Description of markers used for the study of genetic variability of Pinus hartwegii.
Mountain pine (Pinus hartwegii Lindl) is one of the most abundant conifers in the Protection of Flora and Fauna Area Nevado de Toluca in central Mexico; this natural protected area is threatened by urbanization; this has been manifested in forest health; there has been an increase in forest parasites like bark beetles and dwarf mistletoes, making necessary improve forest management and conservation, hence our objective was to study the genetic diversity of mountain pine under the attack of parasites and to generate information that could be used to improve strategies of conservation of these forests. We classified sampled trees into four categories according to the type of parasite present in a tree (bark beetle: BB; dwarf mistletoe: DM; bark beetle and dwarf mistletoe: BM and non-attacked trees or healthy trees: HT). Genetic diversity was low in comparison with other pine species, but we observed an interesting issue: trees attacked by bark beetle and dwarf mistletoe had higher levels of heterozygosis: Henc = 0.1924 and Henc = 0.1993, respectively. These results suggest that trees with bark beetle and dwarf mistletoes may have higher genetic variability and are a highly valuable genetic resource for mountain pine.
- mountain pine
- bark beetle
- dwarf mistletoe
Forests of the Trans-Mexican Volcanic Belt (TMVB) physiographic region are among the most threatened areas in Mexico. The TMVB region encloses a large number of natural protected areas (NPA), the Protection of Flora and Fauna Area Nevado de Toluca (PFFANT) is one of them. The closeness of this NPA to large cities entails environmental pressures that includes the presence of human settlements, illegal logging, introduction of exotic species and the growing incidence of forest parasites like bark beetles and dwarf mistletoes [1, 2, 3].
PFFANT is formed by conifer forests, with the genera Pinus and Abies being the most representative of this zone [4, 5]. Pinus hartwegii Lindl. is forming large forest stands and is the pine species which grows at the highest altitudes in the PFFANT , unfortunately is affected by dwarf mistletoes (Arceuthobium spp.) and bark beetles (Dendroctonus spp.). The incidence of these parasites is growing, probably because of climate change and deforestation [1, 7, 8, 9].
México ranks fourth in terms of deforestation, with approximately 670,000 ha/year causing losses in genetic diversity and changes in locally adapted populations for example giving place to an increase of parasites populations [10, 11, 12, 13]. Results of forest health diagnoses have suggested that bark beetles are a group of parasites that have affected large extension of forests in Mexico (40.5%), followed by parasitic plants (38.7%), both of them leaving negative consequences in the forest, like high mortality rates of trees affected [11, 14, 15].
Bark beetles grow under the cortex and induce weakening of the tree, the construction of galleries and the inoculation of a staining fungi which is carried by female beetle results death . In the PFFANT, Dendroctonus adjunctus is affecting P. hartwegii [6, 14]. Dwarf mistletoes are obligate heterotrophic plants that acquire all their water and nutrients from their host and can significantly inhibit its growth causing permanent deformation of the stem and crown. This parasite weakens trees in such a way that they become more susceptible to attack by insects, particularly bark beetles. In the PFFANT, P. hartwegii is the host of Arceuthobium vaginatum and A. globosum [6, 8, 15]. Bark beetles and dwarf mistletoes epidemics can lead to shifts in forest, forest successional trajectories and susceptibility to future disturbances [16, 17, 18].
Forest trees are key drivers of terrestrial biodiversity because they function as a carbon sink, preserve the water quality and regulate climate [19, 20]; genetic variability studies are crucial to understand the basic biology of these organisms and to obtain insights on evolution, disease resistance and conservation genetics [21, 22, 23]. In conifer species for example, gene flow is mediated by three types of genomes with contrasting inheritance: nuclear (biparental), mitochondrial (maternal) and chloroplast (paternal) this particularity opens avenues to the study of conifer DNA polymorphism, the study of genetic variability with these three types of markers allow making inferences on the distribution of genetic resources and habitat connectivity .
In this study, nuclear DNA (ncDNA), mitochondrial DNA (mtDNA) and chloroplast DNA (cpDNA) were used to assess the genetic variability and population structure of P. hartwegii populations affected by bark beetles and dwarf mistletoes, expecting that genetic variability will be low in the parasitized populations. We want to contribute to the conservation of mountain pine populations of the PFFANT generating information which helps in the identification of populations genetically valuable.
2. Materials and methods
2.1. Study zone
Sampling was carried out in the Protection of Flora and Fauna Area Nevado de Toluca, which forms part of the Trans-Mexican Volcanic Belt physiographic region. The geographical coordinates of the study zone are 18° 51′ 31″ and 19° 19′ 03″ N and 99° 38′ 54″ and 100° 09′ 58″ W; it is a priority region for conservation due to its diversity of ecosystems in which pine-fir forests and high mountain prairie dominate [1, 25].
2.2. Plant material
We sampled a total of 180 individuals of P. hartwegii. A distance of 50 m was between each tree sampled. Samples were classified into four groups (categories): trees with signs of attack by bark beetles (BB), trees with signs of attack by dwarf mistletoe (DM), trees with signs of attack of both parasites (BM), and trees with no signals of any parasite, which were considered as healthy trees (HT). Each sample consisted of young needle tissue. Immediately after collecting the needles, they were placed on ice for transport to a laboratory where they were maintained at −70°C.
2.3. DNA isolation
Cetyltrimethylammonium bromide (CTAB) method was implemented with a few modifications . Needle tissue was ground to a fine powder with a chilled mortar and pestle, making two washes, the first with 100% ethanol (v/v) and the second with 75% ethanol (v/v). DNA was re-suspended in 70 μL TE and stored at −20°C until it was used.
2.4. PCR amplification reactions
PCRs were performed in a reaction volume of 10 μL, containing: ammonium buffer 15 mM, MgCl2 25 mM, dNTP mix 10 mM, primer 20 μM, DNA 10 ng/μL and 0.5 U of Taq polymerase (Sigma). To amplify nuclear (ncDNA), we used anchored microsatellites (ASSR) which proved to be genetically stables and heritable: ASSR-15 and ASSR-29  and one operon: UBC 254 , one cytochrome oxidase sequence: cox3in, was used to amplify mitochondrial (mtDNA)  and one highly polymorphic microsatellite: 10FF/RR, to amplify chloroplast (cpDNA)  (Table 1).
|Primer name||Marker type||Sequence (5′ → 3′)|
|UBC-254||Nuclear operon ||5′-CGCCCCCATT-3′|
|ASSR-15||Nuclear anchored microsatellite ||5′-(CT)7GCA-3′|
|ASSR-29||Nuclear anchored microsatellite ||5′-(CT)7GTA-3′|
|ASSR-20||Nuclear anchored microsatellite ||5′-(CT)7ATG-3′|
|COX3in||Mitochondrial cytochrome oxidase III gene sequence ||5′-GTA GAT CCA AGT CCA TGG CCT-3′|
5′-GCA GCT GCT TCA AAG CC-3′
|10 FF/RR||Chloroplast microsatellite ||5′-CAGAAGCCCAAGCTTATGGC,|
Amplifications were performed in a Master Cycler Gradient (Eppendorf) Thermal Cycler in all 40 cycles. For ncDNA primers: 1 min of denaturation at 94°C, 1 min of annealing at 48°C and 1 min of extension at 72°C. For mtDNA primers: 1 min of denaturation at 94°C, 1 min of annealing at 48–55°C and 1 min of extension at 72°C. For cpDNA: 1 min of denaturation at 94°C, 1 min of annealing at 58–60°C and 1 min of extension at 72°C; all samples with all primers were given a 7 min of pre-amplification denaturation at 94°C and a 5 min of post-amplification at 72°C.
2.5. Electrophoresis in agarose gel
The amplification products were separated by electrophoresis in agarose gel (1.5%) at constant voltage (100 V and 90 mA). Gels were visualized by UV transilluminator (UVP) with ethidium bromide (10 mg/mL).
2.6. Scoring of bands and data analysis
DNA patterns were inferred according to dominant nature of markers used, so each amplified product was scored for all genotypes for its presence or absence, we made a binary matrix in which band presence was assigned a value of one (1) and the absence of a band a value of zero (0). Co-migrating bands were assumed as the same locus and the same band when scoring.
We used PopGene 32 , Genealex 6.5  and TFPGA  to obtain genetic diversity parameters: mean number of alleles per locus (A), mean number of observed alleles (na), effective number of alleles (ne), Nei’s genetic diversity indices (He); as long as we sampled parasitized trees we used Graphpad Prima 7.0 to perform a chi square test and detect if there were differences in heterozygosis according to the categories considered in sampling. The number of polymorphic alleles (LP), percentage of polymorphic alleles (%LP), population structure fixation indices (GST), indicators of heterozygosis (HT: total genetic diversity of the locus, HS: genetic diversity within populations), gene flow (Nm) and Nei’s genetic distance (D) between the four categories we obtained BB, DM, BM, and HT .
We obtained electrophoretic patterns with high reproducibility and clear band resolution. UBC-254: 11 bands, ASSR-15: 7 bands, ASSR-29: 11 bands, Cox3in: 8 bands and 10FF/RR: 7 bands, combined in 139 band patterns, ranging from 200 to 2000 bp; additionally there were bands only present in HT trees.
3.1. Genetic variability
The number of alleles per locus (na) and the number effective alleles (ne) ranged from 1.3 to 2.0 indicating that the number of alleles transferred from one generation to the next is low; these low values of na and ne consequently act on heterozygosis (Nei’s genetic diversity: He), which also was low compared with other Pinus species (Table 2).
|na||1.9 ± 0.3620||2||1.9 ± 0.3620||1.7 ± 0.4237||1.4 ± 0.5175||1.8 ± 0.4629||1.6 ± 0.5175||1.5 ± 0.5345||1.4 ± 0.5345||2||1.4 ± 0.5345||1.6 ± 0.5345|
|ne||1.3 ± 0.2964||1.3 ± 0.3067||1.7 ± 0.2872||1.3 ± 0.2666||1.0 ± 0.1433||1.1 ± 0.1859||1.1 ± 0.2395||1.0 ± 0.1047||1.0 ± 0.0395||1.1 ± 0.2033||1.0 ± 0.1056||1.1 ± 0.1661|
|He||0.1924 ± 0.1648||0.1993 ± 0.1586||0.1877 ± 0.1526||0.1831 ± 0.1552||0.0539 ± 0.1016||0.0810 ± 0.1196||0.0872 ± 0.1382||0.0582 ± 0.0828||0.0295 ± 0.0368||0.1267 ± 0.1260||0.0592 ± 0.0857||ND|
Among categories (BB, DM, BM and HT) we observed, in some cases, a tendency of BB, DM and BM to present higher values of He, for example with ncDNA BB and DM categories showed He values 0.1924 and 0.1993, respectably, higher than in HT trees which was 0.1831, with cpDNA DM trees presented high He compared with HT (Table 2). According to Chi square tests, the distribution of He showed statistically significant differences between the BB, DM, BM and HT categories (Table 3).
3.2. Population structure
Estimated population structure based on GST (fixation index) was very low probably due to high levels of gene flow (Table 4). The rates of gene flow (Nm) derived from GST were very high with all markers, averaging 15.1 migrants per generation (Table 4). Based on these results (low-population differentiation/high gene flow), we assume that inbreeding rates are low.
Genetic distances ranged from 0.003 between DM/BM with mtDNA and DNA cp markers to 0.014 between HT/DM and HT/BB with ncDNA and cpDNA markers; for all markers parasitized trees had the highest genetic distances between nonattacked trees (HT) and attacked trees (BB, DM and BM) (Table 5).
We found low levels of genetic variability in mountain pine, but also that parasitized trees in some cases had highest levels of heterozygosity; this is not rare if we take into account that plants are subject to various abiotic and biotic stresses, especially those long-lived species like conifers; thus, slow-growing plants will invest heavily in defenses against parasites because of high cost of replacing tissue [9, 36]; substantial variation in susceptibility, damage and resistance are well documented in natural plant populations [21, 37, 38, 39].
4.1. Genetic variability
Levels of heterozygosis in mountain pine were in general low, but in spite of this with ncDNA, we observed a tendency of parasitized trees (BB and DM) to have more genetic variability and this is relevant because in a population stressed by an increment of parasites, high genetic diversity individuals will have more chances to adapt themselves to changes in their environment and attack by parasites and pathogens.
Some theories propose that dwarf mistletoes performance is regulated by physiological (genetic) condition of the host and that infections are greater in sites with high stress, also long-term contact and evolutionary history between specific plant and parasites are expected to increase plant defenses, tree species have been co-evolving with mistletoes for 25 million years, so high genetic variability of parasitized organisms is an insight of co-evolution system [16, 40, 41]. The genetics of host resistance due to a co-evolutionary linkage has been reported in some pine species, such as P. edulis, P. lawsonii, P. montezumae and Fagus sylvatica with their associated bark beetles [9, 42], and for P. ponderosa and Douglas-fir (Pseudotsuga menziesii) to dwarf mistletoes [37, 40, 43, 44].
In PFFANT, pine forests are under high abiotic stress like the presence of human settlements and logging which in turn could be causing biotic stress situations like incrementing incidence of parasites (e.g., because of the loss of trees, bark beetles move to sites where there were no infections and the infection spreads in trees which are predicted to have low defenses). The location of populations parasitized and with high genetic variability is a valuable data to be taken into account for conservation science.
P. hartwegii form dense forest stands in the PFFANT, and their associated parasites are also present with a patchy distribution. They are not present in all populations, indeed there are healthy trees in populations highly parasitized (also called “scape trees”) it could suggest that the parasites of mountain pine select their host based on genetic cues. There is a hypothesis called “gene by gene coevolution” which pose that host-parasite relationship had been kept polymorphisms in plants, these has been recognized in the existence of two cases: host-specificity, and variation in host preference [40, 45], we suggest that in view of the levels of He in parasitized trees, it is possible that a gene-by-gene coevolution case is present in the interaction of mountain pine with its parasites, although there is a need for more studies to probe it. It appears that we are facing a coevolution dynamics, where trees with higher genetic diversity are attacked due to its genes; parasites track specific host genotypes under natural conditions [46, 47, 48] not only to phenotypic characteristics. In other words, plant genetic diversity affects enemies and mutualists .
4.2. Population structure
Organelle genomes provide information about the relative capacity of dispersal of males and females; mtDNA is maternally inherited, and cpDNA is paternally inherited in pines, this shapes gene flow and genetic diversity within and among populations in a particular way [22, 50, 51], we found low levels of GST, in comparison with some other pine species at organelle genomes  and registered high levels of gene flow, these data could suggest that mountain pine subpopulations studied at PFFANT are poorly differentiated which in turn could mean that the population as a whole must be managed and conserved as genetically unique; also we registered the highest with mtDNA on this we can infer that P. hartwegii’s most important means of gene flow seems to be the seed (Table 4), but it is also probably due to the reforestation plans performed at PFFANT by government institutions, which in most of the cases is performed with pines from off-site seed carrying foreign DNA.
High levels of deforestation have been reported at PFFANT, mainly due to illegal logging leading to forest fragmentation, which in turn has devastating effects on forest trees, taking off the best conformed trees, which reduce levels of gene flow and allele diversity, promoting inbreeding and genetic drift . Since conifers are wind pollinated and long lived organisms, effects of inbreeding and or genetic drift are not drastic yet (in view of our results) and the levels of genetic variability reported here may only reflect standing genetic variation, when a population passes through selective pressures it adapts mainly from standing genetic variation [53, 54].
Genetic distances differed according to marker type; there has been reported high polymorphism in Dendroctonus ponderosa related to geographic region . Our results may be explained by differences in land use in which trees were sampled, which could impose different selection pressures in parasites and hosts in response to biogeographic discontinuities [42, 56]. We observed, for example that dwarf mistletoe infections response to altitude, at least Arceuthobium vaginatum and A. globossum (parasites of P. hartwegii) disappear above 3600 masl (personal observation at FFPANT). The lowest genetic distances we found were between DM and BB trees with the tree markers (Table 4), and these trees are genetically similar and have high levels of polymorphism, the differences between HT trees and DM, BB trees may consist in those which parasitized trees possess and make parasites choose them [40, 45], while markers we used are not specifically related with defense and resistance pathways in plants, we report a reliable difference which can serve as a first insight to conduce more studies on genetic resistance of conifers to bark beetles and dwarf mistletoes.
4.3. Conservation implications
The presence of probably immune trees (HT) and resistant trees (BM) must be taken into account to conserve mountain pine forests of the FFPANT, specially confronting an increase of infected areas by bark beetles and dwarf mistletoes; BM are trees whose seeds could be used in reforestation programs, especially in areas where infections are growing, and HT seeds could be used to create barriers which impede the spread of outbreaks.
Pinus forests of the FFPANT are home and a potential distribution area of many species of plants and animals some of which also have high levels of genetic variability which in turn makes FFPANT an important place to conservation of species [4, 57, 58, 59, 60]. These forests provide water to urban areas, hence the importance of preserving this natural area should not be dismissed; there is an urgent need to protect these forests, as long as forest fragmentation is increasing.
It has been reported that there are differences in susceptibility to parasites attack among tree species and even among individuals within a species; when different species interact, selective changes may occur as a result of the interaction; models of host–parasite interactions support the idea that variation in host resistance is, at least partially, genetic and assume the presence of genetic variation [23, 46, 48]. In conifers, some theories have been proposed to explain this variability, including variations in terpene or phenolic content , differences in constitutive defenses [62, 63] and differences in growing conditions [64, 65]. Population genetics theory predicts that under many selective regimes, fitness will increase the number of heterozygous loci ; many authors have reported that genetic variability of trees affected by parasites, in terms of heterozygosity, they report that heterozygous individuals were resistant and suggest that heterozygosity and plant resistance are positively correlated [61, 66, 67, 68, 69].
More studies are needed in order to reaffirm the results reported here, may be with other kinds of molecular markers, and to continue the improvement of management and preservation of the FFPANT. Forests worldwide need to be protected in face of an imminent climate change.
The authors wish to thank Consejo Nacional de Ciencia y Tecnología (CONACyT) and the Universidad Autónoma del Estado de México (UAEMéx) for financial support. Also, they thank the CIEAF students who helped in the laboratory and fieldwork.