Increasing human transfer of plants across geographical regions leads to frequent introductions of non-native plant species around the world. If these introduced taxa meet close relatives, hybridization and introgression can occur1. The inadvertent introduction of species closely related to major crop plants may lead to hybridization and the formation of weedy lineages2. Alternatively, weedy taxa can also evolve directly from a domesticated ancestor3, as in the case of some weedy rice populations4, 5. Weedy lineages can incur massive yield losses and cause major costs6.

Maize is the third most important crop plant in Spain with production reaching almost 4.7 million tonnes7. In 2009, farmers in Northern Spain (Aragon) began to observe plants in their maize fields that resembled cultivated maize before the onset of flowering but then developed highly branching phenotypes with small cobs and shattering seeds8. These traits are typical for teosinte, wild relatives of cultivated maize9. Until 2014, this so-called ‘Spanish teosinte’ has spread in Aragon and has also been reported from a neighbouring region in Catalonia10, 11. About 750 ha of maize cultivation have been affected so far, mostly in Aragon11. Due to maize monocropping, density of Spanish teosinte can become high on affected fields and may cause severe maize yield losses and high economic costs12. In some regions of Aragon, this weed has become the prime agronomic problem for maize farmers10.

Maize (Zea mays mays) was domesticated from its wild relative teosinte about 9,000 years ago in southern Mexico13. Domesticated maize, including high-yielding hybrid maize varieties grown in Europe, is strictly monopodic, with non-shattering kernels that remain tightly attached to the cob, in contrast to the shattering kernels of the cob-less, and highly branched, teosinte. Although maize cobs, or parts of them, can remain in the field and do germinate and grow feral, it is considered unlikely that they successfully establish feral populations beyond arable fields and without human support9. Hence, cultivated maize is generally considered to have little to no risk of causing concerns as a volunteer or feral weed14.

Teosinte is the common name of a group of wild grasses (Poaceae) and includes highly variable species and subspecies that occur in scattered populations in many areas across Mexico and Central America (Mesoamerica)15. In many areas of Mesoamerica, teosinte populations have come under serious threat with the expansion of ranching and farming and are facing a massive decline in abundance to the point of extinction of some species16, which forced the Mexican government to install conservation measures for their protection17. Thus, in their centre of origin, many teosinte populations are endangered and require protection measures, although occasionally they can also act as local weeds18.

Teosinte and maize belong to the same genus, Zea, which consists of five species: 1) perennial diploid (2n = 20) Z. diploperennis, 2) perennial tetraploid (2n = 40) Z. perennis, 3) annual diploid (2n = 20) Z. luxurians, 4) annual diploid (2n = 20) Z. nicaraguensis, and 5) the annual species Zea mays. The latter encompasses four annual diploid (2n = 20) subspecies: (i) ssp. mays, the domesticated maize, (ii) ssp. mexicana, (iii) ssp. parviglumis, and (iv) ssp. huehuetenangensis 19. Z.m. ssp. mexicana and Z.m. ssp. parviglumis are most closely related to domesticated maize, the latter subspecies being called ‘Balsas’ teosinte and considered the ancestor of cultivated maize9. All teosintes are believed to be endemic to Mesoamerica20 where cultivated maize and teosintes often grow in geographic proximity and flower synchronously. Overall, Z.m. ssp. mexicana grows in cooler, drier central highlands, mostly above 1800 m, while Z.m. ssp. parviglumis grows in warmer, wetter lower elevations in the river valleys of southern and western Mexico, mostly below 1800 m15.

Although it is known that all teosintes can hybridize with maize, this typically occurs at low rates even when teosinte is abundant21. Hybridization appears to be most common between domesticated maize and Z.m. ssp. parviglumis 15, 22 but gene flow does not occur reciprocally, which may explain why teosintes continue to coexist even when growing in close vicinity to much larger maize populations21. When teosinte pollen is applied to maize silks, resulting hybrids are vigorous and highly fertile23. However, when teosinte is pollinated by maize pollen, plants of Z.m. ssp. mexicana set seed very inconsistently or not at all22. Similarly, Hufford et al. found evidence of adaptive introgression of Z.m. ssp. mexicana alleles into maize during its expansion to the highlands of central Mexico, but observed very little evidence for adaptive introgression in the other direction, from cultivated maize into Z.m. ssp. mexicana 24.

This asymmetrical pollination success is under the control of a gene called the ‘Teosinte crossing barrier’ (Tcb)23 which may reproductively isolate at least some teosinte species from maize. Aylor et al. postulated that gene flow and subsequent introgression of maize alleles into teosinte populations most likely occur when teosinte first pollinates maize20. The resultant hybrids would then backcross with teosinte which could lead to the introgression of maize alleles into the teosinte background. They speculated that the pollination from teosinte to maize most likely represents the rate-limiting step in the introgression of maize alleles into teosinte20. However, this has not been investigated yet to any conclusive extent, even though teosinte and maize have intensively been studied from a population genetics perspective, including studies utilizing DNA samples recovered from archaeological specimens13, 24,25,26,27.

So far, the Spanish authorities have speculated that the introduced teosinte is Z.m. ssp. mexicana 11. Spanish teosinte can produce long-lasting seed banks, and its control either by mechanical means, crop rotation or herbicide treatment has proven difficult10, 11. Knowing the origin of the novel weed in Spain may help to prevent introductions of further seed material, to monitor the spread of this weed, and to take targeted control measures in the future. The taxonomic identity of the Spanish teosinte, its introduction history, as well as its ecology and potential risks for local and neighbouring European farming systems remain largely unknown. To develop effective measures for monitoring, control and prevention, data on these aspects are fundamental. Here, we addressed the following research questions: (i) to what taxon can teosinte-like plants collected in Spain be assigned?, (ii) what is the potential origin of these Spanish teosinte lineages?, (iii) is there evidence for on-going hybridization between Spanish teosinte and commercially cultivated maize in Spain? To answer these questions, we collected Spanish teosinte and hybrid-like seeds (autumn 2014 and 2015) and leaf samples of Spanish teosinte and cultivated maize (summer 2015) in the region of Aragon, Spain, and genotyped these together with teosinte reference plants using the MaizeSNP50 BeadChip, a widely used resource for high-density genotyping of maize and its wild relatives28,29,30.