Field Work

The collection of specimens of Pinus was carried out in various localities of the state of Hidalgo (Table 1).

Leaf sections and staining

The needles were fixed in FAA (formaldehyde: Glacial acetic acid: 50 % ethyl alcohol, in a proportion of 5: 5: 90) for an indefinite period. Transverse sections were obtained manually, with a single-edged razor; of these, we selected the best for histological staining by means of the procedure utilized by ^{Hernández-León (2011)}; Citrisolv 2 (15 min); EtOH 100 % (5 min); EtOH 96 % (5 min); EtOH 70 % (5 min), EtOH 50 % (5 min); ddH₂O (5 min); Alcian blue (30 min); ddH₂O (5 min); EtOH 50 % (3 min); safranin (2 hr); 50 % EtOH (3 min); 70 % EtOH (3 min); 96 % EtOH (3 min); 100 % EtOH (3 min); Citrisolv (15 min). Once the sections were stained, they were mounted in permanent preparations using Entellan (Merk Co., Germany) synthetic resin, and observed at different magnifications under an optical microscope.

Character Encoding

Through a review of the literature and the observation of the permanent preparations, nine characters and their corresponding character states were established, as shown in Table 2 (^{Coulter and Rose, 1886}; ^{Shaw, 1914}; ^{Martínez, 1945}; ^{Lanyon, 1966}; ^{Mirov, 1967}; ^{Farjon and Styles, 1997}; ^{Ortiz, 1999}). The characters and character states were described with observation of the permanent preparations at different magnifications, with the help of a (Carl Zeiss) Axioscope 40 optical microscope. Leaf sections were photographed with a digital camera system coupled to the microscope, and images were edited in Adobe^® Photoshop^® CS6 Extended, version 13.0.1 x32 (2017), in order to highlight the relevant leaf anatomical characteristics. A morphoanatomical data matrix was built based on the observations.

Data analysis

The leaf anatomical data of the 12 species were analyzed with the unweighted pair group method with arithmetic mean (UPGMA) (^{Sokal and Michener, 1958}), using a presence/absence matrix (Table 3). The analysis utilized Jaccard’s Similarity Coefficient with the online tool DendroUPGMA (^{Garcia-Vallvé et al., 1999}).

Field Work

A total of 30 specimens were collected, processed and deposited in the collection of the Herbarium of the Universidad Autónoma del Estado de Hidalgo (Academic Area of Agricultural and Forestry Sciences at the Institute of Agricultural Sciences at the Autonomous University of the State of Hidalgo), campus Tulancingo, and in the National Herbarium of Mexico (MEXU). The specimens correspond to 1 - 3 individuals of the 12 pine species collected in Hidalgo (Table 1).

Leaf sections and staining

The thirty permanent preparations (30) of needle cross-sections were incorporated into the collection of the Herbarium of the Academic Area of Agricultural and Forestry Sciences at the Institute of Agricultural Sciences of the Autonomous University of the State of Hidalgo and are available for subsequent consultations.

Character encoding

The data matrix of characters and character states are presented in Table 3. The shape of the epidermal cells exhibited inter- and intra-specific variation, consistently with previous records (^{Farjon et al., 1997}).

Eight of the studied species exhibited the basal sheath of the permanent fascicle. The taxa with the deciduous fascicle basal sheath were Pinus ayacahuite, P. cembroides, P. leiophylla and P. pinceana, with the exception of P. leiophylla; these taxa correspond to the soft pines or subgenus Strobus. Pinus leiophylla is one of the few species of hard pines or subgenus Pinus with a deciduous fascicle basal sheath (^{Gernandt et al., 2005}). The character state of the fascicle basal sheath is helpful for allotting specimens to groups; however, in combination with other characteristics, it can be effective for the identification of certain taxa (^{Farjon et al., 1997}).

The studied material had two, three and up to five needles per fascicle; this character has been widely used for identifying pine species, generally in combination with other characteristics (^{Farjon and Styles, 1997}; ^{Farjon et al., 1997}; ^{Eckenwalder, 2009}). The needles of a fascicle form a cylinder; the number of needles per fascicle determines its transversal shape, which corresponds to a section of the cylinder; for example, when there is one needle, the transversal shape will be circular; in fascicles with two needles, it will be semi-circular (Figure 1B); in fascicles with three needles, it will be transversally triangular (Figure 1D, G, I, J, L), and in fascicles with four or more needles, it will have a widely triangular shape (Figure 1A, C, E, F, H, K) (^{Martínez, 1953}; ^{Farjon and Styles, 1997}).

A = Pinus ayacahuite; B = P. cembroides; C = P. devoniana; D = P. greggii; E = P. hartwegii; F = P. leiophylla; G = P. montezumae; H = P. oocarpa; I = P. patula; J = P. pinceana; K = P. pseudostrobus; L = P. teocote; * = Indicates the stomatal position; Cr = Resin duct canal; End = Endodermis; Epi = Epidermis; Escl = Sclerenchyma; Flo = Phloem; Hip = Hypodermis; Mes = Mesophyll; Tt = Transfusion tissue; Xil = Xyleme.

Figure 1 Cross-sections of needles of Pinus species of the state of Hidalgo. 

Pinus cembroides was the only species with two leaf shapes in the cross-section. Due to its semi-circular shape, it can be differentiated from the rest of the taxa (Figure 1B); however, when transversally triangular, it is impossible to separate it from P. pinceana (Figure 1J). Pinus ayacahuite was the only one that exhibited the epistomatal distribution (Figure 1A), while the rest of the species exhibited an amphistomatal distribution.

For the hypodermal cell wall thickness, the most frequently registered character state was “evenly thin hypodermal cell thickness” (e.g., P. ayacahuite, P. greggii and P. leiophylla; Figure 2C), while “hypodermal cell wall thickness gradually increasing inward” was less frequently observed (e.g., P. devoniana and P. pseudostrobus; Figure 2A). Pinus patula and P. pinceana exhibited intra-specific variation for this character (Table 3). According to ^{Farjon and Styles (1997)}, the long leaves, with layers of hypodermal cells with thin walls are restricted mainly to temperate or mesic subtropical climates (e.g., P. ayacahuite, P. greggii, P. patula).

A = Pinus devoniana; B, E, I = P. teocote; C-D = P. greggii; F, H = P. cembroides; G = P. oocarpa; Cre = External resin duct canal; Cri = Internal resin duct canal; Crm = Intermediate resin duct canal; Crs = Septal resin duct canal; End = Endodermis; Epi = Epidermis; Flo = Phloem; Hip = Hypodermis; Hv = Vascular bundle; Mes = Mesophyll; Tt = Transfusion tissue; Xil = Xyleme.

Figure 2 Cross-cutting sections of needles of Pinus species of the state of Hidalgo. 

The existence of different degrees of hypodermal intrusions in the mesophyll has been observed (^{Martínez, 1945}). In this research, Pinus devoniana and P. pseudostrobus exhibited hypodermal intrusions (Table 3, figures 1C, K and 2A); no intrusions are present in other species (e.g., P. greggii; Figure 2C). Hypodermal tissue concentrations have been observed to occur in marginal areas, whereas in the rest of the leaf the hypodermis persists in two cell layers (^{Farjon and Styles, 1997}).

In certain taxa, such as Pinus oocarpa, the number of resin duct canals was highly variable, ranging between 1 and 6, while P. pinceana, for example, consistently exhibited two resin duct canals (Figure 1J). In the latter case, the character can be of use for identifying the species at state level (Table 3) (^{Perry, 1991}; ^{Farjon and Styles 1997}; ^{Cole et al., 2008}). Previous studies indicate an interval of 1-12 resin duct canals in the leaves of pine species of Latin America, and in the case of the state of Hidalgo, 1-6 resin duct canals were found in each needle (^{Farjon and Styles, 1997}).

Pinus oocarpa and P. ayacahuite exhibited the largest number of resin duct canals (6); a lower number (1) was observed in sections of P. oocarpa needles. According to ^{Helmers (1943)}, there is a close relationship between the number of resin duct canals and the environment. Eventually, some needles may have no resin duct canals, but this condition is not associated with any particular species. P. donnell-smithii is the exception to specific recognition, since many of its needles have no resin duct canals (^{Perry, 1991}). According to certain authors, the number of resin duct canals is highly variable, and therefore they do not regard it as a systematic element (^{Martínez, 1945}).

The position of the resin duct canals is a characteristic that is commonly used for the classification and identification of species since ^{Engelmann (1880)} and ^{Coulter and Rose (1886)}, because most species are characterized by exhibiting a single position, although there are combinations like the ones cited in this study for Pinus devoniana and P. montezumae; (Figure 1C, G). Nevertheless, combinations with more than two positions have never been registered (^{Farjon and Styles, 1997}). The joint use of the position and number of the resin duct canals is very helpful for identifying many species (^{Perry, 1991}). In taxa with a variable number of canals, those that are largest and whose position is fixed are regarded as primary, and the smaller ones are considered to be subsidiary (^{Farjon and Styles, 1997}). Since their position is less variable than their number, it is more helpful for identification purposes (^{Martínez, 1945}). The intermediate position of the resin duct canals was predominant among the pines of the state of Hidalgo (e.g., Pinus leiophylla, P. montezumae, P. pseudostrobus) (^{Martínez, 1945}; ^{Farjon and Styles, 1997}) (Figure 1F, G, K), while the septal position was observed only in P. oocarpa; therefore, this character state is diagnostic for this species, at least in the state of Hidalgo (figures 1H, 2G).

The external thickening of the endodermal cell walls (Figure 2I) has been used as a diagnostic character (^{Farjon and Styles, 1997}). However, species with multiple individuals, such as those included in the present research, exhibit inter- and intra-specific polymorphism, which is not adequate for taxonomic identification.

The presence of one (Figure 2H) or two (Figure 2I) vascular bundles supports ^{Koehne’s proposal (1893)} of dividing the genus Pinus into two sections - Haploxylon and Diploxylon-, which correspond with the subgenera Strobus and Pinus (^{Gernandt et al., 2005}), consistently with the results documented herein.

Certain taxa -Pinus oocarpa and P. teocote (Figure 1L)- exhibit a sclerenchyma in the vascular region that surrounds and sometimes divides the vascular bundles (^{Farjon and Styles, 1997}). However, due to its variation, this is not a diagnostic character.

Cluster analysis

The analysis of similarity of the morphoanatomical characters of the needles resulted in two main groups (Figure 3). Group one comprises Pinus ayacahuite, P. cembroides and P. pinceana, of the subgenus Strobus, which are more related with one another than with other species; on the other hand, it includes P. greggii, P. patula and P. teocote, of the subgenus Pinus. The second main group consists of taxa of the subgenus Pinus: P. devoniana, P. hartwegii, P. leiophylla, P. montezumae, P. oocarpa and P. pseudostrobus; P. oocarpa was separated from the rest of the species within the group. At the terminals, groups are formed by taxon, except for a sample of Pinus cembroides that is grouped together with P. pinceana. According to ^{Eckenwalder (2009)}, the convergence in the characteristics of the leaves between unrelated species is caused by a strong association between the leaf characteristics and the environmental conditions, so that the former can reflect the mechanisms whereby the plants adapt to their environment (^{Tian et al., 2015}).

Cophenetic correlation coefficient = 0.847018494508054. The number in each terminal indicates the number of specimens per species.

Figure 3 Dendrogram obtained through an UPGMA group analysis using Jaccard’s similarity coefficient in 12 Pinus species distributed in the state of Hidalgo.  

Identification key

Based on the dichotomous key resulting from the research documented herein, it is possible to differentiate 10 of the 12 pine species of the state of Hidalgo. P. cembroides and P. pinceana are separated only when the shape of the transverse section is semi-circular in the first species.