243
Taxonomic significance of palynomorphological 
characteristics of selected Centranthus 
(Caprifoliaceae) species
Abstract
Pollen morphology of herbarium specimens of four Centranthus species (C. ruber, 
C. longiflorus, C. kellereri and C. calcitrapae) was studied using LM and SEM. The 
research aim was to provide data on their pollen characteristics and to evaluate 
the taxonomic value of these data for species-specific identification. Pollen grains 
are tricolpate, suboblate to prolate (P/E = 0.81–1.42); medium- or large-sized 
(P = 49.21–90.44 µm; E = 43.89–93.10 µm). Colpi are long or medium-length, 
wide at equator, tapered to acute or obtuse ends. Exine sculpture is echinate-
microechinate-nanoechinate; echini (1.00–1.39 µm high) and microechini 
(0.55–0.98 µm) are conical, with straight or convex sides and acute apices, 
nanoechini are 0.22–0.46 µm high. Most important characters of taxa diagnostic 
at species level for the taxonomy are: size of pollen and colpi, exine structure, size 
of echini and microechini, and pattern of tectum in areas between echini. Pollen 
grains of C. calcitrapae and C. macrosiphon (sect. Calcitrapa) are generally smaller 
in size than grains of C. ruber, C. longiflorus and C. kellereri (sect. Centranthus). 
Pollen of C. kellereri was analysed for the first time in the current study.
Iz vleček
S svetlobnim in vrstičnim elektronskim mikroskopom smo preučili morfologijo 
peloda herbarijskih primerkov štirih vrst rodu Centranthus (C. ruber, C. longi-
florus, C. kellereri in C. calcitrapae). Namen raziskave je bil ugotoviti značilnosti 
njihovega peloda in oceniti taksonomsko uporabnost za identifikacijo vrst. Pelo-
dna zrna so monade, radialno simetrična, izopolarna, trikolpatna, suboblatna do 
prolatna (P/E = 0.81–1.42); srednja do velika (P = 49.21–90.44 µm; E = 43.89–
93.10 µm). Brazde so dolge do srednje dolge, široke na sredini, zožene, z ostro ali 
topo konico. Eksina je ehinatna-mikroehinatna-nanoehinatna. Večji (ehini, visoki 
1.00-1.39 µm ) in manjši (microehini, visoki 0.55–0.98 µm) izrastki so konični, z 
ravnimi ali konveksnimi stranicami in ostro konico. Najmanjši izrastki (nano-
ehini) so visoki 0.22–0.46 µm. Dodatni znaki vrst rodu Centranthus, značilni za 
posamezne vrste in uporabni za taksonomijo, so: velikost peloda in brazd (kolpi), 
struktura eksine, velikost ehinov in mikroehinov in vzorec krovne plasti (tektuma) 
med ehini. Pelodna zrna vrst C. calcitrapae in C. macrosiphon (sect. Calcitrapa) so 
običajno manjša kot pri ostalih treh vrstah: C. ruber, C. longiflorus in C. kellereri 
(sect. Centranthus). V tej raziskavi smo prvič preučili pelod vrste C. kellereri.
Key words: palynomorphology, 
exine sculpture, taxonomy, 
Centranthus, Valerianaceae.
Ključne besede: palinomorfologija, 
struktura eksine, taksonomija, 
Centranthus, Valerianaceae.
Received: 3. 7. 2020
Revision received: 5. 9. 2020
Accepted: 3. 11. 2020
1 M. G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine, 2 Tereshchenkivska St, Kyiv 01004, Ukraine. E-mail: palynology@ukr.net
2 Department of Plant and Fungal Diversity and Resources, Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Acad. Georgi 
Bonchev St, bl. 23, 1113 Sofia, Bulgaria. E-mail: dani@bio.bas.bg
* Corresponding author: E-mail: dani@bio.bas.bg
Zoya M. Tsymbalyuk1 
, Daniella Ivanova2 ,
*

 & Lyudmila M. Nitsenko1 
DOI: 10.2478/hacq-2021-0021 20/2 • 2021, 243–256

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Taxonomic significance of palynomorphological characteristics of selected 
Centranthus (Caprifoliaceae) species
Introduction
Genus Centranthus DC. has long been regarded as be-
longing to the family Valerianaceae Batsch. However, ac-
cording to APG II (2003), APG III (2009) and APG IV 
(2016) Valerianaceae is included in the family Caprifoli-
aceae Juss. s.l. The phylogenetic position of Valerianaceae 
within the order Dipsacales has been studied extensively 
(Backlund & Donoghue 1996, Bell et al. 2001, Dono-
ghue et al. 2001, 2003, Zhang et al. 2003, Bell 2004, Hi-
dalgo et al. 2004, etc.). According to molecular phyloge-
netic data, within Valerianaceae, Valeriana L., Plectritis 
(Lindl.) DC. and Centranthus form a sister clade to Fedia 
Gaertn. and Valerianella Mill. (Hidalgo et al. 2004, Bell 
& Donoghue 2005, Bell 2007, Winkworth et al. 2008, 
Bell et al. 2012, 2015). 
Valerianaceae traditionally has been subdivided into 
three tribes: Patrinieae Höck, Triplostegieae Höck and 
Valerianeae Höck. The genus Centranthus is included in 
Valerianeae, subtribe Centranthinae Graebn. In general, 
tribe Valerianeae is formed by the genera Valeriana, Cen-
tranthus, Fedia, Plectritis and Valerianella, with the ad-
dition of some small South American genera segregated 
by some authors (Raymúndez et al. 2002, Hidalgo et al. 
2004, Bell & Donoghue 2005). The most recent com-
prehensive taxonomic treatment of genus Centranthus 
was published by Richardson (1975, 1976). Afterwards, 
different number of Centranthus species were used in the 
molecular phylogenetic studies, e.g. two (Bell & Dono-
ghue 2005), four (Raymúndez et al. 2002), five (Hidalgo 
et al. 2004, Bell et al. 2015) or six species of the genus 
(Bell et al. 2012). According to Hidalgo et al. (2004) Cen-
tranthus is monophyletic, and sister to a weakly supported 
group formed by Valeriana (excluding V. celtica L.) based 
on ITS analysis.
Genus Centranthus comprises 8–11 species with cir-
cum-Mediterranean and European distribution, includ-
ing some narrow endemics (Richardson 1975, 1976, 
Mattana et al. 2010, Raab-Straube 2017+, Verloove et al. 
2019, Hassler 2020). Some Centranthus, however, are 
known as introduced or naturalised species in other parts 
of the world. In the flora of Ukraine, Centranthus is repre-
sented by two species, C. ruber (L.) DC. and C. calcitrapae 
(L.) Dufr. (Ilinskaya 1958, Katina 1961, Vovk et al. 1972, 
Mosyakin & Fedoronchuk 1999). Centranthus calcitrapae 
is included in the Red Book of Ukraine (Ryff 2009). In 
Bulgaria, C. ruber is grown as decorative and medicinal 
plant. The only naturally occurring representative of the 
genus is the species C. kellereri (Stoj., Stef. & T.Georgiev) 
Stoj. & Stef. (Delipavlov et al. 1995). It is a Bulgarian 
endemic (Meshinev 2006) of limited distribution and is 
listed in the Red Data Book of the Republic of Bulgaria as 
Critically Endangered (Peev & Tsoneva 2015). In some 
sources this taxon is sometimes accepted as C. longiflo-
rus Steven subsp. kellereri (Stoj., Stef. & T. Georgiev) 
I.  Richardson (e.g., Richardson 1975, 1976, Bilz 2011, 
Bilz & al. 2011, Raab-Straube 2017+), or more recently as 
Valeriana kellereri (Stoj., Stef. & T.Georgiev) Christenh. 
& Byng (e.g., IPNI 2020, POWO 2020). Representatives 
in genus Centranthus are “glabrous, usually glaucous, an-
nual or rhizomatous perennial herbs with erect, usually 
unbranched flowering stems” (Richardson 1976). 
It is well known that morphological characteristics of 
pollen grains as additional diagnostic features are often 
used in the taxonomy (Jacobs et al. 2011, Tsymbalyuk 
et al. 2018, 2019a, 2019b, etc.). Many authors studied 
and discussed the pollen morphology of Valerianaceae, 
including some Centranthus taxa, using light (LM), scan-
ning (SEM) and/or transmission electron microscopy 
(e.g., Clarke & Jones 1977, Clarke 1978, Kupriyanova & 
Alyoshina 1978, Patel & Skvarla 1979, Diez 1984, etc.). 
Despite the relatively numerous publications, the know-
ledge about the structure of pollen grains in Centranthus 
is fragmentary because the available descriptions usually 
have only briefly addressed the pollen morphology of one 
or a few selected taxa, or researchers analyse few selected 
pollen features.
Palynomorphological investigation of selected Centran-
thus species was carried out in order to provide detailed 
quantitative and qualitative data on their pollen charac-
teristics and to evaluate taxonomical value of these data 
for species-specific identification.
Materials and methods
Pollen grains of four species of Centranthus (C. ruber, 
C. longiflorus, C. kellereri, C. calcitrapae) were sampled in 
the National Herbarium of Ukraine (KW – herbarium 
of the M. G. Kholodny Institute of Botany, National 
Academy of Sciences of Ukraine, Kyiv, Ukraine; acronym 
according to Thiers, continuously updated). Data of the 
seven studied specimens are cited below according to the 
label information, in English translation.
The methods used in the present study are described 
in details earlier by the first author (see Mosyakin & 
Tsymbalyuk 2015a, 2015b, 2017). Pollen morphology 
was studied using both LM and SEM. For LM studies 
(Biolar, ×700), the pollen was acetolysed following Erdt-
man (1952), mounted on slides with glycerinated gelatin, 
analysed and photomicrographed. Pollen morphometric 
features of 20 properly developed pollen grains from each 
specimen were measured, and the measurements included 
the following parameters: polar axis (P), equatorial diam-
eter (E), colpus width, apocolpium diameter, mesocol-

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Taxonomic significance of palynomorphological characteristics of selected 
Centranthus (Caprifoliaceae) species
pium diameter, exine thickness. All exine measurements 
were taken in the mesocolpium and apocolpium regions. 
The P/E ratio was calculated in order to determine pol-
len shape. For all the quantitative characters, descriptive 
statistics was applied and the range (minimum and maxi-
mum values), arithmetic mean and standard deviation 
were calculated. The number of echini / microechini / na-
noechini per unit area (100 µm²) was determined. Some 
qualitative features were also analysed: outline, shape, 
exine sculpture. Normality of data distribution for each 
morphological feature measured was checked by the Sha-
piro-Wilk's test. The statistical significance of interspecific 
differences among variables was tested by Wilcoxon rank-
sum test. Statistical data analyses were performed with the 
help of R software (R Core Team 2019). The slides were 
deposited in the palynotheca at the National Herbarium 
of Ukraine (Bezusko & Tsymbalyuk 2011). 
For SEM studies (JEOL JSM-6060LA), dry pollen 
grains were treated with 96%-ethanol, then the samples 
were sputter-coated with gold and investigated at the 
Centre of Electron Microscopy of the M. G. Kholodny 
Institute of Botany. The measurements of the echini, mi-
croechini and nanoechini were made using the program 
AxioVision Rel.4.8.2.
Terminology used in descriptions of pollen grains fol-
lows mainly the glossaries of Punt et al. (2007) and Hal-
britter et al. (2018). Abbreviations of taxon author names 
follow Brummitt & Powell (1992), with additions avail-
able from IPNI (2020). 
Results
Both original and published data on quantitative and 
qualitative pollen characters used in this study are sum-
marised in Tables 1–5. LM and SEM photomicrographs 
of pollen grains are shown in Figures 1–5. Shape of pollen 
grains of the species examined was determined according 
to their P/E ratio (Table 1). Mean P/E ratios are shown 
on Figure 6.
Palynological results
General description of pollen grains of 
Centranthus 
Pollen grains in monads, radially symmetrical, isopo-
lar, tricolpate, suboblate to prolate (P/E = 0.81–1.42), in 
equatorial view elliptic, circular, rarely rhombic, in polar 
view 3-lobate, circular (Table 4); medium- or large-sized: 
P = 49.21–90.44 µm; E = 43.89–93.10 µm  (Tables 1, 3). 
Colpi long or medium-length, 2.66–14.63 µm wide at 
equator (Table 2), with distinct, slightly uneven, un-
dulate and more or less straight margins, tapered to 
acute or obtuse ends; surrounded by distinct or indis-
tinct, narrow halo, 0.66–1.06 µm thick. Mesocolpi-
um = 26.6-65.17 µm, apocolpium = 9.31–25.27 µm 
 (Table 2). Exine 3.99–6.65 µm thick in mesocolpium, 
5.32-9.31 µm in apocolpium (Table 2). Tectum 1.5 or 
three times thinner than infratectum. Columellae simple 
and dense in mesocolpium, in apocolpium thicker, long-
er, sparser, branched above. Exine sculpture echinate-mi-
croechinate-nanoechinate; echini conical, 1.00-1.39 µm 
high, 0.85–1.15 µm wide at base (Table 5); microechini 
0.55–0.98 µm high, 0.57–1.00 µm wide at base  (Table 5), 
both echini and microechini with straight or convex 
sides and acute apices; nanoechini 0.22–0.46 µm high, 
0.31-0.61 µm wide at base (Table 5); tectum psilate or 
psilate-perforate in areas between echini  (Table 4). Col-
pus membranes granulate-echinate (Table 4).
Table 1: Pollen morphometric characters (all measurements given as µm). SD – standard deviation; 1, 2, 3 – specimen number; 
G – general specimens' measurements.
Tabela 1: Morfometrične značilnosti peloda (vse meritve so v µm). SD – standardni odklon; 1, 2, 3 – številka primerka; 
G – splošne meritve primerka.
Taxon Polar axis
Mean ± SD
Polar axis
Range (min-max)
Equatorial diameter
Mean ± SD 
Equatorial diameter 
Range (min-max)
P/E ratio 
(Mean, min-max)
C. ruber 1 64.50 ± 5.41 55.86–73.15 62.11 ± 3.64 54.53–67.83 (1.04) 0.87–1.23 
C. ruber 2 69.09 ± 5.99 58.52–77.14 60.78 ± 3.76 53.20–71.82 (1.14) 0.83–1.38 
C. ruber 3 69.16 ± 6.66 59.85–77.14 62.24 ± 3.70 53.20–69.16 (1.11) 0.91–1.42 
C. ruber G 67.58 ± 6.42 55.86–77.14 61.71 ± 3.76 53.20–71.82 (1.10) 0.83–1.42 
C. longiflorus 1 65.90 ± 5.58 50.54–71.82 61.31 ± 5.76 46.55–71.82 (1.08) 0.81–1.42 
C. longiflorus 2 63.04 ± 6.35 53.20–71.82 61.31 ± 6.20 53.20–71.82 (1.03) 0.91–1.30 
C. longiflorus G 64.47 ± 6.15 50.54–71.82 61.31 ± 5.99 46.55–71.82 (1.05) 0.81–1.42 
C. kellereri 86.45 ± 2.88 79.80–90.44 71.55 ± 7.65 63.84–93.10 (1.22) 0.95–1.39 
C. calcitrapae 54.33 ± 2.60 49.21–59.85 48.34 ± 2.63 43.89–53.20 (1.13) 1.02–1.28 

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Taxonomic significance of palynomorphological characteristics of selected 
Centranthus (Caprifoliaceae) species
Table 2: Pollen morphometric characters (all measurements given as µm). SD – standard deviation; 1, 2, 3 – specimen number; 
G – general specimens’ measurements.
Tabela 2: Morfometrične značilnosti peloda (vse meritve so v µm). SD – standardni odklon; 1, 2, 3 – številka primerka; 
G – splošne meritve primerka.
Taxon Colpi width  
Mean ± SD
(min-max) 
Mesocolpium  
Mean ± SD
(min-max) 
Apocolpium 
Mean ± SD
(min-max) 
Exine in mesocolpium
Mean ± SD
(min-max) 
Exine in poles
Mean ± SD
(min-max) 
C. ruber 1 10.24 ± 3.36 
(3.99–14.63)
43.69 ± 2.31 
(39.9–46.55)
20.74 ± 2.80
(17.29–25.27)
4.05 ± 0.19
(3.99–4.65)
6.65 ± 0
(6.65)
C. ruber 2 9.44 ± 2.26 
(5.32–11.97)
43.35 ± 3.10 
(39.9–49.21)
12.90 ± 2.06
(9.31–15.96)
3.99 ± 0
(3.99)
7.18 ± 0.62
(6.65–7.98)
C. ruber 3 11.43 ± 2.31 
(7.98–14.63)
41.96 ± 3.21 
(37.24–46.55)
13.30 ± 1.68
(10.64–15.96)
4.12 ± 0.26
(3.99–4.65)
7.31 ± 0.66
(6.65–7.98)
C. ruber G 10.37 ± 2.81 
(3.99–14.63)
43.00 ± 3.00 
(37.24–49.21)
15.64 ± 4.24
(9.31–25.27)
4.05 ± 0.19
(3.99–4.65)
7.04 ± 0.60
(6.65–7.98)
C. longiflorus 1 6.78 ± 2.26 
(3.99–11.97)
42.49 ± 2.50 
(39.9–46.55)
16.62 ± 0.66 
(15.96–17.29)
3.99 ± 0
(3.99)
6.91 ± 0.99
(5.32–7.98)
C. longiflorus 2 8.64 ± 0.66 
(7.98–9.31)
39.30 ± 1.75 
(37.24–42.56)
18.35 ± 2.50 
(15.96–22.61)
3.99 ± 0
(3.99)
6.91 ± 0.99
(5.32–7.98)
C. longiflorus G 7.71 ± 1.90 
(3.99–11.97)
40.89 ± 2.68 
(37.24–46.55)
17.48 ± 2.02 
(15.96–22.61)
3.99 ± 0
(3.99)
6.91 ± 0.99
(5.32–7.98)
C. kellereri 5.85 ± 2.16 
(2.66–9.31)
54.72 ± 3.23 
(50.54–65.17)
21.81 ± 1.48
(19.95–23.94)
5.58 ± 0.53
(5.32–6.65)
7.98 ± 1.03
(6.65–9.31)
C. calcitrapae 7.98 ± 1.88 
(5.32–10.64)
30.92 ± 2.72 
(26.6–34.58)
16.62 ± 0.66 
(15.96–17.29)
4.52 ± 0.65
(3.99–5.32)
7.31 ± 0.66
(6.65–7.98)
Table 3: Summary of pollen morphometric characters (original and literature data; all measurements given as µm).
Table 3: Povzetek morfometričnih značilnosti peloda (lastni in literaturni podatki; vse meritve so v µm).
Taxon Polar axis Equatorial  
diameter 
P/E ratio Colpi  
width 
Mesocolpium Apocolpium Exine in  
mesocolpium
Exine in  
poles
C. ruber
(Clarke & Jones 
1977)
(50)52– 
61(65)
(49)54– 
59(62)
(0.86)0.93– 
1.08(1.17)
15 – – 4.0 6.0
C. ruber
(Patel & Skvarla 
1979) 
77 60 1.29 – – – 3.9 6.4
C. ruber 55.86–77.14 53.20–71.82 0.83–1.42 3.99–14.63 37.24–49.21 9.31–25.27 3.99–4.65 6.65–7.98
C. longiflorus 50.54–71.82 46.55–71.82 0.81–1.42 3.99–11.97 37.24–46.55 15.96–22.61 3.99 5.32–7.98
C. kellereri 79.80–90.44 63.84–93.10 0.95–1.39 2.66–9.31 50.54–65.17 19.95–23.94 5.32–6.65 6.65–9.31
C. calcitrapae 49.21–59.85 43.89–53.20 1.02–1.28 5.32–10.64 26.6–34.58 15.96–17.29 3.99–5.32 6.65–7.98
C. calcitrapae
(Kupriyanova & 
Alyoshina 1978)
48.0–49.2 44.8–46.8 – 5.4–6.0 22.8–25.2 16.8–19.2 2.4–3.6 4.8–6.0
C. calcitrapae
(Patel & Skvarla 
1979) 
44–68 29–50 1.38–1.50 – – – 4.4 8.4
C. calcitrapae
(Diez 1984)
44–56 34–48 1.06–1.40 7–8 28 – 4.0 6.0
C. macrosiphon
(Diez 1984)
46–66 38–53 0.91–1.33 7–8 30–35 – 4.0 8.0

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Centranthus (Caprifoliaceae) species
Table 4: Summary of pollen morphological characters (original and literature data).
Table 4: Povzetek morfometričnih značilnosti peloda (lastni in literaturni podatki).
Taxon Apertures Polar  
view
Equatorial  
view
Colpi Colpus 
membrane 
Exine  
sculpture
C. ruber tricolpate 3-lobate, 
circular
elliptic, circular, 
rarely rhombic
long, wide;
ends acute 
granulate-
echinate
echinate-microechinate-nanoechinate, 
psilate-perforate between echini
C. ruber
(Clarke &  
Jones 1977)
tricolpate circular circular to  
slightly elliptic
long;
ends obtuse to acute 
echinate echinate-microechinate
C. ruber
(Patel &  
Skvarla 1979) 
tricolpate circular elliptic, circular long;
ends acute
echinate echinate-microechinate
C. longiflorus tricolpate 3-lobate, 
circular 
elliptic, circular, 
rarely rhombic
long or medium-length, 
medium width;
ends acute
granulate-
echinate
echinate-microechinate-nanoechinate, 
psilate-perforate between echini
C. kellereri tricolpate 3-lobate, 
circular 
elliptic,  
rarely circular
long, narrow;
ends acute or obtuse
granulate-
echinate 
echinate-microechinate-nanoechinate, 
psilate between echini
C. calcitrapae tricolpate 3-lobate, 
circular 
elliptic medium-length,  
medium width;
ends acute or obtuse
granulate-
echinate
echinate-microechinate-nanoechinate, 
psilate, sometimes perforate between 
echini
C. calcitrapae
(Kupriyanova & 
Alyoshina 1978)
tricolpate 3-lobate elliptic short;
ends obtuse
– –
C calcitrapae
(Patel &  
Skvarla 1979) 
tricolpate circular elliptic, circular long;
ends acute
echinate echinate-microechinate
C. calcitrapae
(Diez 1984)
tricolpate, rarely 
dicolpate 
circular elliptic,  
rarely circular
– granulate-
echinate
echinate- microechinate
C. macrosiphon
(Diez 1984)
tricolpate circular elliptic,  
rarely circular
– granulate- 
echinate
echinate- microechinate
Table 5: Comparison between echini, microechini and nanoechini (original and literature data, all measurements given as µm).
Tabela 5: Primerjava med ehini, mikroehini in naonoehini (lastni in literaturni podatki; vse meritve so v µm).
Taxon Echini (SEM) Microechini (SEM) Nanoechini (SEM)
Height Width at the base Height Width at the base Height Width at the base
C. ruber (Clarke & Jones 1977) 1.50 – – – – –
C. ruber 1 1.01–1.08 0.95–1.09 0.71–0.96 0.76–0.83 0.29–0.35 0.44–0.47
C. ruber 2 1.00–1.08 0.91–1.05 0.62–0.89 0.60–0.93 0.34–0.43 0.51–0.56
C. ruber 3 1.01–1.23 0.93–1.07 0.68–0.89 0.95–1.00 0.24–0.46 0.46–0.61
C. longiflorus 1 1.03–1.09 0.89–1.10 0.74–0.96 0.76–1.00 0.31–0.33 0.34–0.59
C. longiflorus 2 1.01–1.39 1.05–1.14 0.62–0.74 0.69–0.76 0.30–0.40 0.39–0.52
C. kellereri 1.01–1.39 0.89–1.15 0.55–0.79 0.57–0.69 0.22–0.37 0.36–0.54
C. calcitrapae 1.00–1.06 0.85–0.93 0.58–0.98 0.66–0.84 0.22–0.35 0.31–0.54
C. calcitrapae (Diez 1984) 1.00 – – – – –
C. macrosiphon (Diez 1984) 1.00 – – – – –

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Centranthus (Caprifoliaceae) species
Descriptions of pollen grains
Genus Centranthus 
Sect. Centranthus 
Centranthus ruber (L.) DC. (Figures 1 A–C, 3 A–D; 
Tables 1–5)
LM. Pollen grains tricolpate, suboblate to prolate 
(P/E = 0.83–1.42), in equatorial view elliptic, circular, 
rarely more or less rhombic, in polar view 3-lobate; 
P = 55.86–77.14 µm, E = 53.20–71.82 µm. Colpi long, 
3.99–14.63 µm wide, sometimes rather sunken, with 
distinct, more or less straight margins, rarely slightly 
uneven, with acute ends, surrounded by distinct, narrow 
halo, 0.66–1.06 µm thick, colpus membrane granulate-
echinate: echini long, often ruptured during acetolysis. 
Mesocolpium = 37.24–49.21 µm, apocolpium = 9.31–
25.27 µm. Exine 3.99–4.65 µm thick in mesocolpium, 
6.65–7.98 µm in apocolpium. Tectum thin, three times 
thinner than infratectum. Columellae simple and dense 
in mesocolpium, in apocolpium thicker, longer, sparser, 
branched above. Nexine nearly as thin as tectum. Exine 
sculpture distinct; LO-analysis: columellae circular, 
densely distributed, in apocolpium larger, sparsely dis-
tributed, echini indistinct.
SEM. Exine sculpture echinate-microechinate- nano-
echinate. Echini conical, 1.0–1.23 µm high, 0.91–1.09 µm 
wide at base, with straight or convex sides and acute 
 apices; tectum psilate-perforate in area between echini. 
Microechini 0.62–0.96 µm high, 0.60–1.0 µm wide at 
base; nanoechini 0.24–0.46 µm high, 0.44–0.61 µm 
wide at base; echini (3–8/100 µm²) and microechini 
(2–4/100 µm²) sparsely distributed, nanoechini denser 
(5–15/µm²). Colpus membranes granulate-echinate. 
Echini long, columellae-like and frequently bifurcated, 
and located at edges and centre of colpus membrane 
(Figure 1 B).
Specimens investigated:1. Crimea, Yalta District, 
Nikitsky Bot. Garden, on the shales by the sea. 15 Aug 
1959. M. Kotov (KW). 2. Crimea. Bakhchisaray, Khan's 
Palace, in the flowerbed. 25 May 1973. O.N. Dubovik 
(KW). 3. Crimea. Yalta Park, Mount Cat. 16 Aug 1974. 
Yu. Shelyag-Sosonko, G. Kukovitsa, Ya. Didukh (KW).
Centranthus longiflorus Steven (Figures 1 D–F, 3 E, F, 
4 A, B; Tables 1–5)
LM. Pollen grains tricolpate, suboblate to prolate (P/E = 
0.81–1.42), in equatorial view elliptic or circular, rarely 
more or less rhombic, in polar view 3-lobate; P = 50.54–
71.82 µm, E = 46.55–71.82 µm. Colpi long or medium-
length, 3.99–11.97 µm wide, sometimes rather sunken, 
with distinct, uneven, undulate margins, with acute ends, 
halo indistinct, narrow, 0.66 µm thick; colpus membrane 
psilate-granulate. Mesocolpium = 37.24–46.55 µm, apo-
co lpium = 15.96–22.61 µm. Exine 3.99 µm thick in me-
socolpium, 5.32–7.98 µm in apocolpium. Tectum thin, 
three times thinner than infratectum. Columellae simple 
and dense in mesocolpium, in apocolpium thicker, longer, 
sparser, branched above. Nexine nearly as thin as tectum. 
Exine sculpture distinct; LO-analysis: columellae circular, 
densely distributed, echini distinct.
SEM. Exine sculpture echinate-microechinate-nano-
echinate. Echini conical, 1.01–1.39 µm high, 0.89–
1.14 µm wide at base, with straight or convex sides and 
acute apices; tectum psilate-perforate in area between 
echini. Microechini 0.62–0.96 µm high, 0.69–1.0 µm 
wide at base; nanoechini 0.30–0.40 µm high, 0.34–
0.59 µm wide at base; echini (4–6/100 µm²), micro-
echini (1–4/100 µm²) and nanoechini (7–8/100 µm²) 
sparsely distributed. Colpus membranes granulate-echi-
nate. Granules and echini often coalesced (Figure 1 E).
Specimens investigated: 1. Italy. Pompeii, on the 
streets and squares of the city, near the rocky walls. 27 
June 1960. D. Dobrochaeva. 2. Caucasus, Georgian SSR, 
surrounding Borjomi, limestones near the Kura River. 11 
Oct 1969. M. Kotov (KW).
Centranthus kellereri (Stoj., Stef. & T.Georgiev) Stoj. 
& Stef. (Figures 2 A–C, 4 C–F; Tables 1–5)
LM. Pollen grains tricolpate, oblate-spheroidal to pro-
late (P/E = 0.95–1.39), in equatorial view elliptic, rarely 
circular, in polar view 3-lobate; P = 79.80–90.44 µm, 
E = 63.84–93.10 µm. Colpi long, 2.66–9.31 µm wide, 
with distinct, slightly undulate margins, with acute or ob-
tuse ends, halo indistinct, narrow, 0.66 µm thick; colpus 
membrane granulate-echinate, echini long, often rup-
tured during acetolysis. Mesocolpium = 50.54–65.17 µm, 
apocolpium = 19.95–23.94 µm. Exine 5.32–6.65 µm 
thick in mesocolpium, 6.65–9.31 µm in apocolpium. 
T ectum thin, three times thinner than infratectum. Colu-
mellae simple and dense in mesocolpium, in apocolpium 
thicker, longer, sparser, branched above. Nexine nearly 
as thin as tectum. Exine sculpture distinct; LO-analysis: 
columellae circular, densely distributed, echini distinct.
SEM. Exine sculpture echinate-microechinate-na-
noechinate. Echini conical, 1.01–1.39 µm high, 0.89–
1.15 µm wide at base, with straight or convex sides and 
acute apices; tectum psilate in area between echini. Mi-
croechini 0.55–0.79 µm high, 0.57–0.69 µm wide at 
base; nanoechini 0.22–0.37 µm high, 0.36–0.54 µm 
wide at base; echini (3–4/100 µm²), microechini (3–
4/100 µm²) and nanoechini (4–9/100 µm²) sparsely dis-
tributed. Colpus membranes granulate-echinate. Echini 
long, located at edges and centre of colpus membrane 
(Figure 2 A). 

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Taxonomic significance of palynomorphological characteristics of selected 
Centranthus (Caprifoliaceae) species
Figure 1: Pollen grains of Centranthus (SEM): A–C – C. ruber, D–F – C. longiflorus; A – equatorial view, E – polar view, B – colpus membrane, 
C, D, F – exine sculpture.
Slika 1: Pelod vrst rodu Centranthus (SEM): A–C – C. ruber, D–F – C. longiflorus; A – ekvatorialni pogled, E – pogled z vrha, B – membrana 
kolpusa, C, D, F – struktura eksine.
Specimen investigated: Institutum Botanicum Aca-
demiae Scientiarum Bulgariae. Pirin: in declivibus saxosis 
calcareis in l. d. “Banski suhodol”. 30 July 1968. B. Kuz-
manov, S. Kožuharov (KW). 
C
A
E F
D
B

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Taxonomic significance of palynomorphological characteristics of selected 
Centranthus (Caprifoliaceae) species
Figure 2: Pollen grains of Centranthus (SEM): A–C – C. kellereri, D–F – C. calcitrapae; A – polar view, E – equatorial view, B – broken pollen 
exine, columellae, C, D, F – exine sculpture.
Slika 2: Pelod vrst rodu Centranthus (SEM): A–C – C. kellereri, D–F – C. calcitrapae; A – pogled z vrha, E – ekvatorialni pogled, B – počena 
eksina peloda, kolumela, C, D, F – struktura eksine.
C
A
E F
D
B

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Taxonomic significance of palynomorphological characteristics of selected 
Centranthus (Caprifoliaceae) species
Sect. Calcitrapa Lange
Centranthus calcitrapae (L.) Dufr. (Figures 2 D–F, 
5 A–D; Tables 1–5)
LM. Pollen grains tricolpate, prolate-spheroidal to sub-
prolate (P/E = 1.02–1.28), in equatorial view elliptic, in 
polar view 3-lobate; P = 49.21–59.85 µm, E = 43.89–
53.20 µm. Colpi medium-length, 5.32–10.64 µm wide, 
with distinct, more or less straight margins, tapering to 
acute or obtuse ends, halo distinct, narrow, 0.66–1.06 µm 
thick; colpus membrane psilate-granulate. Mesocolpium 
= 26.6–34.58 µm, apocolpium = 15.96–17.29 µm. Exine 
3.99–5.32 µm thick in mesocolpium, 6.65–7.98 µm in 
apocolpium. Tectum 1.5 times thinner than infratectum. 
Columellae simple and dense in mesocolpium, in apo-
colpium thicker, longer, sparser, branched above. Nexine 
nearly as thin as tectum. Exine sculpture distinct; LO-
analysis: columellae circular, densely distributed, in apo-
colpium columellae larger and elongated, sparsely distri-
buted, echini distinct.
SEM. Exine sculpture echinate-microechinate-na- 
noechinate. Echini conical, 1.0–1.06 µm high, 0.85–
0.93 µm wide at base, with straight or convex sides and 
acute apices; tectum psilate, sometimes sparsely perforate 
in area between echini. Microechini 0.58–0.98 µm high, 
0.66–0.84 µm wide at base; nanoechini 0.22–0.35 µm 
high, 0.31–0.54 µm wide at base; echini (4–8/100 µm²) 
and microechini (4–7/100 µm²) sparsely distributed, 
nanoechini (16–22/100 µm²) more densely distributed. 
Colpus membranes granulate-echinate.
Specimen investigated: Crimea. South coast, near 
Mount Cat beyond Simeiz, between the stones. 27 May 
1901. C. Golde (KW).
Statistical results
Range, arithmetic mean and standard deviation for the 
quantitative morphological traits of Centranthus taxa are 
detailed in Tables 1 and 2. In addition to descriptive sta-
tistics of the examined morphological characters, the Sha-
piro-Wilk test was used to check the assumptions of data 
normality distribution for each feature measured. Since 
all the variables were found to be not normally distri-
buted (p-value established was < 0.05), they were analysed 
by the non-parametric Wilcoxon rank-sum test. Statisti-
cal significance of differences in the pairwise comparisons 
of metric variables of the pollen grains is presented in 
Table 6. Additionally, box plots showing the variation of 
polar axis and equatorial diameter are given in Figure 7.
Statistically significant differences (p-value < 0.05) be-
tween species were detected in terms of the measurements 
of polar axis, equatorial diameter and exine thickness in 
mesocolpium region, excluding the pair C. ruber–C. lon-
giflorus where the difference was nonsignificant (p-value > 
0.05) for all three characters (Table 6). The differences in 
the length of the polar axis and the equatorial diameter 
are also illustrated by box plots on Figure 7. The species 
C. calcitrapae and C. kellereri differ noticeably; on the 
other hand, the degree of overlap and the similarity be-
tween C. ruber–C. longiflorus is considerable.
Pollen grains differed significantly between all four 
Centranthus species in mesocolpium diameter. Data on 
apocolpium diameter, however, turned out to be sta-
tistically significant only as concerns the pairwise com-
parisons between C. kellereri and the other three species, 
as well as between C. longiflorus and C. ruber. Nonsig-
nificantly different distributions were observed between 
C. calcitrapae–C. ruber and C. calcitrapae–C. longiflorus.
Pairwise comparisons in colpi width between spe-
cies revealed significantly different distributions only in 
the pairs of C. ruber with the other three species. No 
statistically significant differences were found between 
C. calcitrapae–C. longiflorus, C. calcitrapae–C. kellereri 
and C. kellereri–C. longiflorus pairs.
No statistically significant differences were found be-
tween any Centranthus species pairs in terms of the exine 
thickness in apocolpium region.
Table 6: Pairwise comparisons of Centranthus pollen grains metric variables by Wilcoxon rank sum test: ● – statistically significant 
differences (p-value < 0.05), ○ – no statistical significance (p-value > 0.05). Characters are: PA – polar axis, ED – equatorial 
diameter, MD – mesocolpium diameter, AD – apocolpium diameter, CW – colpi width, EM – exine in mesocolpium, EA – exine 
in apocolpium. Species are: CC – C. calcitrapae, CK – C. kellereri, CL – C. longiflorus, CR – C. ruber. 
Tabela 6: Parne primerjave vrednosti meritev peloda vrst rodu Centranthus z Wilcoxonovim testom vsote rangov: ● – statistično 
značilne razlike (p-vrednost < 0.05), ○ – statistično neznačilne razlike (p-vrednost > 0.05). Znaki: PA – polar axis, ED – premer v 
ekvatorju, MD – mezokolpialni premer, AD – apokolpialni premer, CW – širina brazde, EM – eksina v mezokolpiju, EA – eksina 
v apokolpiju. Vrste: CC – C. calcitrapae, CK – C. kellereri, CL – C. longiflorus, CR – C. ruber. 
PA ED MD AD CW EM EA
CC CK CL CC CK CL CC CK CL CC CK CL CC CK CL CC CK CL CC CK CL
CK ● ● ● ● ○ ● ○
CL ● ● ● ● ● ● ○ ● ○ ○ ● ● ○ ○
CR ● ● ○ ● ● ○ ● ● ● ○ ● ● ● ● ● ● ● ○ ○ ○ ○

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Centranthus (Caprifoliaceae) species
Discussion
Comparative pollen morphology
The data obtained in this study demonstrated that pollen 
grains of the studied taxa are morphologically rather differ-
ent, and in most cases they can be distinguished at species 
level. The results confirm and/or supplement the data pro-
vided by other authors (Clarke & Jones 1977, Kupriyano-
va & Alyoshina 1978, Patel & Skvarla 1979, Diez 1984).
Apertures – Pollen grains of all species investigated 
have three apertures. In pollen of C. calcitrapae grains 
with two apertures are rarely observed (Diez 1984). The 
long colpi are characteristic for C. ruber and C. kellereri, 
long and medium-length for C. longiflorus, while brevi-
colpi (according to Kupriyanova & Alyoshina 1978), 
medium-length or long (Patel & Skvarla 1979) colpi are 
observed in C. calcitrapae (Table 4). The wide colpi are 
characteristic for C. ruber, medium-width for C. longiflo-
rus and C. calcitrapae, while narrow colpi are observed in 
C. kellereri (Table 2). In all species colpi are surrounded 
by a narrow halo. Pollen grains of C. ruber and C. cal-
citrapae have distinct and wider halo (0.66–1.06 µm), 
while C. kellereri and C. longiflorus have indistinct and 
narrower halo (0.66 µm).
Shape – According to P/E ratio (Table 1, Figure 6), pol-
len grains are suboblate to prolate in shape. The outline 
in equatorial view is elliptic or circular, in C. ruber and 
C. longiflorus (Figure 3 E) rarely more or less rhombic; in 
polar view – 3-lobate or circular. 
Size – The smallest pollen grains are observed in C. cal-
citrapae (44–68 × 29–53.20 µm) and C. macrosiphon 
(46–66 × 38–53 µm, according to Diez 1984), while the 
largest pollen grains are measured in C. kellereri (79.80–
90.44 × 63.84–93.10 µm). Pollen grains in C. ruber and 
C. longiflorus have similar sizes – (50)52–77.14 × (49)54–
71.82 µm and 50.54–71.82 × 46.55–71.82 µm, respec-
tively (Table 3, Figure 7). Pollen morphometric measure-
ments of C. longiflorus are presented for the first time in 
this investigation.
Sculpture – Pollen grains of all species investigated have 
echinate-microechinate-nanoechinate exine sculpture. Pol-
len grains in C. calcitrapae (Figure 2 F) are characterised by 
small perforations in the tectum area between the echini, 
C. longiflorus (Figure 1 D) and C. ruber (Figure 1 C) have 
psilate-perforate tectum, C. kellereri (Figure 2 C) has psi-
late tectum in area between the echini. The longest echini 
are found in C. kellereri and C. longiflorus – up to 1.39 µm, 
while Clarke & Jones (1977) found echini up to 1.5 µm 
in C. ruber (Table 5). The exine sculpture is an important 
source of taxonomic information. The pollen characters 
allowed distinguishing between morphologically simi-
lar species. Pollen grains of all species investigated have 
granulate-echinate colpus membrane. The data show that 
the echini are long and large columellae-like in C. ruber 
(Figure 1 B), and smaller in C. kellereri (Figure 2 A). In 
other species, colpus membranes are represented by large 
granules and echini. 
Exine – In general, C. kellereri (Figure 4 C) has a thick-
er exine. In pollen grains of C. ruber, C. longiflorus and 
C.  kellereri tectum is three times thinner than infratec-
tum in mesocolpium, while in C. calcitrapae it is 1.5 times 
thinner than infratectum. Columellae in all species are 
distinct, simple and dense in mesocolpium, in apocolpi-
um thicker, longer, sparser, branched above (Figure 2 B). 
In LO-analysis columellae are mainly circular, densely 
distributed; in C. ruber (Figure 3 D) they are larger in 
apocolpium, while in C. calcitrapae (Figure 5 D) they are 
larger and elongated in apocolpium. In pollen grains of 
C. longiflorus (Figure 4 B) and C. kellereri (Figure 4 F) col-
umellae are smaller in apocolpium. The nexine is nearly as 
thin as tectum in pollen grains of all species. 
Figure 3: Pollen grains of Centranthus (LM): A–D – C. ruber,  
E, F – C. longiflorus; A, B, E, F – equatorial view, C, D – polar view, 
D – echini at the edge of colpus. Scale bars = 10 µm.
Slika 3: Pelod vrst rodu Centranthus (LM): A–D – C. ruber,  
E,F – C. longiflorus; A, B, E – ekvatorialni pogled, C, D – pogled z 
vrha, D – ehini na robu brazde. Merilce = 10 µm.
A B
C
E
D
F

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Zoya M. Tsymbalyuk, Daniella Ivanova & Lyudmila M. Nitsenko
Taxonomic significance of palynomorphological characteristics of selected 
Centranthus (Caprifoliaceae) species
Figure 4: Pollen grains of Centranthus (LM): A, B – C. longiflorus,  
C–F – C. kellereri; A, B, E, F – polar view, C, D – equatorial view. 
Scale bars = 10 µm.
Slika 4: Pelod vrst rodu Centranthus (LM): A, B – C. longiflorus,  
C–F – C. kellereri; A, B, E, F – pogled z vrha, C, D – ekvatorialni 
pogled. Merilce = 10 µm.
A B
C
E
D
F
Figure 5: Pollen grains of C. calcitrapae (LM): A, B – equatorial view, 
C, D – polar view, D – LO-analysis: columellae in apocolpium.  
Scale bars = 10 µm.
Slika 5: Pelod vrste C. calcitrapae (LM): A, B – ekvatorialni pogled,  
C, D – pogled z vrha, D – LO-analiza: kolumela in brazda (apokolpij). 
Merilce = 10 µm.
A B
C D
Figure 6: Mean P/E ratio of the pollen grains of Centranthus species: 
1 – C. calcitrapae, 2 – C. kellereri, 3 – C. ruber, 4 – C. longiflorus.
Slika 6: Povprečno razmerje P/E peloda vrst rodu Centranthus:  
1 – C. calcitrapae, 2 – C. kellereri, 3 – C. ruber, 4 – C. longiflorus.
the four Centranthus species studied, outlined in Table 6, 
showed that the number of distinguishing features be-
tween species pairs with distinct differences in the metric 
variables is much greater than species with more similar-
ity. The most important trait turned out to be the meso-
colpium diameter with statistically significant differences 
in all possible pairs. On the other hands, the only feature 
that showed no significant differences between any species 
pair was the thickness of exine in apocolpium region. Cen-
tranthus kellereri was rather distinguishable and showed 
differences with other species in almost all morphological 
characteristics except the exine in apocolpium and (par-
tially) the colpi width. The species C. ruber was signifi-
cantly different from the other three species in colpi width. 
Centranthus longiflorus and C. ruber showed similarities in 
polar axis, equatorial diameter (Figure 7,  Table 6), and ex-
ine thickness, so these features confirm their close affinity.
In general, data in the present investigation are in good 
agreement with the results of previous LM and SEM 
studies (T ables 3–5). However, Kupriyanova & Alyoshina 
(1978) reported pollen grains of C. calcitrapae which had 
a thinner exine and brevicolpi than those found in this 
study and by other authors (Table 3). It is possible that 
the slight variation is due to differences in preparation. 
Earlier, Clarke & Jones (1977) reported colpi with obtuse 
to acute ends in pollen of C. ruber. Clarke (1978) pro-
vided a general description of pollen grains of the genus 
Centranthus, which demonstrates that pollen of all species 
Pairwise comparisons – the results of the test applied in 
this study generally support the above-mentioned obser-
vations. Analysis of the discriminative characters among 












   


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Taxonomic significance of palynomorphological characteristics of selected 
Centranthus (Caprifoliaceae) species
has colpi tapered to broadly acute ends. Data in this study 
and that one of Patel & Skvarla (1979) showed that colpi 
of C. ruber are characterised by acute ends.
Systematic implications of pollen 
morphology
According to Richardson (1976), C. ruber, C. longiflorus 
and C. kellereri are placed in sect. Centranthus. Palyno-
morphological data in this study also suggest a close af-
finity of the three taxa and confirm their placement in 
the same section, because they have similar pollen size, 
mesocolpium length and exine sculpture. But these taxa 
also differ at species level. In particular, C. ruber has wider 
colpi and thinner exine in apocolpium than C. longiflorus 
and C. kellereri. Pollen grains of C. kellereri have a larger 
size, narrower colpi, and thicker exine than those of C. ru-
ber and C. longiflorus. According to Richardson (1976), 
C. kellereri is considered as C. longiflorus subsp. kellereri. 
However, pollen grains of these species are well distin-
guishable by the size, dimensions of microechini and na-
noechini, pattern of tectum in area between echini, and 
thickness of exine. Analysis of P/E ratio showed that pol-
len grains of C. kellereri have the largest P/E, and C. longi-
florus have the smallest P/E, showing significant differ-
ence between them (Table 1, Figure 6). The results of the 
present research support the recognition of C. kellereri as 
a separate species rather than a subspecies of C. longiflo-
rus. However, in order to reach any taxonomic conclusion 
in this case, palynomorphological features should be used 
in combination with other characters of morphology, 
anatomy, cytology, etc. The pollen grains of C. kellereri 
were analysed for the first time in the current study.
Richardson (1976) placed C. calcitrapae and C. mac-
rosiphon in sect. Calcitrapa. The close phylogenetic links 
of these species are also supported by their palynomor-
phological characters, such as pollen size, colpi width, 
exine thickness and sculpture. The present investigation 
showed that pollen grains of the taxa of sect. Centrantus 
are generally larger in size than the pollen grains of species 
in sect. Calcitrapa, which is consistent with Richardson's 
taxonomic classification. 
Thus, pollen morphology proved to be an important 
additional source of information for species-specific iden-
tification within Centranthus. Species could be identified 
based on morphological and morphometric pollen fea-
tures, and especially based on the size of pollen and colpi, 
structure of the exine, size of echini and microechini, and 
pattern of tectum in areas between echini. Moreover, the 
pollen characteristics described here may be used in fu-
ture studies aiming at completing the knowledge on all 
Centranthus species and at understanding the evolution 
of pollen morphology in Valerianaceae. 
Acknowledgements
The authors express their gratitude to Natalia M. Shyian, 
Head Curator of the National Herbarium of Ukraine 
(KW; herbarium of the M. G. Kholodny Institute of 
Botany, National Academy of Sciences of Ukraine) 
for her cooperation. Kind help and cooperation of 
Dmytro O. Klymchuk, Head of the Centre of Electron 
Microscopy (M. G. Kholodny Institute of Botany), 
is greatly appreciated. The authors also express their 
gratitude to Anastasia Davydova for her help with the 
Figure 7: Box plots illustrating the variation in length (in µm) of polar axis (A) and equatorial diameter (B) of Centranthus pollen grains:  
C1 – C. calcitrapae, C2 – C. kellereri, C3 – C. ruber, C4 – C. longiflorus.
Slika 7: Variabilnost dolžine (v µm) polarne osi (A) in premera v ekvatorju (B) peloda vrst rodu Centranthus prikazana s škatlo z brki:  
C1 – C. calcitrapae, C2 – C. kellereri, C3 – C. ruber, C4 – C. longiflorus.




	


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Zoya M. Tsymbalyuk, Daniella Ivanova & Lyudmila M. Nitsenko
Taxonomic significance of palynomorphological characteristics of selected 
Centranthus (Caprifoliaceae) species
Bilz, M. 2011: Centranthus longiflorus ssp. kellereri. The IUCN Red 
List of Threatened Species 2011: e.T165284A5999617. [Downloaded 
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Bilz, M., Kell, S. P ., Maxted, N. & Lansdown, R. V. 2011: European 
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graphs. Thanks are also extended to Dr. Nikolay Velev 
and two anonymous reviewers for valuable comments and 
suggestions made on an earlier version of the manuscript.
Zoya M. Tsymbalyuk  https://orcid.org/0000-0003-2768-0045
Daniella Ivanova  https://orcid.org/0000-0001-5286-030X
Lyudmila M. Nitsenko  https://orcid.org/0000-0003-1945-7409
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