Biodiv. Res. Conserv. 50: 39-52, 2018 BRC www.brc.amu.edu.pl DOI 10.2478/biorc-2018-0008 Submitted 14.05.2018, Accepted 29.06.2018 Taxonomic importance of seed morphology of Veronica L. subsect. Agrestes Benth. (Plantaginaceae) Małgorzata Mazur1*, Magdalena Gawlak2, Elżbieta Sandurska3, Tomasz Kałuski2 & Katarzyna Marcysiak1 1Botanical Garden, Kazimierz Wielki University, Ossolińskich 12, 85-093 Bydgoszcz, Poland 2Research Centre of Quarantine, Invasive and Genetically Modified Organism, Institute of Plant Protection – National Research Institute, Władysława Węgorka 20, 60-318 Poznań, Poland 3Department of Genetics, Kazimierz Wielki University, Powstańców Wielkopolskich 10, 85-090 Bydgoszcz, Poland * correspondence author (e-mail: [email protected]) Abstract. The study was conducted on five species ofVeronica L. subsection Agrestes occurring in Central Europe: Veronica agrestis L., V. polita Fr., V. opaca Fr., V. persica Poir. and V. filiformisSm. These species are very similar morphologically and are often misidentified. Last thorough studies of morphology and micromorphology of their seeds were conducted in 1960s. Based on 48 herbarium specimens, we examined SEM images of 422 seeds. We found that 22 of the 30 studied morphological traits differentiated five species at statistical level and V. agrestis and V. persica differed with the greatest number of features from other species. Our measurements of seed size were not fully congruent with previous studies, suggesting dependence of these features on external conditions. Seeds were usually cochlidiospermous and ovoid with a reticulate-verrucate seed coat, but these features were, at the same time, quite variable. The results of discriminant function confirmed that some characters of the seed size, shape and coat sculpture could be taxonomically useful in distinguishing species of Veronica subsect. Agrestes. In addition, we believe that similarity of the examined seeds may confirm unity of this group and the need of identifying a subsection. Key words: Veronica taxonomy, SEM, micromorphology, seed coat, seed size, seed shape, seed sculpture, testa surface 1. Introduction Fruit and seed traits play a significant role in the taxonomy of many species (Stace 1989; Szkudlarz Veronica L. is the largest genus in the family Plan- 2009; Davitashvili & Karrer 2010; Szkudlarz & Celka taginaceae sensu Angiosperm Phylogeny Group (2009) 2016). The morphology and micromorphology of seeds and comprises 450-500 species, including about 62 are often utilised for taxonomic purposes also in Plan- occurring in Europe (Walters & Webb 1972). In spite taginaceae and related families, like Scrophulariaceae of a great deal of taxonomic research that has been or Orobanchaceae (e.g.: Elisens & Tomb 1983; Dong done on the genus worldwide, several groups still et al. 2015; Richardson Ahedor & Elisens 2015). require detailed investigation of additional characters Studies conducted on some of the species belonging to distinguish species and clarify their taxonomy. One to the subsect. Agrestes showed that seeds of this such group is Veronica subsection Agrestes Benth. subsection differed from the others of subg. Pocilla by The subsect. Agrestes of subgenus Pocilla (Dumort.) their smaller size – the seed length is less than 2 mm; M. M. Mart. Ort., Albach & M. A. Fisch. consists of by the specific cochlidiospermous (concave ventrally) three cosmopolitan species: V. polita Fries, V. persica type of seed, less frequently flattened (V. ceratocarpa); Poiret and V. filiformis Sm. and two representing the by occurrence of wrinkles on the dorsal surface of the European type of range: V. agrestis L., and V. opaca seed; by the shape and size of the raphal ridge and cha- Fries. Other species belonging to the subsect. Agrestes lazal plate and by reticulate-verrucate coat sculpturing are restricted to the hyrcanian-caucasian region (Kulpa with a polygonal cell and variable sizes, their anticlinal 1968; Albach et al. 2005). cell walls irregularly thickened forming, a more or less, AND PHYLOGENY TAXONOMY VARIABILITY, ©Adam Mickiewicz University in Poznań (Poland), Department of Plant Taxonomy. All rights reserved. 40 Małgorzata Mazur et al. Taxonomic importance of seed morphology of Veronica L. subsect. Agrestes Benth. ... convex reticulum and by periclinal cell walls with a part of the country (Żukowski 1959; Zając, Zając central or eccentric papilla (Kulpa 1968; Juan et al. 2001). V. filiformis is regarded as propagating mainly 1994; Muñoz-Centeno et al. 2006; Yilmaz 2013; Hassan asexually and rarely producing seeds, thus, the pres- & Khalik 2014). ence of capsules in some specimens in KRA herbarium The European species belonging to the subsect. should be treated as quite exceptional. This is why we Agrestes are considered as morphologically quite decided to use the small number of seeds of this species similar and, thus, they may be misidentified. Previous that were available in our investigation. analyses of the species within the subsect. Agrestes Observations and measurements of seeds were showed several morphological characteristics which carried out on images from an S-3000N Hitachi Co were useful in distinguishing some of them: the type SEM at the Institute of Plant Protection in Poznań. Dry (procumbent or decumbent) and thickness of the stem, seeds were attached to table mounts using double sided the ratio of the length to the width of the capsule, the adhesive carbon discs. The samples were then sputter type of coverage by hairs and the ratio of the incision coated with gold and photographed using SEM. Seeds in a capsule to the length of its style, the number of of Veronica have a ventro-dorsal construction, with one seeds in a capsule, the shape and overlapping of calyx- axis of symmetry. Therefore, observations were made segments (Tacik & Trzcińska-Tacik 1963; Walters on three sides: the ventral (2-6 seeds per individual), & Webb 1972; Fischer 1981). Fruit morphology and dorsal (1-4 seeds per individual) and lateral (1-3 seeds anatomy (Juan et al. 1997; Saeidi Mehrvarz et al. 2001), per individual). In total, 184 seeds were photographed as well as chemical characteristics (e.g.: Albach et al. on ventral, 125 on dorsal and 113 on lateral surfaces. 2005; Jensen et al. 2005; Saeidi Mehrvarz et al. 2008), We used magnification from 60x to 150x, depending on pollen size (Sánchez Agudo et al. 2009), chromosome the seed size. The coat sculpture was investigated using number (Saeidi Mehrvarz & Kharabian 2005), distri- two magnifications: 500x and 1000x. bution of glycosides (Tomas-Barberan et al. 1988) and In order to statistically characterize seeds we selected molecular data (e.g.: Albach & Greilhuber 2004; Albach 29 quantitative traits, some following already published et al. 2004) also provided important characters within research (e.g.: Tacik & Trzcińska-Tacik 1963; Kulpa the subsection. 1968; Walters & Webb 1972). Thirteen characters were However, none of these researchers examined and measured on the photographs using digiShape software compared all the European species of the subsect. (Moraczewski 2005): the perimeter (P), the length (L), Agrestes, using SEM. Last thorough morphological and the width measured at ½, ¼ and ¾ of the length (W½, micromorphological examinations of seeds of all Euro- W¼, W¾), the thickness at ½, ¼ and ¾ (T½, T¼, T¾), pean species of the subsect. Agrestes were conducted in angles of micropylar and chalazal top (AMi, ACh), the 1960s by Kulpa (1968). This is why we decided to study length of chalaza (LCh) and the length and the width this group using methods that were not accessible at that of chalaza plate (LPl, WPl). Two features were counted time. We conducted our study on examples of Polish directly from the images: the number of wrinkles on the flora assuming they would make good representatives dorsal side (WrN) and the average number of angles in of European plants, due to central European location of the centrally located 5 cells of the seed coat (ACN). In Poland. addition, fourteen traits were obtained from the calcula- Our first aim was to characterize the seed morpho- tions: P/L, P/W½, L/W½, L/T½ L/W¼, L/W¾, L/T¼, L/ logy and seed coat sculpture of the species of subsect. T¾, W¼/W¾, T¼/T¾, LCh/L, LCh/LPl, LPl/WPl and Agrestes. Basing on the results of this examination, we L/WrN. wanted to determine if seed traits can help in identifying To describe the seeds, we also used other features. the subsection as the unity, or if they differ the species The degree of folding of the edge (FE) at the ventral side enough to have a taxonomic value. of seeds was assessed with a three-level scale: 1 – not folded, 2 – medium folded, 3 – strongly folded. Follow- 2. Material and methods ing the nomenclature of other authors (e.g.: Yamazaki 1957; Kulpa 1968), we found all seeds in subsection The research was based on the study of herbarium Agrestes to be cochlidiospermous. We considered specimens. Seeds of the following Veronica species seeds to be deep-cochlidiospermous, when L/T½ < 2.5, were examined: V. persica (130 seeds), V. agrestis whereas L/T½ ≥ 2.5 were shallow-cochlidiospermous. (92), V. polita (90), and V. filiformis (14) (Appendix We also estimated the seed shape in outline from ven- 1). The latter species, as an invasive kenophyte, has tral and dorsal surfaces (e.g.: Martinez-Ortega & Rico been spreading through Europe since 1930s (Müller & 2001; Stearn 2004). We defined three main types of Sukopp 1993; Pielech et al. 2012; Scalone & Albach shapes: obovoid, elliptical, suborbicular and two tran- 2012; Tokarska-Guzik et al. 2012). It is rare in Poland sitional types: broadly obovoid and broadly elliptical. and, at the moment, can be found only in the southern We defined elliptical seed shape when 0.9 < W1/4/W3/4 Biodiv. Res. Conserv. 50: 39-52, 2018 41 < 1.1 and obovoid – when 0.9 > W1/4/W3/4 > 1.1 value. was illustrated by the UPGMA dendrogram constructed Seed shapes were considered as broadly obovoid and on the Euclidean distances.