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ORIGINAL PAPER
Invasion of Eragrostis albensis in Central Europe:
distribution patterns, taxonomy and phylogenetic insight
into the Eragrostis pilosa complex
Anna Wro
´bel .Ewelina Klichowska .Evgenii Baiakhmetov .
Arkadiusz Nowak .Marcin Nobis
Received: 16 April 2020 / Accepted: 20 March 2021
ÓThe Author(s) 2021
Abstract The Eragrostis pilosa complex (Poaceae)
comprises five widely distributed and regionally
invasive species—E. albensis,E. amurensis,E.
imberbis,E. multicaulis, and E. pilosa, distinguished
by tiny and variable morphological characters and
with so far unknown phylogenetic relationships.
Recently, some doubts have been raised about the
status of an invasive glandular morphotype occurring
in Central Europe assigned either to E. amurensis or to
E. albensis. Here, we addressed this issue by analysing
morphology, internal transcribed spacers of nuclear
ribosomal DNA, and five inter-simple sequence repeat
markers. The genetic evidence supported closer rela-
tionship of this glandular morphotype to eglandular E.
albensis, widely established in Central Europe, than to
glandular E. amurensis described from Asia. We
propose to adopt a new taxonomic treatment that E.
albensis includes both eglandular and glandular indi-
viduals, and to classify the glandular ones as E.
albensis var. scholziana M. Nobis & A. Wro
´bel var.
nova. Currently this new taxon is known from a dozen
of localities in Central Europe and is invasive in the
lower section of the Oder River valley, whereas
Eragrostis albensis var. albensis has already spread
widely across Europe in riparian phytocenoses and
anthropogenic habitats. Since probably the first regis-
tered records in 1940s, it has been observed in
European part of Russia, Belarus, Ukraine, Poland,
Slovakia, Czech Republic, Germany, Austria, the
Netherlands, and its further invasion is likely to
proceed. We provided distribution maps concerning
spread dynamics of E. albensis in Europe from 1947 to
2020. In total, the species has been observed on over
1300 localities so far, most of which were found after
2000.
Keywords Alien invasive species Cryptic
invasion Distribution Eragrostis albensis var.
scholziana Integrative taxonomy Lovegrasses
Supplementary Information The online version contains
supplementary material available at https://doi.org/10.1007/
s10530-021-02507-6.
A. Wro
´bel E. Klichowska E. Baiakhmetov
M. Nobis (&)
Institute of Botany, Jagiellonian University, Gronostajowa
3, 30-387 Krako
´w, Poland
e-mail: m.nobis@uj.edu.pl
E. Baiakhmetov M. Nobis
Research Laboratory ‘Herbarium’, National Research
Tomsk State University, Lenin 36 Ave., Tomsk, Russia
634050
A. Nowak
Polish Academy of Sciences Botanical Garden – Center
for Biological Diversity Conservation in Powsin, ul.
Prawdziwka 2, 02-973 Warsaw, Poland
A. Nowak
Institute of Biology, University of Opole, Oleska 22,
45-052 Opole, Poland
123
Biol Invasions
https://doi.org/10.1007/s10530-021-02507-6(0123456789().,-volV)(0123456789().,-volV)
Introduction
Biological invasions are currently considered one of
the major global concerns threatening biodiversity and
triggering economic losses (Pejchar and Mooney
2009; Pys
ˇek and Richardson 2010). To prevent or at
least mitigate such undesirable effects, immediate and
precise identification of spreading newcomers is
necessary in order to take appropriate countermea-
sures. Many alien species, however, belong to taxo-
nomically problematic groups which makes their
detection challenging and, therefore, usually time-
delayed (Verloove 2010;Pys
ˇek et al. 2013). Such
cryptic invaders could be easily misidentified due to
their morphological similarity to other closely related
taxa and may remain unnoticed in the field for a long
time. Moreover, insufficient knowledge about distri-
bution of these organisms as well as routes and time of
their spread may lead to difficulties while classifying
them either as native or introduced in a particular
region (Saltonstall 2002; Pys
ˇek et al. 2013; Morais and
Reichard 2017).
Nowadays, however, development and application
of molecular markers may help to avoid confusion
between invaders and other closely related native taxa,
particularly those of conservation interest, as well as
with other non-native taxa or species of economic
importance (e.g. Newmaster and Ragupathy 2009;
Cseke and Talley 2012; Wong et al. 2018; Martinez
et al. 2020). The molecular approach can also be
successfully applied to unravelling the population
genetic structure of some invasive taxa, determining
their routes of spread and sites of origin, locating their
traces in environmental DNA, examining of their
interactions and impact on native organisms, as well as
identifying the features driving invasion success (e.g.
Briski et al. 2011; Meyer et al. 2016; Hardouin et al.
2018; Fagu
´ndez and Lema 2019). All of these issues
are particularly relevant to biodiversity management
and essential for the effective allocation of efforts and
funding (Pys
ˇek et al. 2013).
Genetic-based tools are thus appropriate candidates
for application to taxonomically problematic groups
such as grasses (Poaceae), a large (ca 12,000 species;
Watson and Dallwitz 1992) monocot family of hard-
to-identify plants occurring on all continents. Many
grasses have been purposely propagated worldwide
due to their agricultural and ornamental properties,
while others have expanded their ranges
spontaneously (David and Baruch 2000; Maillet and
Lopez-Garcia 2000; Canavan et al. 2019). As a
consequence, grasses include many examples of
problematic invasive species, such as representatives
of the following genera: Arundo,Bromus,Echino-
chloa,Eragrostis,Imperata,Pennisetum,Phalaris,
Phragmites,Spartina (CABI 2020; Global Invasive
Species Database 2020).
One of the largest groups of grasses is Eragrostis
Wolf (lovegrasses), which comprises ca 400 species
(Clayton et al. 2006) and is globally distributed from
tropical to temperate regions, being mainly associated
with dry, sandy, or human-disturbed areas. Therefore,
some Eragrostis taxa are widely used as crop plants
and forage (e.g. E. tef (Zucc.) Trotter; Cheng et al.
2017) or for erosion control (e.g. E. curvula (Schrad.)
Nees; Lee et al. 2013), and have been intentionally
introduced into many regions worldwide, where they
have spread within both natural and anthropogenic
habitats (Muranaka and Washitani 2004; Guzik and
Sudnik-Wo
´jcikowska 2005; Michalewska and Nobis
2005; Pagitz 2012). As a result, in many countries, the
representatives of Eragrostis are now classified as
alien casual or naturalised species, and regionally
regarded as invasive (e.g. Pys
ˇek et al. 2009; Yoshioka
et al. 2010; Tokarska-Guzik et al. 2012).
In Central Europe, occurrence of Eragrostis could
be dated back at least to the first half of the XIX
century when, probably for the first time, E. minor
Host was found in Silesia in 1838 within current
borders of Wrocław in Poland (Fiek 1881). Since that
time, over a dozen annual alien Eragrostis taxa have
been observed in Central Europe and some of them
have already become naturalised in this area (S
ˇpryn
ˇar
and Kuba
´t2004; Guzik and Sudnik-Wo
´jcikowska
2005; Scholz and Ristow 2005; Kira
´ly et al. 2011;
Medvecka
´et al. 2012; Hohla 2013). On a regional
scale, two taxa are considered invasive species—E.
minor in the Czech Republic (Danihelka et al. 2012)
and E. albensis H. Scholz in Poland (Tokarska-Guzik
et al. 2012; Dajdok et al. 2018). Other species, such as
E. multicaulis Steud. and E.pilosa (L.) P. Beauv., are
also spreading in Central Europe but have not been
classified yet as invasive in this region (Scholz and
Ristow 2005; Hohla 2006).
Recently, the occurrence of another species, E.
amurensis Prob., was recorded in Central Europe
along the Oder River valley in Germany in 2003 (B
herbarium; Scholz and Ristow 2005) and in Poland in
123
Original Paper
2005 (WRSL, KRA herbarium; Ka˛cki and Szcze˛s
´niak
2009) as well as along the Inn River valley in Upper
Austria in 2013 (LI herbarium; Hohla 2013). Era-
grostis amurensis was described as a new species from
the Amur Province, Russian Far East on the basis of
presence of numerous glands on a whole plant,
especially on leaf sheaths, unlike mostly eglandular
E. pilosa (Probatova and Sokolovskaya 1981). Era-
grostis amurensis (= E. voronensis H. Scholz) as well
as E. albensis,E. imberbis (Franch.) Prob., E. multi-
caulis, and E. pilosa belong to taxonomically prob-
lematic and widely distributed E. pilosa complex
(Seregin 2012a). The most common characters used in
the identification of these species are small differences
in the morphology of the panicle and spikelets as well
as presence or absence of glands and hairs on cauline
leaf sheaths (Probatova 1985;S
ˇpryn
ˇar and Kuba
´t
2004; Seregin 2012a). After delimitation of E.
amurensis as a new species (Probatova and Sokolovs-
kaya 1981), revision of herbarium materials and
observations of its spread in the field have demon-
strated that it occurs in a vast area of temperate Russia,
Mongolia, Kazakhstan (Seregin 2012a), Tajikistan
(Nobis et al. 2015; Wro
´bel et al. 2017), Belarus,
Ukraine (Seregin 2012a; Parfenov 2013), and in
Central Europe (Scholz and Ristow 2005;Ka˛cki and
Szcze˛s
´niak 2009; Hohla 2013).
However, Pagitz (2012) and Hohla (2013) have
recently expressed some doubts about taxonomic
treatment of the glandular E. amurensis specimens
observed in Austria. Pagitz (2012) inclined to the view
that glandular specimens from the North Tyrolean
population found in 2005 (IB herbarium) could rather
belong to E. albensis even though this species at that
time was accepted as eglandular (Scholz 1995; Scholz
and Ristow 2005). Pagitz (2012) suggested that E.
albensis could probably include both eglandular and
glandular morphotypes and put forward such a
hypothesis for further studies. Hohla (2013) finally
followed the taxonomic approach of Seregin (2012a)
and classified the glandular plants from the Inn River
valley in Upper Austria as E. amurensis, however, he
highlighted their probable close relationship with the
glandular specimens from North Tyrol. During our
preliminary research focused on morphology of E.
albensis and its spread in Poland, we observed that
glandular plants from the Oder River valley, so far the
only other known glandular population from Central
Europe and previously also assigned to E. amurensis
(Scholz and Ristow 2005;Ka˛cki and Szcze˛s
´niak
2009), are extremely similar to eglandular E. albensis
occurring in this region. Thus, our findings were in line
with the previous speculations of Pagitz (2012) and
Hohla (2013).
Eragrostis albensis differs from E. pilosa mostly in
having shorter spikelet pedicels, slightly longer lem-
mas and glumes, more prominent lemma veins,
usually no hairs at the apex of upper leaf sheaths,
more scabrid and stiff panicle branches, and no
verticillate branches in the lowest panicle node (Sholz
1995;S
ˇpryn
ˇar and Kuba
´t2004; Nobis and Nobis
2009). Eragrostis albensis was proposed to be
regarded as a Central European young endemic taxon
by Scholz (1995), at that time with known localities
only from the Elbe and the Oder River valleys. Later,
the revision of herbarium materials demonstrated that
the occurrence of E. albensis in Germany could be
dated back to 1982 when it was collected in Berlin
(Scholz and Ristow 2005), while along the Elbe River
valley the species had been documented since 1991
(Scholz and Ristow 2005), and from the Oder River
valley since 1992 (Scholz 1995). Soon after, many
other localities of E. albensis were reported from
Central and Eastern Europe owing to the revision of
herbaria material and new field observations. Probably
the oldest collected specimen of E. albensis in the
region is dated back to 1947 when the species was
discovered along the Vistula River valley within
current borders of Warsaw, Poland (WA herbarium;
Guzik and Sudnik-Wo
´jcikowska 2005). In 1968, E.
albensis was for the first time found in Slovakia along
the Danube River in Bratislava (Portal 2002) and in the
same year in the Czech Republic in Prague (PR
herbarium; S
ˇpryn
ˇar and Kuba
´t2004). These records
are over 10 years older than the first findings in
Germany dated back to 1982 (Scholz and Ristow
2005). Due to such pattern of distribution, S
ˇpryn
ˇar and
Kuba
´t(2004) proposed that E. albensis could rather be
an invader in Central Europe, probably originating in
other, more eastern parts of Eurasia. Nevertheless, the
exact location of the origin spot of E. albensis was not
determined. In Eastern Europe, where less research
has been devoted to E. albensis, the first registered
records of the species could probably be dated back to
1975 when it was collected in Bryansk, European part
of Russia (MW herbarium; Seregin 2012b); later the
species was also confirmed in Ukraine in 1997
(herbarium in Kiev; Gubar 2004) and Belarus (Guzik
123
Invasion of Eragrostis albensis in Central Europe
and Sudnik-Wo
´jcikowska 2005). So far, the species
was also found in Austria (Hohla 2006; Pagitz 2012)
and the Netherlands (L herbarium). Despite increasing
evidence about history of E. albensis spread, the exact
location of its native range remains undetermined and
a question whether it is autochtonous to Central
Europe has not been fully answered yet (S
ˇpryn
ˇar and
Kuba
´t2004; Pagitz 2012; Hohla 2013). Despite such
circumstances, currently the leading approach is to
recognize E. albensis in Central Europe as the alien
invader (S
ˇpryn
ˇar and Kuba
´t2004).
An ongoing discussion about taxonomic treatment
of Central European glandular Eragrostis specimens,
so far either assigned to E. amurensis,E. amurensis-
like plants or to E. albensis, has raised a question about
their phylogenetic relationship as well as the current
distribution and morphological variability of both E.
amurensis and E. albensis (Scholz and Ristow 2005;
Pagitz 2012; Hohla 2013). Due to such uncertainties
and the observed invasive character of this glandular
Eragrostis morphotype in Central Europe, the accu-
rate identification of these grasses is particularly
relevant for better understanding differentiation and
routes of spread of the taxa within the E. pilosa
complex which is essential for biodiversity manage-
ment. Despite the growing interest in studies of
Eragrostis (e.g. Cannarozzi et al. 2014; Carballo
et al. 2019; Somaratne et al. 2019), molecular
evidence concerning variability and relationships
between the taxa from the E. pilosa complex still
remains insufficient. This study aims to clarify the
status of the glandular Central European Eragrostis
morphotype from the complex by means of molecular
markers. Due to the observed morphological character
of these glandular plants as well as their occurrence in
the close vicinity of eglandular E. albensis,we
hypothesise that they are more closely related to
eglandular E. albensis than to glandular E. amurensis
and, therefore, that E. albensis comprises both eglan-
dular and glandular morphotypes. To our knowledge,
this research constitutes the first molecular phyloge-
netic insight into the E. pilosa complex with implica-
tions for its taxonomy and provides the most recent
summary concerning spread of E. albensis in Central
Europe.
Materials and methods
In this study we focused on five taxa from the E. pilosa
complex (E. albensis,E. amurensis,E. imberbis,E.
multicaulis,E. pilosa as well as Central European
glandular Eragrostis specimens so far either assigned
to E. amurensis or E. albensis). We also added five
other Eragrostis taxa occurring in Eurasia: E. cilia-
nensis (All.) Vignolo ex Janch., E. minor,E. pecti-
nacea (Michx.) Nees, E. suaveolens A. K. Becker ex
Claus, and E. virescens J. Presl. Extensive herbarium
material was reviewed in order to determine morpho-
logical patterns among the examined Eragrostis taxa
and to identify the most informative characters for the
taxa from the E. pilosa complex. In total, more than
1500 specimens of Eragrostis were examined (mate-
rials deposited in the herbaria KRA, KRAM, LE, LI,
SZUB, WA, WRSL and the herbarium of the Univer-
sity of Opole; acronyms according to Thiers 2020).
Representative specimens were chosen for the mor-
phological and molecular part of the research. For
taxonomic comparisons, specimens of E. amurensis
were purposely selected from the species0core distri-
bution area in Asia (specimens from Russian Siberia
and Tajikistan).
Morphology
All examined specimens selected for morphological
analyses were listed in ‘‘Online Appendix 1’’. Mea-
surements of panicles, spikelets, upper glumes, lower
glumes, lemmas, and caryopses were carried out
together with analyses of presence of glands, hairs,
and prickles on different parts of culms (Online
Appendix 2). Correlation between quantitative vari-
ables was checked using the Pearson correlation in
RStudio version 1.1.423 (RStudio Team 2016) with R
ver. 3.6.1 (R Core Team 2019). Variables charac-
terised by a strong correlation coefficient (more than
0.90 or less than -0.90) were then individually
assessed; one of each pair was excluded from numer-
ical analysis, since applying both might have influ-
enced the final clustering arrangement unfavourably.
Finally, the 20 most informative quantitative and
qualitative characters (Online Appendix 2) were taken
into consideration. The distance matrix was calculated
by means of Gower0s similarity index using the proxy
package in R (Meyer and Buchta 2019). The UPGMA
(unweighted pair group method with arithmetic mean)
123
Original Paper
cluster analysis was performed in R using the stats
package (R Core Team 2019) to detect morphological
patterns among the examined plants.
Molecular analyses
DNA from dried leaf tissue of herbarium specimens
was isolated using a Genomic Mini AX Plant Spin
(A&A Biotechnology, Poland) kit in accordance with
the manufacturer0s protocol. Where necessary, the
isolated DNA was purified using a gDNA Clean kit
(Syngen, Poland). The purity and concentration of
extracted DNA was assessed using a NanoDrop ND-
1000 spectrophotometer (Thermo Fisher Scientific,
USA). Only pure DNA with an A260/280 (DNA:
protein ratio) value larger than 1.8 was used for the
downstream analyses.
For ITS analysis we chose 25 individuals (Online
Appendix 3). ITS1-5.8 S-ITS2 as a whole was
amplified using forward primer N18L18: 50-
AAGTCGTAACAAGGTTTC-30(Wen and Zimmer
1996) and reverse primer ITS4: 50-
TCCTCCGCTTATTGATATGC-30(White et al.
1990). Amplification reactions were performed in a
total volume of 25 ll, containing 10 ng of genomic
DNA, 1 9final concentration of PCR DreamTaq
Green Buffer (Thermo Scientific, USA), 1 U of
DreamTaq Green DNA Polymerase (Thermo Scien-
tific, USA), 0.12 mmol of dNTPs (Thermo Scientific,
USA), 0.2 pmol of each primer, and 1 lg of bovine
serum albumin (BSA). The PCR mixtures were heated
at 94 °C for 3 min prior to 25–30 cycles of PCR
amplification in a Veriti thermal cycler (Applied
Biosystems, USA); one PCR cycle consisted of
denaturation at 94 °C for 1 min, annealing of primers
at 50 °C for 2 min, and extension at 72 °C for 2 min;
following the last cycle, the PCR mixtures were
incubated at 72 °C for 7 min. Reactions without DNA
were used as negative controls. PCR products were
sent to an external company (Genomed, Poland) for
paired-end Sanger sequencing. The resulting
sequences were manually verified and aligned using
BioEdit ver. 7.0.5.3 (Hall 1999). One sequence of
Eragrostis pectinacea was taken from GenBank
(accession number: GU359301.1) and added to the
dataset. One sequence of Enneapogon desvauxii
(GenBank accession number: GU359339.1) was used
as an outgroup.
As no studies had been published on the use of
chloroplast markers for the E. pilosa complex, here we
tested four cpDNA regions widely used in phyloge-
netic studies of angiosperms. The trnK-matK intron
was amplified using primers trnK-3914F: 50-TGG
GTT GCT AAC TCA ATG G-30(Johnson and Soltis
1994) and matK-AR: 50-CTG TTG ATA CAT TCG
A-30(Osaloo et al. 1999). The trnC
GCA
-rpoB inter-
genic spacer was amplified using primers trnC
GCA
R:
50-CAC CCR GAT TYG AAC TGG GG-30and rpoB:
50-CKA CAA AAY CCY TCR AAT TG-30, modified
by Shaw et al. (2005) after Ohsako and Ohnishi
(2000). The petL-psbE intergenic spacer was ampli-
fied using primers petL: 50-AGT AGA AAA CCG
AAA TAA CTA GTT A-30and psbE: 50-TAT CGA
ATA CTG GTA ATA ATA TCA GC-30(Shaw et al.
2007). The rpl32-trnL
UAG
intergenic spacer was
amplified using primers trnL
UAG
:5
0-CTG CTT CCT
AAG AGC AGC GT-30and rpL32-F: 50-CAG TTC
CAA AAA AAC GTA CTT C-30(Shaw et al. 2007).
As a preliminary screening, selected specimens,
representing E. albensis,E. amurensis,E. multicaulis,
E. pilosa, and the glandular Eragrostis morphotype
from Poland, were used to check the usefulness of
selected cpDNA for genetic delimitation of species
from the E. pilosa complex. All four cpDNA loci were
amplified in a total volume of 25 ll, containing 10 ng
of genomic DNA, 1 9final concentration of PCR
DreamTaq Green Buffer (Thermo Scientific, USA), 1
U of DreamTaq Green DNA Polymerase (Thermo
Scientific, USA), 0.12 mmol of dNTPs (Thermo
Scientific, USA), 0.08 pmol of each primer, and
2lg of BSA. The PCR mixtures were then heated at
80 °C for 5 min prior to 35 cycles of PCR amplifica-
tion in a Mastercycler DNA thermal cycler (Eppen-
dorf, Germany); one PCR cycle consisted of
denaturation at 94 °C for 1 min, annealing of primers
at 46 °C for 1 min, and extension at 72 °C for 2 min;
following the last cycle, the PCR mixtures were
incubated at 72 °C for 5 min. Reactions without DNA
were used as negative controls. PCR products were
sent to an external company (Genomed, Poland) for
paired-end Sanger sequencing.
For analyses of ISSR (inter-simple sequence repeat;
Zie˛tkiewicz et al. 1994) markers, 14 samples of
Eragrostis were chosen (Online Appendix 3, Table 4).
During a preliminary stage, the usefulness of 12
selected primers, including six previously designed for
E. tef (Assefa et al. 2003), was assessed. Finally, five
123
Invasion of Eragrostis albensis in Central Europe
reproducible and scorable primers with polymorphic
bands were chosen and used in PCR reactions in
optimised annealing conditions (Table 1).
The PCR amplification of DNA fragments using the
five ISSR primers was carried out in a total volume of
15 ll, containing 10 ng of genomic DNA, 1 9final
concentration of PCR DreamTaq Green Buffer (for
primers 811, 888, 889; Thermo Scientific, USA) or
Taq Buffer with (NH
4
)
2
SO
4
(for primers ubc836 and
M2; Thermo Scientific, USA), 0.375 U (for 811, 888,
889) or 1 U (for ubc836) or 0.75 U (for M2) of
DreamTaq Green DNA Polymerase (Thermo Scien-
tific, USA), 3 pmol of dNTPs (Thermo Scientific,
USA), 1.33 pmol of primer, 30 pmol of MgCl
2
(Thermo Scientific, USA) for 811, 888, 889, and
ubc836 or 45 pmol for M2.
The PCR mixtures were heated at 95 °C for 3 min
prior to 33 cycles of PCR amplification in a DNA
thermal cycler T100 (Bio-Rad, USA); one PCR cycle
consisted of denaturation at 95 °C for 30 s, annealing
of a primer at 53–58 °C (Table 1) for 30 s, and
extension at 72 °C for 1 min; following the last cycle,
the PCR mixtures were incubated at 72 °C for 10 min.
For each primer, the PCR reaction was repeated to
check reproducibility.
ISSR fragments were separated via electrophoresis
for one hour at 100 V in 2% agarose gel with Midori
Green DNA stain (Nippon Genetics, Germany) for
visualisation. 1X TBE was used as a buffer solution.
Subsequent imaging was carried out under UV light to
confirm the amplification. The size of amplified
fragments was estimated via comparison to a Gen-
eRuler 100 bp Plus DNA Ladder (Thermo Scientific,
USA) and scored in a binary format as either (1)
present or (0) absent.
Molecular data analyses
Maximum-likelihood analysis (ML) of ITS marker was
performed in PhyML 3.0 (Guindon et al. 2010). The
best substitution model for the dataset was determined
using SMS (Smart Model Selection; Lefort et al. 2017)
with AIC (Akaike Information Criterion). The model
GTR ?I (general time-reversible model with a pro-
portion of invariable sites) was indicated as most
appropriate. Support values for the tree nodes were
calculated using the approximate likelihood ratio test
(aLRT; Anisimova and Gascuel 2006).
Bayesian Inference (BI) analysis was performed in
MrBayes 3 (Ronquist and Huelsenbeck 2003). The
most appropriate substitution model was selected
using MrModeltest 2.3 (Nylander 2005) and PAUP*
4.0a166 (Swofford 2002). Model SYM ?G (a sym-
metrical model with gamma distributed rate variation
among sites) was indicated as the best and was set as
lset nst = 6 rates = gamma, prset statefre-
qpr = fixed(equal). An MCMC simulation was set as
a default for 1,000,000 generations, sampling one of
every 500 generations, which sufficed to obtain the
average standard deviation of split frequencies below
0.01 and the potential scale reduction factor of all
parameters close to 1.0. Tracer 1.6.0 (Rambaut et al.
2014) was used to assess convergence and to ascertain
whether all statistics were characterised by effective
sample sizes (ESS) greater than 200. The first 25% of
the iterations were discarded as a 0burn-in0fraction; the
remainder was used to construct the Bayesian con-
sensus tree.
The trees were edited in TreeGraph 2 (Sto
¨ver and
Mu
¨ller 2010) and in MEGA ver. 7.0.26 (Kumar et al.
2016). Bayesian posterior probabilities (BPPs) ranged
from 0.90 to 1 and support values from ML (MLs)
higher than 0.7 (Hillis and Bull 1993) were regarded as
sufficiently strong support for clades. Tree nodes with
weak support, with both BPPs less than 0.9 and MLs
less than 0.7, were collapsed and presented as
unresolved (polytomy). To increase the clarity of the
phylogram, the location of support values for each
node was changed from the default tree output and
adjusted manually using Inkscape ver. 3.
Table 1 Primers used in
ISSR analysis concerning
examined Eragrostis taxa
a
B = Non-A (i.e. C, G or
T); D = Non-C (i.e. A, G or
T); Y = Pyrimidine (C or T)
Code Sequence (50to 30)
a
Length Annealing temperature (°C)
811 GAG AGA GAG AGA GAG AC 17 53
888 BDB CAC ACA CAC ACA CA 17 53
889 DBD ACA CAC ACA CAC AC 17 58
ubc836 AGA GAG AGA GAG AGA GYA 18 53
M2 ACA CAC ACA CAC ACA CYG 18 53
123
Original Paper
A binary ISSR matrix containing only polymorphic
bands was analysed in RStudio version 1.1.423
(RStudio Team 2016) with R ver. 3.5.3 (R Core Team
2019). The distance matrix was calculated by means of
the Jaccard similarity index using the proxy package
(Meyer and Buchta 2019). A neighbour-joining tree
and bootstrap analysis were performed using the ape
package (Paradis and Schliep 2018). Bootstrap values
higher than 0.7 were regarded as sufficiently strong
support for clades (Hillis and Bull 1993). The tree was
rooted by Eragrostis taxa from outside the E. pilosa
complex.
Spread dynamics of Eragrostis albensis
Distribution maps of E. albensis spread in Europe
were based on the specimens of this taxon preserved in
the herbaria KRA, KRAM, LI, POZ, SZUB, WA,
WRSL, information in online databases of herbarium
specimens of B, BRNU, GJO, LZ, PRC, W, WU
(JACQ—Virtual Herbaria; https://www.jacq.org/;
accessed 2020-09-10), and MW (https://plant.depo.
msu.ru/module/itemsearchpublic; accessed 2020-09-
10), published localities (Online Appendix 5), ATPOL
database (Zaja˛c A., Institute of Botany, Jagiellonian
University; accessed 2020-09-14), GBIF database
(https://doi.org/10.15468/dl.qc8cf9; accessed
2020-09-04), PLADIAS database (https://pladias.cz/
en/; accessed 2020-09-10), observations of Wrzesien
´
M. (Maria Curie-Skłodowska University in Lublin),
and unpublished data from our field studies. Original
GPS coordinates from a locality were used if possible.
If GPS data was not available, geographic coordinates
were approximated based on a description of a locality
or a number of a grid unit (ATPOL, PLADIAS). Some
regions including south-western Czech Republic,
south-western Germany, central Poland, Slovakia,
Hungary and Eastern Europe could be to some extent
underestimated due to scarce or none data available.
The list of all E. albensis localities which were
included in the distribution maps is available in
‘‘Online Appendix 6’’. Spread dynamics of E. albensis
was divided into three time intervals and set arbitrarily
to 1979, 1999, and 2020. The distribution maps were
prepared in ArcGIS Desktop 10.8 with the ESRI
basemap World Imagery. https://www.arcgis.com/
home/item.html?id=10df2279f9684e4a9f6a7f08febac
2a9.
Results
Morphology: key characters
Analyses revealed five main distinct morphological
groups within the E. pilosa complex (Fig. 1; Table 2).
Two taxa, E. multicaulis and E. pilosa, were aggre-
gated. Each formed its own cluster, which was clearly
separated from the other taxa (Fig. 1, cluster IV and V,
respectively). Eragrostis amurensis was linked in one
cluster with Central European glandular specimens
from the Inn River valley (Fig. 1, cluster I). Central
European glandular specimens from the Oder River
valley (Fig. 1, cluster III) were resolved as a sister
cluster to an aggregation consisting of E. albensis and
E. imberbis (Fig. 1, cluster II).
The most useful diagnostic characters of E. pilosa
are: the presence of several verticillate branches in the
lowest panicle node (in small individuals sometimes
non-verticillate), presence of tufts of long hairs at the
apex of all leaf sheaths, hairs in the panicle axils, and
usually flexuous panicle branches. Eragrostis amuren-
sis is very similar to E. pilosa, however, it has glands
on leaf sheaths and blades. Eragrostis albensis has
more robust and stiff panicle branches than E. pilosa,
no verticillate branches in the lowest panicle node (if
there are several branches, then they are grouped on
one side of a panicle or in clusters opposite to each
other, never arranged as a whorl), tufts of long hairs at
the apex of usually only lower leaf sheaths, and hairs
in panicle axils. Eragrostis imberbis is similar to E.
albensis, however, it has longer pedicels of the lateral
spikelets [E. imberbis: 2–5 mm long; E. albensis:
(0.5–)1.0–2.5(–3.5) mm] and has usually several
verticillate branches at the lowest panicle node.
Eragrostis multicaulis has no hairs in panicle axils,
no verticillate branches in the lowest panicle node,
usually small robust panicle with branches diverging
even to 90°at maturity, and no tufts of long hairs at the
apex of all leaf sheaths (rarely, at most single long hair
on some leaf sheaths).
The analyses showed that, when morphology was
considered exclusively, Central European glandular
specimens did not represent uniform patterns. Spec-
imens from the Oder River valley referred morpho-
logically to eglandular E. albensis to a greater extent
than to glandular E. amurensis (Fig. 1). These plants
had the longest lower glumes within the entire studied
complex. Moreover, similarly as in E. albensis and E.
123
Invasion of Eragrostis albensis in Central Europe
imberbis (Fig. 1; Table 2), they had no tufts of long
hairs at the apex of the uppermost leaf sheaths.
Specimens from the Inn River valley reflected glan-
dular E. amurensis to a greater extent than eglandular
E. albensis (Fig. 1). These plants had less prominent
glumes than specimens from the Oder River valley. In
addition, they were characterised in part by long hairs
at the apex of the uppermost leaf sheaths (Fig. 1;
Table 2). All of the other morphological characters of
Central European glandular morphotype overlapped
considerably.
ITS region
The ITS sequences numbered 606 bp in all taxa from
the E. pilosa complex and from 600 to 606 bp in other
examined Eragrostis taxa. The tree topologies from
the Bayesian inference method and the maximum-
likelihood analysis based on ITS region were consis-
tent. The Central European glandular Eragrostis
morphotype was grouped in a clade with eglandular
E. albensis,E. imberbis, and E. multicaulis (Fig. 2).
The glandular specimens from the Oder River valley
were identical with E. albensis. They differed,
however, from the glandular specimen from the Inn
River valley in Upper Austria by two substitutions
(Table 3). All representatives of this clade, both
glandular and eglandular differed from glandular E.
amurensis by at least two substitutions (Table 3). In
total, six sites were polymorphic in E. amurensis,E.
albensis,E. imberbis,E. multicaulis, and the glandular
morphotype from Central Europe. Eragrostis vires-
cens appeared to be a sister clade to E. pilosa and other
taxa from the E. pilosa complex. Eragrostis minor,E.
suaveolens,E. pectinacea and E. cilianensis subsp.
starosselskyi were resolved as more distantly related
to the E. pilosa complex and formed a distinct clade
(Fig. 2).
Chloroplast DNA regions
All studied chloroplast regions showed lower level of
variation than ITS. Based only on the acquired
sequences of good quality, several mutations were
detected. In trnK-matK intron, one substitution was
noted in E. amurensis (E2) in comparison to identical
sequences of E. albensis (E6) and E. multicaulis (E17).
In rpoB-trnC
GCA
intergenic spacer, two substitutions
Fig. 1 Dendrogram of the cluster analysis (UPGMA) of the
Eragrostis pilosa complex based on 20 selected morphological
characters (Online Appendix 2) using Gower0s similarity index.
I—E. amurensis (green), glandular Eragrostis morphotype from
the Inn River valley, Austria (red); II—E. albensis (dark blue),
E. imberbis (light blue); III—glandular Eragrostis morphotype
from the Oder River valley, Poland (pink); IV—E. multicaulis
(orange); V—E. pilosa (yellow). A list of examined specimens
can be found in ‘‘Online Appendix 1’’
123
Original Paper
Table 2 Comparison of the most informative morphological characters within the Eragrostis pilosa complex, based on the specimens listed in ‘‘Online Appendix 1’’
Character Taxon
E. albensis E. amurensis Eragrostis
sp. (glandular; from the
Oder River valley,
Central Europe: DE, PL)
Eragrostis
sp. (glandular; from
the Inn River valley,
Central Europe: AT)
E. imberbis E. multicaulis E. pilosa
Tufts of long hairs at
the apex of upper
leaf sheaths
Absent Present; rarely absent Absent Absent or present Absent Absent Present; rarely absent
Tufts of long hairs at
the apex of lower
leaf sheaths
Present; very rarely
absent but then hairs
in the panicle axils
present
Present Present Present Present Absent; rarely with
single long hair (not
tufts)
Present
Hairs in panicle axils Present Present; rarely absent Present Present Present Absent Present
Branches at the
lowest panicle node
1–2, if more then usually
grouped on one side of
panicle axis (not
verticillate)
1–2, Rarely more
verticillate
1–2, If more then usually
grouped on one side of
panicle axis (not
verticillate)
1, Rarely more and partly
verticillate
Usually 3 or more
verticillate
1–2 Usually 3 or more
verticillate; rarely
1–2
Pedicels Scabrous to densely
scabrous
Sparsely to densely
scabrous
Scabrous to densely scabrous Scabrous to densely
scabrous
Scabrous to
densely
scabrous
Glabrous or scabrous
only at uppermost
pedicels
Usually glabrous to
sparsely scabrous;
rarely scabrous at
uppermost pedicels
Glands on the glumes
or lemmas keel
Absent Present (sometimes) Absent Absent Absent Absent Absent
Glands on the leaf
sheaths keel
Absent Present Present Present Absent Absent Absent
Glands on leaf blades
margins
Absent Present (sometimes) Present Present Absent Absent Absent
Glands below culm
nodes (as bands)
Absent Present (sometimes) Absent Absent Absent Absent Absent
Florets in spikelet
(number)
(4–)5–8(-9) 4–7(-9) 5–9(-11) 6–8 (5–)6–10 (5–)6–9(-12) (4–)5–8(-11)
Lower glume length
(mm)
(0.4–)0.6–1.0 (0.4–)0.5–0.7(-0.8) (0.75–)0.9–1.2(-1.3) (0.5–)0.6–0.8 0.7–0.9 0.4–0.7(-0.8) (0.2–)0.3–0.6(-0.7)
Upper glume length
(mm)
(1.1–)1.2–1.5(-1.6) (0.9–)1.0–1.3(-1.7) (1.25–)1.3–1.6(-1.8) 1.2–1.3 1.25–1.4(-1.5) (0.8–)0.9–1.2(-1.3) (0.6–)0.7–1.1(-1.4)
Lemma of the lowest
floret in the spikelet
length (mm)
(1.5–)1.6–1.9(-2.1) (1.2–)1.6–1.9(-2.0) (1.6–)1.8–2.0(-2.2) 1.6–1.8(-1.9) 1.7–1.8(-2.0) (1.4–)1.5–1.7(-1.8) (1.2–)1.4–1.7(-1.9)
Lemma of the central
floret in the spikelet
length (mm)
(1.1–)1.3–1.5(-1.75) 1.3–1.6(-1.7) (1.4–)1.5–1.7(-1.8) 1.4–1.6 (1.4–)1.5–1.6 (1.0–)1.2–1.4(-1.5) (1.1–)1.2–1.4(-1.5)
123
Invasion of Eragrostis albensis in Central Europe
were observed in E. pilosa (E22) in comparison to E.
albensis (E6). In petL-psbE intergenic spacer, one
insertion was noted in E. amurensis (E2) in compar-
ison to glandular morphotype from Central Europe
(E34). In rpl32-trnL intergenic spacer, one deletion
and nine substitutions were observed in E. pilosa
(E22) in comparison to identical E. amurensis (E2), E.
albensis (E6), E. multicaulis (E17), and glandular
morphotype from Central Europe (E34).
ISSR markers
In total, the selected set of primers enabled the
acquisition of 144 unique bands (characters). ISSR
markers indicated that E. amurensis differed from the
Central European glandular morphotype which was
aggregated with eglandular E. albensis (Fig. 3;
Table 4). Eragrostis imberbis appeared to be very
closely related to E. albensis and the Central European
glandular morphotype. Eragrostis amurensis,E. mul-
ticaulis, and E. pilosa were considered distinct
lineages; however, relationships between these three
taxa and E. albensis remained unresolved due to weak
support (Fig. 3). The other three examined taxa, E.
minor,E. virescens, and E. cilianensis subsp.
starosselskyi, were regarded as distinct lineages out-
side the E. pilosa complex (Fig. 3).
Taxonomic treatment
As a result of morphological and phylogenetic anal-
yses of the Eragrostis pilosa complex, we propose to
describe the glandular morphotype of Eragrostis
albensis as follows:
Eragrostis albensis var. scholziana M. Nobis & A.
Wro
´bel, var. nov. (Online Appendix 4).
TYPE: Western Poland, Lubuskie Province,
Słubice, the Oder River valley, sandy banks on the
right side of the river, 52°2104.2800 N/14°33019.3200 E,
17 m a.s.l., 26 August 2017, M. Nobis, A. Nowak, A.
Wro
´bel s.n. (holotype KRA 474060!; isotypes KRA
474059, 474061–474064, 475309, 475310, 475312,
475313, 475315–475326, 475328, 475329, 475331,
475332, 475334, 475335, 475339–475346, 482318,
528738–528761!).
Diagnosis: Eragrostis albensis var. scholziana
differs from eglandular E. albensis var. albensis in
having glands on leaf sheaths keel and blade margins.
Table 2 continued
Character Taxon
E. albensis E. amurensis Eragrostis
sp. (glandular; from the
Oder River valley,
Central Europe: DE, PL)
Eragrostis
sp. (glandular; from
the Inn River valley,
Central Europe: AT)
E. imberbis E. multicaulis E. pilosa
Length (lower glume /
lemma of the lowest
floret in the
spikelet) ratio
(0.25–)0.3–0.55(-0.6) (0.2–)0.3–0.4(-0.55) (0.4–)0.5–0.65(-0.8) (0.25–)0.35–0.45(-0.5) 0.4–0.5 0.3–0.45(-0.5) (0.15–)0.2–0.45(-0.5)
Length (lower glume /
upper glume) ratio
(0.35–)0.45–0.75(-0.9) (0.3–)0.4–0.6(-0.7) (0.5–)0.6–0.8(-0.85) (0.35–)0.45–0.65 0.55–0.75(-0.85) (0.35–)0.4–0.75(-0.8) (0.25–)0.4–0.6(-0.7)
123
Original Paper
Other specimens studied (paratypes): western
Poland, Lubuskie Province, Ługi Go
´rzyckie, near the
Oder River valley, field road, 52°3202.2700N/
14°37020.3300E, 14 m a.s.l., 26 August 2017, M. Nobis,
A. Nowak, A. Wro
´bel s.n. (KRA 473835, 482319!);
western Poland, Lubuskie Province, Ługi Go
´rzyckie,
the Oder River valley, sandy banks on the right side of
the river, 5283208.4200N/14836023.9900 E, 10 m a.s.l., 26
August 2017, M. Nobis, A. Nowak, A. Wro
´bel s.n.
(KRA 473831, 473836–473838, 47473–474075,
474078, 74079, 528718–528721!); north-western
Poland, Zachodniopomorskie Province, between Cze-
lin and Stary Błeszyn, the Oder River valley, sandy
banks on the right side of the river, 52°44041.5600N/
14°21032.9600E, 3 m a.s.l., 26 August 2017, M. Nobis,
A. Nowak, A. Wro
´bel s.n. (KRA 473833, 473834,
474084, 475307, 475308, 482320!); north-western
Poland, Zachodniopomorskie Province, Gozdowice,
the Oder River valley, sandy banks on the right side of
the river, 52°45049.9400N/14°1909.1700 E, 5 m a.s.l., 26
August 2017, M. Nobis, A. Nowak, A. Wro
´bel s.n.
(KRA 475302–475305, 528711–528717!); north-
western Poland, Zachodniopomorskie Province, Osi-
no
´w Dolny, the Oder River valley, sandy banks on the
right side of the river, 52°51026.9400 N/14°8019.6100 E,
1 m a.s.l., 26 August 2017, M. Nobis, A. Nowak, A.
Wro
´bel s.n. (KRA 473823, 474067–474070, 474119,
528722–528738!); Kunice, Odra, alluvia, 3 September
2005, Z. Ka˛ cki s.n. (WRSL!); Porzecze (1), Odra, 3
September 2005, Z. Ka˛ cki s.n. (WRSL!); Czelin, Odra,
alluvia, 2 September 2005, Z. Ka˛cki s.n. (WRSL!);
Szumiłowo, Odra (3), 3 September 2006, Z. Ka˛cki s.n.
(WRSL!); Go
´rzyca (1), Odra, alluvia, 3 September
2005, Z. Ka˛ cki s.n. (WRSL!); Austria, Inn River
region, Hagenauer Bucht St. Peter am Hart [O
¨sterre-
ich, Obero
¨sterreich, Innviertel, St. Peter am Hart,
Hagenauer Bucht, junge Anlandungen an der
Su
¨dwestseite der ‘‘Kellerinsel’’], 13°504700E/
48°1603200, MTB:7744/2, Unscha
¨rferadius: 100 m,
334 m, 15 September 2013, M. Hohla (LI 75143!);
Fig. 2 The phylogram from the Bayesian inference based on
ITS1-5.8 S-ITS2 (nuclear ribosomal DNA). Numbers at nodes
represent Bayesian posterior probabilities (upper) and ort values
from maximum-likelihood analysis (lower). The scale bar
represents substitutions per position. Abbreviations: Austria
(AT), China (CN), Kyrgyzstan (KG), Mexico (MX), Poland
(PL), Russia (RU), Tajikistan (TJ). *Central European glandular
morphotype from the Eragrostis pilosa complex. All examined
specimens are listed in ‘‘Online Appendix 3’’
123
Invasion of Eragrostis albensis in Central Europe
Table 3 Polymorphic sites within ITS1-5.8 S-ITS2 (nuclear ribosomal DNA) in examined taxa from the Eragrostis pilosa complex
vs eglandular E. albensis from the Oder River valley, Poland (first row, sample PL E5)
Taxon Sample Nucleotide position
16 29 51 56 57 76 92 97 112 124 151 178 195 228
E. albensis PLE5 CT AT CTAGA T T G C T
E. albensis PLE6......... . . . . .
E. albensis PLE11......... . . . . .
E. sp.
a
PLE14......... . . . . .
E. sp.
a
PLE3......... . . . . .
E. sp.
a
PLE4......... . . . . .
E. sp.
a
ATE32........G. . . . .
E. imberbis RUE26......... . . . . .
E. imberbis RUE28......... . . . . .
E. multicaulis PLE17........G. . . . .
E. multicaulis PLE18........G. . . . .
E. multicaulis CNE31........G. . . . .
E. amurensis TJE7.....C..G. . . . .
E. amurensis RUE2.....C..G. . . . .
E. amurensis RUE29.....C..G. . . . .
E. pilosa TJ E20 A C G A T C C T G C C A T C
E. pilosa TJ E22 A C G A T C C T G C C A T C
E. pilosa PL E23 A C G A T C C T G C C A T C
Taxon Sample Nucleotide position
350 385 416 420 537 540 541 551 554 555 581 582 586 591
E. albensis PLE5GCT CT CCC ACAAC T
E. albensis PLE6..............
E. albensis PLE11..............
E. sp.
a
PLE14..............
E. sp.
a
PLE3..............
E. sp.
a
PLE4..............
E. sp.
a
ATE32.T............
E. imberbis RUE26...........T..
E. imberbis RUE28...........T..
E. multicaulis PLE17T.............
E. multicaulis PLE18T.............
E. multicaulis CNE31T.............
E. amurensis TJE7............G.
E. amurensis RUE2............G.
E. amurensis RUE29............G.
E. pilosa TJ E20 . . C T G T T T G A T . . C
E. pilosa TJ E22 . . C T G T T T G A T . . C
E. pilosa PL E23 . . C T G T T T G A T . . C
The same nucleotide is marked as a dot
Eragrostis (E.); country: Austria (AT), China (CN), Kyrgyzstan (KG), Poland (PL), Russia (RU), Tajikistan (TJ)
a
Central European glandular morphotype from the E. pilosa complex. All examined specimens are listed in ‘‘Online Appendix 3’’
123
Original Paper
Austria, Inn River region, Hagenauer Bucht St. Peter
am Hart [O
¨sterreich, Obero
¨sterreich, Innviertel, St.
Peter am Hart, Hagenauer Bucht, Insels SE des
Leitdammdurchstiches], 13°403600 E/48°1601400 N,
MTB:7744/1, Unscha
¨rferadius: 100 m, 335 m, 15
September 2013, M. Hohla (LI 751048!).
Fig. 3 Neighbour-joining tree of the ISSR profiles of examined
Eragrostis taxa. The scale bar represents distance between
specimens (calculated by means of the Jaccard similarity index).
Numbers at nodes represent bootstrap values. Abbreviations:
Austria (AT), China (CN), Kyrgyzstan (KG), Poland (PL),
Russia (RU), Tajikistan (TJ).
a
Central European glandular
morphotype from the E. pilosa complex. All examined
specimens are listed in ‘‘Online Appendix 3’’
Table 4 Summary of the ISSR analyses in examined Eragrostis taxa
Taxon Sample E5 E6 E34 E38 E32 E26 E28 E2 E7 E31 E40 E15 E12 E13 Unique bands
E. albensis PL E5 48 46 46 46 43 24 25 28 25 18 27 3 3 3 1
E. albensis PL E6 49 48 48 45 25 26 30 25 18 27 3 3 3 0
E. sp.
a
PL E34 49 49 44 24 25 29 25 17 26 3 3 3 0
E. sp.
a
PL E38 49 44 24 25 29 25 17 26 3 3 3 0
E. sp.
a
AT E32 48 25 26 31 26 17 26 3 4 3 2
E. imberbis RU E26 27 27 17 15 11 17 1 3 2 0
E. imberbis RU E28 28 18 15 11 17 1 3 2 0
E. amurensis RU E2 37 32 19 25 3 6 3 0
E. amurensis TJ E7 35 18 25 3 6 3 2
E. multicaulis CN E31 24 20 3 2 1 2
E. pilosa KG E40 391215
E. virescens TJ E15 24 2 2 17
E. minor KG E12 39 25 9
E. cilianensis KG E13 44 17
Values in diagonal of the table represent the number of bands obtained per specimen; other values represent the number of bands
shared by a pair of specimens. The last column shows the number of bands unique to a specimen in an examined set of samples. All
of the unique bands within the E. pilosa complex were generated using primer M2 (see: Materials and methods). Austria (AT), China
(CN), Kyrgyzstan (KG), Poland (PL), Russia (RU), Tajikistan (TJ)
a
Central European glandular morphotype from the E. pilosa complex. All examined specimens are listed in ‘‘Online Appendix 3’’
123
Invasion of Eragrostis albensis in Central Europe
Spread of Eragrostis albensis in Central Europe
Since the first confirmed record of E. albensis in
Europe (1947, Warsaw, Poland), the species has been
observed on over 1250 localities in Central Europe,
over 50 in Eastern Europe, and over 10 in Western
Europe (Fig. 4). From 1947 to 1979 only three
localities were confirmed in Central Europe. In
1980–1999, a considerable spread of E. albensis was
noted mainly along the Elbe River valley in Germany
and along the Vistula River valley in Poland. The
species was also found on several localities along the
Oder River valley in Germany and on a few other
localities in anthropogenic habitats. In the last
20 years, E. albensis propagated further especially
along the Elbe River and the Oder River (Fig. 5)
valleys. It was also observed in many new localities in
anthropogenic habitats, particularly along roadsides in
Upper Austria, North Tyrol, south and east Poland as
well as along rail in south-eastern and east Poland
(Fig. 4).
Discussion
The place of origin, distribution and differentiation of
the taxa from the E. pilosa complex has been already
lively discussed but so far with no support of
molecular evidence (S
ˇpryn
ˇar and Kuba
´t2004; Guzik
and Sudnik-Wo
´jcikowska 2005; Pagitz 2012; Seregin
2012a; Hohla 2013). This study indicates that glan-
dular specimens of lovegrasses from Central Europe
are more closely related to eglandular E. albensis than
to glandular E. amurensis as was previously suggested
(Scholz and Ristow 2005). Therefore, as opposite to
the typical eglandular E. albensis var. albensis,we
proposed to delineate a glandular variety of this
species under the name E. albensis var. scholziana.
Eglandular E. albensis var. albensis is much more
widespread and occurs widely across Central, Eastern,
and part of Western Europe, and is already invasive
along several Central European rivers, mainly the
Elbe, the Oder and the Vistula. On the other hand, the
glandular E. albensis var. scholziana is so far known
only from a dozen of localities in Central Europe and
currently is invasive only in the lower section of the
Oder River valley.
Results of our work provide another example that
illustrates a wide problem concerning a time-delayed
identification of spreading organisms which have
broad distribution and complicated taxonomy. Such
cryptic invaders may remain overlooked or misiden-
tified for a long time, very often until they start to
attract more attention after becoming established or
problematic in a particular area (Saltonstall 2002;
Gerlach et al. 2009; Wong et al. 2018). Another
challenge is that lack of solid evidence about history of
spread and a place of origin may lead to confusion
while classifying a taxon as either native or alien in a
specific region (Pys
ˇek et al. 2013; Morais and
Reichard 2017).
Unresolved native ranges of E. albensis and E.
amurensis
Eragrostis albensis native range has not been conclu-
sively resolved. Scholz (1995) proposed that E.
albensis could be a Central European young endemic
taxon originated as a result of a rapid evolutionary
process from a single closely related Eragrostis
individual introduced by accident from the eastern
countries. However, the leading hypothesis suggests
that it could rather be an invader, which originated
outside Central Europe and invaded this region from
the east borders (S
ˇpryn
ˇar and Kuba
´t2004).
Like for Eragrostis albensis,E. amurensis native
range has not been resolved yet. Lomonosova (2000)
presumed that its westernmost borders reach only
Novosibirsk Province, Western Siberia (Russia). On
the other hand, Seregin (2012a) proposed to determine
native range of E. amurensis as reaching further to the
west, as far as to SE Belarus and Central and Eastern
Ukraine. Our study indicates that previous records of
E. amurensis from Central Europe are invalid and,
therefore, its known distribution does not reach further
than to Eastern Europe. However, taking also into
account that E. albensis is established in European part
of Russia, Belarus and Ukraine (Sukhorukov 2011;
Seregin 2012a,b), there is a need for a revision of
glandular specimens from Eastern Europe and Wes-
tern Siberia to determine if these specimens indeed
represent E. amurensis or maybe rather E. albensis
var. scholziana, or even both taxa. Such research could
shed more light on actual distribution and native
ranges of these two species in Eurasia.
123
Original Paper
Fig. 4 Spread dynamics of Eragrostis albensis in Europe. Localities recorded to 1979 (a), 1999 (b), and 2020 (c). All localities are
listed in the ‘‘Online Appendix 6’’
123
Invasion of Eragrostis albensis in Central Europe
Eragrostis albensis introduction route to Europe
Probably, the first localities of E. albensis in both
Eastern and Central Europe were most likely over-
looked. Therefore, the data from existing herbarium
material could be biased to some extent and should be
analysed cautiously. Since the first known registered
records in 1947 from Warsaw in Poland, E. albensis
has become widely established in Central Europe and
so far it has been noted in hundreds of localities, most
commonly in Germany and Poland. One probable
scenario of invasion is that E. albensis could have
probably invaded Central Europe from the east
(S
ˇpryn
ˇar and Kuba
´t2004), firstly through anthro-
pogenic habitats and then started to penetrate adjacent
riparian communities most likely near roads and
bridges (Michalewska and Nobis 2005). Taking into
consideration the time of the first confirmed records in
Central Europe, an introduction of the species to this
region could probably be attributed to military actions
during the Second World War in 1940s and accidental
dispersal of E. albensis seeds from the territory of
Russia towards Central European countries. After that,
E. albensis could have spread further simultaneously
through rail, road traffic and along riversides, which
was enhanced by dispersal of seeds in sand and gravel
gathered along rivers and used in building areas as
well as for road maintenance during winter (Micha-
lewska and Nobis 2005). Moreover, floods in XX
century might have promoted its rapid propagation
along rivers (Guzik and Sudnik-Wo
´jcikowska 2005).
In the last 20 years, the species has considerably
spread especially in anthropogenic habitats, most
likely due to constantly growing road traffic and use
of sand from the river banks for road maintenance.
Eragrostis albensis invaded habitats and spreading
in Central Europe
Eragrostis albensis is a therophyte which produces
numerous small seeds, ca 0.8 90.4 mm, which could
be dispersed by watercourse, human activities, ani-
mals or wind over long distances. Along rivers, E.
albensis prefers open and sparse alluvial communities
including sandy, gravelly and muddy areas on flood-
plain terraces or river banks exposed during summer
low water levels (Dajdok et al. 2018). It occurs
abundantly in alluvial habitats and forms dense
extensive stands which considerably limit the area
for other plants. As a result, E. albensis has already
outcompeted some of the native species in the region
mainly along the Oder (Fig. 4), Elbe, and Vistula
River valleys where it has invaded riparian phyto-
coenoses, including those protected by NATURA
2000—Isoe
¨to-Nanojuncetea, code 3130 and Biden-
tetea tripartiti, code 3270 (Guzik and Sudnik-Wo
´jci-
kowska 2005; Krumbiegel 2008;Ka˛cki and
Szcze˛s
´niak 2009; Dajdok et al. 2018). Moreover, the
species could regionally have a negative impact on
other therophytes growing on river banks. Spreading
E. albensis has already been identified as a potential
threat to species such as Corrigiola litoralis L.,
Dichostylis micheliana (L.) Nees, Lindernia procum-
bens (Krock.) Philcox, and Lythrum hyssopifolia L.
(Ka˛cki and Szcze˛s
´niak 2009; Jackowiak et al. 2014;
Dajdok et al. 2018).
According to our research, E. albensis var.
scholziana occurs in much fewer localities compared
to E. albensis var. albensis. Glandular variety occurs
both along roadsides and rivers (Scholz and Ristow
2005;Ka˛cki and Szcze˛s
´niak 2009; Pagitz 2012; Hohla
2013), and so far, it exhibits invasive potential along
the Oder River valley, where it grows abundantly and
usually with no admixtures of the typical eglandular
morphotype. A spread of E. albensis var. albensis has
been widely observed in Central Europe along rivers,
Fig. 5 Riparian communities from the Bidentetea tripartiti
class along the Oder River, invaded by Eragrostis albensis var.
scholziana (white arrow) in Słubice, Poland, 52°2104.2800 N,
14°33019.3200 E(a); mature generative shoots of eglandular E.
albensis var. albensis on sandy banks of the Oder River near
Les
´na Go
´ra, Poland, 52°01052.200 N, 15°38016.900 E(b);
photographs taken 26 August 2017 by A. Wro
´bel
123
Original Paper
mainly the Oder, Elbe, and Vistula River valleys as
well as in anthropogenic habitats including roadsides,
railway tracks, and pavements (Guzik and Sudnik-
Wo
´jcikowska 2005; Michalewska and Nobis 2005;
Pagitz 2012; Wro
´bel and Nobis 2017). Taking into
account the dynamics of E. albensis propagation, it
seems very likely that its spread will continue and new
localities will be reported soon. Moreover, similarly to
other species spreading along motorways and main
roads (e.g. Ambrosia artemisiifolia L., Cochlearia
danica L., Dittrichia graveolens (L.) Greuter, Sagina
maritima G.Don, Senecio inaequidens DC.; Brandes
2009;Zaja˛c and Zaja˛c 2019), it is probably a matter of
time that E. albensis will be observed also in other
countries which are adjacent to its currently known
distribution.
Glandular morphotypes in the E. pilosa complex
The molecular evidence obtained in this study sug-
gests that the presence of glands as well as hairs, in
some cases, may not constitute a species-specific
character in Eragrostis. Instead, it may rather be
regarded as variability within one species. The
species-specificity of the morphology, distribution,
and abundance of glands as well as hairs has been
already the subject of lively debate in botany and is far
from resolved (e.g. Van den Borre and Watson 1994;
Taia 2006; Ciccarelli et al. 2007; Pagitz 2012; Rola
et al. 2019). In the genus Eragrostis, the presence of
glands (crateriform-like structures, pits, or bands on
various parts of a plant) was recognised as a useful and
diagnostic morphological character enabling the iden-
tification of particular taxa, including in the field
(Tutin 1980; Van den Borre and Watson 1994;
Peterson 2003; Shouliang and Peterson 2006; Gir-
aldo-Can
˜as et al. 2012). In the E. pilosa complex, the
typical E. pilosa is considered completely eglandular
or at least eglandular on leaf sheaths and blades. It may
only sporadically have few glands on a panicle axis or
below culm nodes (Koch 1974; Peterson 2003;
Giraldo-Can
˜as et al. 2012; Seregin 2012a). In the
complex, presence of glands on leaf sheaths has been
related to three taxa so far – E. perplexa delimited in
the USA as well as E. amurensis and E. voronensis
described from Russia. Densely glandular plants
observed in America were proposed as Eragrostis
perplexa L.H.Harv., (Harvey 1954), subsequently,
lowered to the rank of a variety by Koch (1974) as
Eragrostis pilosa var. perplexa (L.H.Harv.) S.D.Koch
and regarded by him as restricted to North America.
This taxon was distinguished mainly on the basis of
presence of abundant glands scattered on a whole plant
and characterised by longer glumes, lemmas and
caryopsis in comparison to the typical E. pilosa (Koch
1974; Peterson 2003). In Eurasia, two species with
glandular leaf sheaths were described—E. amurensis
(Probatova and Sokolovskaya 1981) and E. voronensis
(Scholz 2010), however, the latter was considered a
synonym of the former species after a taxonomic
revision (Seregin 2012a). As a consequence, all
individuals morphologically assigned to the E. pilosa
complex in Eurasia but characterised by possession of
glands on leaf sheaths have been identified as E.
amurensis (Scholz and Ristow 2005;Ka˛cki and
Szcze˛s
´niak 2009; Seregin 2012a).
Nevertheless, our findings support the hypothesis of
Pagitz (2012) that E. albensis could also express both
eglandular and glandular morphotypes which implies
that presence of glands on leaf sheaths should no
longer be treated as an exclusive character of E.
amurensis in the E. pilosa complex in Eurasia.
Moreover, E. albensis may be variable across its
range and could comprise more than one genetic
lineage as the glandular specimens from the Inn River
valley in Upper Austria had a slightly different genetic
profile and morphology in comparison to plants from
the Oder River valley. The next step should be to
examine glandular specimens of Pagitz (2012) from
North Tyrol, Austria using the ITS marker to deter-
mine if they represent the same genetic pattern as
Upper Austrian population or form another evolution-
ary lineage of E. albensis.
Although we did not find any hints of recent
hybridisation between taxa from the E. pilosa complex
based on the ITS sequences, more data is needed to test
this possibility. In addition, the potential crossing
between taxa from the E. pilosa complex and more
distant Eragrostis species should be verified. Proba-
tova and Sokolovskaya (1981) speculated about
putative origin of E. amurensis and attributed it to
hybridisation between eglandular E. pilosa and glan-
dular E. minor. However, this hypothesis was not
supported by molecular evidence by Probatova and
Sokolovskaya (1981) and would require further
investigation.
123
Invasion of Eragrostis albensis in Central Europe
Hairy plants in the E. pilosa complex
The characters related to hairs have been widely
adopted as diagnostic in the E. pilosa complex. A
presence of tufts of long hairs on lower leaf sheaths has
been indicated as a diagnostic for E. albensis, whereas
E. multicaulis has been generally accepted as not
having tufts of long hairs and E. pilosa as having them
on all leaf sheaths (Scholz 1995;S
ˇpryn
ˇar and Kuba
´t
2004; Michalewska and Nobis 2005; Nobis and Nobis
2009; Seregin 2012a).During our revision, we found
that all specimens of E. albensis from Poland, both
glandular and eglandular, had tufts of long hairs at the
apex only of lower leaf sheaths and did not have long
hairs at the uppermost leaf sheaths. In addition, they all
had hairs in panicle axils. We also observed that E.
multicaulis examined from different countries did not
have hairs in panicle axils and tufts of long hairs at the
apex of leaf sheaths or at most only single long hairs on
some leaf sheaths. Such pattern is therefore specific for
many populations.
Pagitz (2012) also raised a problem related to the
identification of dwarf specimens of E. albensis which
could be confused with E. multicaulis which is
regularly smaller and has shorter panicles than E.
albensis. Such difficulty was also experienced by
Scholz who suggested to identify small plants from the
Oder River valley as E. multicaulis and bigger ones as
E. albensis (specimens in WRSL herbarium) even
though both morphotypes had tufts of long hairs at the
apex of lower leaf sheaths. However, Pagitz (2012)
noted that E. albensis in North Tyrol, Austria partly
had tufts of long hairs at the uppermost leaf sheath
(which makes these individuals more similar to E.
pilosa than to E. albensis). Similarly, the glandular
specimens from the Inn River valley in Upper Austria
partly had tufts of long hairs at the uppermost leaf
sheaths. That could suggest that there is variability in
the expression of hairs in the species from the E. pilosa
complex which could complicate their identification.
As a consequence, collected evidence implies that
presence of both hairs and glands could represents
regionally variable patterns and, therefore, should
rather be treated more cautiously in the taxonomic
treatment of the E. pilosa complex.
A case of Eragrostis imberbis
The problem concerning classification and distribution
of taxa from the E. pilosa complex becomes even more
complicated if we consider one other taxon, E.
imberbis. It was described from Russian Far East
and was characterised by huge panicles usually longer
than the rest of the culm, distinctly long spikelet
pedicels, scabrid panicle branches, and eglandular leaf
sheaths (Tzvelev 1976; Probatova 1985). Seregin
(2012a,b) suggested that Asian E. imberbis distributed
across Russian Far East and south Siberia might
possibly be conspecific with European E. albensis and
that these two taxa could be one species broadly
distributed across Eurasia. Eragrostis albensis has
never been reported from Asia, however, according to
the latest revision, it appeared that it is not possible to
indicate clear morphological differences between E.
albensis and E. imberbis (Seregin 2012a,b). Eragrostis
imberbis can be distinguished from E. pilosa mainly
by having densely scabrous panicle branches and
longer lemma (Probatova 1985; Seregin 2012a), and
these two diagnostic differences are similar to those
between E. albensis and E. pilosa. This study
confirmed that specimens from Russian Far East,
identified as E. imberbis, resembled morphology of
European E. albensis to a large extent. Eragrostis
imberbis differed only slightly from E. albensis in
longer pedicels of lateral spikelets and by having
usually several verticillate branches at the lowest
panicle node (Table 2). Our molecular analyses
resolved E. imberbis as a distinct although closely
related lineage to E. albensis. The next step should be
to use more sensitive, genome-wide approach and
population sampling across Eurasia to get a better
insight into the evolutionary history and dispersal dy-
namics at the population level of species from the E.
pilosa complex.
Conclusions
Molecular analyses based on ITS and ISSR markers
have indicated that the E. pilosa complex is geneti-
cally variable across Eurasia and there is a possibility
to determine species- and lineage-specific genetic
apomorphies. The first molecular insight to the
complex indicated that E. albensis deviates from E.
pilosa in many traits and, therefore, deserves to be
123
Original Paper
treated as a separate taxon. The present study also
revealed that glandular Central European morphotype,
so far classified as E. amurensis or E. albensis,is
genetically more similar to eglandular E. albensis,
widely established in Central Europe, than to glandu-
lar E. amurensis described from Asia. Here, we
propose to adopt a new taxonomic treatment that E.
albensis includes both eglandular and glandular indi-
viduals, and classify the glandular variety as E.
albensis var. scholziana. Our study implies that
presence of glands on leaf sheaths should not be
treated as an exclusive character of E. amurensis
within the E. pilosa complex in Eurasia and, therefore,
more attention should now be paid to glandular
Eragrostis plants during identification.
Since the first confirmed records in 1947 till now, E.
albensis has spread considerably in Central Europe
along river valleys, roadsides and railway tracks. Its
progressive invasion may potentially pose a threat to
native riparian phytocenoses, therefore, regular
biomonitoring would be recommended in order to
control its impact on local alluvial communities and
populations of rare species occurring there.
This study could be a starting point for further
large-scale research which is needed to get a full
picture of variability within the E. pilosa complex
across Northern Hemisphere. It would be especially
essential to determine whether the glandular speci-
mens recorded from Eastern Europe and Western
Siberia do not actually represent the glandular E.
albensis var. scholziana. Further investigation could
also address the question if E. albensis and E.
imberbis, resolved as distinct lineages in this study,
constitute two closely related but different species.
GenBank accessions
ITS1-5.8 S-ITS2 of nuclear ribosomal DNA.
Eragrostis albensis: MT344699 (E5), MT344698
(E6), MT344694 (E11); Eragrostis albensis var.
scholziana: MT344702 (E3), MT344700 (E4),
MT344691 (E14), MT344678 (E32); Eragrostis
amurensis: MT344701 (E2), MT344697 (E7),
MT344680 (E29); Eragrostis cilianensis subsp.
starosselskyi: MT344696 (E9), MT344692 (E13);
Eragrostis imberbis: MT344682 (E26), MT344681
(E28); Eragrostis minor: MT344695 (E10),
MT344693 (E12); Eragrostis multicaulis:
MT344688 (E17), MT344687 (E18), MT344679
(E31); Eragrostis suaveolens: MT344686 (E19);
Eragrostis pilosa: MT344685 (E20), MT344684
(E22), MT344683 (E23); Eragrostis virescens:
MT344690 (E15), MT344689 (E16).
Chloroplast DNA
petL-psbE intergenic spacer, partial sequence—Era-
grostis albensis var. scholziana: MW019440 (E34);
Eragrostis amurensis: MW019441 (E2).
rpL32 gene, partial cds; and rpl32-trnL(UAG)
intergenic spacer, partial sequence—Eragrostis
albensis: MW036251 (E6); Eragrostis albensis var.
scholziana: MW036254 (E34); Eragrostis amurensis:
MW036250 (E2); Eragrostis multicaulis: MW036252
(E17); Eragrostis pilosa MW036253 (E22).
trnC(GCA)-rpoB intergenic spacer, partial
sequence; and (rpoB) gene, partial cds –
Eragrostis albensis: MW036255 (E6); Eragrostis
pilosa MW036256 (E22).
tRNA-Lys (trnK) gene, intron, partial sequence;
and matK gene, partial cds—Eragrostis albensis:
MW036258 (E6); Eragrostis amurensis: MW036257
(E2); Eragrostis multicaulis: MW036259 (E17).
Acknowledgements We would like to thank the curators of
the herbaria KRA, KRAM, LE, LI, WA, SZUB, and WRSL as
well as the herbarium of the University of Opole for making
their collections available during our research. We would like to
express our appreciation to Martin Pfosser for lending us
specimens labelled as E. amurensis from Upper Austria
(herbarium LI), which greatly enriched our study. We are also
grateful to Adam Zaja˛c (Jagiellonian University), Małgorzata
Wrzesien
´(Maria Curie-Skłodowska University in Lublin),
Zygmunt Dajdok (University of Wrocław), and Zygmunt
Ka˛cki (University of Wrocław) for sharing their data on E.
albensis localities in Poland. We would like to thank two
anonymous reviewers and the associate editor Carla Lambertini
for their insightful and helpful feedback.
Funding This study was partially supported by the National
Science Centre, Poland, Grant Nos. 2018/29/B/NZ9/00313 and
2017/25/B/NZ8/00572.
Data availability Data are available in the electronic
appendices, and are also available from the corresponding
author on reasonable request.
Declarations
Conflict of interest The authors declare that they have no
conflict of interest.
123
Invasion of Eragrostis albensis in Central Europe
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