Two Morphologically Distinct Groups of the Calypogeia Fissa Complex Were Found in Europe

Biodiv. Res. Conserv. 23: 29-41, 2011 BRC www.brc.amu.edu.pl DOI 10.2478/v10119-011-0014-x Two morphologically distinct groups of the Calypogeia fissa complex were found in Europe Katarzyna Buczkowska1*, Jakub Sawicki2, Monika SzczeciÒska2, Stanis≥aw RosadziÒski3, Mariola Rabska1 & Alina Bπczkiewicz1 1Department of Genetics, Adam Mickiewicz University, Umultowska 89, 61-614 PoznaÒ, Poland, e-mail: *[email protected] 2Department of Botany and Nature Protection, Uniwersity of Warmia and Mazury in Olsztyn, Plac £Ûdzki 1, 10-728 Olsztyn, Poland 3Department of Plant Ecology and Environmental Protection, Adam Mickiewicz University, Umultowska 89, 61-614 PoznaÒ, Poland Abstract: Two genetically distinct groups (PS and PB) detected previously within the C. fissa complex in Europe were studied with respect to 47 morphometric characters. The two examined groups differed statistically significantly with respect to 34 morphological traits. The forward stepwise method of discriminant analysis showed that the set of diagnostic characters could be limited to nine. The best diagnostic features were morphological characters describing the shape of leaf: length and width of leaf, height of dorsal part and distance from the apex to the ventral base of the leaf, length of the 3rd coordinate of the leaf, and underleaf width as well as characters of the stem: length of internodes and size of internode cells. Plants of the PS group were smaller (shoot width range from 922-1780 µm) than plants of the PB group (1600-3900 µm). Based on genetically identified samples, classification functions for each group were computed and the derived functions were used for the classification of samples from the herbarium collections. The principal component analysis and dendrogram constructed on the basis of Euclidean distance, using the set of diagnostic characters, divided the examined samples into two groups that corre- lated with groups detected by isozyme markers. Results of multivariable analysis showed that it is possible to satisfactorily characterise morphologically both genetically distinct groups of the C. fissa complex. Key words: Bryophyta, liverworts, Calypogeia, morphology, biometry, classification functions 1. Introduction logical similarity, they cannot be described as taxonomi- cally distinct species, e.g., Pellia epiphylla (ZieliÒski A limited number of good diagnostic qualitative 1987), Conocephalum conicum (Szweykowski & Krzako- traits resulting from reduced, as compared to angio- wa 1979), Aneura pinguis (Bπczkiewicz & Buczkowska sperms, morphology and high phenotypic plasticity of 2005; Bπczkiewicz et al. 2008), Reboulia (Boisselier- the gametophyte is a serious problem in bryophyte tax- Dubayle et al. 1998). A formal taxonomic treatment of onomy. Application of different molecular methods in these species is difficult. Morphological differences, if bryophyte taxonomy revealed that many liverwort spe- they exist, are slight, and concern, mostly, quantitative cies described on the basis of morphological traits, in traits which have continuous phenotypic range. It is fact, are only species complexes composed of several practically impossible to say whether the observed dif- cryptic or semicryptic species (Bischler & Boisselier- ferences have a genetic basis or not (Szweykowski Dubayle 2000; Shaw 2001; Szweykowski et al. 2005; 1984). One way to resolve this problem is to precisely Wachowiak et al. 2007; Bπczkiewicz et al. 2008; characterise morphologically plants that have been Heinrichs et al. 2010; Kreier et al. 2010; Sawicki et al. determined genetically. Morphological and anatomical 2010). Thus, it is expected that some liverwort species studies based on genetically identified plants led to can still be discovered, even in Europe. description of one of cryptic species detected in Cono- Genetic differences between cryptic species are of cephalum conicum complex as a new C. salebrosum the same rank as between morphologically well- species (Szweykowski et al. 2005). Similarly, biometric characterised species, although, due to great morpho- studies based on genetically identified material allowed VARIABILITY, AND VARIABILITY, TAXONOMY PHYLOGENY © Adam Mickiewicz University in PoznaÒ (Poland), Department of Plant Taxonomy. All rights reserved. 30 Katarzyna Buczkowska et al. Two morphologically distinct groups of the Calypogeia fissa complex were found in Europe determination of morphological diagnostic characters 2. Material and methods for a new taxon of the Calypogeia genus (Buczkowska 2010) previously detected by isozyme and molecular 2.1. Plant material markers (Buczkowska & Bπczkiewicz 2011; Bucz- Plants used in biometrical studies originated from kowska & Dabert 2011). different regions of Europe. The studied plants were Calypogeia fissa (L.) Raddi is regarded as a sub- initially determined on the basis of morphological traits oceanic-mediterrean, amphiatlantic species (M¸ller according to M¸ller (1951-1958) and Damsholt (2002). 1951-1958; Damsholt 2002). In Europe, it occurs west In the living plants, just after collection, the oil body of Ireland, Great Britain, Spain, Portugal and France characters were studied. In general, 53 samples of C. fissa across the Balkans to Central Russia in the east as well complex were examined (Appendix 1). The samples as in southern parts of the Nordic countries and Ice- were divided into two parts. Part one contained 40 land. The species is also known from Asia (Turkey and samples identified on the basis of isozyme markers and East Himalayas) and North Africa (Marocco, Tunesia), oil body characters as C. fissa group PS and PB according Mediterranean islands and Macaronesia (Paton 1999; to Buczkowska (2004a). Part two comprised 11 samples Damsholt 2002). C. fissa in Poland occurs mainly in from herbarium collections which were determined the western regions of the country (Ko≥a 1989; Szwey- based on morphological criteria only. Additionally, kowski 2006). This species seems to be morphologi- 2 samples determined in the previous study as group G cally well characterised and typically developed plants (Buczkowska 2004a) were included in the biometrical are readily distinguished from other Calypogeia spe- analysis. Five stems from each sample were measured cies by their characteristic bidentate leaves and by with respect to 47 quantitative traits according to the underleaves armed with teeth on the lateral margin method described by Buczkowska (2004b). (M¸ller 1951-1958). However, some less typical forms can be occasionally found, which create problems for 2.2. Statistical analysis taxonomists (Schuster 1969; Damsholt 2002; Paton Statistical analyses were performed in two steps: the 1999). Currently within C. fissa, two subspecies are analyses in the first step were based on a matrix of 40 sam- formally recognized: C. fissa subsp. fissa occurring in ples genetically identified as group PS and PB (Appen- Europe (Grolle & Long 2000) and C. fissa subsp. neogea dix 1). Descriptive statistics: means, ±95% confidence Schust. known only from North America (Schuster intervals for the mean, standard deviations, minimums, 1969). In Europe, one form ñ C. fissa f. subxeropholia maximums and coefficients of variation were computed Schiffn. was distinguished by Bischler (1957) and two for each group. The significance of the difference be- varieties (var. fissa, var. intermedia) and two forms (f. tween means of the examined groups was tested by one- fissa, f. subintegrifolia) by Damsholt (2002). Moreover, way analysis of variance (ANOVA). The forward several other varieties and forms were described by stepwise method of discriminant analysis and Wilksí Warnstorf (1917); however, none of them was accepted. lambda value was used in order to design the best diag- C. fissa is a very variable species with regard to morpho- nostic features that facilitate discriminating between the logical traits like size, shape of underleaf lateral margin examined groups. The Wilksí Lambda value fit the and leaf apex as well as ecological preferences (Schuster range from 0 (perfect discrimination) to 1 (lack of discri- 1969; Paton 1999; Damsholt 2002). mination). The classification functions for the studied The isoenzyme studies have shown that the species groups were also computed and the derived functions is genetically differentiated and composed of geneti- were used to classify the samples from herbarium cally distinct groups which have been tentatively called collections which were not genetically determined. as PS, PB and G (Buczkowska 2004a). The groups PS, The functions can be used for classifying new cases PB were found in Poland and comprised, respectively, into different groups with a better than chance accu- small and bigger plants, the group G was noted in Ger- racy. A new case can be classified to the group for many. The PS group was genetically most distinct, which it has the highest classification score (Krzyúko whereas genetic distance between groups PB and G were 1990). lower. These groups detected by genetic methods also In the second step, all samples were included in differed in oil body characters and habitat requirements analyses. Principal component analysis and cluster (Buczkowska 2004a). analysis (agglomeration and k-means clustering The main purpose of the present study was: (i) to methods) were performed to examine differences check whether any stable morphological differences between the investigated groups. Normality of the data existed between genetically detected groups; (ii) to was checked by the Shapiro-Wilk

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