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Molecular Study of Selected Taxonomically Critical Taxa of the Genus Iris L. from the Broader Alpine-Dinaric Area

Authors:
  • Institut za poljoprivredu i turizam Porec

Abstract and Figures

Some wild, morphologically diverse taxa of the genus Iris in the broad Alpine-Dinaric area have never been explored molecularly, and/or have ambiguous systematic status. The main aims of our research were to perform a molecular study of critical Iris taxa from that area (especially a narrow endemic accepted species I. adriatica, for which we also analysed genome size) and to explore the contribution of eight microsatellites and highly variable chloroplast DNA (ndhJ, rpoC1) markers to the understanding of the Iris taxa taxonomy and phylogeny. Both the microsatellite-based UPGMA and plastid markers-based maximum likelihood analysis discriminated three main clusters in the set of 32 analysed samples, which correspond well to the lower taxonomic categories of the genus, and support separate status of ambiguous regional taxa (e.g., I. sibirica subsp. erirrhiza, I. x croatica and I. x rotschildii). The first molecular data on I. adriatica revealed its genome size (2C = 12.639 ± 0.202 pg) and indicated the existence of ecotypes. For future molecular characterisation of the genus we recommend the utilisation of microsatellite markers supplemented with a combination of plastid markers.
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Article
Molecular Study of Selected Taxonomically Critical
Taxa of the Genus Iris L. from the Broader
Alpine-Dinaric Area
Tim Weber 1,2 , Jernej Jakše 3, Barbara Sladonja 1, Dario Hruševar 4, Nediljko Landeka 5,
Slavko Brana 6, Borut Bohanec 3, Milenko Milovi´c 7, Dalibor Vladovi´c 8, Božena Miti´c 4, and
Danijela Poljuha 1, *,
1Institute of Agriculture and Tourism, Karla Huguesa 8, HR-52440 Poreˇc, Croatia;
t.weber19@imperial.ac.uk (T.W.); barbara@iptpo.hr (B.S.)
2Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, UK
3Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia;
Jernej.Jakse@bf.uni-lj.si (J.J.); Borut.Bohanec@bf.uni-lj.si (B.B.)
4Department of Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6,
HR-10000 Zagreb, Croatia; dario.hrusevar@biol.pmf.hr (D.H.); bozena.mitic@biol.pmf.hr (B.M.)
5Public Health Institute of the Istrian Region, Nazorova 23, HR-52100 Pula, Croatia; ddd@zzjziz.hr
6Public Institution Natura Histrica, Riva 8, HR-52100 Pula, Croatia; slavko.brana@natura-histrica.hr
7
Medical and Chemical School, Ante Šupuk Street 29, HR-22000 Šibenik, Croatia; milenko.milovic@si.t-com.hr
8
Nature History Museum Split, Poljana kneza Trpimira 3, HR-21000 Split, Croatia; dalibor@prirodoslovni.hr
*Correspondence: danijela@iptpo.hr; Tel.: +385-52-408-336
Joint last authors.
Received: 1 August 2020; Accepted: 16 September 2020; Published: 18 September 2020


Abstract:
Some wild, morphologically diverse taxa of the genus Iris in the broad Alpine-Dinaric
area have never been explored molecularly, and/or have ambiguous systematic status. The main
aims of our research were to perform a molecular study of critical Iris taxa from that area (especially
a narrow endemic accepted species I. adriatica, for which we also analysed genome size) and to
explore the contribution of eight microsatellites and highly variable chloroplast DNA (ndhJ,rpoC1)
markers to the understanding of the Iris taxa taxonomy and phylogeny. Both the microsatellite-based
UPGMA and plastid markers-based maximum likelihood analysis discriminated three main clusters
in the set of 32 analysed samples, which correspond well to the lower taxonomic categories of
the genus, and support separate status of ambiguous regional taxa (e.g., I. sibirica subsp. erirrhiza,
I. xcroatica and I. xrotschildii). The first molecular data on I. adriatica revealed its genome size
(2C =12.639
±
0.202 pg) and indicated the existence of ecotypes. For future molecular characterisation
of the genus we recommend the utilisation of microsatellite markers supplemented with a combination
of plastid markers.
Keywords: Iridaceae; Europe; chloroplast DNA; microsatellites; phylogeny; taxonomy
1. Introduction
Iris L. (family Iridaceae) is a diverse genus with over 300 taxa distributed worldwide, mostly in the
northern hemisphere [
1
,
2
]. In addition to conservational importance, many wild and cultivated taxa
provide great horticultural value [
3
]. Phylogenetic and evolutionary studies of relationships of wild
Iris taxa have long been challenging for several reasons. Namely, wide distribution, morpho-ecological
diversity, multiple hybridisations, and convergent evolution processes, make definitive statements of
the origin and evolution of taxa in the genus Iris very dicult [
4
,
5
]. To resolve a myriad of uncertainties
Plants 2020,9, 1229; doi:10.3390/plants9091229 www.mdpi.com/journal/plants
Plants 2020,9, 1229 2 of 16
and issues related to taxonomic and phylogenetic relationships within the genus Iris, extensive work
was performed on morpho-anatomical features, palynology, phytochemical constituents’ analysis,
cytogenetic traits, and molecular analysis [
6
9
]. Despite dierent approaches to lower (and individual)
taxonomic categories, most authors agree on the classification of the genus Iris into six subgenera,
which are divided into sections and series [1,10,11].
Most of the European native taxa of the genus Iris belong to the subgenus Iris L., section Iris L.
(so-called “Pogoniris”), represented by numerous rhizomatous Iris taxa characterised by bearded outer
tepals. Less prevalent on the European territory are taxa from the subgenus Limniris (Tausch) Spach,
section Limniris (Tausch) Spach (so-called “Apogoniris”), which are rhizomatous irises whose outer
tepals are without a beard [
1
,
3
]. The broad Alpine-Dinaric, as well as the surrounding Mediterranean
and Pannonian area of Europe (where irises for our study were sampled) is characterised by peculiar
eco-climate conditions which have caused a great morphological variability of some Iris populations
and groups. Their variety has resulted in ambiguous systematic status of some regional, especially
endemic, Iris taxa [
5
,
8
]. Some of them are recognised in the national and regional floras [
12
,
13
] and
still have an unclear phylogenetic and classification status. Some of them neither are accepted in the
World Checklist of Selected Plant Families [
2
], nor are molecularly researched in detail. Therefore we
intended to molecularly study some, insuciently researched and/or globally neglected taxa; namely:
I. xcroatica Horvat et M. D. Horvat (endemic in Croatia and Slovenia), I. illyrica Tomm. ex Vis.(endemic
in Croatia, Slovenia, and Italy), I. sibirica L. subsp. erirrhiza (Posp.) Wraber (endemic in Bosnia and
Herzegovina, Croatia, and Slovenia) and I. xrotschildii Degen (endemic in Croatia). However, in this
study we paid special attention to the validly described [
14
] and accepted [
2
], molecularly unexplored
endemic species I. adriatica Trinajsti´c ex Miti´c (Figures 1and 2).
Plants 2020, 9, x FOR PEER REVIEW 2 of 16
morpho-ecological diversity, multiple hybridisations, and convergent evolution processes, make
definitive statements of the origin and evolution of taxa in the genus Iris very difficult [4,5]. To
resolve a myriad of uncertainties and issues related to taxonomic and phylogenetic relationships
within the genus Iris, extensive work was performed on morpho-anatomical features, palynology,
phytochemical constituents’ analysis, cytogenetic traits, and molecular analysis [6–9]. Despite
different approaches to lower (and individual) taxonomic categories, most authors agree on the
classification of the genus Iris into six subgenera, which are divided into sections and series [1,10,11].
Most of the European native taxa of the genus Iris belong to the subgenus Iris L., section Iris L.
(so-called “Pogoniris”), represented by numerous rhizomatous Iris taxa characterised by bearded
outer tepals. Less prevalent on the European territory are taxa from the subgenus Limniris (Tausch)
Spach, section Limniris (Tausch) Spach (so-called “Apogoniris”), which are rhizomatous irises whose
outer tepals are without a beard [1,3]. The broad Alpine-Dinaric, as well as the surrounding
Mediterranean and Pannonian area of Europe (where irises for our study were sampled) is
characterised by peculiar eco-climate conditions which have caused a great morphological
variability of some Iris populations and groups. Their variety has resulted in ambiguous systematic
status of some regional, especially endemic, Iris taxa [5,8]. Some of them are recognised in the
national and regional floras [12,13] and still have an unclear phylogenetic and classification status.
Some of them neither are accepted in the World Checklist of Selected Plant Families [2], nor are
molecularly researched in detail. Therefore we intended to molecularly study some, insufficiently
researched and/or globally neglected taxa; namely: I. x croatica Horvat et M. D. Horvat (endemic in
Croatia and Slovenia), I. illyrica Tomm. ex Vis.(endemic in Croatia, Slovenia, and Italy), I. sibirica L.
subsp. erirrhiza (Posp.) Wraber (endemic in Bosnia and Herzegovina, Croatia, and Slovenia) and I. x
rotschildii Degen (endemic in Croatia). However, in this study we paid special attention to the validly
described [14] and accepted [2], molecularly unexplored endemic species I. adriatica Trinajstić ex
Mitić (Figures 1 and 2).
I. adriatica (Figure 1a–c) is a narrow endemic plant from the I. pumila complex, characterised by
an extremely dwarf stem (one of the smallest species within the genus Iris) and relatively large
yellow, violet, or purple solitary flowers (Figure 1a–c) [14].
Figure 1. Narrow endemic wild Alpine-Dinaric endemic species Iris adriatica: (ac) Individuals of
different colours (Photo: Miroslav Mitić).
It is confined to a few Croatian localities in the wider area of Dalmatia and classified as a NT
(near threatened) species [13]. Given that the localities newly recorded by authors are spatially
distant from the previously catalogued specimens (Figure 2), questions of subspeciation or
higher-level genetic divergence can arise. All the more so as the recent metabolic profiling [15]
Figure 1.
Narrow endemic wild Alpine-Dinaric endemic species Iris adriatica: (
a
c
) Individuals of
dierent colours (Photo: Miroslav Miti´c).
Plants 2020,9, 1229 3 of 16
Plants 2020, 9, x FOR PEER REVIEW 3 of 16
revealed a notable diversity between the ecotypes and their pharmacological and chemotaxonomic
potential.
Figure 2. (a) Distribution map of the narrow endemic Alpine-Dinaric species Iris adriatica included in
our study (all localities are in Croatia, and are incorporated in the national Flora Croatica Database
(https://hirc.botanic.hr/fcd)—FCD; marks: —earlier data from the FCD;
—localities of collected
specimens in our study); (b) habitat on the locality Brnjica-Pokrovnik; (c) habitat on the island of
Cres.
Since the 1990s, when molecular biology techniques have become widely accessible,
taxonomical biology has been driven towards using molecular methods to establish and re-establish
evolutionary relationships between species [16,17]. Tang et al. [18] developed 400 ortholog-specific
EST-SSR (Expressed Sequence Tag—Simple Sequence Repeats) markers, which can be reliably used
to distinguish between the species in the Iris genus, providing a cheap and efficient way to resolve
taxonomical discrepancies. Simple Sequence Repeats or microsatellites are present in most species;
they are usually locus-specific, multiallelic, polymorphic, and co-dominant and are as such ideal
candidates for discriminating between Iris species [19].
AChloroplast gene sequences are often used for plant phylogenetic studies and DNA barcoding
because of the relatively low evolutionary mutation rates, their uniparental inheritance, high level of
genetic diversity, and absence of recombination. Many candidate plastid regions have been
suggested as the plant barcode and have as such been extensively tested [20–22]. However, to this
end, a single marker has not yet been found which could reliably distinguish between a majority of
plant species. Different combinatorial approaches have been used in different instances, to set on a
final consortium [23]. Plastid DNA regions rpoC1 and ndhJ used previously to evaluate plant
phylogeny with low taxonomic variation [22] seemed appropriate for our study.
One of the basic genomic parameters that characterise the species and represent one of the
important plant traits is the total amount of DNA in the unreplicated haploid or gametic cell nuclei,
referred to as the C value or genome size [24]. Genome size data have numerous applications: They
can be used in comparative studies on genome evolution, or as a tool to estimate the cost of
whole-genome sequencing programs [25]. Currently, the largest updated plant genome size
database—Plant DNA C-values database contains data for 12,273 species and among them 65
C-values for 44 species of genus Iris [26]. For most species involved in our study C-values are
measured in several studies [27–29]. Different methods were used for the measurement of plant
Figure 2. (a) Distribution map of the narrow endemic Alpine-Dinaric species Iris adriatica included in
our study (all localities are in Croatia, and are incorporated in the national Flora Croatica Database
(https://hirc.botanic.hr/fcd)—FCD; marks:
Plants 2020, 9, x FOR PEER REVIEW 3 of 16
revealed a notable diversity between the ecotypes and their pharmacological and chemotaxonomic
potential.
Figure 2. (a) Distribution map of the narrow endemic Alpine-Dinaric species Iris adriatica included in
our study (all localities are in Croatia, and are incorporated in the national Flora Croatica Database
(https://hirc.botanic.hr/fcd)—FCD; marks: —earlier data from the FCD;
—localities of collected
specimens in our study); (b) habitat on the locality Brnjica-Pokrovnik; (c) habitat on the island of
Cres.
Since the 1990s, when molecular biology techniques have become widely accessible,
taxonomical biology has been driven towards using molecular methods to establish and re-establish
evolutionary relationships between species [16,17]. Tang et al. [18] developed 400 ortholog-specific
EST-SSR (Expressed Sequence Tag—Simple Sequence Repeats) markers, which can be reliably used
to distinguish between the species in the Iris genus, providing a cheap and efficient way to resolve
taxonomical discrepancies. Simple Sequence Repeats or microsatellites are present in most species;
they are usually locus-specific, multiallelic, polymorphic, and co-dominant and are as such ideal
candidates for discriminating between Iris species [19].
AChloroplast gene sequences are often used for plant phylogenetic studies and DNA barcoding
because of the relatively low evolutionary mutation rates, their uniparental inheritance, high level of
genetic diversity, and absence of recombination. Many candidate plastid regions have been
suggested as the plant barcode and have as such been extensively tested [20–22]. However, to this
end, a single marker has not yet been found which could reliably distinguish between a majority of
plant species. Different combinatorial approaches have been used in different instances, to set on a
final consortium [23]. Plastid DNA regions rpoC1 and ndhJ used previously to evaluate plant
phylogeny with low taxonomic variation [22] seemed appropriate for our study.
One of the basic genomic parameters that characterise the species and represent one of the
important plant traits is the total amount of DNA in the unreplicated haploid or gametic cell nuclei,
referred to as the C value or genome size [24]. Genome size data have numerous applications: They
can be used in comparative studies on genome evolution, or as a tool to estimate the cost of
whole-genome sequencing programs [25]. Currently, the largest updated plant genome size
database—Plant DNA C-values database contains data for 12,273 species and among them 65
C-values for 44 species of genus Iris [26]. For most species involved in our study C-values are
measured in several studies [27–29]. Different methods were used for the measurement of plant
—earlier data from the FCD;
Plants 2020, 9, x FOR PEER REVIEW 3 of 16
revealed a notable diversity between the ecotypes and their pharmacological and chemotaxonomic
potential.
Figure 2. (a) Distribution map of the narrow endemic Alpine-Dinaric species Iris adriatica included in
our study (all localities are in Croatia, and are incorporated in the national Flora Croatica Database
(https://hirc.botanic.hr/fcd)—FCD; marks: —earlier data from the FCD;
—localities of collected
specimens in our study); (b) habitat on the locality Brnjica-Pokrovnik; (c) habitat on the island of
Cres.
Since the 1990s, when molecular biology techniques have become widely accessible,
taxonomical biology has been driven towards using molecular methods to establish and re-establish
evolutionary relationships between species [16,17]. Tang et al. [18] developed 400 ortholog-specific
EST-SSR (Expressed Sequence Tag—Simple Sequence Repeats) markers, which can be reliably used
to distinguish between the species in the Iris genus, providing a cheap and efficient way to resolve
taxonomical discrepancies. Simple Sequence Repeats or microsatellites are present in most species;
they are usually locus-specific, multiallelic, polymorphic, and co-dominant and are as such ideal
candidates for discriminating between Iris species [19].
AChloroplast gene sequences are often used for plant phylogenetic studies and DNA barcoding
because of the relatively low evolutionary mutation rates, their uniparental inheritance, high level of
genetic diversity, and absence of recombination. Many candidate plastid regions have been
suggested as the plant barcode and have as such been extensively tested [20–22]. However, to this
end, a single marker has not yet been found which could reliably distinguish between a majority of
plant species. Different combinatorial approaches have been used in different instances, to set on a
final consortium [23]. Plastid DNA regions rpoC1 and ndhJ used previously to evaluate plant
phylogeny with low taxonomic variation [22] seemed appropriate for our study.
One of the basic genomic parameters that characterise the species and represent one of the
important plant traits is the total amount of DNA in the unreplicated haploid or gametic cell nuclei,
referred to as the C value or genome size [24]. Genome size data have numerous applications: They
can be used in comparative studies on genome evolution, or as a tool to estimate the cost of
whole-genome sequencing programs [25]. Currently, the largest updated plant genome size
database—Plant DNA C-values database contains data for 12,273 species and among them 65
C-values for 44 species of genus Iris [26]. For most species involved in our study C-values are
measured in several studies [27–29]. Different methods were used for the measurement of plant
—localities of collected
specimens in our study); (
b
) habitat on the locality Brnjica-Pokrovnik; (
c
) habitat on the island of Cres.
I. adriatica (Figure 1a–c) is a narrow endemic plant from the I. pumila complex, characterised by an
extremely dwarf stem (one of the smallest species within the genus Iris) and relatively large yellow,
violet, or purple solitary flowers (Figure 1a–c) [14].
It is confined to a few Croatian localities in the wider area of Dalmatia and classified as a NT (near
threatened) species [
13
]. Given that the localities newly recorded by authors are spatially distant from
the previously catalogued specimens (Figure 2), questions of subspeciation or higher-level genetic
divergence can arise. All the more so as the recent metabolic profiling [
15
] revealed a notable diversity
between the ecotypes and their pharmacological and chemotaxonomic potential.
Since the 1990s, when molecular biology techniques have become widely accessible, taxonomical
biology has been driven towards using molecular methods to establish and re-establish evolutionary
relationships between species [
16
,
17
]. Tang et al. [
18
] developed 400 ortholog-specific EST-SSR
(Expressed Sequence Tag—Simple Sequence Repeats) markers, which can be reliably used to distinguish
between the species in the Iris genus, providing a cheap and ecient way to resolve taxonomical
discrepancies. Simple Sequence Repeats or microsatellites are present in most species; they are
usually locus-specific, multiallelic, polymorphic, and co-dominant and are as such ideal candidates for
discriminating between Iris species [19].
AChloroplast gene sequences are often used for plant phylogenetic studies and DNA barcoding
because of the relatively low evolutionary mutation rates, their uniparental inheritance, high level
of genetic diversity, and absence of recombination. Many candidate plastid regions have been
suggested as the plant barcode and have as such been extensively tested [
20
22
]. However, to this end,
a single marker has not yet been found which could reliably distinguish between a majority of plant
species. Dierent combinatorial approaches have been used in dierent instances, to set on a final
consortium [
23
]. Plastid DNA regions rpoC1 and ndhJ used previously to evaluate plant phylogeny
with low taxonomic variation [22] seemed appropriate for our study.
One of the basic genomic parameters that characterise the species and represent one of the
important plant traits is the total amount of DNA in the unreplicated haploid or gametic cell nuclei,
referred to as the C value or genome size [
24
]. Genome size data have numerous applications: They can
Plants 2020,9, 1229 4 of 16
be used in comparative studies on genome evolution, or as a tool to estimate the cost of whole-genome
sequencing programs [
25
]. Currently, the largest updated plant genome size database—Plant DNA
C-values database contains data for 12,273 species and among them 65 C-values for 44 species of genus
Iris [
26
]. For most species involved in our study C-values are measured in several studies [
27
29
].
Dierent methods were used for the measurement of plant DNA content, but flow cytometry has
become the method of choice due to its reliability, simplicity, and relatively low cost [30,31].
A noticeable lack of eorts to molecularly resolve remaining issues in Iris phylogeny and taxonomy
on the Alpine-Dinaric area (including the adjacent areas of Mediterranean and the Pannonian Plain) in
the context of conservation was extremely important when designing the study. Hence, to provide
molecular insights into phylogenetic relationships of selected wild Iris taxa of the wider Alpine-Dinaric
area, with a special emphasis on regional endemics and molecular evidence for their conservation,
the aims of our research were: (i) To characterise representative and critical Iris taxa from the wider
Alpine-Dinaric area by nuclear (SSR) markers; (ii) to clarify the genetic divergence within and between
several wild (local endemic) and cultivated Iris populations through chloroplast DNA (cpDNA)
markers; (iii) to present the first molecular description of a nearly threatened narrow endemic dwarf
species I. adriatica; and (iv) contribute to the eorts of establishing optimal molecular markers for
detecting taxonomic and phylogenetic relationships within critical taxa of the genus Iris.
2. Results
2.1. SSR Analysis
In total, 32 Iris samples across the Alpine-Dinaric region were analysed (Supplementary Table S1).
Parameters of genetic diversity evaluation are presented in Table 1. SSR marker analysis was able
to identify a total of 71 alleles (Supplementary Table S2). The observed number of alleles per locus
ranged from 6 (at locus IM123) to 12 (at loci IM196 and IM327) with an average of 8.8 alleles and
4.3 eective alleles per locus. At locus IM348, out of eight alleles, allele 125 showed a frequency of 0.71;
thus locus polymorphism information content (PIC) was 0.466, while at locus IM164, allele 324 showed
a frequency of 0.68 resulting in locus polymorphism of PIC =0.480. In general, the number of eective
alleles was relatively low, indicating that rare and frequent alleles are present in the examined group
of samples. The highest numbers of eective alleles (5.5 and 6.2) were observed at loci IM196 and
IM327, respectively, where the frequencies of alleles were equally distributed. PIC values ranged
from 0.466 (at locus IM348) to 0.845 (at locus IM391), indicating sucient polymorphism information
content of all loci. Loci IM164 and IM348 were moderately informative (0.25 <PIC <0.5), while the
rest were highly informative (PIC >0.5). The expected heterozygosity varied between 0.490 (IM348)
and 0.877 (IM391), with an average of 0.728. The highest observed heterozygosity (0.871) was found at
locus IM123, and the lowest (0.129) was characteristic of locus IM164. The observed heterozygosity
was lower than expected on all loci except IM123. The probability of identity (PI) values were in a
range from 0.072 to 0.357, and the total PI calculated for all loci was 2.01
×
10
7
, indicating a low
probability of identical genotypes.
The UPGMA clustering analysis (Figure 3) discriminated 28 genotypes and revealed three distinct
groups of samples. The first cluster contained samples of mostly tall bearded Alpine-Dinaric taxa:
I. xcroatica,I. xgermanica L., I. illyrica,I. pallida Lam., I. pumila L., I. reichenbachii Heu., and I. xrotschildii,
grouped in two subclusters. The second cluster (with several smaller subclusters) consists of all samples
of narrow endemic dwarf species I. adriatica, its closely related species I. attica Boiss. & Heldr. as well
as I. barbata cultivar, a horticulturally-widespread variety in the region. All samples of I. sibirica L.
sensu lato grouped in the third cluster, together with I. pseudacorus L. and I. graminea L. within a
separate subcluster.
Plants 2020,9, 1229 5 of 16
Table 1.
Values of observed (H
o
) and expected (H
e
) heterozygosity, number of alleles (n), eective
number of alleles (n
e
), polymorphic information content (PIC), and probability of identity (PI) of
8 microsatellite loci for all studied samples of the Alpine-Dinaric taxa of the genus Iris.
Locus n neHoHePIC PI
IM93 9 4.1 0.452 0.769 0.727 0.131
IM123 6 4.0 0.871 0.763 0.712 0.172
IM164 7 2.0 0.129 0.518 0.480 0.292
IM196 12 5.5 0.500 0.833 0.805 0.079
IM200 8 3.4 0.387 0.721 0.672 0.178
IM327 12 6.2 0.593 0.855 0.821 0.085
IM348 8 1.9 0.194 0.490 0.466 0.357
IM391 9 7.2 0.714 0.877 0.845 0.072
Average 8.8 4.3 0.480 0.728 0.691 -
Total - - - - - 2.01 ×107
Plants 2020, 9, x FOR PEER REVIEW 5 of 16
The UPGMA clustering analysis (Figure 3) discriminated 28 genotypes and revealed three
distinct groups of samples. The first cluster contained samples of mostly tall bearded Alpine-Dinaric
taxa: I. x croatica, I. x germanica L., I. illyrica, I. pallida Lam., I. pumila L., I. reichenbachii Heuff., and I. x
rotschildii, grouped in two subclusters. The second cluster (with several smaller subclusters) consists
of all samples of narrow endemic dwarf species I. adriatica, its closely related species I. attica Boiss. &
Heldr. as well as I. barbata cultivar, a horticulturally-widespread variety in the region. All samples of
I. sibirica L. sensu lato grouped in the third cluster, together with I. pseudacorus L. and I. graminea L.
within a separate subcluster.
Figure 3. UPGMA dendrogram obtained with Dice’s similarity coefficient based on eight SSR
markers for 31 out of 32 collected Iris samples (as explained in the Material and Methods section);
Bootstrap percentages (>50) are shown in the nodes of the dendrogram; labels I–III denote major
clusters.
2.2. Chloroplast Barcodes Analysis
The maximum likelihood (ML) analysis was used in reconstructing phylogenetic relationships
of a heterogeneous group of Iris species based on two plastid markers (rpoC1, ndhJ). ML analysis
discriminated three major clusters of which seven groups of taxa and 10 different genotypes (Figure
4). In the ML dendrogram, three main groups of irises were discriminated, with I. reichenbachii
separated from the rest. The first group consisted of five undiscriminated mostly Alpine-Dinaric
species. Dwarf bearded irises I. adriatica, I. pumila, and I. attica were not separated and grouped with
I. barbata cult. in the second cluster. Both subspecies of I. sibirica grouped in a third cluster, together
with the out grouped I. graminea and I. pseudacorus. The samples accessed from NCBI gene repository
Figure 3.
UPGMA dendrogram obtained with Dice’s similarity coecient based on eight SSR markers
for 31 out of 32 collected Iris samples (as explained in the Material and Methods section); Bootstrap
percentages (>50) are shown in the nodes of the dendrogram; labels I–III denote major clusters.
Plants 2020,9, 1229 6 of 16
2.2. Chloroplast Barcodes Analysis
The maximum likelihood (ML) analysis was used in reconstructing phylogenetic relationships
of a heterogeneous group of Iris species based on two plastid markers (rpoC1,ndhJ). ML analysis
discriminated three major clusters of which seven groups of taxa and 10 dierent genotypes (Figure 4).
In the ML dendrogram, three main groups of irises were discriminated, with I. reichenbachii separated
from the rest. The first group consisted of five undiscriminated mostly Alpine-Dinaric species. Dwarf
bearded irises I. adriatica,I. pumila, and I. attica were not separated and grouped with I. barbata cult. in
the second cluster. Both subspecies of I. sibirica grouped in a third cluster, together with the out grouped
I. graminea and I. pseudacorus. The samples accessed from NCBI gene repository I. missouriensis Nutt.,
I. sanguinea Hornem., and I. gatesii Foster were grouped appropriately, according to their classification
within the genus Iris.
I. reichenbachii
I. rotschildii
I. illyrica
I. germanica
I. gatesii
I. pseudacorus
I. graminea
I. missouriensis
I. sibirica erirrhiza
I. sibirica
I. sanguinea
I. adriatica
I. barbata
I. attica
I. pumila
I. pallida
I. croatica
x
x
x
cult.
subsp.
subsp.
I
III
0.007
sibirica
II
LEGEND:
Iris, Iris
Limniris, Limniris
section
section
subgenus series
Oncocyclus
Iris,
section
Elatae
Laevigatae
Longipetalae
Pumilae
Sibiricae
Spuriae
Figure 4.
Maximum Likelihood (ML) tree of 32 Iris samples and sequences from NCBI (http://www.
ncbi.nlm.nih.gov) (I. missouriensis,I. sanguinea,I. gatesii) based on two plastid markers (rpoC1,ndhJ).
Bootstrap percentages are shown in the nodes of the dendrogram; labels I–III denote major clusters.
2.3. Genome Size
4
0
,6-diamidino-2-phenylindole (DAPI) fluorochrome is known to bind to DNA, specifically,
to AT base pairs and therefore lower values for absolute genome size analysis are found [
32
].
The determination of total DNA content of plants from DAPI stained cells was: For I. adriatica
12.639
±
0.202 pg (2C) and for I. xgermanica 24.249
±
0.163 pg (2C) respectively as compared to the
Pisum sativum cv. Kleine Rheinländerin (9.07 pg/nucleus) internal standard.
3. Discussion
In our study, we applied 8 SSR markers developed by Tang et al. [18] which proved to be highly
polymorphic and amplified alleles across the 39 Iris ecotypes and cultivars. We were not able to utilise
the IM61 marker recommended but the remaining markers provided sucient resolution to distinguish
between our samples. We observed the greatest allelic diversity on IM196 and IM327 in concurrence
with the aforementioned study; however, the observed number of alleles per locus in our study was
significantly lower (average 8.8) suggesting greater phylogenetic similarity across all of our samples.
Although it is comparable with the average number of alleles per locus observed within the group of
13 yellow-flag, Siberian, and tall-bearded Iris cultivars analysed by [
18
]. In our case, a small population
size could be the reason for low allele frequency. Genetic similarity ranged from 0.23 to 0.8 and 0.26 to
1.00 among Alpine-Dinaric taxa from the subgenus Iris (section Iris) grouped in the UPGMA clusters I
Plants 2020,9, 1229 7 of 16
and II, respectively. The highest genetic similarity was intraspecific (Dice =1; I19 and I21; I13 and
I19; I30 and I31), whilst the lowest were interspecific (Dice =0.23; I22 and I41 in cluster I; Dice =0.26;
I16 and I10; I16 and I11 in cluster II). Genetic similarity between endemic dwarf ecotypes of I. adriatica
grouped within a separate subcluster, and correlated with the locations of origin, ranged from 0.55 to
1.00, implying significant and disperse genetic diversity among ecotypes. Taxa from the subgenus
Limniris (section Limniris) displayed genetic similarity in a range from 0.07 to 0.72, the highest between
samples of I. sibirica subsp. erirrhiza. Only a few SSR markers were needed to identify (distinguish)
ecotypes and species.
The unique microsatellite profiles were established as described in the method section below,
nevertheless, we acknowledge that the SSR analysis can dier from lab to lab as the method inherently
produces high numbers of edge cases where a judgment call has to be made. An example of an edge
case is the apparent presence of 3 alleles in what we presumed (and confirmed for I. adriatica) to be
2n =2x species. As described, this was resolved by establishing a common SSR profile for those
particular samples, since our subsequent analysis methods rely on the binary presence or absence
of a particular allele and a presence of 3 alleles would likely confound the result and be factually
incorrect. To resolve such an edge case a full sequencing run could reveal genomic mutations, such as
translocation, or perhaps other properties of the genome at that position which would allow the probes
to bind in this particular way. Further, as I. xgermanica is a suspect tetraploid [
5
,
18
,
33
], the additional
genetic information could skew the subsequent phylogenetic analysis as additional peaks appeared
in positions only in one individual and could thus not be compared to any other values in the study,
carrying an extremely low PIC. For our analysis such peaks were considered to be outliers; however,
we are not suggesting they are not valid data in dierent subsamples.
This means that for any analysis the attribution of a particular profile needs to be internally
consistent and cannot be used at face value form any further studies which want to include the same
dataset. In our case, we employed the algorithm described in the methods to come to a conclusion which
was cross-examined within the research group to preserve the established logic of sorting dierent
cases. The final analysis of genetic relationship relies on the presence and absence of specific alleles so
for our purposes the aim was to obtain the same profiles for the same species when attributing an SSR
profile, without knowing which species the profile belongs to. Since a matching algorithm can only be
established ad-hoc after accessing the reads, there is a potential to introduce some bias into edge-case
decision making. Nevertheless, we are confident in our results several reasons; sample duplicates were
included as an internal control and independently produced the same profiles using the same “blind”
determination method, the chloroplast marker analysis largely produced the same clustering, profile
dierences between presumed same species are minimal, our described SSR relationship mirrors the
relationships which were confirmed or predicted using taxonomic, botanical or other methods.
Dierent combination of chloroplast genome sequences were proposed for species discrimination,
such as rpoC1,rpoB, and matK;rpoC1,matK, and psbA-trnH; [
34
] and rbcL and trnH-psbA [
35
]. In a
recent review [
23
], authors Saddhe and Kumar discussed the utility of plastid markers to dierentiate
between dierent species within plant divisions, where they establish ndhJ as a good candidate marker
for barcoding angiosperms. Additionally, rpoC1 is often used as a supplementary marker to increase
the barcoding depth of samples [
36
]. Plant Working Group (PWG) of the Consortium for the Barcoding
of Life (CBOL) recommended the combination of rbcL and matK as the plant barcode [
20
], while rpoB
and here applied rpoC1 showed markedly lower discriminatory power. Chloroplast marker matK
is recommended as one of the best DNA barcoding candidates for species discrimination [
20
,
37
].
However, this chloroplast region proved to be dicult to amplify and sequence in certain taxa,
and additional universal primers and optimisation of PCR reactions were necessary [
38
,
39
]. In our
study, the preliminary amplification of matK sequences was unsuccessful and the testing of additional
plastid markers is foreseen. However, the combination of ndhJ and rpoC1 revealed to be adequate
for discrimination up to the series taxonomic level, indicating the possibility of applying additional
candidates for the species discrimination. As discussed, a plastid marker with sucient resolution
Plants 2020,9, 1229 8 of 16
would be operationally favourable for widespread utility in discriminating between dierent species.
Up to date a few phylogenetic studies based on chloroplast markers were carried out on Iris [
6
,
40
42
].
Neither ndhJ nor rpoC1 was not tested in any Iris genus study.
Groupings of the Iris taxa from the broader Alpine-Dinaric area, observed in our research by both
sets of molecular markers (Figures 3and 4), mostly correspond to proposed phylogenetic relationships
based on palynological features [
8
]; a clear distinction between the subgenera Limniris and Iris and
within the majority of the lower taxonomic Iris categories of sections and series emerges. The anticipated
exception is the position of analysed NCBI sequence of Middle Eastern species I. gatesii (Figure 4),
which separated within the subgenus Iris in an individual cluster, as it belongs to the dierent series
Oncocyclus (Siemssen) Baker [
1
]. However, the unexpected exceptions are positions of the species
I. pumila based on SSR markers (Figure 3), and of I. reichenbachii based on ML analysis (Figure 4).
Molecular analysis of both sets of markers (Figures 3and 4) in principle resulted in the creation of three
main clusters: Two of three clusters covering rhizomatous taxa from the subgenus Iris, section Iris,
with a beard (“Pogoniris”, [
3
]), while the taxa from the subgenus Limniris, section Limniris, rhizomatous
irises with falls without a beard (“Apogoniris”, [
3
]) were grouped in the third cluster (Figures 3and 4).
For the ML analysis control NCBI sequences: Of I. sanguinea (subgenus Limniris; sect. Limniris, series
Sibiricae (Diels) Lawrence) and I. missouriensis (subgenus Limniris; sect. Limniris, series Longipetalae
(Diels) Lawrence), grouped with other members of the same subgenus (Figure 4); and of I. gatesii
(subgenus Iris; section Oncocyclus) made a separate branch between samples of “Apogoniris” and the
rest of the “Pogoniris” (Figure 4). Such results are in agreement with previous studies and monographs
of the genus Iris [1,3,11,41,43].
Within the subgenus Iris, section Iris, on the series level, one cluster (based on both sets of molecular
markers; Figures 3and 4) comprises the group of mostly tall bearded irises and covers the series Elatae
Lawr. [
10
]. The second cluster covers the group of dwarf bearded irises and matches the series Pumilae
Lawr. [
10
], except for I. pumila grouping in the first cluster based on SSR markers analysis (Figure 3).
However, plastid markers (Figure 4) did not discriminate analysed taxa within neither series Elatae (the
only exception is I. reichenbachii) nor Pumilae. In our study chloroplast markers ndhJ and rpoC1 provide
a weaker resolution into the species, concurrent with other authors [
22
]; however, we acknowledge that
the analysis of sequence data is quicker and much less prone to human error and enables clustering
comparison across dierent studies if the sequences are made publicly available. Further, our study
looked at only two plastid regions, as compared to eight microsatellite loci. Therefore, we would
recommend the utilisation of SSR markers for subsequent analysis supplemented by a plastid marker
combination for the genus Iris, until a single plastid marker combination is established as a convention.
According to SSR markers analysis (Figure 3), within the cluster I, two subgroups were formed:
In the first are two samples of tall bearded I. xcroatica,I. xgermanica, and, unexpectedly, dwarf bearded
I. pumila, whereas one sample of I. xcroatica is grouped with other analysed tall bearded irises within the
second subgroup. Although its taxonomic position is critical and still unresolved, the taxon I. xcroatica
is considered as a native endemic taxon in northern Croatia and Slovenia [
12
,
13
,
44
]. Likely due to
morphological similarities, it is often mixed with and named as a synonym for I. xgermanica [
1
,
2
,
5
,
13
],
which is, in our opinion, distributed worldwide only as a cultivated hybrid species [
1
,
9
]. The fact that
the WCSP [2] wrongly “declares” I. croatica Horvat & M.D. Horvat as an illegitimate name, due to an
incorrect replacement with I. croatica Prodan, provokes further taxonomic confusion [
45
], explained in
detail in [
5
]. The close relationship between I. x croatica and I. xgermanica was noticed by examining
both plant and pollen morphology [
8
] (B. Miti´c, personal observations) and is confirmed with our
results—their joint sub clustering (Figure 3). However, they are both tetraploids of yet unresolved
origin with reported chromosome numbers of 2n =44 for I. xgermanica, and 2n =48 for I. xcroatica [
5
,
46
].
Two earlier speculations about (auto) tetraploid origin of I. xcroatica both agreed that the progenitor
species for that hybrid is I. pallida, although this is yet to be cytogenetically confirmed [
5
,
8
]. Grouping
a sample of I. xcroatica together with I. pallida and I. illyrica within the second subgroup in our results
(Figure 3) confirms the proximity of tetraploid I. xcroatica and presumed progenitor species I. pallida.
Plants 2020,9, 1229 9 of 16
Considering the clear discrimination within lower taxonomic subgroups such as series, obtained
by the applied marker systems (Figure 3), the status of other closely related taxa from the so-called
I. pallida complex could be discussed. Taxonomic relationships within the complex have not been
fully explored and it is not yet clear whether the taxa of this complex have the status of species
or subspecies. Namely, the majority of taxa from this complex (including representatives from our
research—I. pallida and I. illyrica) were defined at the species level and extracted, apart from the series
Elatae into the new series Pallidae (A. Kern.) Trinajsti´c [
47
]. Although earlier taxonomic researches of
I. pallida complex [
48
,
49
] have supported such taxonomic treatment of its taxa, a later palynological
study [
8
] indicated their return into the status of subspecies level (as classified by WCSP [
2
]), and of the
series Pallidae back into the series Elatae. Results of our study are in accordance with the last hypothesis
as both marker systems (Figures 3and 4) grouped members of those series closely together.
The taxon I. xrotschildii from the series Elatae also garners considerable attention in the context of
this study. So far, this narrow endemic iris is known from a single locality on Mt. Velebit (Croatia).
It is described as a natural hybrid between species I. illyrica and I. variegata L. [
1
,
50
] with observed
morphological, palynological, and cytogenetic variabilities [
46
]. Some of the mentioned features
confirm the hybridogenous origin of this taxon. Despite this, no further molecular studies have been
done on the taxon to confirm its claimed status. This is the likely reason it was recently considered as
a synonym of I. xgermanica by WCSP [
2
]. Unfortunately, due to hard-to-reach mountainous terrain
(with mines still present in the area) and the small number of specimens in the only known population
on Mt. Velebit (B. Miti´c, personal observations), only one sample of this taxon was included in our
analysis. Bearing this in mind, the SSR profile of I. xrotschildii that shares at least one allele on all
analysed loci with I. illyrica as well as their position in the same UPGMA subcluster additionally
support their parent-sibling relationship (Figure 3). Moreover, although I. xgermanica and I. xrotschildii
are presumed synonyms [
2
], their discrimination by SSR could disprove that assumption and would
favour the placement of I. xrotschildii within a separate taxonomic position. However, further extensive
detailed molecular study of I. xrotschildii and its presumed parents is needed to confirm both its
separate taxonomic status and its dierence with I. xgermanica.
Furthermore, unexpected discrepancies occur in the placement of I. reichenbachii, which was
positioned in the same UPGMA subcluster as I. illyrica,I. pallida, and I. xrotschildii (Figure 3) and also
as an outgroup in ML dendrogram (Figure 4). Namely, I. reichenbachii is native in mountainous regions
of the Balkan Peninsula and SW Romania, known as parental species of some natural hybrids [
5
],
and according to [
10
] was firstly placed in the series Pumilae. However, according to both chromosome
numbers 2n =24, 48 [
43
] and pollen analyses [
8
] it seemed to fit better in the series Elatae. Nevertheless,
outgrouping of I. reichenbachii in our ML analysis (Figure 4) might indicate its specific position between
two series that still needs to be explored, as it has the same number of chromosomes [
5
] and pollen
grains [
8
] as tall bearded irises and is morphologically quite dwarfish [
43
]. Further, its genome size (1C
value) is intermediate between some members of both series Elatae and Pumilae [33].
Cluster II in our study (Figures 3and 4) covers mostly dwarf bearded irises. However, except for
I. pumila based on SSR markers, grouping within the first cluster (Figure 3), together with tall bearded
I. xcroatica and I. xgermanica. Given current evidence, we speculate that the grouping may have
happened due to the normalisation of the chromosomal content applied, and treatment of SSR data as
codominant, with maximally two alleles counted. An additional element could be genetic variability
of I. pumila, evident from genome size of this tetraploid species (2n =32), diering in several previous
studies (e.g., 1C =13.20 pg [
27
]; 1C =6.81 pg [
33
]; 1C =10.64 pg [
51
]). Furthermore, this taxon is
supposed to have the same hypothetical ancestor as tall bearded irises (with x =4 [
3
,
43
]), and is often
known as the parental species (together with some tall bearded irises as second parents) of many native
and artificial hybrids [43].
On the contrary, all other investigated samples of dwarf bearded irises of the series Pumilae [
10
]
grouped in a separate cluster II based on plastid markers (Figure 4). Such results are in accordance
with pollen morphology of dwarf bearded irises [
8
,
52
] and confirm their separate taxonomic position,
Plants 2020,9, 1229 10 of 16
and belonging to the same I. pumila complex [
14
]. Since I. attica is the only member of the complex with
the status of a subspecies, and with others having equal rank of species in the WCSP [
2
], our results
(Figures 3and 4) suggest that they should be treated at the same taxonomic rank. Therefore, further
research is needed to corroborate (or disprove) our statement about taxonomic relationships within the
whole I. pumila complex.
Meanwhile, special attention in our study was dedicated to one member of the complex—a
relatively-recently described diploid (2n =16) species I. adriatica [
14
], native and endemic to Croatia.
Namely, to prepare the basis for its conservation, because of its nearly threatened species status [
13
],
we were particularly focused on its molecular features. Evidence about taxonomic and phylogenetic
values of palynological and phytochemical features of I. adriatica are well documented [
8
,
15
]. However,
thus far, this species has not been researched on a molecular level. In the present results (Figure 3)
we documented diversity of dierent populations of the species I. adriatica, showing the existence
of geographical ecotypes. In particular, the UPGMA grouping (Figure 3) of established ecotypes
corresponds well with the geographical origins of the samples (Figure 2, Supplementary Table S1):
The island populations (sample numbers I26 island of Braˇc; I10, I11, and I12 island of Cres) have
separated from the land coastal populations (Figure 3, Supplementary Table S1, other samples).
Therefore, we assume that island populations might be a specific ecotype of the typical species.
Within inland populations, we were particularly interested in the population of the hinterland
population “Brnjica-Pokrovnik”, which has been singled out as an ecotype based on phytochemical
analysis [
15
]. In our analysis (Figure 3, sample no. I18) it has a separate branch in the dendrogram,
although it is “surrounded” by other inland populations. Therefore, it is obvious that potential
inland ecotype(s) require additional investigations. One more reason in favour of the separation of
ecotypes is the fact that “Brnjica-Pokrovnik” population is growing on an open calcareous meadows
(mainly belonging to the Festuco-Koelerietum splendentis Horvati´c 1963 association), whilst the rest of the
researched populations grow on limited rocky pastures and hills (mainly belonging to the Stipo-Salvietum
ocinalis Horvati´c 1985 association), very often endangered by the succession, i.e., overgrowth
with macchia.
Furthermore, in this study we present the first genome size estimation of I. adriatica measured
by flow cytometry and expressed according to [
24
] as 2C value =12.639
±
0.202 pg. Observed value
of genome size for I. adriatica we could hardly compare with values of all other members of the
complex I. pumila, since the data are known only for tetraploid species I. pumila [
27
,
33
,
51
]. However,
as previously mentioned, data for this tetraploid species indicates its variability. Our results of genome
size value for diploid species I. adriatica are the first data about genome size for this strictly endemic,
near threatened species and should contribute to its future conservation. The 1C value of I. adriatica is
similar to that of tetraploid I. pumila obtained by [
33
]. Such results should confirm belonging of both
species to the same complex. Additionally, similar deviations in 1C values as in the species I. pumila,
were observed for the species I. x germanica: Our results of 2C =24.249 pg for this “control” species
could be compared to the result (1C =12.45) of [
27
], while the value of 1C =5.87 for the same species
was observed by [
33
]. Therefore, the genome sizes of critical taxa of the genus Iris require further,
more complex research.
In our results within the third cluster (Figures 3and 4) all samples of so-called “Apogoniris” taxa [
3
]
grouped together, further all are representatives of the subgenus Limniris, section Limniris. Such results
are in accordance with some previous research of molecular phylogeny of these taxa [
40
,
53
,
54
].
Additionally, our analysis based on both sets of markers (Figures 3and 4) resulted with dierent
subclusters within the subgenus Limniris. Namely, mentioned subgroups correspond well to the
series as a lower taxonomic level (according to [
1
,
10
]): Laevigatae (Diels) Lawrence (I. pseudacorus),
Sibiricae (Diels) Lawrence (both subspecies of I. sibirica), and Spuriae (Diels) Lawrence (I. graminea).
The analysed NCBI sequences of “Apogoniris” taxa (I. missouriensis and I. sanguinea) additionally
support that distinction (Figure 4), they grouped with other members of the subgenus Limniris,
Plants 2020,9, 1229 11 of 16
section Limniris. Moreover, I. sanguinea, which belongs to the series Sibiricae [
1
], grouped close to other
members of this series.
Furthermore, all samples of I. sibirica sensu lato (series Sibiricae) grouped apart from other members
of the subgenus Limniris (Figures 3and 4), and created further subclusters (Figure 3). This was especially
interesting because of the still unclear position of the Alpine-Dinaric mountain populations described as
subspecies of the typical I. sibirica species [
55
]. Although plastid markers (Figure 4) did not discriminate
I. sibirica subspecies, the results of SSR analysis (Figure 3) confirmed their dierentiation. This is also
in accordance with the presumption that I. sibirica subsp. erirrhiza might be a mountain ecotype [
46
],
which diers from the typical lowland subspecies I. sibirica subsp. sibirica [
55
]. This is particularly
interesting for further conservation of wild, especially endemic irises from that area. Namely, I. sibirica
subsp. erirrhiza was found only in several localities in Bosnia and Herzegovina, Croatia, and Slovenia
where it might be an endemic taxon [
46
,
55
]. The subclustering of I. sibirica subsp. erirrhiza samples in
our research and an extra subcluster of typical I. sibirica subsp. sibirica (Figure 3) additionally confirms
this distinction of subspecies as ecotypes. Unfortunately, in our study we did not have a sample of
the population of I. sibirica subsp. erirrhiza from Mt. Bjelolasica (Croatia), the supposed link between
the subgenera Limniris and Iris in the territory of Southern Europe [
8
]. Further research focused on
broader ecotype samples of I. sibirica sensu lato is needed to give a better insight into the phylogenetic
structure within this complex taxon.
Regarding other representatives of the subgenus Limniris in our study, we can comment on
the specific position of the species I. graminea, which separated in the distinct cluster in both trees
(Figures 3and 4). Therefore, our results might support the hypothesis that the species I. graminea is
probably the most primitive member of the subgenus Limniris on the Southern European territory [
8
].
Besides this, our analysis of microsatellites (Figure 3) might also confirm the opinion based on
palynological observations, that the subgenus Iris is more advanced than the subgenus Limniris [
8
,
56
].
In closure, we can confirm that our results of the molecular study of Alpine-Dinaric taxa of the
genus Iris correspond well with their positions within the subgenera Iris and Limniris, and are in
accordance with some other recent molecular researches of taxa of the genus Iris [
41
,
57
]. Additionally,
our results present the first molecular data on narrow endemic and near threatened species I. adriatica
and also support the separate taxonomic status of investigated ambiguous regional taxa (e.g., I. sibirica
subsp. erirrhiza,I. xcroatica and I. xrotschildii).
4. Materials and Methods
4.1. Plant Material and DNA Extraction
Plants of the genus Iris distributed across the broader Alpine-Dinaric region were collected either
in their natural habitats during the vegetation seasons 2016–2018, retrieved from botanical collections
of the National Botanical gardens in Zagreb (Croatia) and Ljubljana (Slovenia) (Supplementary Table
S2). Most vouchers are live specimens deposited within the Iris collections of the mentioned Botanical
Gardens in Zagreb and Ljubljana, and one in the private garden of the corresponding author. Herbarium
voucher specimens are deposited in the herbarium of the Istrian Botanical Society, Vodnjan, Croatia
(not yet registered in the Index Herbariorum). Total genomic DNA was isolated from 25–100 mg
dried or fresh leaves, depending on the sample, using the commercial kit PureLink
®
Plant Total DNA
Purification Kit (Invitrogen
TM
; Waltham, Massachusetts, USA), in accordance with the manufacturer’s
instructions. One sample (I24; I. sibirica subsp. sibirica; Supplementary Table S2) was excluded from
SSR analysis due to poor imaging signals.
4.2. Microsatellite and Chloroplast Barcodes Amplification
Eight SSR markers [
18
] were used for genotyping (Supplementary Table S2), following the
optimised procedures described in [
18
]. Forward SSR primers were end-labelled with one of three
fluorophores, 6FAM, HEX, or TAMRA (Supplementary Table S3). Briefly, the initial denaturation step
Plants 2020,9, 1229 12 of 16
was performed at 95
C for 3 min, followed by 1 cycle of 94
C for 30 s, 55–64
C (depending on optimal
annealing temperature (T
a
)) for 30 s and 72
C for 45 s. The annealing temperature was decreased
1
C per cycle in subsequent 7 cycles until reaching the optimal T
a
(Supplementary Table S3) at which
35 cycles were carried out, with a final extension at 72
C for 20 min. The PCR products were checked
on 2% agarose gels to confirm amplification. The length of the PCR products was determined through
capillary gel-electrophoresis (Macrogen Europe B.V., Amsterdam, the Netherlands). SSR alleles were
resolved on the ABI3730XL DNA Analyser (Applied Biosystems
TM
; Waltham, Massachusetts, USA),
using GeneMarker
®
Software V2.7.0 (SoftGenetics, State College, Pennsylvania, USA) and 400HD
ROXTM dye-labelled internal size standard marker. SSR peak estimates were determined using inbuilt
software on pre-set settings. Each peak was individually evaluated. False positives were eliminated
by looking at peak values appearing at the same position in reads where no SSR probe was present
for a particular analyte, judged to be innate background. Due to slight shifts occurring at each read,
peaks from dierent runs, which were consistently dierent in length were judged to be the same
SSR profile [
58
]. All samples were described using a maximal value of two alleles at each SSR locus
examined normalised to a 2n =2
×
chromosomal content (Supplementary Table S2). Where more than
two alleles (peaks) were apparent their pattern was cross-examined with other available samples to
determine their unique descriptive allelic values.
A combinatorial approach of ndhJ and rpoC1 plastid markers (Supplementary Table S3) was used
for barcoding according to the procedure of [
59
]. The procedure consisted of an initial denaturation
step at 94
C for 5 min, followed by 35 cycles of 94
C for 30 s, 55
C for 30 s, 72
C for 60 s and a final
extension step at 72
C for 10 min. Before sequencing PCR products were additionally purified using
exonuclease I and shrimp alkaline phosphatase to remove unincorporated nucleotides and primers.
The barcodes were Sanger Sequenced using ABI 3130XL capillary machine (Biotechnology Faculty,
University of Ljubljana, Ljubljana, Slovenia) and submitted to GenBank (Supplementary Table S2).
Further three additional sequences (I. gatesii, GenBank: KM014691.1; I. missouriensis, NCBI Reference
Sequence: NC_042827.1; I. sanguinea, NCBI Reference Sequence: NC_029227.1) were mined from the
NCBI repository. Sequences were aligned using Codon Code Aligner V9.0.1 (CodonCode Corporation,
Centerville, MA, USA).
4.3. Data Analysis
Genetic parameters were calculated for 32 Iris samples over eight microsatellite loci (Table 1).
The number of amplified microsatellite alleles (n), number of eective alleles (n
e
), observed
heterozygosity (H
o
), and expected heterozygosity (H
e
) were calculated using POPGENE, version
1.32 [
60
]. Polymorphic Information Content (PIC) was calculated with the program Cervus, Version
3.0.7 [
61
] and probability of identity (PI) was determined using IDENTITY v.1.0 program [
62
].
Genetic distances between all pairwise combinations of the samples were calculated using Dice’s
coecient of similarity. The dendrogram was constructed from the resultant matrices via the UPGMA
distance-matrix method using the PAST software [
63
]. Statistical support for the tree topology was
assessed by 1000 bootstrap replicates. The two chloroplast loci (rpoC1 and ndhJ) sequence data were
aligned using the “Create Alignment” algorithm implemented in CLC Genomics Workbench 20.0.2.
Alignments were joined together and a Maximum Likelihood Neighbour-Joining tree was constructed
using the “Maximum Likelihood Phylogeny” algorithm of CLC using the Jukes–Cantor nucleotide
substitution model.
4.4. Genome Size Analysis
The DNA content of I. adriatica and I. xgermanica plants were analysed by flow cytometry
analysis according to the method reported in [
32
]. A portion of the fresh young leaves tissue of
approximately 1 cm
2
was used in sample preparation. For an internal standard, the Pisum sativum
cv. Kleine Rheinländerin (9.07 pg/nucleus) was used for reference. Both the sample and the standard
were chopped finely using a razor and released into 0.1 M citric acid containing 0.5% Tween 20.
Plants 2020,9, 1229 13 of 16
The homogeneous mixture was filtered through a 30-
µ
m nylon filter removing larger particles.
4
0
,6-diamidino-2-phenylindole (DAPI) was used as the genome staining dye. A 3–4-fold volume of
staining buer containing 4
µ
g ml
1
of DAPI in 0.4 M Na
2
HPO
4×
12H
2
O was added to the specimens.
Samples were analysed with a Partec CyFlow
®
Space flow cytometer using linear scale. FloMax
®
software (Partec, Münster, Germany) was used for the calculation of relative nuclear DNA content.
5. Conclusions
In the present molecular study of selected representative and critical Iris taxa from the wider
Alpine-Dinaric area, we enhanced the current knowledge and understanding of the genus Iris
taxonomy and phylogeny of the area; important for their further protection and conservation in
the study area. Our research showed taxonomic positions of investigated taxa within the genus Iris,
which is mostly in accordance with previous comprehension of the genus Iris. We were especially
focused on getting the first molecular data on the nearly threatened narrow endemic dwarf species
I. adriatica, hitherto molecularly unexplored. The results of molecular analysis showed that the 2C
value for this species is 12.639
±
0.202 pg, pointing to its relationship with other dwarf irises from
the I. pumila complex, and indicating the existence of ecotypes. Additionally, we stressed some,
presently unresolved, key taxonomic questions about certain critical groups and/or taxa of the genus
Iris from that area, and the most pertinent are: Taxonomic and phylogenetic relationships of some
complex Iris groups from this area (e.g., I. x germanica,I. pallida,I. pumila and I. sibirica groups) and the
taxonomic status of regionally recognised, but globally neglected endemic taxa: I. sibirica subsp. erirrhiza,
and natural hybrids I. xcroatica and I. xrotschildii. For mentioned groups and taxa our study establishes
baseline taxonomic and phylogenetic relationships across the Alpine-Dinaric region, but more precise
confirmation of their phylogenetic and taxonomic status require further, more complex molecular
analysis on a broader set of Iris samples. Regarding the contribution to the eorts of establishing
optimal molecular markers for detecting taxonomic and phylogenetic relationships within critical
taxa of the genus Iris, we would recommend the utilisation of SSR markers for subsequent analysis
supplemented with a combination of plastid markers until a plastid marker combination for the genus
is established and fully validated as convention. Chloroplast markers ndhJ and rpoC1 provide a weaker
resolution into the species; however, analysis of sequence data is quicker and much less prone to
human error. Further, our SSR study looked at 8 microsatellite loci as compared to two plastid regions.
Chloroplast markers can give further context to SSR analysis and provide independent control despite
their lower resolution as they can confirm broader clusters. For future studies of the genus Iris we
would additionally recommend the inclusion of other appropriate barcoding regions to serve the same
purpose and hopefully increase the sequencing resolution.
Molecular evidences obtained in this study, besides contribution to the knowledge on taxonomy
and phylogeny of the genus Iris in the Alpine-Dinaric, Mediterranean and Pannonian area, should also
help in further understanding about the importance of wild, especially endemic Iris taxa and encourage
their more intensive conservation eorts.
Supplementary Materials:
The following are available online at http://www.mdpi.com/2223-7747/9/9/1229/s1,
Table S1: Alpine-Dinaric taxa of the Genus Iris used in the present molecular study; Table S2: Genotypes of the
analysed Iris samples at eight microsatellite loci (allele sizes in bp); Table S3: SSR and chloroplast markers used in
the present molecular study of the Alpine-Dinaric taxa of the genus Iris.
Author Contributions:
Conceptualization, D.P., B.S., B.M., and T.W.; methodology, D.P., J.J., B.B., T.W.; sampling,
B.M., D.H.; S.B., N.L., M.M., and D.V.; formal analysis, T.W., D.P., J.J., B.B.; writing—original draft preparation,
D.P., T.W., B.M., B.S., and D.H.; writing—review and editing, J.J., B.B., B.S., B.M., T.W., D.P., D.H., S.B., N.L., M.M.,
and D.V.; visualisation, T.W., D.P., J.J., and D.H.; All authors have read and agreed to the published version of
the manuscript.
Funding:
The parts of this research were funded by the Slovenian Research Agency (SRA–ARRS), grant number
P4-0077, and the University of Zagreb, grant number 106-F20-00025. University of Westminster Distant Horizons
Award fund provided mobility support.
Plants 2020,9, 1229 14 of 16
Acknowledgments:
The authors would like to thank Miroslav Miti´c, Radni´c family, and Nediljko Ževrnja for
fieldwork assistance. We would also like to thank Jože Bavcon and Janja Makše, University Botanic Gardens
Ljubljana for their help in collecting selected Iris samples, and Botanical Garden, Faculty of Science, University
of Zagreb for providing us the living plant material of some taxa of the genus Iris from its collections. Thanks
are also extended to anonymous reviewers, whose valuable comments contributed to the final appearance of
the manuscript. We also thank the Institute of Agriculture and Tourism, University of Zagreb, and University
of Ljubljana for in kind support. Field research was performed with the permission of the Croatian Ministry of
Environment and Nature Protection (Decision no. UP/1-612-07/15-48/23).
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
... In addition to the 50 populations whose genome sizes were assessed in this study, we gathered published data for 53 species ( [14,[33][34][35][36][37][38][39][40][41][42] from the Plant DNA C-values database release 7.1. [15], and more recently published estimates [43,44]; Table S2). We did not include two accessions of imprecise species identification, I. aff. ...
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