Seed management and selection in ancient maize
landraces from the French Pyrenees: ethnobotanical
survey and selection experiment

Brigitte Gouesnard, Yacine Diaw, Laurène Gay, Joëlle Ronfort, Jacques David

► To cite this version:

Brigitte Gouesnard, Yacine Diaw, Laurène Gay, Joëlle Ronfort, Jacques David. Seed management and selection in ancient maize landraces from the French Pyrenees: ethnobotanical survey and selection experiment. Genetic Resources and Crop Evolution, In press, 10.1007/s10722-025-02555-0. hal-05210925

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# Seed management and selection in ancient maize landraces from the French Pyrenees: ethnobotanical survey and selection experiment

Brigitte Gouesnard* · Yacine Diaw · Laurène Gay* · Joëlle Ronfort* · Jacques David*

Received: 9 April 2025 / Accepted: 2 July 2025
© The Author(s) 2025

**Abstract** Maize landraces (*Zea mays* subsp. *mays*) have evolved under the joint action of environmental factors and of the farmers who cultivated them. In this study, we aim to quantify the selection gradients exerted by farmers by proposing them a selection test consisting in choosing the ears they would select if they were to grow maize landraces the following year. The study focused on the Pyrenees region of France, where landraces were cultivated until the arrival of hybrids in the 1960s and conserved ex-situ ever since. We interviewed former Pyrenean farmers or their children who were cultivating landraces 60 years ago. The survey documented seed management practices

and know-how. Our selection test showed that their selection was based solely on ears: old farmers selected healthy and productive ears by using ear length and volume as the first two selection criteria. Both were highly correlated with the kernel weight per ear. Heritabilities of ear traits at an individual plant level were estimated in one trial for four landraces and were found variable between traits and landraces (average 0.36 ranging between 0 and 0.76). We calculated the expected genetic change after one generation of mass selection, following farmer selection criteria. For ear length, genetic change was expected to reach about 3.4% (from 1 to 7.5% over the 17 selection tests). We investigated seed selection practices both east and west of the Pyrenees and compared them qualitatively with those of native American farmers.

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s10722-025-02555-0.

B. Gouesnard (✉) · L. Gay · J. Ronfort · J. David UMR AGAP Institut (Amélioration génétique et adaptation des plantes méditerranéennes et tropicales), CIRAD (Centre de Coopération internationale en recherche agronomique pour le développement), INRAE (Institut national de recherche pour l'agriculture, l'alimentation et l'environnement), Institut Agro, Univ Montpellier, F-34398 Montpellier, France e-mail: brigitte.gouesnard@inrae.fr

L. Gay e-mail: laurene.gay@inrae.fr

Y. Diaw Centre pour le Développement de l'Horticulture (CDH), Institut Sénégalais de Recherches Agricoles (ISRA), Dakar, Sénégal

**Keywords** *Zea mays* subsp. *mays* · Ethnobotanical survey · Landraces · Seed management · Heritability · Mass selection

## Introduction

Landraces from small-scale farmers contribute to a large amount to the genetic diversity of maize (*Zea mays* subsp. *mays*, WFO (2025)). In developed countries, landraces, with few exceptions, are no longer cultivated, and maize landraces are conserved ex-situ in genebanks (Ortiz et al. 2010).
Indeed, landraces have been progressively replaced by modern hybrid cultivars. In France, the transition took place between 1950 and the 1960s. The main reason for abandoning landraces and adopting hybrids was the low yield of these populations compared to modern hybrid cultivars. To give an order of magnitude, in the publication by Gouesnard et al. (2016), the grain yield of populations was only 52% of the yield of the elite hybrids studied. Yet, in crosses with a tester, landraces showed less difference from the commercial hybrids: yield was on average about 26% lower than modern hybrids (Böhm et al. 2014). A difference of the same order of magnitude was observed in trials in 1984 and 1986 (23%) between landraces crossed with a tester and hybrids (Gallais et al., personal communication). In the same experiment, landraces crossed with testers were evaluated for silage, and the difference was found much smaller, around 6%. Indeed, the interest of maize landraces for silage improvement has been demonstrated for Argentinean and Brazilian-landraces (Bertoia et al. 2006; Bertoia 2001; Incognito 2016; Oliveira et al. 2020). Although maize was recognized early on as an excellent forage plant soon after its introduction to Europe, it was only bred for grain traits for a long time. Consequently, the difference between modern hybrids and landraces is lower for forage than for grain. In addition to forage yield, other adaptive traits are of interest in landraces. For example, sources of tolerance to water deficit have been found in dryland maize landraces (Gouesnard et al. (2016) for European landraces, Maafi et al. (2021) for Algerian landraces), and landraces tend in general to have a high capacity to tolerate biotic and abiotic stresses (Zeven 1998). Advances in molecular biology provide new tools for exploring and exploiting the genetic variability of landraces to improve maize drought tolerance (McMillen et al. 2022). Barbosa et al. (2021) for example performed Genome-Wide Association Study (GWAS) on 1326 landrace progenies (landraces x elite inbred line backcrosses) developed by the Centro Internacional de Mejoramiento de Maíz y Trigo (CIMMYT) Genetic Resources Program, originating from 20 landraces populations collected in arid regions. Landraces can also be used for their nutritional qualities: phytonutrients are abundant in these

cultivars, contributing to the health and well-being of both livestock and humans (Landoni et al. 2024).

The difference in yield between landraces and commercial hybrids increased over time, partly because progeny selection in hybrid cultivars is more efficient than mass selection performed in population cultivars (Hallauer and Miranda 1981). For example, the genetic progress in US landraces between the 1900s and 1930s was much lower than that achieved later with hybrids (Duvick 2005). This can be explained by heterosis in hybrid formulas, which enables a gain in productivity and a lower inbreeding depression (Charcosset 2002). However, the high levels of diversity available in landraces (Lefortbuson et al. 1991; Rebourg et al. 1999) should favor their evolution, and little is known about the response to selection of these crop populations with a broad genetic base.

Besides ex-situ conservation, maize landraces can also be preserved in-situ, on-farm. (Vetelainen et al. 2009). Many European networks and associations of farmers and gardeners maintain landraces, or promote their on-farm conservation (Raggi et al. 2024). As previously reported, the custodian farmer plays a fundamental role in on-farm conservation. According to the International Treaty of Plant Genetic Resources for Food and Agriculture (ITPGRFA), this figure is described as “a farmer who conserves landraces’ seeds over time, having great knowledge about these landraces in their environment (often inherited from their parents or grandparents), as well as the way of cultivating and their use as food. This farmer holds the responsibility for seeds, which constitute not only genetic but also cultural heritage” (FAO/ITPGRFA 2019; Landoni et al. 2024). In France, only a small fraction of farmers who still practice traditional agriculture, often in organic farming, still cultivate landraces. Associations such as the Reseau Semences Paysannes and a farmers’ association in Aquitaine (AgroBioPerigord) play an important role for on-farm preservation of landraces (Bocci and Chable 2008; Fenzi and Couix 2021).

However, in developing countries, landraces are still cultivated, often along with some modern cultivars. It is notably true in Mexico, the center of domestication and diversity for maize, where several investigations examined why and how farmers keep cultivating local cultivars while improved cultivars have been introduced (Bellon and Brush 1994; Bellon
and Risopoulos 2001; Louette and Smale 2000; Rice et al. 1998). They analyzed the traditional farming system associated with landraces. The human component is important to take into account as Villa et al. (2005) defined a landrace as "a dynamic population of a cultivated plant that has historical origin, distinct identity and lacks formal crop improvement, as well as often being genetically diverse, locally adapted and associated with traditional farming systems". The farmers' management practices were studied by analyzing the choice of cultivar, the seed flows and the management of seed selection. These investigations showed that farmers select complete ears rather than seeds (Louette and Smale 1998; Moreno et al. 2006; Rice et al. 1998) and that, despite significant cross-pollination and gene flow between landraces, the landrace's ear characteristics are maintained thanks to the homogenizing practices of farmers' selection (Louette and Smale 1998).

As maize landraces represent a highly valuable reservoir of diversity for crop improvement (Zeven 1998) that is no longer adequately conserved on-farm in France, an initiative was started to preserve this diversity ex-situ. In the 1960s, researchers from INRAE (National Research Institute for Agriculture, Food and the Environment) collected 265 landraces in France in order to build germplasm collections and to preserve the genetic diversity available in these cultivars. After 1983, the corresponding collections were managed through an association of public research laboratories and private maize breeders called "PROMAIS" (Dallard et al. 2000). The genetic diversity of this collection has been studied through many investigations supported by INRAE and the PROMAIS breeders association (Gallais et al. 2000). These investigations highlighted large levels of within-population diversity (75% of the total diversity), both in an enzymatic study on a sample of 30 populations (Lefortbuson et al. 1991) and in an restriction fragment length polymorphism (RFLP) survey of 65 populations (Rebourg et al. 1999).

This collection included 194 landraces from the South-West of France (SWF), out of which 119 originated from the Pyrenees and were collected for their cold tolerance and their early flowering. Molecular analyses using simple sequence repeat (SSR) markers showed that these landraces belong to the Pyrenees-Galicia genetic group, one of the seven genetic groups identified in a set of 275 American and

European populations (Camus-Kulandaivelu et al. 2006). The study of the South-Western populations (SWF) with SNP markers confirmed this origin and detected two distinct genetic groups in these populations, one located in the eastern part (E-SWF) and the other in the western part of SWF (W-SWF) (Diaw et al. 2021). The data suggested the occurrence of a hybrid zone between these two groups in the districts of Haute-Garonne and Ariège, near the Garonne valley (Diaw et al. 2021). This genetic structure could be due to demographic factors (random genetic drift in each group and restricted gene flow between them). Maize landraces grown east and west of the Pyrenees may also have differed for a set of genes related to climate adaptation or differential selection criteria by farmers east and west of the Pyrenees. As the effect of farmer selection had not been investigated in Diaw et al. (2021), our aim here was to document and analyze traditional farmers' selection practices, combining an ethnobotanical approach with a quantitative genetics experiment.

Farmers' seed management practices are considered the main factors influencing the dynamics of genetic diversity at the population level (Badstue et al. 2007; Pautasso et al. 2012). Thomas et al. (2011) advocated a multidisciplinary approach in order to study seed exchanges in traditional farming systems, combining analyses of social, biological and environmental factors. As maize landraces are no longer cultivated in the Pyrenees, information on seed management practices was collected through interviews with old farmers or their children, hoping to gather memories of maize cultivation 40 to 60 years ago. In 2012 and 2013, a first survey was carried out in the Pyrenees to collect data on the knowledge and skills of small-scale farmers (Beigbeder and Carraretto 2018 and http://pro-mais.org/les-populations-de-mais-pyreneennes). This survey documented the sowing techniques and agricultural practices, seed management, human and animal consumption of maize, the associated culture with beans, and the transition related to the introduction of hybrid cultivars. The interviews were carried out with approximately 30 farmers in five Pyrenean valleys using a subsistence agriculture agro-system. This study showed that, to select seeds for the following generation, farmers only relied on ears morphology (as opposed to whole plants in the field). The study reports the verbal description of the ears chosen by farmers. The criteria used were related to the size of the ear: "the prettiest
ears, the most beautiful ears, the largest, very straight ears, with kernels well aligned". These criteria also considered the health status: "attractive grains not crooked", and pollination success "well-finished ear". Practices for seed renewal and seed exchange ranged from no exchange at all, to exchanges with neighbors in the same valley or on the occasion of a trip or social event such as a wedding. We also learned that most farmers grew a single cultivar, the same one for human and animal consumption. Farms were generally small, with the smallest cultivation areas reported for maize as low as 0.2 or 0.3 ha. Fields of such small surfaces can lead to small population size and thereby strong genetic drift, combined with inbreeding effects. However, the interviews could not quantify the selection criteria, nor predict the genetic change expected from this mass selection on ears.

The present study was designed in order to complete the study of Beigbeder and Carraretto (2018). The first objective was to obtain new data on farmers' practices and skills, in order to 1) allow comparisons with practices of the Mesoamerican natives who cultivated maize landraces in traditional agrosystems and 2) get insights into the relative role of random genetic drift and selection in these on-farm management systems. The second objective was to determine whether traditional farming practices on landraces were favorable for on-farm genetic improvement. Yield improvement by mass selection requires that traits related to the yield be heritable and that the selection differential exerted by the farmers on their landraces be strong enough. To quantify these parameters, we set up a field trial to estimate heritability for yield-related traits for four Pyrenean maize landraces. We also asked the farmers surveyed to select ears within a large panel representing four old Pyrenean landraces. This ear choice test mimicked the seed selection for the following generation and was used to estimate selection differentials. Finally, we tested whether selection practices differed between the two zones (E-SWF and W-SWF).

## Material & methods

Introduction to the survey, general questions and requests

The survey was carried out in July 2018 in the Pyrenees. In the previous 3 months, we had contacted

the farmers involved in the 2012–2013 survey (Beigbeder and Carraretto 2018), as well as other farmers identified using information given by local town hall clerks. In total, 18 people participated in the survey: 12 were old farmers (between 85 and 96 years old), and 6 were old farmers’ children (Table 1). Eight were located in the Eastern part of the Pyrenees, and 10 in the Western part (Fig. 1). The survey was individual, narrative and qualitative (Suppl. File 1). We first asked them the number of cultivars they used to grow, and maize uses for human consumption and animal feeding. Second, we obtained information on the size of the plot cultivated with maize in order to estimate the strength of genetic drift in these populations. Third, we enquired about their practices on seed management methods such as selection methods, seed exchanges and seed conservation practices. Farmers also provided us with additional information about agricultural techniques or recipes. At the beginning of the survey, we displayed a set of uni-colored ears from full-sib crosses of the four landraces used for the test of ear choice (see below) and asked the farmers which landrace was most similar to the landrace they used to cultivate. Displaying these maize ears helped refresh their memory.

### Ears choice experiment

#### Selection of landraces

We selected four Pyrenean landraces, two per genetic and geographical area (East vs West), using the following criteria. First, we selected non-introgressed landraces, using the admixture coefficients estimated by Diaw et al. (2021) using more than 20 000 SNPs markers. We then extracted phenotypic data for all Pyrenean landraces from Gouesnard et al. (1997) and plotted the distribution of traits for the two genetic / geographic groups (Suppl. Fig. 1) to ascertain that the selected landraces were close to the average phenotypic value of the group. Finally, for each genetic group, we selected two landraces with either yellow or white kernels, the two main colors found in Pyrenean landraces. To represent the Eastern part of the Pyrenees (E-SWF), we chose Riverenert 1 (PPS68 or FRA0410068), with yellow kernels and Aston A (PPS70 or FRA041070), with white kernels, both collected in the "Ariège" geographic area. To represent the Western part of the Pyrénées (W-SWF), we chose
<table><caption>**Table 1** Description of the farmers who were interviewed during the summer of 2018</caption><thead><tr><th>Village code (legend Fig. 1)</th><th>Name of the village</th><th>District number</th><th>Number of landraces</th><th>Exchange</th><th>Force-feeding geese</th><th>Cultivar type</th><th>Status of the investigated person</th></tr></thead><tbody><tr><td>1</td><td>Aleu</td><td>9</td><td>2 yellow</td><td>neighbors or further</td><td>?</td><td></td><td>Old farmer</td></tr><tr><td>6</td><td>Engomer</td><td>9</td><td>2 white</td><td>?</td><td>Yes</td><td></td><td>Old farmer</td></tr><tr><td>8</td><td>Lacave</td><td>9</td><td>2 white and 1 yellow</td><td>?</td><td>Yes</td><td>PPS68</td><td>Children</td></tr><tr><td>11</td><td>Massat</td><td>9</td><td>1 yellow</td><td>?</td><td>?</td><td></td><td>Old farmer</td></tr><tr><td>12</td><td>Moulin-Neuf</td><td>9</td><td>2 white</td><td>?</td><td>Yes</td><td>PPS1052</td><td>Old farmer</td></tr><tr><td>14</td><td>Riverenert 3</td><td>9</td><td>?</td><td>?</td><td>Yes</td><td></td><td>Children</td></tr><tr><td>14</td><td>Riverenert 2</td><td>9</td><td>?</td><td>neighbors</td><td>?</td><td></td><td>Old farmer</td></tr><tr><td>14</td><td>Rivernevert 1</td><td>9</td><td>?</td><td>?</td><td>?</td><td></td><td>Children</td></tr><tr><td>2</td><td>Bagnères de Bigorre</td><td>65</td><td>1 yellow and few ear of white</td><td>?</td><td>No</td><td>PPS68</td><td>Old farmer</td></tr><tr><td>3</td><td>Beaucens</td><td>65</td><td>1 yellow</td><td>no</td><td>No</td><td>PPS1052</td><td>Old farmer</td></tr><tr><td>4</td><td>Bun</td><td>65</td><td>1 yellow</td><td>village around</td><td>No</td><td>PPS1052</td><td>Old farmer</td></tr><tr><td>5</td><td>Campan 1</td><td>65</td><td>1 yellow</td><td>neighbors</td><td>No</td><td></td><td>Old farmer</td></tr><tr><td>5</td><td>Campan 2</td><td>65</td><td>1 blonde and 1 copper color</td><td>no</td><td>No</td><td></td><td>Old farmer</td></tr><tr><td>7</td><td>Esquieze Serre</td><td>65</td><td>1 yellow</td><td>no</td><td>No</td><td></td><td>Old farmer</td></tr><tr><td>9</td><td>Laloubère</td><td>65</td><td>?</td><td>?</td><td>No</td><td></td><td>Children</td></tr><tr><td>10</td><td>Lesponne</td><td>65</td><td>1 yellow</td><td>neighbors</td><td>No</td><td></td><td>Old farmer</td></tr><tr><td>13</td><td>Oursbellile</td><td>65</td><td>1 white</td><td>?</td><td>No</td><td>PPS70</td><td>Children</td></tr><tr><td>15</td><td>St Jean Pied de Port</td><td>64</td><td>?</td><td>neighbors</td><td>No</td><td></td><td>Children</td></tr></tbody></table>

Districts: 09 "Ariège" representing the eastern part of the Pyrenees, 65 "Hautes Pyrénées" and 64 "Pyrénées Atlantiques" represent the western part of the Pyrenees in this study. The number of landraces refers to the number of landraces cultivated by each farmer; "Exchange" refers to the main type of seed exchanges declared. Cultivar type is the population out of the four presented that most closely resembles the population they were growing

"?", it simply means that we did not get an answer to this question. Given the age of the farmers we interviewed, we had to keep the interviews short and sometimes some answers are missing

<figure><img src="image_2.png" /><figcaption>**Fig. 1** Localization of the villages where the farmers interviewed live. The numbers correspond to the farmers identification number described in Table 1. PPS653, PPS1052, PPS68 and PPS70 refers to the collection site of the four maize landraces</figcaption></figure>
Saint Jean Pied de Port 3 (PPS 653 or FRA0410653) with white kernels collected in the Pyrénées Atlantiques, and Bagnères-de-Bigorre 2 (PPS 1052 or FRA041052), originated from Hautes Pyrénées with yellow kernels. The prospecting sites of the four landraces collected in the 1960s are shown on Fig. 1. Seeds from these four landraces were obtained from the French Maize Biological Resource Center (BRC) located in Montpellier (http://florilege.arcad-proje ct.org/fr/collections/collection-mais) hosted by the Gamet BRC (https://umr-agap.cirad.fr/l-unite/centr es-de-ressources-biologiques/crb-gamet-montpellier). In order to multiply seeds and remove any potential maternal effect, all four landraces were grown in the same field at the INRAE experimental unit "Diascope" at Mauguio, southern France (43°37'02" N, 3°51'18" E) in 2017 to produce 150 ears for each landrace by open-pollinated crosses, so that the two white landraces had multicolored kernels.

### Agro-morphological analysis of the selected cultivars

To characterize agro-morphological variation within and among these four selected landraces, all the ears produced in the 2017 field trial (150 ears per landrace) were measured for the 10 following traits: total ear length (E_length in mm), ear length with kernels (L2 in mm), ear diameter 1/3 down the ear (DIAM1), ear diameter 1/3 up the ear (DIAM2), number of rows (N_row), number of kernels per row (NKR). From the measured traits, we calculated the mean ear's diameter (E_diam estimated by the mean between DIAM1 and DIAM2), the conicity of the ear as (((DIAM1-DIAM2)*3)/(2*E_lenth))*100 following (Ordas and Deron 1988), the length of the top of ear without kernel (EAR_TOP), the volume of the ear estimated as a cylinder (0.001*3.1416*E_length*E_diam/4). To estimate yield, we counted the number of kernels per ear (K_number) and the weight of all the kernels collected on the ear (K_weight) using a subset of 80 ears per landrace produced in 2018. To predict K-number and K_weight, we established a linear regression based on E_length, E_diam, N_row, NKR from a subset of 50 ears per landrace. To validate the estimation of the regression coefficients, we used cross-validation, predicting K_number and K_weight for the rest of the sample (30 ears). Then, we estimated the correlation between these estimated values for K_number and K_weight and the observed

values of the same 30 ears. This procedure of cross validation was repeated ten times. It showed that K_number and K_weight could be predicted with a good accuracy for each of the four landraces: the coefficients of correlation were higher than 0.7 over the 10 replicates for both K_number and K_weight for the four landraces, except for PPS653 for K_weight for which the coefficients were between 0.4 and 0.7 for eight replicates. Nevertheless, all correlations were significant (with p-values <0.05). Finally, K_number and K_weight were predicted for the whole dataset produced in 2017 (150 ears × 4 landraces) using the linear regression equations.

The agro-morphological variation observed for ears in the four landraces was compared using box-plots and mean values were compared using ANOVA and Tukey tests. We also performed a principal component analysis (PCA) on the whole dataset and on the ten traits to visualize the overall morphological variation within and between the landraces (R Development Core Team 2011).

### Ears choice experiment and estimation of selection differentials on ear morphology

During the interview, farmers were provided with ears from one to three of the four selected landraces (each represented by 150 ears), of which they were asked to select 15 they would have sown the following year (i.e. so as to mimic their selection practices). Nine out of the 18 farmers surveyed took part in this selection test for one or several landraces, which provided us with 17 replicates of ear selection tests (Table 2).

To visualize the differentials of selection S, we used PCA on ear morphology and added the mean of the 150 ears sample ($T_0$), along with the mean of the 15 selected ears ($T_1$ to $T_9$, see legend of Table 2) as supplementary data. For each ear selection test and for each trait, we also estimated the differential of selection S as the difference between the average phenotypic value of the 15 ears selected by a farmer and the average phenotypic value of the 150 ears presented ($S = \mu_{15ears} - \mu_{150ears}$). To standardize the selection differentials, we divided them by the standard error and we performed an ANOVA (R Development Core Team 2011) to investigate the effect of three factors: the zone of origin of the farmer (E-SWF vs W-SWF), the farmer's identity (nested within
<table><caption>**Table 2** Landraces presented to the farmers for the ear choice experiment</caption><thead><tr><th>Farmer</th><th>District</th><th>Choice experiment</th><th>PPS1052</th><th>PPS653</th><th>PPS68</th><th>PPS70</th></tr></thead><tbody><tr><td>Aleu</td><td>09</td><td>T3</td><td>x</td><td></td><td>x</td><td></td></tr><tr><td>Beaucens</td><td>65</td><td>T9</td><td>x</td><td></td><td></td><td></td></tr><tr><td>Bun</td><td>65</td><td>T2</td><td>x</td><td></td><td>x</td><td></td></tr><tr><td>Campan1</td><td>65</td><td>T7</td><td></td><td>x</td><td>x</td><td></td></tr><tr><td>Campan2</td><td>65</td><td>T8</td><td></td><td>x</td><td>x</td><td></td></tr><tr><td>Engomer</td><td>09</td><td>T5</td><td>x</td><td></td><td>x</td><td>x</td></tr><tr><td>Esquiezes_serre</td><td>65</td><td>T1</td><td>x</td><td></td><td>x</td><td></td></tr><tr><td>Moulin_Neuf</td><td>09</td><td>T4</td><td>x</td><td></td><td></td><td></td></tr><tr><td>Oursbellile</td><td>65</td><td>T6</td><td>x</td><td></td><td></td><td>x</td></tr></tbody></table>

PPS1052 is a yellow landrace from the western part of the Pyrénées, PPS653 is a white landrace from the western part of the Pyrénées, PPS68 is a yellow landrace from the eastern part of the Pyrénées, and PPS70 is a white landrace from the eastern part of the Pyrénées. The column named “Choice experiment” corresponds to the legend of Fig. 3

zone), and the landraces (PPS68, PPS70, PPS653, PPS1052).

Estimation of narrow sense heritability for ear morphological traits and prediction of response to selection

In order to predict the expected genetic change after one generation of selection, we need to estimate the heritability of the traits considered. For each of the four selected landraces, we built full-sib (FS) and half-sib (HS) families to estimate the additive and dominance components of the genetic variance for different quantitative traits (Gallais 1990). In the field trial of 2017, for each landrace, twenty randomly chosen plants were used as pollen donors and crossed to two mother plants. The plants were randomly sampled from a balanced sample of the gene bank. A total of 40 FS families were thus obtained for each landrace.

In 2018, these 160 FS families (i.e. 40×4 landraces) were grown in the south of France (INRAE Experimental Unit Diascope, Mauguio) in an experimental trial with two replicates arranged in complete blocks. The total trial was organized in 320 rows, with 28 plants belonging to the same FS family in each row. The space between two rows was 0.80 m and the density 70 000 plants / ha. The position of each family within the block was randomized. At flowering time, we measured the number of days to pollen shedding and silking at the plot level. At harvest, a maximum of 15 ears were collected in each plot (12.3 ears per plot on average). We measured the same set

of quantitative traits as the ones measured on the ears used for the survey (as detailed above).

The relationships among individuals within each landrace is either FS, HS or unrelated, which enabled us to write the pedigree of each individual within each landrace. Then, we used a restricted maximum likelihood procedure, implemented in ASReml v1.1 (Gilmour et al. 2009) to fit an “animal model” (Wilson-son et al. 2010) with a fixed effect for the replicate and two random effects for the respective additive and dominance genetic effects:

$$y = X\beta + Z_1a + Z_2d + e,$$

where y is the vector of individual phenotypic values, β is the vector of fixed effects (intercept and replicate effect), a is the vector of individual additive genetic values and d is the vector of individual dominance genetic values. X, Z₁ and Z₂ are the design matrices associated with the fixed effect and the two random effects. e is the vector of residual errors. a, d and e are assumed to be normally distributed with means of zero, and variances of σ²A, σ²D and σ²E, respectively. We assumed no maternal effects. For each components of variance (additive, dominance and residual), we calculated a standard deviation using ASReml. We considered that a variance (additive or dominance) was significant at 95% if its confidence interval, estimated as twice (i.e. 1.96) their standard deviation, excluded zero. Finally, for each trait and each landrace, we used the estimates of the additive genetic variance (σ²A), the dominance genetic variance (σ²D) and residual variance (σ²E) to quantify the narrow sense heritability (h²) as h² = σ²A / (σ²A + σ²D + σ²E). The ratio a² = σ²A / (σ²A + σ²D)
was calculated to quantify the weight of the additive variance in the genetic variance.

We used the breeder's equation to predict the expected genetic response *R* of each quantitative trait to selection, using the estimate of the trait's narrow-sense heritability, *h*², and the selection differential measured for this trait in the farmers' choice experiment *S*. We used the following equation:

$$R = 1/2 * h^2 * S \text{ (Gallais 1990).}$$

## Effective population size of landraces

We used the field description data collected during the interviews to estimate the number of ears needed to sow the next generation. As these values were variable among farmers, we simulated the worst scenario, by using the values of the smallest surface, lowest density of plants, and average number of kernels collected on an ear estimated on a subset of 80 ears. Based on the number of female (*N*<sub>f</sub>) and male (*N*<sub>m</sub>) plants used to sow the field for the next generation of maize, we calculated the effective size (*N*<sub>e</sub>) as:

$$1/N_e = 1/(4N_f) + 1/(4N_m) \text{ (Caballero 1994).}$$

## Results

### Survey analysis

The smallholder farmers we interviewed grew maize for both human and animal consumption in a subsistence agriculture agrosystem. Farm animals (including pigs, poultry, lambs and ewes) were fed with maize and, in some cases, geese and ducks were force-fed. The use of maize varied among farmers, but we found no noteworthy difference between the eastern and the western parts of the Pyrenees. Although the name of the dish prepared for human consumption differed between the two regions, the recipes were similar: the “milhas” (in the eastern part) and the “pastet” (in the western part) were made of maize flour cooked in water or milk, generally on the occasion of the farmer killing a pig. This use of maize flour remains a well-known traditional dish nowadays.

The area under maize cultivation was generally small, but variable between farms, from 0.25 to 2 ha (mean 0.60 ha (sd=0.61)). The cultivation techniques were similar in the eastern and western parts except for sowing: in rows in Ariège (eastern part),

in squares in the western part. Generally, sowing was done by hand after tracing, and covering with a plow. Between two to four kernels were sown together in each hole. Density was also variable, between 50 000 plants/ha and 100 000 plants/ ha before thinning. Four farmers said they sowed beans (*Phaseolus vulgaris*) at the same time as maize. After harvest, braids were made with husked leaves, and ears were dried either in front of or inside the house.

Table 1 summarizes the information collected during our survey. On average, a farmer cultivated 1.4 cultivars. Most of them cultivated one cultivar (9 occurrences), used for both human and animal consumption. In the eastern part of the Pyrenees, about half of the farmers grew a second cultivar (4 occurrences with two cultivars and one with three), generally a white cultivar with small kernels, used for the force-feeding of geese.

As already described in Beigbeder and Carraretto (2018), our survey confirmed that seed selection for the next generation was performed only on ear morphology, without any selection on plants in the field. The selection was performed at different times: at stripping, during shelling, or at the end of the winter in the house. The kernels chosen as seeds for the next generation were shelled carefully, often by old people. One farmer told us that old people selected on seed size. All farmers declared that they eliminated kernels from the top and the bottom of the ears.

We also identified variable seed exchange practices. Some farmers did not exchange seeds (3/9), others declared that they used to exchange seeds within the village only, given that the cultivar was the same inside the village (4/9). Finally, two farmers used to exchange seeds with neighboring villages or with a foreign village while travelling (Table 1).

### Morphological and agronomical variation among the four landraces

A previous study (Gouesnard et al. 1997) showed that the landraces from the eastern group tend to flower later and have a larger number of rows per ear compared to landraces from the western group (Suppl. Fig. 1). In accordance with this result, the data collected for this study (on 150 ears for each of four representative landraces), showed that the two landraces from the eastern part of the Pyrenees (PPS68 and PPS70) indeed flowered later (respectively 748.5 and
727.5 degree days, in the 2018 experiment) and had a higher row number (12 to 14) than the two populations originating from the western part (PPS653 and

PPS1052) (respectively 698 and 726.1 degree days, 8 to 10 rows) (Fig. 2). All four landraces were differentiated for the predicted kernel number, which is

<figure><img src="image_3.png" /><figcaption>**Fig. 2** Boxplot representing mean values and variation of nine traits related to ear morphology and male flowering date for four landraces (PPS1052 and PPS653 in W-SWF, PPS68 and PPS70 in E-SWF). The traits are: ear length (E_length in mm), ear diameter (E_diam in mm), ear conicity (conicity), number of rows composing an ear (N_row), number of kernels per row (NKR), height of a kernel (K_height in mm), ear volume (vol-</figcaption></figure>

ume), predicted number of kernels on the ear (K_number), predicted weight of kernels produced on an ear (K_weight), top of ear without kernel (ear_top in mm), male flowering date (DTA in degree days base 6). n = 150 ears for each trait and each landrace. Different letters on the top of the boxplot indicates two populations with significantly different means
an important component of yield. Kernels of the two eastern landraces tended to be smaller, and the kernel weight per ear was heavier. No difference was observed for conicity and ear diameter (Fig. 2).

The first axis of the PCA on the 10 morphological traits explained 45.8% of variation (Fig. 3). Kernels weight (K_weight) and correlated traits (positively, as K_number, NKR, E_length and E_diam, or negatively, as conicity, Fig. 3C) contributed mostly to this first axis. Considering the two first axes, we observed that the four landraces were distributed along the diagonal, but there was large overlaps between the ellipses representing 70% of the variation within the landraces. Yet, the overlap between ellipses was smaller for landraces from a different region (western part versus eastern part). This distribution shows that the landraces collected in the eastern part of the Pyrenees had more kernels per row (NKR) and per ear (K_number) and smaller kernels (K_height) compared to landraces sampled in the western part of the Pyrenees. The correlations observed among traits (Fig. 3C) highlighted that productive ears generally were long ears. It also showed that if farmers select ears based on their morphology (E_length, volume, N_row, NKR), they should have a positive response on yield (predicted by K_weight).

The additive, dominance and residual components of variance estimated for each trait and each landrace are given in Suppl Table 1. We detected significant additive genetic variance for 15 trait x landrace combinations out of 40. We found little evidence for an influence of dominance variance, it was significant only for ear_top in the landrace PPS68 and the ratios of additive variance on genetic variance ($a^2$) were generally high (> 0.5 for 29 trait x landrace combinations) and highly variable, even among landraces for a given trait (Table 3). Overall, the traits with the lowest additive genetic variance were NRK, K_number, K_height, and ear_top, whereas E_diam, E_length and volume of the ear showed larger value of additive genetic variance. Narrow sense heritabilities ranged from 0.00 to 0.76 (Table 3). On average over the four landraces, the highest heritability was found for traits describing ear morphology (0.57 for E_diam, 0.47 for E_length and 0.53 for the volume of the ear) with the exception of ear_top (heritability of 0.07). We compared the genetic variances ($\sigma_A^2 + \sigma_D^2$) among landraces for each trait by ranking the landraces from the most genetically diverse to the least genetically

diverse. We observed that the ranking was consistent for all traits with the lowest genetic variance always found in landrace PPS68.

## Farmers' selection practices

At the beginning of the interview, we showed the farmers a few representative ears of our four landraces and asked them to indicate which ears were morphologically closer to those they had grown before. We detected no clear influence of the geographic origin of the landraces (W-SWF vs E-SWF) on the choice of the farmers (Table 1). PPS1052 (W-SWF) was the most frequently chosen landrace (by one farmer located in the Eastern part and two from the Western part).

Nine farmers carried out the ear selection experiment on one or several landraces, which added up to a total of 17 replicates with different farmer x landrace combinations (Table 2). For each replicate, we estimated the standardized differentials of selection for 10 traits describing ear morphology. As shown in Table 4, the standardized differentials of selection were generally high. Ear length and ear volume were the traits with the most significant and highest standardized selection differentials, which suggests that these traits guided farmers' selection. The differentials of selection were also very high and positive for the number of kernels per ear and the weight of kernels per ear. Other traits with high and positive differentials of selection were the number of kernels per row and the ear diameter. For the ear-top, the differentials of selection were negative, which means that the farmers selected fully filled ears. Low differentials of selection were found for the number of rows, ear's conicity and kernel height. Selection was generally based on several traits, except for Campan1 with the white landrace PPS653, who selected based on two traits only (the full filling of the ears and the ear's volume).

We performed an ANOVA on standardized selection differentials to test whether there were significant differences between geographic regions (West or East), among farmers within a geographic region and among landraces. We detected no significant difference between geographic regions (Suppl Table 2), nor among farmers except for ear diameter for which the farmer living in Oursbellile selected primarily on this criterion for any landrace he evaluated. The
**Fig. 3** Principal component analysis performed on 10 morphological traits collected on maize ears (n=150) for four Pyrenean landraces. The positions of the means of the 150 ears are indicated with the suffix T0, along with the means of the 15 selected ears (suffix from T1 to T9 referred to the farmer who selected the ears). A: position of the 150 ears X 4 landraces on the two first axes (each color represents one landrace, see legend); B: PCA details for each landrace, C: position of the 10 morphological traits on the correlation circle obtained on axes 1 and 2

<figure><img src="image_4.png" /></figure>
<table><caption>**Table 3** Narrow sense heritability estimates ($h^2$) and ratio of additive variance on genetic variance ($a^2$) for the ten ear morphological traits and the four landraces</caption><thead><tr><th>Trait</th><th>Description of trait</th><th>h<sup>2</sup>_PPS68</th><th>h<sup>2</sup>_PPS70</th><th>h<sup>2</sup>_PPS653</th><th>h<sup>2</sup>_PPS1052</th><th>a<sup>2</sup>_PPS68</th><th>a<sup>2</sup>_PPS70</th><th>a<sup>2</sup>_PPS653</th><th>a<sup>2</sup>_PPS1052</th></tr></thead><tbody><tr><td>Conicity</td><td>Conicity</td><td>0.20</td><td><strong>0.32</strong></td><td><strong>0.32</strong></td><td><strong>0.42</strong></td><td>0.4</td><td><strong>1</strong></td><td><strong>1</strong></td><td><strong>1</strong></td></tr><tr><td>E_diam</td><td>Ear diameter</td><td><strong>0.47</strong></td><td>0.53</td><td>0.54</td><td><strong>0.73</strong></td><td><strong>1</strong></td><td>0.63</td><td>0.54</td><td><strong>1</strong></td></tr><tr><td>E_length</td><td>Ear length</td><td>0.19</td><td><strong>0.54</strong></td><td>0.49</td><td><strong>0.68</strong></td><td>0.57</td><td><strong>1</strong></td><td>0.69</td><td><strong>1</strong></td></tr><tr><td>ear_top</td><td>Top of the ear without kernel</td><td>0.00</td><td>0.15</td><td>0.04</td><td>0.11</td><td>0</td><td>0.37</td><td>0.69</td><td>0.39</td></tr><tr><td>K_height</td><td>Height of the kernel</td><td><strong>0.32</strong></td><td>0.45</td><td>0.43</td><td>0.33</td><td><strong>1</strong></td><td>0.57</td><td>0.77</td><td>0.77</td></tr><tr><td>K_number</td><td>Number of kernel per ear</td><td>0.07</td><td>0.35</td><td>0.20</td><td><strong>0.50</strong></td><td>0.17</td><td>0.88</td><td>0.41</td><td><strong>1</strong></td></tr><tr><td>K_weight</td><td>Weigth of kernel per ear</td><td><strong>0.23</strong></td><td>0.54</td><td>0.14</td><td><strong>0.53</strong></td><td><strong>1</strong></td><td>1</td><td>0.22</td><td><strong>1</strong></td></tr><tr><td>N_row</td><td>Number of row</td><td>0.15</td><td>0.30</td><td>0.34</td><td><strong>0.53</strong></td><td>0.25</td><td>0.46</td><td>0.89</td><td><strong>1</strong></td></tr><tr><td>NKR</td><td>Number of kernel per row</td><td>0.03</td><td>0.45</td><td>0.23</td><td><strong>0.35</strong></td><td>0.06</td><td>0.86</td><td>0.33</td><td><strong>1</strong></td></tr><tr><td>Volume</td><td>Volume of the ear</td><td><strong>0.31</strong></td><td>0.61</td><td>0.44</td><td><strong>0.76</strong></td><td><strong>1</strong></td><td>0.93</td><td>0.44</td><td><strong>1</strong></td></tr></tbody></table>

In bold, $a^2$ and $h^2$ values greater than 2 times their standard deviation

differentials of selection are represented visually in Fig. 3A, B: for each landrace, we represented the mean of the 150 ears (suffix T0) as well as the mean of the 15 ears selected by each farmer (suffix T1 to T9). The direction of the vectors T0-T>0 represents the traits driving selection. These vectors generally point towards selection for larger ear diameter and volume for all four landraces (Fig. 3). The length of the vectors represents the strength of selection and tended to be larger for PPS68 and PPS70 (both E-SWF) than for the two other landraces for which selection differentials were more variable from one farmer to the other. Yet, selection was sometimes remarkably consistent across farmers, for example for the landrace PPS1052, where three farmers out of seven selected the exact same 15 ears.

### Expected response to selection

In our ear selection experiment, we provided the farmers with 150 ears per landrace and they selected 15, giving a selection differential of 15/150 = 10%. We combined this selection differential with our estimates of narrow sense heritability to predict the univariate expected response to selection (R). The expected response in percent of the original value varied from -3.3% to 13.7% (Suppl Table 3). These values were negative only for conicity and K_height.

R was also positive for N_row (on average 1.1%), for NKR (1.7%), for E_diam (1.6%) and for E_length (3.4%) (Suppl Table 3). For predicted traits (K_num-ber and K_weight), R was also positive and higher (2.8% and 4.6% on average, respectively). For the multiplicative trait, such as ear's volume, R was on average equal to 7.3%. The farmers' selection we measured should therefore on average tend to increase the proportion of cylindrical ears with smaller kernels and higher kernel weight. Yet, results varied among landraces and farmers, as shown in Fig. 4. The variability among landraces is largely explained by differences in the amount of additive genetic variance in their traits, for example R values were slightly higher for the landrace PPS1052, where most traits had high additive genetic variance.

### Landraces expected effective population size

Based on the interviews, we estimated that 53 ears were needed each year to sow 0.25 ha of maize at a density of 50 000 plants/ha, considering that 220 kernels were kept on each ear (out of 330 kernels in total on average over the four landraces). These figures are those of the worst situation (small surface, low density). A population with 53 females (the ears used for sowing) and a maximum of 53*220 males (the maximum number of pollen donors if
<table><caption>Table 4 Standardized selection differentials estimated on ten ear morphological traits in 17 farmer x landrace combination samples</caption><thead><tr><th>Landrace</th><th>Farmer</th><th>E_length</th><th>E_diam</th><th>conicity</th><th>ear_top</th><th>N_row</th><th>NKR</th><th>K_height</th><th>volume</th><th>K_number</th><th>K_weight</th></tr></thead><tbody><tr><td>PPS1052</td><td>Aleu</td><td>0.702**</td><td>0.762**</td><td>-0.251</td><td>-0.345**</td><td>0.493*</td><td>0.447**</td><td>0.081</td><td>0.908**</td><td>0.600**</td><td>0.757**</td></tr><tr><td>PPS68</td><td>Aleu</td><td>1.097**</td><td>0.465</td><td>-0.389</td><td>-0.296*</td><td>0.440</td><td>0.909**</td><td>0.064</td><td>1.111**</td><td>1.015**</td><td>1.066**</td></tr><tr><td>PPS1052</td><td>Beaucens</td><td>0.513*</td><td>0.544**</td><td>-0.150</td><td>-0.345**</td><td>0.208</td><td>0.684**</td><td>-0.41*</td><td>0.629**</td><td>0.691**</td><td>0.705**</td></tr><tr><td>PPS1052</td><td>Bun</td><td>0.992**</td><td>0.920**</td><td>-0.172</td><td>-0.345**</td><td>0.777**</td><td>0.644**</td><td>0.119</td><td>1.222**</td><td>0.879**</td><td>1.042**</td></tr><tr><td>PPS68</td><td>Bun</td><td>0.816**</td><td>0.408*</td><td>-0.368*</td><td>-0.103</td><td>0.028</td><td>0.931**</td><td>-0.42**</td><td>0.848**</td><td>0.827**</td><td>0.916**</td></tr><tr><td>PPS653</td><td>Campan 1</td><td>0.503</td><td>0.225</td><td>0.0083</td><td>-0.349**</td><td>0.289</td><td>0.408</td><td>0.001</td><td>0.550**</td><td>0.548</td><td>0.608</td></tr><tr><td>PPS68</td><td>Campan 1</td><td>0.856**</td><td>0.879**</td><td>-0.246</td><td>-0.020</td><td>0.604</td><td>0.920**</td><td>-0.34</td><td>1.233**</td><td>1.124**</td><td>1.190**</td></tr><tr><td>PPS653</td><td>Campan 2</td><td>0.624**</td><td>0.057</td><td>-0.324</td><td>-0.349**</td><td>0.093</td><td>0.503*</td><td>0.028</td><td>0.472</td><td>0.536</td><td>0.577</td></tr><tr><td>PPS68</td><td>Campan 2</td><td>0.664**</td><td>0.748**</td><td>0.0592</td><td>-0.280</td><td>0.111</td><td>0.831**</td><td>-0.38*</td><td>0.978**</td><td>0.815**</td><td>0.983**</td></tr><tr><td>PPS1052</td><td>Engomer</td><td>1.358**</td><td>0.864**</td><td>-0.172</td><td>-0.263*</td><td>0.588*</td><td>0.855**</td><td>0.159</td><td>1.441**</td><td>0.983**</td><td>1.187**</td></tr><tr><td>PPS68</td><td>Engomer</td><td>0.856**</td><td>0.599*</td><td>-0.255</td><td>-0.290</td><td>0.111</td><td>1.031**</td><td>-0.44**</td><td>1.022**</td><td>0.967**</td><td>1.089**</td></tr><tr><td>PPS70</td><td>Engomer</td><td>1.191**</td><td>0.878**</td><td>-0.400</td><td>-0.371**</td><td>0.238</td><td>0.803**</td><td>0.329</td><td>1.349**</td><td>0.802**</td><td>1.264**</td></tr><tr><td>PPS1052</td><td>Esquieze Serre</td><td>0.995**</td><td>1.010**</td><td>0.1804</td><td>-0.263*</td><td>0.683*</td><td>0.578**</td><td>0.177</td><td>1.292**</td><td>0.798**</td><td>1.011**</td></tr><tr><td>PPS68</td><td>Esquieze Serre</td><td>1.127**</td><td>0.615*</td><td>-0.508**</td><td>-0.449**</td><td>0.604</td><td>0.942**</td><td>0.092</td><td>1.242**</td><td>1.134**</td><td>1.191**</td></tr><tr><td>PPS1052</td><td>Moulin-Neuf</td><td>1.092**</td><td>0.813**</td><td>-0.178</td><td>-0.345**</td><td>0.398</td><td>0.934**</td><td>-0.16</td><td>1.200**</td><td>0.987**</td><td>1.108**</td></tr><tr><td>PPS1052</td><td>Oursbellile</td><td>1.025**</td><td>1.096**</td><td>-0.109</td><td>-0.307**</td><td>0.872*</td><td>0.776**</td><td>-0.01</td><td>1.382**</td><td>1.043**</td><td>1.190**</td></tr><tr><td>PPS70</td><td>Oursbellile</td><td>1.191**</td><td>0.953**</td><td>-0.122</td><td>-0.031</td><td>0.494</td><td>1.025**</td><td>0.005</td><td>1.429**</td><td>1.111**</td><td>1.335**</td></tr></tbody></table>

Asterisks indicate the tests' significance (* at 5%, ** at 1%) for the 17 tests of ears identified by informant and landrace
<figure><img src="image_5.png" /><figcaption>**Fig. 4** Distribution of the expected response to selection (in percent of the initial values) for four ear traits for each of the four landraces (PPS68 in blue, PPS70 in green, PPS653 in red; PPS1052 in grey)</figcaption></figure>

all the kernels of the 53 ears come from different male plants) has an effective size of 211 (Caballero 1994). If all the kernels of an ear have the same male parent, the effective size is then divided by about 2 (i.e. 106). According to Crossa (1989), an effective size (referred to as sample size in Crossa 1989) over 90 is sufficient to conserve rare alleles at a frequency of 5% with a probability of 0.99 (Crossa 1989), but an effective size higher than 458 is required to conserve rare alleles at a frequency of 1% with a probability of 0.99. With an effective size generally higher than 106, we therefore, expect that the reduction of diversity due to a drift effect during seed regeneration should be quite low, even in the worst case situation (small field).

## Discussion

We studied maize landraces that were cultivated 60 years ago in the southwest of France as well as farmers’ practices. The agriculture of this mountainous region is devoted essentially to livestock farming, and maize hybrids are now only cultivated in lowlands. Since the seventeenth century, farmers in the southwest of France have developed know-how on maize cultivation that is doomed to disappear as landraces are no longer cultivated. We wanted to collect this knowledge and compare it with that of native Mesoamerican farmers described in the literature.

Farmer's management of landraces and comparison with Mesoamerican agrosystems

We analyzed three components of farmers’ management of maize diversity in this region until 1960: cultivar choice, seed exchange, and seed selection and management (Bellon et al. 1997).

### *Cultivar choice*

Consistent with Beigbeder and Carraretto (2018), our study indicated that the average number of cultivars grown by each farmer was close to one (Table 1). In the survey, some farmers who were feeding geese said they grew a white cultivar with small kernels (Millette). Besides this exception, the same cultivar was used for human consumption and animal feeding, and neither the use nor the characteristics of the landraces such as precocity influenced the choice of the cultivars. This differs from the situation in Mexico, the center of origin of maize, where the farmers grew several cultivars: more than two cultivars per season (Louette and Smale 2000), 2.5 cultivars on average (Moreno et al. 2006) or more than one cultivar (Rice et al. 1998). In Mexico, farmers identified the cultivar by the color of the kernels, and this was similar in the Pyrenees.
## Seed exchanges

As described by the farmers we interviewed, seed exchanges were rare and generally restricted to other farmers within the village or within the family. This contrasts with the practices that have been reported for landraces in Mexico (Louette et al. 1997). In Cuzalapa, farmers' own harvests represented 45% of the maize area in the study zone, whereas 40% were planted using seeds from other Cuzalapa farmers and 15% with foreign introductions. Bellon et al. (2011) reported that 90% of all the seed lots were obtained within 10 km of the community in Central Mexico. In our study, none of the farmers mentioned crop losses forcing them to obtain seeds from another farm. The rare exchanges mostly aimed at trying to increase productivity or simply out of curiosity.

## Seed selection and management

All the farmers told us that their selection was based only on the ears, and never on the whole plant morphology in the field. Beigbeder and Carraretto (2018) had previously reported the same practice in this region. In Mexico, similar practices were reported (Louette and Smale 1998; Moreno et al. 2006; Rice et al. 1998). In our survey, two farmers (Moulin Neuf and St Jean Pied de Port) said that the selection was done by the head of the farm, which was similar to the situation reported for Mexico (Rice et al. (1998), Sierra de la Marta).

All farmers used to remove the top and the bottom of the ear when collecting seeds for the next generation, as mentioned in Beigbeder and Carraretto (2018). This practice was also mentioned in Mexico and Guatemala (Johannessen 1982; Louette and Smale 2000; Perales et al. 2003). It therefore seems to be a common practice, probably due to the fact that kernels at the top of the ear are smaller and often attacked by insects, while kernels at the bottom of the ear are more humid and often misshaped.

The old farmer of Moulin Neuf cultivated white cultivars. When he did the test on the white landrace Aston, he carefully eliminated the multicolored ears. Indeed, Pyrenean farmers did not cultivate multi-colored cultivars (Beigbeder and Carraretto 2018). This was also observed by Louette and Smale (1998) in a farmer choice experiment using a black kernels cultivar contaminated by a white one. This practice

could further contribute to the limited gene flow among cultivars.

In conclusion, the Pyrenean farmers carried out selection in a similar way to Mesoamerican farmers, using similar criteria based on ear morphology only (voluminous, healthy, single-colored, and full ears). However, there were notable differences in the number of cultivars grown by a single farmer, in the practices of seed exchange and in the way flour was extracted, which was done by mills in the Pyrenees (dry milling), and by dry milling and by lime-cooking in American countries where tortillas are eaten.

Ears selection experiment and expected response to selection

To obtain genetic progress through selection on a phenotypic trait (R), two genetic parameters are needed: a high differential of selection (S), and a large narrow sense heritability ($h^2$).

## Heritability of the traits

Narrow sense heritabilities (estimated at the individual level) were variable according to the trait and the landrace (from 0.26 for the number of kernels per row to 0.53 for ear volume). They were slightly higher for ear diameter and volume than for the other traits, in particular yield components (including the number of rows). In their literature review, Hallauer and Miranda (1981) have summarized the published estimates of narrow sense heritability in maize and delimited three classes of traits according to their heritability: $h^2<0.3$ for yield, $0.3<h^2<0.5$ for ear length and diameter, $0.5<h^2<0.7$ for kernel-row number. Similar results were found for two Spanish landraces ((Malvar et al. 1996), $h^2$=0.36 and 0.21 for ear length; $h^2$=0.63 and 0.80 for row number per ear). Our results were within the range of literature estimates, except for the number of rows that had a rather low heritability (except for PPS1052) (Table 3). Note that the variance components estimated had large standard deviations, due to the limited size of our crossing design. Moreover, the heritabilities we estimated here were measured in a field in 2018 where we homogenized the environment as much as possible. As a result, even though we can assume that the farmers also tend to avoid variability in environmental conditions across their fields, our
experimental design may minimize the environmen-
tal variance and thus lead to an overestimation of the
heritabilities compared to the heritabilities that would
occur in the farmers' field. In addition, we cannot
exclude common environmental effects because full-
sib progenies were cultivated in the same plot. This
could also have inflated heritability estimates.

*Farmers' selection criteria and differentials of selection*

All farmers eliminated unhealthy ears in their ear
choice, as previously mentioned by Soleri et al.
(2000). Our selection experiment revealed that the
two main traits under selection were ear length and
ear volume. This is in agreement with the literature.
Johannessen (1982), in Guatemala, mentioned that
2/3 of the farmers selected on ear length. For Sol-
eri (1999), in Mexico, it was also a main criterion.
Louette and Smale (1998) cited as main criteria: ear
weight, ear length, length of the ear presenting ker-
nels, total number of kernels, and kernel filling.
Serpolay-Besson et al. (2014) studied the evolution
of two open-pollinated cultivars in farm in Italy and
France during two generations. They report that farm-
ers applied strong selection pressures on ear quanti-
tative traits and always selected significantly longer
ears. We found a negative differential of selection
for the upper part of the ear without kernel (ear_top)
showing that farmers selected well-filled ears.

The research in Mesoamerica highlighted con-
siderable variability of the selection criteria among
farmers and even a lack of consistency for a given
farmer (Louette and Smale 1998; Rice et al. 1998;
Soleri et al. 2000). Our results also underline this
variability (no farmer effect on standardized selec-
tion differentials). It should nevertheless be pointed
out that our sample size may have been too small to
detect significant differences among farmers.

***Expected response to selection for the different traits***

Because farmers in our survey only selected on ears'
morphology, the selection method resembles mass
selection restricted to female plants. Its expected effi-
ciency depends on both the heritability of the trait
and the selection differential. The univariate pre-
dicted responses after one generation of selection
were positive for most traits (on average over the 17

tests, R=3.4% for E_length), and remarkably high
for the traits most closely related to yield, such as
the kernel weight of an ear (R=4.6%) (Fig. 4, Suppl
Table 3). This implies that old farmers were able to
integrate the visual characteristics of ear morphology
in order to choose ears with a larger number of grains
and having a higher grain weight.

Our experimental selection design simulated a selection rate of 10% (15 ears picked out of 150). This was useful to have a manageable test that did not take too long, but a selection rate of 1% would have been more realistic, as practiced by Louette and Smale (1998) in their selection test. Indeed, assuming a yield of 1500 kg/hectare (average yield in France in 1950, Derieux et al. (1987) and a weight of 1000 kernels of 0.373 kg (as found in our experiment in 2018), we expect that a farmer will harvest around 4 millions of kernels per hectare. With a sowing density of 50.000 plants/hectare, the selection rate can reach 1.25%. Simulating a selection intensity of 1% would therefore have been more realistic. It has been shown that the selection intensity shifts from 1.755 to 2.665 when the selection rate changes from 10 to 1% (Gallais 1990). Therefore, we anticipate the expected response to selection to be around 1.5 times higher with a selection intensity of 1% than what we estimated here with a selection rate of 10%. It is therefore likely that we underestimated the expected change of the traits after one generation of selection due to the limited selection intensity of our experimental design. Yet, other factors may have led to an overestimation. First, heritabilities may have been overestimated in our experimental design, as described above. Second, the expected response *R* was calculated on a single generation, without taking into account potential genotype * environment interactions that could affect the phenotype of the following generation. Strong GxE interactions could also alter the heritabilities of the traits (Pour-Aboughadareh et al. 2022). These two effects could reduce the expected value of *R*.

A literature review showed that mass selection is
effective in modifying highly heritable traits in maize
(Hallauer and Miranda 1981). Divergent popula-
tions for ear length or protein and oil content were,
for example, created in an experimental station using
mass selection (Dudley and Lambert 1992; Lopez-
Reynoso and Hallauer 1998). For yield, Hallauer and
Miranda (1981 p 214) summarized the response to
selection by mass selection for different populations,
number of generations and selection rates: the response to selection is overall positive, but remains low (a few percents). Experiments performed on farms or with farmers, such as ours, are scarce but we found two examples in the literature. In the first one, a selection experiment similar to ours, in which farming households selected 10% from a sample of 100 ears from their own maize population or another population from their community found a low expected response to selection (Soleri 1999). The realized response to selection was assessed in a field trial for three successive generations of farmers’ selection, along with a control in which seeds were selected randomly to form the following generation. This field experiment was realized for populations from six households and 14 traits were measured, related to plant or ear morphology and phenology. A significant change was observed in the selected fields (compared to the random fields) for only 11 trait*population combinations out of 252 in total. The main reason for this limited evidence of phenotypic change induced by selection was the low heritability of the traits used for farmers’ selection in these populations. The second example of on-farm selection experiment is the participatory maize breeding program that was conducted by stratified mass selection in Portugal (Alves et al. 2018). In this study, responses to selection for grain yield were significant in only one of the two populations. Finally, a third example that illustrates the strength of farmers’ selection practices is the study of McLean-Rodriguez et al. (2021), who compared selection signatures in maize landraces across 50 years of in-situ and ex-situ conservation from the state of Morelos, Mexico. Most outliers identified by two detection methods coincided with QTLs known to be associated with ear, kernel and yield-related traits. Over the following generations, genotype * environment interactions reduced the expected genetic progress from one year to the next. Finally, considering the evolution of grain yield over years from 1900 to 1930 in USA, Duvick (2005) reported no clear response to selection of open-pollinated cultivars with a yield under 20q/ha.

As a conclusion, our selection experiment proved that the farmers were able to visually identify the most productive ears. They exerted a high selection differential on ear length and ear volume and the large heritabilities of these traits led us to predict a strong response to mass selection. When we compare

the results of our experiment with the literature, it is therefore striking to see that mass selection seemed promising, even if we keep in mind that the expected responses we estimated may change across generations and environments. This contrast calls for further research of the genetic progress in landraces or open-pollinated populations of maize, in realistic field conditions, and performed across repeated generations and environments.

### Comparison of selection differentials between the 2 genetic groups (E-SWF and W-SWF)

Diaw et al. (2021) showed that Pyrenean landraces in the South-West of France were structured in two genetic groups (E-SWF and W-SWF, Fst=2.6% estimated on 23,412 genome-wide SNPs, p-value < 10⁻¹⁵). Although this low level of differentiation suggests a low effect of genetic drift and a potential gene flow between the two regions, we were interested in the role of adaptation to farmers’ practices. Although the number of informants was very limited (only a few farmers are still alive to testify to their practices) and the analysis was based on memories of a period of more than 50 years, our study was not able to detect any significant difference in the selection differentials between the two areas (E-SWF vs W-SWF). Our survey allowed documenting the effective population sizes of the fields and thereby the potential strength of the genetic drift. We estimated that the number of ears necessary for sowing the next generation was 53 for an area of 0.25 ha. Based on analytical calculation from Crossa (1989), we showed that this can be considered large enough to avoid significant drift effects.

### The transition to hybrids

The transition to hybrids occurred rapidly, between 1950 and 1960 (Beigbeder and Carraretto 2018). Such a rapid abandonment of the landraces is surprising and suggests that the advantages of hybrids in terms of yield, possibilities of genetic improvement, and/or cultivation practices were obvious. In the 1960s, some farmers stopped maize cultivation, notably because of the rural exodus, and other farmers began hybrid cultivation. They were very aware of the differences between hybrids and their local cultivars, as reported by Beigbeder and Carraretto (2018). Hybrid
cultivars flower later, their husks are easily detach-
able, the plants cope with high density better with flat
roots and the kernel is less round and has less flour.
More importantly, the productivity gain of hybrids
compared to landraces is clear (Duvick 2005). Yet,
some of the farmers, such as old farmers or those who
fed geese, kept cultivating local cultivars for longer.
The current situation is that the culture of local maize
populations in the Pyrenees has been abandoned, with
the exception of very few people who still cultivate
landraces in their gardens or on organic farms. How-
ever, a few years ago, associations of farmers culti-
vating maize landraces started appearing in France
and Italy (Fenzi and Couix 2021). Landraces are
also conserved ex-situ, and the results presented here
document the on-farm conservation practices of the
past and the selection criteria associated with these
accessions. The value of these maize landraces for
modern breeding is high, notably for their precocity
and digestibility in silage (Bertoia et al. 2006; Bertoia
2001; Incognito 2016; Oliveira et al. 2020). These
traits, along with other traits related to grain quality
(Landoni et al. 2024) or drought tolerance (Barbosa
et al. 2021; Gouesnard et al. 2016; Maafi et al. 2021),
will have to be evaluated and could be useful to enrich
elite cultivars. Haplodiploidization makes it possible
to rapidly extract lines from the populations that can
then be used in selection (Strigens et al. 2013). More
recently, with genomic selection, methods for iden-
tifying alleles of interest in genetic resources have
flourished and pre-breeding schemes have been pro-
posed (Allier et al. 2020a, b). These methods could
help to value the genetic diversity available in old
maize landraces from the Pyrenees.

Conclusion

The objectives of our study were (1) to document
know-how on maize landrace cultivation and in
particular the management of seeds, (2) to estimate
selection differentials exercised by farmers on the
ears and to estimate the narrow sense heritability
of the traits under selection in order to determine
whether traditional farming practices could lead to
on-farm genetic improvement of the landraces, (3)
to compare the practices in the Eastern and West-
ern part of the Pyrenees and determine whether
these practices could contribute to the genetic and

morphological differentiation observed between the
two geographic zones.

Our results showed that mass selection on ears,
as exerted by these farmers, is likely to have led to
genetic progress, even if our experimental design
may overestimate the response to selection. Nev-
ertheless, landraces were abandoned in favor of
hybrids for which performance and genetic pro-
gress were higher. The criteria for choosing the
ears were variable among farmers but did not dif-
fer significantly between areas. From our study, it
seems that genetic groups in the Pyrenees evolved
mainly through low neutral divergence and genetic
drift, but that the size of maize fields remained
large enough to maintain a high level of diversity.
We found that, in just over two centuries, the farm-
ers of the Pyrenees developed a specific know-how
for maize cultivation, integrating maize farming
into their culture as shown by the list of local words
describing the different parts of maize and related
actions (Beigbeder and Carraretto 2018). We inter-
viewed the last survivors who knew the 60 s maize
landraces and the farming practices associated with
them. This knowledge is invaluable to better know
and use these maize landraces that are now con-
served ex-situ.

**Acknowledgements** This research was supported by the pro-
ject Amaizing ANR-10-BTBR-01 and by West African Agri-
cultural Productivity Program (WAAPP/ISRA). We are grate-
ful to Dr Ndiaga Cissé (http://www.ceraas.org/, ISRA, Thies,
Senegal) for the grant, to Jean Beigbeder (Promais) for discus-
sion, to Maryse Carraretto for her aid for the survey, to Anne
Zanetto and Christophe Raynaud (maize biological resources
center of Montpellier) for the supply of landraces seeds, to
Romain Chapuis, Serge Malavieille and their colleagues for
their help of the experiment at UE Diascope INRAE (Mau-
guio). This work was supported by the CIRAD—UMR AGAP
HPC Data Center of the South Green Bioinformatics platform
(http://www.southgreen.fr/).

**Author contributions** BG prepared and investigated the survey, interpreted the data, wrote the manuscript; YD investigated the survey and corrected the manuscript, LG helped in the interpretation of data and corrected the manuscript, JR and JD corrected the manuscript and supervised the study.

**Funding** This research was supported by the project Amaiz-
ing ANR-10-BTBR-01 and by West African Agricultural Pro-
ductivity Program (WAAPP/ISRA) for the grant of Yacine
Diaw.

**Data availability** All data collected during this study have
been deposited in an archive (a public dataset: https://doi.org/
10.57745/6GJFKY). Landrace seeds are available from BRC GAMeT in Montpellier (crb-gamet@cirad.fr).

## Declarations

**Conflict of interest** The authors declare no competing interests.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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