*2.2.5 Key to the species of Magnolia in Jalisco.*

	- Free petiole stipules, free carpels, fruit with dorsal dehiscence (sect. *Magnolia*)
	- Leaf blades 35.0–45.0 � 23.0–29.0 cm; flowers 16.0 cm in diameter; fruits 14.5 � 8.5–9.0 cm; carpels 37–44; basal carpels 5–5.7 � 1.5–2 cm and their decurrence from 0.8–2.0 cm long (O of Jalisco) … *M. ofeliae*
	- Spataceae bract, peduncular internodes and petioles essentially glabrous or with pubescence limited to the nodes … 4.
	- Narrowly oblong sepals … 6.
	- Flowers 11–14 cm in diameter, with a loose pollination chamber, fruit 5.0–7.5, oblongoid to ellipsoid, carpels 16–22, with seeds scarlet red … *M. pugana*
	- Pollination chamber incipient or narrowly oblongoid, leaves 8.0–17.0 (18.0) � 3.0–6.0 (8.0) cm, elliptical-lanceolate, frequently acute apex, petals 6–7, carpels 17–25 … 7.

*How to Save Endangered Magnolias? From Population Biology to Conservation Action… DOI: http://dx.doi.org/10.5772/intechopen.94346*

	- Sepals narrowly oblongoid, reflexed, opening up to 170 degrees, pollination chamber tight and narrowly oblongoid, inner whorl of petals subequal … *M. talpana*.

#### **2.3 Genetic structure, diversity, and differentiation**

In this section, we review what is known so far of genetic structure and diversity in three western Mexican *Magnolia* species: *Magnolia vallartensis, M. pacifica* s.l. (including the new species *M. talpana*), and *M. pugana* s.l. (including the new species *M. granbarrancae*), all belonging to the so-called *Magnolia pacifica* complex [11] (**Figure 4**). As they are distributed along a continentality and moisture gradient, the influences of their different distances to the Pacific Ocean in their genetic structure and diversity have been tested [18]. In this study, leaf tissues of 278 individuals from 10 localities representative of the *M. pacifica* complex were sampled. Three localities were sampled for *M. vallartensis*: Arroyo Palo María (APM), Las Lajitas (LL), and Provincia (PV); three for *M. pacifica* s.l.: Cerro San Juan (CSJ), San Sebastián del Oeste (SS), and Bosque de Arce (BA, *M. talpana*); and four for *M. pugana* s.l. Arroyo La Virgen (ALV), Arroyo San Lorenzo (ASL), Arroyo Palo

#### **Figure 4.**

*Results of Bayesian clustering based on STRUCTURE; analysis performed on a) the entire data set (278 individuals, 76 loci ISSR); b) the* M. pugana *s.l. group (120 individuals); c) the* M. pacifica *s.l.-*M. vallartensis *group (158 individuals). Relationships between K and Delta K values and structure bar plots are shown. Vertical bars represent each individual analyzed and bars are divided into distinct colors when there is evidence of admixture. From [18].*

Verde (APV), and Río Verde (RV, *M. granbarrancae*) (**Figure 4**). A total of 76 reproducible DNA fragments of six Inter-Simple Sequence Repeats (ISSR) (primers UBC 810, 814, 834, 836, 855, and 857) were amplified and analyzed using Bayesian analysis with the program STRUCTURE, UPGMA clustering, Monmonier's algorithm with Barrier 2.2, AMOVA, and genetic diversity parameters. Also, a Mantel test was done to evaluate the relationship between genetic and geographic distances.

## *2.3.1 Genetic structure*

Results of Bayesian analysis with STRUCTURE (**Figure 4**), UPGMA clustering (**Figure 5**), and the Exact Test for differentiation (**Table 2**) are in accordance that there are two main genetic clusters for the whole of three species, being *M. pugana* s.l. the eastern main group, and the cluster of *M. pacifica* s.l*.-M. vallartensis* the

#### **Figure 5.**

*UPGMA dendrogram showing the genetic relationships of the* M. pacifica *species complex, the number at each node indicates the bootstrap percent having above 50% values. From [18].*


#### **Table 2.**

*Nei's unbiased genetic distance (above diagonal) and exact test differentiation probability values (below diagonal) among sampled localities of the* M. pacifica *species complex. From [18].*

*How to Save Endangered Magnolias? From Population Biology to Conservation Action… DOI: http://dx.doi.org/10.5772/intechopen.94346*

western grouping. But each one of these two main groups has its own structure, having two subgroups each (**Figures 4** and **5**). *M. pugana* s.l. group is segregated in the *M. pugana* s.s. subgroup (ALV-ASL localities), located SW of the Santiago river canyon, and the APV-*M. granbarrancae* (RV) group, located NE of that canyon. In the other main group, *M. pacifica* s.l. subgroup (BA-SS-CSJ) was separated from the *M. vallartensis* one (PV-LL-APM). Additionally, the 900 bp (primer 834) and 850 bp (primer 855) ISSR loci only amplified and were exclusive for *M. pacifica* s.l*.- M. vallartensis* group, these loci did not amplify *M. pugana* s.l. samples. The AMOVA based on the three species and the two Bayesian groups showed that only 8% of the genetic variation was explained by differences among species as well as among the Bayesian groups (**Table 3**), so AMOVA was less informative than the Bayesian analysis. Most of the variation (≥ 82%) was explained by differences within localities, suggesting high levels of cross-pollination [30]. This predominance of outcrossing reproductive mode was also found by [31] in some Neotropical magnolias.

Other evidence for the structure represented by four groups was the results of the test with Monmonier's algorithm (Barrier 2.2), which detected three significant geographical barriers to gene flow, segregating the four groups (**Figure 4**). All geographical boundaries had 100% bootstrap support. One of these is the Trans-Mexican Volcanic Belt (TMVB), which is the main physiographic barrier between *M. pugana* s.l. and the group of *M. pacifica* s.l.-*M. vallartensis*. This mountain range has been identified as an important gene flow barrier for both plants [32–35] and animals [36, 37], even its complex orogenic processes have led to population isolation, speciation, and diversification [38]. The other two significant barriers detected correspond to the Santiago river canyon on the one hand (between the *M. pugana* s. s. and the APV-*M. granbarrancae* group), and the basins of the rivers Ameca, Mascota, Pitillal, and Cuale on the other hand (between *M. pacifica* s.l. and *M. vallartensis*). The effect of the deep Santiago river canyon (3–15 km wide and 500– 700 m deep) and other river basins as significant gene flow barriers also have been documented for birds, reptiles, insects and other plant species [34, 39, 40].

The outcomes of this population genetics study reveal that *M. pugana* s.l., the easternmost and the most continental group, is the most geographically and genetically distinct group of all *M. pacifica* species complex, and clearly support its recognition at the species level [16]. The most likely process of speciation for *M. pugana* s.s. and *M. granbarrancae* is allopatric isolation, being the TMVB the main


#### **Table 3.**

*Molecular variance analysis (AMOVA) for Bayesian analysis and taxonomic groups of the* Magnolia pacifica *species complex. From [18].*

gene flow barrier between them and their western close relatives. This is in congruence with Vázquez-García *et al.* [1], who suggest that allopatric speciation is a major driver of *Magnolia* diversification in Neotropical Magnoliaceae. The genetic divergence between *M. pacifica* s.l. and *M. vallartensis* is lesser than between this main group and *M. pugana* s.l., indicating a more recent process of segregation or current gene flow. There is no clear geomorphological barrier between some localities of *M. talpana* and *M. vallartensis* in their southern ranges (between BA and all the *M. vallartensis* localities). Therefore, genetic differences between these localities might be rather explained by a process of parapatric ecological differentiation [41, 42], as it is common that gene exchange among closely related taxa happens in at least 25% of plant species [43]. Nevertheless, the four identified genetic groups, independently of partially sharing genes, are distinctive evolutive entities and should be considered as separated conservation units.

#### *2.3.2 Genetic differentiation*

Differentiation indices were moderate in general, but higher in *M. pugana* s.l. (GST = 0.120 0.021, D = 0.028 0.007) than in *M. pacifica* s.l.–*M. vallartensis* (GST = 0.106 0.016, D = 0.026 0.006). The Exact Test for population differentiation showed significant genetic differences between localities of *M. pugana* s.s. and the *M. granbarrancae*, and between localities of *M. pugana* s.l. and *M. pacifica* s. l.–*M. vallartensis*, but not within each *M. pugana* subgroup and within the *M. pacifica* s.l..–*M. vallartensis* group (**Table 2**). The higher genetic differentiation of *M. pugana* s.l. is in accordance with its smaller, more fragmented, and more isolated populations. The highest distance to the Pacific Ocean of *M. pugana* s.l. causes a lower environmental humidity and more extreme cyclical temperature changes, that is, a greater continentality [44]. These drier and more extreme conditions result in declines, fragmentation, and isolation of *M. pugana* s.l. populations, which need a constant input of water to survive. In contrast, the more humid maritime environments of *M. pacifica* s.l.–*M. vallartensis* are more favorable to maintain populations of these cloud forest mesophytic species, which is reflected in less isolation and differentiation. Similarly, genetic differentiation has ranged from moderate to high in most Neotropical Magnolia species [31].

#### *2.3.3 Genetic diversity:*

*M*. *pugana* s.l. had lower genetic diversity than the *M*. *pacifica* s.l.-*M*. *vallartensis* group. *M*. *pugana* s.l. exhibited a Shannon Index (I) = 0.268, total heterozygosity (HT) = 0.158 (0.023 SD), and intrapopulation heterozygosity (HS) = 0.134 (0.020 SD). In contrast, *M. pacifica* had an I = 0.272, HT = 0.175 (0.025), and HS = 0.159 (0.023), and *M. vallartensis* an I = 0.275, HT = 0.171 (0.024) and HS = 0.153 (0.022). Genetic diversity also varied among localities; *M. pugana* s.l. localities had the lowest genetic diversity (HE = 0.121–0.140, *M. granbarrancae* having the lowest I = 0.218 and H = 0.121), whereas the locality Bosque de Arce (BA) of *M. talpana* showed the highest genetic diversity (I = 0.280, HE = 0.17) among the western *Magnolia* localities studied. The genetic diversity estimates for all the studied taxa here were lower than the average values reported for plant genetic diversity based on ISSR (H = 0.22) [45], and much lower than those reported for two threatened eastern Mexican *Magnolia* species: *M. sharpii* Miranda [46] (I = 0.56) and *M. schiedeana* Schlecht. [47] (I = 0.50) [48]. As with the higher genetic differentiation, the lowest genetic diversity of *M*. *pugana* s.s. and *M. granbarrancae* is consistent with their smaller populations, higher isolation, and fragmentation, all influenced by a drier and more extreme climate.

*How to Save Endangered Magnolias? From Population Biology to Conservation Action… DOI: http://dx.doi.org/10.5772/intechopen.94346*

#### *2.3.4 Isolation by distance pattern*

The correlation between geographical and genetic distances among all localities of the *M. pacifica* species complex, revealed by Mantel tests, was high and significant (r = 0.80, p < 0.001), however, when the tests were applied to each of the two main genetic groups separately, no significant correlation was detected. Isolation by distance might explain the genetic structure and differentiation pattern within the *M. pacifica* species complex. Isolation by distance has been a strong pattern observed among plant studies, under this and in rapid environmental change, adaptive responses to environmental stress will be constrained by the natural dispersal mechanisms [49]. Pollination by beetles in small and isolated populations of *Magnolia* is not very efficient [50], and habitat fragmentation and other anthropogenic factors may also be troublesome for *Magnolia* seed dispersal by birds [50, 51]. Even if dispersal occurs, seeds may not germinate or seedlings may not survive in places without enough humidity [52].

In summary, based on ISSR genetic variation, the *M. pacifica* species complex exhibits a population genetic structure composed of two main groups, the eastern *M. pugana* s.l more continental group, and the western *M. pacifica* s.l.-*M. vallartensis* group, with the more maritime climate. Both main groups are segregated by the physiographic barrier of the TMVB and isolation by distance, and are at the extremes of a maritime-continental climatic gradient. *M. pugana* s.s*.* and *M. granbarrancae* are subject to a drier and more extreme climate, therefore having more deforested, fragmented, and isolated habitats, which leads to lower genetic diversity and a higher genetic differentiation. This differentiation within *M. pugana* s.l. and the physiographic barrier of the Santiago river canyon have structured this taxa in two genetic subgroups, *M. pugana* s.s. and the *M. granbarrancae*. The *M. pacifica* s.l-*M. vallartensis* group exhibited genetic segregation in two subgroups, having several canyon river barriers between both taxa, but maintaining a partial gene flow at their southern ranges. The three species of the *M. pacifica* complex have lower genetic diversity than eastern Mexican *Magnolia* species which are considered as endangered. Even more, the eastern and more continental *M. pugana* s.s. and *M. granbarrancae* undergo the lowest genetic diversity, which, together with their smaller and more isolated populations, makes these populations more vulnerable to gene drift and bottlenecks, therefore greater risk of extinction. All main genetic groups and subgroups defined in this study should be considered as separate conservation units, and concerted efforts are needed to protect them.

#### **2.4 Phenological divergence**

Phenology aims to characterize the behavior and the biological adaptation of the species in its natural habitats, which may help guide important management conservation strategies. We studied three different species of *Magnolia* (*M*. *vallartensis*, *M*. *pacifica* s.l., *M*. *pugana* s.l.) in western Mexico (**Figure 1**). Phenological observations were made at monthly intervals for one year and four months. The phenophases (flowering and fruiting) in an annual cycle and the intensity of each phenological event were estimated using the Fournier intensity index. The sum of all the intensity categories assigned to each individual was divided by the maximum amount that could be attributed to the population (relativizing the total number of selected individuals with the total sum sampled per month). Circular statistics were used to analyze and interpret phenological patterns. Using the statistical package Oriana version 4.0, the months were converted into angles, with intervals of 0.999° to 30° wide and it was calculated: 1) average angle (μ) or average date that refers to the time of the year around which the phenological activity of individuals is more

concentrated; 2) circular standard deviation; and 3) the vector r, which indicates the intensity of the concentration (0 to 1) around the average angle. The length of the vector r can be considered a measure of the degree of seasonality. In addition, the Rayleigh test was applied to indicate the significance (P < 0.05) of the mean angle.

The synchrony of the flowering phenophase between populations or species is essential to detect possibilities of gene flow between species and existence of temporary barriers in reproductive phenophases. Since the incipient asynchrony observed in the flowering of the three species was linked to their amplitude of flowering (which lasts up to six weeks with significant intersection) it was considered insufficient to prevent gene flow. The hypothesis of non-seasonality in the phenophases of the three species was rejected, which was high in *M*. *pugana*, intermediate in *M*. *pacifica* and low in *M*. *vallartensis*.

The flowering (female-flower) phenophase showed an incipient asynchrony among species, being earlier in *Magnolia vallartensis* (end of May), intermediate in *M. pacifica* (second week of June), and late in *M. pugana* (end of June). Seasonality was high in *M. pacifica* and *M. pugana* [20] (**Figure 6**). This incipient asynchronous female-flower phase differs from some Colombian *Magnolia* species, in which flowering is commonly markedly asynchronous among species and populations of each species [53].

The fruiting phase was asynchronous among species, and showed high seasonality only for *Magnolia pugana* (February) while in *M. vallartensis* the maximum fruiting was in August and for *M. pacifica* in December (**Figure 6**). This pattern is similar to that reported for *Leucaena* sp. and *Guazuma ulmifolia* Lam. [54, 55], a possible strategy to reduce competition among potential seed dispersers of *Magnolia* including birds, rodents and lizards. The intensity of fruiting is concentrated in rainy seasons [53]. Fruit dehiscence of *Magnolia vallartensis*, *M. pacifica* and *M. pugana* showed an asynchronous pattern between species. Similarly, eight

#### **Figure 6.**

*Circular histograms of two phenophases for the three* Magnolia *species. A. Flowering (female-flower) phenophase. B. Fruiting phenophase. Based on monthly averages of frequencies from January to December 2017.* Magnolia vallartensis *(I), left column,* M. pacifica *(II), central column and* M. pugana *(III), right column. The arrows represent significant average angles (Rayleigh test p < 0.001), while the length of the vector r (0 to 1) indicates the concentration around the average angle [20].*

*How to Save Endangered Magnolias? From Population Biology to Conservation Action… DOI: http://dx.doi.org/10.5772/intechopen.94346*

species with an asynchronous pattern are reported for pioneer species in the central Amazon region of Brazil [56].

Differences in productivity were also evident, the higher productivity of reproductive phenophases (flowering and fruiting) of *Magnolia pugana* compared to that of *M. pacifica* and *M. vallartensis* makes it possible to reject the hypothesis of no significant differences. These differences may be due to their evident genetic differences [18], but also to their environmental differences; for instance, *M. pugana* populations thrive in permanent streams, surrounded by tropical deciduous forest and open oak-pine woodlands, a condition that provides more sunlight that can help to keep their reproductive phenophases more productive (flowers and fruits), whereas in the case of *M. vallartensis* and *M. pacifica*, their populations are usually surrounded by high and dense canopy cloud forest and tropical semi-evergreen forest trees that overshadow many *Magnolia* trees, which limits their productivity due to the scarcity of sunlight along the year.

#### **2.5 Floral scents**

More recently, several ecological studies have examined the roles of floral scent in the biology of the plant [57]. Scents, essential oils, or volatiles are secondary metabolites produced by plants to fulfill protection functions against herbivores, phytopathogens, and even other plants [58, 59]. These form a complex matrix with a lipophilicvolatile nature [60], from a chemical point of view, these volatile compounds belong to chemical classes such as terpene derivatives (oxygenated or hydrocarbons), phenylpropanoids, benzenoids, and nitrogen-containing compounds [61, 62]. The combinations of the constituents of this scent mixture give each flowering plant species a unique fragrance [57]. Through a compilation of data, the genus *Magnolia,* has been reported to be an interesting source of secondary metabolites found both in plant extracts and in essential oils, as shown by studies carried out mainly in species distributed in Asia and in which applications in traditional medicine are included [63–65]. In this sense, in many of the endemic species, studies related to describing the metabolic composition as a complement to determine potential uses, contribute to knowledge, and improve the conservation of their habitats. As an example, we extracted and characterized for the first time the essential oils present in flowers of *M. vallartensis*, *M. pacifica* and *M. pugana*, three endemic species of the genus *Magnolia* along a continenrtality and moisture gradient in western Mexico [19].

#### *2.5.1 Methods*

For details of flower collection techniques, essential oils extraction and determination of floran scents chemical composition see Mendeley Data repository [66].

#### *2.5.2 Comparisons*

The yield of essential oils from flowers of these three *Magnolia spp.* (**Table 4**) showed that in general these vegetal parts present a similar low yield between 0.2 to


**Table 4.**

*Essential oils of* Magnolia *flowers: Yield and organoleptic characteristics.*

0.3%. On the other hand, the chemical composition of oils showed qualitative differences among individual components. The chemical profiles of these scents analyzed by GC/MS, observed in **Figure 7**, identified 97 compounds in total between species; 63 in *M. vallartensis*, 53 in *M. pacifica* and 39 in *M. pugana*.

In the chromatograms differences evidenced, in a specific manner, on the major components. Within the chemical composition of floral essential oils the major compounds greater than 3% of the total components obtained for each species from the most abundant were as follows. In the case of *M. pacifica*, (caryophyllene, bicyclo-dec-1-ene, 2-isopropyl-5-methyl-9-methylene, bicyclogermacrene, βelemene and epi-cyclocolorenone); for *M. vallartensis* (caryophyllene, geranyl methyl ether, β-elemene and caryophyllene oxide); and in *M. pugana* (cyclocolorenone; 2Z,6E-farnesol; benzoic acid (5,5-dimethyl-4-oxo-2 cyclohexenyl ester, β-elemene, and caryophyllene oxide). Chemical composition data has an interesting application when essential oils occurrence is studied in the field of taxonomy. This assesses the possible chemotaxonomic relationship between chemical compounds and species, identifying compounds that could act as indicators. Based on this idea, the results obtained by a point-to-point analysis throughout the 90-minutes chromatograms and comparing the mass spectra including trace components, a matrix was constructed (**Figure 8**).

The presence of compounds could be associated to the closely related taxonomic affinity confirmed trough molecular phylogenetic analysis; for example analyzing other three species closely related: *M. schiedeana*, *M. grandiflora* L. [67] and *M. tamaulipana* [11]; the pair *M. schiedeana-M. tamaulipana* (both endemic from Mexico) share the presence of geranyl methyl ether as the major compound up to 87%, while in *M. grandiflora* (widely distributed in the southern North America) shows the presence of its precursor: geraniol [68]. In this way, it was determined that the floral scents of the endemic species *M. pacifica, M. vallartensis* and *M. pugana* share qualitatively 14 components in their chemical profile, which is equivalent to 14.46% of total; these components suggested that could act as chemical markers for a determination at the gender level. Otherwise, floral oil compounds shared by pairs of species were also founded, the pair *M. pacifica-M. vallartensis* shared 27 more compounds of the total (42.47%); *M. vallartensis*-*M. pugana* 2 more compounds of the total (16.49%); and *M. pacifica*-*M. pugana* just one more additional compound to the total (15.46%). In the case of components particularly present in each species, *M. pugana* had 22 compounds, *M. vallartensis* 20 and

**Figure 7.** *Floral scents chemical profiles by gas chromatography:* A) M. vallartensis, B) M. pacifica, *and C)* M. pugana.

#### **Figure 8.**

*Matrix of chemical compounds present in* Magnolia *floral scents.*

*M. pacifica* 11 components. The Jaccard similarity index allowed inferring the qualitative composition, the pair *M. pacifica* - *M. vallartensis* show the strongest similarity about 54.6%, while with *M. pugana* these species showed 19.5% and 18.6% of similarity respectively. The essential oils of flowers of *M. pacifica*, *M. vallartensis* and *M. pugana* showed a composition rich in sesquiterpenes with significant differences in their composition. Most species of *Magnolia* possess distinctive floral scent profiles, even though they may be taxonomically closely related, the chemical differences among these taxa may have arisen from interaction with pollinators or the environment [69]. There are factors that can affect the chemical composition and quality of an essential oil such as the age of the plant, altitude, climate, genetics, geography,

type of soil [70]. For this reason, it is necessary to continue with studies focused on establishing the relationships between the components and relating the chemotaxonomic field including more *Magnolia* species populations to conform a robust database that provides the necessary information to correlate chemical markers and to become a useful tool in species classification in addition to genetic analysis. These applications can help promote strategies in the conservation of this genus and its habitat.

#### **2.6 Pre-germination treatments and seed dormancy**

The most important and critical stage during the development of the plants is germination; therefore, it is also for the natural repopulation of the species and the dynamics of the populations [71]. The ability of a seed to germinate is known as viability, that is, the embryo is alive and can remain so for some time. A viable seed with dormancy is one that cannot germinate under any condition of physical environmental factors (humidity, temperature, light/dark, etc.), which are otherwise favorable for germination [72]. Therefore, the success of germination depends on whether or not the seeds have a dormancy mechanism, on their viability and on the time needed for them to germinate [73].

*Magnolia* seeds have viability percentages ranging from 50–100% [74, 75]. Different studies have shown a low percentage of germination because they can present different types of dormancy (including physical, chemical, mechanical, physiological and morphological) [76]. To eliminate dormancy and increase the percentage of germination and produce the greatest number of *Magnolia* seedlings, different pre-germination procedures have been applied [77–80]. This section will present the research carried out in *Magnolia iltisiana*, *M*. *pacifica*, *M*. *pugana* and *M*. *vallartensis*, to know the viability percentages and the different most successful pregermination treatments, to break the lethargy and increase germination.

#### *2.6.1 Seed management and germination*

Seeds must be collected from 10 different plants of ripe fruit and extracted manually. To prevent fungal infection, a contact fungicide (Captan) was used for the seeds of *M*. *iltisiana* [81], while the other species were rinsed in a 3% sodium hypochlorite solution for 30 minutes. It is recommended that after being collected and disinfected, they are immediately stored in a well-ventilated and humid environment inside a refrigerator at 4–5°C, to avoid dehydration [76].

Were used 100 seeds per treatment with five replicates of 20 seeds per container for the four species. Once the treatments were concluded seeds of *M*. *pacifica*, *M*. *pugana* and *M. vallartensis* were sown inside a greenhouse at the Centro Universitario de Ciencias Biológicas y Agropecuarias (CUCBA) and were buried at 1.5 cm depth in 25 ml plastic containers. The substrate used for planting was "peatmoss" [29]. The study of *M. iltisiana* was carried out in the experimental greenhouse of Las Joyas Scientific Station (ECLJ), and they were planted in a mixture of sand and "germinaza" (1:1) as substrate. The number of germinated seeds was recorded daily for 60 days [75]. Seeds were considered to have germinated when the radicle emerged or when the hypocotyl was observed [82, 83].

#### *2.6.2 Viability tests*

The percentage of viability of all species was determined through tests in a 1% tetrazolium solution; two replicates of 50 seeds were used for *M*. *iltisiana*, while for *How to Save Endangered Magnolias? From Population Biology to Conservation Action… DOI: http://dx.doi.org/10.5772/intechopen.94346*

the other species it was in 30 seeds. The seeds were immersed without aril in this solution for 24 hours at 30°C, in darkness [72]. After this time, they were cut transversely and the tissues were observed in a stereoscope. What were stained in deep red is considered viable, while those not, are considered unviable (**Figure 9**) [84].

The viability is 80% for *M*. *iltisiana*, this result agrees with what was found in [77] where they obtained 80% of viable seeds in *M. schiedeana*. While the seeds of *M*. *pugana* registered 67% [76]. Reference [75] reported that in species of the same genus from southern North America it has an average of 50% viability, similar results are found in *M*. *vallartensis* and *M*. *pacifica* with 50% and 53% respectively (**Figure 10**). These percentages are lower than those recorded in other *Magnolia* species. For example, in [74] found 100% viability in seeds of *M. dealbata* Zucc*.* [85]. In Ref. [80] obtained 92% for *M*. *perezfarrerae* A. Vázquez & Gómez-Dominguez [86] and 87.5% in *M. sharpii*.

#### *2.6.3 Pre-germination treatments, germination tests, and dormancy types*

Four treatments were used for *M*. *iltisiana* [81]. In *M*. *pugana* six treatments were carried out [76]. Three treatments were performed for *M*. *pacifica* and *M*. *vallartensis* [87]. The germination of *M*. *iltisiana*, *M*. *pacifica*, *M. pugana* and *M*. *vallartensis,* is of the epigeal type, the embryo developed a pair of foliaceous cotyledons, a flaccid fatty endosperm, a hypocotyl and a radicle, confined to the micropile area [76, 81, 87], (**Figure 11**).

In general, the percentage of germination obtained for *M. iltisiana, M. pacifica, M. pugana* and *M. vallartensis* are low. The highest germination recorded are 60%, 21%, 52% and 12%, respectively. In these studies, it is found that for *M. pacifica*, cold stratification treatment promoted the highest number of germinated seeds. Similar results have been obtained in [77] for *M. schiedeana*, with this same treatment with 84% germination. Another important finding is that the manual aryl removal treatment for *M. iltisiana, M. pugana,* and *M. vallartensis* are the most successful. These results coincide with those of reference [80] reported for *M. perezfarrerae* (64%) and *M. sharpii* (73%) with the mechanical scarification treatment (i.e., the seeds are placed in purified water and then the aril is manually

#### **Figure 9.**

*Staining of seed tissues with tetrazolium in the viability test in* Magnolia pugana*: a) viable seed, and b) non-viable seed.*

**Figure 10.** *Viability percentages for:* Magnolia iltisiana*,* M*.* pacifica*,* M*.* pugana, *and* M*.* vallartensis*.*

#### **Figure 11.**

*Germination process of* M. pugana: *a) exposure of the endosperm and hypocotyl, b) elongation of the hypocotyl and cotyledon, c) leaf cotyledons and testa flushing, d) seedling with leaf primordia exposed above ground.*

removed). This treatment also proved to be effective with 90–100% germination in seeds of *M. dealbata* [74] and *M. champaca* (L.) Baill. ex Pierre [88] (73%) under the same treatment [89].

Low germination rates (< 70%) may indicate that the seeds are dormant and cannot be broken [90]. The results in these studies suggest that cold stratification treatments and manual aryl removal may indicate the presence of physiological and chemical dormancy, respectively [72]. Taken together, these results are consistent with reference [29] who recommends that *Magnolia* seeds undergo stratification periods so that immature embryos can develop, while in Ref. [91] reported that Magnoliaceae aryls contain inhibitors that delay germination.

On the other hand, it has been proven that the use of phytohormones is a promoter of germination with physiological dormancy [72]. Conversely, it was found that the phytohormone treatments used in the experiments on *M. iltisiana* and *M. pugana* did not increase the germination percentage, suggesting that the seeds of these species do not possess physiological dormancy. Although the seeds of *How to Save Endangered Magnolias? From Population Biology to Conservation Action… DOI: http://dx.doi.org/10.5772/intechopen.94346*


**Table 5.**

*Types of dormancy reported in* Magnolia iltisiana*,* M*.* pacifica*,* M*.* pugana *and* M*.* vallartensis*.*

*M. iltisiana* have chemical dormancy, physical dormancy is also identified, that manifests itself through the development of a lignified testa that prevented the absorption of water, and the third type of dormancy is mechanical, which in this case the head exerted too much pressure on the embryo, delaying germination [81]. In the studies conducted for *M. pacifica*, *M. pugana* and *M. vallartensis* it was found that seeds do not have physical dormancy because they have the capacity to absorb water [76, 87] (**Table 5**).

#### **3. Future scenarios**

#### **3.1 Vulnerability in climate change scenarios**

Climate change is an important driver for future distributions of tree species, as it is expected to modify environmental conditions critical for plant populations maintenance. An analysis of 40 species of North American tree species from the temperate zone [92] revealed the higher vulnerability to climate change for the species growing in a colder climate, including the high-elevation mountain trees. In tropical mountain landscapes of Andes, the changes in habitat suitability are expected to be of a mixed character, while favorable for some tree species they may be prejudicial for others, with an overall general increase of species risk of extinction found in approximately 20% of tree species [93]. The increase of the risk of tree species extinction from climate change is related to the projected scale of the climate zones shift, which is highly variable across the globe [94]. In the situation of the terrain with complex irregular topography the extent of habitat suitable for species may be particularly sensitive to climate change [95], as the habitat tolerances of the mountain flora are generally narrow and the distribution shift upwards in the mountains frequently means the reduction of populations. At the same time, the complex topography may offer the high heterogeneity of habitat, which could provide opportunities for emerging local refugia, detectable at a fine spatial scale [96]. In the case of *Magnolia*, the narrow distribution ranges of species may constitute a risk factor, that could lead to the disappearance of the suitable habitat in some of the climatic projections for the end of the 21st century, as it was found in the case of the Andean species *M. mercedesiarum* D.A. Neill, A. Vázquez & F. Arroyo [97, 98].

An important step in the assessment of tree species vulnerability and extinction risk in the climate change scenarios is the analysis of habitat suitability dynamics. The habitat suitability belongs to the core concepts of the ecological niche theory, particularly of great importance in the field of plant ecology, as plants cannot evade adverse environmental conditions by sheltering or migrating within the single generation [99]. The assumption that healthy plant populations stay in the equilibrium with the contemporary or recent environmental conditions is central for accessing habitat suitability through niche modeling and projecting [100]. The climatic

envelope models are a particular class of habitat suitability and niche models that uses the climatic variables as an approximation to the maximal extent of the habitat suitable to support species [101]. This class of models is widely used for the analysis of species-climate relations in the current climate, and for the heterochronic studies using past and future climate strata, as well as for predicting species distributions.

After accepting the equilibrium assumption, we used the field observations of the presence of three *Magnolia* species to develop the corresponding climatic envelope models, which captured the range of the environmental conditions that are suitable for species persistence in time and space. Further projections of the habitat suitability in the climate change scenarios in the next 80 years allowed us to detect the probable habitat suitability changes for *Magnolia* species, and to evaluate species vulnerability to climate change.

#### *3.1.1 Methods*

The species presence dataset of three taxa of the *Magnolia pacifica* complex [*M. pacifica* s.l. (including *M. talpana*), *M. pugana* s.l. (including *M. granbarrancae*), and *M. vallartensis*] was compiled from herbarium specimens at IBUG, IEB, ZEA, WIS, MEXU, XAL, MO, MICH, field observations cited in taxonomic literature [11, 13, 16], virtual images at Naturalista [102]. Monthly averaged climatic variables from WorldClim 2.1 dataset [103] available for the recent past (1970–2000) were taken as an approximation to current conditions, while the future conditions under two CO2 emission scenarios were taken from the downscaled projections of the general circulation model CanESM5 [104]. The detailed description of the species distribution modeling procedure and predictions are available in Mendeley Data repository [66].

#### *3.1.2 Results and discussion*

As it was expected, the climatic envelope models recovered the suitable habitat extent larger than the known species distributions. The reasons of the overestimation are discussed in Shalisko *et al.* [66]. However, in the continental scale, the high suitability was predicted close to the occurrence records, the most distant grid cells identified as suitable were separated from known species occurrence sites in less than 150 km. The estimated suitable for the species presence zone in 1970–2000 varied from 9560 km<sup>2</sup> for *M. vallartensis* to 23940 km<sup>2</sup> for *M. pugana* s.l. (**Figure 12**). The suitable areas were well separated from another two species in the case of *M. pugana* s.l.,overlapped with *M. pacifica* s.l. In the case of the *M.6vallartensis* the estimated overlap of potentially suitable habitat with that of *M. pacifica* s.l. was of 4053 km<sup>2</sup> , which equals to 42% of the entire high suitability zone for *M. vallartensis*.

Despite the systematic overestimation of the suitable area, the climatic envelope models are useful for the evaluation of the species vulnerability to climate change, as the same bias applies to the prediction of habitat suitability in current conditions and future projections. The changes in the area with suitable conditions may be proportional to the changes in true potential distribution.

The dynamics of suitable area in SSP2–4.5 scenario [105] (**Figure 13**) was favorable for *M. pugana* s.l., as the size of the potentially suitable area in 2080–2100 was about 13% larger than in current conditions, and almost all grid cells labeled as suitable in recent past persist at the end of 21st century. The models for both *M. pacifica* s.l. and *M. vallartensis* predicted suitable habitat reduction which is particularly fast in the second half of the century, with loss of about 45% of the suitable habitat in the case of the former species and 53% in the least. Interestingly, in the

*How to Save Endangered Magnolias? From Population Biology to Conservation Action… DOI: http://dx.doi.org/10.5772/intechopen.94346*

#### **Figure 12.**

*Areas of habitat suitability higher than ESS threshold in at least 50% of cross-validation runs of the climatic envelope model for recent (1970–2000) climatic conditions. The key map on the left part shows the location of the right frame within North America.*

current climatic conditions, the habitat suitability for *M. vallartensis* was found to be high not only in the locations close to the known distribution, but also in the separate coastal zone northwards, and the persistence of the habitat suitability in future scenarios was higher in the northern area, where species observations are unknown. In the case of *M. pacifica* s.l.the future high habitat suitability in SSP2–4.5 scenario was predicted roughly in the same geographic zones as was identified for current conditions.

The baseline SSP3–7.0 scenario [105] produced habitat suitability projections that are concerning in terms of species survival (**Figure 13**). In the case of three species, the fast decline in habitat suitability was predicted from the middle of the century, resulting in a loss for the end of the century of 66% of the suitable area in the case of *M. pugana* s.l., dramatic 92% loss of habitat suitable for *M. pacifica* s.l. and complete disappearance of the habitat of *M. vallartensis*.

#### **Figure 13.**

*Areas of habitat suitability higher than ESS threshold in at least 50% of cross-validation runs of the climatic envelope model for projected future (2080–2100) climatic conditions under SSP2–4.5 and SSP3–7.0 scenarios. Symbology and extent is the same as in Figure 12.*

The uncertainty associated with habitat loss projections remained high due to the limitations of climatic envelope modeling and the uncertainty from global circulation models and CO2 emission scenarios. However, the general trend of probable habitat loss for *M. pacifica* s.l.and *M. vallartensis* detected in the analysis was not compromised by accounting for the uncertainty, as the similar trend of change between current conditions and projections for the end of the 21st century was found in the 95% confidence intervals. In the case of *M. pugana* s.l. the likely habitat suitability change cannot be interpreted as the sign of significant risk when taking into consideration the prediction uncertainties.

Similarly to reference [95] interpretation of the effect of habitat suitability reduction in the species survival, we consider that the risk of species extinction from habitat loss may be overestimated when the data was analyzed in coarse-scale, as the local small size refugia were excluded from consideration. The true vulnerability of species to climate change depends on several factors outside of the scope of the current analysis. Tree populations may have a lag in reaction to climate change as the long-living sessile organisms [106]. The result of this lag could be the absence of the immediate disappearance risk for adult tree individuals, that could successfully tolerate significant environmental stress, but the reduction of the reproductive success required for the populations maintenance. In many species of the North American trees, the observed distribution is not entirely concordant with the current climate, as the long-living organisms may present the 'extinction debts' and 'colonization credits' at some parts of their actual or potential ranges [106]. In the case of *Magnolia* species, the lag between the loss of the climatic suitability and the actual population disappearance may be at least several decades required for the eventual death of adult representatives of the populations. Another aspect that was not accounted for in the vulnerability analysis is the possibility of the wide environmental tolerance within the populations that were not manifested in the current biotic and abiotic conditions, but that could contribute to species survival in climate change scenarios.

#### **4. Conservation strategy**

#### **4.1 Conservation status**

More than 350 *Magnolia* species have been described worldwide, and of a total of 314 that have been evaluated in their conservation status, 48% (147 sp.) have been classified as threatened with extinction in the wild [107]. However, there are still 34% of *Magnolia* species that have not been evaluated mainly due to data deficiencies, despite that *Magnolia* conservation has become a major international task [108]. Therefore, in this section, we present an evaluation of the conservation status of the three western Mexican *Magnolia* species (section *Magnolia*) that have been evaluated so far [18], to propose and implement conservation measures.

#### *4.1.1 Methods*

*Conservation status assessment.* The threatened status of *M. pacifica* s.s., *M. talpana*, *M. pugana* s.s*.*, *M. granbarrancae*, *M. vallartensis,* and *M. iltisiana* were assessed here using the IUCN Red List Criteria (criteria B1ab + B2ab, [109]) and the GeoCAT cloud software tool [110]. The Extent of Occurrence (minimum convex polygon, EOO) and the Area of Occupancy (grid cell area with occupancy, AOO) of each taxon were delimited from georeferenced records obtained from IBUG herbarium specimens, the GBIF, Tropicos.org, REMIB-CONABIO, REBIOMEX

databases, and field data. AOO was based on the IUCN default cell width of 2 km. The criterion C-2 (Genetics) of the Method of Extinction Risk Evaluation of Plants in Mexico (MER) from the Mexican Official Norm 059 [111] was used to contribute partially to the MER assessment. Criterion C-2 proposes that (1) if the population has heterozygosity (He) < 10–20% (depending on the molecular marker used) and (2) a genetic differentiation (Gst or Fst) > 20%, it has a higher threatened status or extinction risk.

### *4.1.2 Results and discussion*

The estimated EOO (km2 ) and AOO (km2 ) were: for *M. pugana* s.s. *=*1,259.3, 96, respectively; *M. granbarrancae =* 0.7, 12; *M. pacifica* s.s. =1,216.2, 72; *M. talpana* = 91.8, 32; *M. vallartensis* = 124.0, 44; and *M. iltisiana* = 19,444.2,196 (**Table 6**). These western Mexican *Magnolia* species are in an endangered status because of their higher fragmented populations, lower genetic diversity, and narrower extent of occurrence when compared with other threatened *Magnolia* species [48].

*M. granbarrancae* and *M. talpana* have extremely narrow geographical ranges (EOO < 100 km<sup>2</sup> ) that are in accordance with the IUCN criteria for the category of Critically Endangered species: B1ab (iii, v). Populations of these species are small and severely fragmented (criterion B1a), and present a continuing decline in the area, quality of habitat [criteria B1b (iii)], and number of mature individuals [criteria B1b (v)] [109]. Moreover *M. granbarrancae* fulfill with the IUCN criterion C1 for Critically Endangered species, having a known total number of 74 mature individuals for the species, and an estimated or projected continuing decline of at least 25% in the next three years because the flooding of its habitat by the construction of the El Zapotillo damp on the Río Verde.

The genetic diversity of a species is an important indicator of its conservation status due to its positive correlation with the capacity to adapt and overcome abiotic and biotic changes. The genetic diversity of the three species studied here is considered lower than the average (H = 0.22) [45], and it is even lower than that of *M. sharpii* (I = 0.56), a species categorized as endangered because of its narrow EOO (2,228 km<sup>2</sup> ), severely fragmented and degraded habitats, and the fact that it is known from only five locations [48, 107]. *M. pugana* together with *M. granbarrancae* had a total heterozygosity HT of 0.158, *M. pacifica* together with *M. talpana*, of 0.175, and *M. vallartensis* of 0.171, this low genetic diversity of the three species is in accordance with criterion C-2 (intrinsic biological vulnerability with genetic heterozygosity <20%) of the Mexican Standard NOM-059-SEMARNAT MER for being considered in the category of Endangered species [111].

The IUCN criteria do not consider the levels of genetic diversity and differentiation for assessing extinction risk, but *M. pugana* and *M. granbarrancae* suffer of very low genetic diversity [11] and very low number of individuals, so these two species should be categorized as Critically Endangered even when *M pugana* s.s. has an area of occupancy of 96 km<sup>2</sup> . It is noteworthy that despite *M. pugana* s.s. EOO and AOO are in accordance with the IUCN Endangered category, their highly fragmented and isolated small populations suffering a seasonal dry and extreme climate, and its low genetic diversity make consider this species as Critically Endangered. *M. pugana* s.l. (including *M. granbarrancae*) has even lower genetic diversity and higher genetic differentiation than the other western Mexican *Magnolia* species studied. This fact, together with the fact of having more fragmented, more isolated, smaller populations, and being surrounded by a seasonally drier environment [103], make *M. pugana s.s.* and *M. granbarrancae* be proposed to be included in the category of Critically Endangered of extinction, as it had been previously cataloged by [112] for *M. pugana* s.l.



**Table 6.** *Relevant conservation data to guide establishing conservation priorities for* Magnolia *section* Magnolia *in western México. EOO: Extent of occurrence, AOO: Area of occupancy.*

#### *How to Save Endangered Magnolias? From Population Biology to Conservation Action… DOI: http://dx.doi.org/10.5772/intechopen.94346*

*Magnolia vallartensis* should also be considered as Critically Endangered, as it has been categorized by [107], due to having an EOO of only 124 km<sup>2</sup> , low number of individuals, high deforestation rates, forest plagues and fires, fragmentation, climate change, cattle raising, roads and urban growth. Both, the EOO and the AOO of *M. pacifica* s.s. are in accordance with the IUCN category of Endangered (EOO < 5,000 km<sup>2</sup> and AOO <500 km<sup>2</sup> ). Furthermore, in the case of *M. pacifica* s.l. (including *M. talpana*) and *M. vallartensis* the extent of occurrence is projected to suffer a severe reduction in the next 80 years as a consequence of the shrinkage of the areas of high habitat suitability, estimated under the highly probable SSP3–7.0 and less probable SSP2–4.5 scenarios. The scope of the projected extent of occurrence reduction in the case of *M. vallartensis* may bear this species into the critically endangered status by the end of the century [criteria B1b (i, iii)]. In the case of *M. pacifica* s.l. the projected reduction of the area of suitable habitat for the end of the century is highly significant, however, may be not enough to move species to the critically endangered category by using only this criterion.

Despite *M. iltisiana* has an AOO <500 km<sup>2</sup> (196), and that it has some populations with severe fragmentation, as those near Morelia city (in Michoacán), several populations of this species do not have severe fragmentation and inhabit in the protected area of Sierra de Manantlán Biosphere Reserve. Therefore *M. iltisiana* should maintain its category of Vulnerable, as it has been suggested by [107].

*Magnolia granbarrancae* which is located to the northeast side of the Santiago river canyon (composed by the RV locality), *M. pugana* s.s. (composed by ALV, ASL and APV), and the populations of *M. pacifica* s.s*., M. talpana* and *M. vallartensis* should be considered as five separate units of conservation. The implementation and enforcement of *in situ* and *ex situ* conservation actions should protect and preserve at least one locality of each population of *M. pugana* s.s., and *M. granbarrancae*, as well as the most divergent localities of *M. pacifica* s.s*.* (CSJ and SS), *M. talpana* (BA) and *M. vallartensis* (PV and APM)*.* The BA locality of *M. talpana* ("Bosque de Arce", maple forest) conserve the highest genetic diversity, which coincides with being one of the most important forests in terms of plant species richness, endemism, and floristic composition for western Mexico [113, 114]. Greater efforts must be made to preserve all of these taxa, and a higher focus is required to protect *M. pugana* s.s and *M. granbarrancae*, characterized by low levels of genetic variation and highly fragmented and small populations. All these *Magnolia* species must be fully evaluated with the MER method for inclusion in the list of endangered species of the official Mexican Standard NOM-059. Education, conservation, management, and ecological restoration plans are badly needed to decrease their threatened status and raise awareness of the fate of these important species in extinction risk.

#### **4.2 Propagation and reintroduction**

Magnolias, like other plant species, may be reproduced both sexually (by seeds) and asexually (through plant tissues).

#### *4.2.1 Sexual reproduction*

This kind of propagation involves genetic recombination, which provides a genetic variability that improves the plant's ability to adapt to its environment [29, 115], ensuring that long-term survival by reducing the risk of suffering a bottleneck effect, which puts a species in danger of extinction [116]; Also, more vigorous seedlings are generated and the propagation is easier and cheaper than asexual reproduction, on the other hand, the plants take longer to reach maturity and bloom. In section "2.6 Pre-germination and seed dormancy treatments" of this *How to Save Endangered Magnolias? From Population Biology to Conservation Action… DOI: http://dx.doi.org/10.5772/intechopen.94346*

chapter, the aspect of sexual reproduction in magnolias of Western Mexico is addressed more extensively.

## *4.2.2 Asexual propagation*

Asexual reproduction has been only reported for *M*. *tamaulipana* of sect. *Magnolia* and for species of sect. *Macrophylla*. Asexual propagation occurs through stems, shoots and roots and is more convenient for horticultural purposes, where it is sought to preserve certain characteristics through generation of clones and flower faster than those propagated by seeds [29], however, the methods used are more expensive and transportation is more complicated [117]. Although no studies have been conducted on vegetative propagation in the Mexican magnolias of the sect. *Magnolia*, we list the most used horticultural methods for the *Magnolia* genus [29, 117]:

#### *4.2.2.1 Cuttings*

This approach has the advantage of reducing costs in large-scale production and the plants obtained are more uniform, but to ensure survival it is necessary to have better-equipped facilities and develop specific protocols for Neotropical magnolias. Method for cuttings in *Magnolia* could be found in the following references: [29, 117, 118].

#### *4.2.2.2 Layering*

It is recommended when you want to get only a few plants. Produces larger plants in a shorter period of time, but requires more space and labor [29]. More information about this technique can be avaible in the following references [29, 117, 118].

### *4.2.2.3 Harnpariphan's technique.*

Here named as such, is an intermediate cutting-layering technique. In the cutting a reed incision is made 5 cm above the basal cut, a small piece of plastic is inserted between the reed and the stem to ensure an acute angle separation. A paste with fungicide and rooting hormone (1:1) is applied to the exposed sides of the incision. Subsequently, the wound area is covered with a substrate, securing it as layering with a piece of plastic tied at the ends with ropes. A small segment is cut from the basal part of the cutting-layer in water, to ensure that the air does not interrupt the flow of water through the conductive vessels. The basal part must be in contact with the water and the covered region of the wound must remain in the air, taking care to always keep the layering moist, making sure that the plastic has small holes that allow the excess water to drain. Leaf areas with lesions or infections should be trimmed and the water always kept fresh by making replacements when necessary [117].

#### *4.2.2.4 Grafting*

It is an easy and cheap method but requires more space, labor and time. Recommended for species and cultivars difficult to root [29], the graft should be done between genetically closest species since there is better long-term compatibility [119]. It is a great alternative for threatened species with low fertility [117]. For major types of grafts of magnolias see the following references: chip-budding [29, 118], side-grafting [29], wedge or cleft grafting [117], crown grafting [117],

canutillo graft [117], shield budding [117], patch budding [117], and approach grafting [117].
