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\DOI{10.5802/crbiol.164}
\datereceived{2024-08-14}
\daterevised{2024-09-19}
\dateaccepted{2024-09-23}
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\begin{document}

\begin{noXML}

\CDRsetmeta{articletype}{review}

\title{The milpa, from Mesoamerica to present days,\unskip\break a
multicropping traditional agricultural system serving agroecology}

\alttitle{La milpa, de la M\'{e}so-Am\'{e}rique \`{a} nos jours, un
syst\`{e}me agricole traditionnel au service de l'agro\'{e}cologie}

\author{\firstname{Noa} \lastname{Vazeux-Blumental}\CDRorcid{0009-0001-1971-1019}}
\address{Universit\'{e} Paris-Saclay, INRAE, CNRS, AgroParisTech, GQE-Le Moulon, 91190, 
Gif-sur-Yvette, France}

\author{\firstname{Domenica} \lastname{Manicacci}\CDRorcid{0000-0002-6779-113X}}
\addressSameAs{1}{Universit\'{e} Paris-Saclay, INRAE, CNRS, AgroParisTech, GQE-Le Moulon, 91190, 
Gif-sur-Yvette, France}

\author{\firstname{Maud} \lastname{Tenaillon}\CDRorcid{0000-0002-0867-3678}\IsCorresp}
\addressSameAs{1}{Universit\'{e} Paris-Saclay, INRAE, CNRS, AgroParisTech, GQE-Le Moulon, 91190, 
Gif-sur-Yvette, France}
\email[M. Tenaillon]{maud.tenaillon@inrae.fr}

\shortrunauthors

\keywords{\kwd{Domestication}
\kwd{Crop diffusion}
\kwd{Traditional farming}
\kwd{Maize-bean intercropping}
\kwd{Plant--plant interactions}
\kwd{Agricultural practices}}

\altkeywords{\kwd{Evolution des plantes cultiv\'{e}es}
\kwd{Polyculture}
\kwd{Interactions plante-plante}
\kwd{Pratiques agricoles}
\kwd{Association ma\"{i}s-haricot}}

\thanks{SPS through the Doctoral school ``Sciences du V\'{e}g\'{e}tal
ED567'', European Union's Horizon 2020 research and innovation
programme under grant agreement No. 862862 (INCREASE Project).}

\begin{abstract} 
The association of maize (\textit{Zea mays} ssp. \textit{mays}), common
bean (\textit{Phaseolus vulgaris}), and squash (\textit{Cucurbita}
ssp.) within the milpa represents the most emblematic multi-cropping
subsistence system of Mesoamerica. This system was likely established
in the Guerrero-Jalisco area in southwestern Central Mexico shortly
after---or perhaps even before---the domestication of the three
taxa. Its success relies on several factors: complementarity of
nutritional intakes, resilience to biotic and abiotic constraints, and
the mobilization of positive interactions between the three taxa,
enabling the system to be productive under input-limited conditions.
Higher yields compared to sole-cropping have frequently been described
and attributed to the complementarity between the aerial and root
systems of the different taxa of the milpa, as well as to direct and
indirect facilitation processes involving root exudates, bacterial
symbioses, and the mycorrhizal network. In Europe, while practiced
until recently, the milpa has gradually been abandoned in favor of
maize sole-cropping, except in some isolated regions (such as
Transylvania) where this traditional agricultural system has persisted.
The question of whether varieties of the three taxa used in
multi-cropping systems were co-introduced to Europe at the time of the
discovery of the Americas, as opposed to being re-associated later in
Europe, remains open. It is important to note that maize usage
differed: maize of flint type is coarsely ground for the preparation of
polenta in Europe, while in Mesoamerica, tropical varieties are soaked
in alkaline solution to improve nutritional quality before being finely
ground to make tortilla dough. Recently, maize-bean intercropping has
been reintroduced into modern European agricultural systems. However,
the use of elite varieties and chemical inputs in conventional conducts
prevents full exploitation of positive interactions between species. We
argue here that milpa has an important role to play in the
agroecological transition. In this context, we propose avenues for the
selection of varieties that promote synergies between species and
discuss the constraints linked to its mechanization.
\end{abstract}

\begin{altabstract} 
L'association du ma\"{i}s (\textit{Zea mays} ssp. \textit{mays}),  du
haricot commun (\textit{Phaseolus vulgaris}) et de la courge 
(\textit{Cucurbita} ssp.) au sein de la milpa repr\'{e}sente le
syst\`{e}me de polyculture vivri\`{e}re le plus embl\'{e}matique de la
M\'{e}so-Am\'{e}rique. Ce syst\`{e}me a \'{e}t\'{e} probablement
\'{e}tabli dans la r\'{e}gion du Guerrero-Jalisco, dans le centre
sud-ouest du Mexique, peu apr\`{e}s --- voire m\^{e}me avant --- la
domestication des trois taxons qui le composent. Son succ\`{e}s repose
sur plusieurs facteurs : des apports nutritionnels compl\'{e}mentaires,
sa r\'{e}silience face aux contraintes biotiques et abiotiques, ainsi
que la mobilisation d'interactions positives entre les trois taxons,
permettant au syst\`{e}me d'\^{e}tre productif dans des conditions
limitantes en intrants. Ainsi, un rendement plus \'{e}lev\'{e} par
rapport aux cultures monosp\'{e}cifiques a fr\'{e}quemment \'{e}t\'{e}
d\'{e}crit, attribu\'{e} aux compl\'{e}mentarit\'{e}s entre les
syst\`{e}mes a\'{e}riens et racinaires des diff\'{e}rentes esp\`{e}ces
ainsi qu'aux processus de facilitation directs et indirectes impliquant
des exsudats racinaires, des symbioses bact\'{e}riennes et le
r\'{e}seau mycorhizien. En Europe, la milpa a \'{e}t\'{e} pratiqu\'{e}e
jusqu'\`{a} r\'{e}cemment puis peu \`{a} peu abandonn\'{e}e au profit
la culture du ma\"{i}s seul, \`{a} l'exception de quelques r\'{e}gions
isol\'{e}es comme la Transylvanie o\`{u} ce syst\`{e}me d'agriculture
traditionnel a perdur\'{e}. La question de la co-introduction en Europe
de vari\'{e}t\'{e}s des trois taxons d\'{e}j\`{a} utilis\'{e}es en
polyculture au moment de la d\'{e}couverte des Am\'{e}riques, par
opposition \`{a} leur r\'{e}association ult\'{e}rieure en Europe, reste
ouverte. Il est cependant important de noter que les usages du ma\"{i}s
ont diff\'{e}r\'{e} : le ma\"{i}s de type corn\'{e} est
grossi\`{e}rement moulu pour la pr\'{e}paration de la polenta en
Europe, tandis qu'en M\'{e}so-Am\'{e}rique, les vari\'{e}t\'{e}s
tropicales sont tremp\'{e}es dans une solution alcaline pour
am\'{e}liorer leur qualit\'{e} nutritionnelle avant d'\^{e}tre finement
moulues pour confectionner des tortillas. R\'{e}cemment, la culture
associ\'{e}e ma\"{i}s-haricot a \'{e}t\'{e} r\'{e}introduite dans les
syst\`{e}mes agricoles modernes en Europe. L'utilisation de
vari\'{e}t\'{e}s \'{e}lites et d'intrants chimiques dans les pratiques
conventionnelles ne permettent cependant pas de tirer pleinement profit
des interactions positives entre esp\`{e}ces. Nous d\'{e}fendons ici
l'id\'{e}e que la milpa a un r\^{o}le important \`{a} jouer dans la
transition agro\'{e}cologique. Nous proposons dans ce contexte des
pistes de r\'{e}flexions pour la s\'{e}lection de vari\'{e}t\'{e}s
favorisant les synergies entre les esp\`{e}ces et nous discutons des
contraintes li\'{e}es \`{a} sa m\'{e}canisation.
\end{altabstract}

\maketitle

\end{noXML}

\twocolumngrid

\section{Mesoamerican agriculture}\label{sec1}
The development of agriculture is arguably the most significant element
of the so-called Neolithic\unskip\break 
package~\cite{Cilingiroglu2005}. This
package includes the Neolithic  Revolution~\cite{Childe1936} with the
domestication of plants and animals, technological advancements, and
the shift to sedentary lifestyles, along with the accompanying
economic, social, and ideological changes that led to the emergence of
complex  societies~\cite{Weisdorf2005}. The transition to agrarian
societies independently arose in different regions across the globe,
recognized as centers of  domestication~\cite{Vavilov1992}. This shift
was likely in part driven by major climate changes at the end of the
Pleistocene, which included a reduction in climate variability, a rise
in atmospheric CO$_{2}$ levels, and an increase in rainfall, all of
which dramatically transformed the Earth's 
environment~\cite{Richersonetal2001}. One well-documented example is
the Mesoamerican domestication center, which refers to the
pre-Columbian cultural region encompassing central Mexico to the
western regions of Honduras, Nicaragua, and Costa 
Rica~\cite{Kirchhoff1943}. Several archaeological discoveries in
Mesoamerica attest to human occupation in the region at the
late-Pleistocene and  early-Holocene~\cite{Chattersetal2014}, with the
earliest evidence found at Chiquihuite cave in the state of Zacatecas
in Mexico dating from the last glacial maximum 
(26,500--19,000~yr~BP)~\cite{Ardeleanetal2020}. Cave excavations in
Mexico indicated that between the sixth and the five millennia BP,
survival was ensured by fishing, megafauna hunting, and plant gathering
activities~\cite{Niederberger1979}. A progressive increase of food
production along with an increased sedentarization between 10,000 and
3000~yr~BP is documented in 
Mesoamerica~\cite{Rosenswig2015,Acostaetal2018}.

Mesoamerica was the cradle of major staple crops, including among other
species maize  (\textit{Zea mays}  ssp.\ 
\textit{mays}~L.)~\cite{Matsuokaetal2002}, multiple bean species 
(\textit{Phaseolus}~spp.)~\cite{Bitocchietal2012}, multiple squash
species 
(\textit{Cucurbita}~spp.)~\cite{Eguiarteetal2018,Barrera-Redondoetal2021}, 
avocado  (\textit{Persea americana}~Mill.)~\cite{Solaresetal2023}, and
chili peppers  (\textit{Capsicum annuum}~L.)~\cite{Kraftetal2014}.
Beyond its role in social and economic development in Mesoamerica,
agriculture was and still is deeply intertwined with cultural diversity
in the region. 

Maize, in particular, was revered as a sacred plant by Mesoamerican
societies, as evidenced by the etymology of maize's wild ancestor in 
Nahuatl---the  language spoken by the Aztecs---``teocintle'' meaning
``sacred maize ear''~\cite{Carrascoetal2022,Haugen2009}. Analyses of
jade caches in Maya temples of Guatemala indicated the central role of
the ``Maize God'' in sacred  rituals~\cite{Estrada-Belli2006}. In
various ethnic groups in Mesoamerica, maize is anthropomorphized and
believed to possess a sacred soul  (Figure~\ref{fig1}). The
significance of maize beliefs among different ethnic groups in
Mesoamerica  (Teenek, Nahua, Xi'iuy and Totonac) was also 
highlighted~\cite{Carrascoetal2022}. Moreover, as a central element of
the local populations' diets, maize has been and continues to be the
subject of\unskip\break 
numerous pilgrimages and prayers to ensure 
successful
harvests~\cite{Sellen2011}.

\begin{figure}
\vspace*{4pt}
\includegraphics{fig01}
\caption{\label{fig1}Milpa mythology. Mural painted by  Fernando Castro
Pacheco in~1971 at the Palacio de Gobierno in M\'{e}rida (Yucat\'{a}n,
Mexico), embodies the Mayan mythology around maize. At its center is a
Mayan man emerging from the fertile ear of maize, held in the hand of
the southern divinity (bacab) as depicted in the  ``Popol Vuh'', the
sacred book of the Maya. Overseeing this scene is the northern bacab,
symbolized by a white hand at the top. Both the western (left) and
eastern (right) bacabs contemplate their creation, represented by the
rich foliage of the maize plant, symbolizing the essence of life (Photo
by M. Tenaillon).}
\end{figure}

Archaeological remains show that agricultural systems developed in
Mesoamerica were diverse and sophisticated. Mesoamerican farmers
developed techniques such as irrigation, terracing, crop rotation and
mixed  cropping~\cite{Redman1999,Angelakisetal2020}. 
Perhaps the most
emblematic intercropping system is the tripartite milpa system, often
composed of squash  (\textit{Cucurbita}~spp.), common bean 
(\textit{Phaseolus vulgaris}~L.)---hereafter bean---and, maize. The
word ``milpa'' originates from the Nahuatl language, where
``\textit{milli}'' means planted plot and ``\textit{pan}'' means 
upon~\cite{Bierhorst1985}. 

The milpa was likely developed in the Guerrero-Jalisco area, in the
Western part of Central Mexico, where agri-food technology became
highly advanced between 4500 and 3500~yr~BP. This 
is\unskip\break 
evidenced by the
use of clay pots for steaming and cooking, technologies that were not
present in other parts of Mesoamerica at the 
time~\cite{Zizumbo-Villarrealetal2012}. Milpa subsequently became the
cornerstone of pre-Columbian agriculture, spanning a vast region from
northeastern North  America~\cite{Lewandowski1987} to northern South 
\mbox{America}~\cite{Locquevilleetal2022}. The complementary nutritional
intake provided by the three crops likely contributed to its widespread
success~\cite{Zizumbo-Villarrealetal2012}.

As it spread and was adopted through Mesoamerica and beyond, the milpa
system diversified in various ways. First, in terms of terminology,
milpa is referred to as  ``\textit{Diohe'ko}''---things that sustain
us---in Seneca  language~\cite{Lewandowski1987,ParkerParker1910}, as
``\textit{qu\`{e}ela}'', ``\textit{yela}'' or ``\textit{yel}'' in
Zapotec  dialects~\cite{Broadwell2004} or
``\textit{\~{n}aa}''~\cite{Pickett2013} while the Mayans designate it
as  ``\textit{Kool}''~\cite{TeranContreras2023}. Second, milpa was also
the basis of a rich diversity of mythology and beliefs. For the
Iroquoian people, the three plants were considered as a sacred gift as
they miraculously sprouted from the body of Sky Woman's daughter, thus
granting the gift of agriculture to the Iroquois 
nations~\cite{MacDougall2005}. Third, the milpa system diversified
through the inclusion of various associated crops, such as other
species of the \textit{Phaseolus} genus  (\textit{Phaseolus
lunatus}~L., \textit{Phaseolus acutifolius}~A.~Gray and, more rarely,
\textit{Phaseolus coccineus}~L.\ and \textit{Phaseolus dumosus} 
Macfadyen---see~\cite{Locquevilleetal2022}), agaves 
(\textit{Agave}~ssp.), chilis  (\textit{Capsicum}~spp.), husk tomatoes 
(\textit{Physalis}~spp.), chan (\textit{Hyptis  suaveolens} [L.] Poit),
hog plums, jocote  (\textit{Spondias purpurea}~L.) among other 
species~\cite{Zizumbo-Villarrealetal2012,Illsley2003}.

In this review, we first detail the domestication processes of maize,
beans, and squash, and the subsequent development of the milpa system
in the Guerrero-Jalisco region. We then examine how milpa, a
traditional agri-food system, was adapted by small-scale European farms
and later integrated into modern agricultural practices. Finally, we
highlight the beneficial interactions between these three crops,
emphasizing their potential as key elements in promoting agroecology.

\section{The emergence of milpa in the Guerrero-Jalisco
area}\label{sec2}
Archaeological findings and genetic data indicate that the simultaneous
growing of the three crops appeared not very long after their
respective 
domestication~\cite{Doebley2004,PipernoFlannery2001,Ranereetal2009}. In
fact, it is possible that the wild relatives of maize, bean and squash
were consumed together even before domestication in the pre-ceramic
period, as evidenced by contemporary consumption of wild forms using
tools and techniques from the Archaic time period in 
\mbox{Mesoamerica}~\cite{Zizumbo-Villarrealetal2012}. The co-occurrence of the
wild and early-domesticated forms of the three taxa in southwestern
Central Mexico would have probably facilitated their association 
(Figure~\ref{fig2}). Interestingly, the domestication of the three
crops tells very different stories. These differences are rooted in the
distributions of their wild relatives and are further shaped by
biological differences. Maize, for example, is a monoecious,
outcrossing and wind-pollinated plant. In contrast, common beans are
predominantly self-pollinating, while squash species, which are mostly
monoecious, rely on insect pollination for outbreeding. Moreover,
human-directed selection has resulted in distinct domestication
syndromes for each species.

\begin{figure*}
\includegraphics{fig02}
\caption{\label{fig2}Domestication areas, routes, and introductions
into Europe of the three crops composing milpa. In the figure (left
panel), colors indicate each species. In gold, maize was domesticated
in Mesoamerica around 9000~years before present (yr~BP). Maize spread
northward and southward across the American continent before being
introduced to southern Spain following Christopher Columbus'
expeditions in~1492 and to northern Europe before~1539. In orange,
squash (\textit{C.~argyrosperma}) was domesticated in Mesoamerica
around 10,000~yr~BP, but little is known about the routes it took
before being introduced to Europe. In green, beans underwent two main
independent domestication events around 8000~yr~BP in Mesoamerica and
the Andes, resulting in two distinct gene pools: M1 and~A2. A secondary
domestication from M1 led to the M2 group, while two secondary
domestication events from A2 led to the A1 and A3 groups. All groups,
except~A2 due to its photoperiod sensitivity, were introduced into
Europe after Pizarro's explorations in~1529 for the Andean groups.
Today, intercropping of maize-bean-squash is still practiced in some
traditional European farming systems (right panel, photo from
southwestern France by Maud Tenaillon).}
\end{figure*}

Maize ($2n = 2X = 20$) was domesticated in the Rio Balsas drainage
basin located in northern Guerrero  (Figure~\ref{fig2}) from the annual
subspecies of teosintes, \textit{Zea mays} ssp.\ \textit{parviglumis}
around 
9000~yr~BP~\cite{Pipernoetal2009,Ranereetal2009,Matsuokaetal2002}.
Recent results have also found that ssp.\ \textit{mexicana} has also
greatly contributed to maize make-up through  admixture---the genetic
mixing of these two subspecies through 
interbreeding~\cite{Yangetal2023}. Many traits differentiate maize from
teosintes (reviewed in Tenaillon and
Manicacci~\cite{TenaillonManicacci2011}).  A major one is the softer
glume, which leads to kernel exposition and accessibility, making maize
a valuable  crop~\cite{Wangetal2005}; starch properties were also
modified in  maize~\cite{Whittetal2002}. Regarding the architecture,
the prevention of axillary meristem development led to the suppression
of the elongation of lateral ear 
branches~\cite{Iltis1983,Doebleyetal1997,Whippleetal2011}, which
translated into strong apical dominance and short ear-tipped branches
in  maize~\cite{Doebley2004,Studeretal2011}. Additionally,
domestication has resulted in increased yield with dramatic increase in
female ear length and  width~\cite{StitzerRoss-Ibarra2018}, as well as
a reduction in seed
shattering~\cite{DoebleyStec1991}.

The genus \textit{Phaseolus} ($2n = 2X = 22$) is composed of 70
species. Among them, five are domesticated including
\textit{P.~vulgaris}  (common bean), which is by far the one with the
broadest geographic distribution. This species was domesticated twice 
independently~\cite{Chaconetal2005}. The wild species
\textit{P.~vulgaris} originated in Mexico and spread into South
America, leading to two distinct and geographically isolated wild gene
pools in Mesoamerica and the  Andes~\cite{Bitocchietal2017,Gepts1990}.
Domestication occurred independently in these gene pools around 
8000~yr~BP~\cite{Mamidietal2011}, with ongoing debate about the
precise locations  (Figure~\ref{fig2}). Mesoamerican domestication is
supported by both molecular and archaeological evidence that pinpoint
the Rio Lerma--Rio Grande de Santiago basin in Jalisco, in West Central
Mexico~\cite{Kwaketal2009}. However, nucleotide data suggest an earlier
domestication in the Oaxaca Valley in southern central 
Mexico~\cite{Bitocchietal2013}, an area where maize also spread early
through human 
migration~\cite{Zizumbo-VillarrealColunga-GarciaMarin2010}. A recent
study proposes that initial domestication has occurred in high-altitude
regions of Jalisco and Durango  (Figure~\ref{fig2}, M1), with a
secondary domestication of low-altitude races  (Figure~\ref{fig2}, M2)
in  Mesoamerica~\cite{Belluccietal2023}. In the Andean gene pool,
potential domestication sites include central-southern Peru based on
chloroplast  DNA~\cite{Chaconetal2005}, while nuclear nucleotide data
and archaeological findings from Huachichocana in Argentina suggest
southern Bolivia and northern  Argentina~\cite{Beebeetal2001}. Recent
findings indicate a primary domestication in Peru  (Figure~\ref{fig2},
A2), followed by two secondary domestications  (Figure~\ref{fig2}, A1
and A3), defining three distinct Andean gene 
pools~\cite{Belluccietal2023}. Multiple domestications may have been
facilitated by the selfing mating system of this species. Domestication
resulted in morphological changes including loss of pod dehiscence,
traits limiting seed dispersal and seed dormancy, fewer nodes on the
main stem, larger pods and  seeds~\cite{Koinangeetal1996}. 

The genus \textit{Cucurbita} ($2n = 2X = 40$) includes 14~species,
with six independent domestications from various wild ancestors present
across the 
Americas~\cite{Castellanos-Moralesetal2018,Eguiarteetal2018,Nee1990}. 
Kate et~al.~\cite{Katesetal2017} established the first squash phylogeny
based on and suggested different domestication centers: 
\textit{C~pepo}~L.\ ssp.~\textit{pepo}  (pumpkin) and
\textit{C.~argyrosperma} Huber ssp.~\textit{argyrosperma} (cushaw
pumpkin) in Mexico, \textit{C.~moschata} Duchesne  (butternut squash or
calabaza) in Colombia, \textit{C.~pepo}~L.\ ssp.~\textit{ovifera} (L.)
Harz (summer squash) in eastern North America, and
\textit{C.~ficifolia} Bouch\'{e}  (figleaf gourd or chilacayote) and
\textit{C.~maxima} Duchesne ssp.~\textit{maxima} (Hubbard squash) in
South 
\mbox{America}. More recent results, however, suggest domestications of
\textit{C.~moschata} and \textit{C.~ficifolia} in 
Mexico~\cite{Eguiarteetal2018,Hernandez-Rosalesetal2020,Aguirre-Duguaetal2023}. 
The domestication center of \textit{C.~argyrosperma} was pinpointed in
the lowlands of Jalisco  (Figure~\ref{fig2}) using genomic data from
192 wild and domesticated  individuals~\cite{Barrera-Redondoetal2021}.
In milpa agriculture, commonly used \textit{Cucurbita} species include
\textit{C.~pepo}, \textit{C.~moschata}, \textit{C.~argyrosperma} and
\textit{C.~ficifolia}~\cite{Eguiarteetal2018}. The earliest
archaeological evidence for \textit{C.~maxima} and
\textit{C.~argyrosperma} dates back to 4000--5000~yr~BP, while
evidence for \textit{C.~pepo} dates back to
8000--10,000~yr~BP~\cite{Smith1997}. The domestication of pumpkins and
squashes in Mesoamerica likely began with human selection favoring
less-bitter  seeds~\cite{Liraetal2016}. Today, while all major
\textit{Cucurbita} crops are cultivated for their fleshy fruit,
\textit{C.~argyrosperma} is still mainly valued for its 
seeds~\cite{Barrera-Redondoetal2021,Eguiarteetal2018}. The evolution of
edible flesh involved the reduction of bitter compounds known as 
cucurbitacins~\cite{Zhouetal2016}. Despite distinct primary usages,
many domestication traits are common among the domesticated
\textit{Cucurbita} species. They include loss of bitter compounds,
reduction of physical defenses such as urticating trichomes,
elimination of seed dormancy, enlargement of fruits and seeds, and
diversification of fruit\break
morphology~\cite{Barrera-Redondoetal2021,Chomickietal2020,Paris2017}. 

\section{The milpa, from Mesoamerica to Europe}\label{sec3}
Maize spread northward into southwestern United States around 
4000~yr~BP~\cite{Merrilletal2009}; and southward from Mexico 
(Figure~\ref{fig2}), reaching southwestern Amazonia by 
6500~yr~BP~\cite{Bruggeretal2016}. Southwestern Amazonian basin may
have acted
as a secondary center of improvement before the 
subsequent diffusion of this\unskip\break 
``improved'' maize to lowland South America east of
the Amazonian basin and to the Andes in the 
west~\cite{Kistleretal2018}. In Europe (Figure~\ref{fig2}), Caribbean
maize adapted to warm climates was introduced to southern Spain after
Columbus's 1492  expedition~\cite{BrandoliniBrandolini2009}. Flint
maize from North America reached northern Europe, as noted by Bock 
in~1539~\cite{Finan1948} and later confirmed by genetic 
studies~\cite{Rebourgetal2003}. Genetic analyses revealed that northern
European landraces are genetically linked to their North American
counterparts, while southern European landraces are closer to Caribbean
lowland tropical 
maize~\cite{TenaillonCharcosset2011,Brandenburgetal2017}. 

Similarly, \textit{P.~vulgaris} arrived in Europe following early
European exploration of the Americas  (Figure~\ref{fig2}). All groups
except~A2, which is sensitive to photoperiod, were introduced to 
Europe~\cite{Belluccietal2023}. The prevalence of Andean accessions and
smaller Andean introgression segments in European varieties suggests
that the Andean gene pool was introduced before the Mesoamerican gene
pool to  Europe~\cite{Belluccietal2023}, following Francisco Pizarro's
1529~expedition to northern  Peru~\cite{Perale2001}. 

As for \textit{Cucurbita} species, there is currently limited knowledge
about their spread outside their centers of origin 
(Figure~\ref{fig2}). 

Admixture played a crucial role in shaping specific genetic groups in
Europe. In maize, similar to the emergence of Corn Belt dents in the
mid-latitudes of the United States, European Flints likely originated
from admixture between northern and southern 
\mbox{European} genetic 
materials~\cite{Brandenburgetal2017,Rebourgetal2003}, adapting maize to
European climates. In common beans, the distribution of genetic groups
in Europe reflects their adaptive traits, particularly in terms of
photoperiod sensitivity and climate adaptation. Hybridization between
Andean and Mesoamerican \textit{P.~vulgaris} gene pools after
introduction to Europe resulted in new allele combinations, evident in
the higher level of admixture among European bean 
varieties~\cite{Bitocchietal2017}. However, the dissemination of common
beans in Europe was influenced not only by local adaptation, but also
by political and cultural  factors~\cite{Belluccietal2023}.

The three crops (maize, bean, \textit{Cucurbita}) spread in Europe
following their introduction. The question of whether varieties of the
three taxa used in multi-cropping systems were co-introduced to Europe
at the time of the discovery of the Americas, as opposed to being
re-associated later in Europe,\break remains open. 

European farming practices
resembled traditional Mesoamerican milpa systems, characterized by
low-input field management and the use of traditional landraces.
However, maize was  (and still is) used differently: in Mesoamerica,
tropical maize landraces underwent nixtamalization---a process of
soaking and cooking maize kernels in an alkaline solution---before
being ground into so-called masa dough for 
tortillas~\cite{Katzetal1974}. In Italy, and likely across Europe,
maize, mostly of flint types, was typically ground into coarse meal for
polenta. Maize was first mentioned on the Italian peninsula in~1495 and
initially cultivated in the Veneto region, helping northern Italian
farmers avoid food shortages and  famine~\cite{GinnaioJacobs2011}. 
However, without the knowledge of the nixtamalization process, which releases
niacin  (vitamin~B3) and essential amino acids, maize consumption leads
to nutritional deficiencies. It is possible that the early adoption of
the milpa system helped mitigate these deficiencies by providing
nutritional complementarity. From the late  18th~century onward,
however, northern Italy saw a gradual shift toward a maize-based diet,
which led to outbreaks of  pellagra~\cite{GinnaioJacobs2011}. In contrast, in
the Hautes-Pyr\'{e}n\'{e}es department in France, the coculture of
maize and bean was common covering 18,500 hectares in the 19th 
century~\cite{Bonnain-DulonBrochot2004}. In this region, the combined
cultivation of all three taxa was actually frequently found in gardens
and small farms for subsistence 
farming~\cite{BeigbederCarraretto2018}. Traditional multi-cropping
farming practices were abandoned in Europe after the Green Revolution,
largely due to the introduction of highly productive maize 
hybrids~\cite{DeRonetal2018,Vazeux-Blumentaletal2024}. 

Today, milpa in Europe is restricted to a few areas such as the 
Pyrenees (Figure~\ref{fig2}), Transylvania and southern 
Italy~\cite{Piergiovannietal2000},
where maize landraces are selected for their flint type for polenta and
bean is also used for self-consumption. Recently, there has been a
renewed interest in integrating multi-crop farming into modern
agricultural practices. This revival is evident in the maize-bean
intercropping system adopted in the Tarbes region of France in 
the~1980's~\cite{Bonnain-DulonBrochot2004}, in the Potenza region of
Italy  (personal communication from Elisa Bellucci), and areas of 
Germany~\cite{Fischeretal2020}. These systems involve combining bean
cultivars with maize hybrids, often using chemical inputs. Maize in
these modern setups is often utilized for animal feed.

\section{Beneficial mechanisms of traditional milpa}\label{sec4}
Several studies have observed an overall performance advantage of the
milpa system compared to crops grown alone, due to positive
interactions. This is assessed using the Land Equivalent Ratio  (LER)
index, the most commonly used metric for evaluating land use efficiency
in intercropping systems relative to 
sole-cropping~\cite{MeadWilley1980}. Findings consistently show that
milpa systems have LER values greater than~1 compared to monocultures,
indicating superior land use efficiency of the mixture, a phenomenon
also known as 
overyielding~\cite{Ebeletal2017,Lopez-Ridauraetal2021,MtPleasantBurt2010,Zhangetal2014}.

The overyielding observed in milpa systems can be attributed to two
essential non-exclusive beneficial mechanisms underlying positive
interspecific interactions: niche partitioning and 
facilitation~\cite{Brookeretal2015}. On the one hand, niche
partitioning maximizes the exploitation of light and soil resources
through spatial complementarity in the architecture of above-ground and
root parts, temporal  complementarity~\cite{Yuetal2015}, or
complementarity in the types of resources, hence reducing competition
for light, water, and nutrients among  plants~\cite{Bedoussacetal2015}.

On the other hand, facilitation mechanisms occur either directly or
indirectly. Direct facilitation describes the physicochemical
modifications of the local environment  (e.g., increased temperature,
enhanced soil moisture, pH~modification) induced by one component of
the multi-cropping system. It also includes the physical support
provided to other plants, which reduces the risk of lodging and hence
promotes the growth and development of neighboring plants. Indirect
facilitation occurs when the intercropped plants attract external
species that provide new ecosystem services, such as soil bacteria or
mycorrhizal fungi, that benefit the multi-cropping 
\mbox{system}~\cite{Frevilleetal2022}.

\begin{figure*}
{\vspace*{1pt}}
\includegraphics{fig03}
{\vspace*{2pt}}
\caption{\label{fig3}Beneficial mechanisms found in milpa.
Mechanisms demonstrated in the species composing milpa (maize, common
bean, and squash) are written in bold, while those shown in other
species belonging to the same families are in italics. The color of the
mechanism indicates the species providing the service: gold for maize,
green for the bean (and related crops from the same family), orange for
the squash. The services provided by all three species of milpa are
written in black.}
\end{figure*}

In milpa, complementarity mechanisms are observed both above and below
ground among the three crops. For the aerial parts, plant architectural
complementarities tend to reduce competition for light 
(Figure~\ref{fig3}). An experimentally validated model of radiation
interceptions of maize-bean association simulated a higher radiation
use efficiency  (RUE) for intercropped beans than sole cropped beans,
with no RUE difference between intercropped and sole cropped maize. The
model also showed a competitive advantage for the maize compared to
bean with higher photosynthetically active radiation \break
(PAR)~\cite{TsuboWalker2002}. 

Optimization of space utilization in intercropping systems tends to
enhance competitive resistance against weeds. Intercropping maize and
bean has been shown to result in lower weed pressure compared to 
sole-cropping~\cite{WorkayehuWortmann2011}. This difference was
attributed to the greater absorption of PAR by the canopy of
intercropped maize and beans than by canopies of either crop alone,
thereby reducing weed  growth~\cite{Bilalisetal2010}. Similarly,
intercropping maize with squash reduces weed pressure, primarily
due to shading provided by the  squash~\cite{Fujiyoshietal2007}.


Above ground, root system complementarities in milpa decrease
competition for soil nutrients and water 
(Figure~\ref{fig3}). Hence, simulations of associations of the three
crops for nutrient uptake and resource use indicate root
architecture complementarity, which results in increased biomass
production on nitrogen  (N)-deficient  soils~\cite{PostmaLynch2012}.
Differences in root architecture among these crops indeed reflect a
diversity of nutrient foraging strategies, with shallow, more vertical
and, deep soil exploration for maize, bean and squash, 
respectively~\cite{Zhangetal2014}. There is also \mbox{evidence} that the
higher transpiration observed in the associated maize and bean plants
compared to maize alone is the result of increased water assimilation,
due to higher root proliferation and a more complete exploitation of
the water resource in the soil
\mbox{profile~\cite{WalkerOgindo2003}.}

In milpa, direct facilitation primarily occurs through the physical
support provided by maize for the growth of bean  (Figure~\ref{fig3}).
Moreover, optimized crop canopies in maize-bean and maize-bean-squash
associations reduce soil temperature, increase water availability 
and electron transport rates compared
to maize grown  alone~\cite{Perez-Hernandezetal2021}. In milpa systems,
plants are known to release root exudates that can locally modify
rhizosphere  properties~\cite{Gentetal2005,Lietal2016a,Shenetal2002}.
Results on maize and faba bean  (\textit{Vicia faba}) intercropping for
example, show that the bean's phosphorus  (P) uptake strategy acidifies
the rhizospheric soil via root exudate production  (Figure~\ref{fig3}).
This acidification enhances the availability of inorganic~P,
facilitating uptake by  maize~\cite{Lietal2007,Zhangetal2016}. Whether
these results apply to maize-common bean intercropping remains to be
tested. 

Indirect facilitation also occurs in milpa  (Figure~\ref{fig3}). Most
food legumes form nodules to host N-fixing rhizobial bacteria, a
process initiated by plant-released flavonoids and rhizobial 
Nod~factors~\cite{Oldroydetal2011}. This signaling pathway, crucial for
N~fixation in legumes, also supports symbiosis with mycorrhizal 
fungi~\cite{Parniske2008}. It has been shown that in common bean, this
fixation can reach 60\% of its total 
N~acquisition~\cite{Tsaietal1993}. In intercropped maize-bean, the
symbiotic N~fixation can also benefit  maize~\cite{Latatietal2016}. In
a first step of the culture, when the nodules are not yet developed,
the two species compete for soil mineral~N. The advantage of the cereal
due to its faster root development triggers symbiotic fixation in the 
legume~\cite{Laietal2022}. Hence a greater proportion of the soil~N is
available to the cereal in intercropping than in sole-cropped stands,
limiting the need for additional  fertilization~\cite{Latatietal2013}.
Additionally, flavonoids found in maize root exudates enhance
nodulation in faba bean and improve  N~uptake~\cite{Lietal2016b}.
Moreover, the high level of synchrony between N~inputs and crop
N~uptake in the maize-soybean association tends to avoid the build-up
of excess~N in soils leading to a reduction of nitrate 
leaching/lixiviation~\cite{Nieetal2012}. Whether these results hold for
maize-bean intercropping remains to be
\mbox{determined.}

Common mycorrhizal networks (CMN) also play a key role in indirect
facilitation  (Figure~\ref{fig3}). It was demonstrated that
N~assimilation in maize intercropped with bean increased due to
N~transfer via interplant hyphal connections of arbuscular mycorrhizal 
fungi~\cite{Dawoetal2009,Gutierrez-NunezGavito2024}.

Crop assemblage in milpa also reduces pest and disease pressure through
complementarity and facilitation  (Figure~\ref{fig3}). This involves
barrier effects (resistant plants restraining pathogens), dilution
effects  (lower concentration of potential targets) and compensation
effects  (poor growth of susceptible plants is compensated by the
growth of non-sensitive ones that better utilize resources). In
maize-bean intercropping, common bacterial blight and rust diseases
decreased by 23\% and 51\%, respectively, compared with 
sole-cropping~\cite{Fininsa1996}. Likewise, both decrease of the maize
pest \textit{Rhopalosiphum maidis} and reduction of herbivorous beetles
were observed in intercropped maize-squash-faba 
bean~\cite{Trujillo-ArriagaAltieri1990} compared to maize alone, and
similarly in  maize-bean-squash~\cite{Risch1980} compared to
sole-cropped plants of each of the three species. 

The success of milpa results from ecosystemic services delivered by
crop interactions within agricultural systems. These services,
extensively studied in natural ecosystems, include net primary
production, N~mineralization, soil moisture retention, water flow
regulation, carbon storage, soil formation, and soil 
retention~\cite{DiazCabido2001}. Insights from these studies have
inspired agroecology. Conversely, experiments in agronomic contexts,
that involve fewer plant species, can serve as valuable models for
understanding more complex natural ecosystems.

\section{The milpa, a lever for the agroecological
transition}\label{sec5}
Conventional agriculture has significantly boosted food production, but
it has also caused severe environmental drawbacks, including increased
greenhouse gas emissions, soil degradation, and extensive freshwater 
withdrawals~\cite{Tudietal2021}. A sustainable agricultural model is
needed to improve resource efficiency and reduce 
inputs~\cite{Gliessman2014}. 

Crop mixtures enhance productivity by harnessing beneficial plant--plant
interactions instead of relying on chemical inputs. In this context,
the well-studied milpa system offers valuable nutritional
complementarities among species and serves as an exemplary
agroecological model. A study involving 989 Guatemalan small farms
practicing traditional and derived forms of milpa showed that one
hectare of milpa produces more calories than one hectare of sole
cropped  maize~\cite{Lopez-Ridauraetal2021}. Maize primarily provides
carbohydrates  (68--75\%), squashes are rich in lipids (41--49\%),
and beans offer substantial proteins (19--28\%)
(S\'{a}nchez-Vel\'{a}zquez et~al.~\cite{Sanchez-Velazquezetal2023}  and
references herein). The proteins obtained from milpa can serve as a
high-quality substitute for animal proteins, with an amino acid score
considering protein digestibility often exceeding FAO/WHO 
recommendations~\cite{Sanchez-Velazquezetal2023,vanLoonetal2023}.
Additionally, milpa boasts significant micronutrient content, supplying
11~different vitamins and eight different minerals with interesting
complementarities. For example, maize contains a substantial amount of
vitamin~C, almost absent from bean and pumpkin seeds, while bean is
notably rich in vitamin~B4 compared with maize and pumpkin seeds, and
pumpkin seeds provide greater amounts of vitamins~A 
and~B3~\cite{Sanchez-Velazquezetal2023}. Milpa also contains diverse
bioactive compounds such as polyphenols, phytosterols, saponins, fiber,
bioactive peptides, and carotenoids, which are linked to various health
benefits~\cite{RodriguezMadreraetal2021}. 

To advance the use of milpa in agroecology, there is a crucial need to
identify phenotypic traits and their genetic determinants that foster
positive plant--plant and plant--microorganism interactions. These traits
include those related to aerial and root architectures, to resource
acquisition, to root exudation, and to symbiotic relationships with
bacteria or mycorrhizal fungi. In maize-bean associations, maize acts a
dominant competitor that can affect the overall 
productivity~\cite{Nassaryetal2020,Santallaetal2001,WilleyOsiru1972},
but can also suffer from lodging due to weight of the bean,
highlighting the need to find a balance between low-competitive but
robust maize  varieties~\cite{Vazeux-Blumentaletal2024}. 

Recent research explores two primary strategies for selecting
multi-cropping: trait-blind and trait-based 
approaches~\cite{Barotetal2017}. Trait-blind selection tests various
genotype combinations directly in the field to find the best
performers. Trait-based selection focuses on specific traits that
enhance desired outcomes, such as efficient N fixation in beans for
milpa  systems~\cite{Gabaetal2015,MartinIsaac2015}. The latter method
requires\unskip\break 
understanding genetic and phenotypic traits that foster
complementary interactions between species. This would help breeders to
target beneficial traits increasing yield predictability and
sustainability. In both approaches, selection should be conducted in
low-input settings to foster synergistic interactions and to allow the
improvement of efficient systems based on positive biological
interactions rather than chemical 
inputs~\cite{Maestreetal2009,Maitraetal2021}. Note that it is possible
to optimize the selection schemes by combining the trait-blind and
trait-based methods. Initial trait-blind selection can identify
promising combinations, which can then be further refined using
trait-based methods to enhance specific 
desirable\unskip\break  
characteristics~\cite{Barotetal2017}.

A promising avenue to foster beneficial interactions and improving
selection for efficient multi-cropping systems would be to remobilize
underutilized genetic pools, such as wild relatives, early domesticated
forms, and landraces, that can expand the range of potential plant
architectures and functions in multi-cropping systems. Landraces,
selected for efficiency in intercropping, likely carry valuable
co-adaptive alleles that modern breeding has often 
neglected~\cite{Frevilleetal2022}. In addition, native maize landraces
used in milpa systems possess a more diverse seed-endophytic microbiome
with greater antagonistic effect against soil-borne bacteria compared
to hybrid  varieties~\cite{Gastelumetal2022}. Germplasms of traditional
maize, bean, and squash varieties have been preserved both {ex
situ} in American and European GenBanks and {in situ} in
small-scale farms such as in Transylvania. Both {in situ} and
{ex situ} resources are key to exploring the full range of
potential interactions in the milpa systems.

Selecting suitable varieties for multi-cropping systems is complicated
by significant genotype x cropping system interactions, as well as
practices x genotype x cropping system 
interactions~\cite{Beckeretal2023,Santallaetal2001,Vazeux-Blumentaletal2024}. 
Practices include soil management, sowing date, plant density and
inputs. Together, they significantly impact productivity and seed
quality of intercropping systems. For example, results from alternating
rows of maize and common beans indicate that reduced tillage preserved
the mycorrhizal network, leading to earlier root colonization and
improved nutrition for the beans, which in turn enhanced nitrogen
transfer to the  maize~\cite{Gutierrez-NunezGavito2024}. Additionally,
sowing beans before maize proved\unskip\break  
beneficial, as it allowed the beans to
quickly connect to the mycelial network and reduced competition between
the two crops~\cite{Gutierrez-NunezGavito2024}. Regarding inputs, a
meta-analysis of maize intercropped with legumes, including common
beans, found that high nitrogen inputs reduce the Land Equivalent Ratio
(LER) by shifting the balance from complementarity to competition
between crops. Under these conditions, the benefits of complementarity
diminish, making the competitive interactions more 
dominant~\cite{Mudareetal2022}. Note that the natural presence of
inoculum, or artificial inoculation of bean seeds, is key to take
advantage of the symbiotic nitrogen fixation. Likewise, complementarity
in phosphorus uptake also occurs in P-deficient soils, resulting in
significant overyielding in maize/faba bean 
intercropping~\cite{Lietal2007} but also in 
chickpea/maize~\cite{Lietal2004}, white  lupin/wheat~\cite{Cuetal2005},
common  bean/wheat~\cite{Lietal2008}, and faba\break 
bean/wheat~\cite{Lietal2016b}. 

Finally, the large-scale adoption of intercropping is hindered by the
lack of suitable mechanization  tools~\cite{Feikeetal2012}. Harvesting
and sorting intercropped plants is challenging, as separation depends
on species and on combined harvester 
settings~\cite{Bedoussacetal2021}. There are two main possibilities to
explore, either converting row-intercropping to strip-intercropping to
use existing  machinery~\cite{Gutierrez-NunezGavito2024} or developing
new machines for row-intercropping. The former may impact beneficial
interactions, while the latter will require significant investment.
Recently, adjusting harvester settings has enabled successful
harvesting and sorting of multi-cropping systems such as wheat-lupin
and barley-pea using standard  equipment~\cite{Bedoussacetal2021},
which could be in principle applied to maize-bean intercropping.
However, an additional challenge lies in the need for multiple harvest
passes in beans.

\section*{Declaration of interests}
The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

\section*{Funding}
NV-B was financed by a doctoral contract from
SPS through the Doctoral
school ``Sciences du
V\'{e}g\'{e}tal ED567''. This work was funded by
the \mbox{European} Union's Horizon 2020 research and innovation programme
under grant agreement No.\ 862862 (\mbox{INCREASE} Project).

\section*{Acknowledgements}

We sincerely thank Tudor Ursu and Anamaria Roamn for their insightful
discussions on milpa in Transylvania, and Luis E. Eguiarte for his
helpful feedback and detailed corrections on the manuscript. We also
greatly appreciate Doyle McKey's valuable comments and edits, which
significantly improved the manuscript. We acknowledge
the use of OpenAI's ChatGPT for providing assistance in correcting the
text in this manuscript without generating original\break content.

\CDRGrant[SPS]{ED567}
\CDRGrant[EU]{862862}

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