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

Abstract

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.

Full text

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
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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
kingdom1–7. 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 backcrossing4,8–11. 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 extinction14–16.
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 genera34–38.
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 communities47–50,
can be used for their classication51, and in studies related to climate change52–54. Moreover, the species are of
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Research
Botanical
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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 hybridisation57–60. 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.
Results
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 ://plot.ly/~eugen 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 ct.org/packa ge=facto extra /, and
plotly v.4.9.2 (Fig. b), https ://plotl y.com/r/getti 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 ://plot.ly/~eugen ebaya hmeto v/5/), but not in SNPs markers (Fig.4d, https
://plot.ly/~eugen 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 y.com/r/getti 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 io.com/produ 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 tion.org/packa ges/stats /versi ons/3.6.2/.
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Discussion
Although many interspecic hybrids have been described in the genus Stipa43,57–59, 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 ://tree.bio.ed.ac.uk/so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 species72–78 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°5′47.07′’ / E 76°39′6.22′’, 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°5′53.97′’ / E 76°38′37.28′’,
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 platforms96–100. 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 hybridisation104–107. Contributions from each cluster in a range between 45 and 55% were considered as F1
hybrids, while rst‐ and second‐generation 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
request.
Received: 18 February 2020; Accepted: 31 July 2020
References
1. Grant, V. Plant Speciation 2nd edn. (Columbia University Press, New York, 1981).
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Acknowledgements
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 ://doi.org/10.1038/s4159 8-020-70582 -1.
Correspondence and requests for materials should be addressed to E.B.orM.N.
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