Petiole indumentum types of the genus Alchemilla (Rosaceae) from Iran

Introduction

Alchemilla L. (Rosaceae) was primarily introduced by Linnaeus (1753) and classified as Eualchemilla byFock (1988), Lagerheim (1894) and Haumann and Balle (1936). The genus thought to be related to the subtribe Sanguisorbinae (Hutchinson, 1964) because of its inconspicuous flowers. However, its relation to the tribe Potentilleae (Notov and Kusnetzova, 2004) was confirmed by DNA sequence data (Eriksson et al., 1998, 2003) and based onanther structure (anthers with one elliptic theca on the ventral side of the connective) it has been placed in the subtribe Alchemillinae (Soják, 2008).

The genus Alchemilla comprises more than 1000 species (Fröhnerm, 1995) and has Holarctic distribution, with a main center of species richness in west of Eurasia, but they also may grow in south India, Sri Lanka, Java, China, Japan and on the mountains of Africa and Madagascar (Gehrke et al., 2008). Species of Alchemilla are distributed mainly in north and northwest of Iran. However, some species may occur in west and center of the country (Frohner, 1969; Khatamsaz, 1993). They have woody rhizome (Pawlowski and Walters, 1972), commonly growing in open meadows, stony slopes, shady places, river banks and forest edges of alpine and subalpine regions, from 1700 to 3300 m altitudes (Juzepczuk, 1941; Frohner, 1969; Khatamsaz, 1993). Frohner treatment’s (1969) of Alchemilla, in Flora Iranica recognized 31 species from Iran, Afghanistan, parts of west Pakistan, north Iraq, Azerbaijan and Turkmenistan, but Khatamsaz (1993) considered 24 species of the genus for Flora of Iran.

Alchemilla is a taxonomically difficult group (Izmailow, 1981) and a well-known example of polyploidy, autonomous apomixis (in the sense of agamospermy), in the Rosaceae (Czapik, 1996). Many representatives of this genus display heteroblastic plasticity, variability and instability in morphological characters (e.g. flowers, leaves and indumentum). This causes great difficulties in their identification and results in formation of several species complexes and micro-species (Asker and Jerling, 1992; Horandl, 2004). The present micromorphologic research, aims to find new consistent diagnostic characters (e.g. petiole micromorphological) for a precise delimitation of various species and to evaluate the extent to which the petiole indumentum micromorphology can be used for classification of the species of Alchemilla in Iran.

Materials and Methods

In this study, petiole morphology of 24 species (including different taxa from several populations) of Iranian species of Alchemilla was examined. We used both freshly collected samples (from 2010 to 2012, during spring and late summer) and dried herbarium specimens of the Guilan University Herbarium (GUH), the Tehran University Herbarium (TUH) and the Herbarium of the Research Institute of Forests and Rangelands of Iran (TARI). All the examined petioles were belonged to well-developed basal leaves. The voucher specimen of each newly collected species deposited in the Guilan University Herbarium (GUH). Species sampled are listed in Appendix 1. For scanning electron microscopy (SEM), the petioles (a portion of them, constantly taken from distal end) were selected, washed and then kept for drying (Eriksen and Yurstev, 1999; Faghir et al, 2010). Dried materials were mounted on SEM stubs by double-sided adhesive tape of silver paint and finally coated with gold in a sputter coater. Scanning electron microscopy was carried out using a Vega Tescan Razi instrument. The petiole photograph also were taken by digital microscope, Dino-Lite, AN-413T model. Flora Iranica (Frohner, 1969), Flora of Iran (Khatamsaz, 1993) and Flora of USSR (Juzepczuk, 1941), were the principal references for identification and terminology of indumentum in this paper.

Result

Our findings revealed two main types of petiole indumentum including: the straight cylindrical and flat ribbon shape hairs (Figure 1).

Figure 1.Cylindrical and flat ribbon shape trichome of A. microscopica(yellow arrow indicates the ribbon shape hair). Scale Bar = 200 µm

The first type is found in all the 24 species but, the second type is observed only in the five studied species (A. kurdica, A. hessii, A. fluminea, A. pseudo-cartalinica and A. sericata). Seven subtypes were identified as following:

Subtype I: Densely appressed-subappressed: this subtype was recorded in A. amardica (Figures 2A and 2B), A. pseudo-cartalinica and A. sericata.

Subtype II: Subappressed-erecto-patent: this subtype was identified in A. citrina, A. farinosa,A. plicatissima,A. valdehirsutaand A. gigantadus (Figures 2C and 2D).

Subtype III:Erecto-patent: this subtype was recognized in A. kurdica, A. persica,A. retinervisand A. sedelmeyeriana (Figures 2E and 2F).

Subtype IV: Densely erecto-patent: this subtype was found in A. rigida, A. condensa, A. surculosa (Figures 2G and 2H) and A. pectiniloba.

Figure 2. A and B. A. amardica; C and D. A. gigantodus;E and F. A. sedelmeyeriana; G and H. A. surculosa, Scale Bars: A, C, E and G = 1 mm, B, D, F and H = 200 µm

Subtype V: Densely horizontal: this subtype was present in A. caucasica (Figures 3A and 3B), A. hyrcana and A. microscopica.

Subtype VI: Glabri-horizental-erecto-patent-declinate: this subtype was common in A. fluminea, A. hessii and A. rechingeri (Figures 3C and3D).

Subtype VII: Declinate pilose: this subtype was found in A. erythropoda (Figures 3C and 3D) and A. meloncholica.

Figure 3. A and B. A. caucasica; C and D. A. rechingeri;E and F. A. erythropoda. Scale Bars: A, C and E = 1 mm, B, D and F =200 µm

The indumentum length was measured (Table 1). A. erythropoda with 1.76-1.80 mm and A. persica with 0.82-0.84 mm had the maximum and minimum hair length, respectively. In many species scattered secretory glands, wax granules and platelets were visible on the petiole surfaces.

Discussion

Our observations confirmed the taxonomical usefulness of petiole trichome micromorphological characters within the studied taxa. The diagnostic application of petiole indumentum types is well known in the family Rosaceae and especially in closely related genera of the tribe Potentilleae (Rosaceae) (Notov and Kusnetzova, 2004; Soják 2008) like Alchemilla and Potentilla L. (Eriksen and Yurstev, 1999; Faghir et al., 2010, Faghir et al., 2011). Juzepczuk treatment’s (1941), in the Flora USSR, used petiole indumentum evidences (along with other diagnostic criteria) at different taxonomic ranks (section, series and species level). Hayirlioglu-Ayaz and Beyazoglu (1997) identified different species of Turkish Alchemilla (e.g. A. plicatissima, A. stevenii, A. monticola) using hair types of petiole and other parts (e.g. stem and hypanthium). In Flora Iranica (Fröhner, 1969) and Flora of Iran (Khatamsaz, 1993) several species of Alchemilla were separated based on petiole indumentum types.

This study deals with the micromorphological details of petiole indumentum types and according to our findings, two main types, seven subtypes of petiole hairs were recorded in the genus Alchemilla. However, presence of flat ribbon shape trichome has not been reported earlier (Fröhner, 1969; Khatamsaz, 1993). The cylindrical hair was the most dominent type and erecto-patent was the most aboundent subtype in the studied species. The erecto-patent indumentum was found either alone or mixed with sub-appressed trichome of the subtype II and horizontal and declinate hairs of the subtype IV. In contrast, densely appressed-subappressed of the subtype I and declinate pilose of the subtype VII had minimum occurrence within the studied species.

In the current analysis, the petiole hairs of several populations of the same species were examined. The petiole indumentum type was constant within the populations of the same species (e.g. A. caucasica, A. farinosa, A. hessii, A. hyrcana and A. persica). Erecto-patent hair types were recorded from all the studied populations of A. persica (Appendix 1), the most widely distributed (north, northwest, center and west provinces) species of the genus in Iran. However, petiole hair density of the studied taxa, varied from young to old plants and the leaves produced in early and later growing seasons (Juzepczuk, 1941).

Based on the current results, petiole indumentum data supported the previous classification of Fröhner (1969) and Khatamsaz (1993) and was reliable criterion for distinguishing the species. Based on petiole indumentum features, an identification key has been prepared as followings:

Key to species

1- Petiole covered by densely appressed-subappressed or sub-appressed-erecto-patent hairs ……………………………………………………………............. 2
- Petiole glabrous or covered by erecto-patent, horizontal or declinate hairs .... 9
2- Petiole with densely appressed-subappressed hairs .….…….……................ 3
- Petiole with subappressed erecto-patent hairs …………...….........……..…... 5
3- Hairs argenteo-sericatae, 0.85-0.90 mm long ..……..........……. 1. A. sericata
- Hairs appressed-subappressed, more than 1 mm in length ….......…............ 4
4- Appressed-subappressed, pilose hairs of 1.50-158 mm long ....................................................................................... 2. A. pseudo-cartolonica
- Appressed-subappressed, sericeo-pubscence hairs of 1.30-1.47 mm long ....................................................................................................... 3. A. amardica
5- Subappressed-erecto-patent hairs 0.85-1 mm long ..……......…………....… 6
- Subappressed-erecto-patent hairs of 1->1.50 mm long .….…………......… 7
6- Densely pilose hairs of 0.94-1 mm long …..….…..…….… 4. A. plicatissima
- Pilose-subsericeo hairs of 0.85-0.90 mm long .….…………..…… 5. A. citrina
7- Hairs densely pilose, 1.58-1.64 mm long …….……….... 6. A. valdehirsuta
- Hairs sericeo-pilose or pilose > 1.50 mm .…....……...……………………… 8
8- Hairs sericeo-pilose, 1.38-1.41 mm long ..…..…………… 7. A. gigantodus
- Hairs pilose, 1.28-130 mm long ………..……………….…..….. 8. A. farinosa
9- Petiole covered by only declinate hairs ……………………..…...……..…. 10
- Petiole glabrous or covered by erecto-patent, horizontal or declinate hairs .. 11
10- Petiole with densely pilose hairs of 1-1.1 mm long ..…… 9. A. melancolica
- Petiole with densely subhirsute hairs of 1.76-1.80 mm long ................................................................................................. 10. A. erythropoda
11- Petiole covered by sparsely to ± densely erecto-patent hairs …........….… 12
- Petiole glabrous or covered by erecto-patent, horizontal and declinate hairs ........................................................................................................................... 19
12- Petiole with only sparsey to subdensely erect-patent hairs .........…..…… 13
- Petiole with densely erecto-patent hairs ……….…........….…...………...…. 16
13- Hairs shorter than 1 mm long .………..………..……...………..……… 14
- Hairs longer than 1 (1.35-1.40) mm long ..………...…..….....… 11. A. kurdica
14- Hairs 0.82-0.88 mm long .…………………….……….………………… 15
- Hairs 0.91-0.95 mm long …...………………………….….… 12. A. retinervis
15- Hairs 0.85-0.88 mm long ………………….……...… 13. A. sedelmeyeriana
- Hairs 0.80-0.83 mm long .…………….……........……..………. 14. A. persica
16- Petioledensely hirsute ………………..…………….………..….…......... 17
- Petiolesdensely sericeo-subsericeo grayish ……………..………………… 18
17- Hairs 1.40-1.42 mm long ……………………….…...…... 15. A. surculosa
- Hairs 1.08-1.11 mm long ………….………….………..… 16. A. pectinoloba
18- Hairs 1.41-1.43 mm long ……….……….…………….… 17. A. condensa
- Hairs 1.53-1.58 mmlong ………………..…………………..…... 18. A. rigida
19- Petiole covered by horizontal hairs …………………………..………….. 20
- Petiole glabrous or covered by erecto-patent, horizontal and declinate hairs of >1 mm long ……….................…………………………………………….. 22
20- Petiole densely pilose ……...……………………….…...…….……….. 21
- Petiole covered by cylindrical and ribbon shape hairs ..…. 19. A. microscopica
21- Petiole densely hairy; hairs 1.53-1.58 mmlong .……….….. 20. A. hyrcana
- Petiole pubescent; hairs 1.70-1.73 mmlong …………….….. 21. A. caucasica
22- Petiole pilose, with flat ribbon shaped hairs ...………………………… 23
- Petiole pilose …….………………………………….…….… 22. A. rechingeri
23- Hairs 0.82-0.85 mm long ………..…………………………..… 23. A. hessii
- Hairs 0.92-0.95 mm long …………………………..………… 24. A. fluminea

Acknowledgement

The authors are grateful to Dr. Farideh Attar (Central Herbarium of Tehran University and School of Biology, University College of Science, Tehran,) and the curator of the Herbarium of the Research Institute of Forests and Rangelands, Tehran (TARI), for their cooperation and allowing us to access the herbarium specimens.