Vol. 79, No. 2: 117-123, 2010 ACTA SOCIETATIS BOTANICORUM POLONIAE 117 VARIATION OF SEED MORPHOLOGY OF TROLLIUS EUROPAEUS L. AND TROLLIUS ALTISSIMUS CRANTZ (RANUNCULACEAE) WOJCIECH ANTKOWIAK1, IRMINA MACIEJEWSKA-RUTKOWSKA2, ANDRZEJ M. JAGODZIÑSKI3, 4, DARIUSZ KAYZER5, MA£GORZATA KLIMKO1 1 Department of Botany, University of Life Sciences in Poznañ Wojska Polskiego 71c, 60-625 Poznañ, Poland 2 University of Life Sciences in Poznañ Wojska Polskiego 71d, 60-625 Poznañ, Poland e-mail: [email protected] 3 Department of Forest Protection, University of Life Sciences in Poznañ Wojska Polskiego 71c, 60-625 Poznañ, Poland 4 Institute of Dendrology, Polish Academy of Sciences Parkowa 5, 62-035 Kórnik, Poland 5 Department of Agricultural Economics and Management, University of Life Science in Poznañ Wojska Polskiego 28, 60-637 Poznañ, Poland (Received: June 7, 2009. Accepted: November 18, 2009) ABSTRACT The aim of this study was to investigate seed morphology and intra- and inter-population variation of seeds of Trollius europaeus L. and Trollius altissimus Crantz., two controversial species regarding their taxonomical posi- tion. We analyzed seed-coat microsculpture and some biometrical traits (length and width, width/length ratio, vo- lume and projected perimeter). Seed sculpture did not differ between species, but seeds of T. altissimus were usu- ally larger than seeds of T. europaeus. Although species differed significantly in seed morphology, it was possible to show the populations of both species that were similar regarding the analyzed seed traits. We noted a signifi- cant inter-population differentiation of seeds in both species with respect to seed-coat microornamentation and biometrical traits. We conclude that T. europaeus and T. altissimus are probably not two distinct species, but T. europaeus should be divided into two lower taxa in the rank of variety or subspecies. KEY WORDS: morphology, seeds, Trollius europaeus, T. altissimus, variation. INTRODUCTION extensive areas of northern and central lowland areas of Europe, having large and generally continuously distribu- Trollius L. (Ranunculaceae) (globeflower) consists of ted populations. Towards the south of the continent, its po- arctic-alpine perennial herbs, native to the cool, temperate pulations appear to be more scattered, growing only in the regions of the northern hemisphere. The greatest diversity mountains. As with other Trollius species, it is also charac- of Trollius species has been noted in Asia, especially wi- terized by substantial morphological variation, especially thin southern China (Després et al. 2003). They usually in its foliage and generative organs, even within a given grow in moist meadows and in heavy and clay soils. Thirty population (Doroszewska 1974). Similarly, high genetic globeflower species have been described, but the systema- variability of T. europaeus has been also documented (De- tic position of some is still controversial due to the highly sprés et al. 2002). As Doroszewska (1974) pointed out,all variable intrageneric morphological characters of Trollius. these factors have resulted in challenges in its intraspecific Additionally, some species are not strictly differentiated, classification; numerous taxa have been described as their ranges at least partly overlap and there exist interme- forms, varieties, subspecies or even separate species. We diate forms between them (Doroszewska 1974). believe that only two varieties of this species should be re- Among three European species of this genus, T. europa- cognized, T. europaeus var. europaeus (grows within the eus L. has the widest distributional range. It occupies whole range of species in Europe) and T. europaeus var. 118 SEED VARIATION OF TROLLIUS Antkowiak W. et al. TABLE 1. The origin of analyzed populations of Trollius species and the number of seeds, of which the morphology was investigated. No Species Origin in Poland Longitiude Altitude No of population and latitude (m a.s.l) of measured seeds 1 T. europaeus Kujawsko-Pomorskie Province Sitowiec 53°22N 141 337 17°46E 2 T. europaeus Wielkopolska Province Ferdynandowo 53°15N 118 112 17°22E 3 T. europaeus Wielkopolska Province Liszkowo 53°14N 119 153 17°19E 4 T. europaeus Wielkopolska Province Popówek 53°17N 112 158 17°18E 5 T. europaeus Wielkopolska Province £ob¿enica 53°16N 113 94 17°16E 6 T. europaeus Wielkopolska Province Mnichy I 52°33N 72 168 15°58E 7 T. europaeus Wielkopolska Province Mnichy II 52°33N 72 132 15°58E 8 T. europaeus Wielkopolska Province Mnichy M³yn 52°32N 71 804 15°59E 9 T. europaeus Wielkopolska Province Uciskowo 52°37N 75 153 16°45E 10 T. europaeus Wielkopolska Province Dziewicza Góra 52°28N 76 163 17°01E 11 T. europaeus Wielkopolska Province Trzcieliñskie Bagno I 52°18N 71 149 16°42E 12 T. europaeus Wielkopolska Province Trzcieliñskie Bagno II 52°18N 71 145 16°42E 13 T. altissimus Ma³opolska Province Tatra Mts Bobrowiec 49°15N 2246 144 19°47E 14 T. altissimus Podkarpackie Province Bieszczady Mts Po³onina Wetliñska 49°09N 1255 108 22°33E 15 T. altissimus Podkarpackie Province Bieszczady Mts Po³onina Bukowska 49°03N 1253 185 22°46E 16 T. altissimus Dolnol¹skie Province Bystrzyckie Mts Zieleniec 50°21N 760 464 16°25E 17 T. altissimus Dolnol¹skie Province Stolowe Mts Rogowa Kopa 50°28N 770 384 16°20E transsilvanicus (Schur) B³. (occurs in Central Europe). Tu- rent geographical habitats, particularly in mountain ranges. tin (1993) and Pankhurst (2006) identified two subspecies Trollius europaeus s.s. appears only in lowlands and in the of European globeflower: T. europaeus subsp. europaeus, lower mountain levels whereas T. altissimus is a plant of which corresponds to Doroszewskas var. europaeus, and the mountain regions, above forest zone. Morphological di- T. europaeus subsp. transsilvanicus (Schur) Domin, which stinction between both species is based on the shape and si- is basically equivalent of Doroszewskas var. transsilvani- ze of the stigma and the size of follicle beaks. cus and naturally occurs from the Southern Alps to the The seeds of the Trollius genus, as with most of Ranun- Carpathians. Chrtek and Chrtkova (1979) recognized the culaceae, develop from anatropous, bitegmic and crassinu- above-mentioned subspecies as a separate species, T. euro- cellate ovules (Corner 1976; Brückner 1994; Endress and paeus and Trollius altissimus Crantz. This view is shared Igersheim 1999). The inner structure is also typical of Ra- by other botanists in Central and Eastern Europe (Mosya- nunculaceae. While in dormancy, the seeds consist of kin and Fedoronchuk 1999; Mirek et al. 2002; Piêko-Mir- small, rudimentary embryos, surrounded by a living endo- kowa and Mirek 2003; Zicha 2009). Both taxa have diffe- sperm containing lipids and protein bodies. The testa is de- Vol. 79, No. 2: 117-123, 2010 ACTA SOCIETATIS BOTANICORUM POLONIAE 119 eply pigmented, multi-layered and incomplete at the micro- ed morphology variation both at the species and population pylar end of the seed (Hepher and Roberts 1985a, b). The level. The following seed traits were measured: length seeds are small, not more than 2 mm long, but relatively (mm), width (mm), width/length ratio, volume (mm3) and heavy, without any apparent adaptations for vector disper- projected perimeter (mm). The arithmetical mean, standard sal (Hitchmough 2003). A portion of the seeds are general- deviation and coefficient of variation for each mentioned ly eaten by larvae of Chiastocheta Pokorny species, of trait were calculated at the population level. In addition, which the imago pollinate Trollius flowers (e.g. Yu and Ri- multivariate analysis of variance in one-way classification dley 2003; Després et al. 2007 and others). was used (Morrison 1976). Our null hypothesis was that The details of seed morphology of species within the there were no differences among the mean values of the Trollius genus have not been documented. In recent years, traits of investigated populations with regard to the mean however, seed traits, especially seed-coat micromorpholo- values of biometrical traits. It was rejected at the á=0.05 le- gy, have proven to be a useful taxonomic criterion within vel of significance. many different taxonomic groups at species level, inclu- Mean values of traits of particular populations were com- ding the species of Ranunculaceae (e.g. Cappelletti and Po- pared with the overall mean values of all populations. 2 2 ldini 1984; Feng-Xia 2003). Using T0 ,i and T0 ,j statistics, populations and traits were The goal of this study was to investigate seed morpholo- determined to be responsible for rejection of general hypo- gy of T. europaeus and T. altissimus, especially seed-coat thesis (Lejeune and Caliñski 2000). Our analysis allowed microsculpture, as well as intra- and inter-population varia- us to position the Trollius populations in canonical coordi- tion of both species. Additionally, we wanted to investigate nates. the utility of seed morphology in differentiating the two Multivariate differences among the means of the studied species. seed traits were investigated to estimate similarities of the populations within species. The Mahalanobis distance was assumed as a measure of those differences; it was tested METHODS using the Laweley-Hotelling statistic. In addition, the si- gnificance of contrast between T. europaeus and T. altissi- We collected samples of ripe seeds of T. europaeus of 12 mus was investigated (Lejeune and Caliñski 2000). populations and T. altissimus of five populations from na- tural localities in Poland in the years 1999-2001 (Table 1). Every gathered sample consisted of seeds originated from RESULTS at least 30 specimens. The samples were kept separate at room temperature and were analyzed at the same time. The seeds of T. europaeus and T. altissimus are superfi- They were not dissected, but only cleaned. The using of cially very similar. Seeds of both species are elliptical or a scanning electron microscope (LEO 435 VP, Leo Elec- ovate. The transversal sections of seeds of each species are tron Microscopy Ltd., Cambridge, U.K.) enabled us to in- triangular.