Genetic Conservation and Use of Genetic Resources of 18 Mexican Pine Species

: Genetic conservation is key to maintaining forests for the future; however, these face several threats. Mexico is an example of the degradation of forest genetic resources during the past three decades due to its deforestation rates. This country is considered a center of pine diversity but its genetic conservation efforts are not enough. To deﬁne genetic conservation units (GCUs) and propose measures for the conservation and sustainable use of 18 Mexican pine species, we analyzed the distribution of the species at the national level and in germplasm transfer zones, and evaluated the species with a set of minimum requirements for conservation and indicators from the EUFORGEN program. We determined that 13 to 15 genetic zones harbored the target species, in which Pinus teocote , Pinus cembroides , Pinus devoniana , Pinus maximinoi , Pinus douglasiana , and Pinus leiophylla were the most widely distributed. We deﬁned 173 areas for establishing GCUs for the total of the species studied; 50% of them were selected from areas with genetic information, 5% were selected from seed stands, and 45% from natural forests. We detected that most of the forest reproductive material used is collected from seed stands, and the use of seeds from breeding trials is scarce.


Introduction
Facing continuous environmental changes, genetic conservation is a key to maintaining forest resources for the future. The main objective of genetic conservation is to preserve the genetic integrity and natural levels of genetic diversity [1]. However, conservation activities are limited by the financial, temporal, and technical resources available [2]. For this reason, activities must be selected by keeping in mind the species with ecological, economic, and social potential uses by country based on current conservation status, potential economic use, threat of genetic erosion, genetic distinction, ecogeographic distinction, national or conservation agency priorities, biological importance, cultural importance, relative cost of conservation, conservation sustainability, and ethical and aesthetic considerations [3].
Forest genetic resources have been used for testing of tree species and provenance for different uses and under different environmental conditions [4]. The purpose of a provenance test is to establish the most healthy and fastest growing population among many others of the same tree species, in order to select such a population as a seed source for planting at appropriate sites [5]. This is a tool for improving forest genetics. In common garden experiments, also known as provenance trials, it is possible to test which provenances have adapted to expected climate conditions [6] because tree species possess The species distributions were determined based on geographic data (latitude and longitude) using the plots of the National Forest and Land Inventory (NFLI, 2004(NFLI, to 2007), which covers Mexico in a systematic stratified random sampling with a grid of 5 km (Supplementary Materials Figure S1). Plots which considered forest plantations were removed. The geographic data were presented using QGIS software 3.28.2. [35].

Genetic Zones
The germplasm transfer zones (equivalent to seed zones) were used as a proxy because genetic zones are still undefined for the country. Seed zones are homogeneous areas in terms of climate and latitudinal or longitudinal distribution [36] for regulating seeds, fruits, and vegetative material. These seed transfer zones were previously defined by the Mexican National Forest Commission (CONAFOR by its Spanish acronym), and are available at https://www.gob.mx/conafor (accessed on 8 August 2022). The genetic zones were obtained for the 18 Mexican pines species by overlapping their distribution with the germplasm transfer zones. All genetic zones with less than 20 trees for each species reported in the NFLI were excluded from subsequent analysis. For the geographic data, QGIS software 3.28.2. was used [35].

Conservation Units
Genetic conservation units (GCU) were defined as an in situ conservation strategy. It was followed a set of minimum requirements for defining the units based on population size, management, monitoring, and ownership [37] (Supplementary Materials Table S2). We selected at least one GCU per genetic zone and species, but in fragmented distribution, one unit was selected from each patch. During the selection, it was prioritized extensive and centered populations in the genetic zone or patch. For each genetic zone, first, we chose the populations characterized in previous genetic studies or provenance or progeny trials [12,17,18,[22][23][24][25][26][27][28][29] (Table S3), which were selected for possessing representative trees within the zones. The previous studies and trials were obtained from an exhaustive documentary review from printed and digital scientific journals. Secondly, they were chosen populations located in seed stands defined by the Mexican National Forest Commission (CONAFOR, information available for 2021 at https://www.gob.mx/conafor, accessed on 5 September 2022). Thirdly, we selected populations from protected areas for biodiversity conservation established by the Mexican Commission for Biodiversity (CONABIO, information for 2016 available at http://www.gob.mx/conabio, accessed on 7 September 2022), instead private ownership. Finally, we selected populations situated in private forests. The geographic data were represented using QGIS software 3.28.2 [35].

Use of Genetic Resources
For each genetic zone, the presence of germplasm production units (seed stands or seed orchards) were used in order to define the use and importance of genetic resources. We assumed that the effort and investment during the establishment and maintenance of those units was an economic indicator of the species in the area. Then, genetic trials established by different institutions, that is, provenance and progeny trials, were used, as they report information about populations identified for choosing forest reproductive material with well-known genetic backgrounds.

Importance of Genetic Zones for Conservation and Sustainable Use
Based on the previous information, six species indicators were collected in all genetic zones: the number of trees sampled by the NFLI (n i ), the number of populations with molecular data (n mk ), the number of populations with seed stands (seed stand plus seed area) (n st ), the number of individuals selected for progeny tests (n is ), the number of populations present in provenance tests (n pt ), and the number of seed orchards (n so ). Finally, we estimated the number of GCUs (n g ). For each species, their status was calculated by applying indicators assigned and adapted from the EUFORGEN program [90] (Table 1).  The importance of the genetic zones was calculated using the previous information published by studies [91][92][93], which defined their reforestation and restoration potential, and timber production by genetic zone. In these studies, a proportional assignment to each genetic zone was made using as a basis the distribution of the 18 species and those harbored in genetic zones. We also determined a value for conservation and a value for breeding for the different genetic zones of the species in a subjective scale from 1 to 4 [31], with 4 being the highest priority. For this, the expert knowledge and the recommendations established by the papers or reports previously analyzed were considered.

Use of Genetic Resources
For the target species, the conservation efforts and use of genetic resources by genetic zone were quite contrasting (Supplementary Materials Tables S5-S9). The genetic characterization was null or narrow for some species and regions, that is, P. jeffreyi, P. lawsonii, P. maximartinezii, P. quadrifolia, and P. cembroides subsp. orizabensis, but broader for others, that is, P. arizonica. The efforts to use forest reproductive material were reduced and concentrated on selecting seed stands (Table S7), for example, P. arizonica and P. durangensis had more seed stands than other species (26 and 20, respectively), but few or no seed orchards (2 and 0, respectively) or provenance/progeny trials (0 for both species). We detected many gaps in different regions for all of the species, but some zones had no efforts to use forest reproductive material; that is, I.

Conservation and Management of Genetic Resources
The conservation efforts, characterization, and use of forest genetic resources for the 18 species were incomplete ( Table 2). Most of the genetic zones had gaps for the characterization of forest genetic resources or even seed production. Molecular studies were null for P. jeffreyi, P. lawsonii, P. maximartinezii, P. quadrifolia, and low for P. devoniana; the rest of the species ranged from 100 to 22.22% where P. strobiformis and P. ayacahuite possessed the higher values (100 and 85.71%, respectively). P. arizonica and P. leiophylla were the only species that had seen seed orchards established (I6 > 0). Provenance and progeny trials had been stablished for a few species, in which their efforts were limited (I3 and I4 > 0). P. arizonica and P. durangensis had more forest reproductive units, covered several states, and were used for reforestation and restoration of degraded lands and timber production.

Use of Genetic Resources
For the target species, the conservation efforts and use of genetic resources by genetic zone were quite contrasting (Supplementary Materials Tables S5-S9). The genetic characterization was null or narrow for some species and regions, that is, P. jeffreyi, P. lawsonii, P. maximartinezii, P. quadrifolia, and P. cembroides subsp. orizabensis, but broader for others, that is, P. arizonica. The efforts to use forest reproductive material were reduced and concentrated on selecting seed stands (Table S7), for example, P. arizonica and P. durangensis had more seed stands than other species (26 and 20, respectively), but few or no seed orchards (2 and 0, respectively) or provenance/progeny trials (0 for both species). We detected many gaps in different regions for all of the species, but some zones had no efforts to use forest reproductive material; that is, I.

Conservation and Management of Genetic Resources
The conservation efforts, characterization, and use of forest genetic resources for the 18 species were incomplete ( Table 2). Most of the genetic zones had gaps for the characterization of forest genetic resources or even seed production. Molecular studies were null for P. jeffreyi, P. lawsonii, P. maximartinezii, P. quadrifolia, and low for P. devoniana; the rest of the species ranged from 100 to 22.22% where P. strobiformis and P. ayacahuite possessed the higher values (100 and 85.71%, respectively). P. arizonica and P. leiophylla were the only species that had seen seed orchards established (I6 > 0). Provenance and progeny trials had been stablished for a few species, in which their efforts were limited (I3 and I4 > 0). P. arizonica and P. durangensis had more forest reproductive units, covered several states, and were used for reforestation and restoration of degraded lands and timber production.

Importance of Genetic Zones for Conservation and Use of Genetic Resources
For reforestation and restoration areas, XII.4, XII.5, X.3, X.2, XII.1, V.3, XII.2, and XV.1 zones were the most important due to having the potential of seedling production, germplasm production units, and seed conservation banks (see [91]); whereas for timber production, X.3, V.3, XII.5, XII.3, X.1, XII.2, III.1, and III.2 were the best zones due to their amount of timber harvested and used for the industry (see Tables 1 and 2 from [93]). Based on this information, it was defined that V.3, X.2, X.3, XII.1, XII.2, XII.4, XII.5, and XV.1 were the most suitable areas of germplasm for the target species studied (Figure 3), which presented the highest potential for conservation and breeding. Although they were not the genetic zones with the greatest number of trees inventoried, they were the most diverse in the number of species and the ones where the actions dedicated to the conservation and use of genetic resources were more intensive (Table 3).
R PEER REVIEW 7 of 17 amount of timber harvested and used for the industry (see Tables 1 and 2 from [93]). Based on this information, it was defined that V.3, X.2, X.3, XII.1, XII.2, XII.4, XII.5, and XV.1 were the most suitable areas of germplasm for the target species studied (Figure 3), which presented the highest potential for conservation and breeding. Although they were not the genetic zones with the greatest number of trees inventoried, they were the most diverse in the number of species and the ones where the actions dedicated to the conservation and use of genetic resources were more intensive (Table 3).

Discussion
The priority zones were defined to help the conservation and the use of forest genetic resources in 18 Mexican pine species. Based on the germplasm transfer zones [36] as a proxy for genetic zones, 173 areas have been identified for establishing conservation units into 22 genetic zones. Additionally, it was considered that eight genetic zones could supply the forest reproductive material in order to be used for the species' afforestation, restoration, and for establishing plantations.
We detected that some species were distributed more than others, and have high levels of genetic variation due to their biological characteristics; for example, P. cembroides is one pine that harbors more than one temperate mountain range, and P. teocote is the most abundant and widespread in Mexico, both having a high level of genetic diversity [15,51]. It is possible that the species' distributions are influenced by the environment and their ecological factors. This is in line with the authors of [39], who determined that elevation is an important factor that drives the geographical location and genetic diversity of the species. Additionally, they pointed out that P. cembroides possesses the ability to adapt to its growth conditions. On the other hand, some species have a more restricted distribution but possess high genetic variation as P. arizonica [15,52], which is according to the authors of [22] who studied forests in Chihuahua state. Based on the results of study [38], this is possible due to their populations having enough individuals and sufficient outcrossing to prevent genetic drift.
Most of the species have potential for restoration of land; consequently, they are used during reforestation or conservation programs, for example, seedlings of P. cembroides and P. devoniana are the most produced in the nurseries for the reforestation of areas across the country [91], while P. douglasiana, P. maximinoi, and P. teocote are species most harvested and used in the forest industry [93]. P. cembroides is a tree to tolerate drought and is able to grow in sites in poor soils [94], while P. devoniana is a pine to resist drought [95]. These characteristics have been attractive for nurserymen during seedling production and for planters in the restoration of semi-arid regions and dry zones. However, sometimes hard conditions in the sites selected (hydric stress and low mineral nutrition) can strongly reduce their survival [96]. For conservation, P. cembroides has been suggested to promote the natural functions of forest dynamics in reforested areas [97].
Although there are efforts to conserve Mexican pine species, the preservation of their genetic resources is limited because the current legislation only points out the use of forest genetic resources [98,99] but does not define conservation strategies. The little conservation actions made for governmental agencies depend on their budget, which is more scarce each year. These conditions demand some activities during forest management proposed by the authors of [100] that help to maintain the populations of target species as (i) limiting livestock grazing, (ii) controlling forest fires, (iii) eliminating competing vegetation, (iv) expanding the present populations by reforestation outside the priority populations' boundaries using local reproductive material, (v) finding new and potentially suitable locations and establishing new populations that are sufficiently large to be genetically viable, (vi) establishing provenance trials and traditional progeny tests as genetic archives, and (vii) storing seeds in gene banks.
The results showed that the information about genetic characterization for most species is not enough; however, the results can help start a national conservation program for the species most used in forest management. Some of the target species have been more characterized than others which could be due to their economic importance, for example, P. douglasiana or P. arizonica, which produce an average of 56 554 and 13 410 m 3 year -1 timber, respectively [93]. The conservation of genetic diversity must be a priority because it is the basis for the eventual adaptation and resilience of tree species studied regarding environmental stress and change [101]. On the other hand, although there is a Mexican network of Natural Protected Areas (NPA) that harbor some of the tree species and could be used as genetic diversity reserves, this is insufficient for future conservation needs. According to the authors of [102], the protection of pine species is inadequate because they possess a small percentage of the area of the total species, therefore, they suggest directing this strategy (NPA) to species with limited distribution such as P. cembroides subsp. cembroides.
The purpose of defining genetic conservation units is to generate a network in the country in order to maintain the Mexican pines which are facing current environmental changes in other parts of the world; for example, in Europe, the establishing of genetic conservation units is a tool to assess, monitor, and support the conservation of forest genetic resources [90,103]. This action can allow the future use of pines from Mexico in three programs: breeding, production, and conservation.
The target species have high genetic diversity. According to the authors of [104], the Mexican pines possess high levels of genetic variation and relatively little genetic differentiation among populations. However, there are other species with low levels of diversity such as P. greggii Engelm. ex Parl., which is used in national or international breeding programs [12,80]. P. ayacahuite, P. leiophylla, and P. teocote are some pines that have provenance trials to define their potential for commercial plantations, for example, P. ayacahuite is one of the species most used for Christmas tree producers [105] while P. leiophylla and P. teocote are managed in natural forests for timber production [93]. Facing climatic changes, adaptive trials have been tested in the studied species: P. ayacahuite, P. arizonica (water availability and water stress) [106,107]; P. arizonica, P. leiphylla, and P. teocote (fire damage) [108]; P. arizonica, P. ayacahuite, P. cembroides, P. chiapensis, P. devoniana, P. durangensis, P. engelmannii, P. hartwegii, P. jeffreyi, P. lawsonii, P. maximartinezii, P. maximinoi, P. montezumae, P. strobiformis, P. teocote (frost resistance) [109], and Pinus cembroides (salt tolerance) [110].
Some of the areas selected could be affected by illegal extraction of timber [111] or other factors that limit genetic conservation, for example, land use change for agriculture [112]. However, the establishing of GCUs in protected areas can help with maintaining them in the long term because the rate of forest loss is lower [113]. In these areas, the financial resources, human capacity, and appropriate equipment are the main aspects that have the greatest effect on forest land loss [114]. For this reason, administration and finance must be continually evaluated during the management of the protected areas in order to preserve the UCGs selected.
On the other hand, the diversity of pines from Mexico across the mountain regions promotes habitats that harbor local wildlife. This is an element to add more importance to forest conservation. This is in line with the authors of [115], who defined that natural forests dominated by P. montezumae and P. pseudostrobus conform to the best habitat to comprise the highest bird species richness from Northeast Michoacán, Mexico, for example, Hylocharis leucotis, Archilochus alexandri, Selasphorus platycercus, Trogon mexicanus, and Melanerpes formicivorus.
Finally, we suggest incorporating these GCUs into a national management program based on three steps. Firstly, the legislation must promote and pay for the conservation of the forest resources to the forest owners as this action will motivate the society to protect the GCUs against fires, illegal extraction, grazing, or changes in land use. Secondly, there must be education provided for the communities regarding banning the extraction of forest resources near and inside of GCUs, for example, wood, land, and resin. Thirdly, government institutions, universities, and research institutes must work together to develop a plan to establish the UCGs defined in this work, install common garden experiments in order to evaluate the future effects of climatic changes and analyze adaptation strategies, and promote the conservation ex situ of germplasm in seed banks, for example, in the National Center for Genetic Resources (CNRG by its Spanish acronym). This plan must be accompanied mainly by silvicultural guidelines, activities for the prevention and control of pests and diseases, use of local seed sources for stand renewal, and control of the germplasm movement.

Conclusions
In this study, 173 genetic conservation units were defined for 18 pine species from Mexico in their natural range of distribution and some actions determined for their conservation and sustainable use. P. leiophylla, Pinus teocote, P. cembroides (with two subspecies: bicolor and cembroides), P. devoniana, and P. douglasiana were the species with more genetic conservation units due to their broad distribution. These harbored similar or different genetic zones due to their adaptation to different ecological requirements (temperature, rainwater, soil). For the number of areas detected for genetic conservation units, half of them contain genetic information which was reported in journals, and almost 40% of them were proposed in protected areas based on our selection. We found that most of the forest reproductive material used currently is collected from seed stands, and the use of seeds from breeding trials is scarce. The genetic characterization of the target species has gaps in the genetic zones. Therefore, this demands, in the short term, future studies regarding those species most used in the forest industry, breeding programs, and restoration of degraded lands.