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Morphological and genome-wide evidence for natural hybridisation within the genus Stipa (Poaceae)

  • PAS and University of Warmia and Mazury in Olsztyn
  • Tomsk State University, Altay State University

Abstract and Figures

Hybridisation in the wild between closely related species is a common mechanism of speciation in the plant kingdom and, in particular, in the grass family. Here we explore the potential for natural hybridisation in Stipa (one of the largest genera in Poaceae) between genetically distant species at their distribution edges in Mountains of Central Asia using integrative taxonomy. Our research highlights the applicability of classical morphological and genome reduction approaches in studies on wild plant species. The obtained results revealed a new nothospecies, Stipa × lazkovii, which exhibits intermediate characters to S. krylovii and S. bungeana. A high-density DArTseq assay disclosed that S. × lazkovii is an F1 hybrid, and established that the plastid and mitochondrial DNA was inherited from S. bungeana. In addition, molecular markers detected a hybridisation event between morphologically and genetically distant species S. bungeana and probably S. glareosa. Moreover, our findings demonstrated an uncertainty on the taxonomic status of S. bungeana that currently belongs to the section Leiostipa, but it is genetically closer to S. breviflora from the section Barbatae. Finally, we noticed a discrepancy between the current molecular data with the previous findings on S. capillata and S. sareptana.
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SCIENTIFIC REPORTS | (2020) 10:13803 | 
Morphological and genome-wide
evidence for natural hybridisation
within the genus Stipa (Poaceae)
Evgenii Baiakhmetov1,2*, Arkadiusz Nowak3,4, Polina D. Gudkova2,5 & Marcin Nobis1*
Hybridisation in the wild between closely related species is a common mechanism of speciation in
the plant kingdom and, in particular, in the grass family. Here we explore the potential for natural
hybridisation in Stipa (one of the largest genera in Poaceae) between genetically distant species
at their distribution edges in Mountains of Central Asia using integrative taxonomy. Our research
highlights the applicability of classical morphological and genome reduction approaches in studies
on wild plant species. The obtained results revealed a new nothospecies, Stipa × lazkovii, which
exhibits intermediate characters to S. krylovii and S. bungeana. A high-density DArTseq assay
disclosed that S. × lazkovii is an F1 hybrid, and established that the plastid and mitochondrial DNA was
inherited from S. bungeana. In addition, molecular markers detected a hybridisation event between
morphologically and genetically distant species S. bungeana and probably S. glareosa. Moreover,
our ndings demonstrated an uncertainty on the taxonomic status of S. bungeana that currently
belongs to the section Leiostipa, but it is genetically closer to S. breviora from the section Barbatae.
Finally, we noticed a discrepancy between the current molecular data with the previous ndings on S.
capillata and S. sareptana.
Hybridisation in the wild between closely related species is a common mechanism of speciation in the plant
kingdom17. Due to the prevalence of polyploidy found in angiosperms it has been estimated that around 11%
of owering plants may have arisen through hybridisation events4. In addition, speciation via hybridisation can
lead to an equal ploidy number within parental and newly formed species3. In general, hybridisation is oen
accompanied by introgression and causes gene transfer between species via repeated backcrossing4,811. On the
one hand it may have contributed to species diversity and speciation5,12,13, on the other, deleterious consequences
of hybridisation such as decreased tness, genetic assimilation and gene swamping may drive populations toward
the brink of extinction1416.
In the grass family (Poaceae) hybridisation and introgression are well studied mainly for economically impor-
tant plants, such as wheats17,18, maize19,20, rice21, 22, barley23,24, oats25,26, rye27,28, sugarcanes29,30, and sorghums31,32.
Nowadays new molecular markers and technologies that rst came to the eld of agriculture are becoming widely
used in studies of wild populations with little or no previous genomic information. For instance, genotyping-by-
sequencing (GBS) and GBS-like approaches that were initially developed for maize and barley33 help to detect
hybridisation and introgression events in many wild plant genera3438.
e genus Stipa L. belongs to the subfamily Pooideae and alongside with Bambusoideae (bamboos), and
Oryzoideae (rices) form the so-called BOP clade39. e BOP species are known as the "cool season" or "pooid"
grasses and all are C3 and distributed in temperate climates40. Following Tzvelev (1974), the genus Stipa includes
six main sections Barbatae Junge, Leiostipa Dumort, Pseudoptilagrostis Tzvelev, Regelia Tzvelev, Stipa, and Smirno-
via Tzvelev41, and comprises over 150 species native to Asia, Europe and North Africa42,43. In its strict sense,
the genus is monophyletic44,45, but subdivisions within the genus are not consistently supported by available
molecular data43,46. Species of the genus are dominants and/or subdominants in steppe plant communities4750,
can be used for their classication51, and in studies related to climate change5254. Moreover, the species are of
           
        * ;
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great economic importance mainly as pasture and fodder plants, especially in the early phases of development55,
they can be used for soil remediation processes56, and as ornamental plants (e.g. S. capillata L., S. pulcherrima
K. Koch, S. pennata L.).
For decades it has been hypothesised that some Stipa taxa arose via hybridisation5760. According to our
observations, Stipa hybrids reproduce vegetatively and, less frequently, sexually60. It recently was shown that
hybrids in Stipa can produce fertile pollen grains and therefore are able to backcross with both parental species61.
In addition, based on morphology, a hybrid origin can be attributed to ca. 30% of Stipa species where only in
Middle Asia 23 of 72 species are regarded as nothospecies43. For instance, to such taxa belong S. × czerepanovii
Kotukhov (= S. orientalis Tri n. × S. richteriana Kar. & Kir.); S. × fallax M. Nobis & A. Nowak (S. drobovii (Tzvel.)
Czer. × S. macroglossa P. A. Smirn. subsp. macroglossa); S. × gegarkunii P. A. Smirn. (= S. caucasica Schmalh. × S.
pulcherrima K. Koch); S. × hissarica M. Nobis (= S. lipskyi Roshev. × S. orientalis Trin.); S. × tzveleviana Kotukhov
(= S. orientalis × S. macroglossa subsp. kazachstanica); and S. × zaissanica Kotukhov (= S. orientalis × S. hohenack-
eriana Trin. & Rupr.)43,60,62,63.
Heretofore, all putative hybrid taxa within Stipa were described based exclusively on morphological com-
parison. e only exception is Stipa × heptapotamica Golosk., whose origin has been established using molecular
methods61. Although its parental species Stipa richteriana Kar. & Kir and S. lessingiana Trin. & Rupr. were mor-
phologically distant and aliated to dierent sections Leiostipa and Subbarbatae Tzvelev41,58,64, genetically they
are closely related65,66 and able to hybridise with each other61.
During eld studies in eastern Kyrgyzstan in 2015 and 2017, interesting specimens of Stipa, combining
characters not observed in the previously described taxa, were found on the south shore of Lake Issyk-Kul
(Fig.1). Due to these specimens seeming to be morphologically intermediate between two species from the same
locality, we hypothesised that they can be hybrids between S. krylovii Roshev. and S. bungeana Trin. Although,
traditionally both putative parental taxa were assigned to the section Leiostipa58, they are distant phylogeneti-
cally and belong to two dierent clades61,65. Both of them have wide distribution ranges, Stipa krylovii occurs
in the Russian Far East and Southern Siberia, Mongolia, China, Northern Nepal, Southern Tajikistan, Eastern
Kazakhstan, and Eastern Kyrgyzstan43,67, whereas S. bungeana is distributed in Southern Mongolia, China, and
Eastern Kyrgyzstan68,69 (Fig.1a).
Since hybrids between genetically distant Stipa species have not been observed previously in nature, in the
current study by using integrative taxonomy based on morphology and high density genome wide genotyping-by-
sequencing data, we aim to (1) obtain insight into the extent of hybridisation between S. krylovii and S. bungeana
on macro- and micromorphological levels; (2) assess levels of inter-species gene ow (if present) between the
examined Stipa taxa; (3) analyse the usefulness of SilicoDArT and SNPs markers for genomic studies in Stipa.
Numerical analysis. e factor analysis of mixed data (FAMD) revealed six markedly dierentiated groups
of OTUs in accordance with the taxonomic classication of the examined taxa (Fig.2). e rst three dimen-
sions explained 41.71%, 13.64%, and 10.14%, of the total variability, respectively. e rst dimension is com-
posed, in order of descending contribution, by the quantitative variables AL, Col1L, CL, LG, CvH (Supple-
mentary TableS2, for character abbreviations see Table1). e second dimension is composed, in order of
descending contribution, by the quantitative variables DDL, LHTA, LigIV, WVS, LHD, SL, and the qualitative
variable HTTA (Supplementary TableS2). e third dimension is composed, in order of descending contribu-
tion, by the quantitative variables HLCol2, HLCol1, WCol1, CBW, and the qualitative variable AdSVL (Sup-
plementary TableS2). e two dimensional plot revealed the overlapping of OTUs belonging to S. breviora
and S. bungeana, whereas OTUs of S. sareptana are slightly overlapped with OTUs of S. krylovii and S. capillata
(Fig.2a). A clear dispersal of the OTUs could be seen in the three-dimensional plot, where dierences between
Figure1. Distribution map represents (a) general ranges of S. krylovii (green) and S. bungeana (red) with the
dashed line indicating the hypothetical border, (b) localities of the examined specimens used for the molecular
analysis. e current map is based on Google Maps.
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the studied species are explained by the third principal axis (Fig.2b and in the interactive three-dimensional plot
available at https :// ebaya hmeto v/3/). In particular, the third axis dierentiates S. breviora and S.
bungeana as clear non-overlapped clouds of OTUs.
In addition, the notch plots of variables showed signicant dierences between means and the strong evidence
of diering medians within all the taxa for CL; AL demonstrates the dierence within all the taxa except the pair
S. krylovii and S. sareptana; Col1L exhibits the dierence within all the taxa except the pair S. bungeana and S.
breviora; LG indicates the dierence within all the taxa except the pairs S. capillata and S. sareptana, as well
as S. breviora and the putative hybrid (S. bungeana × S. krylovii), here and below named as S. × lazkovii; the SL
variable shows the dierence within all the taxa except the pairs S. × lazkovii and S. capillata, and S. krylovii and
S. sareptana (Supplementary Fig.S1).
Figure2. Factor analysis of mixed data performed on 22 quantitative and three qualitative characters of the six
examined species of Stipa. (a) Plot of the two principal axes. (b) Plot of the three principal axes. e gure was
created using the R-packages factoextra v.1.0.6 (Fig. a), https ://CRAN.R-proje ge=facto extra /, and
plotly v.4.9.2 (Fig. b), https ://plotl ng-start ed/.
Table 1. Morphological characters used in the present study.
Character Abbreviation
Quantitative characters (mm)
Width of blades of vegetative shoots WVS
Length of ligules of the middle cauline leaves LigC
Length of ligules of the internal vegetative shoots LigIV
Length of lower glume LG
Length of anthecium AL
Width of anthecium AW
Length of callus CL
Length of hairs on the dorsal part of callus CdH
Length of hairs on the ventral part of callus CvH
Length of callus base CBL
Width of callus base CBW
Length of hairs on the dorsal line on lemma LHD
Length of hairs on the ventral line on lemma LHV
Distance from the end of dorsal line of hairs to the top of lemma DDL
Distance from the end of ventral line of hairs to the top of lemma DVL
Length of hairs on the top of lemma LHTA
Length of lower segment of awn Col1L
Length of middle segment of awn Col2L
Length of seta SL
Length of hairs on lower segment of awn HLCol1
Length of hairs on middle segment of awn HLCol2
Width of lower segment of awn WCol1
Qualitative characters
Character of abaxial surface of vegetative leaves (glabrous, with prickles) AbSVL
Character of adaxial surface of vegetative leaves (short hairs, long hairs, mixed) AdSVL
Type of hairs on the top of anthecium (glabrous, poor developed, well developed) HTTA
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Seven notch plots of variables show signicant dierences between means and the strong evidence of dif-
fering medians within S. bungeana, S. krylovii, and their putative hybrid: AL, CL, Col1L, SL, WCol1, LG, and
WVS (Supplementary Fig.S1). At the same time, S. bungeana, S. krylovii and S. × lazkovii share six characters
that have no signicant dierences between their means: CvH, CBL, CBW, DVL, HLCol1, and HLCol2. Further,
S. × lazkovii and S. krylovii share seven characters with no signicant dierences between their means, but dif-
fer with S. bungeana: CdH, DDL, Col2L, LigC, LigIV, AW, LHTA. Finally, only two characters LHD and LHV
have no signicant dierences between means within pairs S. × lazkovii and S. krylovii, and S. × lazkovii and S.
bungeana, but have signicant dierences between means of S. krylovii and S. bungeana (Supplementary Fig.S1).
Micromorphology. e micromorphological examination of Stipa bungeana, S. krylovii and their putative
hybrid revealed the pattern of lemma that is typical for the genus Stipa (Fig.3)45,60,62,70,71. In all three taxa, the
fundamental long cells are rectangular to more or less square in shape. e side walls of long cells are raised
and undulate. Silica bodies are sparse or absent, but if present, they are reniform to ovate, whereas cork cells are
absent. Hooks are frequent and oriented towards the lemma apex, whereas prickles are present mostly near the
lemma apex (Fig.3). Macrohairs are straight or bent near the base, cylindrical and/or string-like, with a bulbous
base and a needle-like apex. ey are organised in seven lines. e lemma apex is scabrous due to abundant
hooks, prickles and short macrohairs (present especially in S. bungeana and in the hybrid), surpassed by a ring
of unequal macrohairs. e pattern of lemma apex shows clearly intermediate character of S. × lazkovii between
the two putative parents (Figs.3a, 3f, 3k).
Figure3. Micromorphological patterns of Stipa krylovii (a-e), S. × lazkovii (f-j) and S. bungeana (k–o): top of
lemma (a, f, k), lemma abaxial surface (b-c, g-h, l-m), adaxial surface of leaf blade (d, i, n), abaxial surface of
leaf blade (e, j, o). Abbreviations: h – hooks, lc – long cells; mh – macrohairs, pr – prickles; sb – silica bodies.
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DArTseq analysis. A total of 137,437 SilicoDArT and 125,850 SNPs markers were obtained using a DArT-
seq high-density assay, of which 76,604 silicoDArT and 19,133 SNPs markers were kept aer the ltering steps.
e rst two axes of principal coordinates analysis (PCoA) explained 77% and 91% of the total genetic diver-
gence within the studied taxa based on the SilicoDArT and SNPs markers, respectively, whereas the third axes
explained only 6.3% and 3% (Fig.4).
In general, based on genetic similarities both markers revealed six markedly dierentiated groups (Fig.4).
Most of the specimens are grouped together accordingly to their taxonomical classications. However, one
sample (ID0494394), which morphologically was somewhat similar to S. breviora, is grouping together with S.
bungeana OTUs and far distant to the rest of OTUs belonging to S. breviora. All S. × lazkovii specimens have
an intermediate position between S. bungeana and S. krylovii, suggesting an admixed origin. In addition, on
the basis of two axes both markers are not allowed to dierentiate two taxa, S. capillata and S. sareptana. On the
other hand, the dierence can be marked in the three-dimensional plot based on SilicoDArT markers (Figs.4b,
the interactive plot available at https :// ebaya hmeto v/5/), but not in SNPs markers (Fig.4d, https
:// ebaya hmeto v/7/).
Figure4. Principal Coordinates Analysis plot based on genetic distances between samples. (a) Plot of the two
principal axes based on SilicoDArT markers. (b) Plot of the three principal axes based on SilicoDArT markers.
(c) Plot of the two principal axes based on SNPs markers. (d) Plot of the three principal axes based on SNPs
markers. e gure was created using the R-packages ggplot2 v.3.3.0 (Figs a and c), https ://ggplo t2.tidyv erse.
org/, and plotly v.4.9.2 (Figs b and d), https ://plotl ng-start ed/.
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A fastSTRU CTU RE analysis of the SilicoDArT markers revealed the most likely number of clusters at K value
of 5 (Fig.5a). For the SNPs markers, the ’best’ K was inferred in fastSTRU CTU RE as K = 4 (Fig.5b). Both analyses
dened S. breviora, S. bungeana, and S. krylovii as clear taxa with the exception of the specimen ID0494394
(Fig.5) that shares 73% of markers with S. bungeana and 27% with probably S. glareosa, indicating their rst
backcross generation progeny (Fig.5a). e last-mentioned taxon was not present in the analyses, however, it
is common in the locality, where the specimen ID0494394 was growing. In case of SNPs markers, the specimen
ID0494394 has 75% of markers with S. bungeana, 19% with S. capillata/S. sareptana, and 6% with S. krylovii,
suggesting a possible hybridisation between these species followed by backcrossing with S. bungeana (Fig.5b).
e fastSTRU CTU RE analyses revealed F1 hybrid specimens between S. krylovii and S. bungeana due to
samples of S. × lazkovii have admixture between these clusters in a range of 55% and 45% for the SilicoDArT
markers (Fig.5a), and 50/50% for the SNPs markers (Fig.5b). e fastSTRU CTU RE output for the SilicoDArT
markers exhibits no dierence between S. capillata and S. sareptana resulting in clustering them together (Fig.5a),
whereas the analysis of the SNPs shows an admixture between S. capillata/S. sareptana and S. krylovii in a range
of 53% and 47%, respectively (Fig.5b).
e results of the UPGMA cluster analyses revealed a clear division of samples into two major clades (Fig.6).
According to the clustering obtained with the SilicoDArT markers, the rst clade is subdivided into four smaller
clusters, specically, comprising samples of: (1) S. × lazkovii; (2) S. krylovii; (3) S. sareptana; (4) S. capillata
(Fig.6a). e rst two species are genetically closely related to each other and distant to S. sareptana and S.
Figure5. FastSTRU CTU RE results based on (a) SilicoDArT markers for K = 5 and (b) SNPs markers for K = 4.
e gure was created using an in-house R script in RStudio v.1.1.463, https ://rstud cts/rstud io/.
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capillata that together form one sub-cluster. e second clade is composed of three clusters comprising samples
of: (1) S. breviora; (2) S. bungeana; (3) the sample ID0494394 that is genetically closer to S. bungeana than to
S. breviora. e UPGMA cluster analysis of the SNPs markers demonstrated the subdivision of samples into
the same number of clusters as were obtained for the SilicoDArT markers. However, in this case, specimens of
S. × lazkovii are genetically closer to S. bungeana, but not to S. krylovii.
Genetic mapping onto chloroplast genomes of Stipa species and mitochondria of specimens from the Poaceae
family (Supplementary TableS3) revealed 11 SilicoDArT markers assigned to chloroplast DNA and 27 loci
assigned to mitochondrial DNA. e downstream neighbour-joining cluster analysis showed grouping of Stipa
taxa into two main clades (Fig.7). In the rst clade three species could be dened: S. krylovii, S. capillata (boot-
strap support 90%), S. sareptana (bootstrap support 84%), with an exception of the specimen ID0494394 that is
grouped together with S. krylovii (Fig.7). e second clade comprises a group of S. bungeana and S. × lazkovii
(bootstrap support 79%), and the rest of S. breviora specimens with a good bootstrap support of 87% (Fig.7).
All S. × lazkovii samples are grouping alongside with S. bungeana, and one S. bungeana specimen (ID0459867)
is placed outside the main group of S. bungeana and S. × lazkovii with a bootstrap support of 79% (Fig.7).
Figure6. Unweighted Pair Group Method with Arithmetic Mean cluster analyses based on Jaccard’s similarity
coecients generated from (a) SilicoDArT markers and (b) SNPs markers. e gure was created using the
R-package stats v.3.6.2, https ://www.rdocu menta ges/stats /versi ons/3.6.2/.
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Although many interspecic hybrids have been described in the genus Stipa43,5759, so far only a single molecular
investigation was performed to verify the origin of one such species, Stipa × heptapotamica61 that appeared to
be a hybrid between genetically closely related species65,66. e current study is the rst report of hybridisa-
tion between two genetically distant Stipa species, S. krylovii and S. bungeana61,65, at their distribution edges in
Mountains of Central Asia (Fig.1a).
Analyses of morphological variation resulted in a clear delimitation of the studied species (Fig.2b). Par-
ticularly, the main morphological characters (Table1) show that species S. capillata, S. sareptana, S. krylovii, S.
bungeana, S. × lazkovii representing the section Leiostipa are quite distant to S. breviora which traditionally has
been aliated to the section Barbatae41. As expected, the hybrid specimens of S. × lazkovii were mostly charac-
terised by intermediate morphological traits between the parental taxa S. krylovii and S. bungeana (Figs.2 and 3,
Supplementary Fig.S1). In addition, some OTUs of S. sareptana were slightly overlapped with OTUs of S. krylovii
and S. capillata. However, S. sareptana and S. krylovii are easy to distinguish based on morphology of leaves
(scabrous in S. sareptana and glabrous in S. krylovii) and the lemma apex (with a poorly developed ring of hairs
in S. sareptana and with a well-developed ring of hairs in S. krylovii)43,58,67. As for S. sareptana and S. capillata,
these taxa can be delimited by characteristics of their vegetative leaves (scabrous in S. sareptana and glabrous in
S. capillata) and characters of lemma (hairs on the top in S. sareptana and glabrous in S. capillata)43,58,67.
Both PCoA and fastSTRU CTU RE analyses conrmed that S. × lazkovii is the F1 hybrid of S. krylovii and S.
bungeana (Figs.4 and 5). In addition, the neighbour-joining cluster analysis identied S. bungeana as the source
of maternal DNA for all hybrid specimens, suggesting unidirectional hybridisation (Fig.7). However, due to the
small sample size, we cannot exclude either an opposite combination or interspecic gene ow through intro-
gression that could exist in nature, especially since in this area of Issyk-Kul Lake populations of both parental
species are extremely large.
Although morphologically S. bungeana is considered as a member of the section Leiostipa58, molecular
analyses demonstrated that it is quite distant from S. krylovii, S. capillata, and S. sareptana from the same sec-
tion (Figs.4, 6 and 7). ese ndings support our previous molecular results for these taxa based on a nuclear
region61,65. In addition, the results of the distance based clustering algorithms UPGMA and NJ revealed that
S. bungeana is closer to S. breviora then to the rest of Leiostipa taxa from the study (Figs.6 and 7). is result
demands further investigations on S. bungeana to establish its proper taxonomic place in the genus Stipa.
Analyses of molecular markers also revealed that the genetic relationships within some studied taxa are
more complex than expected. Firstly, the sample ID0494394, which morphologically was somewhat similar to
S. breviora, appeared to be an introgressive hybrid that shares 73% of markers with S. bungeana and 27%, more
likely, with S. glareosa (Fig.5a). Here, we presume that hybridisation events are happening between S. bungeana
Figure7. Neighbor-joining tree reconstructed based on the SilicoDArT markers derived from chloroplast
and mitochondrial genomes. e bootstrap values > 50% obtained from 10,000 replicates are shown above the
branches. e gure was created using Figtree v1.4.4, https ://w are/gtr ee/.
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and S. glareosa, because the introgressive hybrid was found on the north shore of Lake Issyk-Kul, where only
three Stipa taxa were recorded (S. bungeana, S. breviora, and S. glareosa). However, due to S. glareosa was
absent in the analyses, a new study focusing on hybridisation should be performed to verify if the gene ow is
a common event within these taxa. Secondly, our research demonstrates the discordance between the results of
fastStructure and PCoA analyses from one side and the UPGMA and NJ from the other. e rst two represent
no or almost no dierence between S. capillata and S. sareptana (Figs.4 and 5). Notwithstanding, the UPGMA
and NJ dendrograms show that genetically these taxa can be delimited (Figs.6 and 7) that supports our previ-
ous molecular investigations on these taxa61,65. However, in the current research S. capillata and S. sareptana are
grouped together, whereas based on the nuclear Intergenic Spacer (IGS) the last taxon is closer to S. krylovii61,65.
Due to the limited number of analysed specimens in the present and previous studies, we believe that a bigger
sample size combining genetics and traditional taxonomy should be undertaken in order to better resolve the
relationship between these species.
Until now the DArTseq approach has been used mostly in commercially important plant species7278 and its
implication in genomic studies in wild species is still limited79,80. us, the current study highlights the applicabil-
ity of genome reduction approaches such as DArTseq in studies on natural hybridisation in wild, and specically
in a grass genus Stipa. e high density genome wide genotyping-by-sequencing resulted in a total of 137,437
silicoDArT and 125,850 SNPs markers, of which 76,604 silicoDArT and 19,133 SNPs provided robust information
of the Stipa genome in the absence of the reference sequence information. Such number of markers is several
100-fold higher than was achieved in our previous study on natural hybridisation in Stipa61. In particular, by
using inter simple sequence repeat markers (ISSR) we were able to detect only 105 polymorphic bands for the
S. heptapotamica hybrid complex. In addition, dominant markers were used in several genomic studies in Stipa
and resulted in 372 polymorphic ISSR bands for S. bungeana81, 34 polymorphic ISSR bands for S. ucrainica and
S. zalesskii82, 212 polymorphic ISSR bands for S. tenacissima83, 231 polymorphic random amplied polymor-
phic DNA (RAPD) bands for S. krylovii84, 310 polymorphic RAPD bands for S. grandis85, and 504 polymorphic
sequence-related amplied polymorphism bands for S. bungeana81. us, both silicoDArT and SNPs markers
may better suit for genetic diversity studies in Stipa. Furthermore, the current study demonstrated the useful-
ness of silicoDArT markers as a tool to detect chloroplast and mitochondrial loci and thus may help to clarify
the maternal inheritance of hybrid species.
Taxonomic treatment. Stipa × lazkovii M. Nobis & A. Nowak, nothosp. nov. (Fig.3f-j, Supplementary Figs
S2 and S3). TYPE: Kyrgyzstan, between Kongurlen and Kultor, 17km SW from coast of Issyk-Kul, semidesert, N
42°547.07’ / E 76°396.22, elev. 1940m, wp. 930, 6 July 2017, M. Nobis, E. Klichowska, A. Wróbel, A. Nowak sn.
(holotype KRA 495,093! (specimen in the middle part of the sheet); isotypes KRA 487,067!, 487,066!, 481,608!).
Diagnosis: Stipa × lazkovii diers from S. krylovii Roshev. by having shorter anthecium (7.3–8.5mm vs.
9.0–11.5), shorter callus (1.8–2.2 vs. 2.3–3.8mm long), shorter glumes (15–17 vs. 18–28mm long) as well as by
having long prickle-hairs below the top of the anthecium (Fig.3). Having long prickle-hairs below the top of the
anthecium Stipa × lazkovii is also similar to S. bungeana, however diers from it by longer anthecium (7.3–8.5
vs. 4.8–6.0mm long), longer callus (over 1.8 vs. up to 1.3mm long), longer glumes (over 15 vs. up to 15mm
long) and narrower leaves (0.5–0.6 vs. 0.6–1.0mm wide).
Description: Plants perennial, densely tued, with a few culms and numerous vegetative shoots; culms
35–55cm tall, 3-noded, glabrous at and below the nodes. Leaves of vegetative shoots: sheaths glabrous, at mar-
gins ciliate; ligules truncate, up to 0.2mm ciliate at margins; blades convolute, up to 25cm long, 0.5–0.6(–0.7)
mm in diameter, adaxial surface densely pubescent with up to 0.1mm long hairs (prickles), adaxial surface
glabrous, rarely very slightly scabrous. Cauline leaves: sheaths glabrous and with white edge, shorter than inter-
nodes; ligules 0.5–5mm long, acute and glabrous; blades glabrous, up to 12cm long. Panicle up to 25cm long
contracted, at base enclosed by sheath of uppermost leaf, branches erect, setulose, single or paired. Glumes
subequal, 15–25mm long, narrowly lanceolate, tapering into long hyaline apex. Anthecium 7.3–8.5mm long
and 0.7–0.9mm wide. Callus 1.8–2.2mm long, densely pilose on ventral and dorsal surfaces, callus base acute,
cuneate, scar elliptic. Lemma pale green, on dorsal surface with abundant hooks and with 7 lines of ascending
hairs, hairs up to 0.5mm long, ventral line of hairs terminates at 1.3–1.7mm below top of lemma and dorsal line
terminates at 1.5–2.2mm below top of lemma; top of lemma scabrous due to hooks and prickles and at apex with
a ring of hairs up to 0.5mm long. Palea equals to lemma in length. Awn 95–118mm long, bigeniculate; lower
segment of column 19–25mm long, twisted, scabrous due to prickles and short hairs up to 0.15; upper segment
of column 11.5–13mm long, twisted, scabrous due to prickles and short hairs up to 0.2mm in long; seta exu-
ous 65–80mm long, hairs in the lower part of the seta 0.1–0.2mm long, gradually decreasing in length towards
apex. Anthers yellow, 4–5mm long, glabrous.
Etymology: e name of the taxon honours prof. dr Georgy A. Lazkov (Academy of Sciences, Bishkek, Kyr-
gyzstan), the eminent botanist, taxonomists and expert of vascular plants of Middle Asian Mountains.
Other specimens studied (paratypes): Kyrgyzstan, western Tian-Shan, Kongurlen Valley, steppe grasslands
near the road, 3km E of Kongurlen settl., to the S of SW part of Issyk-Kul Lake, N 42°553.97’ / E 76°3837.28’,
elev. 1945m, wp. 644, 10 July 2015, M. Nobis, A. Nowak sn. (KRA 476,871, 476,870!, 476,869!, WA!).
An identication key to central Asian species of Stipa that have scabrous awns or awns that are throughout
covered by 0.1–0.3mm long hairs is given in Supplementary S4.
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Materials and methods
Plant material. Morphological examination is based on plant specimens deposited in the KRA herbarium
(the acronym from iers86). In total, 188 fully developed Stipa samples were studied under a light microscope
SMZ800 (Nikon, Japan) including 40 specimens of S. krylovii, 40 of S. bungeana, 6 of S. × lazkovii, 22 of S. brevi-
ora, 40 of S. capillata, and 40 of S. sareptana.
For molecular analysis, we collected leaves of plants from localities where S. krylovii and S. bungeana grow
together with their putative hybrid, as well as from areas where S. krylovii and S. bungeana grow separately from
each other (Fig.1b). Additionally, we included Stipa taxa that frequently occur in the area near of Issyk-Kul Lake.
In total, we selected 20 specimens of S. krylovii, 20 specimens of S. bungeana, 6 specimens of S. × lazkovii, 10
specimens of S. breviora, 2 specimens of S. capillata, and 2 specimens of S. sareptana. Only one taxon, S. glareosa,
is not presented in the study due to it was not found in the locality of S. × lazkovii. Moreover, S. glareosa belongs
to the section Smirnovia41 and exhibits unique characters (e.g. long and pilose awns with a single geniculation),
which were not observed in any Stipa taxa in this region.
All voucher specimens used in the molecular analysis are preserved at KRA (Supplementary TableS1). e
names of plants were adopted from the WCSP87.
Macromorphological analyses. For the morphometric analyses, 188 specimens were used as operational
taxonomic units (OTUs)88. As a rst step, the Shapiro–Wilk test was used in the R-package MVN89 to assess the
normality of the distribution of each character. e non-parametric Spearman’s correlation coecient was used
in the R-package MVN to examine relations between the studied characters. e 22 most informative quantita-
tive and three qualitative morphological characters, commonly used in keys and taxonomic descriptions were
chosen for the analyses (Table1).
A Factor Analysis of Mixed Data (FAMD)90 was performed in the R-package FactoMineR91 to characterise
variation within and among groups of taxa without a priori taxonomic classication and to extract the variables
that best identied them. e number of principal components included in the analysis was chosen based on
Scree’s test92. e R-package factoextra93 was used to visualise the rst two components, whereas the R-package
plotly94 was chosen to illustrate the rst three.
Notch plots were created in the R-package ggplot295 to explore distributional relationships between each
response variable and the studied taxa (Supplementary Fig.S1). e notched box plots display a condence
interval around the median, which is normally based on the median ± 1.57 × interquartile range/square root of
n. According to this graphical method for data analysis, if the notches of the two boxes do not overlap, there
is "strong evidence" (95% condence) that their medians dier. Additionally, to reveal signicant dierences
between means of particular characters across all examined taxa the nonparametric Kruskal–Wallis test followed
by the Wilcoxon rank sum test for post hoc group comparisons were calculated. To address the multiplicity of
comparison, the Bonferroni method was applied to calculate corrected p-values.
Micromorphological examination. e lemma and lamina micromorphology within Stipa × lazkovii, S.
krylovii, and S. bungeana were examined using scanning electron microscopy (SEM). e dried samples were
coated with a gold layer using a Quorum Q150R S coater (Quorum, UK). e SEM images were obtained by a
scanning electron microscope S-4700 (Hitachi, Japan). Further, we examined the adaxial and abaxial surfaces
of lamina, and ve sets of diagnostic characters of lemma micromorphology: (1) long cells, (2) silica bodies, (3)
hooks, (4) prickles, (5) macrohairs.
DNA extraction, amplication, and DArT sequencing. Isolation of genomic DNA was performed
from dried leaf tissues using a Genomic Mini AX Plant Kit (A&A Biotechnology, Poland). Quality check, quan-
tication and concentration adjustment for sequencing and genotyping were accomplished using a NanoDrop
One (ermo Scientic, USA) and agarose gel electrophoresis visualisation. e concentration of each sample
was adjusted to 50ng/μL. Puried DNA samples (1μg for each sample) were sent to Diversity Arrays Technology
Pty Ltd (Canberra, Australia) for sequencing and marker identication.
DArTseq represents a combination of a DArT complexity reduction methods and next generation sequenc-
ing platforms96100. e technology is optimised for each organism and application in order to select the most
appropriate complexity reduction method (both the size of the representation and the fraction of a genome
selected for assays). Based on testing several enzyme combinations for complexity reduction Diversity Arrays
Technology Pty Ltd selected the PstI-MseI method for Stipa.
DNA samples were processed in digestion/ligation reactions as described previously97, but replacing a sin-
gle PstI-compatible adaptor with two dierent adaptors corresponding to two dierent Restriction Enzyme
(RE) overhangs. e PstI-compatible adapter was designed to include Illumina owcell attachment sequence,
sequencing primer sequence and "staggered", varying length barcode region, similar to the sequence previously
reported33. Reverse adapter contained owcell attachment region and MseI-compatible overhang sequence. Only
"mixed fragments" (PstI-MseI) were eectively amplied by PCR using an initial denaturation step of 94°C for
1min, followed by 30 cycles with the following temperature prole: denaturation at 94°C for 20s, annealing at
58°C for 30s and extension at 72°C for 45s, with an additional nal extension at 72°C for 7min. Aer PCR
equimolar amounts of amplication products from each sample of the 96-well microtiter plate were bulked
and applied to c-Bot (Illumina, USA) bridge PCR followed by sequencing on Hiseq2500 (Illumina, USA). e
sequencing (single read) was run for 77 cycles.
Sequences generated from each lane were processed using proprietary DArT analytical pipelines. In the pri-
mary pipeline, the fastq les were rst processed to lter away poor quality sequences, applying more stringent
selection criteria to the barcode region compared to the rest of the sequence. In that way the assignments of
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the sequences to specic samples carried in the "barcode split" step were very reliable. Approximately 2.5 mln
sequences per barcode/sample were identied and used in marker calling.
DArTseq data analysis. DArTseq produce two types of data: (1) co-dominant single nucleotide polymor-
phisms (SNPs) markers, and (2) dominant SilicoDArT markers that represent the presence or absence of restric-
tion fragments. All molecular analyses with the DArTseq data (SNPs and SilicoDArT) sets were performed aer
ltering steps in the R-package dartR101 with the following parameters: (1) a scoring reproducibility of 100%,
(2) at least 95% loci called (the respective DNA fragment had been identied (= called) in greater than 95% of
all individuals), (3) monomorphic loci were removed, (4) SNPs that shared secondaries (had more than one
sequence tag represented in the dataset) were randomly ltered out to keep only one random sequence tag.
ree approaches were used to analyse genetic structure of the studied taxa: (1) Principal Coordinates Analy-
sis (PCoA), (2) fastSTRU CTU RE analysis, and (3) Unweighted Pair Group Method with Arithmetic Mean
(UPGMA). e PCoA analyses based on Euclidean distance matrices were performed using R-packages dartR
and visualised by using ggplot2 to show the rst two components, and plotly to illustrate the rst three com-
ponents. Genetic structure was then investigated using the fastSTRU CTU RE soware, which implements the
Bayesian clustering algorithm STRU CTU RE, assuming Hardy–Weinberg equilibrium between alleles, in a fast
and resource-ecient manner102. A number of clusters (K-values) ranging from 2 to 10 were tested using the
default convergence criterion of 10−6 and priors. e most likely K-value was estimated with the best choice
function implemented in fastSTRU CTU RE. In case of a range of K values, the true K was determined as a value
between the estimates predicted by fastSTRU CTU RE and based on what made most biological sense. e out-
put matrices for the best K-values were reordered and plotted using an in-house R script in RStudio (Version
1.1.463)103. e threshold of 0.10 < q < 0.90 was applied as the most widely utilised measure for the assessment
of hybridisation104107. Contributions from each cluster in a range between 45 and 55% were considered as F1
hybrids, while rst and secondgeneration backcrosses with one parent were considered at values 0.25 and
0.125, respectively108. e UPGMA cluster analyses based on Jaccard’s distance matrices were performed using
R-packages dartR and visualised with stats109.
Finally, the SilicoDArT tags were used to determine maternal inheritance of the putative hybrid Stipa × laz-
kovii. e trimmed sequences of the parental species S. krylovii and S. bungeana, and the rest of studied taxa
were mapped onto chloroplast genomes of Stipa species and mitochondrions of specimens from the Poaceae
family (Supplementary TableS3) by using Minimap2110. e nal binary data matrix was used to generate a
neighbor-joining tree (NJ) derived from Jaccard’s genetic distances in the ngerprint analysis with missing data
soware v1.31 (FAMD)111 with a set of 10,000 bootstrap replicates. e resulting tree was visualised and edited
using Figtree v1.4.4112.
Data availability
e datasets used and/or analysed during the current study are available from the corresponding authors upon
Received: 18 February 2020; Accepted: 31 July 2020
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e study was supported by a RSF grant (project no. 19-74-10067); partially by a DS grant of the Jagiellonian Uni-
versity (DS/MND/WB/IB/1/2018) and by the National Science Centre, Poland (grant no. 2018/29/B/NZ9/00313).
e authors would like to thank Dr. Ewelina Klichowska and Anna Wróbel (Jagiellonian University, Kraków)
for their help in conducting the eld studies. We would like to express our gratitude to Daria Kareva (Tomsk
State University, Russia) for preparing the line drawing of the nothospecies. Finally, we thank two anonymous
reviewers for providing valuable comments on the manuscript.
Author contributions
E.B. and M.N. designed and supervised the study. M.N. and A.N. performed the eld studies. M.N. collected
the specimens for the morphological and molecular analyses and performed the micromorphological analysis
on SEM. E.B. and P.D.G. performed the macromorphological analysis. E.B. performed the molecular analysis.
E.B. and M.N. analysed all the data and wrote the manuscript with the other authors’ helps. All authors revised
the dra, provided comments, and approved the nal version of the manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary information is available for this paper at https :// 8-020-70582 -1.
Correspondence and requests for materials should be addressed to E.B.orM.N.
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Supplementary resource (1)

... To assess the relationship between genetic diversity and geographical distribution, previous studies have reported that the genetic diversity of S. bungeana is higher at the species level than at the population level, and which is no significant relationship between the genetic distance and geographical distance (Jing et al. 2013). The wild Stipa species in the natural ecosystem are prone to hybridization, the studies have found that the offspring of this natural hybridization had a great variation in morphology, and the taxonomic status of S. bungeana is not certain from the genome, it should belong to the section Leiostipa, but from the perspective of genetics, it is closer to S. breviflora of the section Barbatae (Baiakhmetov et al. 2020). A study of the hybrid-complex for S. heptapotamica showed that the hybridization between the wild Stipa each other of Poaceae is an introgression event (Nobis et al. 2019). ...
... A study of the hybrid-complex for S. heptapotamica showed that the hybridization between the wild Stipa each other of Poaceae is an introgression event (Nobis et al. 2019). However, the uncertainty of the taxonomic status of S. bungeana (Baiakhmetov et al. 2020) and the evidence for the chloroplast (cp) whole genome and its phylogenetic status still need to explore. Angiosperm Phylogeny Group (APG) classification system facilitates our understanding of plant evolution and classification (Chase et al. 2016), such as gene markers. ...
... Krawczyk et al. (2018) analyzed 21 newly sequenced complete plastid genomes from 19 groups of Stipa through the gene fragment analysis, and they found that the multilocus barcodes composed of ndhH, rpl23, ndhF-rpl32, rpl32-ccsA, psbK-psbI and petA-psbJ for Stipa were performed the best, but the effectiveness was less than 70% of the analyzed taxa, indicating that these markers did not apply to all Stipa. Hybridization among species of Stipa in different habitats may have resulted in different highly variable gene segments (Baiakhmetov et al. 2020). In the view of the phylogenetic clustering tree, although it was related to other Stipa, it clustered in a single clade (Figure 3). ...
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Stipa bungeana Trin. 1833 is an important forage grass in Poaceae, widely distributed in the temperate steppe of Northern China, with strong grazing tolerance and feeding value. In this study, we performed the complete chloroplast (cp) genome sequence of S. bungeana to explore its phylogenetic position with other Stipa. The results showed that the circular complete cp genome of S. bungeana was 137,759 bp in length, including a large single copy (LSC) of 81,652 bp, a small single copy (SSC) of 12,817 bp, and two inverted repeats (IR) of 21,645 bp. The GC content accounts for 43.71% and annotated 134 single genes, which include 87 protein-coding genes, eight rRNA genes, and 39 tRNA genes. Maximum-likelihood (ML) phylogenetic tree suggested that the S. bungeana was closely related to other Stipa except for S. purpurea.
... The genus Stipa L. is represented by over 150 species distributed in warm temperate regions throughout Europe, Asia, and North Africa (Nobis et al., 2020). Currently, the taxonomic treatment of the Stipea Dumort. ...
... Additionally, the issue of the phylogeny of the genus is greatly complicated by a large number of hybrids, many of which were previously recognized as separate species. Molecular studies have proven that hybridization and introgression processes exist in feather grasses (Nobis et al., 2019;Baiakhmetov et al., 2020Baiakhmetov et al., , 2021, and they are more common than previously believed. ...
... Within grasses, the genus Stipa is undoubtedly one of the largest and the most taxonomically difficult genus. In Central Asia, the genus Stipa comprises 72 species, including 23 of the putative hybrid origin (Nobis et al., 2020). Kazakhstan has the largest number of feather grass species among Central Asian countries and within the whole range of the genus (Bor, 1970;Freitag, 1985;Nobis et al., 2017Nobis et al., , 2020. ...
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The genus Stipa is one of the largest genera of Poaceae. The phylogeny of the genus is still poorly resolved, and one of the main problems is linked to the extensive inter and intrasectional hybridization. Disclosure of hybridization processes is a key to understanding relationships between species within the genus. During a floristic study in feathergrass steppe ecosystems of central Asia, we found challenging specimens of Stipa with an interesting combination of morphological characters suggesting their origination through hybridization between morphologically and phylogenetically distant species. To confirm our hypothesis, we applied a combination of classical morphological and genome-wide SNP genotyping methods. Using such an approach, we determined that the new taxon named Stipa × smelanskyi arose from crossing S. richteriana and S. drobovii and confirmed that it is an F1 hybrid. Moreover, we found a S. drobovii specimen with a minor admixture of S. richteriana loci that may indicate putative introgression events among these taxa.
... In addition, hybridisation may lead new organisms not only to intermediate traits of parental species but also to extreme, or transgressive, phenotypes [31] that complicate their proper taxonomic treatment. In feather grasses, the hypothesis of hybrid origin of some species was initially tested using multivariate morphological analyses [23,32] and, more recently, applying molecular markers among genetically closely related species in the Stipa heptapotamica complex [33] as well as within two genetically distant species, S. krylovii and S. bungeana [34]. Furthermore, due to the usage of integrative taxonomic approaches, it was shown that some Stipa taxa, previously assigned to S. richteriana and S. grandis, appeared to be cryptic species [33,35]. ...
... For instance, high-throughput techniques based on restriction enzymes, e.g., RADseq [36] and genotyping-by-sequencing [37], which have been foremost used in agricultural species [38], are currently widely applied in phylogenetics and studies related to hybridisation in many wild plant genera with little or no previous genomic information [39][40][41]. Recently, a promising result has also been demonstrated in Stipa where the usage of the DArTseq technique resulted in an increased number of markers that was several 100-fold higher than in the previous genomic studies [34]. ...
... In grasses, hybridisation and introgression phenomena are still mainly studied in crop species, e.g., rice [58], wheat [59] and sugarcane [60]. To date, we have detected hybrids not only between genetically closely related species [33], but also among genetically distant Stipa taxa [13,34]. The results present here and our previous findings helps us to shift toward thinking of the Stipa phylogeny as reticulate webs rather than a strictly bifurcating tree. ...
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Background The proper identification of feather grasses in nature is often limited due to phenotypic variability and high morphological similarity between many species. Among plausible factors influencing this issue are hybridisation and introgression recently detected in the genus. Nonetheless, to date, only a bounded set of taxa have been investigated using integrative taxonomy combining morphological and molecular data. Here, we report the first large-scale study on five feather grass species across several hybrid zones in Russia and Central Asia. In total, 302 specimens were sampled in the field and classified based on the current descriptions of these taxa. They were then genotyped with high density genome-wide markers and measured based on a set of morphological characters to delimitate species and assess levels of hybridisation and introgression. Moreover, we tested species for past introgression and estimated divergence times between them. Results Our findings demonstrated that 250 specimens represent five distinct species: S. baicalensis , S. capillata , S. glareosa , S. grandis and S. krylovii . The remaining 52 individuals provided evidence for extensive hybridisation between S. capillata and S. baicalensis , S. capillata and S. krylovii , S. baicalensis and S. krylovii , as well as to a lesser extent between S. grandis and S. krylovii , S. grandis and S. baicalensis . We detected past reticulation events between S. baicalensis , S. krylovii , S. grandis and inferred that diversification within species S. capillata , S. baicalensis , S. krylovii and S. grandis started ca. 130–96 kya. In addition, the assessment of genetic population structure revealed signs of contemporary gene flow between populations across species from the section Leiostipa , despite significant geographical distances between some of them. Lastly, we concluded that only 5 out of 52 hybrid taxa were properly identified solely based on morphology. Conclusions Our results support the hypothesis that hybridisation is an important mechanism driving evolution in Stipa . As an outcome, this phenomenon complicates identification of hybrid taxa in the field using morphological characters alone. Thus, integrative taxonomy seems to be the only reliable way to properly resolve the phylogenetic issue of Stipa . Moreover, we believe that feather grasses may be a suitable genus to study hybridisation and introgression events in nature.
... The availability of genome-wide datasets helps to understand the demographic processes underlying spatial patterns of genetic variation in non-model organisms (Baiakhmetov, et al., 2020;Baiakhmetov, et al., 2021;Perriigo et al., 2020;Tonzo and Ortego, 2021). In our study, by combining population genomic data and niche modeling we shed light on the contemporary genetic structure and ecological niches of the coldadapted, endemic-vicariants S. gracilis and S. zeravshanica as well as their response to Quaternary climatic oscillations. ...
... 350,000 BP. Our findings are in line with previous research on mountain species of the genus Stipa that mostly emerged less than a million years ago (Krawczyk et al., 2017;Krawczyk et al., 2018, Baiakhmetov et al., 2020Baiakhmetov et al., 2021). The origin of many neoendemic species, including Stipa, was related to climate oscillations in the Pleistocene. ...
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Understanding species distribution, genetic diversification and evolutionary history is extremely important for mountainous regions with a high diversity of endemic species, which are particularly sensitive to climate change. In this study, we use environmental and molecular data obtained from genome-wide analyses to infer the genetic variability, demographic processes, and response of the cold-adapted, endemic geographical-vicariants Stipa gracilis (distributed in the Tian Shan Mts) and S. zeravshanica (distributed in the western Pamir-Alai Mts) to Quaternary climatic oscillations in a Central Asian mountain biodiversity hotspot. Genomic-based reconstructions of demographic history indicate that the examined endemics presented larger effective population sizes during the Last Glacial Maximum (LGM) period and experienced parallel demographic declines afterward. The results of fastSTRUCTURE analysis revealed three genetic clusters within S. gracilis populations and two within S. zeravshanica. The past distribution models reveals the glacial connectivity of both species, resulting in the detection of an admixture of S. zeravshanica genes in the specimens from the westernmost 'Alaian' population of S. gracilis. Although the occurrence of both species is closely associated with calcareous rocks, the differences in the ranges of the species distributions depend mostly on climatic factors, especially temperature and precipitation. The wider realized ecological niche of S. gracilis allows it to better adapt to global warming and potentially extend its range in the future, while S. zeravshanica, with its narrower niche, is more susceptible to environmental changes and potentially at risk of extinction. The findings will contribute to a better understanding of the factors shaping the distribution and genetic differentiation of mountain endemic species and provide a theoretical basis for their conservation by identifying areas sensitive to climate change in biodiversity hotspots.
... Stipa is a large and taxonomically challenging genus, with some species highly variable in morphology [3], sometimes with very subtle diagnostic features between species [13] and in which formation of interspecies hybrids has been confirmed [14,15]. All of this resulted in disorder in the taxonomic names that have recently been reviewed by the development of the Middle Asian Stipa synopsis, published along with the key for species identification [3]. ...
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Background The study presents results of research on the evolution of plastid genomes in Stipa L. which is a large genus of the Poaceae family, comprising species diverse in terms of geographic distribution, growing under highly variated habitat conditions. Complete plastome sequences of 43 taxa from Stipeae and Ampelodesmae tribes were analyzed for the variability of the coding regions against the background of phylogenetic relationships within the genus Stipa. The research hypothesis put forward in our research was that some of coding regions are affected by a selection pressure differentiated between individual phylogenetic lines of Stipa, potentially reducing the phylogenetic informativeness of these CDS. The study aimed to answer the question, which genes evolve in Stipa most rapidly and what kind of changes in the properties of encoded amino acids this entails. Another goal of this research was to find out whether individual genes are affected by positive selection and finally, whether selective pressure is uniform within the genus or does it vary between particular evolutionary lines within the genus. Results Results of our study proved the presence of selective pressure in 11 genes: ccsA, matK, ndhC, ndhF, ndhK, rbcL, rpoA rpoC1, rpoC2, rps8 and rps11. For the first time the effect of positive selection on the rps8, rps11, and ndhK genes was documented in grasses. The varied pace of evolution, different intensity and effects of selective pressure have been demonstrated between particular phylogenetic lines of the genus tested. Conclusions Positive selection in plastid genome in Stipa mostly affects photosynthetic genes. The potential strongest adaptive pressure was observed in the rbcL gene, especially in the oldest evolutionary group comprising Central Asian high-mountain species: S. basiplumosa, S. klimesii, S. penicillata and S. purpurea, where adaptive pressure probably affected the amino acids directly related to the efficiency of CO2 assimilation.
... Binks, Steane & Byrne, 2021), identification of nothotaxa (e.g. Baiakhmetov et al., 2020;Binks et al., 2020;Bradbury, Binks & Byrne, 2021;Robins et al., 2021) and disentangling species complexes (e.g. Baumsteiger et al., 2017;Rutherford et al., 2018;Diaz et al., 2021). ...
Species delimitation is challenging in rapid radiations because the typical markers of speciation are often obscured. Here, we use comprehensive sampling and genome-wide single nucleotide polymorphisms to assess species boundaries in a radiation of nine morphologically similar Leptospermum taxa that failed to be discriminated in previous phylogenomic analyses. Our data recovered clear separation of L. maxwellii, L. sericeum and L. inelegans as currently circumscribed. A phrase-named taxon, Leptospermum. sp. Peak Charles/Norseman, was not distinct from L. incanum, and we recommend their synonymization. Another pair, L. nitens and L. roei, were also indistinct and differ by a single morphological character that also varies in L. inelegans without taxonomic recognition. We recommend synonymization of L. nitens and L. roei and consistent treatment of this character as a non-diagnostic, variable trait. Difficulty arose in discriminating L. erubescens and L. oligandrum; we make three suggestions and recommend further morphological investigation to determine the most appropriate taxonomic outcome. As expected, hybridization was common across the complex, but, unexpectedly, many individual plants were genetically identical within, and sometimes between, populations of most species. We hypothesize that this is due to apomixis. Overall, this study demonstrates the value of population genomics in the integrative taxonomy toolbox for disentangling species in rapid radiations, while also offering insight to the evolution of this poorly known group of Australian Leptospermum.
... Binks, Steane & Byrne, 2021), identification of nothotaxa (e.g. Baiakhmetov et al., 2020;Binks et al., 2020;Bradbury, Binks & Byrne, 2021;Robins et al., 2021) and disentangling species complexes (e.g. Baumsteiger et al., 2017;Rutherford et al., 2018;Diaz et al., 2021). ...
Species delimitation is challenging in rapid radiations because the typical markers of speciation are often obscured. Here, we use comprehensive sampling and genome-wide single nucleotide polymorphisms to assess species boundaries in a radiation of nine morphologically similar Leptospermum taxa that failed to be discriminated in previous phylogenomic analyses. Our data recovered clear separation of L. maxwellii, L. sericeum and L. inelegans as currently circumscribed. A phrase-named taxon, Leptospermum. sp. Peak Charles/Norseman, was not distinct from L. incanum, and we recommend their synonymization. Another pair, L. nitens and L. roei, were also indistinct and differ by a single morphological character that also varies in L. inelegans without taxonomic recognition. We recommend synonymization of L. nitens and L. roei and consistent treatment of this character as a non-diagnostic, variable trait. Difficulty arose in discriminating L. erubescens and L. oligandrum; we make three suggestions and recommend further morphological investigation to determine the most appropriate taxonomic outcome. As expected, hybridization was common across the complex, but, unexpectedly, many individual plants were genetically identical within, and sometimes between, populations of most species. We hypothesize that this is due to apomixis. Overall, this study demonstrates the value of population genomics in the integrative taxonomy toolbox for disentangling species in rapid radiations, while also offering insight to the evolution of this poorly known group of Australian Leptospermum.
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Kraków Geobotanical School is considered one of the oldest and longest-operating botanical schools in Europe; it was active from 1859 to 2020. The purpose of this article is to summarize the achievements of the school. We divided the history of the school into six periods: (i) the Formation period (1859–1903); (ii) the Romantic period (1903–1917), wherein long-term research projects were completed and with M. Raciborski considered as the first headmaster of the school; (iii) the Classical period (1917–1970), wherein new ideas and research techniques were developed with W. Szafer as the headmaster; (iv) the Duumvirate period (1971–1993), with J. Kornaś and A. Jasiewicz as the headmasters; (v) the Descendant schools period (1994–2010), wherein traditional (morphological) methods were utilized; and (vi) the Decline period (2011–2020). Each of these periods was characterized by the names of the headmasters or leaders, their roles, and their main achievements. We suppose that Kraków Geobotanical School, in its present structure, has finished its scientific activity, for which we present a few arguments. We have attached to the main text of the article, an extensive tables containing the topics of geobotanical research carried out in each of the six periods, along with publication samples. e most important scientific achievements of Kraków Geobotanical School are the following: several thousand publications, including monographs and syntheses of knowledge on Polish flora and vegetation; introduction of new disciplines in Poland (e.g., paleobotany, nature conservation, phytosociology, palynology, study on synanthropization); description of new plant and fungus taxa; and identification of syntaxonomic units in Poland and abroad.
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Citation: Nobis M, Krzempek M, Nowak A, Gudkova PD, Klichowska E (2022) Resurrection of Stipa tremula and taxonomy of the high-alpine species from the Stipa purpurea complex (Poaceae, Pooideae). PhytoKeys 196: 21-47. Abstract Stipa purpurea is a high-alpine species that occurs in cryophilous steppes, semi-deserts and stony slopes, from the Tian Shan and Pamirian Plateau through Qinghai-Xizang Plateau to the Himalayas and is characterised by a great morphological variability. During the revision of specimens of the taxon, we observed that the pattern of this variability is linked to the geographical distribution of the specimens. Numerical analyses (PCA and UPGMA) revealed three groups of OTUs corresponding to three morphotypes within the S. purpurea complex. A set of macro-and micromorphological characters, supported by a map of general distributional ranges, are presented to distinguish each of the three taxa within the complex and we reassess the status of Lasiagrostis tremula described by Ruprecht in 1869. As a result, Stipa tremula, S. purpurea and S. arenosa were distinguished within the complex. The intermediate characters of S. arenosa may suggest its putative hybrid origin (S. tremula × S. purpurea), whereas the presence of extremely long florets may be an expression of the gigas effect. We propose two new combinations (S. tremula and S. arenosa), describe a new nothospecies (S. ×ladakhensis) that originated from hybridisation between S. klimesii and S. purpurea s.l. and designate the lectotype for Ptilagrostis semenovii. An identification key and detailed morphological description of species from the S. purpurea complex are also presented.
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Despite their evolutionary relevance, multispecies networks or syngameons are rarely reported in the literature. Discovering how syngameons form and how they are maintained can give insight into processes such as adaptive radiations, island colonizations, and the creation of new hybrid lineages. Understanding these complex hybridization networks is even more pressing with anthropogenic climate change, as syngameons may have unique synergistic properties that will allow participating species to persist. The formation of a syngameon is not insurmountable, as several ways for a syngameon to form have been proposed, depending mostly on the magnitude and frequency of gene flow events, as well as the relatedness of its participants. Episodic hybridization with small amounts of introgression may keep syngameons stable and protect their participants from any detrimental effects of gene flow. As genomic sequencing becomes cheaper and more species are included in studies, the number of known syngameons is expected to increase. Syngameons must be considered in conservation efforts as the extinction of one participating species may have detrimental effects on the survival of all other species in the network.
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The genus Stipa L. comprises over 150 species, all native to the Old World, where they grow in warm temperate regions throughout Europe, Asia, and North Africa. It is one of the largest genera in the family Poaceae in Middle Asia, where one of its diversity hotspots is located. However, identification of Middle Asian Stipa species is difficult because of the lack of new, comprehensive taxonomic studies including all of the species recorded in the region. We present a critical review of the Mid-Asian representatives of Stipa, together with an identification key and taxonomic listing. We relied on both published and unpublished information for the taxa involved, many of which are poorly known. For each taxon, we present a taxonomic and nomenclatural overview, habitat preferences, distribution, altitudinal range, and additional notes as deemed appropriate. We describe four new nothospecies: S. ×balkanabatica M. Nobis & P. D. Gudkova, S. ×dzungarica M. Nobis, S. ×pseudomacroglossa M. Nobis, S. ×subdrobovii M. Nobis & A. Nowak, one subspecies S. caucasica Schmalh. subsp. nikolai M. Nobis, A. Nobis & A. Nowak, and eight varieties: S. araxensis Grossh. var. mikojanovica M. Nobis, S. caucasica var. fanica M. Nobis, P. D. Gudkova & A. Nowak, S. drobovii (Tzvelev) Czerep. var. jarmica M. Nobis, S. drobovii var. persicorum M. Nobis, S. glareosa P. A. Smirn. var. nemegetica M. Nobis, S. kirghisorum P. A. Smirn. var. balkhashensis M. Nobis & P. D. Gudkova, S. richteriana Kar. & Kir. var. hirtifolia M. Nobis & A. Nowak, and S. ×subdrobovii var. pubescens M. Nobis & A. Nowak. Additionally, 12 new combinations, Achnatherum haussknechtii (Boiss.) M. Nobis, A. mandavillei (Freitag) M. Nobis, A. parviflorum (Desf.) M. Nobis, Neotrinia chitralensis (Bor) M. Nobis, S. badachschanica Roshev. var. pamirica (Roshev.) M. Nobis, S. borysthenica Klokov ex Prokudin var. anomala (P. A. Smirn.) M. Nobis, S. holosericea Trin. var. transcaucasica (Grossh.) M. Nobis, S. kirghisorum P. A. Smirn. var. ikonnikovii (Tzvelev) M. Nobis, S. macroglossa P. A. Smirn. var. kazachstanica (Kotuchov) M. Nobis, S. macroglossa var. kungeica (Golosk.) M. Nobis, S. richteriana var. jagnobica (Ovcz. & Czukav.) M. Nobis & A. Nowak, and S. zalesskii Wilensky var. turcomanica (P. A. Smirn.) M. Nobis are proposed, and the lectotypes for 14 taxa (S. arabica Trin. & Rupr., S. bungeana Trin. ex Bunge, S. caspia K. Koch, S. ×consanguinea Trin. & Rupr., S. effusa Mez, S. ×heptapotamica Golosk., S. jacquemontii Jaub. & Spach., S. kungeica Golosk., S. margelanica P. A. Smirn., S. richteriana, S. rubentiformis P. A. Smirn., S. sareptana A. K. Becker, S. tibetica Mez, and Timouria saposhnikovii Roshev.) are designated. In Middle Asia the genus Stipa comprises 98 taxa, including 72 species, four subspecies, and 22 varieties. Of the 72 species of feather grasses, 23 are of hybrid origin (nothospecies). In Middle Asia, feather grasses can be found at elevations from (0 to)300 to 4500(to 5000) m, but most are montane species. The greatest species richness is observed at altitudes between 1000 and 2500 m. Nineteen species grow above 3000 m, but only nine above 4000 m. The number of taxa (species and subspecies) growing in each country also varies considerably, with the highest noted in Kazakhstan (42), Tajikistan (40), and Kyrgyzstan (35). Of the 76 taxa of Stipa (species and subspecies) recorded in Middle Asia, 41 are confined to the region, with some being known only from a single country or mountain range. Distribution maps of selected species are provided.
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Understanding the genetic diversity of Aegilops biuncialis, a valuable source of agronomical useful genes, may significantly facilitate the introgression breeding of wheat. The genetic diversity and population structure of 86 Ae. biuncialis genotypes were investigated by 32700 DArT markers with the simultaneous application of three statistical methods— neighbor-joining clustering, Principal Coordinate Analysis, and the Bayesian approach to classification. The collection of Ae. biuncialis accessions was divided into five groups that correlated well with their eco-geographic habitat: A (North Africa), B (mainly from Balkans), C (Kosovo and Near East), D (Turkey, Crimea, and Peloponnese), and E (Azerbaijan and the Levant region). The diversity between the Ae. biuncialis accessions for a phenological trait (heading time), which is of decisive importance in the adaptation of plants to different eco-geographical environments, was studied over 3 years. A comparison of the intraspecific variation in the heading time trait by means of analysis of variance and principal component analysis revealed four phenotypic categories showing association with the genetic structure and geographic distribution, except for minor differences. The detailed exploration of genetic and phenologic divergence provides an insight into the adaptation capacity of Ae. biuncialis, identifying promising genotypes that could be utilized for wheat improvement.
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Abstract Climate change, characterized by warming and precipitation variability, restricted the growth of plants in arid and semiarid areas, and various functional traits are impacted differently. Comparing responses of functional traits to warming and precipitation variability and determining critical water threshold of dominate steppe grasses from Inner Mongolia facilitates the identification and monitoring of water stress effects. A combination of warming (ambient temperature, +1.5°C and +2.0°C) and varying precipitation (−30%, −15%, ambient, +15%, and +30%) manipulation experiments were performed on four Stipa species (S. baicalensis, S. bungeana, S. grandis, and S. breviflora) from Inner Mongolia steppe. The results showed that the functional traits of the four grasses differed in their responses to precipitation, but they shared common sensitive traits (root/shoot ratio, R/S, and specific leaf area; SLA) under ambient temperature condition. Warming increased the response of the four grasses to changing precipitation, and these differences in functional traits resulted in changes to their total biomass, with leaf area, SLA, and R/S making the largest contributions. Critical water thresholds of the four grasses were identified, and warming led to their higher optimum precipitation requirements. The four steppe grasses were able to adapt better to mild drought (summer precipitation decreased by 12%–28%) when warming 1.5°C rather than 2.0°C. These results indicated that if the Paris Agreement to limit global warming to 1.5°C will be accomplished, this will increase the probability for sustained viability of the Stipa steppes in the next 50–100 years.
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Background Bread wheat is one of the most important and broadly studied crops. However, due to the complexity of its genome and incomplete genome collection of wild populations, the bread wheat genome landscape and domestication history remain elusive. Results By investigating the whole-genome resequencing data of 93 accessions from worldwide populations of bread wheat and its diploid and tetraploid progenitors, together with 90 published exome-capture data, we find that the B subgenome has more variations than A and D subgenomes, including SNPs and deletions. Population genetics analyses support a monophyletic origin of domesticated wheat from wild emmer in northern Levant, with substantial introgressed genomic fragments from southern Levant. Southern Levant contributes more than 676 Mb in AB subgenomes and enriched in the pericentromeric regions. The AB subgenome introgression happens at the early stage of wheat speciation and partially contributes to their greater genetic diversity. Furthermore, we detect massive alien introgressions that originated from distant species through natural and artificial hybridizations, resulting in the reintroduction of ~ 709 Mb and ~ 1577 Mb sequences into bread wheat landraces and varieties, respectively. A large fraction of these intra- and inter-introgression fragments are associated with quantitative trait loci of important traits, and selection events are also identified. Conclusion We reveal the significance of multiple introgressions from distant wild populations and alien species in shaping the genetic components of bread wheat, and provide important resources and new perspectives for future wheat breeding.
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Large collections of the forage and bioenergy grass Dactylis glomerata were made in northwest (NW) Europe along east to west and north to south clines for genetic resource conservation and to inform breeding programmes of genetic diversity, genepools, and ploidy. Leaves were sampled for genetic analysis and seed and rhizome for ex-situ conservation. Genotyping by sequencing (GBS) was used to assay nuclear DNA diversity and plastome single nucleotide polymorphism (SNP) discovery was undertaken using a long-read PCR and MiSeq approach. Nuclear and plastid SNPs were analysed by principal component analysis (PCA) to compare genotypes. Flow cytometry revealed that all samples were tetraploid, but some genome size variation was recorded. GBS detected an average of approximately 10,000 to 15,000 SNPs per country sampled. The highest average number of private SNPs was recorded in Poland (median ca. 2000). Plastid DNA variation was also high (1466 SNPs, 17 SNPs/kbp). GBS data, and to a lesser extent plastome data, also show that genetic variation is structured geographically in NW Europe with loose clustering matching the country of plant origin. The results reveal extensive genetic diversity and genetic structuring in this versatile allogamous species despite lack of ploidy variation and high levels of human mediated geneflow via planting.
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Background: The spread of maize cultivation to the highlands of central Mexico was accompanied by substantial introgression from the endemic wild teosinte Zea mays ssp. mexicana, prompting the hypothesis that the transfer of beneficial variation facilitated local adaptation. Methods: We used whole-genome sequence data to map regions of Zea mays ssp. mexicana introgression in three Mexican highland maize individuals. We generated a genetic linkage map and performed Quantitative Trait Locus mapping in an F2 population derived from a cross between lowland and highland maize individuals. Results: Introgression regions ranged in size from several hundred base pairs to Megabase-scale events. Gene density within introgression regions was comparable to the genome as a whole, and over 1,000 annotated genes were located within introgression events. Quantitative Trait Locus mapping identified a small number of loci linked to traits characteristic of Mexican highland maize. Discussion: Although there was no strong evidence to associate quantitative trait loci with regions of introgression, we nonetheless identified many Mexican highland alleles of introgressed origin that carry potentially functional sequence variants. The impact of introgression on stress tolerance and yield in the highland environment remains to be fully characterized.
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In sugarcane (Saccharum spp. hybrid) breeding, introgression of useful genes via intergeneric hybridization is a powerful strategy for improving the crop productivity. Erianthus arundinaceus shows great potential in terms of useful traits; however, little is known about the cytogenetic and agronomic characteristics of intergeneric hybrids between these two species. Here, we examine the cytogenetic and agronomic characteristics, and relationships between the two in intergeneric F1 hybrids between modern sugarcane cultivar and E. arundinaceus identified by amplification of 5S rDNA markers and morphological characteristics. The nuclear DNA content of the hybrids varied from 6.07 to 8.94 pg/2C, with intra-clonal variation in DNA content and 5S rDNA sites. Genomic in situ hybridization revealed 53 to 82 chromosomes in the hybrids, with 53 to 56 derived from sugarcane and 1 to 29 from E. arundinaceus. There were significant positive correlations between the number of E. arundinaceus chromosomes and dry matter yield, millable stalk weight, single stalk weight, and stalk diameter, but not sucrose content, reducing sugar content, sucrose/reducing sugar ratio or fiber content. This detailed information on intergeneric F1 hybrids between modern sugarcane cultivar and E. arundinaceus will contribute to effective utilization of E. arundinaceus in sugarcane breeding.
Hybridisation, considered one of the major drivers of speciation in plants, commonly occurs among many genera of grasses, including the genus Stipa L. one of the largest in the family Poaceae in the Old World. In the course of research on Stipa, we studied S. richteriana, S. lessingiana, and their putative hybrid S. heptapotamica, all of which grow in south-eastern Kazakhstan. Stipa heptapotamica is treated as a local endemic, known only from the eastern part of the country. We used an integrative approach, combining morphological and molecular data, to validate the status of the hybrid taxon and present for the first time clear data documenting natural hybridisation in Stipa. The macro- and micromorphological analyses presented here show S. heptapotamica to be intermediate to its parental species in the characters studied; pollen analysis demonstrates low viability of pollen grains (below 50% compared to 94% and 87% in S. lessingiana and S. richteriana, respectively); molecular analyses based on inter simple sequence repeat markers (ISSR) and next-generation sequencing (NGS) data prove that S. heptapotamica originated strictly from hybridisation events involving S. richteriana and S. lessingiana. ISSR markers reveal possible introgression events between S. lessingiana and S. heptapotamica, whereas NGS data analyses confirm the maternal inheritance of a plastome from S. lessingiana in S. heptapotamica and support the finding of cryptic lineages within S. richteriana.
The grass subfamily Pooideae dominates the grass floras in cold temperate regions, and has evolved complex physiological adaptations to cope with extreme environmental conditions like frost, winter and seasonality. One such adaptation is cold acclimation, wherein plants increase their frost tolerance in response to gradually falling temperatures and shorter days in the autumn. However, understanding how complex traits like cold acclimation evolve remains a major challenge in evolutionary biology. Here, we investigated the evolution of cold acclimation in Pooideae, and found that a phylogenetically diverse set of Pooideae species displayed cold acclimation capacity. However, comparing differential gene expression after cold treatment in transcriptomes of five phylogenetically diverse species revealed widespread species-specific responses of genes with conserved sequences. Furthermore, we studied the correlation between gene family size and number of cold responsive genes, as well as between selection pressure on coding sequences of genes and their cold responsiveness. We saw evidence of protein-coding and regulatory sequence evolution, as well as origin of novel genes and functions contributing towards evolution of a cold response in Pooideae. Our results reflect that selection pressure resulting from global cooling must have acted on already diverged lineages. Nevertheless, conservation of cold induced gene expression of certain genes indicates that the Pooideae ancestor may have possessed some molecular machinery to mitigate cold stress. Evolution of adaptations to seasonally cold climates is regarded particularly difficult. How Pooideae evolved to transition from tropical to temperate biomes sheds light on how complex traits evolve in the light of climate changes.