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Revista Chapingo serie ciencias forestales y del ambiente

versión On-line ISSN 2007-4018versión impresa ISSN 2007-3828

Rev. Chapingo ser. cienc. for. ambient vol.29 no.1 Chapingo ene./abr. 2023  Epub 02-Mar-2024

https://doi.org/10.5154/r.rchscfa.2022.05.038 

Scientific articles

Ability of morphological traits from needles and cones to identify Pinus pseudostrobus Lindl. varieties

Sebastián Escobar-Alonso1 

J. Jesús Vargas-Hernández* 

Javier López-Upton1 

1Colegio de Postgraduados, Postgrado en Ciencias Forestales. km 36.5 carretera México-Texcoco. C. P. 56230. Montecillo, Texcoco, Estado de México, México.


Abstract

Introduction:

Pinus pseudostrobus, a species of ecological and economic importance in Mexico, has a wide distribution and intraspecific variation, which generates controversy regarding the identification of its operational taxonomic units (OTU).

Objectives:

To evaluate morphological traits of cones and needles of P. pseudostrobus to distinguish varieties and identify traits with greater discriminatory capacity.

Materials and methods:

Twelve traits in cones and 20 in needles were measured on samples from 153 trees identified in four putative OTU. Univariate and multivariate ANOVA, variance component estimation and canonical discrimination analysis (CDA) with cross-validation were carried out.

Results and discussion:

Thirteen needle and 12 cone traits showed significant differences between OTU. The variance between OTU increased 6.8 %, on average, when considering the varieties oaxacana and apulcensis as distinct OTU. Multivariate analysis confirmed significant differences in foliage and cones between OTU. The variation explained by the CDA increased from 0.35 to 0.74 and 0.76 when using foliage, cones, and overall variables, respectively. Eight cone traits allowed discrimination among OTU with average error of 9.2 %.

Conclusions:

Despite the wide intraspecific variation, it was possible to differentiate the main OTU of P. pseudostrobus with cone morphological markers, with important implications for a better management of genetic resources of this species. The present study shows evidence of morphoanatomical differences between oaxacana and apulcensis varieties.

Keywords: canonical discrimination; operational taxonomic unit; morphological variation; oaxacan variety; apulcensis variety

Resumen

Introducción:

Pinus pseudostrobus, especie de importancia ecológica y económica en México, tiene amplia distribución y variación intraespecífica, lo que genera controversias en la identificación de sus unidades taxonómicas operativas (UTO).

Objetivos:

Evaluar características morfológicas de conos y acículas de P. pseudostrobus para distinguir variedades e identificar caracteres con mayor capacidad de discriminación.

Materiales y métodos:

Se midieron 12 caracteres en conos y 20 en acículas en muestras de 153 árboles identificados en cuatro UTO putativas. Se realizaron análisis de varianza univariada y multivariada, estimación de componentes de varianza y discriminación canónica (ADC) con validación cruzada.

Resultados y discusión:

Trece características de acículas y 12 de conos mostraron diferencias significativas entre UTO. La varianza entre UTO aumentó 6.8 %, en promedio, al considerar las variedades oaxacana y apulcensis como UTO distintas. El análisis multivariado confirmó diferencias significativas en el follaje y conos entre UTO. La variación explicada por el ADC aumentó de 0.35 a 0.74 y 0.76 al utilizar variables de follaje, conos y en conjunto, respectivamente. Ocho caracteres de conos permitieron la discriminación entre UTO con error promedio de 9.2 %.

Conclusiones:

A pesar de la amplia variación intraespecífica fue posible distinguir las principales UTO de P. pseudostrobus con marcadores morfológicos de conos, con implicaciones importantes hacia un mejor manejo de los recursos genéticos de la especie. El trabajo muestra evidencias de diferencias morfoanatómicas entre las variedades oaxacana y apulcensis.

Palabras clave: discriminación canónica; unidad taxonómica operativa; variación morfológica; variedad oaxacana; variedad apulcensis

Highlights:

  • The wide phenotypic variability complicates the identification of Pinus pseudostrobus varieties.

  • Multivariate methods identified four putative varieties of P. pseudostrobus.

  • Cone traits were more efficient for discriminating the varieties.

  • The study showed morphoanatomical differences between oaxacana and apulcensis varieties.

Introduction

Pinus pseudostrobus sensu lato is one of the most important native species in Mexico due to its wide natural distribution, ecological conditions of its habitat, productivity, and wood quality (Viveros-Viveros, Sáenz-Romero, López-Upton, & Vargas-Hernández, 2005). Its distribution extends from the highlands of Honduras and Guatemala to the states of Chiapas, Oaxaca, Guerrero, Jalisco, Michoacán, Mexico, Morelos, Puebla, Hidalgo, Tlaxcala, and Veracruz (Perry, 1991), with some populations in Durango and Nuevo León (Farjon & Styles, 1997) Mexico. A 250 km wide strip in the Isthmus of Tehuantepec (Oaxaca and Veracruz) divides the populations of Chiapas and Central America from those of central Mexico; the northern populations are separated from the central region by a similar gap (Farjon & Styles, 1997). Because of the extent and discontinuity of its natural distribution, the species has wide intraspecific variation (Viveros-Viveros, Sáenz-Romero, Vargas-Hernández, & López-Upton, 2006), an important issue for the management and use of its genetic resources (Des Roches et al., 2018).

Pinus pseudostrobus was described in 1839 by John Lindley with botanical samples collected in Angangueo, Michoacán (World Checklist of Vascular Plants [WCVP], 2020). Several varieties or operational taxonomic units (OTU) have been proposed, considering the wide intraspecific variation it harbors. Some of these OTUs are accepted by some authors and rejected by others, which has generated significant controversy. In 1839, Lindley also described P. apulcensis, which in 1909 George Shaw considered a variety of P. pseudostrobus (var. apulcensis Shaw), and which currently has several heterotypic synonyms such as P. oaxacana, P. pseudostrobus var. oaxacana and P. oaxacana var. diversiformis (WCVP, 2020). Pinus pseudostrobus var. oaxacana was classified as a species (P. oaxacana) due to its resin chemistry (Mirov, 1958). Maximino Martínez proposed it as a variety in 1948, believing that the pronounced apophysis distinguish the populations of Oaxaca and Veracruz from the samples used to describe var. apulcensis, originally from Apulco, Hidalgo (Farjon, 1995; Farjon & Styles, 1997). On the other hand, var. coatepecensis (Perry, 1991), also proposed by Martínez, was rejected by Farjon and Styles (1997) and is considered a synonymy of var. pseudostrobus (WCVP, 2020). Other authors claim that only var. apulcensis is worthy of being considered a variety of P. pseudostrobus and its main distinctive feature is the apophysis height (Farjon & Styles, 1997; Farjon & Filer, 2013).

There is uncertainty and controversy over the definition of the UTOs of this species, while, in the field, collectors and academics in Mexico use the varieties proposed by Martínez since 1948. Identification and characterization of intraspecific OTUs of P. pseudostrobus is difficult in part due to their large variability in morphological traits (Stead, 1983; Stead & Styles, 1984; Farjon, 1995), associated with the extent and geographic discontinuity of their habitat and interaction of evolutionary forces influencing magnitude and structure of genetic variation, such as natural selection and genetic grift (Bussotti, Pollastrini, Holland, & Brüggemann, 2015; Caruso, Maherali, & Martin, 2020). In addition, the phylogenetic and reproductive proximity with other pines favors natural hybridization in overlapping areas of its geographic distribution (Delgado et al., 2007; Hernández-Velasco et al., 2021), which makes intraspecific OTUs difficult to characterize and describe; for example, introgressive hybridization has been detected with Pinus montezumae Lamb. (Delgado et al., 2007), P. douglasiana Martínez (López-Reyes, Pérez de la Rosa, Ortiz, & Gernandt, 2015) and possible hybridization with P. arizonica var. stormiae (Martínez) Silva (Gernandt, Hernández-León, Salgado-Hernández, & Pérez de la Rosa, 2009).

Over and above the taxonomic approach, it is important to identify morphological markers that facilitate the differentiation of the species’ varieties because some of them may have adaptive value, useful for the more efficient management, use and conservation of genetic resources of the species and its congeners. By using multivariate analysis techniques, it is possible to integrate variations and interrelationships of these markers. Canonical discrimination analysis allows maximizing the variance between groups and minimizing the variance within groups with linear functions or canonical variables, which can increase the ability to discriminate between individuals belonging to different OTU (Faisal, Haq, & Iqbal, 2021). This approach has been used in studies for the analysis and interpretation of variation in cone and foliage traits in species such as Pseudotsuga menziesii (Mirb.) Franco (Reyes-Hernández, Vargas-Hernández, López-Upton, & Vaquera-Huerta, 2005, 2006), species of the Pseudostrobus group (Stead, 1983; Stead & Styles, 1984), Pinus hartwegii Lindl. (Iglesias, Solís-Ramos, & Viveros-Viveros, 2012) and the P. strobiformis-P. ayacahuite complex (Leal-Sáenz et al., 2020).

The objectives of this study were (i) to evaluate the potential of morphological and anatomical traits of needles and cones to identify P. pseudostrobus varieties, especially between the varieties oaxacana and apulcensis; and (ii) to identify traits with greater capacity to discriminate the OTUs of this species in the south-central region of its natural range in Mexico. The hypotheses were: (i) the morphological traits of cones are more efficient than those of needles to discriminate the OTU of P. pseudostrobus, but the discrimination capacity increases when using both groups of traits; and (ii) in addition to apophysis height, there are other morphological and anatomical traits that facilitate discrimination between varieties of the species.

Materials and methods

Plant material

Samples of foliage and mature female strobili (cones) from the upper part of the crown were collected from 153 trees from various localities (Figure 1). We sampled 66 individuals of the typical variety P. pseudostrobus var. pseudostrobus (Chiapas, Mexico, Oaxaca, Puebla, Tlaxcala, and Veracruz), 60 of oaxacana (Chiapas, Oaxaca, Puebla, and Veracruz), 17 of coatepecensis (Veracruz) and 10 of apulcensis (Hidalgo).

Figure 1 Geographical location of trees sampled for each variety of Pinus pseudostrobus

Morphological and anatomical traits evaluated

Five healthy and developed fascicles (i. e., formed the previous year) and five mature, well-developed cones without apparent defects or malformations were selected from each tree. Information on 20 needle and 12 cone traits was collected using the methodology described by Stead (1983).

In foliage, the number of needles per fascicle (NN), sheath length (SL, cm), length (NL, cm) and thickness (NT, mm) of the longest needle in the fascicle, needle shape coefficient (NSc = NT/NL, mm∙cm-1) and sheath/needle ratio (SR = SL/NL) were measured. Also, number of 'teeth' (NT), lines with stomata (LS), number of stomata per line (NS), and total stomata (TS = LS x NS) were counted in the mid-section of the longest needle, 5 mm from the dorsal side. In cross sections of the middle section of the same needle, the total number of resin canals (RCT) with external (RCE), internal (RCI), middle (RCM) and septal (RCS) position was counted, and the proportion of resin canals with middle position was calculated (PRCM = RCM/RCT). Similarly, the number of intrusions of the hypodermis into the endoderm, number of hypodermal cells touching the endoderm (CHE), number of endodermal cells on the dorsal surface of the vascular bundle (EC) and the RCH ratio (RCH=CHE/EC) were counted.

In closed cones, the length of the peduncle (PL, mm), length (CLC, mm) and maximum diameter (CDC, mm) of the cone were measured and their shape coefficient was calculated (CSC = CLC/CDC); they were then dried at room temperature to open the scales and extract the seeds. Once the cones were opened, the maximum diameter (CDO) was measured, and their degree of openness was calculated (ODC = CDO/CDC). Three fertile scales were separated from the central part of each cone and the width (WA, mm), depth (DA, mm) and height (HA, mm) of the apophysis were measured and the ratios between them were calculated (RWD = WA/DA, RWH = WA/HA and RDH = DA/HA).

Statistical analysis

Univariate and multivariate analysis of variance was performed to determine the magnitude of trait variation between and within the OTUs, and to evaluate their ability to discriminate the putative varieties of P. pseudostrobus. In the univariate analysis, the MIXED procedure of SAS® version 9.4 (Statistical Analysis Software Inc., 2015) was used to estimate mean values for each putative variety and, when there were significant differences (α = 0.05), comparisons between varieties were made with Tukey's test. The discrete variables were transformed with the square root function to meet the criteria of normality and homoscedasticity; however, the results were similar to those of the original variables, so it was decided to present the analyses of the non-transformed values. In the multivariate analysis, carried out with the GLM procedure of SAS, the statistical significance of the variety effect was determined and the oaxacana and apulcensis varieties were compared by a contrast, using needle and cone traits separately.

To evaluate the effect of considering the oaxacana and apulcensis varieties as different UTOs, variance components were estimated for the variables that were significant in the univariate analysis. The VARCOMP procedure of SAS® (2015) was used with two alternative models: the first one considered the four putative OTU, while the second one grouped the samples of oaxacana and apulcensis in the same OTU.

The discriminatory ability of needle and cone traits was determined using a canonical discriminant analysis (CDA) with each group of variables separately and together. The 'candisc' function (Friendly, Fox, & Friendly, 2021) of the R software v. 3.6.1 (R Core Team, 2020) was used to estimate the canonical variables (CV) and the graphical representation of the canonical structure. Linear functions (discrimination criterion) and their cross-validation were calculated with the 'lda' and 'predict' functions of the MASS package with the same “a priori” probability (25 %) of membership (R Core Team, 2020; Ripley et al., 2021). Prior to CDA, the STEPDISC procedure (SAS, 2015) was used for the selection of variables from each group with significant contribution (P ≤ 0.05) to the discrimination model.

Results and discussion

Morphological differentiation for needles and cones

Table 1 shows that the four P. pseudostrobus OTUs under study were differentiated by needle morphology, as differences were found in 13 of the 20 traits evaluated (P ≤ 0.05); variables related to resin canals and hypodermal cells were not significant (P > 0.1617). Although the coefficient of needle shape, sheath length, number of needles, number of intrusions of the hypodermis into the endoderm and the proportion of resin canals with middle position were significant (P ≤ 0.05), the mean comparison test showed no differences in the average values of varieties, probably due to the imbalance in the sample size of the varieties compared. In the case of stomata per line and total stomata, the values were higher for apulcensis variety compared to oaxacana variety.

Table 1 Statistical significance (P) of the effect of varieties (Fvar) and average values of the morphological traits of needles and cones in Pinus pseudostrobus varieties. 

Traits P(Fvar) coatepecensis pseudostrobus oaxacana apulcensis
Needles
Number of needles 0.0193 5.05 a 4.97 a 5.00 a 5.06 a
Length of needle (cm) <0.0001 20.44 b 21.93 ab 24.88 a 24.92 a
Needle thickness (mm) 0.0003 0.87 c 0.92 bc 0.95 ab 1.04 a
Sheath length (cm) 0.0175 2.23 a 2.10 a 2.25 a 2.34 a
Needle shape coefficient (mm∙cm-1) 0.0059 0.04 a 0.04 a 0.03 a 0.04 a
Ratio sheath length/needle length 0.0003 0.11 a 0.09 ab 0.09 b 0.09 ab
Number of teeth 0.0248 22.64 ab 23.00 a 21.08 b 22.51 ab
Stomata lines <0.0001 3.38 c 4.46 ab 4.22 b 5.18 a
Stomata per line <0.0001 64.10 a 63.60 a 60.30 b 64.50 a
Total stomata <0.0001 215.60 d 282.10 b 254.20 c 333.30 a
Total resin canals 0.2422 3.32 3.3 3.13 3.45
External resin canals 0.1854 0.18 0.17 0.09 0.24
Internal resin canals 0.1758 0.06 0.15 0.07 0.15
Middle resin canals 0.6839 3.09 2.98 2.96 3.06
Septal resin canals 0.4312 0 0 0.01 0
Hypodermal intrusions 0.0345 0.09 a 0.22 a 0.36 a 0.09 a
Hypodermal cells 0.1686 0.11 0.34 0.5 0.14
Endodermal cells 0.0007 9.80 b 10.50 a 10.58 a 11.28 a
Middle resin canals ratio 0.0389 0.95 a 0.92 a 0.96 a 0.90 a
Ratio of hypodermal cells 0.1617 0.01 0.03 0.05 0.01
Cones
Peduncle length (mm) <0.0001 17.98 a 11.44 c 7.57 d 14.44 b
Closed cone length (mm) 0.011 91.80 b 99.51 ab 99.59 ab 110.27 a
Closed cone diameter (mm) <0.0001 43.74 c 45.30 c 59.52 a 51.83 b
Open cone diameter (mm) <0.0001 62.05 b 63.60 b 79.12 a 61.29 b
Cone shape coefficient <0.0001 2.11 a 2.23 a 1.70 b 2.12 a
Cone opening degree <0.0001 1.42 a 1.41 a 1.34 a 1.18 b
Apophysis width (mm) <0.0001 14.50 b 15.49 b 17.26 a 15.42 b
Apophysis depth (mm) <0.0001 11.46 c 13.61 b 14.88 a 11.23 c
Apophysis height (mm) <0.0001 2.97 c 5.00 b 9.29 a 4.52 cb
Apophysis width/depth ratio 0.006 1.29 ab 1.17 b 1.19 b 1.37 a
Apophysis width/height ratio <0.0001 5.08 a 3.47 b 2.03 c 3.44 b
Apophysis depth/height ratio <0.0001 4.03 a 3.00 b 1.72 c 2.51 b

Mean values (n = 153) in the same row with different letters are significantly different between varieties according to the Tukey's test (P = 0.05).

Table 1 indicates that cone morphology also showed differences between the variants of the species. Variety had a significant effect on all variables evaluated for cones (P ( 0.0110). Except for closed cone length, in all cases significant differences were found between oaxacana and apulcensis with contrasting mean values for peduncle length, open cone diameter, cone shape coefficient, depth and height of the apophysis. Multivariate analysis on needles and cones showed significant differences between varieties according to Wilks' Lambda, Pillai's trace and Hotelling's trace statistics (P < 0.0001), as well as the contrast between oaxacana and apulcensis, with a Wilks' Lambda of 0.0411 for needle traits and <0.0001 for cone traits.

Variance structure between and within varieties

The variance structure changed when differentiating var. oaxacana from apulcensis. According to Table 2, when considering the four varieties, the variance between OTU (σ2 var) increased between 0.1 and 14.7 % (average 6.8 %) with higher increases in the stomata per line and total needles variables, and in apophysis height, peduncle length and open cone diameter. The increase was most evident in traits with significant variation between varieties (Table 1). The increase in between-group variance and reduction in within-group variance (σ2 arb(var)) (i. e., greater within-variety homogeneity) when subdividing one or more groups is interpreted as evidence of needle and cone morphological differentiation between the two OTUs that were separated in the model. Perry (1991) discriminated the varieties because oaxacana has a longer, more prominent, and erect cone umbo than var. apulcensis; he also mentions that the needles are shorter and the cones longer in apulcensis, although no differences were found in this study (Table 1).

Table 2 Variance components between (σ2 var) and within varieties (σ2 arb(var)) of needle and cone traits of Pinus pseudostrobus in the alternative models with three and four varieties. 

Traits Three varieties Four varieties
σ2 var (%) σ2 arb(var)(%) σ2 var (%) σ2 arb(var)(%)
Needles
Length 22.5 77.5 20.8 79.2
Thickness 8.4 91.6 5.5 94.5
Sheath length/needle length 8.8 91.2 8.9 91.1
Number of teeth 8.2 91.8 9.6 90.4
Lines of stomata 5.9 94.1 8.0 92
Stomata per line 13 87 18.5 81.5
Total stomata 7.1 92.9 13.1 86.9
Endodermal cells 3.0 97.0 3.3 96.7
Cones
Peduncle length 33.5 66.5 48.2 51.8
Closed cone length 1.3 98.7 1.0 99
Closed cone diameter 62.9 37.1 69.1 30.9
Open cone diameter 44.7 55.3 58.9 41.1
Cone shape coefficient 53.7 46.3 63.8 36.2
Cone opening degree 14.9 85.1 11.0 89.0
Apophysis width 23.1 76.9 29.1 70.9
Apophysis depth 9.4 90.6 21.5 78.5
Apophysis height 54.1 45.9 68.7 31.3
Apophysis width/depth ratio 1.7 98.3 3.4 96.6
Apophysis width/height ratio 48.0 52.0 57.0 43.0
Apophysis depth/height ratio 57.2 42.8 62.7 37.3

Important traits for variety discrimination

According to Table 3, the STEPDISC analysis considered seven needle traits and eight cone traits as the most important for discriminating putative OTU. Among the selected cone traits, apophysis height stands out, which is important in the taxonomic identification of the OTU, especially for var. oaxacana (Farjon, Pérez de la Rosa, & Styles, 1997; Perry, 1991); however, peduncle length, cone shape coefficient and degree of opening, apophysis depth and depth/height ratio, length of closed cone and diameter of open cone were also important in differentiating varieties, which had not been reported in previous studies.

In the joint analysis, 11 variables were selected, all eight from cones and three from needles: total stomata, middle resin canals and endodermal cells, the latter was not selected in the separate analysis (Table 3). Although NL was not important in the joint analysis, this trait and those related to resin canals were useful in the study by Delgado et al. (2007) to differentiate between putative hybrids of P. pseudostrobus; also, needle number and length, and middle resin canals showed wide variation with a clinal pattern in P. patula (Dvorak, Jordan, Romero, Hodge, & Furman, 2001). The variance explained by unselected needle traits in the joint analysis, especially needle length, may be contained in the cone variables by the correlations observed with the apophysis depth/height ratio (r = -0.42), apophysis height (r = 0.41) and open cone diameter (r = 0.37).

Table 3 Partial coefficient of determination (r2) and significance (λ = Lambda Wilks; CCC = Canonical correlation coefficient) of needle and cone traits selected with the STEPDISC procedure, separately and together, for discrimination of Pinus pseudostrobus varieties. 

Order Characteristic Partial r2 Significance indicators
Pr > F Pr < λ Pr > CCC
Needles
1 Total stomata 0.1962 <0.0001 <0.0001 <0.0001
2 Needle length 0.1648 <0.0001 <0.0001 <0.0001
3 Middle resin canals 0.0967 0.0018 <0.0001 <0.0001
4 Sheath length/needle length 0.0702 0.0137 <0.0001 <0.0001
5 Stomata per line 0.0684 0.0162 <0.0001 <0.0001
6 Number of needles 0.0602 0.0296 <0.0001 <0.0001
7 Lines of stomata 0.0606 0.0296 <0.0001 <0.0001
Cones
1 Apophysis height 0.5760 <0.0001 <0.0001 <0.0001
2 Peduncle length 0.3314 <0.0001 <0.0001 <0.0001
3 Cone shape coefficient 0.2200 <0.0001 <0.0001 <0.0001
4 Apophysis depth/height ratio 0.2117 <0.0001 <0.0001 <0.0001
5 Cone opening degree 0.1392 <0.0001 <0.0001 <0.0001
6 Apophysis depth 0.1183 0.0004 <0.0001 <0.0001
7 Closed cone length 0.1160 0.0005 <0.0001 <0.0001
8 Open cone diameter 0.1141 0.0006 <0.0001 <0.0001
Needles and cones
1 Apophysis height 0.5760 <0.0001 <0.0001 <0.0001
2 Peduncle length 0.3314 <0.0001 <0.0001 <0.0001
3 Cone shape ratio 0.2200 <0.0001 <0.0001 <0.0001
4 Apophysis depth/height ratio 0.2117 <0.0001 <0.0001 <0.0001
5 Cone opening degree 0.1392 0.0002 <0.0001 <0.0001
6 Apophysis depth 0.1183 0.0004 <0.0001 <0.0001
7 Closed cone length 0.1160 0.0014 <0.0001 <0.0001
8 Open cone diameter 0.1141 0.0023 <0.0001 <0.0001
9 Total stomata 0.0799 0.0088 <0.0001 <0.0001
10 Endodermal cells 0.0746 0.0119 <0.0001 <0.0001
11 Middle resin canals 0.0616 0.0302 <0.0001 <0.0001

Canonical discrimination analysis

According to Table 4, the canonical correlation (Cr) was significant (P ≤ 0.0271 for all canonical variables (CV). In needle CV1 explained 58 % of the variance between varieties, while in cone CV1 explained 72 %, and 68 % in the joint analysis (Table 4; Figure 2). The cr value between the canonical values of individuals and the first two canonical variables were higher (0.64 ≤ cr ≤ 0.87) in the cone and joint analysis compared to the needle analysis (0.48 ≤ cr ≤ 0.59). That is, needle traits showed lower ability to separate varieties (Figure 2a). In contrast, the first two CV of cone traits separate three of the varieties, but there is overlap between var. pseudostrobus and apulcensis (Figure 2b). Although the inclusion of needle traits does not add additional information to that of cones in discriminating the first three varieties, it does reduce the overlap between var. pseudostrobus and apulcensis (Figure 2c).

Table 4 Summary of canonical variables (CV) for Pinus pseudostrobus variety discrimination based on needle and cone traits selected in the separate and joint analyses. 

CV rc * r2 c ** Eigenvalue Proportion explained Cumulative proportion Pr > F
Needles
1 0.59 0.35 0.53 0.58 0.58 <0.0001
2 0.48 0.23 0.29 0.32 0.90 <0.0001
3 0.29 0.08 0.09 0.10 1.00 0.0271
Cones
1 0.86 0.74 2.97 0.72 0.72 <0.0001
2 0.64 0.41 0.68 0.17 0.89 <0.0001
3 0.55 0.31 0.44 0.11 1.00 <0.0001
Needles and cones
1 0.87 0.76 3.08 0.68 0.68 <0.0001
2 0.70 0.49 0.94 0.21 0.89 <0.0001
3 0.59 0.34 0.52 0.11 1.00 <0.0001

*C r = canonical correlation. **c2 r = squared canonical correlation.

The canonical structure matrix shows the raw relationship between needle and cone traits with CV (Table 5). The coefficients represent the relative contribution of each trait to CV. In needles, the traits total stomata (TS) and lines with stomata (LS) are the most important associated with CV1, and needle length (NL) and number of stomata (NS) with CV2; in cones, the traits apophysis height (HA) and depth (DA), as well as their ratio (RDH) are the most important associated with CV1, while open cone diameter (CDO) and cone shape coefficient (CSC) are important in CV1 and CV2. In the joint analysis, no needle trait is relevant for CV1, but TS and endodermal cells (EC) are important in CV2 and contribute to the separation of var. apulcensis from the others (Figure 2c).

Figure 2 Structure of canonical variables (CV1 and CV2) of morphoanatomical traits of needles (a), cones (b) and as a whole (c) differentiating four putative varieties of Pinus pseudostrobus. Needles: LS = lines with stomata, TS = total stomata, SR = sheath /needle ratio, NS = number of stomata, NN = needles per fascicle, NL = needle length, EC = endodermal cells. Cones: CDO = open cone diameter, HA = apophysis height, DA = apophysis depth, CLC = closed cone length, CSC = cone shape coefficient, RDH = apophasis depth/height ratio, PL = peduncle length, ODC = openness degree. 

Table 5 Within-class standardized canonical coefficients (SCC) and total canonical structure coefficients (TSC) associated with Pinus pseudostrobus needle and cone traits in the separate and joint analyses to estimate canonical variables (CV). 

Characteristics CCE CECT
CV1 CV2 CV3 CV1 CV2 CV3
Needles
Total stomata 5.705 2.734 -2.673 0.672 -0.329 -0.426
Needle length -0.134 0.699 -0.527 0.314 0.745 -0.221
Middle resin canals -0.632 -0.008 -0.055 -0.096 -0.093 -0.241
Sheath length/needle length -0.527 0.066 -0.533 -0.432 -0.454 -0.263
Stomata per line -1.808 -1.554 0.483 -0.023 -0.713 -0.384
Number of needles -0.296 0.138 -0.608 -0.188 0.215 -0.709
Lines of stomata -4.651 -3.108 2.502 0.668 -0.113 -0.279
Cones
Apophysis height 0.143 0.933 -0.109 -0.852 0.244 -0.167
Peduncle length 0.529 0.437 -0.129 0.84 0.189 -0.053
Cone shape coefficient -1.943 -1.117 2.599 0.599 -0.567 0.272
Apophysis depth/height 0.705 1.007 0.237 0.825 -0.02 0.443
Cone opening degree 1.447 0.439 -1.225 0.122 -0.025 0.672
Apophysis depth -0.569 -0.598 0.457 -0.554 -0.066 0.27
Closed cone length 2.186 0.32 -2.945 -0.072 -0.258 -0.366
Open cone diameter -1.937 0.137 2.285 -0.692 0.409 -0.098
Needles and cones
Apophysis height 0.223 0.506 -0.594 -0.848 0.143 -0.256
Peduncle length 0.568 0.261 -0.33 0.836 0.141 -0.133
Cone shape coefficient -1.912 0.493 3.084 0.598 -0.375 0.495
Apophysis depth/height 0.759 0.76 -0.319 0.821 0.136 0.388
Cone opening degree 1.432 -0.404 -1.438 0.121 0.208 0.586
Apophysis depth -0.614 -0.145 0.741 -0.552 0.035 0.262
Closed cone length 2.111 -1.101 -2.952 -0.071 -0.337 -0.193
Open cone diameter -1.881 1.305 2.171 -0.69 0.303 -0.273
Total stomata 0.203 -0.465 -0.169 0.035 -0.634 -0.039
Endodermal cells -0.156 -0.329 -0.097 -0.093 -0.443 -0.134
Middle resin canals -0.036 0.529 0.333 0.101 0.034 -0.068

The morphological similarity of cones between var. pseudostrobus and apulcensis may be due to the semi-sympatric relationship they share (Carvajal & McVaugh, 1992) and the wide variability of the former, especially the apophysis height, which sometimes hinders its distinction (Farjon et al., 1997); however, when foliage traits are added, differentiation capacity increases (Figure 1c). It appears that the number of stomata and endodermal cells, associated with the size of the vascular bundle, are useful for distinguishing varieties. On the one hand, stomata density is an important functional trait in the control of water balance and CO2 capture per unit leaf area (Hetherington & Woodward, 2003); moreover, the size of the vascular bundle (xylem and phloem) affects water efficiency and safety, which is a fundamental basis for the regulation of gas exchange (Domec, Palmroth, & Oren, 2016). In this regard, the results support findings that var. apulcensis occupies part of the natural habitat of the typical variety but is absent in more xeric environments (Farjon & Styles, 1997). Cone traits clearly separate var. oaxacana from apulcensis, so these two OTUs should not be considered synonyms.

Cross-validation results reflected the reduced contribution of needle traits to the discrimination between varieties. Table 6 shows that, when using both groups of traits, similar hit percentages were achieved to those obtained with cone traits alone, with average errors of 7 % and 9.2 %. Cone traits were more efficient in the identification and correct discrimination of the UTOs. The greater simplicity and lower cost of measuring cone traits, compared to foliage traits, compensates for the limitations and costs of cone collection, which is only possible at certain time of the year. Nevertheless, the usefulness of some functional traits of adaptive value in needles, such as total stomata and endodermal cells should not be excluded. This is the case of distinguishing P. patula var. longipedunculata in the Sierra Madre del Sur with morphological markers of adaptive value (Dvorak et al., 2001).

Table 6 Percentage of observations (n) classified for each variety with linear discriminant functions (LDF) from needle and cone traits of Pinus pseudostrobus used in separate and joint analyses. 

Putative variety LDF Percentage classified as:
coatepecensis pseudostrobus oaxacana apulcensis
coatepecensis needles 88.2 5.9 5.9 0
cones 94.1 5.9 0 0
(n = 17) joint 100 0 0 0
pseudostrobus needles 13.6 39.4 27.3 19.7
cones 3 75.8 10.6 10.6
(n = 66) joint 1.5 81.8 9.1 7.6
oaxacana needles 10 13.3 66.7 10
cones 1.7 5 93.3 0
(n = 60) joint 1.7 8.3 90 0
apulcensis needles 10 10 20 60
cones 0 0 0 100
(n = 10) joint 0 0 0 100

In the cross-validation analysis, var. pseudostrobus presented the lowest percentage of hits with the two groups of traits (Table 6). The difficulty in its differentiation is due to the high morphological variability because of the wide geographic distribution and interaction with other processes such as diversification, followed by gene flow and low rates of evolution (Gernandt & Pérez-de la Rosa, 2014), which cause greater phenotypic variation compared to the other varieties and complicate its taxonomic delimitation (Figure 1). The wide overlap observed in the morphological variation of this variety compared to the others may be due to individuals in intertaxa transition zones or in the process of speciation, because of introgressive hybridization with other species of the same phylogenetic group (Gernandt et al., 2009). Delgado et al. (2007) were only able to classify 40 % of the putative hybrid individuals of P. pseudostrobus, due to the great variability of morphological traits caused by hybridization with other species

The results of the study confirm the wide intraspecific variation of P. pseudostrobus, coinciding with findings in other phenotypic traits such as seedling performance and survival in different growth environments (Villegas-Jiménez, Rodríguez-Ortíz, Chávez-Servia, Enríquez-del Valle, & Carrillo-Rodríguez, 2016; Viveros-Viveros et al., 2005; Viveros-Viveros et al., 2006), frost tolerance (Viveros-Viveros, Sáenz-Romero, López-Upton, & Vargas-Hernández, 2007) and isoenzyme variation (Viveros-Viveros, Tapia-Olivares, & Sáenz-Romero, 2014). The identification of P. pseudostrobus OTUs and the recognition of the extent of intraspecific variation in traits of adaptive value are indispensable requirements for the proper management and sustainable use of the genetic resources of the species.

Conclusions

The study demonstrated the wide phenotypic variation in morphoanatomical traits of needles and cones in P. pseudostrobus and the feasibility of discriminating its varieties based on these characters; however, the complex interaction of evolutionary forces with intra- and interspecific hybridization processes makes precise identification difficult. Cone traits were more efficient than needle traits for discriminating the OTU of the species, but including both increases precision, especially for distinguishing the typical variety from var. apulcensis. Although apophysis height was important to distinguish the varieties, the inclusion of functional traits of adaptive value, especially those related to stomata density and vascular bundle size in needles, helps in the identification of OTU with a perspective to a better use of their genetic resources. This study shows evidence of morphoanatomical differences between oaxacana and apulcensis varieties.

Acknowledgments

This work is part of the research project “Establishment of regional asexual seed orchards and progeny trials of Pinus pseudostrobus for genetic evaluation of seed trees” (Code CONAFOR-2016-4-277784), financed by the Sectoral Fund for Research, Development and Technological Innovation in Forestry, CONAFOR-CONACYT. The authors thank the support provided by the collaborating researchers of the project for the collection of foliage and cone samples from natural populations of the species during the selection of the superior seed trees in the field.

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Received: May 26, 2022; Accepted: November 08, 2022

*Corresponding author: vargashj@colpos.mx; tel.: +52 595 114 9020

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