Uses, Knowledge and Extinction Risk Faced by Agave Species in Mexico

We compiled an updated database of all Agave species found in Mexico and analyzed it with specific criteria according to their biological parameters to evaluate the conservation and knowledge status of each species. Analyzing the present status of all Agave species not only provides crucial information for each species, but also helps determine which ones require special protection, especially those which are heavily used or cultivated for the production of distilled beverages. We conducted an extensive literature review search and compiled the conservation status of each species using mainstream criteria by IUCN. The information gaps in the database indicate a lack of knowledge and research regarding specific Agave species and it validates the need to conduct more studies on this genus. In total, 168 Agave species were included in our study, from which 89 are in the subgenus Agave and 79 in the subgenus Littaea. Agave lurida and A. nizandensis, in the subgenus Agave and Littaea, respectively, are severely endangered, due to their endemism, lack of knowledge about pollinators and floral visitors, and their endangered status according to the IUCN Red List. Some species are at risk due to the loss of genetic diversity resulting from production practices (i.e., Agave tequilana), and others because of excessive and unchecked overharvesting of wild plants, such as A. guadalajarana, A. victoriae-reginae, A. kristenii, and others. Given the huge economic and ecological importance of plants in the genus Agave, our review will be a milestone to ensure their future and continued provision of ecosystem services for humans, as well as encouraging further research in Agave species in an effort to enhance awareness of their conservation needs and sustainable use, and the implementation of eco-friendly practices in the species management.


Introduction
Mexico is well-known for its high levels of biodiversity, and agaves or magueyes belonging to the family Asparagaceae are a very important example of that biodiversity. The genus Agave is endemic to the American continent (although introduced Agave are now found in many places), mainly in arid and semi-arid areas of Mexico. There are approximately 200 species of Agave, of which 166 are distributed in Mexico and 119 are endemic [1,2]. The subgenus Littaea contains about 78 species (Figure 1) while the subgenus Agave includes around 92 species (Figure 2), making it the subgenus with the greatest distribution throughout Mexico [3]. Subgenera are distinguished by their large inflorescence, either a spike for the subgenus Littaea species or branched with panicles for Agave [4]. The succulence and rigidity of its leaves for water storing, the efficient water absorption system throughout its roots and the limited water loss through transpiration, are some of the adaptations agaves possess for life in dry ecosystems. Their CAM-type photosynthetic metabolism (Crassulaceae Acid Metabolism) allows them to grow in poor soil and weather conditions, withstanding severe environmental pressures [2]. These morphological and physiological adaptations have allowed agaves to live in a wide variety of environments, expanding their geographical distribution [3,5].  From a historical perspective, agaves have played a crucial role in the daily lives of human beings, providing a wide range of resources, from food and the production of alcoholic beverages to a source of fiber. The procurement of fibers from these plants was perhaps the first use given to agaves, since it was used for clothing, baskets, nets, rope, and more [4]. Several species have been used for food, the flowers boiled and eaten, and earthen ovens to cook agave cores or "piñas". This method was an ancestral practice that is nowadays utilized for the production of distilled beverages [6]. The use of Agave to produce the distilled liquors known today as tequila, mezcal, and others was apparently not known to past civilizations, since distillation was introduced after the conquest, but the production of pulque and mead or "aguamiel" has been used since pre-Hispanic times, making these beverages the ones that promoters of the agave cultivation in Mexican civilizations. Currently over 70 traditional uses have been known and recorded for the species of the genus Agave [7], classified under 22 categories [8].
Additionally, the residues derived from the elaboration of distilled beverages, including the flowering stalks, bagasse (solid waste) and stillage (liquid waste) have various uses. The leaves, removed from the core of the agave rosette are commonly used in the preparation of Mexican dishes, such as in the "barbacoa" where the leaves are roasted and used to wrap the meat with spices for cooking while bagasse and stillage residues have been proven as a good alternative for compost [9]. These wastes are polysaccharide-rich byproducts that can be used for the production of fructans, which can be used in supplement products to treat diabetes or obesity [2].
Agave fibers have a wide variety of uses: as food for livestock and the production of different handcrafts, or common products such as nets, ropes or textiles. In rural areas, agaves are widely used as living fences to keep livestock away from crops or houses, to indicate roads, and even for construction [10]. Agave sisalana (sisal) and A. fourcroydes (henequen) are the most useful species for obtaining fiber and are widely used for struc-Agaves are greatly known in the distilled beverages industry. Tequila and mezcal have become symbols of Mexico throughout the world, while pulque, the pre-Hispanic fermented alcoholic beverage, is considered to be one of the first traditional drinks originated from agave. Bacanora and raicilla are some of the least known agave distillates as they have a low level of industrialization compared to the rest of the distilled beverages [6,8].
Pulque is a white fermented beverage that can be obtained from a wide variety of agave species such as A. atrovirens, A. americana, A. salmiana and A. mapisaga. This drink is made through the fermentation of mead or aguamiel, a sugary extract that accumulates inside the core of the ripe agave and that can also be consumed fresh without fermenting [13]. Pulque is consumed both in rural areas, where it may even be considered as an important meal in a daily diet, and in urban areas, where it can be found in pulquerias and Mexican restaurants. Currently, the consumption of pulque is promoted partly so as not to lose a pre-Hispanic tradition [14,15].
Tequila is considered the most representative drink in Mexico, a job and currency generator of foreign exchange for its sales abroad. In 2016, Jalisco obtained $1.6 billion dollars in tequila exports abroad growing to $2.3 billion dollars in 2020 while the industry generated 70,000 jobs [16,17]. Tequila is obtained from Agave tequilana or blue agave, which can only be grown in regions known as Denomination of Origin of Tequila (DOT). This includes the states of Jalisco (126 municipalities) with 90% of production, Michoacán (30 municipalities), Tamaulipas (11 municipalities), Nayarit (eight municipalities) and Guanajuato (seven municipalities) [18]. Since A. tequilana is not common in the wild, tequila is sourced entirely from cultivated plants. In addition, to avoid a perceived waste if the plants are allowed to flower, agave fields are re-planted with young agave plants that are the result of asexual reproduction from a subset of the original plants in the field, thus resulting in a severe loss of genetic diversity generation after generation [19]. In addition, tequila has the most industrialized production processes of all distilled beverages originated from agave. The use of autoclaves (steam-fueled pressure-cookers) to cook the heads of A. tequilana is the most production-efficient method for Agave distillates, having control of the pressure-temperature ratio and its highly reduced cooking time compared to handcrafted methods [2,20,21]. Yet this process has serious costs for the quality and organoleptic characteristics of the final product as using diffusers for the thorough extraction of the sugars causes the addition of other secondary metabolites to the must, the fluid subjected to distillation, lowering the quality of the final product [22].
Mezcal, unlike tequila, is obtained from more than 50 agave species mostly found in the wild in Mexico. In 2007, Colunga-GarcíaMarín stated that at least 56 taxa are used for the production of this beverage. There is a wide diversity in mezcal production, which is due both to the diversity of the agave species used, and the variety of production processes. Thus, while tequila production is standardized and industrialized, the production of mezcal is generally handcrafted varying between regions so its flavor, aroma, alcohol content and quality depends on the region of production, the species of agave used and the mezcal manufacturer in charge of it [23]. Although mezcal production is generally handcrafted, industrial and so-called "ancestral" production processes are also used. Yet generally, traditional mezcal usually uses pit ovens or masonry pools for fermentation, grinding is carried out with tahona or Chilean mill, and distillation is made in copper stills or clay pots.
Bacanora is the local mezcal of the state of Sonora and is obtained from a variety of A. angustifolia, from which mezcal espadín is derived [24]. The variety A. angustifolia var. pacifica differs mainly in the cultivation of the agave as it is a wild agave with no use of fertilizers or pesticides and its distillation replaces the copper stills with metal barrels heated with mesquite wood. Currently, few producers are dedicated to the production and marketing of bacanora [25,26].
Coastal raicilla is obtained from A. rhodacantha and A. angustifolia, while the highland raicilla is acquired from A. maximiliana. This was a popular mezcal among miners of the mountains of the Sierra Madre Occidental of Jalisco and its production is currently only locally handcrafted by around 70 producers in 2014 [2,27].
The consumption of agave-derived products has increased in recent years and therefore its demand is rapidly growing nationally and internationally, mainly that of the distilled beverages. This demand implies a greater quantity of supply and modern technology in production processes, thus decreasing traditional methods and craftsmanship. That being said, it is important to emphasize that depending upon the Agave species and the local conditions of soil, humidity and nutrients, it may take between seven, ten, or 36 years or more (i.e., A. tequilana, A. potatorum, A. seemanniana, respectively) [4,[28][29][30] for them to reach harvest maturity, so if sustainable practices and adequate environments are not secured for wild individuals, the future of their populations could be in jeopardy.
In the case of mezcal, this evolving tradition and increased demand has forced some producers to incorporate new technologies and infrastructures to be capable of adapting to a rapidly growing and demanding market, yet if proper management practices of agave populations are lost, it could result in a reduction of population size and genetic variability. Species such as A. tequilana for making tequila or A. fourcroydes to manufacture henequen, use clonal propagation, where suckers of the same genotype are grown and thus their genetic diversity is eroded generation after generation. The loss of genetic diversity carries a reduction of its adaptability to environmental and climate changes, pathogens and pests [2,23,[30][31][32][33].
As with any species, genetic diversity of Agave species is crucial to secure its evolutionary processes and adapt to a changing environment. Most species of Agave exhibit sexual reproduction and are usually cross-pollinated, contributing to greater genetic diversity in the wild. However, many species also exhibit asexual reproduction, which can also be useful if there are few pollinators available, but it could be detrimental to genetic diversity in agave crops [23,30,31].
The objective of this study is to describe an updated compilation of the species of both subgenera of the genus Agave (Agave and Littaea) found in Mexico, analyzing available data for each species on its uses, biology and extinction risk, and suggesting alternative strategies to avoid extinction or detrimental uses of particular species.

Methodology and Analysis
A review using different digital searches, with emphasis in databases, dissertations and literature in Spanish, several English digital researches, Howard Scott Gentry's physical Agaves of Continental North America book and previous reviews published on the Agave species found in Mexico divided by the two subgenera, Agave and Littaea, was carried out using the following criteria: distribution in Mexico, common names, pollinators and floral visitors, common uses, type(s) of reproduction, endemism, extinction risk according to the IUCN Red List [34], subspecies, and a calculation was made, based on the total importance of each species supported by the parameters mentioned above. The five criteria regarding the importance score of each species have similar weight and are explained below. It is important to emphasize that although the species from the subgenus Littaea are of less economic importance than the subgenus Agave, it is relevant to include them in the analysis because of their biological importance.  [35]. Graphics were made with the package ggplot 2 [36]. A summary of the analysis of both subgenera is listed below with Table 1 showing the subgenus Agave and Table 2 the subgenus Littaea. The last column titled Importance Score is based on five criteria obtained from the analysis, which are considered to be very relevant regarding the conservation status and biological environment of each species; the higher the number, the greater the extinction risk a species faces.           Table 3 is the criteria and corresponding weight, or score, of each species used to create Table 4 where the species with the highest total importance scores were placed under three possible categories mentioned below. The higher the score of the parameters in Table 3, the higher the risk of extinction for the species, therefore those species require more attention, although precautionary measures should also be taken with the rest of the species. Table 3. Weighted score criteria regarding the data collected from the Agave species.  The species with the greatest importance score were arranged by subgenera and separated into three categories as follows: 1. Severely endangered: those species facing the possibility of extinction and that should be subjected to an urgent rescue and recovery program, considering the possibility of stopping harvest from the wild altogether. 2. High risk of extinction: these species need a recovery program and their harvest should be subjected to a strong conservation program. 3. Threatened species: management and recovery plans must be prepared and implemented for their harvest.

Risk Level Based on the IUCN Red List
In order to evaluate the influence of score criteria in the Agave species, a Principal Component Analysis (PCA) was performed for each subgenus with the package Fac-toMineR [98], with the variables and categories used for the Importance score reported in Table 3.

Results and Discussion
The species distribution for the subgenera Littaea and Agave by state of Mexico are shown in Figures 1 and 2, respectively. It is noteworthy that there are no records in Baja California or Yucatán for Littaea, and for Agave there are only three reported species in Yucatán, while for both subgenera, there are no published records for Quintana Roo, Campeche and Tabasco, which can be due to a lack of sampling in these areas. In Littaea the highest richness occurs in the southern state of Oaxaca, while for Agave also Oaxaca is the richest state, along with Sonora and Chihuahua in the northwestern part of the country. Both subgenera have lower numbers of species in the center of the country.
In both subgenera, the proportion of plants used for food is similar (Littaea 18.6% and Agave 20.7%, Figure 3), while Agave species are much more used for making distilled beverages (mainly mezcal and tequila) (Littaea 8.9% and Agave 18.8 %). The better-known species used for the production of distilled beverages were not considered at high risk, due to the proportionally greater knowledge available, although many of them might be endangered because of the lack of proper production management. For instance, these popular Agaves are now cultivated in massive monocultures, while preventing any sexual reproduction, and thus their genetic diversity is rapidly declining, such is the case of A. tequilana [6,18].  It is important to note that most of the species included in Table 4, which need of specific conservation efforts, are used for fiber, ornamentals or have no k uses. There is very little information regarding pollinators and visitors in both subg for most species (more than 75%, Figure 4). Obviously, this is important inform necessary to develop adequate conservation and management strategies. Bat polli records are much greater in Agave than in Littaea ( Figure 5). For visitors, available mation shows an important difference between subgenera ( Figure 5), but more s are needed to disentangle if these visitors are acting as true pollinators. There is The use for fibers is proportionally similar for Littaea and Agave (18.6% vs. 16.5%, respectively, Figure 3), compared to the use of ornamentals, where Littaea has a higher use (28.3% vs. 18.8%, respectively, Figure 3). Species used for fiber, such as A. parrasana, A. albopilosa, A. pelona and A. pintilla were categorized at a high risk of extinction, while A. lurida and A. nizandensis are considered severely endangered and used as ornamentals. It is outstanding that in both subgenera the vast majority of the reported species have some kind of use (Litteae 88.9% and Agave 93.03%, Figure 3).
It is important to note that most of the species included in Table 4, which are in need of specific conservation efforts, are used for fiber, ornamentals or have no known uses. There is very little information regarding pollinators and visitors in both subgenera for most species (more than 75%, Figure 4). Obviously, this is important information necessary to develop adequate conservation and management strategies. Bat pollination records are much greater in Agave than in Littaea ( Figure 5). For visitors, available information shows an important difference between subgenera ( Figure 5), but more studies are needed to disentangle if these visitors are acting as true pollinators. There is ample information for the general type of reproduction in both subgenera ( Figure 5), which may be due to the fact that as mentioned above, species from both subgenera are widely used, so there is an interest in having this information.   The PCA analyses of the different variables reported in Table 3 for both subgenera (Figures 6 and 7) were divided into four quadrants: I. Species with higher importance scores are in Quadrant I. Many of these species are endemic and have many uses, so they can be considered species of main concern for conservation and management.
II. This quadrant contains endemic species with little use and with little or no reproductive information, which is mainly associated with a lack of interest, in contrast to species in quadrants I and III.
III. Species with many uses, so much more reproductive information is available, but a lower importance score than species in Quadrant I.
IV. Species that apparently have no use and are not endemic, but reproductive information is lacking for them.
Having an understanding of the status of each species of the genus Agave in Mexico is fundamental to species conservation and sustainable use, and to create awareness of the practices used nowadays on the utilization of the different species of the genus, averting the possible tragic outcome of extinction. Artisanal and industrial producers are invited to maintain or opt for biodiversity-friendly alternatives for the conservation of agaves, practicing sustainable methods in their practices with agaves. Suggested approaches include the use of species considered at low risk of extinction for the production of distilled beverages (for instance A. americana, A. duranguensis, A. karwinskii or A. macroacantha) or ecofriendly production methods for agaves and their pollinators, such as the Bat Friendly Tequila and Mezcal project [99] created by UNAM (Universidad Nacional Autónoma de Mexico) and the Tequila Interchange Project [19,100]. This project seeks to preserve genetic diversity, seed production and pollinator populations, including bats, by allowing 5% of the agaves in agave fields destined for production of distillates to flower and produce seeds.
Restricting the use of agave species that are at risk may not be an adequate method for the conservation of some species, except in the most critically endangered cases, given that many Mexicans depend on their production to survive [2] and such restrictions might trigger a wave of poaching plants from wild populations. The great potential of sustainable management practices to ensure the future of particular agave species such as the maguey papalote (A. cupreata) and its derivatives and products has already been demonstrated. By providing greater visibility to sustainable practices by the local farmers, knowledge exchange between producers and science and preserving ancestral traditional production practices, agaves clearly have a profitable, sustainable, environment-friendly, socially responsible, and economically viable future [101,102].  .   Tables 1 and 2 for details. productive information, which is mainly associated with a lack of interest, in contrast to species in quadrants I and III. III. Species with many uses, so much more reproductive information is available, but a lower importance score than species in Quadrant I.
IV. Species that apparently have no use and are not endemic, but reproductive information is lacking for them. Figure 6. Principal Component Analysis of the Agave subgenus Agave using the IUCN category, uses, endemism, and reproductive data as variables (see Table 1 for details). Dot color represents the Importance Score obtained. Figure 6. Principal Component Analysis of the Agave subgenus Agave using the IUCN category, uses, endemism, and reproductive data as variables (see Table 1 for details). Dot color represents the Importance Score obtained. . Principal Component Analysis of the Agave subgenus Littaea using the IUCN category, uses, endemism, and reproductive data as variables (see Table 2 for details). Dot color represents the Importance Score obtained.
Having an understanding of the status of each species of the genus Agave in Mexico is fundamental to species conservation and sustainable use, and to create awareness of the practices used nowadays on the utilization of the different species of the genus, averting the possible tragic outcome of extinction. Artisanal and industrial producers are invited to maintain or opt for biodiversity-friendly alternatives for the conservation of Figure 7. Principal Component Analysis of the Agave subgenus Littaea using the IUCN category, uses, endemism, and reproductive data as variables (see Table 2 for details). Dot color represents the Importance Score obtained.
It is also important to mention that the taxonomy of the genus Agave has been a complicated task for scientists, given that the genus is one of the most species-rich in the plant kingdom, and therefore recent discoveries that indicate synonymies and cryptic species are constant events, resulting in ongoing changes and descriptions of new species (see [31]). We must emphasize that there is still a great amount of data to collect on recently described species along with their distributions, natural history, current uses and pollinators to fully understand their biology, potential as providers of services and products, and conservation needs [31]. This study is just the beginning of an effort to update the knowledge about the uses, information available, and conservation needs of Agave species, and we hope to continue to facilitate updates in the future.
Agaves are among the most ecologically and economically important plants of Mexico. From an economic perspective, the role of Agave in production of distilled beverages and fibers, their ornamental relevance, and rural uses as living fences and building materials are very important for the country. In particular, exports of tequila and mezcal are undergoing a huge upward trend. Tequila exports went from 209 million liters in 2018 [103] to 249.4 million liters in 2021 with a net value of $2.3 billion dollars [17]. Mezcal exports went from 2.7 million liters to 7.1 million liters only from 2016 to 2018, with a value of $53 million dollars [103][104][105]. People directly or indirectly involved in the production of mezcal and tequila depend on the income generated by these distilled beverages. Additionally, Mexicans throughout the country depend on agaves for different products such as handcrafts, baskets or ropes made from their fibers, their use in Mexican cuisine for many typical dishes, and the preparation of the pre-Hispanic fermented drink, pulque.
Agaves are emblematic plants for Mexico and have been represented in codex, archeological vestiges and cultural traditions since pre-Hispanic times, being nowadays an extensive source of income and everyday life uses. We must prevent the extinction of agave species due to greedy, industrialized production and cultivation practices. Respect for traditional procedures, clear recognition and incorporation of traditional indigenous knowledge and preserving, creating, and implementing the best sustainable production practices is absolutely crucial for the future of agaves and their pollinators. Local communities demand this with reason, Mexico deserves this, and the world should expect nothing less. The future of Agave and its pollinators should be one of appreciation, respect, and responsible consumption of agave products, solidly anchored in environmental sustainability, social justice, and economic viability.

Conclusions
Our analysis highlights the great diversity of species of the genus Agave in Mexico, the intense level of use that some species are undergoing, the species-specific extinction risk level, and the lack of knowledge about most species. In the process of constructing this diagnostic analysis, it was clear that most of the available information for most species is scarce given their usefulness and distribution.
For the agaves used in mezcal production, we believe that the most convenient solution is to opt for strict sustainable management practices and favorable reproduction methods, while avoiding monoculture procedures that could affect genetic variability, and reduce or avoid the use of pesticides that affects pollinators thus affecting gene flow and connectivity between species. It is important to favor the natural sexual reproduction of the species, promoting natural pollination and protecting their pollinators. Traditional practices should be encouraged for the management of agaves, recognizing monocultures and high industrialization processes as inadequate and only happening under highly controlled conditions, ensuring the future of biodiversity and emphasizing the knowledge of local, traditional producers.