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Flora 222 (2016) 68–85

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Flora

j o ur nal ho me page: www.elsev ier .com/ locate / f lora

orphological and molecular variation in selected species ofrysimum (Brassicaceae) from Central Europe and their taxonomicignificance

neta Czarna a,1, Barbara Gawronska b,1, Renata Nowinska a,∗, Maria Morozowska a,iotr Kosinski a,c

Department of Botany, Faculty of Horticulture and Landscape Architecture, Poznan University of Life Sciences, Wojska Polskiego 71C, 60-625 Poznan,olandDepartment of Biochemistry and Biotechnology, Faculty of Agronomy and Bioengineering, Poznan University of Life Sciences, Dojazd 11, 60-632 Poznan,olandInstitute of Dendrology, Polish Academy of Sciences, Parkowa 5, 62-035 Kórnik, Poland

r t i c l e i n f o

rticle history:eceived 6 February 2015eceived in revised form 10 July 2015ccepted 13 March 2016dited by Hermann Heilmeiervailable online 18 March 2016

eywords:arpology

a b s t r a c t

Carpological and molecular variation among the 14 species of Erysimum distributed in Central Europewas examined. Special attention was paid to the group of six wallflowers (E. hungaricum, E. pieninicum, E.wahlenbergii, E. virgatum, E. durum and E. hieracifolium) that are morphologically difficult to distinguishforming a complex taxonomic group. It was found that the sculpturing of seed testa and the micromorpho-logical patterns regarding the surface of silique septum obviously vary among the studied wallflowers,while seed dimensions and shapes overlap and are not adequately informative. The highest carpolog-ical dissimilarity was noted between E. cheiri and E. crepidifolium, whereas the taxa in the mentionedtaxonomic complex were carpologically very similar, only E. virgatum was slightly different. The molec-

ryptic speciesEMCR-RAPD

TSSCP

ular techniques used here including SSCP and restriction endonuclease-digested rDNA amplified by PCRand combined with RAPD, strongly support the genetic distinctiveness of all Erysimum species studied.Moreover, the results obtained here indicate that the group of five out of six taxonomically problem-atic Erysimum species consist of distinct, closely related species and may constitute a complex of crypticspecies.

© 2016 Elsevier GmbH. All rights reserved.

. Introduction

Erysimum L. is a genus of Brassicaceae that includes wild and cul-ivated plants originating from Europe, the Mediterranean region,outhwest Asia, and North America. As of yet, there is no estab-ished uniform list of species in the genus. Depending on theifferent taxonomical standpoints, the range of wallflower rich-ess on a global scale is between 150 (Al-Shehbaz, 1988, 2010)o 350 (Polatschek and Snogerup, 2002). Such a huge taxonomical
iscrepancy results mainly from the considerable morphologicalimilarity between Erysimum spp., which generates problems inerms of distinction between some species, as well as with dispen-
∗ Corresponding author.E-mail addresses: [email protected] (A. Czarna), [email protected]

B. Gawronska), [email protected] (R. Nowinska), [email protected]. Morozowska), [email protected] (P. Kosinski).

1 These authors contributed equally to this work.

ttp://dx.doi.org/10.1016/j.flora.2016.03.008367-2530/© 2016 Elsevier GmbH. All rights reserved.

sation to proper taxonomical rank (Ancev and Polatschek, 2006;Ghaempanah et al., 2012; Latowski, 1975; Mutlu, 2010; Polatschek,2010, 2011, 2012). On the other hand, Erysimum, when comparedto other genera, is rich in species and subspecies that are endemicto small areas (Brullo et al., 2013; Melendo et al., 2003; Quarmimet al., 2013; Piekos-Mirkowa and Mirek, 2003; Roberson, 2001).Some of the wallflowers are also included in the local and regionalRed Lists (Bilz et al., 2011; Colling, 2005; Korzeniak, 2001, 2008;Turis et al., 2014). Furthermore, the small number of diagnosticmorphological characteristics useful for the separation of criticalspecies contributes significantly to this complexity. These problemscould reflect rapid radiations in the genus that might have gen-erated cryptic species of almost identical morphology but whichare ecologically and/or geographically isolated (Moazzeni et al.,2014).

Erysimum and the entire Brassicaceae family exemplify the tax-onomical groups wherein distinguishing taxa only by traditionalmorphological description is especially difficult. The presence of

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ssemblages, called “species complexes”, is an additional difficultyn correct species identification. According to different authorspecies complexes may be characterized either by recent spe-iation or the speciation among them is still incomplete (Nosilt al., 2009; Schluter and Conte, 2009). Substantial taxonomiconfusion occurs in many plant genera, especially in the case oflosely related species, when the differences in morphologicalharacteristics are few. As a result, the question is which of the tax-nomic ranks (species/subspecies) is most adequate, or whetherryptic species may underlie many of these subtle morphologi-al variants. The assessment of the possible and correct status of

given plant can be complicated. A study by Perny et al. (2004)n Cardamine acris Griseb. (Brassicaceae) provided an example ofllopatric differentiation when classification at the level of sub-pecies was most adequate. All subspecies of C. acris had more oress overlapping values of morphological characteristics and, withhe exception of one, no diagnostic AFLP fragments were presentn them. Moreover, neither morphological characteristics nor habi-at preferences suggest adaptive differentiation. Similar patternsf such classification (including Erysimum) were found in sev-ral other species or species complexes in Brassicaceae (Akeroydnd Ball, 1993; Strid and Tan, 2002). Erysimum parnassi (Boiss.nd Heldr.) Hausskn. (Jalas and Suominen, 1994), or E. cheiri (L.)ranz subsp. inexpectans Véla, Quarmim and Dubset, described byuarmim et al. (2013) are the most known examples. The rank of

ubspecies is appropriate if the separation of populations has beenoo short to result in a distinct morphological divergence (Gregor,003; Lihova et al., 2000), or when the differentiation is counter-cted by occasional gene flow or introgression (Hodalova et al.,002).

On the contrary, cryptic species complexes are groups of closelyelated species that are difficult or impossible to distinguish by

orphological traits. Cryptic species (also called sibling species)ere defined by different authors, in a way that reflects their partic-

lar approach to the species question. Stebbins (1950) defined thems “population systems, which were believed to belong to the samepecies until genetic evidence showed the existence of isolatingechanisms separating them”. According to Wiley (1981): “Cryp-

ic species are species that cannot be diagnosed by morphology,ut that act as independent evolutionary lineages in nature”. Theyave proven to be common in different organisms, comprising ani-als, plants and bacteria. They are characterised by the presence of

istinct gene pools (which is reflected in different multilocus geno-ypes) and by the absence of morphological differences (if present,uch differences are slight only). Genetic variation patterns at thentraspecific and interspecific level differ considerably; therefore,nalysis of allele frequency in populations enables the determi-ation of whether these populations have a common gene poolr not, i.e. whether they represent the same or different biologi-al species. Populations representing separate gene pools (separateiological species), differ with regard to the presence of alternativelleles at some loci, being their diagnostic alleles (Baczkiewicz et al.,008).

At present, several DNA-based identification techniques haveeen developed. Molecular markers have provided an alternativestimate of genetic distinctiveness, and they provide a test for thepecies-level taxonomic status of many suspected cryptic plantpecies that are morphologically indistinguishable (Blouin, 2002;asuyama et al., 1994; Waycott et al., 2002; Yatabe et al., 2001).ne approach has been to use random PCR primers to amplifyenomic DNA fragments and look for species-diagnostic bandsmong the polymorphic DNA fragments (RAPD PCR) (Williams
t al., 1990). This method provides a quick and effective means ofdentifying genetic markers to distinguish closely related species.APD has been used in many studies of genetic relatedness of plantultivars and plant populations, as well as in the study of genetic
22 (2016) 68–85 69

relationships between very different plant species (Adam et al.,2002; Ahmad, 1999; Bartolozzi et al., 1998; Brown-Guedira et al.,2000; Galderisi et al., 1998; Lakhanpaul et al., 2000; Landry et al.,1994; Nkongolo et al., 2002; Rieger and Sedgley, 1998) and can beuseful for the initial detection of a cryptic species (Wilkerson et al.,1995).

Another strategy has been to analyse the structure and sequenceof a hypervariable region of the genome shared by all species. Theribosomal DNA (rDNA), which encodes the rRNA, has been a pre-ferred target. Both the architecture of the rDNA and the sequenceof certain domains of the genes are highly conserved; thus, rRNAgene sequences have been important tools for systematic stud-ies of highly diverged taxa. However, certain regions of the rDNAare especially variable between even closely related species (Abd-Elsalam et al., 2003; Downie and Katz-Downie, 1996; Salariatoet al., 2013; Wesson et al., 1992). Methods such as single-strandconformation polymorphism analysis (SSCP) may facilitate theidentification of diagnostic sequences and could be used to deter-mine whether a region exhibits polymorphisms between species(Kong et al., 2003). Then, restriction enzyme analysis (RFLP) orsequence analysis can be performed to detect differences amongmembers of cryptic species complexes.

Studies combining morphometric analyses with DNA-fingerprinting techniques are still infrequent, but they allowresearchers to draw taxonomic conclusions with a high degree ofconfidence (Perny et al., 2004; Whittall et al., 2004). Examples ofsuch analyses combining different methods have also been foundin the cases of closely related taxa within Erysimum, such as E. lini-folium J. Gay (Bellardi et al., 2013), E. capitatum Greene (Lay et al.,2013), and E. mediohispanicum Polatschek (Munoz-Pajares et al.,2011). A combination of morphological and molecular markerswas also successful in determining the genetic distinctiveness ofan endangered cryptic species (Whittall et al., 2004), including anexample of the occurrence of two cryptic species (E. nervosum s.s.and E. riphaeanum sp. nov.) within E. nervosum Pomel in Morocco(Abdelaziz et al., 2011).

The current study focuses on six taxonomically problematicCentral European wallflowers: E. hieracifolium L., E. durum J.Presland C.Presl (= E. marschallianum Andrz. ex M.Bieb.), E. hungaricumZapal., E. wahlenbergii Simonk., E. pieninicum Pawl., and E. virgatumRoth. Relations between them are interpreted against a backgroundof some other Central European wallflowers that are easier to dis-tinguish by morphological characteristics and, at the same time, areclosely related to this difficult group. They are: Erysimum diffusumEhrh., E. cheiranthoides L., E. cheiri (L.) Crantz, E. crepidifolium Rchb.,E. odoratum Ehrh., E. repandum L., E. witmannii Zaw., and Erysimumsylvestre (Crantz) Scop.

E. hieracifolium is common throughout much of Europe, whereasthe other species show a much more restricted range. Erysimumpieninicum is narrowly endemic in the Pieniny Mountains, whereasE. wahlenbergii is endemic in the Tatra Mountains. A short reviewof the systematic history of six problematic species in the 20th cen-tury shows how complex this problem is (Table S1). Until now, thetaxonomic identification of these species mainly relied on the mor-phology of leaves, flowers, and siliquae. The morphological studyof Pawłowski (1946) resulted in separation of these six species intotwo series, Virgata and Euhieracifolia. The only carpological studythat has been completed to date (Latowski, 1975) provides a simi-lar taxonomical classification. According to other results, these sixwallflower taxa were either classified as separate species (Latowski,1985; Mirek et al., 2002; Szafer et al., 1986) or as a species complex(Ball, 1964), or they retained the status of a species, while oth-
ers were reclassified as one of those species (Ball, 2010; Dostál,1989).
In this study, we evaluate different taxonomic concepts usinga combination of morphometric analyses with molecular variation

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RAPD and nuclear ribosomal ITS regions). We test two hypotheses:ne concerning whether problematic species should be treated as

genetically unique cryptic species and second regarding whetherhey should be treated as a species complex wherein a classifica-ion at the level of subspecies would be more adequate. We balancevidence for or against an each hypothesis. Significant molecularifferences with subtle differences of all carpological characteris-ics seem to support first hypothesis that the group of problematicpecies is possibly an example of complex cryptic species.

. Material and methods

.1. Plant material

This study focused on six problematic species. Most wallflowershat form this group have a restricted or scattered distribu-ion and low number of local populations. Due to difficultiesith identification of these wallflowers on the basis of theiracromorphological traits as well as due to the fact that the

pecimens from the same localities had been variously classi-ed in the past, we used in this study only such plant material

hat was unquestionable. We collect specimens from the mostnown localities, from the oldest noted localities of the taxar herbarium specimens collected by the author of the taxaame. Eight species that formed the comparison group occuruch more extensively, but for the sake of comparisons theyere collected in similar amounts as a group of problematic

pecies.Altogether, 19 populations of 14 species were included in the

tudy. Plant material was collected in a field, as well as was obtainedrom two herbaria and two botanical gardens in an attempt toeceive a complete list of taxa (Table 1). In the population originat-ng from field locations and garden collections, fruits and seeds (for

orphological observations) and leaves (for molecular study) wereollected from 5 to 10 specimens, depending on their availabil-ty. In case of the rest of the examined populations, plant material

as obtained from herbarium vouchers represented by one or twopecimens of each species, depending on their availability.

.2. Morphological analyses

Seed morphometric analyses were performed on samples ofpproximately 30 seeds (29–32) for each population. For each sam-le, seed length and width were measured and seed shape wascored according to the length-to-width ratio. In order to avoidorphological polymorphism, the seeds were collected from theiddle part of the siliqua.

Seed testa, the siliqua valve, and the outer and inner surface, asell as the sculpturing patterns of the fruit septum, were examined

sing scanning electron microscopy (SEM). Character states wereefined for the primary sculptural features (cell shape, anticlinalalls) and some secondary sculpture character states (periclinalall curvature and ornamentation). SEM observations were per-

ormed on a sample of five seeds and fruits for each species. Theerminology for seed and fruit features follows Barthlott’s (1981)ecommendations.

.3. Molecular analyses

.3.1. RAPDTotal genomic DNA was extracted from dried leaves following

he CTAB protocol (Doyle and Doyle, 1987) with minor modifi-
ations. Apart from herbarium specimens (1–2 individuals), fivendividuals per species in each population were sampled for thesolation of DNA (Table 1). For RAPD, each of the species was rep-esented by a DNA bulk. In order to detect differences in the rDNA
22 (2016) 68–85

region, 14 species (single individuals) were individually analysedwith the SSCP and RFLP techniques. This kind of sampling wasdesigned to cover a wide range of total variation present amongparticular species, rather than to assess intrapopulational variation.

PCR amplifications (Williams et al., 1990) were performed withdecamer oligonucleotides primers (Operon Technologies, Almeda,CA, USA). A total of 40 random primers (kits B and H) were screened,of which 14 were selected for further evaluation based on the qual-ity and number of bands amplified (they were polymorphic andproduced reproducible profiles). Amplifications were carried out ina reaction volume of 16 �l containing 2 × PCR Master Mix (Fermen-tas), 20 pmol RAPD primer, and 50 ng of template DNA. Reactionswere performed in an MJ Research Thermal Cycler PTC-100 pro-grammed for 40 cycles, divided into two stages, differing in termsof annealing temperature. The first 20 cycles were preceded by aninitial denaturation at 95 ◦C for 300 s, then each cycle was com-posed of a denaturation step at 92 ◦C for 90 s, an annealing stepat 35 ◦C for 90 s, and an extension step at 72 ◦C for 120 s, afterwhich, in order to enhance specificity, the annealing temperaturewas increased to 38 ◦C. Another 20 cycles followed, in which theother stages remained unchanged. The reaction was completed byfinal synthesis at 72 ◦C for 300 s and storage at 4 ◦C until turned off.Part of each reaction mixture, for the control of PCR, was separatedon 1.5% agarose gel in the presence of ethidium bromide, in 1 × TBEbuffer (100 mM Tris-HCl, pH 8.3, 83 mM boric acid, 1 mM EDTA) at70 V. Selected reactions (the rest of PCR mixtures) were loaded on8% polyacrylamide gel and separated in a 0.5 × TBE buffer at 70 V.The gels were visualised under UV after staining with ethidium bro-mide and were documented using a gel documentation and imageanalysis system (GBox Syngene, UK). The sizes of the amplificationproducts were determined by comparison to 50 bp or 100 bp laddermarkers from Fermentas (Germany).

Only those bands that could be unequivocally scored were con-sidered for analysis. The molecular size of each fragment wasestimated using the Gene Tools software programme from Syn-gene Biotech. Each population/taxon was scored for the presence(1) or absence (0) of every amplification product, and the data wereentered into a binary data matrix. The obtained genetic profiles alsowere analysed in terms of polymorphic RAPD markers that werecommon between the analysed populations and those that wereunique to each species.

2.3.2. ITS amplification and SSCP analysisAmplification of the ITS region (ITS1, the 5.8S gene and ITS2)

was performed with the two pairs of universal primers (Whiteet al., 1990), ITS-1 and ITS-2, and ITS3 and ITS-4. The PCR reactionswere performed on a PTC-100 Thermal Cycler in a total volumeof 25 �l containing 2 × PCR Master Mix (Fermentas), 50 pmol ofeach primer, and 50 ng of template DNA. PCR was programmedwith an initial denaturing at 95 ◦C for two minutes, followed by25 cycles of 95 ◦C for 30 s, 55–60 ◦C (depended on primer used)for 30 s, and 72 ◦C for 1–2 min, with a final extension at 72 ◦C for10 min.

To obtain an equal amount of DNA loading for SSCP elec-trophoresis, three microliters of PCR products were resolved byelectrophoresis in 1.5% agarose gels in the presence of ethidiumbromide (estimation by its band intensity). Images were capturedas described above. Then, three to five microliters of PCR productswere mixed with 6–7 �l of a denaturing buffer (95% formamide,20 mM EDTA, and 0.05% bromophenol blue) to a total volume of12 �l. The mixtures were heated at 96 ◦C for 10 min and then chilledon ice. Eight microliters of the denatured PCR products and 4 �l
of the DNA ladder were loaded on an 8% or 10% non-denaturing,acrylamide:bis (49:1) containing glycerol gel. The denatured PCRproducts were electrophoresed overnight in a 0.5 x TBE buffer ata constant voltage (70 V) at room temperature. After electrophore-

A. Czarna et al. / Flora 222 (2016) 68–85 71

Table 1List of Erysimum species analysed in the study, they geographic origin and sources from which they were obtained.

Species Abbreviations Locality/habitat Latitudes,longitudes

Collection data Collector/authorof determination(verification)

Field collection(F)/herbariumsymbol

E. durum du PL, Mieszków, Jarocincommunity, on railway track

51◦58′N, 11◦30′E 05.07.2009 A. Czarna/A. Czarna F

E. wahlenbergii wa1 PL, Zakopane, MountainousBotanical Garden of INC PAS


E. wahlenbergii wa2 SK, Slovak Tatra Mountains,Zdiar, Monkowa Valley, amongrocks by the foot trail


E. pieninicum pi1 PL, Pieniny Mountains, HomoleGlen, at the foot of the rocks


E. pieninicum pi2 PL, Poznan, systematic gardencollection of Adam MickiewiczUniversity Botanical Garden inPoznan

52◦25′N, 16◦53′E 2010 Index seminum F

E. pieninicum pi3 PL, Pieniny Mountains, on rocksbelow the Czorsztyn castle


E. hungaricum hu UA, Łostun −1653 above sealevel, Czywczynskie Mountains

48◦44′N, 24◦51′E 16.08.1912 H. Zapałowicz/H. Zapałowicz(A. Polatschek)

KRAM

E. hieracifolium hi PL, Rogusko, Nowe Miastocommunity by theWarta river,on railway embankment


E. virgatum vi AT, Tirol, Innsbruck Landdistrict

47◦15′N, 11◦23′E 1881 M. Marr/M. Marr POZ

E. cheiranthoides chs PL, Poznan/city green area 52◦25′N, 16◦58′E 28.08.2001 A. Czarna/A. Czarna FE. odoratum od1 PL, Olkusz, Jura

Krakowsko-Czestochowska,Jewish Cemetary


E. odoratum od2 PL, Olsztyn, JuraKrakowsko-Czestochowska, onlimestone rocks


E. diffusum di UA, Dniester River valley,Kołodróbka, Babince(Babuchów)

48◦38′N, 26◦01′E 10.08.1913 M. Sychowa/T. Wilczynski KRAM

E. repandum re PL, Czarnów, Kieleckie province 50◦88′N, 20◦′57′E 15.07.1922 K. Kazanowski/K. Kazanowski POZE. sylvestre sy SI, Gorenjska district, on the

slope of the at 850 above sealevel

46◦28′N, 13◦′58′E 30.06.1957 Tonelveber/Tonelveber POZ

E. crepidifolium cr SK, Flora Polonica Ex., Sároscounty, bushes on limestonerocks

49◦17′N, 21◦16′E 12.07.1894 E. Wołoszczak/E. Wołoszczak POZ

E. cheiri chi PL, Poznan, garden collection ofPoznan University of LifeSciences


E. witmannii wi1 PL, Pieniny Mountains,Szczawnica, by the bridge on


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Dunajec riverE. witmannii wi2 PL, Pieniny Mountains, Sokola

Perc foot track, on rocks49◦2

is, silver staining was carried out using the standard procedure.he gels were dried and documented as pictures using a scan-er or gel documentation and image analysis system, as describedbove.

.3.3. RFLPFor enzyme digestions, amplification was performed in 100 �l of

eaction mixture using ITS-3 and ITS-4 primers, as described above.hen, to remove excess of dNTPs and primers, the reaction mixtureas precipitated by adding 0.1 vol of 3 M sodium acetate and 2.5

ols of absolute ethanol, after that, DNA was resuspended in ster-lised distilled water. The amplified products (8 �l) were digestedor two to four hours at an appropriate temperature with the fol-owing nine restriction enzymes: EcoRI, PstI, HinfI, HaeIII, MboI,

seI, DraI, AluI, and MspI. Fragment patterns were analysed on% agarose gels containing ethidium bromide and documented, asescribed above. Restriction fragments were sized using the Geneools software from Syngene.

0◦26′E 25.07.1953 H. Piotrowska/H. Piotrowska POZ

2.4. Statistical analyses

The differences in seed length, width and shape were exam-ined with the Mann–Whitney U test. Six taxonomically problematicspecies were tested on the populations level (each population wastreated as one sample), as well as on the species level (all popula-tions of the same species were treated as one sample).

Binary matrices from RAPD and ITS RFLP data were constructedand Jaccard indices were calculated with the combined Gene Toolsand Gene Directory software from Syngene Biotech. The genetic dis-tance (obtained by subtracting the Jaccard coefficient from 1) andmorphological distance (Euclidean) matrices were used for the con-struction of minimum spanning network graphs with SplitsTree4 v.4.13.1 software (Huson and Bryant, 2006).

A Mantel test was performed to analyse the autocorrelationbetween morphological and genetic distance matrices in GenAlEx
v. 6.5 software (Peakall and Smouse, 2012). Significance of the rela-tionship between morphological and genetic distances was testedwith 999 randomly permutations.

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Flora 222

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Table 2Seed coat sculpturing and the characters of epidermal cells. Six problematic species are in bold.

Species Cellular pattern Primary sculpture Secondary sculpture

Outlineof cells

Anticlinalwalls

Relief of cellboundary

Periclinal walls striations ofanticlinal walls

cuticular bandsjoining thecentral structure

A* B C D E Curvature Shape of thecentralprotuberances

mashroom cone other

E. hungaricum (hu) blister + + channelled concave + +E. wahlenbergii (wa) blister + + raised concave + +E. pieninicum (pi) blister + + raised concave + +E. virgatum (vi) blister + + raised concave + +E. durum (du) blister + + raised concave + +E. hieracifolium (hi) blister + + raised concave + +E. cheiranthoides(chs) blister + + raised concave + +E. cheiri (chi) reticulate + + raised flat +E. crepidifolium (cr) blister + + raised concave + +E. diffusum (di) blister + + raised concave + +E. odoratum (od) blister + + raised concave + +E. repandum (re) blister + + raised concave + + +E. sylvestre (sy) occelate + + raised flat/concave + +E. witmannii (wi) blister + + raised concave + +

* A—isodiametric, penta- or hexagonal; B—isodiametric, some penta- or hexagonal, some almost circular; C—straight; D—straight and narrow ridge-shaped; E—straight and broad roll-shaped.

Table 3Fruit valve and septum micro-ornamentation pattern and the characters of epidermal cells. Six problematic species are in bold.

Species Fruit valve Fruit septum

Outer surface Inner surface Cellular pattern Hairs Shape ofepidermal cells

Hair density Type of hairs Cellular pattern Outer walls Lateral walls Presence of rugose striate L S M

1* 2 3 A** B C striate reticulate rugose CX CV X Y Z hairs secondary cuticlestriations

E. hungaricum (hu) + + + + + + +E. wahlenbergii (wa) + + + + + + +E. pieninicum (pi) + + + + + + +E. virgatum (vi) + + + + + + +E. durum (du) + + + + + + + +E. hieracifolium (hi) + + + + + + + +E. cheiranthoides (chs) + + + + + + +E. cheiri (chi) + + + + + + + +E. crepidifolium (cr) + + + + + +E. diffusum (di) + + + + + + +E. odoratum (od) + + + + + + +E. repandum (re) + + + + + + + + +E. sylvestre (sy) + + + + + + +E. witmannii (wi) + + + + + + + +

* 1—strong; 2—moderate; 3—weak.** A—many-armed hairs; B—two-armed hairs; C—mixed types of hairs; CX—convex; CV—concave; X—lateral walls running parallel to the fruit axis are convex and those running perpendicularly to it are concave; Y—lateral walls

running parallel to the fruit axis are concave and those running perpendicularly to it are convex; Z—all lateral walls convex; L—most of cells are long; S—most of cells are short; M—similar participation of long and short cells.

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. Results

.1. Morphological analyses

Lengths of the seeds of the investigated Erysimum species rangedrom 1.01 mm to 3.04 mm, whereas the width of the seeds rangedrom 0.50 mm to 2.13 mm. The length-to-width ratio (regarded aseed shape) ranged from 1.00 to 3.32. However, despite the facthat the minimum and maximum values of each trait overlappedetween populations (Tables S2–S4), the examined populationsiffered significantly in seed length (Kruskal–Wallis test k = 18,

= 572, H = 437.4, p < 0.001), width (H = 415.9, p < 0.001), and shapeH = 380.4, p < 0.001).

Detailed analyses (using the Mann–Whitney U-test) were pre-ared for problematic wallflowers at the population level (Tables2–S4) and next, at the species level (Table S5). The first showedhat problematic species were characterised by the medium-sizednd large-sized seeds, as compared to most of representativesf the other species. Size and shape of seeds changed graduallyetween populations. The seed length ranged from 1.25 mm to.44 mm, dividing species into two aggregations (Table S2). Therst aggregation (with species that had shorter seeds) clusteredost of problematic species. Erysimum wahlenbergii (wa1) and E.

ieninicum (pi3) were in the second aggregation. The width of seedsanged from 0.50 mm to 1.00 mm. Significantly narrower seeds had. hieracifolium, E. virgatum and E. durum, as compared to otherroblematic species (Table S3). The length-to-width ratio ranged

rom 1.79 to 3.32. The most oblong seeds were characteristic for E.ieracifolium and E. durum, while less oblong seeds were found inther problematic species (Table S4). Additionally, two other Cen-ral European species, i.e. E. sylvestre and E. witmannii showed a veryimilar size and shape of seeds to what was observed in problematicpecies.

Studies performed at the species level showed that most prob-ematic species differed from each other in length, width and shapef seeds. There were, however, some exceptions. Erysimum hungar-

cum did not differ from E. pieninicum and E. wahlenbergii in any ofhe analysed characteristics. Two other similar pairs of species were. hieracifolium and E. durum (which differed only in seed length),s well as E. hieracifolium and E. virgatum, which differed only ineed shape (Table S5).

Six problematic Erysimum species were found to be very sim-lar according to their seed-coat microsculpturing and siliqua

icromorphology. The testa sculpture, as well as the microorna-entation pattern of the fruit valve and septum surface, were

onstant for seeds and fruits from different populations of the samepecies. The blister type of the seed-coat sculpture characterisedll six problematic species. Some differences concerned anticlinalalls of the testa surface cells that were raised, broad and roll-

haped for E. durum and E. hieracifolium and raised, narrow andidge-shaped for E. wahlenbergii, E. pieninicum and E. virgatum. Asegards E. hungaricum, the anticlinal walls of the epidermal cellsf the testa were channelled. Central protuberances present on theuter periclinal walls of the testa surface cells were mushroom-haped in seeds of five out of six examined problematic species.he cuticle on the lateral sides of the central protuberances andericlinal walls was striated mainly in radial orientation (Fig. 1;able 2).

The outer surface of the fruit valve was characterised by theugose micro-ornamentation pattern with irregular wrinkles. Withhe fruits of E. hungaricum, E. wahlenbergii, E. pieninicum and E.ieracifolium, the outline of the outer layer cells were isodiametric
r polygonal with somewhat concave outer walls and were weaklyarked, with slightly protruding lateral walls. With respect to the
. durum fruits, the outer walls of the surface cells were convex andheir lateral walls were depressed. Additionally, epidermal cells of

22 (2016) 68–85 73

the outer surface of the fruit valve of five out of six problematicspecies were characterised by the presence of the striatae cuti-cle with striae running parallel to the fruit axis. With respect toE. virgatum fruits, it was difficult to describe the characteristics ofepidermal cells because very many two-armed hairs covered them.Fruit surface of all other problematic species was covered by rarelyscattered many-armed hairs of pimple texture, and the presence ofsome stomata scattered on their surface was also observed (Table 3,Fig. 2). With respect to the epidermal cells on the inner surfaceof the fruit valve and septum, many similarities were found withregards to their cellular pattern, outer periclinal and lateral walls,as well as the shape of cells. Some differences among the exam-ined features concerned the fruits of E. virgatum, E. durum and E.hieracifolium. Both of the last two mentioned species were charac-terised by the presence of very rarely scattered 2-, 3-, or 4-armedstellate hairs of pimple texture on the inner surface of the fruit valve(Table 3; Figs. 3 and 4).

According to seeds of the examined representatives of otherCentral European species, three types of the testa sculpture and thecentral protuberances present on the outer periclinal walls wereobserved. With respect to seeds of E. repandum and E. witman-nii, some of their central structures were joined to one another bythe thick cuticle bands (Fig. 1; Table 2). The fruits of most of thesespecies were strongly or moderately covered mainly by two-armedhairs. Differences in siliqua micromorphology within species of thatgroup also concerned the lateral walls of the epidermal cells on theinner surface of the fruit valve, as well as the shape of the epidermalcells on the fruit septum surface (Table 3; Figs. 2–4).

The network, based on Euclidean distance matrix (combinedseeds measurements and SEM observations), showed that prob-lematic species were separated from representatives of the otherCentral European wallflowers (Fig. 5). On the left side, the prob-lematic species joined with E. cheiranthoides (similarly as inRAPD-based network below), whereas on the other side, they linkedthrough E. virgatum with other studied wallflowers. The closestposition in a group occupied E. wahlenbergii and E. hungaricum, aswell as E. pieninicum (pi3). Close relationships were also observedbetween E. durum and E. hieracifolium and between two popula-tions of E. pieninicum (pi1, pi2). Erysimum virgatum clearly took theoutlying position.

3.2. Molecular analyses

3.2.1. RAPDFourteen primers, producing clear, scorable, and polymorphic

bands, were chosen for the analysis of populations of the Erysimumspecies. Selection was based on the quality of the amplifications,level of polymorphism, and consistency of the pattern of ampli-fication, at least in the two replications. Amplification productswere resolved in polyacrylamide gels (PAGE). As expected, PAGEshowed bands that were better separated and more defined than inagarose gels. This improved resolution capacity also resulted in theappearance of new scorable bands. The increase in polymorphismled to a greater discrimination capacity of the primers with PAGE(compared to agarose). The amount of unique markers obtainedalso increased with the use of polyacrylamide. For example, primerOPH-7 produced a total of 26 unique bands, differentiating betweenspecies in polyacrylamide, but only six in agarose gel.

The amplification of genomic DNA of the 19 genotypes (14species) using RAPD analysis yielded 1245 fragments that could bescored. All of the chosen primers amplified fragments across all thegenotypes studied, with the number of amplified fragments rang-
ing from 59 (OPH-7) to 125 (OPH-4), with an average of 92 bandsper primer that varied in size from 140 to 1868 base pairs. Thelevel of observed polymorphism between Erysimum species washigh, a total of 1245 amplified fragments were polymorphic across

74 A. Czarna et al. / Flora 222 (2016) 68–85

Fig. 1. Seed-coat micro-morphology of examined Erysimum species: (A)—E. hungaricum, (B)—E. wahlenbergii, (C)—E. pieninicum, (D)—E. virgatum, (E)—E. durum, (F)—E.hieracifolium, (G)—E. cheiranthoides, (H)—E. cheiri, (I)—E. crepidifolium, (J)—E. diffusum, (K)—E. odoratum, (L)—E. repandum, (M)—E. sylvestre, (N)—E. witmannii; blister seeds culptufl tral sr

aawrEpDio

otnb1slbfwochrw

culpturing (A–G, I–L, N), reticulate seed sculpturing (H), reticulate-ocellate seed sat on tops (E–H, J, K, L, N), channelled anticlinal walls (A); mushroom-shaped cenounded (J) apices, and central structure of other shape (I, K, M).

ll of the species analysed, indicating a large degree of genetic vari-tion among them. Among 1245 distinguished bands, 862 (69.2%)ere present in the group of problematic species. Fig. S1 is rep-

esentative of the extent of polymorphism observed among therysimum genotypes, as revealed by OPH-18. Table 4 shows therimers used, their sequences, the total number, and the size ofNA fragments and number of exclusive bands (fragments present

n a given taxon/population but absent in others) produced by eachf the RAPD primers.

The highest number of polymorphic bands (195 and 192) wasbserved in two populations of E. pieninicum (pi2, pi3) in contrasto E. odoratum and E. durum (133 and 138 bands, respectively). Theumber of polymorphic markers in other species was compara-le (150–185). The average number of bands per population was64. For details, see Table 5. Most of the polymorphic bands werehared between different populations. Within the complex of prob-ematic species, the highest number of common bands was sharedetween populations of E. wahlenbergii and E. pieninicum (42–46),ollowed by E. hieracifolium and E. hungaricum (39). Similar values

ere also observed for the pair of E. wahlenbergii with E. durumr E. wahlenbergii with E. hungaricum (38). The lowest number of
ommon bands in this group was observed for E. virgatum and E.ieracifolium, as well as for E. virgatum and E. hungaricum: 16 and 18espectively. It is worth noting that a significant number of bandsas shared between the populations of E. pieninicum and E. cheiran-
ring (M); ridge-shaped narrow anticlinal walls (B–D, I, M), anticlinal walls broadtructures (A–C, E, F, L, N), cone-shaped central structures with flat (D, G) or gently

thoides (45–47), as well as E. pieninicum and E. crepidifolium (44),and these values were equal or even higher than between specieswithin the complex. The number of bands, common between par-ticular populations is shown in Table 6.

3.2.2. Genetic similarities and species-specific DNA markersThe estimated similarity using Jaccard coefficient, among

species of Erysimum populations, was based on an analysis of 1245RAPD markers. Values of genetic similarities between analysedpopulations at the intraspecific level (at least for part of them)were not very high (Table S6). The highest similarity (0.634) wasfound between two populations of E. wahlenbergii. A higher dif-ferentiation was observed in the case of two populations of E.odoratum (I = 0.58). Compared to the species mentioned above,three populations of E. pieninicum were much more differenti-ated − the genetic similarity coefficient for them ranged from0.467 to 0.578, and among them, the smallest values of similar-ity were found for population pi1. The differentiation between twopopulations of E. witmannii was also high (I = 0.439) and compa-rable with E. pieninicum (pi1). The genetic similarities were muchlower at the interspecific level. Pairwise values of Jaccard coefficient
similarity in all 14 species ranged from 0.200 to 0.661. Similar val-ues were observed within the complex of six problematic species(0.397–0.661), but they were more comparable and often exceededI = 0.50. The only exception in this group was E. virgatum − the I


Fig. 2. Ultrastructure of the outer surface of siliqua’ valve of examined Erysimum species: (A)—E. hungaricum, (B)—E. wahlenbergii, (C)—E. pieninicum, (D)—E. virgatum, (E)—E.d , (J)—m K, M, N(

vi

sosemfvnh

tpswtuOblap

urum, (F)—E. hieracifolium, (G)—E. cheiranthoides, (H)—E. cheiri, (I)—E. crepidifoliumany-armed appressed hairs of pimple texture (A–C, E–G), two-armed hairs (D, H, J,

F, G, I, N), week pubescence (A–C, E, L).

alue between this and other species ranged from 0.215 to 0.382,ndicating its more distinct position.

RAPD markers applied in the study revealed a total of 439pecies-specific bands (35.3%) in populations of selected speciesf Erysimum, including 187 in six closely related species. In eachpecies, exclusive products were observed. The highest number ofxclusive RAPD bands was observed in E. cheiri (31), where theyake up 19.4% of all polymorphic fragments scored for this species,

ollowed by E. odoratum (30), as well as E. repandum, E. witmannii, E.irgatum and E. pieninicum (pi3), with 29 specific bands. The lowestumber of species-specific bands was found in two populations: E.ieracifolium and E. durum, at 11 and 13 respectively.

Additionally, in the obtained profiles for different populations ofhe same species, putative diagnostic fragments (exclusive bandsresent only in populations of a given taxon) were observed. Twouch bands (671 bp and 206 bp), obtained with primer OPH-12,ere present in the profiles of E. wahlenbergii (wa1, wa2). Another

wo putative diagnostic bands, OPH-41208 and OPH-8181, for pop-lations of E. witmannii (wi1, wi2), were amplified with primersPH-4 and OPH-8. The highest number (7) of putative diagnosticands was observed in at least two of the three analysed popu-

ations of E. pieninicum. One of them, OPH-8507, was present in
ll three populations. Three (OPH-18413,826, and OPH-19585) wereresent in the profiles of two populations (pi1, pi3), while the next
E. diffusum, (K)—E. odoratum, (L)—E. repandum, (M)—E. sylvestre, (N)—E. witmannii;), mixed types of hairs (I, L); strong pubescence (D, H, J, K, M), moderate pubescence

three (OPB-5930, OPH-5248, and OPH-8389) were characteristic forthe populations of pi1 and pi2.

When analysis was limited to a group of problematic species(nine populations, six species), the number of species-specificbands within closely related species significantly increased for eachtaxon, confirming their distinction. The highest number of suchproducts was observed for E. virgatum. Additional putative diag-nostic bands for E. pieninicum (17) and E. wahlenbergii (4) were alsoobserved. On the other hand, among the 862 products counted forclosely related species, 103 (12.0%), were limited (absent in profilesof other species studied here). Some bands were shared simultane-ously by three (99) or more than three different species (21). Thisobservation as well as the presence of set of bands (9) observed inmost species in this group (almost monomorphic) indicated a closerelationship in this complex.

The minimum spanning network graph, based on Jaccard dis-tances from binary RAPD data, showed that the 19 populationgenotypes (14 species) could be resolved into four main groups(Fig. 6). The extreme position was occupied by “good species”(groups I and IV), while problematic species created two looselyconnected groups in the centre of the graph (II and III). Only E. virga-tum was placed among non-problematic species from the left side
of the network. Two populations of E. odoratum were assigned todifferent but adjacent groups. All of the populations of E. pieninicumclustered together with E. cheiranthoides creating left group in the


Fig. 3. Ultrastructure of the inner surface of siliqua’ valve of examined Erysimum species: (A)—E. hungaricum, (B)—E. wahlenbergii, (C)—E. pieninicum, (D)—E. virgatum, (E)—E.durum, (F)—E. hieracifolium, (G)—E. cheiranthoides, (H)—E. cheiri, (I)—E. crepidifolium, (J)—E. diffusum, (K)—E. odoratum, (L)—E. repandum, (M)—E. sylvestre, (N)—E. witmannii;2–4- or 3 armed hairs of pimple texture (E, F), many-armed sublepidote hairs (L); rugose ornamentation with rugulose cuticle (I), reticulate ornamentation, covered by3–5-rayed hairs (G).

Table 4List of primers used in the RAPD analysis, their respective oligonucleotide sequences,numbers and sizes of amplified fragments, number of polymorphic bands and thepolymorphism produced by each primer. All fragments were polymorphic.

Primer Fragmentlength (bp)

Number offragments

Number ofpolymorphicfragments

OPB-05 TGCGCCCTTC 1868−171 85 85OPB-12 CCTTGACGCA 1320−174 70 70OPB-17 AGGGAACGAG 1175−109 91 91OPB-18 CCACAGCAGT 1379−148 80 80OPH-03 AGACGTCCAC 1300−161 86 86OPH-04 GGAAGTCGCC 1220−144 125 125OPH-05 AGTCGTCCCC 1346−145 95 95OPH-07 CTGCATCGTG 1058−175 59 59OPH-08 GAAACACCCC 1116−152 85 85OPH-12 ACGCGCATGT 1235−140 102 102OPH-15 AATGGCGCAG 1229−169 94 94OPH-17 CACTCTCCTC 1217−238 60 60OPH-18 GAATCGGCCA 1041−150 102 102OPH-19 CTGACCAGCC 1356−180 111 111

ciii

Table 5Number of RAPD bands scored in selected species of Erysimum. BT (bands total) –total number of bands present in given taxon (population); EB (exclusive bands) –bands present in a given taxon/population, but absent in other taxa/populations. Sixproblematic species are in bold.

Species Bands (total) EB number/(%) to BT

E. durum (du) 138 13 9.42E. wahlenbergii (wa1) 185 24 12.97E. wahlenbergii (wa2) 165 18 10.91E. pieninicum (pi1) 174 19 10.92E. pieninicum (pi2) 192 20 10.42E. pieninicum (pi3) 195 29 14.87E. hungaricum (hu) 171 24 14.03E. hieracifolium (hi) 179 11 6.14E. virgatum (vi) 160 29 18.12E. cheiranthoides (chs) 179 27 15.08E. odoratum (od1) 133 20 15.03E. odoratum (od2) 159 30 18.87E. diffusum (di) 153 17 11.11E. repandum (re) 168 29 17.26E. sylvestre (sy) 150 25 16.67E. crepidifolium (cr) 168 21 12.5E. cheiri (chi) 160 31 19.37

Total Average: 1379–140 1245 1245

entral part of the graph (II). The rest of problematic species (and,
n addition, the above-mentioned population of E. odoratum) werencluded in the right group in the centre of the network (III). Its noteworthy that distances between populations within species
E. witmannii (wi1) 161 29 18.01E. witmannii (wi2) 175 23 13.14

were comparable with the ones on the species level, which mightsuggest a relatively close relationship between all studied species.


Fig. 4. Ultrastructure of siliqua’ septum surface of examined Erysimum species: (A)—E. hungaricum, (B)—E. wahlenbergii, (C)—E. pieninicum, (D)—E. virgatum, (E)—E. durum,(F)—E. hieracifolium, (G)—E. cheiranthoides, (H)—E. cheiri, (I)—E. crepidifolium, (J)—E. diffusum, (K)—E. odoratum, (L)—E. repandum, (M)—E. sylvestre, (N)—E. witmannii; short andlong surface cells present (A–C, E, F), mostly long surface cells present (G, H, K, L, M, N), mostly short surface cells present (D, I, J); many-armed sublepidote hairs present (L).

Fig. 5. Minimum spanning network inferred from the pairwise Euclidean distance matrix based on morphological and micromorphological traits (19 populations representing14 taxa of Erysimum). For abbreviations of samples refer to Table 1.


F (19 poT

3

l(pItipd4h

gwobaTisi8

itHemphfs

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ig. 6. Minimum spanning network based on Jaccard distance matrix of RAPD dataable 1.

.2.3. ITS region (SSCP and RFLP analyses)Amplification with chosen pairs of ITS primers generally yielded

arger fragments compared to those described by White et al.1990). Clear differences in the length of PCR products betweenarticular species were also observed. The PCR with primers

TS-1 and ITS-2 yielded fragments differing in size from 320o 359 bp, whereas primers ITS-3 and ITS-4 resulted in approx-mately 400–431 bp of fragment. The fragment produced byrimer pair ITS-3 and ITS-4 was approximately 400 bp in E.urum and E. wahlenbergii, 423 bp in E. sylvestre and E. cheiri,31 bp in E. witmannii and 416 bp in all other species studiedere.

The first fragment was resolved poorly in 10% polyacrylamideel. Therefore, only the results of SSCP for PCR products amplifiedith primers ITS-3 and ITS-4 are presented here. Electrophoresis

f the denatured PCR products resulted in one to four major sta-le bands. The locations of bands and distances between the uppernd lower bands differed among the examined Erysimum species.hese variations constituted simple and distinct SSCP patterns for

ndividual species, allowing for distinguishing almost all wallflowerpecies analysed in this study. All SSCP patterns were reproduciblen repeated analyses (regardless of gel electrophoresis, 10% versus% gel used).

Six of the nine restriction enzymes tested had restriction sitesn the ITS region of the selected species of Erysimum. Restric-ion fragments were obtained with the endonucleases EcoRI, HinfI,aeIII, MboI, MseI and AluI. According to the patterns generated,ach species was defined by its own pattern of restriction frag-ents (scores of restriction fragments are shown in Table S7). RFLP

rofiles of the ITS-2 region divided the studied accessions intoomogenous groups that reflected exact taxonomic species. There-

ore, further interpretation of obtained results was conducted on apecies level.

The minimum spanning network, based on Jaccard distancesrom binary ITS RFLP data, revealed three main groups branchedrom E. diffusum, occupying the central position in the graph (Fig. 7).he general pattern was similar to the RAPD-based network −early all problematic species created one loose cluster, apart from. virgatum, which was grouped together with E. cheiranthoides and. odoratum. The third cluster consisted of the rest of the non-roblematic species. It should be emphasised that interspecific
istances within groups of problematic and non-problematic taxaere similar.
pulations representing 14 taxa of Erysimum). For abbreviations of samples refer to

3.2.4. Comparison of genetic and morphological dataThe Mantel test revealed statistically significant but very weak

correlation between RAPD and morphological distance matrices(R2 = 0.14, p = 0.003) on the species/population level. The samecorrelation was insignificant when only six problematic specieswere compared (R2 = 0.24, p = 0.072). There was no correlationbetween ITS RFLP and morphological distances on a species level(R2 = 0.0008, p = 0.43).

4. Discussion

While species identification continues to be based on morphol-ogy, a rising number of studies suggest that sole reliance on thisapproach may lead to the omission of a significant number of rele-vant species (Bickford et al., 2007). As Heinrichs et al. (2011) argue,DNA sequences may improve species identification although thisapproach may not be sufficient in cases when recently emergedspecies have not accumulated mutations in barcode regions. Onthe other hand, as in the young age as Erysimum genus, most of therecently described species are based on single or trivial and poorlyunderstood differences. It is likely that the number of such ‘species’is overstated. The taxonomic complexity suggests recent and rapidradiations that probably did not allow sufficient time for completesorting of ancestral polymorphisms among ITS copies to diverge(Moazzeni et al., 2014). Molecular methods allow to determinespecies without special morphological skills but only combinedmolecular and morphological approach will make possible deeperunderstanding of phenomena of cryptic lineages (Heinrichs et al.,2011).

Cryptic species are probably common in many groups of organ-isms and will be detected with increasing frequency as sensitivemolecular methods, such as sequence analysis, are applied tosystematic problems. Cryptic species identification has becomethe subject of extensive research due to its importance for suchresearch fields as for example population genetics or biogeogra-phy (Dilon and Quipuscoa, 2014; Gomez et al., 2002; Heinrichset al., 2010; Gaudel et al., 2012; Niwa et al., 2014; Saunders andLehmkuhl, 2005; Yu et al., 2013).

4.1. Morphology

The Brassicaceae family is characterised by considerablesomatic seed polymorphism (Lu et al., 2010; Maun and Payne,1989), which means that seeds produced by the same plant or by


Fig. 7. Minimum spanning network using a Jaccard distance matrix based on ITS data representing 14 species of Erysimum. For abbreviations of samples refer to Table 1.

Table 6The number of common bands, shared between particular species of Erysimum. Six problematic species are in bold. Number of fragments specifically shared by every pairamong the six problematic species are marked in grey.

scp1eBHZifude

pecimens from the same population vary in size, shape, weight, orolor. In the case of Central European Erysimum species, polymor-hism refers especially to seed dimensions and shape (Latowski,975). The presented measurements of seed length and width gen-rally coincided with the literature (Ancev and Polatschek, 2006;all, 2010; Bojnansky and Fargasová, 2007; Dvorák et al., 1975;egi, 1906; Kasem et al., 2011; Latowski, 1975; Polatschek, 2010;apałowicz, 1913 − for details, see Table S8). In some cases, min-

mum values were lower than those cited in previous papers (e.g.or E. pieninicum, E. wahlenbergii, E. cheiri) or the maximum val-
es exceeded the values recorded in the literature (e.g. for E.urum, E. sylvestre). However, the differences observed were notspecially meaningful. Previous studies (Bojnansky and Fargasová,
2007; Latowski, 1975; Polatschek, 2010) described the shape ofErysimum seeds in a qualitative way as being oblong or prolon-gated and ovate, ellipsoid, cylindrical, or almost rectangular. Wefound no concurrence of our quantitative measurements with thesequalitative descriptions (Table S9). In our opinion, in the case ofmorphologically similar species, quantitative measurements aremore useful than traditional descriptions that may be biased bysubjectivity.

Among the others, the only E. cheiri was clearly discriminatedfrom the other species by seed measurements. The morphologi-
cal distinction of E. cheiri was also proven by Polatschek (2010)and could be explained by the origin of this horticultural plant,which was probably derived by selection and hybridisation from

8 lora 2

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pecies native to distant regions of Southern Europe (Ball, 2010).opulations of six problematic species did not form one cohesiveroup, but rather, two subgroups, since E. virgatum, E. hieracifoliumnd E. durum had smaller and more oblong seeds as comparedo three other wallflowers. Moreover in the case of problematicpecies, seeds collected from distinct populations of the samepecies differed significantly from each other, whereas no differ-nces between the average values of seed length and shape wereound in comparison with the seeds of other species. In light ofuch results, it seems that seed biometric measurements should bepplied only as auxiliary traits for species identification.

Results of SEM studies within Brassicaceae concerning fruits andeeds micromorphology proved many times that the type of theirculpturing is of taxonomic significance (Kasem et al., 2011; Kayat al., 2011; Maciejewska-Rutkowska et al., 2007; Moazzeni et al.,007). For most of the examined species, seed-coat sculpturing wasf the blister type. Some authors examining other Erysimum speciesEl-Naggar, 2005; Tantawy et al., 2004) named such seed coat sculp-uring as a reticulate with the central structure. Ghaempanah et al.2013) described the scalariform sculpture pattern for E. repan-um seeds and, according to Kasem et al. (2011), the seed coaticroornamentation pattern of E. cheiri was ocellate with flat cen-

ral portions. These results were not confirmed in the present study.he observed differences might be the result of the putative hybridrigin of E. cheiri (for details see above). According to Latowski1975), the central structures present in testa cells of the exam-ned Erysimum species are of a mucous character, and their shapend size are of taxonomic importance. Our results are in agree-ent with Maciejewska-Rutkowska et al. (2007), who examined

culpturing of E. pieninicum seeds, as well as with the findings ofatowski (1975) who described the presence of mushroom-shapedentral protrusions on the seeds of E. hieracifolium, E. pieninicum,. wahlenbergii, E. hungaricum and E. durum. Latowski (1975) alsoescribed the presence of the same type of central structures on E.irgatum seeds, but that was not confirmed in the present study. Asar as the rest of the examined species are concerned, the describedhapes of the central structures were in agreement with the find-ngs of Latowski (1975). The only exception was E. crepidifolium,n which the presence of cone-shaped central protrusions was notonfirmed in this study.

The Brassicaceae family is characterised by a great diversity ofair forms that provide crucial taxonomic characteristics of the epi-ermis. In many cases, these characteristics are more valuable thanny other feature in the morphology of the taxa (Khalik, 2005).ccording to the obtained results, three categories of fruit among

he examined Erysimum species were distinguished with the usef trichome types and the pubescence density on the outer surfacef the fruit valves. These findings are generally in agreement withhe results obtained by other authors (Ancev and Polatschek, 2006;all, 2010; Latowski, 1975; Mutlu, 2010; Zhou et al., 2001). How-ver, some inconsistencies between the obtained results and theiterature data were noticed. According to Latowski (1975), on theruit of E. virgatum, many-rayed hairs were present, and E. witman-ii was characterised by siliquae covered only by medifixed hairs.oreover, Ancev and Polatschek (2006), as well as Mutlu (2010),

lso observed 3- and sometimes 4-rayed hairs on fruit of E. diffusum,ut that was not confirmed in our study.

SEM analyses concerning the micromorphology of the innerurface of siliqua valves showed two groups of species differingccording to the presence or absence of trichomes on the surfacef that part of the fruit. The first group was composed of E. hieraci-

olium, E. durum, E. repandum and E. cheiranthoides, and the rest
f the examined species had a glabrous inner surface of the siliquaalves. Referring to the first group of species, our findings confirmedhe previous results of Latowski (1975), Zhou et al. (2001), and
utlu (2010). However, Latowski (1975) also observed the pres-

22 (2016) 68–85

ence of single hairs on the inner surfaces of valves in the fruits of E.virgatum, and Mutlu (2010) described the presence of many-armedhairs on the inner surfaces of valves in the siliquae of E. cheiri, butthese results could not be confirmed by our study. The presenceof numerous trichomes on the siliqua septum in E. repandum wasdescribed in the presented work for the first time.

Our studies revealed the existence of some differences accordingto examined micromorphological traits within six taxonomicallyproblematic Erysimum species, however the more distinct micro-morphological dissimilarity was noted only for E. virgatum. Thisspecies contrasted with other species in the cone-shaped centralstructures present on the outer periclinal walls of testa cells, thedomination of the short cells on the surface of siliqua septum, andwith the presence of numerous two-rayed hairs on the outer surfaceof the siliqua valve. Erysimum pieninicum did not differ in any char-acteristics from E. wahlenbergii. On the other hand E. hungaricumwas characterised, unlike these two species, by possessing cellswith channelled anticlinal walls in the testa. Among the numerousmorphological traits common to E. durum and E. hieracifolium, onlythe intensity of pubescence on the outer surface of siliqua valves(less intensive for E. durum), as well as in the participation of longand short cells in the fruit septum (long cells clearly dominated inseptum of E. durum), differentiated these two species. Summing upthe carpological differences between problematic species (exceptfor E. virgatum) are present, however, they are relatively small.In such circumstances, the molecular techniques can be helpfulin determining whether we deal with separate species (Jorgensenet al., 2003; Judd et al., 2008; Knowles and Carstens, 2007).

4.2. Molecular analyses

In this study, DNA fingerprinting techniques, including RAPD,SSCP, and digesting patterns (RFLP) of the internal transcribedspacers of 18S-26S rDNA (ITS), were combined with detailedmorphological analyses to seek characteristics that discriminatebetween closely related Erysimum species. The results indicatedthat both techniques were applicable in assessing relatednessamong the examined species. Moreover, the resolution of amplifi-cation products obtained with RAPD primers using polyacrylamidegels resulted in increasing the revealed polymorphism (e.g., muchmore scorable bands appeared). Polyacrylamide gels (PAGE), com-pared to agarose gels (AGE), provide a higher resolution of the bandswith lower molecular weight and were used for resolving DNA frag-ments in the identification of various taxa, such as Malus (Haradaet al., 1993) or tobacco (Filippis et al., 1996). Belaj et al. (2001) com-pared the capacity of two separation matrices and showed that theincrease in polymorphism led to a greater discrimination capacityof the primers with PAGE than with AGE. Forty-four of the 46 olivecultivars studied with PAGE could be identified with the combina-tion of only two RAPD primers, while with AGE, only six cultivarswere identified by the same combination of primers. The results ofthe present study fully confirmed their observations.

RAPD detected a high level of polymorphism among the 19genotypes, representing 14 taxa of Erysimum. The data obtainedstrongly support the genetic distinctiveness, as well as the closerelationship of six problematic species of Erysimum. Between 862polymorphic products scored for this group, 103 (12.0%) fragmentswere shared only among the members of this group and werenot observed in the remaining species studied here. Some bandswere shared by three or more species simultaneously, and somewere present in the profiles of most of them (almost monomor-phic bands). Interestingly, E. cheiranthoides was the only species
that shares the other subset of bands with them, especially with E.pieninicum, which may explain its position in the network, wherethis species clustered with populations of E. pieninicum in a separatesubgroup. Although these observations and the highest number of

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ommon bands between them supported the hypothesis that theroup of six taxonomically problematic species constitutes a com-lex of closely related species, species-specific bands present in therofile of each species identified the distinctiveness of the partic-lar species. RAPD markers applied in the study revealed the 187pecies-specific bands in six closely related species that make up2.6% of all species-specific fragments observed in the analysedopulations. The number of species-specific bands for particularaxon significantly increased when the analysis was limited onlyor species in the complex (nine populations, six species). Generally,he highest number of exclusive bands was present in the profilesf species with clear taxonomic status as E. cheiri or E. odoratum30–31). Within the complex, a similarly high number of such bandsas observed for E. virgatum, for which a distinct taxonomic status

eems to be confirmed by morphological examination, and whichas unexpected for E. pieninicum (29). Moreover, in the profiles

f some species represented by two or more populations, puta-ive diagnostic fragments were observed. Two such bands wereresent in the profiles of E. wahlenbergii, while up to seven werebserved in three populations of E. pieninicum. When the analysisas comprised only of members of the complex, additional puta-

ive diagnostic bands for E. pieninicum (17) and E. wahlenbergii (4)ere found.

RAPD markers could provide systematically meaningful infor-ation on similarities between taxa at presumably different levels

f relatedness. They have proven to be a valuable tool in studyingnter and intraspecific genetic variations (Viccini et al., 2004). Inhe present study, values of genetic similarities at the intraspecificevel (in species represented by two or more populations) were notery high (0.439–0.634), which may indicate that differentiationetween them is significant. It is worthy to note that differenti-tion found among three populations of E. pieninicum was higher,specially taking into account population pi1. Erysimum pieninicumelongs to the rarest wallflower species noted in Poland (endemico the Pieniny Mountains). Despite having small population sizesnd restricted distribution, these populations showed high level ofariation. According to Rucinska et al. (2010; unpublished data),nalysis using ISRR markers indicated low level of variation athe intraspecific level between populations of E. pieninicum, buthowed significant differences in the genetic structure of partic-lar populations (79% of variation). A comparison of our presentesults is difficult because population pi1 was not included in theirnalyses but it is known that species with a narrow occurrencehow sometimes relatively high intraspecific diversity compared toidespread species (Tansley and Brown, 2000; Viccini et al., 2004).

t remains not clear how small populations, e.g., E. pieninicum (pi1)aintain quite high level of genetic diversity. Viccini et al. (2004)

bserved similar results for Lippia diamantinensis, postulating thatn understanding of possible mechanisms involved is important foruccessful conservation management of such populations. Further-ore, if a population has always occurred in small numbers, it may

e that it has adapted to its rare condition (Milligan et al., 1994).The genetic similarities were much lower at the inter-specific

evel and ranged from 0.200 to 0.661 in all analysed popula-ions, confirming their distinction. Apart from E. virgatum (0.215 to.382), differentiation within a group of populations of problematicpecies was smaller and the values of similarity were comparable,hich may confirm the hypothesis that they create the complex of

losely related, and probably cryptic, species. Similar and compa-able values of genetic similarity between other cryptic and closelyelated species in different plants were also reported (Nkongolot al., 2003; Viccini et al., 2004).

Recently, analyses of ribosomal, nuclear, and mitochondrialNAs show promise in establishing new systematic positions forroblematic species. DNA sequence analysis of ribosomal RNAenes is widely used in phylogenetics over a wide range of taxo-

22 (2016) 68–85 81

nomic levels for many organisms (Cooke and Duncan, 1997; Cookeet al., 2000; Crawford et al., 1996; Gomez et al., 2002; Hibbett, 1992;Lee and Taylor, 1992; Redecker, 2000; White et al., 1990). In thepresent study, the internal transcribed spacer region of the nuclearribosomal DNA (ITS) was chosen for analysis because this regionoften displays relatively high levels of variation and, thus, could beuseful for studies below the generic level (Baldwin, 1993). Further-more, ITS is a much-studied region that has been widely used forphylogenetic studies, which has also recently encompassed Erysi-mum species (Moazzeni et al., 2014). Moreover, fixed differences atindels are a particularly useful feature of ITS, hence ITS sequencesremain an excellent diagnostic tool in searching for individuals thatmight be cryptic species (Blaxter, 2004; Blouin, 2002). To determineif DNA polymorphisms in the ITS-2 region contained diagnosticallyuseful information, single PCR products that had been amplifiedwith primers ITS3 and ITS4 from 14 species of Erysimum wereanalysed. Chen et al. (2001) showed that polymorphism in thePCR product length permitted distinguishing of most clinical iso-lates of yeast species. Remaining species with products of similarsizes could be distinguished using restriction enzyme analysis. SSCPanalysis, known as one of the most commonly used mutation detec-tion methods (Sambrook and Russell, 2001), is another methodthat has been useful for the detection of polymorphism. Recently,it has also become a powerful molecular fingerprinting methodfor studying taxonomy at the level of both species and subgroupswithin some species complexes (Kong et al., 2003). Alteration ofthe nucleotide sequence of the molecule by as little as a single basecan reshape the secondary structure, with consequent change inelectrophoretic mobility through native gel (Kong et al., 2003). Sin-gle base changes in 300–400 nucleotides produced distinguishablechanges in migration patterns (Hiss et al., 1994).

Both of the above-mentioned methods were used in the presentstudy. The size of the PCR products differs between species andallows us to preliminarily distinguish part of them. The bandingpattern of ITS-2 region obtained with using the SSCP method dis-tinguished almost all of the 14 examined species of Erysimum. Toconfirm the detected differences, especially among members of aproblematic group, restriction enzyme analysis of the amplifiedregion (RLFP) was performed. The species-specific banding patternsof SSCP and restriction endonuclease-digested rDNA amplified byPCR, combined with RAPD results, confirmed the distinctivenessof the particular species and suggest that these procedures mayprovide a reliable taxonomical tool for identifying the speciesof Erysimum, including discrimination of closely related taxa inspecies complexes.

The problematic species created a more or less loose clus-ter, both in RAPD and ITS RFLP based networks. Only E. virgatumdiffered distinctly from the other species from this group. Interspe-cific distances within groups of problematic and non-problematictaxa were similar, but it was interesting to note that differencesbetween populations within problematic species were relativelylarge (similar to between-species distances). Genetic results, ingeneral, concurred with the conclusions drawn from morphologi-cal data, but morphological distances between species/populationsin problematic group were noticeably shorter (E. virgatum was con-nected to this group, although it was distinctly more distant fromthe well-defined cluster of other problematic species). Congruity onthe species/population level was confirmed, to some extent, by theMantel test (statistically significant but weak correlation betweenRAPD and morphological distance matrices), but taking into consid-eration only six problematic species, the same test did not revealany relationship. There was also no correlation between genetic
(ITS RFLP) and morphological distances on the population level. Inother words, the problematic species constitute a morphologicallymore homogenous group than would have resulted from geneticdata.

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It is worthy to note that some species/populations took differentositions in the network based on molecular approaches com-ared to that from morphological data, suggesting a discordancef phenotype and genotype. According Yu et al. (2013), such con-ict between phenotype and genotype may indicate the occurrencef cryptic species. The occurrence of cryptic speciation as one fromossible mechanisms is thought to create these conflicts. Incon-ruence between morphological and molecular evidence has beeneported in various plant lineages (Martinez-Ortega et al., 2004).

. Conclusions

As mentioned in the introduction and as demonstrated in Table1, in the past, problematic Erysimum species were treated differ-ntly by several authors (from one species, to groups of subspeciesnd finally to separate distinct species). Thus, first, we explored allf these possibilities. Although the obtained results did not fullyeflect any of the existing systematic classifications, we confirmedhe close relationship among E. wahlenbergii, E. hungaricum and E.ieninicum according to morphological characteristics and molecu-

ar results is coinciding with the actual taxonomy proposed by Ball2010). Moreover, we also denoted the analogous and close simi-arity between E. hieracifolium and E. durum, which is not reflectedn Ball’s (2010) study. Results of carpological studies also showedhat E. virgatum is morphologically distinct from the remainingroblematic species, which ought to be reflected by the distantosition of this wallflower in systematics. This observation, as wells the subtle morphological differentiation noted in all other mem-ers within the problematic group, was fully supported by theirenetic distinction. At the time, the possibility of the existence ofne species complex was excluded.

Many lines of evidence obtained in the present study indicatehat the group of problematic Erysimum species consists of distinct,ut closely related species (for details, see above). On the otherand, the high morphological similarity of members in this groupreates questions about their taxonomic rank (cryptic species orubspecies). The existence of both subspecies as well as crypticpecies has been already demonstrated in other wallflowers (Jalasnd Suominen, 1994; Quarmim et al., 2013,) and other genera ofrassicaceae (Grundt et al., 2006; Perny et al., 2004).

Among taxonomists, definitions of species, and especially sub-pecies, are a source of considerable disagreement. There are twopecies concepts. A biological species is a group of reproducingopulations which are isolated from other such groups as far asheir reproduction is concerned. In turn, according to Paris et al.1989) an evolutionary species (sometimes defined as morpholog-cal) “is a single lineage of ancestor-descendant populations that

aintains its own identity from other such lineages and fits into itswn ecological niche, having a unique evolutionary history”. Whileany species fit both biological and evolutionary concepts this is

ot the case with cryptic species. In their case a species definedccording to morphological criteria consists of two or more genet-cally isolated evolutionary species (Paris et al., 1989). Subspeciess the rank recognised below species. Both are populational con-epts in the sense that gene flow is at least potentially possiblemong members of the taxon, but with the subspecies concept,here is a defined geographic component to the definition. Mayr andshlock (1991) formulated a definition that is widely accepted: “Aubspecies is an aggregate of phenotypically similar populationsf a species inhabiting a geographic subdivision of the range ofhat species and differing taxonomically from other populations of
hat species.Moreover, in recent decades, taxonomists have appliedubspecies names to geographic races without evidence of partialsolation. With this practice, in studies provided independently byifferent authors, the subspecies category is used for taxa that are
22 (2016) 68–85

geographically as well as morphologically distinct (Perny et al.,2004; Quarmim et al., 2013; Turner and Nesom, 2000). In otherwords, classification on this level describes morphological varia-tion. Traditionally, subspecies have been defined by morphologicaltraits or color variations, but recent critics are concerned that thesetraits may not reflect underlying genetic structure and phyloge-nies (Haig et al., 2006). Because adaptive divergence can take placedespite gene flow, it requires the use of multiple sources of informa-tion (addressing the questions of reproductive isolation, adaptivedivergence, and spatial patterns of local adaptation) when evalu-ating a taxon’s status (Fraser and Bernatchez, 2001). Thus, higherlevels of confidence can be obtained in classifications based onthe concurrence of multiple morphological, molecular, ecological,behavioural, and/or physiological characteristics (Haig et al., 2006).

In light of our results, we may confirm similarity of problem-atic species, as well as their genetic distinctiveness, but we did notfind evidence for diagnostic morphological variation, characteristicfor rank of subspecies. The analysed species differ morphologi-cally, but not in features that are interpretable and/or recognised bytaxonomists as being species level attributes. Because of the impor-tance of the geographic component in the case of the subspeciescategory, the answer to the question of whether taxonomicallydifferent populations occur sympatrically anywhere during thebreeding season with no evidence of free interchange of geneticmaterial may be very helpful; if so, then they are likely sepa-rate species (Haig et al., 2006). In the present work, the studiedspecies, apart from E. hieracifolium (cosmopolitan species), showa restricted range. Moreover, two of them are endemic. There isno information about sympatry between them but our moleculardata indicate distinct taxonomic positions (supported by pres-ence of specific/diagnostic RAPD fragments) of all Erysimum speciestreated as problematic. In the case of lack of evidence confirmingthe hypothesis that they might be a subspecies complex, we testedthe other one concerning whether problematic species should betreated as a genetically unique cryptic species complex.

We deal with cryptic species when as a result of advanced spe-ciation the gene flow between the populations has been stoppedbut the visual differences between the populations are not yetapparent (Bickford et al., 2007; Schlick-Steiner et al., 2007). Manyspecies traditionally seen as a cosmopolitan are now recognisedas being cryptic species assemblages of regionally more restrictedtaxa. They remained undetected by traditional taxonomic methodsdue to a lack of morphological characteristics of taxonomic utilityand the frequent confounding effects of high phenotypic plasticityor hybridisation (Gomez et al., 2002). The often subtle morpho-logical differences between the species currently recognised wereonly reliably detected when individuals were cultured in identi-cal conditions in the laboratory and cohorts of the same age wereanalysed using scanning electron microscopy and biometrical sta-tistical tools. In wild caught samples, individuals of these speciesare often impossible to discriminate (Gomez et al., 2002).

Although different definitions are described in the publishedpapers, several elements are common to most of them. Crypticspecies have the following characteristics: (1) they poorly differ-entiated morphologically, (2) they represent distinct evolutionarylineages because they are reproductively isolated and (3) they havehistorically been misinterpreted as members of a single species(Paris et al., 1989). Reproductive isolation is used as the primary cri-terion for determining whether or not two morphologically similarpopulations represent cryptic species. A more currently definitionby Bickford et al. (2007) did not regard this as an essential feature ofcryptic species. The reproductive barrier is probably a by-product
of genetic differences between species (Avise, 2004). Populationsbelonging to different biological species have separate multilocusgenotypes, which are not subject to recombination due to the exis-tence of a reproductive barrier. Thus, the lack of experimental data

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n the existence a barrier is not a factor that excludes the possibilityf distinguishing cryptic species since their presence may be con-rmed directly by the differences in their gene pools (Szweykowskit al., 1981).

The analysed species of the Erysimum complex consist of closelyelated and morphologically similar populations, which are dif-erentiated by some micromorphological and carpological traits.owever, except for E. virgatum, the differences were relatively

mall. The RAPD and ITS data (SSCP and RLFP patterns) stronglyupport the genetic distinctiveness of each of six Erysimum species,ven in the presence of other closely related species. The correla-ion of a few cryptic morphological characteristics with a distinctive

olecular fingerprint has also supported the clear taxonomic dis-inction of E. virgatum in the complex of all close relatives. Theested potential cryptic species have distinct gene pools, as con-rmed by the presence of specific/diagnostic DNA markers andifferent multilocus genotypes, as well as by low values of geneticimilarity between populations at the interspecific level, comparedith the intraspecific level. The number of unique bands identified

n cryptic species could be a good indicator of their reproductivesolation (Wolfe et al., 1998). A total of 862 polymorphic RAPDands were scored for members in the complex. Each speciesas genetically distinct, separated by 11–30 species-specific RAPDarkers. Alleles at loci that are private (species-specific) to one

r other group imply that they are not exchanging genes at anyignificant level. Thus, they are reproductively isolated.

According to Okuyama and Kato (2009) relatively small preva-ence of cryptic species within angiosperms may be due to theimited number of studies dealing with this problem. In theiriew this could be attributed to the general perception of theorphology-based taxonomic system as highly reliable. In Erysi-um, there are such examples, and within E. nervosum s.l. Pomel,

wo cryptic species were described by Abdelaziz et al. (2011) as E.ervosum s.s. and E. riphaeanum Lorite, Abdelaziz, Munoz-Pajares,erfectti and J. M. Gómez.

In this study, we evaluated different taxonomic concepts usingultiple data obtained from morphology, as well as molecular

nalyses. Using our molecular approaches, we illustrate that therysimum complex is possibly an example of a complex of cryp-ic species. Apart from E. virgatum, the problematic species, in theresent study, differ at best by subtle morphological differenceshat earlier might have been regarded as infraspecific variation.

oreover, observed discordance of phenotype and genotype (net-orks) may indicate the occurrence of cryptic species. Our study

rovides a framework for future investigations. Nevertheless, fur-her surveys, based on more extensive population sampling, andurther data are required to provide better understanding of theiversification of Erysimum species in the mentioned complex.

cknowledgements

This research was supported by Ministry of Research and Highducation Research Capacity Grants for Poznan University of Lifeciences (No. 508.641.00 and 508.181.00).

ppendix A. Supplementary data

Supplementary data associated with this article can be found,n the online version, at http://dx.doi.org/10.1016/j.flora.2016.03.08.

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