RESEARCH ARTICLE Using multiple microscopic techniques for the comparative systematic of Spergula fallax and Spergula arvensis (Caryophyllaceae) Fazal Ullah 1,2,3 | Wajid Zaman 2,3,4 | Alessio Papini 5 | Muhammad Zafar 3 | Syed Nasar Shah 3 | Mushtaq Ahmad 3 | Saddam Saqib 2,3,4 | Saba Gul 3 | Aamir Sohail 6 1 CAS Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China 2 University of Chinese Academy of Sciences, Beijing 100049, China 3 Department of Plant sciences, Quaid-i-Azam University, Islamabad, Pakistan 4 State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China 5 Department of Biology, University of Florence, Italy 6 Depratment of Botany, University of Peshawar, Peshawar, Pakistan Correspondence Wajid Zaman, State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. Email: wajid@ibcas.ac.cn and Fazal Ullah, CAS Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China. Email: fazalbotanist@gmail.com Review Editor: Peter Saggau Abstract In this study, comparative morphology, foliar anatomy and palynology of Spergula fallax and Spergula arvensis (Caryophyllaceae) were studied using multiple microscopic techniques. Genus Spergula includes worldwide five species, while in Flora of Pakistan the genus has two species. In this research, the comparative morphological, anatomical, and palynological characters of the two Pakistani Spergula species were studied. We examined some distinguishing morphological features, in both species, such as plant size, habitat, leaf morphological characters, inflores- cences, flowers outer whorls, sepals and petals, and flowers number. These characters species were studied analyzing their comparative systematic significant. The foliar anatomical features also provided distinctive characters as the epidermal cell shape, the wall of the epidermal cell, lobes per cell. The differences in quantitative characters were also examined. The palynological characters showed difference in echini arrangement, echini density, and numbers of pore. Quan- titative characters were variations in size of polar, equatorial, exine thickness, pore length, and width and P/E ratio. The multiple microscopic techniques provided sufficient evidence about the systematics of the genus Spergula. Based on morphological, anatomical, and palynological characters, analytical keys were developed for the identification and distinction of the species S. fallax and S. arvensis. KEYWORDS foliar anatomy, microscopy techniques, morphology, palynology, Spergula 1 | INTRODUCTION The genus Spergula L., together with the closely related genus Spergu- laria (Pers.) J.Presl & C.Presl, belongs to family Caryophyllaceae and used to be considered included in subfamily Paronychioideae (Kool & Thulin, 2017). More recently, a relationship also with members of Car- yophylloideae and Alsinoideae was observed (Kool & Thulin, 2017; Turner, 2017). Spergula is a small genus of five species distributed in centered and temperate regions. In Flora of Pakistan, the genus Sper- gula L. is represented by two species (http://www.eflora.org/). The species of the genus are distributed in Western Europe, North America, North Africa, China, India, Pakistan, Japan, Northern and central regions of European, part of Union of Soviet Socialist Repub- lics (USSR), Western and Eastern Siberia, Caucasus, and Far East (New, 1958; Singh, Malik, Balyan, & Singh, 1995). Systematic relationship of Spergula L. species was mainly dis- cussed by morphological data and, more recently, on the basis of DNA sequences (Kool & Thulin, 2017). Very little work has been done on the anatomy and palynology of the genus yet. Morphological, anatom- ical and palynological characters are significant in the identification and species delimitation of Caryophyllaceae (Ataslar, 2004; Perveen & Qaiser, 2006; Ullah et al., 2018; Ullah et al., 2018). The anatomical Received: 23 September 2018 Revised: 18 October 2018 Accepted: 30 October 2018 DOI: 10.1002/jemt.23176 Microsc Res Tech. 2018;1–9. wileyonlinelibrary.com/journal/jemt © 2018 Wiley Periodicals, Inc. 1