Muhaidat et al. Bot Stud (2018) 59:18 https://doi.org/10.1186/s40529-018-0234-x ORIGINAL ARTICLE Open Access Integrating morphological characters, molecular markers, and distribution patterns to assess the identity of Blepharis species from Jordan Riyadh Muhaidat1*, Mohammad H. Brake2, Mazhar Al Zoubi3, Robert I. Colautti4, Amjad Al‑Nasser5, Muheeb Awawdeh6, Khalid Al‑Batayneh1, Wesam Al Khateeb1, Athena D. McKown7, Jamil Lahham1 and Ahmad El‑Oqlah1 Abstract Background: Blepharis constitutes an important part of the vegetation of the Jordanian arid and semi-arid regions, yet whether one or more species of this genus occurs in the Jordanian area is uncertain. We addressed this question by assessing morphological characters and testing Inter-Simple Sequence Repeat (ISSR) markers from three popula‑ tions of Blepharis: two northern (lower slopes of Kufranjah valley and the Dead Sea region) and one southern (Wadi al Yutm). Results: Shoots from randomly chosen Blepharis plants were harvested from each of the three populations for morphological and molecular analyses. In the northern populations, spikes were lax and bract width was signifcantly shorter than length of the longest lateral spine compared to the southern population. A multivariate linear discri‑ minant analysis distinguished the northern populations from the southern one by internode length, bract width, longest lateral spine length, and bract width to spine length ratio. The ISSR analysis revealed that 44 markers across eight primers were polymorphic with major allele frequency of 83.6% and an average of 5.5 polymorphic markers per primer. The genetic resemblance among individuals ranged from 0.27 to 0.96. The three Blepharis populations were accordingly clustered into two distinct groups, similar to the analysis of morphological diferences and corresponding with the “northern” and “southern” population designations. Conclusions: Our results strongly indicate the occurrence of two discrete Blepharis species in Jordan and reject the hypothesis that the genus is represented by only one species. We propose that the Blepharis species in Jordan are B. attenutata Napper (represented by the northern populations) and B. ciliaris (L.) B. L. Burtt (represented by the southern population). These fndings are important for informing and revising foristic work within the region and an updated key has been included in our fndings. Keywords: Blepharis attenuata, Blepharis ciliaris, Inforescence, Spike, Bract, Dimorphic stamens, Linear discriminant analysis, Inter-Simple Sequence Repeat *Correspondence: [email protected] 1 Department of Biological Sciences, Faculty of Science, Yarmouk University, P. O. Box 21163, Irbid, Jordan Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Muhaidat et al. Bot Stud (2018) 59:18 Page 2 of 15 Background it would exist across multiple types of environments, as is Blepharis Juss. is a genus in the family Acanthaceae s.l. the case for some plant species in the region. By compari- (subfamily Acanthoideae, tribe Acantheae) comprising son, multiple Blepharis species in Jordan may indicate 129 species that are broadly distributed in hot, arid and biogeographical preference or habitat specialization. semi-arid regions of the old world tropics and subtropics, Tere is a striking diversity of morphological charac- with the center of diversity in eastern and southern Africa ters in Blepharis, with life forms ranging from annual (Vollesen 2000, 2002; Sage 2004; Fisher et al. 2015). In the and perennial herbs to subshrubs and shrubs (Vollesen Middle East, including Jordan, Blepharis constitutes an 2000). Characters related to habits, inforescences, and important component of the desert vegetation. Te genus foral characteristics were regarded the most useful for is highly specialized to inhabit extreme desert environ- discerning Blepharis species (Vollesen 2000). Te genus ments that receive little rain in winter and experience is distinctive in having inforescences ranging from spikes high temperatures and light intensities during summer (or pseudo-spikate cymose) to a reduced cyme or a soli- (Gutterman 2002). In the Middle East region, two spe- tary terminal fower and leaves are arranged in a pseudo- cies of Blepharis were originally recorded and described whorled phyllotaxy. Bracts are leathery (coriaceous) with in regional foras, and both were regarded as distinct. spiny margins or thin and papery (glumaceous) with Tese included B. attenuata Napper and B. ciliaris (L.) either entire or bristly margins. Bracteoles, when present, B. L. Burtt (Boulos and Lahham 1977; Feinbrun-Dothan are linear to lanceolate with bony midribs, glumaceous 1978; Gutterman 2002). Yet, debate over the number and and generally have entire margins. Stamens are notably identify of Blepharis species occurring in this region has dimorphic and occur as an anterior and posterior pairs. arisen following the taxonomic revision of the genus by Te anterior pair bears laterally fattened flaments, is Vollesen (2000). Based largely on assessments of herbar- hairy toward the base, and appendaged distally, while ium materials, Vollesen (2000) argued that B. attenuata is the posterior pair is slightly narrower with linear fla- the sole member that migrated north and colonized the ments that are “knee-like” and unappendaged. Pistils of desert areas of Egypt, Israel and West bank, and Jordan. Blepharis fowers possess lobed stigma, styles that are fl- Vollesen (2000) also considered B. attenuata as the only iform, glabrous (or rarely hairy with tufty basal glandular species in the Acantheae that grows below the sea-level. trichomes), and a bilocular ovary bearing two ovules per In a survey of leaf anatomy and photosynthetic bio- locule. Fruits are ellipsoid capsules that are lignifed and chemistry in C4 species, Muhaidat et al. (2007) identifed explosively dehiscent, enclosing fat seeds coated with Blepharis samples from Jordan as B. ciliaris. Akhani et al. hygroscopic branched hairs borne on hook-like struc- (2008) brought attention to the possible misidentifcation tures called retinacula (Vollesen 2000; Gutterman 2002). of Blepharis from Jordan in the literature and foras of Feinbrun-Dothan (1978) outlined a number of diag- the Middle East. A subsequent study by Muhaidat et al. nostic and discriminatory morphological traits among (2012) identifed plants collected from the lower parts of Blepharis taxa and used these in a taxonomic key for the Jordan valley as B. attenuata through morphological distinguishing species. Te laxness of the inforescence, characters relating to the general growth habit and info- length and width of bracts, number of bract veins and rescence (Feinbrun-Dothan 1978; Vollesen 2000). None lateral spines, length of the longest lateral spine, and of these studies, however, were able to clearly identify in the ratio of spine length to bract width were noted as a comparative context whether one or more Blepharis important characters for identifying B. attenuata and B. species exist within the Jordanian fora. ciliaris. Variability in morphological features of bracts In Jordan, the distributional range of Blepharis is eco- and the degree of spikes compactness were particularly logically broad. Te genus occurs along desert, rocky important in distinguishing between B. attenuata and slopes and wadi beds (dry riverbeds) of west Irano- B. ciliaris and were highlighted in the taxonomic key Turannian, east Saharo-Arabian and Sudanian (or tropi- (Feinbrun-Dothan 1978). To the best of our knowledge, cal penetration) biogeographical regions (Fig. 1). Tese further assessments of these diagnostic morphological biogeographical zones of Jordan were circumscribed features have not been pursued. based on variations in altitude, temperature regimes, Te purpose of this study was to clearly identify the amount and patterns of rainfall, types and textures of species of Blepharis in Jordan and to test whether the soils, and the very diverse fora of the region (Long 1957; hypothesis that only one species of the genus occurs Feinbrun-Dothan 1978; Elesawi 1985; Palmer 2013). Te in the region is supported. We assessed morphological ecological and climatic diversity ofers a great opportu- features, including those previously regarded as dis- nity to explore and assess Blepharis species identities tinctive characters (Feinbrun-Dothan 1978; Vollesen in relation to geography as a test region for the Middle 2000), and used linear discriminant analysis (LDA) to East. If a single Blepharis species occurs in Jordan, then address diferentiation of three distinct populations Muhaidat et al. Bot Stud (2018) 59:18 Page 3 of 15 Fig. 1 Map of Jordan biogeography and locations of sampling sites for Blepharis. Sampling sites are indicated by red circles. K, Kufranjah valley; D, Dead Sea region; Y, Wadi al Yutm of Blepharis in this study. Te morphological analyses Khateeb et al. 2013; Khierallah et al. 2014; Brake et al. were complemented by Inter-Simple Sequence Repeat 2014; Ng and Tan 2015; Salazar-Laureles et al. 2015; (ISSR) analysis and non-metric multidimensional scal- Yuan et al. 2015;