Applying integrative taxonomy as a tool for revealing hybridisation events in Stipa (Poaceae)

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 show the usefulness of integrative taxonomy as a tool for revealing hybridisation events in Stipa (one of the largest genera in Poaceae). We demonstrate the usage of the classical morphological approach combining with scanning electron microscopy data, as well as analyses of pollen grains, and the application of molecular techniques including data derived from next generation sequencing. We illustrate the application of this integrative tool by specific examples: (1) in the S. × heptapotamica hybrid complex comprising morphologically distant but genetically closely related species, and (2) in the S. × lazkovii hybrid complex containing genetically distant species.

Hybridisation in the wild between closely related species is a common mechanism of speciation in the plant kingdom (Goulet et al., 2017). Due to the prevalence of polyploidy found in angiosperms, it has been estimated that around 11 % of flowering plants may have arisen through hybridisation events (Arnold, 2006). In general, hybridisation is often accompanied by introgression and causes gene transfer between species via repeated backcrossing (Suarez-Gonzalez, 2018). On the one hand, it may have contributed to species diversity and speciation, on the other, deleterious consequences of hybridisation such as decreased fitness, genetic assimilation and gene swamping may drive populations toward the brink of extinction (Mota, 2019).
The genus Stipa L. belongs to the subfamily Pooideae and alongside with Bambusoideae (bamboos), and Oryzoideae (rices) form the so-called BOP clade. The BOP species are known as the "cool season" or "pooid" grasses and all are C3 and distributed in temperate climates. Following Tzvelev (1974), the genus Stipa includes six main sections Barbatae Junge, Leiostipa Dumort, Pseudoptilagrostis Tzvelev, Regelia Tzvelev, Stipa, and Smirnovia Tzvelev, and comprises over 150 species native to Asia, Europe and North Africa (Nobis, 2014;Nobis et al., 2020). In its strict sense, the genus is monophyletic, but subdivisions within the genus are not consistently supported by available molecular data (Kellogg, 2015).
In the present study, we demonstrate the usefulness of integrative taxonomy as a tool for revealing hybridisation events in Stipa by specific examples: (1) in the hybrid complex S. × heptapotamica comprising morphologically distant but genetically closely related species, and (2) in the hybrid complex S. × lazkovii containing genetically distant species.
The S. × heptapotamica hybrid complex includes a very rare and endemic species, S. heptapotamica Golosk. that always grows within the contact zones of S. richteriana Kar. & Kir. and S. lessingiana Trin. & Rupr. in eastern and southern Kazakhstan (Nobis et al., 2019). We hypothesised that S. heptapotamica could be a hybrid of these two taxa, which, according to Tzvelev (1974), belong to two different sections, sect. Leiostipa Dumort. and sect. Subbarbatae Tzvelev, respectively.
To verify our hypothesis, we applied the classical morphometry using the measurement of the 14 most informative morphological characters commonly used in keys and taxonomic description of Stipa. In addition, we performed an ultrastructural analysis of the lemma and lamina using scanning electron microscopy (SEM). Both analyses demonstrated that S. heptapotamica has intermediate characters between S. richteriana and S. lessingiana. Further, we accomplished a viability test using Alexander's stain. The test showed that the frequency of viable pollen grains was very high in S. lessingiana and S. richteriana (94 % and 87 % respectively), while for S. heptapotamica it was less than 50 % (Figure 1). Furthermore, to explore the differences at the molecular level, we performed analyses based on inter simple sequence repeat markers (ISSR) and next-generation sequencing (NGS) data derived from whole chloroplast genomes and the nuclear Intergenic Spacer (IGS). The ISSR markers revealed hybridisation events between S. lessingiana and S. richteriana, and demonstrated possible introgression processes between S. lessingiana and S. heptapotamica, whereas NGS data analyses confirmed the maternal inheritance of a plastome from S. lessingiana in S. heptapotamica and supported the finding of cryptic lineages within S. richteriana.
The S. × lazkovii hybrid complex includes a very rare and endemic species, S. × lazkovii M. Nobis & A. Nowak, that was found in eastern Kyrgyzstan only in one locality (Baiakhmetov et al., 2020). Due to all specimens of S. × lazkovii 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 Leiostipa (Tzvelev, 1976), they are distant phylogenetically and belong to two different clades (Nobis et al., 2019;Krawczyk et al., 2017).
For this hybrid complex, we performed an analysis of macro-characteristics based on the 22 most informative quantitative and three qualitative morphological characters. Next, we used an ultrastructural analysis of the lemma and lamina using SEM. Both analyses suggested that S. × lazkovii has intermediate characters between S. krylovii and S. bungeana. Further, we applied the DArTseq approach representing a combination of DArT complexity reduction methods and next generation sequencing platforms (Sansaloni C. et al., 2011). In total, 76,604 silicoDArT and 19,133 SNPs markers revealed that all S. × lazkovii specimens are F1 hybrids. In addition, molecular markers detected an introgression event between S. bungeana and S. glareosa (Figure 2).
The study of the S. × lazkovii hybrid complex highlighted the applicability of genome reduction approaches such as DArTseq in studies on natural hybridisation in the wild, and specifically in a grass genus Stipa. The 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 the study on the S. × heptapotamica hybrid complex. The figure adapted from Baiakhmetov et al., 2020 Thus, currently, in our studies on hybridisation in Stipa within integrative taxonomy we successfully apply: (1) classical morphometry, (2) SEM, (3) pollen viability tests, (4) molecular markers including ISSR, Silico DArT, as well as SNPs derived from DArTseq, and NGS data of cpDNA and nDNA loci.