Biologia, Bratislava, 59/1: 53—60, 2004 Cytotaxonomy of some species of Acanthophyllum (Caryophyllaceae) from Iran Seyed Mahmood Ghaffari Institute of Biochemistry and Biophysics, University of Tehran, PO.Box 13145-1384, Tehran, Iran; tel.: ++98-21-6113379, fax: ++98-21-6404680; e-mail: ghaffari@ibb.ut.ac.ir GHAFFARI, S. M., Cytotaxonomy of some species of Acanthophyllum (Caryophyllaceae) from Iran. Biologia, Bratislava, 59: 53—60, 2003; ISSN 0006-3088. Original observations on meiotic cells in 17 species of the genus Acanthophyllum are reported. Of these, the chromosome numbers, for 8 taxa viz, A. laxiusculum, A. heratense, A. caespitosum, A. pachycephalum, A. khuzistanicum, A. mucronatum, A. verticillatum and A. crassinodum are new observations. The basic chromosome numbers for the genus are x = 14 and x = 15. Three ploidy levels were observed for x = 15, diploidy (2n = 2x = 30), tetraploidy (2n = 4x = 60) and hexaploidy (2n = 6x = 90). Comparison of the morphological characters, chiasma average and chromosome configuration have shown some similarities between three tetraploid species of A. microcephalum, A. mucronatum and A. verticillatum and two hexaploid species of A. crassinodum and A. glandulosum. A. caespitosum is quite different from the others, especially in chromosome number (n = 14) and the morphology of inflorescence. Therefore, it seems that this taxon should be placed in a new section. The occurrence of polyploidy in some species indicates that this phenomenon plays an important role in the evolution of the Acanthophyllum genus. Chromosome configurations for polyploid species and chiasma average for all species are reported here for the first time. Key words: Caryophyllaceae, Acanthophyllum, meiotic analysis, chromosome number, chiasma formation, Iran. Introduction Acanthophyllum C. A. MEYER is a genus with a total of c. 61 species in the world. Of these 33 occur in Iran of which 23 species are endemic. All species of Acanthophyllum are divided into 7 sections of which 4 sections, namely Oligosperma, Macrostegia, Acanthophyllum, and Pleiosperma, occur in Iran. According to literature (BOISSIER, 1867; HUBER-MORATH, 1967; KOMAROV, 1970; GHAZANFAR & NASIR, 1986; PARSA, 1951; LEONARD, 1986; SCHIMAN-CZEIKA, 1988) the highest number of species has been recorded in east of Iran (Khorrasan province) and in the adjacent area i.e., Turkmenistan and Afghanistan. The east of Afghanistan towards China and the west of Turkey towards Syria are poor in species, where only a single species occurs in China (A. punges ) and Syria (A. verticillatum). Considering the floristic regions according to TAKHTAJAN (1986), all the species of this genus belong to Irano-Turanian region. Chromosome studies serve as a source of data for biologists concerned with systematic and evolutionary investigations. Unlike the morphological characters of specimens that can be measured and remeasured by succeeding genera- 53 Table 1. The species and origin of material examined. Taxon Section Oligosperma A. caespitosum BOISS. A. elatius BUNGE A. heratense SCHIM.-CZEIKA A. korshinskyi SCHISCHK. A. laxiusculum SCHIM.-CZEIKA A. lilacinum SCHISCHK. Section Macrostegia A. bracteatum BOISS. A. gracile BUNGE ex BOISS. A. khuzistanicum SCHIM.-CZEIKA A. pachycephalum SCHIM.-CZEIKA Section Acanthophyllum A. crassifolium BOISS. A. microcephalum BOISS. A. mucronatum C.A. MEY. A. verticillatum (WILLD.) HAND.-MZT. Section Pleiosperma A. crassinodum YUKHAN. & EDMONDSON A. glandulosum BUNGE ex BOISS. A. sordidum BUNGE ex BOISS. Collection No. Origin 10666 9966 10166 7366 7866 8066 11766 3165 8366 8266 7266 8466 9366 7666 5666 2566 4065 12364 6466 6064 6966 Hamadan; Toyserkan Ilam: Eslamabad Bakhtaran: between Sahneh & Kangavar Khorrasan: betweeb Sabzevar & Shahroud Khorrasan: between Mshhad & Torbat-Heydariyah Khorrasan: between Kashmar & Totbat-Heydariyah Khorrasan: Bojnourd Khorrasan: between Mshhad & Shandiz Khorrasan: between Sabzevar & Mehr Khorrasan: between Nayshabour & Kashmar Semnan: Shahroud, 60 Km. Towards Sabzevar Khorrasan: between Sabzevar & Mehr Ghazvin: between Boinzahra & Takestan Khorrasan: between Mashhad & Nayshabour Semnan: Garmsar, Behbar Semnan: Garmsar, Behbar Semnan: Shahroud, Mojen Tehran: 60 Km. Towards Saveh Tehran: Ab-Ali Tehran: NW Semnan: Shahroud, Cheldokhtar, Tang-e-Olang 167 5665 1267 673 2467 14164 Zanjan: between Avaj & Abgarm Khorrasan: between Mashhad & Torbat-Haydariyeh Khuzestan: Dezful Khuzestan: Omidiyeh Tehran: towards Karj Tehran: Karaj, 5 Km. towards Chalus 16564 17464 14764 15364 15064 16364 13267 10266 16664 Tehran: Jajroud Tehran: Firouzkouh Markazi: between Arak & Qom Tehran: Karaj, 5 Km. towards Chalus Qom: towards Arak Tehran: Sorkhehesar Zanjan: 2 Km. N Hamadan: Assadabad Tehran: Jajroud 12466 12066 17164 5265 8166 4965 2965 5966 3465 Khorrasan: between Ghoochan & Dareh-Gaz Khorrasan: Dareh-Gaz, Tandureh park Tehran: between Firouzkouh & Damavand Khorrasan: Ghoochan Khorrasan: Kashmar, Rivash Gorgan: Golestan park Qom: 55 Km. towards Tehran Semnan: Garmsar, Behbar Gorgan: Golestan park tions of botanists, the chromosomal information is ephemeral and it is not directly retrievable from dried plants (herbarium specimens). Consequently, the documentation of karyological analy- 54 sis and meiotic behavior serve as a unique source of information for both current and future investigations. Except of my previous reports (GHAFFARI, Table 2. Chromosome numbers of Acanthophyllum species. Present count Previous count Taxon References (n) (n) 14 15 15 15 15 15 – 15 GHAFFARI 1988 GHAFFARI 1988 – GHAFFARI 1988 15 + 0-3B 15 – 15 – GHAFFARI 1988 15 15 15 15 15 15 – – GHAFFARI 1986 GHAFFARI 1987 – – 30 30 30 30 A. mucronatum A. verticillatum Section Pleiosperma A. crassinodum A. glandulosum 30 30 – – 45 45 – 45 A. sordidum 30 30 Section Oligosperma A. caespitosum A. elatius A. heratense A. korshinskyi (syn. A. khorasanicum) A. laxiusculum A. lilacinum Section Macrostegia A. bracteatum A. gracile A. khuzistanicun A. pachycephalum Section Acanthophyllum A. crassifolium A. mirocephalum 1986, 1987, 1988), chromosome counts on the Acanthophyllum are limited to 6 species (NUSSBAUMER, 1964; ARYAVAND & FAVARGER, 1980). Because of the occurrence of some indistinct species in this genus, SCHIMAN-CZEIKA (personal communications) believed that the chromosome studies are useful and that they may help to clear up the status of indistinct species. The present study describes the meiotic chromosome numbers of 45 collections in Iran, representing 17 species. The data on mean chiasma frequency and meiotic behaviour are reported here for the first time. Material and methods The origin of the plant material studied here is shown in Table 1. Floral buds of plants found in nature were collected and immediately fixed in Piennr’s fluid containing ethanol 96% – chloroform – propionic acid, 6: 3: 2 (v/v/v) for 24 hours at room temperature. Anthers dissected out from the buds were squashed and stained with 2% acetocarmine. Chromosome counts obtained from a minimum of 50 pollen mother cells within each collection (at different stages of meiosis). Because of the difficult spreading of the meiotic chro- (2n) 60 90 GHAFFARI 1986 ARYAVAND & FAVARGER 1980 GHAFFARI 1986 NUSSBAUMER 1964 – – – GHAFFARI 1986 NUSSBAUMER 1964 GHAFFARI 1987 mosomes, the pairing analyses were conducted on limited number of cells (Tabs 2–3). All slides were made permanent by the Vanetian turpentine (WILSON, 1945). Photographs of chromosomes were taken by Olympus Photomicroscope at initial magnification of 330X. Voucher specimens were deposited in the Central Herbarium of Tehran University (TUH). Results The previous and present counts and the results of the analysis of metaphase I of meiosis in pollen mother cells are summarized in Tables 2, 3 and 4. In addition, each section is discussed bellow in details. Section Oligosperma SCHISCHK. Section Oligosperma has the highest number of species amongst other section of Acanthophyllum genus. This section possesses 25 species, from which nine are narrow endemic (SCHIMANCZEIKA, 1988; KOMAROV, 1975). Twenty-one collections representing 6 species were studied in this section. They were uniformly diploid with n = 15 55 Table 3. Types of bivalents, chiasma average per bivalent and positions in diploid taxa of Acanthophyllum Bivalents situation Taxon Section Oligosperma A. caespitosum A. elatius A. heratense A. korshinskyi A. laxiusculum A. lilacinum Section Macrostegia A. bracteatum A. gracile A. khuzistanicum A. pachycephalum Chiasma position No. of cells Ring Rod 22 7 5 8 13 12 89 76 44 86 90 142 241 29 31 34 105 39 419 181 119 206 285 323 1 4 – 25 4 53 1.36 1.76 1.59 1.92 1.48 2.08 6 7 5 14 49 41 43 99 41 64 32 111 139 146 118 309 2 3 1 7 1.57 1.42 1.59 1.60 Terminal Interstitial Chiasma average Table 4. Summary of the analysis of meiotic metaphase I in tetraploid and hexaploid species of Acanthophyllum. Taxon A. crassifolium A. microcephalum A. mucronatum A. verticillatum A. crassinodum A. glandulosum A. sordidum No. of cells analyzed Configurations I II III IV V VI 6 % 5 % 10 % 7 % 6 % 10 % 9 % 6 4.19 12 8.33 24 8.42 16 8.04 2 .90 4 1.07 – – 127 88.81 120 83.33 234 82.10 164 82.41 183 82.80 307 82.30 202 85.59 – – – – – – – – 2 .90 4 1.07 – – 10 6.99 12 8.33 27 9.47 19 9.54 17 7.69 39 10.45 34 14.40 – – – – – – – – – – – – – – 1.43 – – – – – – 17 7.69 19 5.09 – – (except A. caespitosum) (Tabs 1–2, Figs 1A–J,). Chromosome counts for A. caespitosum in 3 different populations were n = 14, which was different from my earlier report (GHAFFARI, 1988). I have reported the haploid chromosome number for this taxon to be n = 15. The erroneous count has been due to the false recording of two univalents at first metaphase that considered incorrectly as bivalents. Many specimens which SCHIMAN-CZEIKA (1985, 1988) named as A. laxiusculum had previously been determined as A. squarrosum . Therefore, my earlier report (GHAFFARI, 1986) about A. squarrosum corresponds to A. laxciusculum. In the samples numbered as 5666 and 2566 (Tab. 1) 56 Chiasma average Level of ploidy 1.37 4X 1.55 4X 1.55 4X 1.54 4X 1.62 6X 1.61 6X 1.31 4X in addition to 15 bivalents, 0 to 3 B-chromosomes were found in many cells at meiosis and mitosis stages. These B-chromosomes showed a tendency to lag at first anaphase. The similar phenomenon in Centaurea kandavanensis has been reported by GHAFFARI (1998). B-chromosomes appeared as univalents without pairing with each other. Chiasmata average in samples without B-chromosomes was 1.47 for one bivalent (see Tab. 3). The results of behaviour of B-chromosomes in this taxon will be published separately. Chromosome counts for A. laxiusculum and A. heratense, and also chiasma average and positions of bivalents for all species in this study, are reported here for the first time. Fig. 1. Meiosis. A – A. caespitosum, metaphase II, n=14; B – A. elatius, metaphase I, n = 15; C – A. heratense, metaphase I, n = 15; D – A. heratense, diplotene, n = 15; E – A. korshinski, metaphase I, n = 15; F – A. laxiusculum, metaphase I, n = 15; G – A. laxiusculu, metaphase I, showing B-chromosome (arrow); H – A. laxiusculum, anaphase II; I – A. lilacinum, metaphase I, n = 15; J – A. lilacinum, late anaphase I, showing laggard bivalent chromosome (arrow). Scale bar 10 µm. Section Macrostegia BOISS. Section Macrostegia is characterized by board hyaline margin of bracteole, it is comprised of 9 species in the world (SCHIMAN-CZEIKA, 1988; KOMAROV, 1975). Six collections studied in this work represented 4 species that were diploid with ga- metic chromosome number of n = 15 (Tabs 1–2, Figs 2A–D). Many specimens which were named by SCHIMAN-CZEIKA (1988) A. pachycephalum had previously been determined as A. bracteatum. Therefore, my previous report (GHAFFARI, 1986) about A. bracteatum refers to A. pachycephalum. 57 Fig. 2. Meiosis. A – A. brcteatum, diakinesis, n = 15; B – A. gracile, diakinesis, n = 15; C – A. khuzistanicum, diakinesis, n = 15; D – A. pachycephalum, metaphase I, n = 15; E – A. crassifolium, diakinesis, n = 30; F – A. microcephalum, metaphase I, n = 30; G – A. mucronatum, metaphase I, n = 30; H – A. verticillatum, metaphase I, n = 30; I – A. crassinodum, metaphase I, n = 45; J – A. glandulosum, metaphase I, n = 45; K – A. glandulosum, metaphase I, showing multivalents configuration; L – A. sordidum, metaphase I, n = 30. Scale bar 10 µm. Chromosome counts for A. pachycephalum and A. kuzistanicum are also repotted here for the first time. 58 Section Acanthophyllum Section Acanthophyllum has 7 species in the world (SCHIMAN-CZEIKA, 1988). Nine collections stud- ied here represented 4 species which were uniformly tetraploid with gametic chromosome number of n = 30 (Tabs 1–2, Figs 2E–H). The similarity in chiasma average and chromosome configurations in A. microcephalum, A. mucronatum and A. verticillatum shows an affinity between them (Tab. 4), but more cell analysis is needed. Chromosome counts for A. mucronatum and A. verticillatum are reported here for the first time too. Section Pleiosperma BOISS. This section has 7 species in the world, two of which are narrow endemic (SCHIMAN-CZEIKA, 1988, KOMAROV, 1975) Nine collections represented 3 species, two of them, A. crassinodum and A. gladulosum, were hexaploid with gametic chromosome number of n = 45 and the third one, A. sordidum, was tetraploid with n = 30 (Fig. 2I). The results of chromosome pairing and chiasma average in A. crassinodum and A. glandulosum may indicate a similarity between them (Tab. 4), but more material examined is needed. Chromosome count for A. crassinodum is reported here for the first time. Discussion The results obtained from the chromosome studies on pollen mother cells, showed an equal basic chromosome number, x = 15, in all species of four sections except for A. caespitosum. Table 2 indicates that nearly all the members of sections Macrostegia and Oligosperma are diploid with 2n = 2x = 30 (except for diploid A. caespitosum with 2n = 2x = 28), members of section Acanthophyllum are tetraploid with 2n = 4x = 60, and members of section Pleiosperma are hexaploid with 2n = 6x = 90 (except for A. sordidum). Comparison of the morphological characteristics (see SCHIMAN-CZEIKA, 1988) and the results of meiotic analysis (Tab. 3) between A. microcephalum, A. mucronatum and A. verticillatum have shown similarities between them. These similarities are especially pronounced in the mean chiasma frequency, rate of univalents, bivalents and tetravalents formation (Tab. 4). It seems that, these three species present a single species with differences in alleles which carry a few characters. For this reason, in some Floras (HUBER-MORATH, 1975; POST, 1933) these species have been mentioned as synonyms. Also in Iran, they have been grown in a single locality. Further, MOBAYEN (1979) did not find any clear differences to separate these species. Acanthophyllum crassinodum is mophologically very close to A. glandulosum (SCHIMAN- CZEIKA, 1988; YUKHANANOV & EDMONDSON, 1977). As it is shown in Table 3, similarities are especially pronounced in the mean chiasma frequency, and bivalents formation. MOBAYEN (1979) believed that characters such as little branches of the stem and swollen nodes are not enough to separate them. On the other hand, we found a great morphological variation for A. glandulosum in nature (especially in different altitudes). Acanthophyllum caespitosum differs from the others in morphology and in karyology as well. All species of Acanthophyllum follow the basic chromosome number of x = 15, whereas the A. caespitosum has a basic number of x = 14. SCHIMANCZEIKA (personal communications) believes that this taxon is quite separated from the others. In her personal letter to me she says “Although in the Flora Iranica, I compared it with A. pulchrum, the two are quite different, for instance A. pulchrum has beautiful blossoms. I do not know any species which has such inconspicuous inflorescence like A. caespitosum. All of the others have distinct inflorescenes separated from the leaves”. Therefore it seems that this taxon must be placed in a new section. In the Acanthophyllum genus, polyploidy appears to have played an important role in its evolution and speciation. Up to now in two sections, Acanthophyllum and Pleiosperma, tetraploidy and hexaploidy were recorded. Also most of the species which were found in the western and in the central parts of Irano-Turanian region (Turkey, Syria and many parts of Iran), are tetraploid and those at eastern and northern parts (north-eastern of Iran, South of Turkmenistan and Pamir) are hexaploid. This wide geographic distribution supports the previous reports of REESE (1958), STEBBINS (1972) and EHRENDORFER (1980). They have considered that the polyploids have greater ability to colonize in a new and wider geographic distributions than their diploid ancestors. Acknowledgements This work was supported by grant (31303241) from the national research council and the research council of the University of Tehran. I would like to thank Dr. SCHIMAN-CZEIKA for her help in the identification of the plants. References ARYAVAND, A.& FAVARGER, C. 1980. Contribution a l etude cytotaxonomique des Caryophyllacees de l Iran. Rev. Biologie. Ecol. Mediterra. 3(1): 15–26. 59 BOISSIER, E.1867. 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