Bo&nualJoumal oJ!he Ltnnean Sock& (2000), 133: 181-194. With 3 figures

doi:10.1006/boj1.1999.0315, available online at http://www.idealibrary.corn on llEal @

Phenetic analysis of the Mexican midland pinyon pines, Pinus cembroides and Pinus johannis

A. ROMERO*, M. LUNA AND E. GARCIA

Especialidad de Botcinica, Instituto de Recursos Naturales, Colegio de Postgaduados, Montecillo, Edo. Mkmico, C.R 56230, Mixico

M. F. PASSINI

Laboratoire de Biologie Hgktale Tropicale, Universite' Pierre et Marie Curie, Paris Vl, 12 rue Cuvier 75252, Paris Cedex 05, France

&ceivedJu$ 1998; occeptedfor publication November 1999

This account presents an analysis of allopatric, sympatric and juxtaposed populations of the pinyon pines, Pinw cembmides Zucc. and I! johannb M.-F. Robert, based on phenetic analysis of morphological data, and draws on a comparison and differentiation of I? culminicofa Andresen & Beaman. A qualitative and quantitative database for populations has been assembled and used for a cluster and ordination analysis using NTSYS. It was found that individual populations of the taxa were distinguishable. The emergence of morphological intermediate populations between I! cembmides and I! johannk was confirmed. The pattern of variation in I? johannb was found to be discrete, showing three geographical groups. The phenetic similarity between I! johannb from Zacatecas and I! johannb from San Luis Potosi and Queretaro was lower than that of I? johannb from Zacatecas and I! culminicola from Nuevo Leon. The following traits distinguish I? cembmides and the other two species: colour and fertility of seeds; cone shape; colour and width of needles; physiognomy; seed dimensions. This last trait can be used to distinguish three subgroups of pines with white endosperm in the northern, central and southern regions of the Mexican highlands.

0 2000 The Linnean Society of London

ADDITIONAL KEY WORDS:--cluster analysis - morphological differentiation - principal component analysis.

CONTENI'S

Introduction . . . . . . . . . . . . . . . . . . . . . . . 182 Material and methods . . . . . . . . . . . . . . . . . . . 184 Results and Discussion . . . . . . . . . . . . . . . . . . . 185

Cluster analysis . . . . . . . . . . . . . . . . . . . . 185 Principal component analysis (PCA) . . . . . . . . . . . . . 189

Conclusions . . . . . . . . . . . . . . . . . . . . . . . 192

* Corresponding author. E-mail: dahly@colpos.colpos.rm

0 0 2 ~ 7 4 / 0 0 / 0 6 0 1 8 1 + 14 $35.00/0 181

0 2000 The Linnean Society of London

I82 A. ROMERO E T A .

Acknowledgements . . . . . . . . . . . . . . . . . . . . 192 References . . . . . . . . . . . . . . . . . . . . . . . 192

INTRODUCTION

Finus subsection Cembmides was extant in Mexico during the Early Tertiary (Axelrod, 1983; Millar, 1993). The Sierra Madre Oriental in the Mexican highlands is the centre of survival and diversity, and the site of origin of the most recent taxa. Malusa (1992) considered that there is insufficient fossil evidence to support the hypothesis of the vicariance of the Mexican pinyon pine, although there are fossil records from the Eocene which indicate a greater area of distribution than the present one. The antiquity of the American fossils closest to the Cembmides subsection is based on the revision and description of Axelrod & Raven (1 985). Axelrod (1986) takes the record back 47 Myr for R sanjuanensis Axelrod (Creeden flora), 13 Myr for R hlloggii Webber (Stewart Spring flora), and 5-6Myr for II lim&enii Knowlt. (Bruneau flora). Citation of authorities follows Brummitt & Powell (1 992).

Pinus monophylla Torr. et FrCm., R edulis Engelm. and I( cembmides Zucc. were the dominant species of the pinyon pine woodlands in North America 37Myr ago (Connant, Thomas & Raush, 1945; Brown, 1957; Smith, 1957; Martin & Harrell, 1957; Van Devender & Spaulding, 1979). It is likely that the development of the ancestral taxon of I( cembmides originated in an era with a stable climate and substrate, quite opposite to the environment of present-day pinyon pine woodlands (Malusa, 1992).

Gelrod (1 986) considered that the prevailing climatic conditions of the Mid Tertiary, which favoured the speciation of this subsection, were not different from those observed in the Mexican highlands today.

Van Devender, Thompson & Betancourt (1987) proposed that the Pinus cembmides varieties recognized are recent geographic races, probably of Quaternary origin, i.e. 2 Myr old.

During the radiation process of the pinyon pines, the most obvious changes have occurred in size and height of ramification, needle number per fascicle and the location of stomata, since all these characters show immediate response to climate changes (McCunne, 1988; Malusa, 1989, 1992). Recently, a trend towards dioecism has been observed (Floyd, 1983; Ada , 1985; Floyd & Kohler, 1990; Passini, 1994). Such traits, among others, have served to distinguish the taxa belonging to the Cembroides group, which includes 19 culminicola Andresen et Beaman, I? johannis Rob.-Pass., R catarinae Rob.-Pass., R monophylla, R edulis, R quadnilia Parl., R discolor D.K. Bailey & Hawksw., R lagunae Passini and R cembmides (Robert, 1978, 1981; Passini, 1982a,b, 1987, 1993). II discolor would be excluded since it is synonym off!

johannis (Passini, 1994). The most morphologically and palynologically variable of these taxa is II cembmides (Axelrod, 1986; Passini, 1982a, 1993).

Zuccarini described R cembmides in 1832. More recently, Passini (1 982a,) described R cembmides Zucc. S.S. (R cembmides var. cembmides) as trees 5-15 m in height, with 2, 3 or 4 needles per fascicle, amphistomatic, with two resiniferous channels and seeds with pink endosperm. The colour of the endosperm differs from the other two species of the Cembroides group. The basic chromosomic number is x = 12, 2n = 24 (Garcia, 1985, 1992). This species (s.s.) is found in Texas at an elevation between 1500 and 2800m. It is also found in Mexico, in a belt extending north-west to

PHENETIC ANALYSIS OF MEXICAN PINES I83

south-east, in Sonora, Chihuahua, Coahuila, Nuevo Leon, Durango, Zacatecas, Aguascalientes, San Luis Potosi, Jalisco, Guanajuato, Queretaro and Hidalgo (Romero, Garcia & Passini, 1996).

Robert (1978) described Pinusjohannis from materials collected in Concepcion del Oro, Zacatecas. She showed that the main differences between l? cembmides s.1. and Rjohannk are in habit and size, colour of the endosperm and cotyledon number. She also mentioned that the greater similarity appeared to be between l? johannis and l? cembroides var. bicolor. This variety was recognized by Little in 1968; 1 1 years later Bailey & Hawksworth (1 979) elevated its rank to species, modifjrlng the epithet bicolor to discolor. However, Passini (1994), along with Little, agreed that l? discolor and Rjohannis are the same species, and by the priority principle established the correct name as Pjohannis Rob.-Pass. with l? discolor Bailey & Hawks. and l? cembroides var. bicolor Little as synonymous.

Robert (1978) and Passini (1 993, 1994) have emphasized the unequivocal difference between l? culminicola and Pjohannis. Pinusjohannis is found between 2500 and 2800 m in a region extending from Arizona and New Mexico to Queretaro, Mexico. Romero, Garcia & Passini (1996) published a map of its distribution.

The observed variation in the morphological characters of l? cembmides and l? johannis led us to formulate the following questions. Is each taxon morphologically differentiated and unequivocally identified? Are there morphologically intermediate individuals within the range of variation? A study was conducted to confirm morphological differentiation among the taxa using additional characters to those proposed by Passini (1 982a), especially those derived from sympatric and parapatric populations of P cembroides and I! johannis. The objectives of this study were: (1) to analyse the patterns of morphological variation among populations of different regions; (2) to distinguish, using a phenetic classification scheme, the relationships between species; and (3) to identify populations with morphologically intermediate characters. This paper presents the morphological evidence. A further paper will present the genetic evidence based upon isozyme studies.

Traditional procedures and multivariate analysis were used by Clausen & Crisci (1989) and Jensen et al. (1993) to analyse the phenetic differences, and classify the populations sampled. In the case of pinyon pine woodlands Snajberk, Zavarin & Debry (1 982) and A d a ( 1 985) produced morphometric studies using l? cembmides and related species to suggest hypotheses relating to the possibility of hybridization.

The patterns of variation of species have been studied by multivariate methods. Jeffers & Richens (1970) used principal components and cluster analysis to classify Ulmus sp. in England and France. Stead (1983) applied Canonical Discriminant Analysis to distinguish between Pinus patula Schltdl. subsp. tecunumanii and II oocarpa Schiede in mixed forest of Central America. A d a (1 985) used Fisher’s Discriminant Analysis to classifir morphological characters of mixed and pure populations of l? cembroides and l? johannis in La Amapola, San Luis Potosi, Mexico, and showed a greater phenotypic plasticity in l? cembmides. Favela (1988) used one form of dis- criminant analysis to establish relationships between groups of I! pseudostmbus Lindl., I! montezumue Lamb., l? hartzoegii Lindl. and l? rudis End. in northeastern and central Mexico. Classifications using numerical taxonomy also revealed patterns of leaf anatomical variation in subsection Oocarpae (Dyer, 1989). Perez (1993) used principal components and canonical discriminant analysis to describe the inter- and intra- specific variations of Pinus ayacahuite Ehrenb.-P strobijiwnis Engelm. complex in northern and southern Mexico.

184 A. ROMERO ETAL.

TABLE 1. Studied sites

Reference Location Elevation Temperature Substrate Soil type Vegetation Populations (m) Precipitation

Concepcion 24'37" 2700 16OC Calcareous Lithosol Pjohannis 1 (22) del Oro, Zac. IOI"28W 400 mm IIcembmide~ 1 (23) Galeana, N.L. 24'53" 3500 3.5OC Calcareous Lithosol II culminuoola 1 (24)

C harcas, 23'2 1'N 2475 14OC Calcareous Lithosol II johannis 1 (25) S.L.P. l o l o l IW 363 mm and Igneous Amapola, 22'04" 2400 1 8 O C Igneous Lithosol II cembmides 13

100' 15%' Rendzina

S.L.P. 101'08W 524 mm (6-8, 10, 12-16, 18-21)

(1-5,9, 11, 17) Rjohannis 8

Cadereyta, 20"5 I 'N 2500 16.8"C Calcareous Rendzina Pjohunnk 1 (26) Qro. 99'40W 454 mm II cembmides 1 (27)

TABLE 2. Traits used for the phenetic study of pinyon pines

Growth form Branching pattern Stem height (m) Branching height (m) Trunk diameter 30 cm above the ground Bark shape Adaxial's needle colour Needle length Needle width Fascicule sheath length Cone length (cm) Cone diameter (cm) Pedicel length (mm) Umbo size (mm) Umbo form Apophysis shape Scale number Fertile seeds number Sterile seeds number Pericarp colour Endosperm colour Seed length (mm) Seed width (mm) Pericarp thickness (mm) Cotyledon number

GRFO BRPA STHE BRHE TRDI BAFO ADNE NELE NEW FASH COLE COD1 PELE UMSI UMFO APSH SCNU FESE STSE PECO ENCO SEW SEW1 PETH CONU

I . Tree. 2. Shrub 1=3,2=4-5 ,3=6

I=0-1.9rn, 2=2-3.9m, 3 = 4 4 . 9 m , 4 = 2 5 m

1 =fissured to the large, 2 =fissured in size plates 1 =dark green, 2 =white Mean of 200 needles Mean of 200 needles Mean of 200 sheaths Mean of 20 cones Mean of 20 cones Mean of 20 cones Mean of 20 umbos of larger scales 1 =convex, 2 =concave 1 =depressed, 2 =elevated Mean of 20 cones Mean of 20 cones Mean of 20 cones I =black, 2=yeUow, 3=grey 1 =pink, 2 =white Mean of 10 seeds Mean of 10 seeds Mean of 10 seeds Mean of 30 seedlings

1 =4-5.9, 2 = 6 7 . 9 , 3=8-8.9, 4=9-10.9

1=10, 2=10.1-20, 3=20.1-30, 4 = 230.1

MATERIAL AND METHODS

The study area is part of the Mexican highlands (Table 1). The reports we consulted were those of Robert (1978), Garcia & Gonzalez (1991), Avila, Garcia & Reyes (1992), Romero, Luna & Garcia (1989) and Zavala & Campos (1993). The 'last three referred to f? j ohnn i s as Z? discolor.

Samples from three individuals in each population in each location were collected. They were described morphologically and 25 qualitative and quantitative characters were used for the numerical analysis. A mean/variable/individual was estimated (Table 2). The study considered 27 Operational Taxonomic Units (OTUs). Two

PHENETIC ANALYSIS OF MEXICAN PINES I85

types of data analysis were carried out: Cluster and Principal Component Analyses. The clustering method included the calculation of the similarity index between each pair of possible OTUs, with the average taxonomic distance coefficient, the construction of the similarity matrix between OTUs with the weight mean group, using the arithmetical mean and the graphic representation of the similarities in a phenogram. The coefficient of cophenetic correlation (r) as a measure of the bias and the matrix of the original distance were calculated. The Principal Components Analysis was carried out to observe the relation between the OTUs, and to estimate the importance value of the variables, which discriminate the groups formed. For that matter, a correlation matrix between characters and the eigenvalues and eigenvectors were calculated; later on, the OTUs were projected upon the axes which represent the first three principal components. The above analyses were done using NTSYS (Rohlf, 1992).

RESULTS AND DISCUSSION

Clustm anahsis

Figure 1 shows the phenogram derived from the analysis in which two large groups are distinguished. In the upper part (0, A) there is a group of shorter (1.5 m), bushy plants, multistemmed from the ground, with bicoloured needles and white endosperm. The lower part (H) contains taller individuals 5 m and above, tree and/or tree like, with branching above 2 my green needles and pink endosperm.

The upper group corresponds to the Pjohannis populations. Within it there are subgroups corresponding to the populations from Charcas (25) and La Amapola (1, 2,4,5,9,11,3,17) San Luis Potosi State and Cadereyta (26) Queretaro State. The second is formed by the topotype of Pjohannis in Concepcion del Oro, Zacatecas (22) which is phenetically related to I! culminicola, from Galeana, Nuevo Leon. The h s t subgroup shows a discrete pattern of variation.

The lower group consists of Z? cembmides populations where the pattern of variation is rather continuous, as measured by the values of the distance indexes, although it is possible to distinguish three subgroups. (1) La Amapola (6,13,15,2 1,7,14,16,8,12) which includes the samples from Concepcion del Oro (23); (2) the populations located southeast of La Amapola in the areas of the Sierra with the greatest densities (10,18,19,20); (3) the populations located in the southernmost area of its distribution, in Cadereyta (27). The cophenetic correlation coefficient (r= 0.8562) seems good.

Analysis of Figure 1 indicates that in sympatric areas, the morphological difference of the taxa studied is confirmed. This is substantiated by the position of Pjjohannis and I! cembmides in the samples from El Sombrerito (UTOs 9, lo), from El Quelital in La Amapola (UTOs 17, 18); from Cadereyta (UTOs 26, 27) and Concepcion del Oro (UTOs 22, 23). Furthermore, three OTUs somewhat separated from the previously described groups apparent include 17, grouped with I! johannis and 10 and 27, grouped with I! cembmides.

OTU 17 reveals a phenetic pattern which combines the characteristics of I! cembmides (mainly vegetative) and I! johunnis (reproductive). Thus the trees are tall with a mean height of 10 my a branching height at 5 m, three orders of branching, a trunk >35 cm diameter and seeds with pink endosperm. Such traits belong to I!

I86

-

I

A. ROMERO ET AL.

Average taxonomic distance coefficient

2.0 1.5 1.0 0.5 0.0

r 0 25 0 02 04

0 05 0 09 0 11 03 -

-

r l '

0 26 0 17 0 22 l ~ 2 4 8 06 8 13 15

8 21 8 07 8 14 8 16 8 08 8 12 8 23 8 18 8 19 8 20 8 10 827 -

l? johannis

l? culminicola

l? cembroides

Figure 1 . Distance phenogram of the relationships between pinyon pine populations (r=0.8562). The numbers correspond to the populations referred to in the text. (0) Pinus johannis, (A) I? culminicola, (m) I? cembmides.

cembmides, but there are other characters indicating R johannis: larger needles (4.89 cm), glaucus abaxial face, larger sheath fascicle (5.47 mm), small cone (2.88 cm long and 3.86 cm wide), small umbo (1.88 mm), umbo and apophysis compressed, abundant scales (19 as a mean), and few fertile seeds (5).

OTU 10, identified as Z? cembmides by its reproductive traits, more closely resembles individuals from Cadereyta than those from La Amapola. The trend in the distribution range of this marginal population is accentuated by the smaller size of the individuals and the branching configuration which relates them phenetically to Z? johannis; the rest of the characters are typical of Z? cembmides. It is important to mention that these populations occupy sympatric areas. In El Sombrerito (OTU 10) and El Quelital (OTU 17), both 49 cembmides and Fjohannis coexist as they do in Cadereyta.

In the first subgroup of Pjohannis, OTU 25 is the northernmost population, followed by La Amapola (OTUs 2,3,4,5,9,11); nearby is OTU 26 from Cadereyta,

PHENETIC ANALYSIS OF MEXICAN PINES

25"

24a

23'

22'

21'

102" 101" 100" 99"

Figure 2. Geographical locations of the phenetically related groups.

which represents the southernmost group. The geographical locations are shown in Figure 2, below the dotted line.

I! johannts of the Zacatecas subgroup and I! culminicola from Nuevo Leon (Fig. 2, above the dotted line), with only 14' latitudinal difference between them, have multistem branching at ground level; the white endosperm phenotype may have separated from the first subgroup due to other traits considered in the analysis of characters. I! johannis from Zacatecas and I! culminicola from Nuevo Leon appear to be phenotypically related, but differ from the subgroup off? johannis which includes the populations of San Luis Potosi and Queretaro.

The two well-defined groups in the phenogram can be differentiated by the colour

187

25"

24"

23"

22"

21"

188 A. ROMERO ETAL.

of the endosperm and growth habit, as was previously mentioned by Passini ( 1982a, 1994).

These results agree with what has been reported by Perry (1 99 1) for the Cembroides subsection. R cembmides is predominantly tree-like, while I? johannis and P culminicola have a largely shrubby or prostrate growth habit. It ought to be borne in mind, however, that it is sometimes possible to find individuals of Rjohannis with a tree- like form.

The first subdivision of the pinyon pines with white endosperm forms a compact group found in the north-central region and in the southern ranges of San Luis Potosi. This also has traits in common with the pinyon pine woodlands of Queretaro. They are somewhat different to the Rjohannis specimens reported by Robert (1 978). Such pinyons were originally referred to as R discolor for the southern ranges of San Luis Potosi by Bailey & Hawksworth (1979, 1983), Bailey, Snajberk & Zavarin (1982), A d a (1985), Zavarin & Snajberk (1986), Avila, Garcia & Reyes (1992), Reyes (1992) and Reyes, Gonzalez & Garcia (1996); and for Queretaro by Zavala & Campos (1993). The evidence of the phenetic analysis shows that this subgroup may be a variant, possibly at subspecific level.

Pinus culminicola and Rjohannis are phylogenetic relatives which have been restricted to calcareous substrates, and considered as the ancestral taxa of the Cembroides section (Malusa, 1992). However, Rjohannis has been found in igneous substrate (rhyolite) in San Luis Potosi. Pinus discolor (Rjohannis is the correct name) appears in the cladograms reported by Malusa (1992) as a taxon derived from R johannis by differentiation of populations, which have developed a wide tolerance to many substrates and occupy drier environments. Malusa’s (1 992) cladograms confirm our own results, since Rjohannis from San Luis Potosi and Queretaro is the phenetic group with more variation: bushy, sub-arboreal and tree forms, number of needles (four in Charcas, three in the rest of highlands, up to two in Queretaro), broader distribution, greater diversity of substrates (calcareous and rhyolitics) and climatic subtypes of arid zone.

The populations with sub-arboreal or tree-like form and pink endosperm cor- respond to R cembmides; they are the groups with wider but gradual variation between their populations, without the contrast shown by Rjohunnis. It is clear that even in sympatry, the species of Rjohannis and R cembmides, from El Sombrerito, El Quelital and La Amapola are differentiated. The same happens in Concepcion del Oro. It should be mentioned that in La Amapola, the populations of these species coexist, which means that the populations occupy different niches in the same general habitat (Grant, 1989). Whereas Rjohannis is less tolerant to higher temperatures and is frequently localized in north and north-eastern exposures (Benavides & Garcia, 1987), R cembmides is tolerant of drought, has higher water use efficiency (Yafiez & Garcia, 1985) and prefers southern facing exposure (Bailey & Hawksworth, 1983; Benavides & Garcia, 1987). It is observed that there has been interspecific selection to take advantage of each of those micro-environmental conditions. Moreover, R johunnis and R cembmides share contiguous areas (parapatry) where there are igneous outcrops overlying sedimentary substrates, in spite of their substrate preferences. However, there is a sympatry of both species, mainly on the igneous substrate.

Figure 1 also shows the possibility of individuals having been produced by hybridization, since there are populations morphologically intermediate (UTOs 1 7 and 10 of La Amapola), where both R cembmides and Rjohannis coexist. There is a phenological lag which limits the outcrossing between both species (Romero et al.,

PHENETIC ANALYSIS OF MEXICAN PINES

TABLE 3. Morphometric variables for the first three principal components

189

Component Eigenvalue Percentage Accumulated

1 10.5362 42.14 42.14 2 3.3875 13.55 55.69 3 2.6080 10.43 66.13

1996), although flowering, which occurs over 15 days in July, does overlap. This same phenomenon is observed in Cadereyta, where populations with ‘intermediate morphology’ are present. OTU 27 shows this phenomenon. Avila (1985) had reported the presence of individuals morphologically intermediate between both species in La Amapola due to overlap in the allometry and length of the fascicles of both species.

Zavala & Campos (1993) also reported the presence of morphologically inter- mediate individuals, based on seed traits. As we do not have information in relation to genetic flux, it is still difficult to connect the intermediate morphology to some hybridization process (see Bailey & Hawksworth (1 979); Bailey et al. (1 982); Zavarin 81 Snajberk (1986)).

It seems that the degree of disturbance (logging, gathering of piiiones, grazing) breaks up the homogeneity of the macro-environment and modifies the humidity of the micro-environment affecting the size of individuals. Populations 1 and 25 are localized in slopes and extend down to agricultural fields; the trees are small, sparse, young or sprouting after logging. OTUs 2 and 3 are populations with a sub-arboreal form which inhabit a wetter environment; these become increasingly dense on the slopes of hills as they approach the peaks. OTU 26 of Cadereyta has smaller individuals in a forest located higher up the hills, mixing with legumes and other families with neotropical affinities.

In summary, the populations of I! johannis from San Luis Potosi show phenotypic plasticity related to the ecological state of the site, while those of Queretaro are affected by the neartic-neotropical transition based on the plants that surround them.

The species differentiation between Rjohannis and I? culminicola is clear; it is likely that allopatry has been the main barrier favouring the maintenance of their separate status.

Principal component anabsis (Pa)

The variation demonstrated by the three first components is shown in Table 3 and Figure 3. The cophenetic coefficient is 0.9408.

Principal components 1 and 2 explain 55.6% of the variation (Fig. 3A). The PCA is consistent with the grouping analysis, since it identifies the two major groups in Figure 1: towards the positive side the I! cembroides populations (m) are located, characterized by the pink colour of the endosperm, larger size of the cones and greater number of viable seeds. Towards the negative side I! johunnis (0) and I! culminicola (A) are located, which have white endosperm, smaller cones and fewer viable seeds. These two species are separated from I! cembroides in the second PCA

I90

0.5

A. ROMERO E T A .

A 0

0 -

-0.5 O * O I n

d P 0 0

A .

-1.0 1 I I I -1.2 -0.6 0 -0.6 1.2

PC1

1.0

0.5

m 0.0

-0.5

-1.0 -

B 0

n o I .2 -0.6 0 -0.6 1.2

PC 1

Figure 3. Principal component analysis (PCA): (A) PCl vs. PC2, (B) PCl vs. PC3. PCl distinguishes species on the basis of the colour of the endosperm (white-pink), the morphometry in the cones (small-tlarge), and number of viable seeds. PC2 distinguishes on the basis of physiognomic attributes (height, arboreal habit and ramification height), and PC3 on seed dimensions (smd+large). (0) Pinus johannl, (A) II culminicola, (w) l? cmzbmidcs.

(Fig. 3B), which concerns physiognomic traits such as total height and height of branching.

There are five populations of P cembroides which are distinguished by the effect of the second component (Nos. 21, 15, 19, 20 and 18), all of them located in La Amapola. They contrast with those from Cadereyta (27), Concepcion del Oro (23) and La Amapola (10). The other populations marked in Figure 3 form the char- acteristic group of II cembmides.

Principal components 1 and 3 explain 52.37% of the variation (Fig. 3B); again we see a separation of the two major groups. On the negative side, near the crossing of the axes, can be found the populations with intermediate characters, L a Amapola (17) and Cadereyta (27). Component 1 groups the populations, while component 3 segregates the populations of PjohannL from Concepcion del Oro, which have small seeds with a thin pericarp (8.57 mm long, 6.10 mm wide and 0.52 mm thick), from those of Pjohannis from Charcas and Cadereyta, which have larger seeds with a thick pericarp (1 2.76 mm long, 8.23 mm wide and 1.05 mm thick). In an intermediate

PHENETIC ANALYSIS OF MEXICAN PINES 191

TABLE 4. Loading of the variables for the first three principal components. The primary ten variables are vegetative characters, the rest are reproductive

Principal components Variables 1 2 3

GRFO BRPA STHE BRHE TRDI BAFO ADNE NELE N E W FASH COLE COD1 PELE UMSI UMFO APSH SCNU FESE STSE PECO ENCO SELE S E W PETH cow

-0.53 0.25 0.65 0.58 0.66

-0.74 -0.79 -0.44 -0.81

0.23 0.79 0.88

0.80

0.33 0.3 I 0.89 0.14

-0.85 -0.93

0.56 0.10

-0.69 0.45

-0.80

-0.80

- 0.47 0.52 0.65 0.61 0.50

-0.26 -0.03

0.60

0.28 -0.23 -0.22

0.45 -0.42

0.53 -0.22 -0.09 -0.09

0.55 0.19 0.08 0.09 0.10 0.11 0.26

-0.28

-0.34 -0.53

0.14 0.40

-0.28 - 0.29 -0.11 -0.23 -0.00

0.13 -0.27 -0.28 -0.04 -0.03 -0.01

0.17 -0.50 -0.17 -0.26

0.05 -0.02 - 0.56 -0.74 -0.47

0.44

position in relation to component 3 are found I? johannG from La Amapola, and El cembroides. The latter species shows less variation in seed size.

The variables defining the axes of the PCAs are shown in Table 4. In PC 1 the most important variables concern reproductive traits; in PC 2 they concern physiognomic characters, while in PC 3 they concern seed size.

The PCAs confirm what is shown in Figure 1. The important morphometric characters for PC 1 showed at the species level: (1) colour of the endosperm, which differentiated the pifiones with a white endosperm from those with a pink one; (2) the number of fertile seeds, high for 49 cembroides and low for other species; (3) cone diameter, greater in El cembroides than in the rest of the individuals; (4) colour of the pericarp, which has three colours for l? cembroides (yellow, grey and black), and one colour for the other species (grey); (5) length of the pedicel, mainly sessile in I? cembroides, sessile to pedunculate in the other species; (6) size and the form of the umbo, large (3.16 mm mean) and protruding in El cembroides and small (2.17 mm mean) and depressed in the other species.

The following vegetative characteristics were important for the separation of species: (1) needles with uniform colour or bicoloured (the first corresponding to El cernbrozifes and the second to the other species with white endosperm); (2) the width of the needle, important for El cembroides but not for the other species.

For PC 2, the physiognomic traits were important, since they help to distinguish not only the species, but also the morphological and the ecotypic variants. Characters such as stem height, growth form and the height of the branching separated: ( 1 ) l? cembroides from the other species; (2) populations of one species in the differential

I92 A. ROMERO ETAL.

belt of 2'09' at northern latitude; and (3) height of individuals in neartic environments vs. those from the neartic-neotropical ecotone. PC 3 was important for relating the seeds with a latitudinal gradient. For Z? johannis, the largest seeds were common in Zacatecas, of intermediate size in San Luis Potosi, and smallest in Cadereyta. The seeds also revealed morphological variation, at least in the pifiones with white endosperm. For IT cembroides, homogeneity was observed in the size of seeds; PCs 1 and 2 defined which traits were the most important.

It remains to be established whether the morphological differences have a genetic origin, or whether it can be proved experimentally that phenotypic plasticity and ecotypic differentiation occur as reported in this article.

CONCLUSIONS

(1) Pinus cembroides and R johannis are readily distinguished based on morphological

(2) The two areas of sympatry, San Luis Potosi and Queretaro, have mor-

(3) The pattern of variation shows geographical subgroups in Pjohannis. (4) The phenetic similarity is smaller between I! johannis and IT cembroides than

among Z? johannis and I! culminicola. (5) The distinctive characters of Z? cembmides and the other species are, according

to degree of importance: colour and fertile seeds, morphometry of the cone, colour and breadth of needle, and characters related to physiognomy. For the piiiones with white endosperm, the seed dimensions separated the northern, central and southern highlands pinyon pines.

characters in allopatric and sympatric populations.

phologically intermediate populations of Z? cembroides and IT johannis.

ACKNOWLEDGEMENTS

We thank Dr Robert Bye, Universidad Nacional Autonoma de Mexico, and Dr Rafael Lira Sade from the Colegio de Postgraduados, Mexico, for their help with the manuscript.

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