing:

Type 1- Reticulate: surface finely reticulate includes: S. hypoleuca, S. nemorosa and S. virgata. (Figure 1- j,k,l). Type 2- Foveolate includes: S. atropatana, S. hydrangea, S. macrosiphon, S. multicaulis, S. limbata, S. verticillata and S. xanthocheilla, (Figure 1- a- g).Type 3- Psilate surface: surface is rather smooth lacking any particular ornamentation: S. sclareae and S. reuterana (Figure 1- h,i). The shape of attachment scar is triangular or orbicular (Figure 1). DISCUSSION The shape, size, sculpturing and color of nutlets examined varied and character combinations identified taxa. Moon et al. (2009) found that position and shape of attachment scar appeared as systematically useful characters. The attachment scar of the taxa examined positioned in the typical basal position but the shape of them is different (Figure 1). Moon et al. (2009), said that

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Table 3. Quantitative characters of nutlet in Salvia species.

Species width of attachment scar (mm)

length of attachment scar (mm)

width of nutlet (mm)

length of nutlet (mm)

S. atropatana 0.14± 0.031 0.34 ± 0.022 1.39 ± 0.021 2.62± 0.014 S. hydrangena 0.50 ± 0.031 0.50 ± 0.032 1.89 ± 0.021 2.54 ± 0.022 S. hypoleuca 0.28 ± 0.019 0.27 ± 0.036 1.70 ± 0.033 2.40 ± 0.029 S. limbata 0. 24 ± 0.023 0.25 ± 0.030 1.39 ± 0.025 2.28 ± 0.020 S. macrociphon 0. 14 ± 0.021 0.29 ± 0.023 2.39 ± 0.024 2.39 ± 0.024 S. multicaulis 0.60 ± 0.031 0.74 ± 0.026 1.91 ± 0.038 2.64 ± 0.025 S. nemorosa 0.09 ± 0.022 0.29 ± 0.021 1.00 ± 0.035 1.54 ± 0.021 S. reuterana 0.20 ± 0.029 0.28 ± 0.020 1.45 ± 0.029 2.14 ± 0.024 S. sclarea 0.10 ± 0.030 0.15 ± 0.029 1.75 ± 0.030 1.53 ± 0.018 S. verticillata 0.10 ± 0.029 0.29 ± 0.022 1.63 ± 0.020 1.63 ± 0.020 S. virgata 0.25 ± 0.031 0.29 ± 0.028 1.10 ± 0.028 1.54 ± 0.022 S. xanthocheila 0.15 ± 0.028 0.24 ± 0.021 1.64 ± 0.025 1.99 ± 0.021

Figure 1. Scanning electron micrographs of nutlets in Salvia species consist of a- shape (x25) ; b- abscission scar (x75) ; c- surface ornamentation (x500): a- S. atropatana; b - S. hydrangea; c- S. limbata ; d- S. macrosiphon

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Figure 1. Continued: e- S. multicaulis; f- S.verticillata; g. S. sclarea ; h. S. reuterana

in Menthaea tribe, nutlets have an areolate, triangular or round attachment scar and in Salviniae as a member of the subtribe of Mentheae, nutlets are round or triangular without an extend area. In this research we found two types of the attachment scar in Salvia species, so that shape and size are be useful in systematic studies. Also, the attachment scar in some members of tribe Westrigieae and Ajuga is baso-lateral and occupies a significant fraction of the length of the mericarp (Guerin et al., 2005). So, the nature of the attachment scar may be of systematic value in other genera or tribes in Lamiaceae.

However, some researchers found that the nutlet micromorphology is an important taxonomic character in flowering plants as well as Lamiaceae (Ryding, 1994; Jamzad, 2000; Salmaki, 2008). In our study, nutlet

morphology was useful for separating closely related species. For instance, S. limbata and S. hypoleuca have many similarities in leaf shape, leaf indumentum, color of corolla and stamen type, but our data suggests that they have different nutlet characters. Özkan et al. (2007) suggested that the stamen type is correlated with the nutlet characters. In this research, S. hydrangea and S. multicaulis with stamen type A have similarities in flower characters. In both of them their calyces clearly campanulate in fruit, their corolla is pink and upper lip of corolla is straight, but they are different in nutlet characters such as sculpture, strip band and their size (Table. 1,2; Figure 1c, 1g). Taxa with stamen type B such as S. sclarea, S. hypoleuca did not show the same shape or ornamentation of nutlet. Also, S. macrosiphon and S. reuterana with stamen type B, have tubular calyx.

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Figure 1. Continued- i- S. xanthocheila; j- S. hypoleuca; k- S. nemorosa; l- S. virgata

The upper lip of corolla in these two species is the same. But they are different in nutlet. In S. reuterana, nutlet has ovoid shape, orbicular scar with psilate ornamentation but the nutlet shape in S. macrosiphone is spherical with triangular scar and foveolate sculpture (Figure 1. d, h). S. xanthocheila is closely related to S. atropatana especially in an early flowering state. In flora Iranica, the main different characters between these two species are narrow leaves. Both of them have campanulate calyx, white corolla that upper lip of it is tridentate, so distinguished them are too hard. These two species are different in qualitative and quantitave characters of nutlet. In S. atropatana, the ornamentation of nutlet is foveolate but S. xanthocheila has reticulate sculpture (Figure 1. a, i). Also the nutlet size is different in two of them (Table 3). On the other hand, S. virgata and S. nemorosa are

similar especially in inflorescence, color of corolla and shape of bracts.

In both of these two species, verticillasters have 2-6 flowered and distant, bracts are ovate- acuminate with pale lilac corolla, but their size and ornamentation of nutlet are different. In S. verticillata with stamen type C, the nutlet characters are very similar to species with stamen type B. So detailed comparisons with the conclusions of the previous study are contradictory. In conclusion, we observed variation in the external morphology of nutlets in selected species Salvia representing the morphological diversity of the genus in Iran. Characters didn’t correlate with stamina types; however, shape, size, ornamentation, attachment scar and color varied and were useful in distinguishing species.

REFERENCES Bojnansky, V. and Fargasova, A. (2007). Atlas of seeds and fruits of

central and east European flora (The Carpathina mountains region). Springer, Stuttgart.

Davis, P.H. (1969). Flora of Turkey. Edinburgh at the University Press. Habibvash, F.N., Rajamand, M.A., Sarghein, S.H., Heidari, R. and

Heidari, R.M. (2007). Anatomical observation on nutlets of some Salvia species (Lamiaceae) from West Azarbaijan in Iran. Pak. J. Biol. Sci. 10: 3385-3389.

Hye- Kyoung, M. and Hong, S. (2006). Nutlet morphology and anatomy of the genus Lycopus (Lamiaceae: Mentheae). J. Plant Res. 119: 633-644.

Jamzad, Z., Harley, M.M., Ingrouille, M., Simmomds, M.S.J. and Jalili, A. (2000). Pollen exine and nutlet surface morphology of the annual species of Nepeta L. (Lamiaceae) in Iran in M. M. Harley, G. M. Morton and S. Blackmore (Editors). Pollen and Spores: Morphology and Biology, pp. 385-397. Royal Botanical Gardens, Kew.

Moon, H.K., Hong, S.P., Smets, E., Huysmans, S. (2009). Micromorphology and character evolution of nutlets in Tribe Menthaea (Nepetoideae, Lamiaceae). Systematic Bot. 34(4): 760-776.

Mozafarian, V. A. (1996). Dictionary of Iranian Plant Names. Farhang Mosavar, Tehran.

Rechinger, K. H. (1987). Salvia, In: Flora Iranica, Labiatae, No 150. Edits, K. H. Rechinger and I.C. Hedge. Akademische Druck and Verlagsanstalt, Graz, Austria.

0zkan, M., Aktas, K., Ozdemir, C. and Guerin, G. (2009). Nutlet morphology and its taxonomic utility in Salvia (Lamiaceae: Mentheae) from Iran. Acta Bot. Croat. 68(1): 105-115.

Scholarly J. Agric. Sci. 102 Pobedimovea, E. G. (1978). Flora of Russia. Nauka Publishers,

Leningrad. Ryding, O. (2001). Myxacarpy in the Nepetoideae (Lamiaceae) with

notes on myxodiaspory in general. Syst. Geogr. Pl. 71: 503- 514. Ryding, O. (1994). Phylogeny of leucas group (Lamiaceae). Sys. Bot.

23 (2): 235- 237. Salmaki, Y., Zarre, Sh. and Jamzad, Z. (2008). Nutlet micromorphology

and its systematic implication in Stachys L. (Lamiaceae) in Iran. Feddes Repertorium 119 (7-8): 631-645.

Stearn, W. T. (1978). Botanical Latin. Redwood Burn Limited, London. Vural, A. and Adiguguzel, N. (1996). A new species from Central

Anatolica: Salvia aytachii M. Vural et N. Adiguguzel (Labiatae). Turk. J. Bot. 20: 531-534.

Walker, J.B. and Sytsma, K.J. (2007). Staminal Evolution in the Genus Salvia (Lamiaceae): Molecular Phylogenetic Evidence for Multiple Origins of the Lever. Annals of Bot. 100: 375- 391.

Walker, J.B., Sytsma, K.J., Treutlinn, J. and Wink, M. (2004). Salvia (Lamiaceae) is not monophyletic: implications for the systematic, radiation, and ecological specializations of Salvia and tribe Mentheae. American J. Bot. 91: 1115-1125.