18/2 • 2019, 313–322 DOI: 10.2478/hacq-2019-0001 Species delimitation and inter-specific gene flow in Tamarix L. (Tamaricaceae) Masoud Sheidai1 , Tahmineh Shagholi2, Maryam Keshavarzi2 , Fahimeh Koohdar1,* & Habibollah Ijbari3 Key words: Tamarix, ITS, HGT Abstract tree, Species delimitation. Tamarix L. play important role in preventing deforestation in Iran. Tamarix species exhibit wide range of morphological variation therefore, the species Ključne besede: Tamarix, ITS, delimitation become difficult. This is further complicated due to similarity of drevo HGT, razmejitev vrst. morphological characters in closely related species and the occurrence of inter- specific hybridization. The present study was performed to identify Tamarix species and their potential hybrids in Semnan Province of Iran. We used ITS and ISSR and 42 morphological characters for our investigation. Molecular phylogeny of the studied species and their relationship was not in agreement with the species tree of morphological characters and with taxonomic treatment of the genus. HGT tree of ITS and morphological data obtained revealed the occurrence of inter-specific hybridization or introgression between Tamarix species. Izvleček Vrste rodu Tamarix so v Iranu pomembne za preprečevanje krčenja gozdov. Zanje je značilna široka morfološka variabilnost, s katero so sposobne preživeti v različnih ekoloških razmerah, zato je razmejitev vrst težavna. Dodatne težave predstavljajo podobni morfološki znaki pri ozko sorodnih vrstah in prisotnost medvrstnega križanja. V članku želimo določiti vrste rodu Tamarix in njihove potencialne križance iz province Semnan v Iranu. V raziskavi smo uporabili ITS in ISSR molekulske markerje in 42 morfoloških znakov. Molekularna filogenija obravnavanih vrst in njihova razmerja niso bili v skladu z dendrogramom Received: 24. 7. 2018 morfoloških znakov in s taksonomsko členitvijo rodu. Z HGT dendrogramom Revision received: 21. 1. 2019 podatkov iz ITS in morfološko analizo smo pokazali obstoj medvrstnega križanja Accepted: 21. 1. 2019 oziroma introgresije med vrstami rodu Tamarix. 1 Faculty of Life Sciences & Biotechnology, Shahid Beheshti University, Tehran, Iran. 2 Department of Biology, Alzahra University, Tehran, Iran. 3 Department of Biology, Faculty of Sciences, University of Zabol, Zabol, Iran. * Corresponding author. Faculty of Life Sciences & Biotechnology, Shahid Beheshti University. E-mail: [email protected] 313 Masoud Sheidai, Tahmineh Shagholi, Maryam Keshavarzi, Fahimeh Koohdar & 18/2 • 2019, 313–322 Habibollah Ijbari Species delimitation and inter-specific gene flow in Tamarix L. (Tamaricaceae) Tamarix species hybridize and may form different 1. Introduction taxonomic forms due to inter-specific hybridization and introgression (Gaskin & Schaal 2003, Gaskin & Kazmer The genus Tamarix L. contains about 54 species that 2019, Mayonde et al. 2019). Due to different degree of mainly grow in saline areas of deserts and semi-deserts in gene flow amongTamarix species, plants with variable Asia, Europe, North-east and South-west of Africa. Tama- morphological characters occur in the same area. There- rix species play important role in preventing deforestation fore, high gene flow in Tamarix species caused by inter- in Iran (Sheidai et al. 2018). Tamarix species have limited specific hybridization resulting in phenotypic variations benefits to human but have been used as ornamental plant have rendered taxonomic classification of the genus in gardens or public plantations. For example, T. gallica problematic (Baum 1978, Ijbari et al. 2014). T. tetran- L., T. chinensis Lour., T. ramosissima Ledeb. are frequently dra Pall. ex M.Bieb., T. gallica L., T. chinensis, T. ramo- used as ornamental plants for their feathery appearance sissima, and T. parvifloraDC. are considered to be one and their catkin-like inflorescences (Gaskin 2003). Tama- variable species or hybridizing group, designated by the rix species are good for windbreak (for example T. aphylla hybrid name T. pentandra Pall. (Sudbrock 1993). T. chin- (L.) Karst. and T. Africana Poir.) or for erosion control ensis and T. ramosissima are morphologically alike and and easily grow in poor soils (Baum 1967, Gaskin and differ only in some microscopic characters and geneti- Schaal 2002, Ijbari et al. 2014). cally distinct in Asia (Mayonde et al. 2016). However, Ardebil Azerbaijan-e sharghli Caspian Sea Khorasan-e Shomali Gilan Golestan Azerbaijan-e gharbi Zanjan Mazandaran Qazvin Alborz Tehran Semnan Kordestan Khorasan-e Razavi 35°N Hamadan Qom Kermanshah Markazi Lorestan llam Esfahan Khorasan-e Chahar Yazd Jonubi Mahal o Khuzestan Bakhtiari Kohgilayeh va Boyer Ahmad 30°N Kerman Fars Bushehr Sistan va Hormozgan Baluchestan Persian Gulf 45°E 50°E 55°E 60°E Figure 1: Distribution map of Tamarix species in Iran. Slika 1: Karta razširjenosti vrst rodu Tamarix v Iranu. 314 Masoud Sheidai, Tahmineh Shagholi, Maryam Keshavarzi, Fahimeh Koohdar & 18/2 • 2019, 313–322 Habibollah Ijbari Species delimitation and inter-specific gene flow in Tamarix L. (Tamaricaceae) they have been proven to be genetically different and are 2008). ITS sequences are useful to construct phylogenies known to hybridize in North America and South Africa of angiosperms at lower taxonomic levels (Baldwin et (Gaskin & Schaal 2002, Gaskin & Kazmer 2009, May- al. 1995) and reveal polymorphisms (double base read- onde et al. 2016). ings) within plant individuals (Campbell et al. 1997). Morphological characteristics are important in Tamar- Polymorphisms in some individuals can occur because ix species delimitation. Tamarix leaves are taxonomically concerted evolution is not fast enough to homogenize useful and they show variation in shape and attachment repeats of mutations among the multiple copies in the modes in different species (Baum 1978). Tamarix flow- genome, and/or because of recent hybridization events ers are bisexual, rarely unisexual and plants are either (Campbell et al. 1997). ISSR molecular markers were monoecious or dioecious. The flowers either have five or shown to be informative for genetic diversity and popu- four sepals with corresponding number of petals. Flow- lation structure studies (see for example, Sheidai et al. ers have five or numerous stamens that are free or fused 2012, 2013, Azizi et al. 2014). and are inserted into a fleshy, glandular, hypogynous disc Our study was conducted in the Semnan Province of (Obermeyer 1976, Baum 1978). The presence of bisexu- Iran because species of this area have not been identified. al flowers and cross-pollination in Tamarix lead to the After morphological identification of Tamarix species, occurrence of high genetic diversity and hybrid forma- their identification was also checked by BLAST using tion in these species (Gaskin & Schaal 2002, Gaskin & ITS (Internal transcribed sequences) of the nuclear DNA Kazmer 2009, Gaskin et al. 2012, Mayonde et al. 2015, (nrDNA). Furthermore, the morphological and ITS 2016). The intraspecific genetic variability may be used analyses of identified species were carried to reveal the for local adaptation and also prevents homozygosity and species delimitation and relationship (Sheidai et al. 2013, genetic extinction of the studied Tamarix taxa (Ijbari Minaeifar et al. 2016) and we carried out introgression et al. 2014). patterns in populations of in T. szowitsiana Bge. and T. Thirty-five Tamarix species occur in Iran as reported androssowii Litv. species by using Inter-simple sequence by Schiman-Czeika (1980). These species have been used repeats (ISSR) and in T. Androssowii, T. Meyerii Boiss. in plantation to prevent deforestation in Iran. However, and T. Szowitsiana by ITS molecular markers. our general survey and extensive collections in different provinces may suggest the occurrence of more number of species/ sub-species in the country. The correct identity 2. Materials and methods of our Tamarix collections can be verified at population level through detailed taxonomic investigations using 2.1 Morphological investigation both morphological and molecular approaches (Arian- manesh et al. 2014, Ijbari et al. 2014). Eighty plants were randomly collected from 22 geograph- Tamarix species occur in 21 provinces of Iran (Fig- ically areas in Semnan Province in Iran and used for mor- ure 1). Ecological and climatic differences may influence phological investigations. The voucher specimens were the morphological appearance of Tamarix but will not deposited in Herbarium of Shahid Beheshti University affect the identity of the species (Sheidai et al. 2018). De- (HSBU) (Table 1). spite the phenotypic differences in our plant collection Morphological characters (Table 2) used are according and the previous studies observed in different localities, to Ijbari et al. (2014). Morphological data were stand- we hypothesized that the identities are most likely to be ardized (Mean = 0, Variance = 1) and used to estimate the same. Due to co-occurrence of two or three Tama- Euclidean distance. Grouping of the species was done by rix species in overlapping areas, it is very important to UPGMA (Unweighted paired group using average meth- identify and delimit these species. Moreover, due to fre- od) clustering and principal coordinate analysis (PCoA) quent gene exchange between species in the same area (Podani 2000). These analyses were done using PAST ver. there is high probability of hybrid formation. Therefore, 2.17 (Hammer et al. 2012). it is necessary to highlight gene flow among the species growing within each locality (Ijbari et al. 2014,