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A new species of Crocus ser. Verni (Iridaceae) with 2n = 12 chromosomes from

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A new species of Crocus ser. Verni (Iridaceae) with

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Irena Raca , Dörte Harpke , Lulëzim Shuka & Vladimir Ranđelović

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PLANT BIOSYSTEMS - AN INTERNATIONAL JOURNAL DEALING WITH ALL ASPECTS OF PLANT BIOLOGY
https://doi.org/10.1080/11263504.2020.1829735

A new species of Crocus ser. Verni (Iridaceae) with 2n ¼ 12 chromosomes from

the Balkans
€rte Harpkeb, Lul€ezim Shukac and Vladimir Rand-elovica
Irena Racaa,b, Do
a
Department of Biology and Ecology, Faculty of Sciences and Mathematics, University of Nis, Nis, Serbia; bLeibniz Institute of Plant Genetics
and Crop Plant Research (IPK), Gatersleben, Germany; cDepartment of Biology, Faculty of Natural Sciences, University of Tirana,
Tirana, Albania

ABSTRACT ARTICLE HISTORY

A Crocus population in the Northern Albanian Alps obviously belonging to C. ser. Verni was referred to Received 18 March 2020
C. heuffelianus, but closer field investigations revealed that it could not be identified with this or any Accepted 23 September 2020
other Verni species. The detailed description of a new species, Crocus bertiscensis, including morph-
KEYWORDS
ology, leaf anatomy, cytology, and phylogenetic relationship is provided and illustrated. Phylogenetic
Taxonomy; morphology;
analyses based on genome-wide single nucleotide polymorphism data obtained through genotyping- anatomy; GBS
by-sequencing placed C. bertiscensis as clearly distinct from C. heuffelianus, supporting its rank as a
species within C. ser. Verni. Morphologically, it differs from C. heuffelianus by showing paler and flatter
perigone segments, short perigone tubes and leaf white stripe width of 1/7 compared to the leaf
diameter. Finally, karyological and molecular analysis revealed that C. bertiscensis applies to the

2n ¼ 12 cytotype of C. heuffelianus from Cakor Pass area reported by earlier studies.

Introduction (Mather 1932; Karasawa 1950, 1951; Brighton et al. 1973;

Within the genus Crocus L., Crocus heuffelianus Herb. (C. ser.
Verni B. Mathew) represents one of the most critical and
challenging taxa. It is distributed from 41˚ (Brighton 1976;
Dietrich 2002; Shuka 2008) to at least 49˚ (Maw 1881;
 ski and Passakas 1976) latitude and from 13˚ (Peruzzi
Rafin
2016) to 29˚300 (Maw 1881; Mihaly and Kricfalusy 1997) lon-
gitude, inhabiting a wide range of habitats. Crocus heuffelia-
nus can be found in elevations from 162 to 2,320 m a.s.l. in
pedunculate oak, beech, and Balkan pine forests as well as in
subalpine and alpine grasslands (Gjeta and Hallaçi 2018; Raca
et al. 2019). Depending on the authors, this species is some-
times split into different taxa mainly based on throat hair
presence/absence (e.g. C. scepusiensis (Rehmer & Wol.)
Borbas ex Kulcz., C. vittatus Schloss. & Vuk., nom. illeg.,
Mosolygo  et al. 2016). However, besides the plethora of pop-
ulations showing conspicuously bearded perigone throats,
some populations show individuals lacking hairs. In the ori-
ginal protologue description, C. heuffelianus was described as
a species with hairless throat from the Banat region (Heuffel
1835; Ko €rnicke 1856; Schlosser and Vukotinovic 1857; Schu €r
1866; Maw 1881). Additionally, as a species belonging to C.
ser. Verni, C. heuffelianus is characterized by filamentous
corm tunics, presence of one bract and can be distinguished
from its relatives by having heart-shaped patches in the
upper regions of the perigone segments (Harpke et al. 2015).
This species is characterized by a wide range of reported
chromosome numbers, 2n ¼ 10, 12, 18, 19, 20, 22, and 23
Brighton 1976; Rafin  ski and Passakas 1976; Dietrich 2002;
Mosolygo  et al. 2016), comprising diploids and tetraploids
(Harpke et al. 2015; Mosolygo  et al. 2016). To infer if the dif-
ferent cytotypes of C. heuffelianus can be distinguished
based on morphology, a thorough investigation is required
since the diagnostic characters used so far were
not sufficient.
Moreover, also molecular studies up to now failed to dis-
entangle the complex relationships, because the applied
markers could not resolve the relationships (Harpke et al.
2015) and not all cytotypes were included (Harpke et al.
2015; Mosolygo  et al. 2016). To resolve the relationships,
genotyping-by-sequencing (GBS) was applied to obtain gen-
ome-wide single nucleotide polymorphism data.
As a consequence of the above mentioned problems, we
designed a study comprising a thorough sampling, where
we combine morphological and leaf anatomical characters,
chromosome counting with a multi-locus phylogeny. Herein,
we investigated in particular a Crocus population in the
Northern Albanian Alps (locality Sylbice€-Doberdol) obviously
belonging to C. ser. Verni (prophyll present, bracteoles
absent, corm tunic showing parallel fibres (Mathew 1982;
Rand-elovic et al. 1990; Dietrich 2002; Shuka et al. 2009), but
couldn’t be identified as a typical C. heuffelianus, or any
other Verni species at first sight. Based on the detailed mor-
pho-anatomical, cytological, and molecular analyses of col-
lected material, a unique set of features is revealed, allowing
the description of a new species.

CONTACT Irena Raca irena.raca@pmf.edu.rs Department of Biology and Ecology, Faculty of Sciences and Mathematics, University of Nis, Nis,
18000 Serbia
ß 2020 Societa Botanica Italiana

Published online 19 Oct 2020

2 I. RACA ET AL.
Materials and methods
Morphological investigations
Observations were based on fresh material, including 28 indi-
viduals. A total of 11 parameters were measured (corm
height and width, leaf width, perigone tube length, outer
and inner perigone segment height and width, stigma
length, anther length and stigma/anther length ratio).
Moreover, three quantitative (number of cataphylls, leaves,
flowers) and eight qualitative characters (tunic type, the pres-
ence of the heart-shaped apical perigone mark, flower,
anther, style and stigma color, hair abundance in perigone
throat and leaf surface) were checked for their constancy.
Herbarium vouchers are deposited at the Institute of Plant
Genetics and Crop Plant Research-IPK Gatersleben GAT (herb-
arium acronyms follow Thiers 2016), in Herbarium
Moesiacum Nis HMN and National Herbarium, the Faculty of
Natural Sciences at the University of Tirana TIR. In order to
define diagnostic parameters, we compared morpho-anat-
omy of C. bertiscensis with C. heuffelianus population col-
lected from locality Muntele Mic, Carpathians, Romania. This
population is in the former Banat region, where the type was
collected (Heuffel 1835; Herbert 1847; Ko€rnicke 1856; Dietrich
2002). Considering the plethora of C. heuffelianus cytotypes,
followed by pretty complicated taxonomical status, we refer
to them as C. cf. heuffelianus. The comparison also included
the literature data from the relevant species descriptions
(Maw 1881; Mathew 1982; Harpke et al. 2015), except the
one published by Herbert (1847) since it was based on quali-
tative morphological characters only. Hence, Table 1 contains
11 distinguishing morpho-anatomical parameters.
Anatomical investigations
For the investigation of leaf anatomy, ten individuals were
collected during the flowering time (14 June 2017) and pre-
served in 50% alcohol. Transversal cross-sections were made
by a manual microtome (Gligorijevic and Pejcinovic 1983),
stained with safranin and alcian blue, dehydrated and there-
fore examined by Leica DM 1000 (Leica Microsystem,
Germany) microscope (Raca et al. 2017, 2019). Anatomical
features considered in leaf cross-sections were section height
and length, section length/white stripe width ratio and the
number of vascular bundles. Measurements were done in
ImageJ (Schneider et al. 2012).
Karyological analysis
Ten individuals were temporarily cultivated in pots and har-
vested in late autumn at the IPK. For chromosome analysis,
squash preparations of their root tips were made according
to the following procedure, already successfully applied in
Crocus (Miljkovic et al. 2016): pretreatment of root tips with
distilled water for 24 h on ice, was followed by fixing with
Carnoy’s Solution (1 volume of glacial acetic acid: 3 volumes
of 96% ethanol); a 1% solution of carmine in 45% acetic acid
for 1 h was used to stain the chromosomes. Chromosomes
were counted in more than 10 plates in order to define the
chromosome number. Genome size of silica dried leaf mater-
ial of five individuals were measured in a flow cytometer
(Meudt et al. 2015), with Pisum sativum L. as standard
(2 C ¼ 9.09 pg; Dolezel et al. 2007).
Molecular methods
The genomic DNA extraction from silica dried leaf material
was conducted according to the protocol described in detail
by Nemati et al. (2019).
For all taxa of C. ser. Verni (49 individuals), except for C.
siculus Tineo, genome-wide single-nucleotide polymorphisms
(SNPs) were obtained using genotyping-by-sequencing (GBS)
analyses (Elshire et al. 2011) as described by Nemati et al.
(2019). Crocus malyi Vis. was used as an outgroup. Read files
of GBS Illumina runs were submitted to the European
Nucleotide Archive (ENA) and are accessible through the
study ID PRJEB38682 and through the accession numbers
ERS4789664–ERS4789698.
Phylogenetic analysis
Barcoded reads from the 14 samples were de-multiplexed
using the CASAVA pipeline 1.8 (Illumina, Inc.). Adapter trim-
ming of GBS sequence reads was performed with CUTADAPT
1.15 (Martin 2011) and reads shorter than 60 bp after adapter
removal were discarded. Raw GBS reads were clustered using
the IPYRAD 0.7.5 (Eaton and Overcast 2016). The minimal
number of samples per locus was set to 20, the clustering
threshold of reads within and between individuals was set to
0.85. For the other parameters the default settings of param-
eter files generated by IPYRAD were used. For the analyses
of GBS data, due to the restriction to 100 kbp alignment
length in MRBAYES, 1038 loci were concatenated and BI ana-
lysis (running for 1  106 MCMC generations) were using the
MRBAYES 3.2 (Ronquist et al. 2012) pugin in Geneious R8.1.9
(Kearse et al. 2012).
Taxonomic treatment
Crocus bertiscensis Raca, Harpke, Shuka & V. Randjel.
sp. nov
Type
Albania. Northern Albanian Alps: Tropoja district, between
Sylbic€e and Dashi lakes, ca. 16 km far from Old Tropoja vil-
lage, alpine pastures (Seslerion comosae; zone of alpine grass-
land above the forest) on siliceous substrate, 2000–2270 m
a.s.l., 14 June 2017, Rand-elovic V, Raca I, Harpke D. and Shuka
L. (holotype GAT-57010! isotypes HMN-13488! and
TIR-12157).
Description
Corm subglobose, 4.9–8.4 mm in diameter (mean 6.75 ± 0.83),
3.3–8.9 mm in height (5.18 ± 1.09); corm tunic brown with
thin mostly parallel fibres, slightly reticulated near the apical
region (Figure 1(a)); thinner fibres 0.06–0.09 mm, thicker
0.18–0.20 mm wide; neck indistinct. Cataphylls (sheathing
 PLANT BIOSYSTEMS - AN INTERNATIONAL JOURNAL DEALING WITH ALL ASPECTS OF PLANT BIOLOGY 3

Table 1. Morphological comparison of C. bertiscensis with C. heuffelianus populations from Muntele Mic and the relevant literature data.
Alb. Alps Prokletije Muntele Mic Maw 1881 Mathew 1982 Harpke et al. 2015
Corm width (mm) 6.8 ± 0.8 8.6 ± 1.6 9.2 ± 1.9 12.7 8-10(-13) 11.8 ± 1.6
Perigone tube length (mm) 33.1 ± 5.9 47.5 ± 8.5 54.1 ± 16.3 50-150 66.0 ± 14.0
Outer perigone segment length(mm) 27.2 ± 3.1 35.9 ± 3.9 38.3 ± 5.5 38.1 (25-)30-55 38.4 ± 4.9
Outer perigone segment width (mm) 25.7 ± 1.2 34.6 ± 1.6 34.9 ± 7.6 9-20
Anther length (mm) 8.1 ± 1.1 10.7 ± 1.5 13.2 ± 1.8 10-18 15.3 ± 2.0
Stigma lobes length (mm) 2.3 ± 0.7 3.3 ± 0.7 3.6 ± 0.7
Leaf width (mm) 3.2 ± 0.5 4.3 ± 0.6 5.7 ± 0.8 (2-)4-6(-8)
Wh. stripe/leaf width ratio (mm) 1/7 1/8 1/14

18.6–33.5 mm (25.67 ± 3.11) in length and 5.6–11.1 mm

(7.87 ± 1.32) in width. Throat conspicuously hairy (Figure
1(b)). Filaments white, broader and stiff at the base, thinner
and filiform at the connection to the anthers; anthers
5.7–11.4 mm (8.13 ± 1.11) long, yellow. The 60% of styles lon-
ger, 22% equal and 18% shorter compared to the stamens
(usually styles 1.11 mm longer than stamens). Styles orange,
in apical part divided into 3 lobes 1.2–4.3 mm (2.35 ± 0.72)
long. Capsules and seeds not seen.

Etymology
Crocus bertiscensis is herein named after Bertiscus, an ancient
name for Prokletije Mountains (Montenegrian) and Northern
Albanian Alps (Albanian part of the mountain), originated
from the Greek language with literal meaning “hill, moun-
tain.” First mentions appeared in Fragments of Strabo’s
Geographica (ca 7 BC) as one of the mountains in the north-
ern part of the ancient kingdom of Macedonia. “Bertiscus”
was the root for nomenclature of several other species such
as Draba bertiscea Lakusic & Stevan. (1995), Valeriana bertis-
cea Pancic (1875), Crepis bertiscea Jav. (1923), and Onobrychis
bertiscea Sirj. & Rech.f. (1935).

Ecology and distribution

Figure 1. Morphological features of Crocus bertiscensis: (A) the tunic, (B) the
hairy throat.
leaves) 2–3, mostly 3, whitish and skinny. Leaves synanthous
and linear, 1–3, mostly 2, without ribs; leaf papillae not so
abundant (Figure 2(c)); white median stripe on the adaxial
surface width approximately 1/5 to 1/8, mostly 1/7, in rela-
tion to the leaf diameter (Figure 2(a)); leaf diameter meas-
ured out from the cross-sections 2.0–3.9 mm (3.77 ± 0.54);
section (keel) height 0.33–0.65 mm (0.55 ± 0.08); palisade tis-
sue 2–, spongy 3-layered (Figure 2(b)); vascular bundles
12–24, mostly 19. Prophyll present; bracts present at the
base of perianth; bracteoles lacking. Both prophyll and bracts
silvery white, thin and membranous. Perigone tube short,
22.7–51.2 mm (33.11 ± 5.94). Flowers 1, perigone segments
flatter to slightly concave, elliptic to obovate, lilac, with
darker marking at the base and dark violet heart(or V-
)shaped-patch at the distal end (Figure 3). Outer segments
usually longer 19.6–34.2 mm (27.22 ± 3.14) and broader
6.0–11.7 mm (8.88 ± 1.25). Moreover, inner segments
The type locality of Crocus bertiscensis is in the alpine pas-
tures of Northern Albanian Alps, Tropoja district, around Lake
of Dashi, ca. 2,220 m a.s.l. This species was noticed in the
upper part of Gashi Valley, Markofça and in the adjacent
parts of Montenegro (Prokletije Mt), appearing in the similar
habitat. It grows in Seslerion comosae association of alpine
pastures on the siliceous substrate. The following species
were noticed as associated with C. bertiscensis: Soldanella pin-
dicola Hausskn., Geum montanum L., Gentiana acaulis L.,
Gentiana punctata L., Gentiana verna L., Plantago gentianoides
Sibth. & Sm., Primula elatior (L.) L., Primula minima L.,
Homogyne alpina (L.) Cass., Dryas octopetala L., Ranunculus
crenatus Waldst. & Kit., Erigeron uniflorus L., Narthecium scar-
dicum Kosanin or inbetween individuals of Vaccinium myrtil-
lus L. Crocus bertiscensis was also observed in meadows in

the lower part (1,000 m a.s.l.) of the Cakor Pass region
in Montenegro.
Karyological analysis
Crocus bertiscensis has a chromosome number of
2n ¼ 2x ¼ 12 (Figure 4) and genome size of
2 C ¼ 6.51 ± 0.31 pg (CV standard: 2.91 to 4.81, CV samples:
3.7 to 4.48).
4 I. RACA ET AL.
Figure 2. Anatomical features of Crocus bertiscensis: (A) the leaf cross section, (B) the arm details, (C) the big bundle (ad – adaxial side, ab – abaxial side, la –
lacuna area, vb – vascular bundles, e – epidermis, pp – palisade parenchyma, sp – spongy parenchyma, p – papilla, sc – sclerenchyma, ph – phloem, xy – xylem).
Discussion
Phylogenetic analyses point out that C. bertiscensis belongs
to C. ser. Verni, where it groups as sister to a clade compris-
ing C. heuffelianus, C. tommasinianus Herb., C. vernus (L.) Hill,
C. neglectus Peruzzi and Carta, C. etruscus Parl., and C. ilvensis
 before the revision of the series (Harpke et al. 2015) or artifi-
Peruzzi & Carta (Figure 5). Samples collected on the Cakor
Pass and in one other location in Montenegro grouped
together intermingled with the studied individuals described
here as C. bertiscensis indicating also gene flow between
these geographically close populations. We therefore assume
that they share the same cytotype, since they not only group
together but also share the same genome size. Additionally,
the herein described C. bertiscensis is referring to the 2n ¼ 12
cytotype (Figure 4) of C. cf. heuffelianus from Cakor Pass
area, reported by Brighton (1976). Based on a comparison of
morphology and chromosome numbers Dietrich (2002)
already suggested that the 2n ¼ 12 cytotype might represent
a separate species and informally treated it as Crocus

“ Cakor Pass.”
The measured genome size is in the range of the meas-
urements of Brandizzi and Grilli Caiola (1996) for other dip-
loid crocuses including the closely relative C. etruscus
(2 C ¼ 7.25 pg). However, another much higher genome size
was measured for C. etruscus (2 C ¼ 19.30) and C. ilvensis
(2 C ¼ 18.90) by Carta et al. (2015) as well as for ornamental
cultivars of C. vernus (2 C ¼ 23.1 pg; Olszewska and Osiecka
1982) and a white cultivar of C. vernus (2 C ¼ 22.1 pg;
Zonneveld et al. 2005). The latter two likely represent the
alloploid C. negelectus, which was considered as C. vernus
cial ornamentals. However, it needs additional investigations
to clarify the inconsistencies of measured genome sizes and/
or to infer if massive chromosomal rearrangements resulted
in a reduction of the chromosome number while nearly tri-
pling the genome size.
The taxonomic status of this species is supported by mor-
phological criteria common for the representatives of C. ser.
Verni (prophyll presence, bracteole absence, tunic showing
parallel fibres (Mathew 1982; Rand-elovic et al. 1990; Harpke
et al. 2015). Furthermore, heart or V-shaped marks positioned
at the upper regions of perigone segments suggests that C.
bertiscensis is closely akin to C. heuffelianus (Figure 3).
However, such marks can also be sporadically found in cro-
cuses belonging to other groups within the genus (pers.
obs.). As all the other species belonging to C. ser. Verni, C.
bertiscensis leaf cross-sections are consisting of an almost
squared keel (with its central part filled by parenchyma cells,
frequently called lacuna area – Figure 2(a)) and two lateral
 PLANT BIOSYSTEMS - AN INTERNATIONAL JOURNAL DEALING WITH ALL ASPECTS OF PLANT BIOLOGY
Figure 3. The habitus of a Crocus bertiscensis individual from the type locality Sylbic€e-Doberdol in the Northern Albanian Alps (photographs by L. Shuka).
Figure 4. Somatic chromosomes of Crocus bertiscensis (2n ¼ 2x ¼ 12).
arms with their margins recurved towards the keel (Raca
et al. 2017, 2019). Leaf mesophyll contains two layers of pol-
ygonal palisade cells and three or four-layered spongy paren-
chyma (Figure 2(b)); sclerenchyma sheaths are prominent
and papillae can occur at the end of the arms (Figure 2(c))
5
which is in accordance with Raca et al. (2017, 2019). The
study by Raca et al. (2019) have already indicated that, from
the morpho-anatomical aspect, Balkan C. cf. heuffelianus
rather represents an aggregate of species. However, C. bertis-
censis differs from other C. cf. heuffelianus taxa by showing
short perigone tubes ( 2 longer in other populations),
and leaf white stripe width of 1/7 compared to the leaf
diameter (this ratio is 1/14 in C. cf. heuffelianus population)
(Table 1, Figure 2(a)). The ratio of the white stripe is also
higher in comparison to all other up to now investigated C.
cf. heuffelianus populations on the Balkans (Raca et al. 2019).
Compared to the population from Muntele Mic, showing
intense purple goblet-shaped flowers, C. bertiscensis is char-
acterized by paler and flatter perigone segments. Moreover,
perigone throat of C. bertiscensis is conspicuously hairy
(Figure 1(b)) like in other populations from the Central and
Western Balkans (Raca et al. 2019) while it is declared as
glabrous in the first description referring to C. heuffelianus
(Heuffel 1835).
6 I. RACA ET AL.

Figure 5. Phylogenetic tree derived from Bayesian phylogenetic inference (BI) of the concatenated 1,038 loci obtained by GBS, 99,412 bp. Posterior supports (pp)
are shown at the nodes. Locations are attached to the taxon names (CHE – Switzerland, BIH – Bosnia and Herzegovina, ALB – Albania, MNE – Montenegro, ITA –
Italy, SVK – Slovakia, SRB – Serbia, ROU – Romania, XKX – Kosovo region; This designation is without prejudice to positions on status, and is in line with UNSCR
1244/1999 and the ICJ Opinion on the Kosovo declaration of independence, HRV – Croatia).

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