**2. Materials and Methods**

*2.1. Plant Material*

To assess the nuclear (ITS1, ITS2, and 5--ETS) and plastome (*trnL-F*) barcodes' capability to identify grass pollen and create a local reference database of barcode sequences, a broad spectrum of Poaceae species widespread in Central Russia were selected: *Alopecurus pratensis* L., *Arrhenatherum elatius* (L.) P.Beauv. ex J.Presl & C.Presl., *Briza media* L., *Bromus inermis* (Leyss.) Holub (syn. *Bromopsis inermis*), *Calamagrostis epigeios* (L.) Roth, *Dactylis glomerata* L., *Elymus repens* (L.) Gould (syn. *Elytrigia repens*), *Festuca pratensis* Huds., *Lolium perenne* L., *Phleum pratense* L., *Poa annua* L., *Poa pratensis* L., *Poa supina* Schrad., and *Poa trivialis* L. Additionally, *Festuca arundinacea* Schreb. and *Poa palustris* L. were collected for the ETS primers' design. Fresh leaf material of the morphologically identified grass plants was sampled in the field (Moscow region) and from the Moscow State University Herbarium collection specimens.

Pure single-species pollen for a subset of the selected reference species was manually collected to make artificial pollen mixes: *Calamagrostis epigeios*, *Phleum pratense*, *Bromus inermis*, *Festuca pratensis*, *Elymus repens*, *Alopecurus pratensis*, and *Lolium perenne*. These species pollinate in abundance and are easy to collect in enough quantities to create pollen mixes of various complexity. Therefore, pollen was collected during summer in the active pollination time of these species. The collected pollen was weighed, and a sample of 10 mg of each species was suspended in 100 μL TE-buffer. From each sample, 2 μL of the suspension was analyzed using light microscopy, and pollen grains were counted to estimate the number of pollen grains for each species. Each sample was diluted in TE buffer to achieve an equal pollen count per mL based on the observed number of pollen grains. Then single-species pollen samples were mixed by volume to create a 100 μL pollen mix that contained pollen from different species in equal abundance (approx. 10,000 pollen grains per mix in total). The species composition of each artificial mix is presented in Table 1.


**Table 1.** Pollen artificial mixes' composition.


**Table 1.** *Cont.*
