Characterization of Grapevine Genetic Resources in the Comunitat Valenciana (Spain)

ABSTRACT Grapevine cultivation is of historic importance arround the Mediterranean basin. The Spanish Levant is an area of notable grapevine diversity, where ancient varieties destined for producing wine, table grapes and raisins can still be found. For some of these neglected varieties, however, no complete description has ever been made; current legislation does not, therefore, allow their commercial use. The botanical characterization of old varieties is an important step toward their identification and can also help to clarify synonymies and homonymies, problems very commonly encountered with this crop. The present work provides ampelographic descriptions of seven genotypes for which microsatellite marker profiles were already available, and discusses the possible origins of their names, as well as instances of synonymy and homonymy. Seven accessions of ancient grapevine genotypes from the Spanish Levant were subjected to in situ ampelographic analysis following official methods. The characteristics of their leaves, bunches and berries were recorded. The ampelographic descriptions made in the present work provide insight into the history of cultivation of the examined genotypes and would pave the way for their inclusion in the Spanish national catalog, which would allow their commercial use. The information gathered on the possible origin of these genotypes’ names, and instances of synonymy and homonymy, enriches our knowledge of this recovered germplasm. The present results contribute toward our understanding of Europe’s grapevine genetic diversity, and highlight the need to conserve it.


Introduction
According to the International Organization of Vine and Wine (OIV), Spain has the largest area under grapevine (Vitis vinifera L.) cultivation, and despite the broad grapevine cultivar diversity that is found, just two varieties occupy over 43% of the area given over to viticulture: the white variety Airén, and the red variety Tempranillo. Airén occupies a total 22.3% (217,000 ha) of this surface (International organization of vine and wine, 2017) (it is grown almost exclusively in Spain, especially in the region of Castilla-La Mancha, where it has its origins (Cabello et al., 2011)), and Tempranillo 20.8% (203.000 ha; it is also grown in 17 other countries worldwide) (International organization of vine and wine, 2017). This heavy favoring of a small number of varieties is seen in grapevine-growing areas all over the world; out of an estimated 6000-10000 varieties available (Lacombe et al., 2013;Wolkovich et al., 2018), 33 cover half of all viticultural surface, and just 13 occupy one third of it

Ampelographic Characterization of Bunches and Berries
The characteristics of the bunches and berries of each grapevine variety were recorded following the OIV list of descriptors (International Organisation of Vine and Wine, 2009). According to the OIV definition norms (International Organisation of Vine and Wine, 2009), 5-8 bunches were measured and characterized when mature and eight characteristics were analyzed for each bunch: OIV-202, OIV-    (Tables 3 and 4). The modal value for each descriptor for bunches and berries was selected as the final result. After the notation, the berries were then opened and the seeds extracted; 30 well developed seeds were then dried to constant weight in a stove at 35°C and afterward their length and weight were recorded.

Statistical Analysis
In order to avoid errors from data of ampelographic characterization referred to plants grown into different environments, principal component analysis (PCA) was performed using only the calculated relationships described above for mature leaf measured parameters. This will allow to summarize and more easily visualize the dataset. All data were analyzed using the SAS System v.9.3 software package (SAS Institute, Cary, NC, USA).

Ampelographic Characterization of Adult Leaves
Supplementary Table S2 shows the results for the relationships calculated from the different leaf lengths and angles. Table 1 summarizes the ampelographic description of the adult leaves of each genotype. Some of the genotypes had clearly different leaves characteristics (Table 1 and Figure 2). For example, Esclafagerres´ leaves had a strongly overlaid petiole sinus; the Mamella de Vaca leaves were very large; Macabeo Negro´s leaves had teeth with both sides straight and no upright hairs on the veins, although they were present between the veins and were very long (data not shown); Mondragón ´s leaves had teeth with both sides straight; leaves in genotype Montalbana had prostrate hairs (that tended to fall off) on the upper side of the leaf; in genotype Morsí the leaves had large and notably pale green leaves, teeth in the petiole sinus (not always) and upper lateral sinuses (not always), a strongly overlaid petiole sinus, a medium density of prostrate hairs between the main nerves on the lower leaf surface, and upright hairs on those nerves; and Trepadell´s leaves had deep, open lateral sinuses, sometimes containing a tooth.
PCA performed using the relationships calculated from the leaf measurements and angles recorded (Suppl . Table S2) showed the first two axes to account for 84.21% of the total variance, and the first three to account for 94.02% (Suppl. Table S3). The variables with the most weight associated with the first axis (prin1) were Rel.6, Rel.7, Rel.8, Rel.9, Rel.14 and Rel.15, all of which are related to the depth of the upper and lower lateral sinuses (Suppl. Table S3). The variables with the most positive weight associated with prin2 were Rel.10, Rel.11, Rel.12 and Rel.13, all of which refer to the angles formed by the main veins; those with the most negative weight were Rel.2, Rel.3, Rel.4 and Rel.5, all related to the shape of the leaf. The variables with the most positive weight associated with prin3 were Rel.3 and Rel.5, which refer to the shape of the left side of the leaf; that with the most negative weight was Rel.2, which refers to the relationship between L1d and the main vein.
In the diagram obtained using the first three PCA axes (Figure 3), the genotypes with very small lateral sinuses (e.g., Macabeo Negro and Montalbana) cluster to the left with respect to prin1; those with deep lateral sinuses (such as Trepadell and Esclafagerres) cluster toward the right. The genotypes with leaves showing the largest sum values of their angles (particularly Morsí) appear toward the upper part of the diagram with respect to prin2, while those with the smallest (Trepadell and Mondragón) appear toward the bottom. With respect to prin3, the genotypes with the smallest Rel 3 and Rel 5 and largest Rel 2 values (Morsí and Trepadell), which are characterized by their asymmetric leaves in which the nerves L1 and L2 on the left side are smaller than those on the right, gather toward the front of the diagram. Esclafagerres and Mamella de Vaca, with their more symmetrical leaves, appear toward the back. Table 2 shows the results for the bunch quantitative variables, and Table 3 reports the qualitative descriptors. Trepadell had the largest and heaviest bunches, while Macabeo Negro had the smallest and lightest. Those of Mondragón were often cylindrical whereas all other genotypes had conical bunches ( Figure 4). Morsí had the least compact bunches, and Macabeo Negro the most tightly packed bunches. All the genotypes had bunches with 1-2 wings (Figure 4). Table 3 also reports the qualitative descriptors recorded for the berries, while Table 4 shows the values for their measured quantitative variables. All the genotypes had berries of a uniform size throughout their bunches ( Figure 5), and all had almost spherical berries ( Figure 6) except for Macabeo Negro which had flattened spherical and inverse ovoid berries, and Trepadell which had short, elliptical berries. Berry color varied (Table 3); Mamella de Vaca had non-uniform pink berries, Montalbana had berries with a red epidermis, Esclafagerres, Morsí and Trepadell had yellow-green berries, and those of Macabeo Negro and Mondragón were blue-black. The berries of all the genotypes showed a medium level of bloom, except for Macabeo Negro, which showed a high level of bloom accorfing to the OIV-227 characteristic. The berries of Mamella de Vaca and Macabeo Negro had a patent hilum. The berry pulp of all the genotypes was juicy; none had any notably different flavor. The pulp of Esclafagerres, Mondragón and Morsí berries was soft, while that of Mamella de Vaca, Macabeo Negro, Montalbana and Trepadell was firm. The berry epidermis was thin in Mondragón, Morsí and Trepadell, but thick and with a bitter taste in the remaining genotypes (data no shown). Finally, the seeds of all the genotypes were well formed (Table 3 and Figure 6). Those of Montalbana were the smallest and lightest, and those of Mamella de Vaca the largest and heaviest.

Discussion
With the exception of Macabeo Negro, references exist to the prior cultivation of varieties with the same names as the present material before the arrival of phylloxera in this region in 1912 (Piqueras, 2005). For example, in the 19th century, Esclafagerres, Mamella de Vaca, Mondragón, Montalbana and Trepadell were names mentioned by the Dirección General de Agricultura, Industria y Comercio (Dirección General de Agricultura, Industria y Comercio, 1891); Esclafagerres (written as "Esclafacherris"), Montalbana and Morsí appear in an ancient book from 1885 (Abela y Sainz de Andino, 1885); and Montalbana (written as "Muntalbana") appears in the famous 15th century manuscript book Llibre de l'Espill ("Book of the Mirror") written by Jaume Roi which is one of the   (209) (2) 1-2 wings (2) 1-2 wings (2) 1-2 wings (2) 1-2 wings (2) 1-2 wings (2) 1-2 wings (2) 1-2 wings BERRY DESCRIPTORS Uniformity of size OIV (222) (2) uniform (2) uniform (2) uniform (2) uniform (2) uniform  great works of medieval literature in Valencian language. In Cabello et al. (2011), Mondragón is mentioned as a native minority cultivar; it is also cited by García de Los Salmones (1914) as being cultivated in the Province of Castellón. According to Viard (1892), the Romans made their best wine from vines cultivated in the Mondragone Hills in Campania (Italy), and the name of the present Mondragón genotype may have its roots in this, but nevertheless Mondragón is an offspring of Marufo x Monastrell (D'Onofrio et al., 2021) and Marufo is a putative Portuguese variety with female flowers and that bears chlorotype D, with a major role in the development of cultivars in the Iberian Peninsula (Cunha et al., 2020); therefore, the Italian origin for this cultivar name seems unlikely. Also, according to Favà (2001), the origin of the name Montalbana may lie in a Tuscan wine known as Montalbano made in the Montalbano Hills area of Pistoia. The name Esclafagerres comes from the Valencian words "esclafar" meaning to squash, and "gerres" meaning clay wine vats. The name might therefore be 'equivalent' to the names Quebrantatinajas, Rompetinajas or Revientatinajas in Castilian Spanish (meaning vat-cracker, vat-breaker, and vat-ripper, respectively). However, the microsatellite profile of Esclafagerres (Gisbert et al., 2018) does not coincide with that of any of these other varieties, which in any event are recorded as having red-or black-skinned berries, not green-yellow like those of Esclafagerres. According to local growers, Esclafagerres was not a good winemaking variety, but was used alongside others, such as Merseguera, to reduce the final alcohol content -a characteristic appreciated in the area since the high temperatures cause the grapes to accumulate large amounts of sugar that becomes converted into alcohol during fermentation. No historic reference was found for any variety with the name of Macabeo Negro -the name given known by the grower on whose vineyard the single accession of this genotype was discovered (in El Pinoso, Province of Alicante). The SSR profile (Jiménez et al., 2019) of Macabeo Negro indicates it to be quite unlike the well known variety Macabeo, also, these two genotypes have different ampelographic characteristics, fr instance, Macabeo Negro has blue-black berries and short bunches, while Macabeo has yellow berries and medium-to-large bunches.
Macabeo Negro, Montalbana and Mondragón are strongly related, all three have blackish berries and are of chlorotype D andtheir SSR profiles indicated that they could all be hybrids of the variety Crujidera (synonymous with the Portuguese variety Marufo, has female flowers and belongs to chlorotype D), and Monastrell (the most grown variety in the Province of Alicante) (Jiménez et al., 2019). The three varieties are distinguished in that Macabeo Negro has virtually no lateral sinuses leaving it with 'entire' leaves, more compact bunches, and its berries are not uniform in shape throughout the bunch. Montalbana, in contrast, is distinguished by the red-violet color of its berries, and the small size of its seeds. Finally, the pulp of Mondragón berries is softer than that of the other two genotypes (Figures 3 and 6).
Among the white genotypes, Esclafagerres and Trepadell have bigger and more compact bunches than Morsí, but all coincide in their conical shape and the presence of wings. In addition, Morsí has bunches of variable weight (CV = 48.1%) which could be related to the tendency of some table grape varieties to show millerandage (Ibáñez et al., 2020). Thus, in some years the bunches may be uniform in weight, and in others vary variable. Esclafagerres and Trepadell belong to chlorotype A (Jiménez et al., 2019), as do 75% of all varieties from Spain and Portugal (Arroyo- García et al., 2016;Castro et al., 2011). Morsí, however, belongs to chlorotype C and has the same SSR profile as Sbaa Tolba, which is recorded in the VIVC database as originating in Algeria. Morsí and Sbaa Tolba are therefore synonyms (García et al., 2020;Jiménez et al., 2019). Galet (2000) cites Sbaa Tolba as a white table grape variety from Morocco, and Laiadi et al. (2009) suggest it may be found in different parts of the Maghreb and that it might be related to Sultanina, with which it shares the C chlorotype. This chlorotype indicates a possible origin in the Near East/North Africa (Arroyo- García et al., 2016). In an ampelographic and molecular analysis of Algerian genotypes, Zinelabidine et al. (2014) reported an analyzed accession under the name of Sbaa Tolba (from the M'zej Edchiche Institut Téchnique d'Arboriculture Fruitière (ITAF) grape collection in Algeria) to have the same profile as Planta Fina (an accession held in the IMIDRA collection (Martín et al., 2003)). The SSR profile of this last accession coincides with that for Planta Fina in the VIVC database, and with those reported for this same genotype by García et al. (2020) and Jiménez et al. (2019). In addition, the foliar morphology presented in Zinelabidine et al. (2014) for the accession in the ITAF does not match with that of Morsí (synonym Sbaa Tolba), for example the petiole of the first is clearly open while in Morsí is strongly overlapped.Thus, the accession stored as "Sbaa Tolba" in the ITAF from Zinelabidine et al. (2014) is a case of misnaming.
Finally, Mamella de Vaca is differentiated by its non-uniform pink berries. (Jiménez et al., 2019) detected the same SSR profile in accessions collected from the area but with no name, in an accession that was said to belong to the variety Cambril -an identification rejected since Cambril is an early ripening variety (Favà, 2001) -and in an accession collected under the name of Mamella de Vaca. All these materials were named as "Mamella de Vaca" by Jiménez et al. (2019). Plants with the same profile were later found under the name Roget Tardà and Raïm de Tot Sants (García et al., 2020). Unfortunately, the name Mamella de Vaca has also been given to other varieties. For example, the variety Ahmeur bou Ahmeur has also been found under this name. Ahmeur bou Ahmeur is a widespread table grape variety from North Africa (Galet, 2000) with "rouge" berries and, to complicate matters, many synonyms (Zinelabidine et al., 2014), including the Castilian Spanish name "Teta de Vaca" (cow teat) which is linguistically equivalent to Mamella de Vaca despite it not actually being the same variety (Cabello et al., 2011;Clemente, 1807;Martín et al., 2003). To  complicate things even further, in the Comunitat Valenciana, Ahmeur bou Ahmeur is known as Botó de Gall (García et al., 2020). Although Jiménez et al. (2019) originally called the accessions they examined by the name Mamella de Vaca, it might be better to reduce confusion by using the synonym Roget Tardà (García et al., 2020); this means 'late red' in the Valencian language, which describes the berry color and the ripening time very well.

Conclusions
The ampelographic descriptions of the studied genotypes made in the present work pave the way for their inclusion in the Spanish national catalog, which would allow for their commercial use. The information gathered on the possible origin of their names, and the recognition of synonymies and homonymies, enriches our knowledge of the recovered germplasm and of Europe's viticultural heritage as a whole. A program has begun to conserve these genotypes and their contribution to grapevine diversity.