Diversity of plant communities with Carex flava agg. in Poland and their relationship with soil properties

The Carex flava aggregate belongs to one of the most taxonomically difficult groups of sedges which colonize diverse habitats, from organic to sandy, from acidic to alkaline, usually humid and moist. The study included 129 vegetation plots and ten soil variables (organic matter, phosphorus, potassium, magnesium, calcium, carbonates, carbon, nitrogen, pH, and the ratio between organic carbon and nitrogen). The main aim was to determine the relationships between the various plant communities C. flava agg. occur in and their soil properties. With the aid of the two-way indicator species analysis and cluster analysis, we delimited nine vegetation types from the Scheuchzerio palustris-Caricetea fuscae, Littorelletea uniflorae, Molinio-Arrhenatheretea and Alnetea glutinosae classes differing in their response to soil properties. The CCA revealed pH, N, K, C, CaCO3, P and Ca to be statistically significant, and to account for 11.55% of the total variance in species composition. The largest differences, both in terms of species composition and in soil conditions, were revealed between communities with C. lepidocarpa and C. demissa. Carex lepidocarpa occurred in calcareous and extremely rich fens (Caricion davallianae) whereas C. demissa was found to occur in poor and moderately rich fens (Sphagno-Caricion canescentis, Caricion canescenti-nigrae). Carex flava grew mostly in calcareous, rich fens and wet grasslands (Caricion davallianae, Calthion palustris). Carex viridula was found in both calcareous, extremely and moderately rich fens and wet grasslands, and in nutrient-poor habitats such as dunes and sandy lake shores. The ecological niche of C. viridula is very wide and this species showed no affinity to any specific syntaxon.


Introduction
The Carex flava complex belongs to one of the most taxonomically difficult groups because of a lack of clear morphological discontinuities (e.g. Havlíčková 1982;Crins and Ball 1989a,b;Pykälä and Toivonen 1994;Jiménez-Mejías et al. 2012) and the presence of numerous hybrids (Schmid 1982;Więcław and Koopman 2013;Więcław and Wilhelm 2014). The Carex flava complex in Europe is considered consisting of six species (Więcław 2017). These taxa are usually small in size and have short rhizomes; they usually have a solitary subcylindrical male spike on top of the inflorescence, above globose to oblong female spikes with more Hájek 1999;Hájek et al. 2005) and Carici flavae-Cratoneuretum (Hájek et al. 2005). On the other hand, because of the morphological similarity of the sedges and difficulties in their identification, some phytosociological publications treat them as a broadly-defined aggregate; for example, Jarolímek and Šibík (2008) mentioned Carex flava agg. as diagnostic for the class Scheuchzerio palustris-Caricetea fuscae and the alliance Caricion davallianae (see also Hájek 2002;Peterka et al. 2017). Moreover, most phytosociological and ecological studies were focused on C. flava and C. lepidocarpa, and less so on C. demissa and C. viridula (e.g. Hájek 1999Hájek , 2002. In addition, closely related species which are poorly morphologically differentiated can be easily overlooked or the differences observed may be regarded as a manifestation of habitat-dependent variation. Therefore, this study was aimed at identifying types of plant assemblages capable of supporting species of the C. flava complex and at determining relationships between soil properties and species composition and diversity of vegetation with C. flava agg.
The results presented are part of a comprehensive study on the taxa of the C. flava complex in Poland, with a particular reference to inter-and intra-specific differentiation, taxonomy and hybridisation within the group (Więcław and Podlasiński 2013;Więcław and Koopman 2013;Więcław and Wilhelm 2014;Więcław 2014a,b, Więcław 2017.

Field sampling
Field data were collected in 2012-2014 in Poland from various habitats, for example from upland and lowland, coastal and inland, from calcareous, extremely and moderate-rich fens and wet grasslands to poor habitats such as dunes and sandy lake shores ( Fig. 1; Online Resource 1). A total of 129 plots were sampled in patches with the presence of species from C. flava agg., specifically 16 with C. demissa subsp. demissa, 57 with C. flava s.str., 19 with C. lepidocarpa subsp. lepidocarpa and 37 with C. viridula var. viridula. The sedges were identified using the keys of Pykälä and Toivonen (1994) and Więcław (2014a).

Soil analysis
Soil samples were dried at room temperature, and then rubbed through a sieve to remove fractions larger than 1 mm. The following properties were determined in the thus-prepared material: organic matter content (as loss on ignition at 550°C), pH (potentiometrically in 1 M KCl), contents of assimilable nutrients (phosphorus P, potassium K, magnesium Mg and calcium Ca, using the American Society of Agronomy method; Sparks et al. 1996), carbonates (Scheibler's method; Tatzber et al. 2007) and total carbon C and nitrogen N (CHNS analyser, Costech Analytical Technologies Inc.; Sparks et al. 1996). The latter assay provides data for calculating the C/N ratio. The ratio between organic carbon and nitrogen (C/N) is one of the indicators of soil fertility. The lower the ratio, the more fertile the soil.

Data processing
Two multivariate statistical techniques were used to analyse the vegetation data: the two-way indicator species analysis (TWINSPAN package, v. 2.3;Hill and Šmilauer 2005) and the cluster analysis (MVSP package, v. 3.1;Kovach 1985Kovach -1999. Each of these approaches provides a somewhat different view on the structure of the data, and when employed together they can be used to better understand the diversification of the vegetation. Clusters resulting from divisions (TWINSPAN) with eigenvalues greater than 0.3 are treated as the final clusters which, however, do not necessarily constitute communities. The cluster classification (MVSP) was performed using the minimum variance, based on the squared Euclidean distance.
The statistical significance of differences between empirical distributions of the data analysed and the theoretical normal distribution was examined using the Shapiro-Wilk test. Since distributions of most data sets deviated from normality, the non-parametric U Mann-Whitney test, Kruskal-Wallis test and Dunn's multiple comparisons test were used to examine whether differences between sites of C. flava agg. species and between relevés (Shannon diversity index, the evenness and number of species) were significant. Calculations were performed using the software package Statistica v. 13.1 for Windows (StatSoft, Inc. 2013). The Shannon diversity index and the evenness were calculated for each relevé using the MVSP package (Kovach 1985(Kovach -1999. Relationships between vegetation plots and environmental factors were determined using the software package CANOCO v. 4.51 (ter Braak and Šmilauer 2002). Vegetation distribution patterns in relation to environmental variables were explored using canonical correspondence analysis (CCA), after detrended correspondence analysis (DCA) detected a unimodal structure of the species data (the gradient length represented by the first ordination axis was greater than 3 SD). The Monte Carlo permutation test was further applied to test for statistical significance of environmental variables.

Nomenclature
The nomenclature of vascular plants, Carex taxa and mosses follows World Flora Online (WFO 2020), Koopman (2015) and Ochyra et al. (2003), respectively. The phytosociological units are in compliance with the syntaxonomic nomenclature of the vegetation of Europe (Mucina et al. 2016). Assignment to phytosociological units below the class level follows Matuszkiewicz (2018) and Dierßen (2001). The fen terminology is in accordance with Hajek et al. (2006). Specimens collected in the field have been deposited in the Herbarium Stetinensis (SZUB).

Results
Floristic composition and diversity of plant communities with taxa representing Carex flava agg The TWINSPAN resulted in eight final clusters (I-VIII; see Online Resource 2 and 3). The first division of the dataset separated plots with C. lepidocarpa and C. viridula from C. flava and C. demissa. The division was based on indicators: C. viridula with a coverage of 3 or more, and C. lepidocarpa with a coverage of 2 or more. All the relevés with C. lepidocarpa coverage of more than 2 were assigned to a single vegetation unit (cluster I) whereas the relevés with C. viridula coverage of 3 and more were divided into three groups (Online Resource 2). The division of the three groups was based on indicators: Bryum pseudotriquetrum with a coverage of 2 and more (cluster III) and Pohlia nutans with a coverage of 1 and more (cluster IV). Cluster II contained relevés dominated by species of the classes Scheuchzerio palustris-Caricetea fuscae (e.g. Carex nigra, C. panicea, Campylium stellatum) and Molinio-Arrhenatheretea (e.g. Molinia caerulea, Lythrum salicaria, Deschampsia cespitosa). All the relevés with C. demissa were assigned to a single vegetation unit (cluster V), with a C. demissa coverage of 3 and more (Online Resource 3), as an indicator. The relevés with C. flava were divided into three clusters. Clusters VI and VIII were identified by the presence of Valeriana dioica subsp. simplicifolia and Alnus glutinosa, respectively. Cluster VI covers relevés taken exclusively in the mountainous area in south-eastern Poland whereas cluster VII groups relevés from both lowlands throughout the country and mountains and uplands, mainly in the South-West of Poland. These relevés are distinct by the absence of Valeriana dioica subsp. simplicifolia, while V. dioica s.str. is frequent (Online Resource 3).
The MVSP resulted in the division of the entire dataset into two main groups; the first one contained relevés with C. lepidocarpa and C. viridula, while relevés in the other group contained relevés with C. flava and C. demissa (Fig.2a). Further analyses of the main groups showed that some relevés with C. viridula were included in the group with C. lepidocarpa, while some of the relevés with C. flava were assigned to the group with C. demissa (Fig. 2b, c). The MVSP demonstrated a separate nature of relevés with C. viridula from dunes (cluster IV versus clusters III and II), shores of water bodies and the vicinity of a chalk mine (cluster IIIa versus cluster IIIb), as well as relevés with C. demissa (cluster Va versus Vb) and those with C. flava (cluster VI versus cluster VII), see Fig. 2b,c; Online Resource 2 and 3).
Relevés with C. lepidocarpa (cluster I) were similar in terms of their floristic composition and proportions of species (Online Resource 2). Such a species composition is characteristic of the association Caricetum paniceo-lepidocarpae. It is dominated by diagnostic species such as C. lepidocarpa and C. panicea; there are also calciphilous vascular plants (e.g. Carex flacca, Epipactis palustris and Parnassia palustris) and mosses (e.g. Campylium stellatum and Fissidens adianthoides, see Online Resource 2). The number of species per relevé averaged 22; Shannon's diversity index and evenness averaged 4.22 and 0.96, respectively (Table 1).
Patches featuring C. viridula observed in depressions between dunes (cluster IV) were characterized by the presence of species representing the classes Oxycocco-Sphagnetea (e.g. Drosera rotundifolia, Vaccinium oxycoccus, Aulacomnium palustre) and Nardetea strictae (Calluna vulgaris, Pohlia nutans, Empetrum nigrum), and the order Scheuchzerietalia palustris (Drosera anglica, Lycopodiella inundata, Sphagnum teres). Relevés with C. viridula from wet grasslands and fens (cluster II) showed a presence of species from the class Molinio-Arrhenatheretea (e.g. Lysimachia vulgaris, Ranunculus acris, Lotus pedunculatus) and the order Caricetalia fuscae (e.g. Hydrocotyle vulgaris, Carex nigra, Veronica scutellata, see Online Resource 2). The fen and wet grassland relevés differed significantly from those of dunes in terms of the Shannon diversity index and the species richness (P = 0.0193 and P = 0.0091, respectively) as well as from those from lake shores (P = 0.0048 and P = 0.0040, respectively). The species richness in fen and wet grassland averaged 21 per relevé (Shannon index of 4.12) and 11 (Shannon index of 3.29) and 14 species (Shannon index of 3.56) in dune and lakeshore relevés, respectively.
The relevés with C. demissa were divided into two groups (Va and Vb; see Fig. 2c and Online Resource 3). Those included into cluster Va were distinct by the presence of species representing the classes Nardetea strictae (e.g. Nardus stricta, Potentilla erecta) and Fig. 2 Results of hierarchical cumulative classification using Euclidean distance-based minimum variance techniques. a -Results for the entire dataset; b -Analysis for plots of C. lepidocarpa (cluster I) and C. viridula (IIsamples from fens and wet grasslands, IIIasandy lakeshores, IIIbsites near a chalk mine, and IVdunes); c -Analysis for C. demissa (Vasamples from poor acidic fen and Vbmoderate-rich fens and fen grasslands) and C. flava (VIsamples with C. flava from fens and wet grasslands with Valeriana dioica subsp. simplicifolia from Bieszczady Mts, VIIfens and wet grasslands without Valeriana dioica subsp. simplicifolia, and VIIIfrom Alnus forest).
Oxycocco-Sphagnetea (Sphagnum fallax, Polytrichum strictum), as well as by a lower contribution of wet grassland species, compared to relevés in cluster Vb (Online Resource 3). The two groups identified by the MVSP were significantly different in their numbers of species (23 and 31 in clusters Va and Vb, respectively; Z = −2.2229; P = 0.0262), the Shannon diversity index (4.35 and 4.80 for clusters Va and Vb, respectively; Z = −2.1170; P = 0.0343) and evenness (0.97 and 0.98 for clusters Va and Vb, respectively; Z = −2.4346; P = 0.0149).
The C. flava relevés proved most species-rich, the Shannon index and the evenness being at their highest as well (Table 1). Relevés acquired in the Bieszczady Mountains (cluster VI) were significantly different from the remaining relevés (clusters VII and VIII) in terms of their Shannon index, evenness, and the number of species (5.07 vs 4.77 and 4.75, P = 0.0042; 0.98 vs 0.97 and 0.97, P = 0.0208; 38 vs 30 and 30, P = 0.0042, respectively). The Bieszczady Mts support a floristically rich variant of the Valeriano simplicifoliae-Caricetum flavae which featured, fairly frequently, Valeriana dioica subsp. simplicifolia and Eriophorum latifolium, while in other regions the assemblages with C. flava featured fairly abundant Valeriana dioica s.str. All the relevés with C. flava showed an abundance of Juncus articulatus and small sedges such as C. panicea and C. nigra (Online Resource 3). Moreover, species primarily characteristic of wet grassland associations (e.g. Anthoxanthum odoratum, Briza media, Cirsium palustre, Climacium dendroides, Festuca rubra, Geum rivale, Myosotis scorpioides and Ranunculus acris) were also frequent. Some relevés from cluster VII were dominated by wetland species, for example Blysmus compressus (relevés 15 and 21), Carex hartmanii (40 and 41) and Equisetum palustre (12, 14 and 43, see Online Resource 3.

Distribution of plots and species along environmental gradients
The CCA results indicate that all the variables analysed accounted for 14.36% of the total variance in the species data ( Table 2). The first axis and all the canonical axes were significant as tested by the unrestricted Monte Carlo permutation test (first axis: P = 0.026; all axes: Table 1 Diversity of species occurrence in phytocoenoses with Carex flava agg.; results of Kruskal-Wallis test and post hoc Dunn's multiple comparisons test, showing significance of differences in Shannon's index, evenness and number of species. F -C. flava s.str.; D -C. demissa; L -C. lepidocarpa; V -C. viridula; ×mean; Pstatistical significance. The Significance level of P ≤ 0.05 is denoted in bold.

Species
Kruskal-Wallis test Dunn's multiple comparisons test Percentage of explained species data variance 14.36 P = 0.006). The results of forward selection in the CCA revealed seven variables (pH, N, K, C, CaCO 3 , P and Ca) to be statistically significant and to account for 11.55% of the total variance in species composition (Table 3).
In the ordination space (CCA), all the C. lepidocarpa relevés occupy the right-hand part of the diagram and are associated with relatively high pH values and soil Ca, Mg, CaCO 3 , C and N, as well as with a relatively high C/N ratio ( Fig. 3; Table 4). The C. lepidocarpa habitat differed significantly from habitats of C. demissa, C. flava and C. viridula by being relatively rich in carbonates, calcium and magnesium in the soil, as well as showing a high soil pH ( Table 5).
Relevés of C. viridula are scattered in the ordination space; however, most samples collected from dunes (cluster IV) and lake shores (cluster IIIa) are positioned in the left-hand side of the diagram and are associated with relatively low values of most soil parameters studied ( Fig. 3; see also Table 5). The relevés taken from fens and wet grasslands (cluster II) are concentrated mainly in the central part of the diagram whereas samples collected from sites near the chalk mine (cluster IIIb) are placed in the right-hand part of the diagram together with samples of C. lepidocarpa. Statistically significant differences, primarily concerning pH and soil contents of organic matter, C, N, Mg, Ca and CaCO 3 were detected between relevés from dunes (IV) and fens and wet grasslands (II), as well as between dunes (IV) and sites near the chalk mine (IIIb), see Fig. 4a-h.
All the relevés with C. demissa occupy the left-hand part of the CCA diagram and are associated with relatively low values of most soil parameters studied (Fig. 3). The upper part of the diagram contains the relevés from poor acidic fens (cluster Va), while the relevés from moderate-rich fens (cluster Vb) are grouped in the central part. Significant differences between them concerned pH and the soil Ca content (Fig.  4d,g). Table 3 Forward selection results with the test of variable significance for vegetation samples collected at sites with the presence of species from the C. flava complex. Ccarbon concentration; N nitrogen concentration; C/Nratio between organic carbon and nitrogen concentration; org. mat.organic matter content; pHsoil pH; Cacalcium concentration; CaCO 3carbonates concentration; Mgmagnesium concentration; Pphosphorus concentration; Kpotassium concentration; * significance level P ≤ 0.05.
Plots of C. flava are scattered on the CCA diagram; however, relevés from the Bieszczady Mts (cluster VI) are situated in the upper part whereas the remaining relevés from fens and wet grasslands (cluster VII), as well as all the forest relevés (cluster VIII), are grouped in the lower part (Fig. 3). Statistically significant differences between the Bieszczady relevés and the other relevés were revealed for soil organic matter content, as well as for concentrations of carbon, nitrogen, magnesium and potassium (Fig. 4a-c,f,e).

Discussion
Vegetation types with taxa representing Carex flava agg We delimited nine vegetation types with taxa included in the Carex flava group that differed in their species composition and response to soil properties.
Carex lepidocarpa is a characteristic species of the assemblage Caricetum paniceo-lepidocarpae from the alliance Caricion davallianae (Kwiatkowski 1999). In Poland C. lepidocarpa grows only in calcareous sites (calcareous, extremely rich and rich fens). Those habitats are severely threatened due mainly to general and wide-scale disturbances in hydrological conditions (Wołejko et al. 2005;Šefferová Stanová et al. 2008;Grootjans et al. 2015). Moreover, C. lepidocarpa is sensitive to changes in hydrology and becomes increasingly rare. It is regarded as threatened in some Central European countries (Pykälä 1994).
Carex flava s.str. is found in calcareous, rich and moderate-rich fens and wet grasslands as well as in alder thickets and woods. In Poland, it grows on soils with a wide pH range, from (4.3-)5.0 to 7.6. Carex flava is one of the diagnostic species of the Valeriano simplicifoliae-Caricetum flavae, found in mountainous regions, throughout the flysch zone of the Slovak, Polish and Ukrainian Carpathians, and occasionally in Czechia (Pawłowski et al. 1960;Hájek 1999;Hájková 2002, 2011;. In Poland, Koczur and Nicia (2013) reported a substantial variability in the assemblage, mainly produced by the wide range of the habitat parameters, notably the soil pH, varying from (5.8-)6 to (7.6-)8 (see also Mazurek and Nicia 2006); our study, however, showed the Valeriano simplicifoliae-Caricetum flavae to be a rather homogenous fen community (see also Hájek 1999), despite the habitat varying   from acidic to slightly alkaline (from 5.0 to 7.6). The assemblage showed a fairly constant floristic composition, but a high number of species from the class Molinio-Arrhenatheretea points to its potential to become transformed into a grasslands community. In Czechia, the Valeriano simplicifoliae-Caricetum flavae is vicariant to the Valeriano dioicae-Caricetum davallianae in the mountainous regions of the Western Carpathians (Hájek and Hájková 2011). In Poland we found patches featuring Carex flava and Valeriana dioica s.str. as well as other species diagnostic for the Valeriano dioicae-Caricetum davallianae, for example Carex panicea, Potentilla erecta, Succisa pratensis, Calliergonella cuspidata (Hájek and Hájková 2011); however, C. davalliana was not found at any of our sites and the proportion of wet grasslands species was considerable. In Poland, V. dioica subsp. simplicifolia and V. dioica s.str. are vicariants; the former occurs mainly in the East and South-East whereas the latter is known from the central and western part of the country (Zając and Zając 2001). According to Zarzycki et al. (2002), the taxa are similar in their habitat requirements, but are different in terms of their continentalism and, to a lesser extent, in acidity preference: V. dioica subsp. simplicifolia is subcontinental and usually grows on alkaline soils (more seldom on soil with neutral pH), whereas V. dioica s.str. is a sub-Atlantic species and grows on neutral pH soils. Our study confirmed the presence of the Valeriano simplicifoliae-Caricetum flavae in the Bieszczady Mountains, while the remaining patches with C. flava (outside of the Bieszczady) are heterogenous and represent various associations, usually the wet grasslands ones from the class Molinio-Arrhenatheretea. Carex demissa usually occurs in poor and moderaterich fens as well as along forest roads and paths. In Poland, it was usually reported from acidic sites (pH 3.4-5.5) with usually a very low calcium concentration and with a carbonate-poor soil. It was only at a single D -C. demissa; L -C. lepidocarpa; V -C. viridula; the significance level of P ≤ 0.05 is marked with bold. For abbreviations of soil properties, see Table 3.
In Poland, C. viridula is regarded as one of the species characteristic of the Cyperetum flavescentis from the class Isoëto-Nanojuncetea, which appears spontaneously on emergent bottom and shores of water reservoirs (Popiela 1997;Matuszkiewicz 2018). However, our study found C. viridula to grow, along lake shores, in Ranunculo-Juncetum bulbosi communities from the class Littorelletea uniflorae (see also Szańkowski and Kłosowski 2006;Hrivnák et al. 2011;Šumberová et al. 2011). All the sites with C. viridula located along lake shores were supported by the bryophyte Bryum pseudotriquetrum, which is common in wet and moderately infertile sites (Ellenberg et al. 1991). According to Dierßen (2001) this species can also occur on slightly acidic habitats such as fens and lake shores.
Communities featuring C. viridula and occurring in humid dune depressions along the Polish Baltic Sea coast are similar to marsh assemblages of the class Scheuchzerio palustris-Caricetea fuscae. Theurillat et al. (2015) distinguished, within the class Scheuchzerio palustris-Caricetea fuscae, the alliance Caricion viridulo-trinervis, which covers low-sedge vegetation of wet, phosphorus-limited dune slacks of Atlantic coasts of Western Europe (see also Peterka et al. 2017). On the other hand, Sival and Grootjans (1996) frequently recorded C. viridula on dune slacks of the island of Schiermonnikoog in the northern part of the Netherlands in a pioneer assemblage with Littorella uniflora, Samolus valerandi, Radiola linoides and Juncus bulbosus. In Poland, the relevés made in between-dune depressions were distinct in showing the presence of the bryophyte Pohlia nutans, common and often abundant on wet or dry, acidic, peaty, sandy or gravelly soil on heaths and moors, in sand dunes, acidic grassland, bogs, and woods. It avoids base-rich or calcareous sites (Ellenberg et al. 1991).
Carex viridula is fairly frequently found also in fens and wet grasslands across a wide spectrum of soil conditions (e.g. Więcław and Podlasiński 2013), in communities representing both the class Scheuchzerio palustris-Caricetea fuscae and the Molinio-Arrhenatheretea. According to Hájek et al. (2008), C. viridula reached a high cover in disturbed moderately rich fens in Bulgaria and occurred with some calciphilous species, for example Eriophorum latifolium and Parnassia palustris. In Poland, C. viridula can also grow at sites with a high soil calcium carbonate level (see also Więcław and Podlasiński 2013) and is capable of colonizing heavily disturbed sites (Więcław 2017). Thus, the ecological niche of C. viridula is very wide, compared to that of C. flava, C. lepidocarpa and C. demissa. Carex viridula grows in various plant communities without any tight syntaxonomic affinity.

Ecological differentiation within closely related species
In Poland, the Carex flava complex includes four closely related sedge species (Więcław 2014a), which, as shown in this study, occupy diverse habitats and have diverse ecological requirements. In general, many closely related taxa may be distinctly different in their ecological preferences (e.g. Dakskobler et al. 1999;Hölzel 2003;Koutecká et al. 2011;Budzhak et al. 2016). A clear differentiation, both in the community composition, soil properties, and habitat types was revealed within the Bolboschoenus maritimus group (Hroudová et al. 1999). According to Hroudová et al. (1999), the closely related taxa of the Bolboschoenus maritimus group show a much stronger ecological than morphological differentiation (no distinct diagnostic characters). Our investigation has shown that C. lepidocarpa and C. demissa are the most ecologically isolated species of the C. flava complex. The former was found on calcareous and extremely rich fens (Caricion davallianae) whereas the latter was reported from poor and moderately rich fens (Sphagno-Caricion canescentis and Caricion canescenti-nigrae). However, earlier morphological studies on the C. flava group in Poland showed C. lepidocarpa and C. demissa to be fairly distinctly separated morphologically, mainly in the dimensions of the generative characters (Więcław 2014a). Similarly, taxa within the Festuca varia group were well separated both morphologically and ecologically, syntaxonomic studies on the group showing the presence of five vicariating regional associations and one edaphically determined association, growing on ecologically different sites (Wallossek 1999). Other studies, addressing species of the Molinia caerulea complex in Central Europe (Molinia caerulea s.str. and M. arundinacea), showed differences in their morphology and chromosome number (Dančák et al. 2012) as well as in habitat preferences (Budzhak et al. 2016). Comparative studies on three related species of the genus Viola, combined with their phytosociology and environmental parameters showed the species to differ in terms of their association with the soil moisture gradient and chemistry (Hölzel 2003). Koutecká et al. (2011) reported results of phytosociological research which clearly separated three Myosotis species from the Myosotis palustris group into different types of habitats and plant communities. They found only two locations with the simultaneous presence of two Myosotis species (M. caespitosa and M. nemorosa). These examples show closely related species to differ fairly distinctly in terms of micro-species morphology and to be clearly separated by ecological preferences. This pattern is, however, not so pronounced within the C. flava complex.
Our study showed the C. flava taxa to be capable of sharing a habitat, except for C. lepidocarpa and C. demissa, distinctly separated ecologically in Poland.
Carex lepidocarpa and C. flava s.str. Can co-occur on calcareous and rich fens (Caricion davallianae). They are clearly different morphologically (e.g. Schmid 1984;Crins and Ball 1989a,b;Pykälä and Toivonen 1994;Więcław 2014a), but mixed populations frequently support hybrids, whereby an unequivocal species identification is challenging (Więcław and Wilhelm 2014). The ecological amplitude of Carex flava s.str. is wider than that of C. lepidocarapa; the former species can also grow on wet grasslands and moderate-rich fens, where frequently co-occurs with C. demissa and their hybrid (C. ×alsatica). Carex flava s.str. and C. demissa are rather well separated morphologically, but the problem of intermediate hybrids present in their mixed populations appears here as well (Więcław and Wilhelm 2014).
Carex viridula is encountered both in calcareous, extremely and moderately rich fens, wet grasslands, and in very poor habitats such as dunes and sandy lake shores. Carex viridula frequently co-occurs with both C. lepidocarpa and C. flava, and much more seldom with C. demissa (Więcław 2014a,b) and hybridizes with all three mentioned species (Więcław and Wilhelm 2014;Schmidt et al. 2018). Carex viridula is morphologically different from both C. flava and C. lepidocarpa, mainly in its much smaller utricles and shorter beaks, but is morphologically very close to C. demissa (e.g. Pykälä and Toivonen 1994;Hedrén 2003;Więcław 2014a). Noteworthy is the fact that Carex viridula is the only species of the C. flava complex which grows on sandy lake shores and in dune depressions.
To sum up, detailed knowledge on closely related species, combining results of taxonomic and environmental studies, allows us to fairly precisely determine the ecological amplitude of narrowly-defined species. The combination of an ecological and taxonomic approach may lead to a better understanding of the evolutionary ecology of plants.

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
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