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Brassica and Sinapis Seeds in Medieval Archaeological Sites: An Example of Multiproxy Analysis for Their Identification and Ethnobotanical Interpretation

The genus Brassica includes some of the most important vegetable and oil crops worldwide. Many Brassica seeds (which can show diagnostic characters useful for species identification) were recovered from two archaeological sites in northern Italy, dated from between the Middle Ages and the Renaissance. We tested the combined use of archaeobotanical keys, ancient DNA barcoding, and references to ancient herbarium specimens to address the issue of diagnostic uncertainty. An unequivocal conventional diagnosis was possible for much of the material recovered, with the samples dominated by five Brassica species and Sinapis. The analysis using ancient DNA was restricted to the seeds with a Brassica-type structure and deployed a variant of multiplexed tandem PCR. The quality of diagnosis strongly depended on the molecular locus used. Nevertheless, many seeds were diagnosed down to species level, in concordance with their morphological identification, using one primer set from the core barcode site (matK). The number of specimens found in the Renaissance herbaria was not high; Brassica nigra, which is of great ethnobotanical importance, was the most common taxon. Thus, the combined use of independent means of species identification is particularly important when studying the early use of closely related crops, such as Brassicaceae.


Introduction
The Brassicaceae are a large family of flowering plants with a circumpolar distribution, comprising 338 genera and 3700 species, including several major agricultural crops [1,2]. The type genus, Brassica, is by far the most important economically, and is currently grown in over 150 countries as a vegetable or oilseed crop [3]. Six Brassica species dominate current global production, viz.: Brassica oleracea L., Brassica rapa L., Brassica napus L., Brassica carinata A. Braun, Brassica nigra (L.) K. Koch, and Brassica juncea (L.) Czern [4]. Polyploidy has played a key role in the evolution of the cultivation of Brassica species, with allopolyploid hybridization between the three diploid species (B. rapa, AA, B. nigra, BB, and B. oleracea, CC) giving rise to three allotetraploids (B. juncea, AABB, B. napus, AACC, and B. carinata, BBCC- [5]). The domestication of these species is thought to have originated around the Mediterranean region of Europe [6][7][8]. The family also contains several genera that feature as minor crops, including (among others): Raphanus (R. sativus L., the radish), Armoracia (A. rusticana G. Gaertn., B. Mey. et Scherb., horseradish), Camelina (C. sativa (L.) Crantz, gold of pleasure), Nasturtium (N. officinale R. Br., watercress) and Sinapis (e.g., S. alba L., white mustard). These species have a long history of cultivation, particularly the Brassicas and

Results
The Emilia-Romagna archaeological sites dating from the 6th to the 17th century AD included 19 contexts in which seeds were found, although in the majority of these (about 58%), the seeds were present at concentrations below 1 seed/L. Furthermore, in the vast majority of the contexts (about 84%), Brassica and Sinapis seeds/fruits accounted for <1% of all the seeds/fruits recovered. In 7 contexts (nos. 3, 7, 12a, 13, 15, 16a and 16b), the concentration of seeds/fruits varied between 1 and 13 seeds/L, although in all these cases, the Brassicaceae seeds represented ≤1% of all the seeds found. The sole context that showed a notable divergence from these trends was the brick tank in Ferrara (no. 16ccontext I in Materials and methods), where the concentration of Brassica/Sinapis exceeded 1700 seeds/L and accounted for more than 10% of all the seeds found. An intermediate condition was observed in the coeval context no. 15, where Brassica/Sinapis were found in modest concentrations (8 seeds/L) and represented < 1% of all the seeds collected (Table 1).

Archaeobotanical Analysis
The individual plant taxa varied in the distribution of the contexts in which they were found. The most widely found of the taxa identified to species rank were Rapistrum rugosum (L.) All. (11 of 19 contexts) and Brassica rapa (11 contexts), although the latter was far more abundant throughout. Most of the other species featured only in a minority of the contexts surveyed. For example, Sinapis alba seeds were identified with high certainty in several contexts, although few such seeds were found overall (present in 4 of the 19 contexts). The seeds of the genus Brassica were more abundant, with well-preserved representatives of 5 distinct species recorded in 11 of the 19 contexts (B. rapa, B. napus, B. juncea, B. nigra, and B. oleracea). Of these, the vast majority (ca. 95%) were discarded as B. rapa, but the residual Brassica seeds were far more restricted and subdivided between B. nigra (1.6%; 2 contexts), B. napus (1.4%; 5 contexts), B. oleracea (<1%; 1 context), and B. juncea (<1%; 1 context). Most of the other confirmed species were similarly restricted to 5 contexts or less, the sole exception being Myagrum perfoliatum L. (8 contexts).
The presence of less well-preserved or abnormal/intermediate samples meant that many of the samples could only be identified at higher taxonomic levels. For instance, around 2.5% of the seeds could only be provisionally identified as either unknown Brassica seeds (Brassica sp.) or as Brassica/Sinapis seeds, and species identity could not be determined for the seeds of the genera Lepidium (3 contexts), Rorippa (2), or Sisymbrium (1); the genus even remained unidentifiable for some seeds.
In addition to Brassica sp.pl. and Sinapis alba, some other taxa may also have had ethnobotanical value (e.g., Camelina sativa and Isatis tinctoria L.); others were probably simple instances of weeds s.l.

a-DNA Barcoding
As expected, the ancient seed samples invariably provided DNA yields that were too low and degraded to allow detection by agarose-gel electrophoresis or conventional spectral photometry. Similarly, all attempts using standard barcode primers and protocols used for conventional rbcL or matK barcoding failed to generate detectable amplicons. The application of the MT-PCR method did nevertheless succeed in producing visible amplicons for both barcoding genes. Success was modest for rbcL. The first of the two regions targeted (rbcL1) yielded only 8 products of the expected 218 bp, but none of the sequences generated from these amplicons matched either the Brassica or the Sinapis species expected from the seed morphology. However, 39 seeds produced strong amplification products for the second region (rbcL2, 203 bp), and most of these yielded sequences of variable quality in both directions. BLASTn searches of the trimmed sequences against the NCBI database invariably identified the appropriate region of rbcL. Subsequent searches of the BOLD Systems database revealed matches with sequence homologies above 95% for all but 8 of the samples ( Table 2). Most of the remaining samples matched the species that were clearly incongruent with the seed morphology and were therefore deemed contaminant amplicons. Most of these matched species or genera that were likely to have been in the vicinity of the excavation sites, such as Citrus, Juniperus, Pinus, Picea or Cedrus. Thus, only nine samples were matched most closely to the three Brassica species (B. rapa, B. napus and B. oleracea) in the BOLD Systems database and the reference panel (Table 2).  Greater success was achieved using primers that targeted the matK gene. Here, all six loci targeted within the matK gene produced sequences that corresponded to one of several members of the Brassicaceae. These are each described below, in turn.
MatK1. The different matK primers for this amplicon varied in their capacity to generate viable products and in the ability of the resultant sequences to differentiate between species held on public databases. The conventional matK1 primers performed the least well. These primers yielded only 12 sequences of modest quality, and none matched significantly with any reference barcode held on either the BOLD Systems or on the NCBI database. The performances of the matk1a primers were substantially better, with modest quality sequences of 42-182 bp in length (after trimming) secured from 64 samples. Here, matches were recovered for 41 of the 65 samples. Most of these matched equally well to multiple species in terms of percentage identity, score quality, and e-value, but in all cases, the most strongly matched species were all members of the Brassicaceae family. A finerlevel diagnosis than family was not possible for 7 samples, but of the remaining sequences, 16 were assigned to the tribe Brassiceae, 10 to the genus Brassica, one to another genus in the family, four to a single species of Brassica, and three to another species within the family (Supplementary Information Table S1). The matk1b primers yielded fewer sequences (26), but these were generally longer (150-212 bp) than the matk1a. Again, matches were secured for all 26 using NCBI BLASTn searches (Supplementary Information Table S1). Of these, 14 matched two or more species of Brassica (genus-level diagnosis) and six matched a single Brassica species. The remaining six samples matched species, genera, or families outside the Brassicaceae and were therefore deemed contaminant sequences. The performance of matk1c was slightly improved compared with the other matK1 combinations. Trimmed sequences of between 118 and 181 bp were secured from 36 samples. Following BLASTn searches of the NCBI database, 26 species were matched to two or more species of Brassica and one was identified to a single Brassica species. The remaining samples were identified to the same level as the BOLD systems.
MatK4. These primers generated 19 sequences of generally good quality that varied in length between 189 and 279 bp, with 17 sequences falling in the range of 262-279 bp. Searches using these sequences produced matches with both databases (Supplementary  Information Table S1). Following sequence-homology searches, all except the shortest sequence matched two or more members of the genus Brassica (genus-level diagnosis). The shortest sequence failed to match any species above the 95% similarity threshold.
Matk5. This combination of primers generated the most extensive set of sequences (90 samples), which varied in length from 75 to 225 bp. These sequences generated diagnoses at various taxonomic levels when searched against both public databases ( Table 3). The finest level of diagnosis was obtained when these sequences were subject to BLASTn searches on the NCBI database. Here, no sequences matched the reference barcodes of a single species. However, the number of hits on the NCBI database was high enough that the frequency of hits to one target frequently allowed the provisional identification of the sample. For example, 11 samples matched equally with 52 reference sequences of B. oleracea, but also to four other sequences representing three other species. Given the scope for misidentification and the extent of hybridization in the genus, these 11 sample sequences were therefore designated as provisional B. oleracea. Similar frequency distributions were also noted for 17 sample sequences matching B. nigra and its allotetraploid sister species, B. carinata. However, given that the latter is native to Eastern Africa [40], these samples were provisionally identified as B. nigra only. The diploid-allotetraploid sister pairing of B. napus/B. rapa could not be distinguished using this amplicon, but collectively produced another highly skewed distribution, with 11 sample sequences falling into this category (provisional B. napus or B. rapa). Thus, samples assigned to Brassica species belonging to the cultivated U-triangle group [5] accounted for 43% (39/90) of the sequences retrieved. Among the remainder, a further 7 samples matched multiple Brassica species equally (genus level diagnosis). The residual 45 samples (50%) were identified at tribe or family level or with a single species of the family outside the type genus Brassica, and were therefore deemed inaccurate diagnoses on the basis of incongruence with seed morphology (species-level diagnosis, Brassicaceae; see Table 3). Table 3. Taxonomic diagnosis using matK5. Best match results recovered on BOLD Systems v4 and NCBI databases using amplicon sequences of matK5 recovered from the study sites (only the highest match is shown). The percentage sequence identity and similarity score are shown, along with whether probability of a match was lower than 10 −5 . The species sharing the highest number of hits are provided, along with the lowest level of taxonomic diagnosis possible based on aDNA (given that all seeds possessed Brassicaceae-type seed morphology).

Information from Ancient Herbaria
The search for samples of Brassica and Sinapis produced results only for 3 of the 7 assessed herbaria and made it possible to find 25 specimens ( Table 4). The species recognized with certainty were B. napus, B. nigra, B. oleracea, B. rapa and S. alba; also in addition, some of the specimens were not clearly identifiable, and were therefore doubtfully attributed to the cited species, or simply to Brassica sp.

Discussion
Our archaeobotanical analyses of the seed and fruit morphology were internally consistent and suggestive of the presence of several taxa belonging to the Brassicaceae. Notably, the presence of Sinapis alba was found to be recognizable among the samples in which the external seed tegument was well preserved [15]. Perhaps the most interesting aspect of these data was the presence of several species of Brassica. The large quantity of seeds available for this genus [50] in the two contexts examined allowed the unequivocal separation of the samples into morphological groups associated with species descriptions. On this basis, the dominant species among our sites seems to have been B. rapa s.l., with representatives of B. nigra and B. napus also well represented. However, several studies have reported extensive intraspecific variability in the key diagnostic features of seed-surface architecture, seed size, and seed shape for species belonging to this genus [51][52][53][54]. Despite this known variability, none of the widely used diagnostic keys attempt to estimate the frequency of erroneous diagnosis [15,50,[55][56][57]. We therefore sought to support identifications based on seed morphology through the use of DNA sequencing as an independent mode of species diagnosis. DNA yields from ancient biological samples (aDNA) are typically low and highly degraded [58]. However, several groups have successfully used the DNA extracted from ancient plant material to confirm species identity using short amplicons of the core DNA barcodes [59,60] or else elected to target supplementary or non-coding barcode markers [61]. Such works typically use either large volumes of starting materials [62] or next-generation sequencing platforms [63]. Currently, we are unaware of any study using aDNA for the identification of individual Brassica seed samples. Here, we were not able to secure high-quality barcode sequences in either rbcL or matK by direct PCR, but we were able to do so using a modified form of nested PCR [64]. Several other works have reported similar success in recovering Sanger sequence data from materials containing low levels of template DNA, including DNA from animal feces [65], adulterated food [66], formalin-preserved specimens [67], and dried museum specimens [68].
In this study, we found that our ability to use aDNA-derived sequences to differentiate between members of Brassica varied considerably between amplicons, with the best results deriving from the matK5 primer set. Reference to the seed morphology allowed us to discount all barcode-based diagnoses that fell outside the genus Brassica, most of which were relatively short sequence reads. It is perhaps notable that the finest level of taxonomic match was achieved after searches were performed using NCBI BLASTn searches. This may be at least partly attributable to the fact that the NCBI database contains a far higher proportion of data originating from next-generation sequencing than from Sanger sequencing, and the former is noted for its lower levels of technical errors [69]. Considering only the search results that were deemed reliable (i.e., those within Brassica) provided some support for the morphological identifications. Specifically, there was evidence of the significant presence of B. nigra in both sites, as well as of B. oleracea. The inability of the matK5 sequences to distinguish between B. napus and one of its progenitor species, B. rapa [5], precluded the confirmation of the distributions of these individual species, but was nevertheless congruent with the overall inferences of the presence made from the morphological analyses.
Both sources of plant diagnosis suggested a significant presence of B. nigra. The efficacy of morphological diagnosis partly depends on the characteristics of the seeds of this species. In fact, in the genus Brassica, originally, dark-seeded species (e.g., B. nigra and B. napus) and yellow-seeded species (e.g., B. rapa and B. juncea) are present [70]: the first have significantly more lignin than the latter [71], especially in the inner epidermis of the seed-coat (testa) [72,73], perhaps also because the seeds of B. nigra must undergo a necessary dormancy period before germination [72]. This feature surely makes the seeds of B. nigra more resistant than others, and the careful choice of seeds made in view of the aDNA analyses probably caused a slight over-representation of this taxon.
The total number of specimens found in the Renaissance herbaria was not high if considered in the light of the economic importance of these species, and the presence of samples was limited to the three herbaria richest in exsiccata (Erbario Aldrovandi, Erbario ex Cibo B and Erbario Cesalpino). The B. oleracea was probably more easily available than the others due to its widespread cultivation as a vegetable; in fact, featured 7 specimens, plus one of uncertain attribution, comprising 1/3 of the total number of specimens found in the herbaria. In this regard, Mattioli [74] referred that various cabbage "species" exist, and Durante [75] noted that cabbages were cultivated and transplanted in all kitchen gardens and vineyards. By contrast, B. napus and B. rapa were only marginal presences among the herbarium samples, whereas B. rapa was the most abundant species among the archaeobotanical remains. It is worth noting that during the 16th century, B. napus was commonly known, as attested by Mattioli [74]; therefore, it was probably commonly cultivated, and various "species" of it existed [75]; B. rapa was also extremely common in Italy, especially in the north [74,75]. B. nigra, another medicinal plant [76,77], was present in all three herbaria, even if with only one sample each; finally, Sinapis alba also had few records, in two herbaria only, despite its recognition as the common quality of mustard, cultivated in the kitchen gardens [74,78].

Ethnobotanical Considerations in the Possible Uses of Brassica and Sinapis Seeds
In the two pits, the significant abundance of seeds of the Brassica sp.pl. and Sinapis alba makes it possible to hypothesize a specific use of these taxa: we observed two contexts that evidently collected waste from food preparation [24,79].
It is known that, during the Middle Ages, various species of Brassica were cultivated in Europe to obtain oil [80,81]. During this period, in fact, for economic, cultural, and religious reasons, the use of "minor" vegetable oils in food preparation became quite widespread and nearly dominant in several territories, principally in those that were (at least partially) outside the range of Olea europaea L. [82]. In particular, the oil of rapeseed (B. rapa/campestris var. oleifera), was used up to the 20th century, particularly in northern Italy [83], where the taxon was commonly cultivated, even in the mid-1500s [74,75]. On the other hand, the oil of swede (B. napus) was frequently used as a condiment, as a fuel for oil lamps, to make soap, and in wool crafts [75]. Thus, the numerous seeds of B. rapa s.l. and B. napus found in the archaeological contexts of Ferrara and Lugo probably represent the waste of gentle squeezing (since not many fragments are present) to obtain oil. It should be noted that they were also used as components in complex pharmaceutical preparations, such as the theriaca: the seeds of B. napus and B. rapa were regarded as excellent counterpoisons and, therefore, inserted in the composition of numerous antidotes [74].
On the other hand, various seeds of the genera Brassica and Sinapis are used as spices and ingredients in sauces, in particularly B. nigra, B. juncea, and S. alba [84,85]. In the mid-15th century Michele Savonarola, ancestor of the more famous Girolamo, as a physician of Duke Borso d'Este in Ferrara, wrote a dietetics treaty, listing and commenting on the foods that were more or less commonly present on the tables of that epoch [86]. Among various features, Savonarola notes that in Ferrara, "ogni contrada" (every neighborhood) had two or three sales counters for "senava" (mustard), which was consequently widely used in kitchens. Here, the term "senava" much probably identifies the mostarda, which in its current form was likely codified in the Middle Ages but was subsequently differentiated in diverse regional recipes [87,88]. Mostarda can be a sauce made of crushed mustard seeds only, or a very rich and complex product, with the addition of fruits (grapes or their derivatives, figs, apples, pears, quinces, blackberries, walnuts etc.) and various spices in seeds (anise, coriander, fennel, pepper etc. [88]). It is interesting to observe that nearly all these potential ingredients were found among the botanical remains of the two pits [38,79]. Mustard is considered as one of the "universally" widespread elements of the cuisine of the late Western Middle Ages [89,90]; the contemporaneous presence of B. nigra and S. alba in both in Ferrara and Lugo suggests that this product could also have been produced in both contexts.

Archaeological Context
The seeds considered in this study come from two sites in Ferrara and Lugo (Emilia-Romagna- Figure 1a) with two particular contexts, described below.
(I) Ferrara-Corso Porta Reno/via Vaspergolo (Figure 1b). In a key position of the medieval city, an excavation (about 300 m 2 ) was carried out in 1993-1994, which exposed a stratification of the city of early medieval foundation [91]. The chronological range of the archaeobotanical material analysed was from the 10th to the 15th century AD; at the beginning, it was a zone of peri-urban kitchen gardens with wooden structures, which subsequently became part of the historical city centre with brick houses [36,37]. The excavation context that produced the greatest number of seeds of Brassica/Sinapis was a brickwork pit (3.5 × 1.5 × 1.4 m- Figure 1d) for domestic waste, collocated under the floor of a house and used for a few years in the middle of the 15th century. Quality and typology of the artifacts found within this tank indicate that the dump was used by an upper/middle-class family [92,93]. The pit was probably a place to discharge foodpreparation waste [38,39,79].
(II) Lugo (Ravenna)-Piazza Baracca/via Magnapassi (Figure 1c). The urban area, in the center of the town, excavated in 2009, revealed a zone with productive activities dating to between 14th and 16th century. The area featured numerous wells, which were later reused to dispose of waste, and a brickwork pit (3.7 × 2.0 × 1.9 m- Figure 1e), analogous to the pit of Ferrara, whose filling dated from the 15th-16th century (this information was provided by Chiara Guarnieri-Soprintendenza Archeologia, Belle Arti e Paesaggio Bologna-Modena-Reggio Emilia-Ferrara). A significant number of Brassica/Sinapis seeds were also found in this context (still unpublished).

Archaeobotanical Analysis
Archaeobotanical analyses were performed in the Laboratory of Palynology and Palaeobotany of the University of Modena and Reggio Emilia.
Identification keys for the seeds of these two genera, defined as "globose, spherical or irregular in shape" [15], take into account the structure (although scar, chalaza, and the grooves of the radicle ridge do not have significant diagnostic value [50]), the shape, and the size. However, the most discriminant features are the type of reticulation, the size of the interspace, and the character of the stippling, which can be observed overall in the middle part of the seed (on the seed coat; for further details, see Table 5). For the seeds of the genus Brassica, "while it is not possible in all cases to identify an individual seed with certainty, it is usually possible to make a fairly reliable separation of a mixture of species" [50]. Observations were made with a stereomicroscope ( Figure 5) with up to 80 magnifications. Nomenclature was updated following [10,49].

Ancient DNA Analysis
The overall strategy for the molecular characterization of ancient seed samples was to use established chloroplast barcode markers, supplemented where necessary with chloroplast Simple Sequence Repeat (SSR) markers that were initially screened against a worldwide Brassica reference panel.

Plant Materials and DNA Extraction
Archaeological seeds with a Brassica-type phenotype were transported to Aberystwyth, UK, for subsequent DNA extraction. Here, the following precautions were taken to minimize the probability of exogenous contamination during the extraction process. First, the seeds were not handled directly and were moved to an isolated microbiology laboratory (no previous history of plant molecular biology) for DNA extraction. Once there, seeds were initially exposed to UV light (20 min) to break and immobilize any contaminant DNA on the seed surface. Seeds were then immobilized on a sterile glass slide using nail varnish and air-dried under positive pressure. Central tissues (endosperm and embryo tissues) were then carefully removed from the seeds under sterile conditions using a dissecting microscope, and the testa (seed coats) discarded ( Figure 6). The isolated internal tissues were transferred into a sterile tube containing DNeasy lysis buffer (400 µL) and RNase (20 µL) (both Qiagen, UK) and mechanically disrupted, and DNA was extracted according to the manufacturer's instructions, except for the use of 100 µL elution buffer (rather than 50 µL).

Primer Selection
The two universal barcode loci for plants (rbcL and matK [94]) were targeted as sites to enable species identification. For the archaeological seed samples, given the expectation of extensive degradation, a wide range of forward and reverse primers were screened to generate amplicons covering at least part of the barcoding locus (Supplementary Information,  Table S2). All primers were designed using reference sequence from the BOLD database downloaded onto the Geneious software.

DNA Sequencing
Amplification products were submitted for sequence analysis to Macrogen (http://www.macrogen.com, accessed on 20 June 2012). Here, cycle-sequencing reactions were carried out according to [96]. Manual editing of raw traces and subsequent alignments of forward and reverse sequences enabled us to assign edited sequence for most species. The 3 and 5 termini were clipped to generate consensus sequences for each taxon. Nucleotide sequences were then translated into amino-acid sequence using ExPASY (http://www.expasy.ch/tools/dna.html, accessed on 28 November 2021).

Sequence Analysis and Verification
Consensus sequences were produced for each taxon at each locus by alignment of the forward and reverse sequences using ClustalW (http://www.ebi.ac.uk/clustalw/, accessed on 1 December 2021). All sequences were searched on BLASTn (http://www.ncbi. nlm.nih.gov/BLAST/, accessed on 5 December 2021) or the BOLD Systems V4 database (https://www.boldsystems.org/, accessed on 5 January 2022, last visited 1 February 2022) to verify taxon (or close taxonomic group) and locus.
Following the method described here, aDNA of 242 Brassica seeds was analysed ( Table 6): 161 from Ferrara (context I) and 81 from Lugo (context II).

Research in Italian Renaissance Herbaria
To compare the results of the archaeobotanical analyses with other contemporary sources, we decided to search samples of species attributable to the two genera under examination in the Italian Renaissance herbaria, the oldest in Europe (16th century- [97]); the information contained in these collections is vital to address questions related to the species or varieties cultivated and used during this epoch, especially if these data are integrated in a combined approach involving various disciplines, as already demonstrated in previous studies [98][99][100][101][102][103].

Conclusions
The multiproxy approach in this research was proven to be of interest. The two waste pits of Ferrara and Lugo offered a notable and unusual quantity of seeds of Brassica sp.pl. and Sinapis alba. Thus, in this case, optimal conditions were available in which to perform the traditional morphometric analyses on the seeds in the most effective way possible, showing non-negligible species diversity within the genus Brassica in both contexts.
In addition, despite the aforementioned limitations, the availability of such a large volume of remains made it possible to attempt research on the aDNA for a taxon normally not studied in this sense (in contrast to the frequency with which other important economic plant species, such as Vitis vinifera L. or cereals, are studied [111][112][113]). Results were obtained that could form the basis for new and more in-depth investigations in this field.
Our research on the ancient herbaria (which are datable to an epoch slightly later than the contexts studied) and other historical sources of the Middle Ages/Renaissance period allowed to understand how these species were frequent and widespread among the cultivated food plants. Furthermore, the seeds of these species were used to obtain oil and other seasonings, which, from the medieval period onwards, became typical elements of all European cooking.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants11162100/s1. Table S1: Taxonomic diagnosis using matK1, 1a, 1b, 1c, 4. Best match results recovered on BOLD Systems v4 and NCBI databases using amplicon sequences of matK1, 1a, 1b, 1c, 4 recovered from the study sites. The percentage sequence identity and similarity score shown, along with whether probability of a match was lower than 10 −5 . The species sharing the highest number of hits are provided, along with the lowest level of taxonomic diagnosis possible based on aDNA (given that all seeds possessed Brassicaceae-type seed morphology). Table S2: Sequences of barcode primers used to amplify material in this study. Target sequence from Brassica napus L. strain ZY036 chloroplast complete genome (Genbank: GQ861354).