MORPHOMETRIC ANALYSIS OF NECTARIES AND THEIR … · phological characteristics of nectaries in...

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Arch. Biol. Sci., Belgrade, 67(2), 511-524, 2015 DOI:10.2298/ABS140911014N

MORPHOMETRIC ANALYSIS OF NECTARIES AND THEIR POTENTIAL USE IN THE TAXONOMY OF THE JOVIBARBA HEUFFELII COMPLEX (CRASSULACEAE)

Danijela Nikolić1,*, Milica Spasić1, Jasmina Šinžar-Sekulić2, Vladimir Ranđelović1 and Dmitar Lakušić2

1 Department of Biology and Ecology, Faculty of Sciences and Mathematics, University of Niš, Višegradska 33, Niš, Serbia2 Institute of Botany and Botanical Garden Jevremovac, Faculty of Biology, University of Belgrade, Takovska 43, Belgrade, Serbia

*Corresponding author: [email protected]

Abstract: The aim of this study was to quantify the morphological variation of nectaries among 14 populations of Jovibarba heuffelii based on multivariate statistics, and to establish whether nectaries possess taxonomic signifi-cance for differentiating taxa within the J. heuffelii complex. To this end, we measured the width of the nectary, its height, the angle between the carpels and nectary, the shape of the nectary and the distance between nectaries were measured and analyzed. Descriptive statistics, the Tukey HSD (honest significant difference) of homogenous groups for the unequal N post-hoc test, canonical discriminant analysis (CDA) and multiple correspondence analysis (MCA) were used. Morphometric analysis showed that the quantitative and semiquantitative character-istics of nectaries in the J. heuffelii complex are highly morphologically variable, both within and between popula-tions, and that they are unreliable as taxonomic characters for taxon differentiation within the J. heuffelii complex.

Keywords: Jovibarba heuffelii; floral nectaries; morphology.

Received September 11, 2014; Revised October 27, 2014; Accepted November 3, 2014

INTRODUCTION

Floral nectaries are relatively simple structures of diverse origin, partaking in the pollination process. They may appear in almost all parts of a flower and produce sweet-tasting exudates with different quantitative and qualitative character-istics (Bernadello, 2007). Although the shape, structure and position of nectaries have been

used for taxonomic and phylogenetic studies for more than a century (Behrens, 1879; Bonnier, 1879; Knuth, 1906; Brown, 1938; Norris, 1941; Fahn, 1953; Brown, 1961), the value of nectaries in systematics was recognized only fairly recent-ly (Zandonella, 1977; Fahn, 1979; Vogel, 1981; Cronquist, 1981, 1988; Smets, 1986, 1988; Smets and Cressens, 1988; Smets et al., 2000, 2003). Fahn (1979) classified floral nectaries into 9 types

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according to their position within the flower. Representatives of the Crassulaceae (Sempervi-vum) family have nectaries of the 4th type, char-acterized as “nectary as a disc surrounding base of ovary”. Bernadello (2007) criticizes the use of the term “disc” as it has been used for various structures in the form of a ring within the flower, and they may or may not belong to the nectar-ies (e.g., Daumann, 1931; Fahn, 1979; Cronquist, 1981; Smets, 1986; Schmid, 1988; Smets and Cresens, 1988; Vogel, 1998). Schmid (1988) has based his classification of floral nectaries solely

on the position of the nectaries, explaining that more general classification systems are better un-derstood and more practical for use. According to his criteria, nectaries in the J. heuffelii complex belong to the type of floral receptacular nectaries. Bernadello (2007) supplements this classification with the terms intrastaminal, extrastaminal and interstaminal, depending on whether the nec-taries are situated on the receptacle between the staminal whorl and the ovary (intrastaminal), or between the perianth and the androecium, respec-tively (extrastaminal). The receptacular nectaries

Table 1. Essential characteristics of the localities from which populations of J. heuffelii were obtained. Vouchers were deposited in the Herbarium at the Institute of Botany, Faculty of Biology, University of Belgrade (BEOU).

Population N_lat E_long Substrate Altitude

[m] Voucher

1. SR-Pljačkovica 42 34 492 21 53 758

silicate 674 BEOU, 16465

2. SR-Gradac 44 13 251 19 51 937

limestone 490 BEOU, 16458

3. SR-Suvaja 44 09 726 19 53 196


4. SR-Studenica 43 29 220 20 32 390

serpentinite 486 BEOU, 16461

5. SR-Nebeske Stolice 43 15 645 21 50 031


6. SR-Treska 43 15 615 20 47 395


7. SR-Basarski kamik 43 09 337 22 42 398


8. SR-Radan 42 55 039 21 33 450


9. SR-Besna Kobila 42 31 437 22 14 105


10. SR-Stara planina 43 23 234 22 38 196


11. MA-Treskavec 41 24 147 21 32 144


12. MA-Mavrovo 41 38 143 20 42 299


13. RO-Domogled 44 52 417 22 25 541


14. BU-Trojanski prolaz 42 46 016 24 31 020


MORPHOMETRIC ANALySIS OF JOVIBARBA HEUFFELII NECTARIES 513

located exactly between the stamens may be re-ferred to as interstaminal. According to this crite-rion, the nectaries in J. heuffelii are intrastaminal receptacular nectaries.

Molecular analyses have shown that the Crassulaceae family is monophyletic (Morgan and Soltis, 1993; Soltis and Soltis, 1997), while the presence of nectary scales in this family is a synapomorphic characteristic together with suc-culent leaves and anysocitic stomata. Jovibarba heuffelii (Schott) A. Löve & D. Löve is a complex of insufficiently clarified taxa, distributed in the central part of the Balkan Peninsula and central and southern parts of the Carpathians (Meusel, 1965; Jalas et al., 1999).

Numerous taxa have been described with-in this complex (Diopogon stramineus Jord. & Fourr., Sempervivum reginae-amaliae Heed. &

Sart. ex Baker, S. transylvanicum Baker, S. vele-novskyi Cheshm., S. heuffelii Schott., var. glabrum Beck & Szyszyl., S. heuffelii var. albanicum Ki-tanov, S. heuffelii var. bulgaricum Cheshm. and S. heuffelii var. vestitum Domin). Their taxonomic positions are not completely elucidated, and they are presently treated as synonyms of the broadly defined species J. heuffelii. The characters most commonly used for differentiation of taxa within this complex include the presence of glandular hairs on the rosette leaves and stem, in inflores-cences, on the carpels, the ratio of calyx/corolla length, as well as the shape and position of nec-taries (Pančić, 1874; Domin, 1932; Kitanov, 1948; Cheshmedjiev, 1969; Vălev, 1970; Gajić, 1972; Müssel, 1977; Micevski, 1998).

Pančić (1874) used the shape and position of nectaries to differentiate the taxon Sempervivum kopaonikense from Mt. Kopaonik in Serbia from

Fig. 1. Distribution of the population of J. heuffelii in Serbia, Macedonia, Bulgaria and Romania. 1. SR-Pljačkovica; 2. SR-Gradac; 3. SR-Suvaja; 4. SR-Studenica; 5. SR-Nebeske Stolice; 6. SR-Treska; 7. SR-Basarski kamik; 8. SR-Radan; 9. SR-Stara planina; 10. SR-Besna Kobila; 11. MA-Treskavec; 12. MA-Mavrovo; 13. RO-Domogled; 14. BU-Trojanski prolaz. For details see Table 1.)

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Table 2. Results of descriptive statistics for the analyzed characters of nectaries.

Descriptive statisticsPopulation Acr. Valid N Mean Min Max Std. Dev. St. Er. CV %

W 21.00 0.56 0.41 0.76 0.09 0.02 15.39SR-Pljačkovica H 21.00 0.50 0.36 0.63 0.08 0.02 16.92 A 19.00 29.18 14.73 44.81 6.97 1.60 23.90

SR-GradacW 21.00 0.67 0.46 0.87 0.10 0.02 15.41H 21.00 0.52 0.27 0.64 0.10 0.02 19.27A 15.00 21.34 11.53 36.29 7.32 1.89 34.33

SR-SuvajaW 19.00 0.62 0.42 0.78 0.09 0.02 14.24H 19.00 0.42 0.27 0.56 0.07 0.02 16.88A 12.00 44.37 21.82 92.70 22.26 6.43 50.17

SR-StudenicaW 20.00 0.64 0.50 0.82 0.10 0.02 15.44H 20.00 0.35 0.23 0.43 0.05 0.01 15.02A 16.00 49.06 22.27 76.59 15.21 3.80 31.01

SR-Nebeske StoliceW 22.00 0.72 0.57 0.89 0.08 0.02 11.12H 22.00 0.48 0.31 0.85 0.11 0.02 22.88A 21.00 42.39 18.59 80.12 14.49 3.16 34.18

SR-TreskaW 20.00 0.85 0.66 1.04 0.10 0.02 11.81H 20.00 0.50 0.30 0.65 0.08 0.02 16.01A 20.00 42.34 24.79 59.96 9.23 2.06 21.79

SR-Basarski kamikW 25.00 0.83 0.62 1.16 0.14 0.03 17.18H 25.00 0.55 0.46 0.71 0.07 0.01 12.03A 25.00 38.80 17.97 60.55 10.68 2.14 27.52

SR-RadanW 20.00 0.73 0.60 0.85 0.08 0.02 11.59H 20.00 0.49 0.31 0.63 0.08 0.02 15.66A 13.00 30.86 22.05 53.43 9.08 2.52 29.42

SR-Besna KobilaW 20.00 0.72 0.55 0.90 0.11 0.02 14.61H 20.00 0.52 0.37 0.62 0.06 0.01 11.38A 20.00 32.89 21.29 50.85 7.16 1.60 21.77

SR-Stara planinaW 20.00 0.74 0.57 0.97 0.11 0.02 14.66H 20.00 0.47 0.40 0.64 0.06 0.01 13.34A 20.00 42.86 14.99 85.03 17.13 3.83 39.97

MA-TreskavecW 20.00 0.51 0.39 0.64 0.07 0.02 14.03H 20.00 0.48 0.34 0.64 0.08 0.02 16.46A 20.00 37.50 13.13 64.42 16.08 3.60 42.89

MA-MavrovoW 20.00 0.77 0.55 1.05 0.13 0.03 17.21H 20.00 0.50 0.36 0.64 0.08 0.02 15.74A 17.00 36.38 18.07 86.90 16.54 4.01 45.46

RO-DomogledW 20.00 0.76 0.54 0.96 0.11 0.03 14.83H 20.00 0.43 0.23 0.56 0.08 0.02 19.55A 20.00 34.92 26.83 51.17 6.20 1.39 17.77

BU-Trojanski prolazW 20.00 0.86 0.66 1.10 0.11 0.02 12.44H 20.00 0.53 0.36 0.65 0.08 0.02 15.46A 20.00 40.30 20.74 70.51 13.34 2.98 33.09


the taxon Sempervivum patens from the south-ern Carpathians in Romania. For S. kopaonik-ense, he claimed that its nectaries are “at right angles, rounded or tridented at the top”, while in S. patens they are “directed toward the top, cut off straight or shallowly indented”. Preager (1932) also believed that nectaries are a significant trait of the population from Kopaonik, which he treat-ed as a form of the species J. heuffelii. The men-tioned author stated that S. kopaonikense differs from the typical forms by “hypogynous scales spreading instead of erect with a corresponding bulge at the base of the calyx and corolla, giving the flower a ventricose appearance.” The mor-phological characteristics of nectaries in repre-sentatives of the genus Jovibarba Opiz have never been studied before.

The aim of this study was to quantify the morphological variation of nectaries between populations of J. heuffelii based on multivari-ate statistics, and to find out whether nectar-ies may have taxonomic significance in the differentiation of taxa within the J. heuffelii complex.

MATERIALS AND METHODS

Plant material

289 individuals from 14 populations of Jovibar-ba heuffelii s.l. were collected from their natural habitats for morphological analysis. The samples were taken from Serbia, Macedonia, Bulgaria and Romania (Table 1., Fig. 1). The plants in the flow-ering stage were collected in July and August dur-ing two vegetation seasons (2010, 2011). Voucher specimens were deposited in the Herbarium at the Institute of Botany and Botanical Garden “Jevremovac”, at the Faculty of Biology, Univer-

sity of Belgrade - BEOU (Thiers, 2014). The in-florescences were stored in glycerol:96% ethanol (50:50 v/v) until measurements were taken.

Morphometric analysis

For morphometric analysis, 4 nectaries from 4 different flowers from the inflorescence were separated per individual (1156 nectaries in total). The nectaries were photographed using a LEICA DM 1000 microscope. For measuring, the angle of the nectary with a carpela was isolated per in-dividual and photographed on a LEICA MZ16 A stereomicroscope. All the characters were mea-sured using the LeicaQWin image analyzing pro-gram (Leica image software).

The characters used in analysis: width of nec-tary (W), height of nectary (H), angle between carpel and nectary (A), shape of the nectary: 1 - flat surface of nectary (Fsurf), 2 - convex sur-face of nectary (Csurf), 3 - two teeth of nectary (TwoT), 4 - three teeth of nectary (ThreeT), distance between nectaries: 1 - space between nectaries less than width of the nectary (Sless), 2 - space between nectaries greater or equal to width of a nectary (Sgrate).

Statistical analysis

Descriptive statistics (mean, maximum, mini-mum, variance, standard error and standard de-viations, coefficient of variation) for the quanti-tative characters were done. The Tukey HSD of homogenous groups for the unequal N post-hoc test was used to analyze the differences in the nectary morphology of 14 populations. Canoni-cal discriminant analysis (CDA) was used to test the hypothesis for the separation of the analyzed populations. CDA analyses were performed on individuals for 4 characters (width of the nectary, height of nectary, the angle between carpel and

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nectary, shape of the nectary), and two different data sets. The first data set included only popula-tions of S. kopaonikensis and S. patens from lo-calities Kopaonikand Domogled, while the sec-ond included all analyzed populations. Canonical scores for each case were calculated in order to estimate the distances between individuals that were used to visualize the relationship among the 14 populations, which were defined as a priori groups. The qualitative characters of all the in-vestigated populations were statistically analyzed by multiple correspondence analysis (MCA) and presented on a scatterplot diagram. All statistical analyses were performed using the Statistica 5.1 (Statsoft, 1996) package.

RESULTS

Detailed morphological analysis has shown that the nectaries in individuals from J. heu-ffelii populations are whitish, somewhat lighter in color than the carpels, and situated on the receptaculum in the base of the apocarpous gynoecium. Their upper surface varies from flat to convex to indented, with two recogniz-able teeth (Figs. 2A-C). The two teeth may be symmetrical or one may be larger than the oth-er. In some individuals, the teeth are slightly rounded and close to each other, so the upper side of the nectary is cordate (Fig. 2C), while in other individuals the teeth converge at the top, forming a beak shape (Fig. 2D). Certain individuals have nectaries with widely spaced, slightly rounded teeth (Fig. 2F). Some individu-als within the populations have three more or less pronounced teeth on the upper side of the nectaries (Fig. 2E). These populations include SR-Treska with only one individual with such nectaries, SR-Basarski kamik with 2 individu-als, MA-Treskavec with 4 individuals, MA-Ma-

vrovo and RO-Domogled with one individual each, and BU-Trojanski prolaz with 3 individu-als bearing 3-teeth on the nectaries. The dis-tance between nectaries also varies from one population to another. It may be greater than the width of the individual nectary (Fig. 3A) or smaller than the width of the nectary (Fig. 3B). There are records of the upper part of the nec-tary curving toward the inner side, toward the carpel (Fig. 3C). The results have shown that most individuals have two teeth on the upper part of the nectary, but in certain populations, there are also individuals with level surfaces on the nectaries or with three teeth.

Descriptive statistics

The width of the nectary varies from 0.39 to 1.16 mm. The maximum value of this character was registered in the SR-Basarski kamik population. The smallest width of the nectary was noticed in the MA-Treskavec population. This charac-ter showed a moderate coefficient of variability ranging from 11.12 to 17.18% (Table 2). The height of the nectary varies from 0.23 to 0.85 mm. The minimum value of this character was reg-istered in the RO-Domogled and SR-Studenica populations, while the maximum value was no-ticed in the SR-Nebeske Stolice population. The coefficient of variability of this character was in a range of 11.38% to 22.87%. The angle between the carpel and nectary is the most variable char-acter, ranging from 11.53° to 92.70°.

The maximum angle was registered in the SR-Suvaja population and the minimum value in the SR-Gradac population. The coefficient of variability for this character was in a range of 17.77% to 50.17%. Very high variability of this character was shown by the SR-Suvaja popula-tion (50.17%).


Fig. 2. Light micrographs of the shape of nectaries: (A) flat surface of nectary (sample from MA-Mavrovo), (B) convex surface of nectary (sample from MA-Basarski kamik), (C) cordate shape (sample from SR-Besna Kobila), (D) beak shape (sample from MA-Mavrovo), (E) three teeth on surface (sample from SR-Suvaja), (F) widely spaced, slightly rounded teeth (sample from SR-Treskavec), x50.

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Tukey HSD post-hoc test

This test showed statistically significant variability in the mean values of characters (p<0.05) between certain pairs of populations (Table 3). Within the first homologous group, the MA-Treskavec, SR-Pljačkovica and SR-Suvaja populations stand out, as they do not show statistically significant differ-ences in the mean values of the character width of nectary (W). Each of these three populations shows statistically significant differences to the other analyzed populations. For the character height of nectary (H), the unequal N HSD test showed separation of the SR-Studenica, SR-Suvaja and RO-Domogled populations as the most simi-lar, while these populations show statistically sig-nificant differences to other analyzed populations within the first homologous groups. The angle between the carpel and nectary (A) is a character showing statistical similarities for the SR-Gradac, SR-Pljačkovica, SR-Radan, SR-Besna Kobila, RO-Domogled and MA-Mavrovo populations. Each of these populations shows statistically significant dif-ferences to the other analyzed populations. The un-equal N HSD test for the character shape of nectary showed the least differences among populations within the J. heuffelii complex for all the analyzed characters. There are no statistically significant differences in variations of this character in most populations, except those from Macedonia (MA-Treskavec, MA-Mavrovo) and Bulgaria (BU-Tro-janski prolaz), which show statistically significant differences from the other analyzed populations in the mean values of this character (Table 3).

Canonical discriminant analysis (CDA)

Analysis of populations from Kopaonik (type lo-cality for taxon S. kopaonikense) and Domogled (type locality for taxon S. patens), taken as a priori groups, showed a complete lack of differentiation between these two groups of populations accord-

ing to the quantitative characters of nectaries (Fig. 4B, Table 4). The CDA including all analyzed pop-ulations also resulted in no statistically significant differentiation of populations (Fig. 4A, Table 4).

Multiple correspondence analysis (MCA)

This analysis showed that the first axis carried 16.17% of the total variability in the sample, while the second axis carried 9.52% (Fig. 5). Based on the character distance between nectaries, the SR-Suvaja, SR-Nebeske Stolice, SR-Gradac and SR-Basarski kamik populations were arranged into one group in the positive part of first correspon-dent axis (Fig. 5). The nectaries of these popu-lations are spaced at a distance greater than the width of an individual nectary (Fig. 3A). The ad-ditional characters responsible for grouping these populations were convex surface of the nectary (Csurf: 1) and flat surface of the nectary (Fsurf: 1) (the surface of nectaries in individuals from these populations is either flat or convex).

All the other populations were grouped around the characters two teeth of nectary (TwoT: 1) and space between nectaries less than the width of the nectary (Sless: 1) on the negative part of the first MCA axis, indicating that these characters had the greatest impact on population grouping. Indi-viduals from these populations are characterized by the presence of two teeth on the surface of the nectaries and the distance between the nectaries is smaller than the width of a single nectary (Fig. 3B).

DISCUSSION

The more recent literature such as Parnell and Far-vager (1993), Jalas et al. (1999) and Hart et al. (2003), does not support the separation of several taxa within the J. heuffelii complex. On the other hand,


Table 3. Results of Unequal N HSD test for the variables: width, height, angle and shape of nectaries. Different letters (a, b, c, d) indicate statistically significant differences of variable means at p<0.05.

Unequal N HSD

Population Width Height Angle Shape1 SR-Pljačkovica 0.57 a b 0.51 b c d 29.18 a b 2.37 a b

2 SR-Gradac 0.66 b c 0.52 b c d 21.34 a 2.60 a b c

3 SR-Suvaja 0.64 a b c 0.43 a b c 44.37 b c 2.50 a b c

4 SR-Studenica 0.65 b c 0.35 a 49.06 c 2.88 a b c

5 SR-Nebeske Stolice 0.71 c 0.49 b c d 42.39 b c 2.76 a b c

6 SR-Treska 0.85 d e 0.50 b c d 42.84 b c 2.89 a b c

7 SR-Basarski kamik 0.84 d e 0.55 d 39.22 b c 2.21 a

8 SR-Radan 0.75 c d e 0.53 b c d 30.86 a b 3.00 a b c

9 SR-Besna Kobila 0.72 c 0.52 c d 32.89 a b 2.60 a b c

10 SR-Stara planina 0.74 c d 0.47 b c d 42.86 b c 2.70 a b c

11 MA-Treskavec 0.51 a 0.48 b c d 37.50 b c 3.05 b c

12 MA- Mavrovo 0.77 c d e 0.50 b c d 36.38 a b c 3.00 b c

13 RO-Domogled 0.76 c d e 0.43 a b 34.92 a b c 2.90 a b c

14 BU-Trojanski prolaz 0.86 e 0.53 c d 40.29 b c 3.16 c

Table 4. Factor loadings of the variables and eigenvalues of the first two axes of canonical discriminant analysis (CDA) for A – all analyzed populations and B – Sempervivum kopaonikensis vs. S. patens.

Canonical discriminant analysis (CDA)

Width Height Angle Shape Eigenvalue

ACD1 -0.99 -0.25 -0.14 -0.03 0.98

CD2 -0.08 -0.84 0.53 0.08 0.45

BCD1 0.86 0.36 0.29 0.10 0.38CD2 0.46 -0.49 -0.68 0.34 0.18

Marhold (2011) recognizes two infraspecies taxa: J. heuffelii subsp. heuffelii and J. heuffelii subsp. glabra (Beck & Szyszył.) Holub. The main char-acter used to distinguish subsp. heuffelii and subsp. glabra has been the absence or presence of hairs on the adaxial and abaxial sides of the rosette leaves (Gajić, 1972). Lectotypification of all names within the J. heuffelii complex (Nikolić et al., 2014) was performed in order to solve the taxonomic issues within this group. Nectaries, as systematic characters useful for taxonomy in the

J. heuffelii group, were first mentioned by Pančić (1874) and Praeger (1932), while in other flora they were neglected.

This morphometric analysis has shown that the quantitative and semiquantitative character-istics of nectaries in the J. heuffelii complex are highly morphologically variable, both within and between populations. In certain cases, as for ex-ample in the species Brassica rapa L., there is a particularly high variability in the morphology of the nectaries due to ploidy, so within the same species haploid, diploid and tetraploid individu-als vary according to the morphological charac-teristics of their nectaries (Davis et al., 1996). In the species J. heuffelii only cases of diploidy have been recorded (Uhl, 1961), which indicates that ploidy is not the cause of such high variability. On the other hand, ecological factors may cause high morphological variability in nectaries, as well as variability in the other qualitative char-acteristics of nectaries such as production and chemical composition of the nectar. A positive correlation between the intensity of nectar se-cretion and nectary size was recorded in certain genera, e.g. Citrus and Lamium (Fahn, 1949; Gulyas, 1967). High temperatures and favorable soil moisture increase nectar secretion (Bonnier, 1879; Zander, 1921; Ostashenko-Koodryavzeva, 1928; Fahn, 1949; Kartashova, 1965). Environ-mental factors such as water availability may be very important for nectary shape in certain rep-resentatives of the Lamiaceae family in the Medi-terranean, since in areas that are more arid the nectar-excreting stomas decrease in size, the vol-ume of nectaries is altered and they are generally smaller than in temperate regions (Petanidou et al., 2000). Petanidou et al. (2000) also remarked that nectary volume is positively correlated with the volume of nectariferous tissue. As J. heuffelii is a succulent plant with a CAM metabolism, in an ecological sense a xerophyte that may survive

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Fig. 3. Micrographs of nectary position: A − distance between nectaries greater than or equal to width of a nectary (sample from SR-Besna Kobila), B − distance between nectaries less than width of the nectary (sample from SR-Stara planina), C − upper part of nectary curving toward the inner side, toward the carpel (sample from SR-Pljačkovica), D − right angle between nectary and carpels (sample from SR-Nebeske Stolice), d-distance, n-nectary, c-carpel, x8.


without water for a long period of time, the water regime of the habitat should not play a crucial role in the size of the nectaries. Our previous studies have shown that the size of the vegetative and reproductive organs in Jovi-barba heuffelii increases as altitude decreases, and in serpentinite and silicate substrates as compared to limestone substrate (Dimitrijević et al., 2011; Spasić, 2012). The significance of altitude on the composition of the nectar in bee-pollinated flowers was recorded by Andre-jeff (1932), Hocking (1968) and Heinrich and Raven (1972), as flowers in plants growing at higher altitudes produce nectar of greater en-ergy value than flowers at lower altitudes, indi-cating that altitude may influence the qualitative characteristics of nectaries and thereby also in-directly influence the quantitative morphologi-cal characteristics. The amount of nutrients in the soil may also influence nectar production, since nectar secretion is higher under low nitro-gen supply (Shuel, 1955). The high level of vari-ability in nectaries and the position of nectaries within the flower are very commonly caused by the type of pollination and behavior of pollina-tors (Nepii, 2007).

The quantitative characters width of nectary (W) and height of nectary (H) showed moderate variability, which is in accordance with previous-ly obtained results for the morphometric char-acters of reproductive organs. The coefficient of variability for these characters had low to moder-ate values (Dimitrijević et al., 2011).

It is important to note that populations with the greatest angles between the nectaries and car-pels at the same time also show the greatest varia-tion quotient for that character. This means that within the same populations there are individuals with right angles (Fig. 3D) and very sharp angles between the nectaries and carpels, indicating that this character is not stable and that it lacks the taxonomic significance assigned by Pančić (1874) and Preager (1932). According to the character-istic shape of nectaries and the distance between two nectaries, it can be concluded that there are two distinct morphological types of nectar-ies that can be used for grouping the analyzed populations and that are commonly observed in individuals of these analyzed populations. The first type of nectaries is those with two teeth, very close to each other at a distance smaller

Fig. 4. Results of canonical discriminant analysis (CDA) for: A) all analyzed populations; B) Sempervivum kopaonikensis vs. S. patens.

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than the width of an individual nectary (SR-Studenica, SR-Besna Kobila, SR-Stara planina, MA-Treskavec, MA-Mavrovo, RO-Domogled, BU-Trojanski prolaz, SR-Pljačkovica, SR-Radan and SR-Treska). The second type is those with a convex or flat surface, far from each other at a distance greater than the width of an individual nectary (SR-Basarski kamik, SR-Nebeske Stolice, SR-Gradac and SR-Suvaja). The total variability of nectaries, also partially connected to variations in the nature of habitats, indicates that according to the characteristics of nectaries there was no differentiation in the analyzed population.

In a taxonomic sense, the characteristics of nectaries were not the cause of differentiation be-tween the taxa Sempervivum kopaonikense and S. patens, but the semiquantitative characters shape

of nectary and distance between nectaries were shown as the most important in the separation of populations into two groups, indicating that they may be used as additional characters in the analysis and taxonomy of the J. heuffelii group. Were the study area increased to include the whole range of the species, then knowledge about the variability of nectaries would definitively be better. Further research would need to include the qualitative characteristics of nectaries (pro-duction of nectar and its quantitative-qualitative characteristics) as well as the pollination process.

Acknowledgments: We gratefully acknowledge the finan-cial support provided by the Serbian Ministry of Education and Science and Technological Development, Grant No. 173030. We are grateful to two anonymous reviewers for valuable comments on this manuscript, and Sonja Stoja-novic (UK) for the final English corrections.

Fig. 5. Results of multiple correspondence analyses (MCA) for qualitative characters.(PLA, SR-Pljačkovica; GRA, SR-Gradac; SUV, SR-Suvaja; STU, SR-Studenica; NES, SR-Nebeske Stolice; TRE, SR-Treska; BAS, SR-Basarski kamik; RAD, SR-Radan; STP, SR-Stara planina; BEK, SR-Besna Kobila; Mak 1, MA-Treskavec; Mak 2, MA-Mavrovo; Rum, RO-Domogled; Bug, BU-Trojanski prolaz)


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