Redalyc.POPULATION VARIATION in the ENDEMIC PINUS CULMINICOLA DETECTED by RAPD

Polibotánica ISSN: 1405-2768 [email protected] Departamento de Botánica México

Favela Lara, Susana POPULATION VARIATION IN THE ENDEMIC PINUS CULMINICOLA DETECTED BY RAPD Polibotánica, núm. 30, septiembre, 2010, pp. 55-67 Departamento de Botánica Distrito Federal, México

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POPULATION VARIATION IN THE ENDEMIC PINUS CULMINICOLA DETECTED BY RAPD

Susana Favela Lara

Departamento de Ecología, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, San Nicolás de los Garza, Nuevo León. Correo electrónico: [email protected]

ABSTRACT high but differentiation between them low. This suggests that even though this tree- Pinus culminicola, the dwarf pinyon, line pine has a restricted and fragmented is an endangered species endemic to distribution through the Sierra Madre northeastern Mexico, where it grows Oriental, gene fl ow between populations at the highest altitude of any of the has been suffi cient to prevent a dramatic Cembroides group. In order to determine loss of genetic variation and genetic drift. the degree of genetic isolation between populations of P. culminicola and the Key words: Pinus culminicola, genetic amount of gene flow between them, variation, RAPD. samples were obtained from Cerro El Potosi and Sierra La Viga, two localities RESUMEN within its restricted area of distribution in the Sierra Madre Oriental, and analyzed Pinus culminicola, piñonero enano que cre- using random amplifi ed polymorphic DNA ce en las partes más elevadas que cualquier (RAPD). The fi ve primers tested for the pino del grupo Cembroides, es endémico analysis showed banding patterns with del Noreste de México y está enlistado very high reproducibility and clear band como especie amenazada. Con la inten- resolution. These fi ve primers produced ción de examinar los niveles y patrones de a total of 72 distinct bands, 52 of which variación genética entre poblaciones de were polymorphic across the whole P. culminicola para determinar su grado sample. The genetic diversity in the two de aislamiento genético y la ocurrencia populations was high with a percentage de flujo genético entre poblaciones, se of polymorphism of 53.7% and degree of tomaron muestras en dos localidades de su diversity measured by the Shannon index área de distribución restringida en la Sierra of 56%. The total variation found between Madre Oriental y se analizaron utilizando the two populations was 5.98% (P = RAPDs. 0.0001). Most of the variation was found within populations (94.02%). Contrary to Los cinco primers probados para el expectations, the level of genetic variation análisis de P. culminicola mostraron pa- found in the two isolated populations was trones de bandeo con un alto grado de

55 Núm. 30: 55-67 Septiembre 2010 reproducibilidad y una resolución clara is that they are more highly fragmented de las bandas. Estos primers arrojaron than those in northern latitudes (Ledig et un total de 72 bandas distintas, de las al., 2000). However, this pattern can be cuales 52 fueron polimorfas a través de affected by historical events. Many conifers todas las muestras. La diversidad gené- of the northern latitudes expanded into their tica fue alta en las dos poblaciones con present range within the last 10 000 years, un porcentaje de polimorfi smo de 53.7% leaving little time for differentiation to oc- y un grado de diversidad medido por el cur. The ancestors of the present-day Pinus índice de Shannon de 56%. El total de banksiana and Pinus contorta, for example, la variación encontrada entre las dos po- were in contact in the geologically recent blaciones fue de 5.98% (P = 0.0001). La past; the lack of differentiation within these mayor variación fue dentro de poblaciones species refl ects recent gene fl ow (Ledig et (94.02%). Contrario a lo esperado, los al., 2000). niveles de variación entre las poblaciones aisladas de P. culminicola fue baja. Esto su- The geographical range of a species is giere que a pesar de que la especie presenta considered to be one of the best predictors una distribución restringida y aislada en of the level of genetic variation found in la Sierra Madre Oriental, el fl ujo genético natural populations (Hamrick et al., 1992). entre las poblaciones ha sido sufi ciente An example is provided by the genus Pinus. para prevenir una pérdida dramática de Pine species that are distributed as scattered variación genética y una deriva. isolated populations have more genetic diversity among populations, while more Palabras clave: Pinus culminicola, varia- widespread and continuous distributed ción genética, RAPD. pines have less among-population diversity (Hamrick et al., 1992, Ledig et al., 2001, INTRODUCTION Molina-Freaner et al., 2001).

Molecular studies have revealed that coni- Fragmentation into small, scattered popula- fers have high levels of genetic variation tions is expected to lead to genetic isolation, and relatively little genetic differentiation loss of genetic diversity, differentiation of among populations (Ledig, 1998). The the populations and increased probability of common explanation for the low genetic extinction (Ledig et al., 2001). Thirty fi ve differentiation found in conifers species of 47 pine species reported to Mexico are is the reproductive system: seed and po- endemic to this country (Perry et al., 1998). llen are basically wind dispersed and this Many of these species are represented by allows a more effi cient gene fl ow among small, scattered populations and also are distant populations (Delgado et al., 1999). presumed threatened or endangered. Nine Levels of diversity in Mexican conifers are of these Mexican pine species are listed by generally twice as those in species from the International Union for Conservation northern temperate latitudes (Ledig et al., of Nature and Natural Resources (IUCN, 2000). The most obvious reason for higher 2001) as species of concern (Farjon and levels of differentiation in Mexican species Page, 1999).

56 Favela Lara, S.: Population variation in the endemic Pinus culminicola detected by RAPD

Pinus culminicola, the dwarf pinyon, grows tions; or alternatively whether it represents at the highest altitude of any of the Cem- a relict of an ancient plant community. broides group, in the Northeast of Mexico. It occurs at the top of the two highest moun- Pinus culminicola has a very restricted tains of the Sierra Madre Oriental, separated range and during dry periods is very sus- from each other by about 72 km (straight- ceptible to fi re. In 1975 more than 5 ha of line distance) and is listed as an endangered a large community on the northern slope of species. It grows in an altitudinal range of Cerro el Potosi were completely destroyed 3300-3 650 m. It is a shrub or small tree 1-5 by fi re. In 1996 another big fi re destroyed m high, commonly multi-stemmed from the more than the 40% of the remaining com- base, usually spreading with branches ex- munity in Cerro el Potosi. P. culminicola tending outward from the base from 3-4 m, is classifi ed as “EN” (A3c + B2a,b(i-v)) forming dense vegetation (“matorral”). The on the IUCN Red List, and is considered soils are shallow, rocky, gravely limestone. endangered based principally on its low Rainfall is high since rain, sleet and snow area of occupancy and estimated continuing occur frequently throughout the year. Un- decline of at least 50% within 10 years or fortunately at the very high, isolated locatio- three generations (IUCN, 2001). ns where this taxon grows, temperatures and rainfall measurements are not available. At The aim of this study is to use RAPD mar- the summit of Cerro el Potosi, Nuevo León, kers to examine the levels and patterns of one of the populations where this species genetic variation between two populations grows, it occurs with Pinus hartwegii. In of Pinus culminicola to determine their Sierra La Viga, Coahuila, P. culminicola has degree of genetic isolation and if there is been found with P. hartwegii and also with evidence that gene fl ow occurs between the P. strobiformis and in association with other two populations. conifer species (Pseudotsuga menziesii and Abies vejarii). MATERIAL AND METHODS

Pinus culminicola is an interesting pine be- Sample collection cause it is so different from the other pinyon pines species. The ecological conditions of Two populations were sampled from their moist, cool, high-altitude environments is restricted distribution in the Sierra Madre in marked contrast to the semiarid condi- Oriental (Fig. 1, Table 1). Within each tions generally associated with the other population, 30 individuals were randomly pinyon pines species. P. hartwegii and P. selected and 10 g of mature needle samples strobiformis, the associated species, are collected from each tree. These were dried well adapted to the high altitude conditions; and stored in plastic bags containing 10 g however, they do not have “close relatives” of silica gel. that are adapted to warmer semiarid and arid conditions. The taxon P. cembroides grows DNA extraction around the base of Cerro el Potosi under hot and dry conditions. One question is how P. DNA was extracted from 0.5g of dried culminicola adapted to such different condi- needles following the method of Doyle

57 Núm. 30: 55-67 Septiembre 2010

Fig. 1. Populations of Pinus culminicola sampled in the Northeast of Mexico.

58 Favela Lara, S.: Population variation in the endemic Pinus culminicola detected by RAPD

Table 1. Sampled populations of P. culminicola in the Northeast of Mexico.

Species Coordinates Population Altitude number/Locality

P. culminicola 25º 21’ 25” 1. Sierra La Viga, Ramos 3 240 m 100º 31’ 59” Arizpe, Coahuila P. culminicola 24º 47’ 20” 2. Cerro El Potosí, Galeana, 3 450 m 100º 11’ 38” Nuevo León*

and Doyle (1990) with the addition of an using these primers. RAPD products were ammonium acetate wash to remove excess separated alongside 1 Kb ladder on 1.6% carbohydrates (Hollingsworth et al., 1999). agarose gels, stained with ethidium bromide DNA extractions were further purified (0.5 μg/ml) gel in TBE buffer and visualized using a DNeasy plant mini kit (QIAGEN) using a Genesnap 4.0 (Syngene UK). with slight modifi cations to the published instructions. The quality and quantity of PCR conditions DNA was assessed by running samples alongside a HyperLadder 1 concentration 25μl PCR reactions were prepared with the marker (Bioline UK) on 1.0% agarose following reagents: 1X reaction buffer (16 in TBE Buffer. DNA was visualized via mM (NH4)2SO4, 67 mM Tris-HCl (pH ethidium bromide staining (0.1 mg/ml) 8.8), 0.01% Tween-20); 2.5 mM MgCl2, 0.2 under UV light and images captured using mM dNTP’s; 0.5 μM primer 2% formamide, Genesnap 4.0 (Syngene UK). DNA samples 1Unit BioTaq (Bioline), ~10 ng template were stored at -20°C. DNA. Amplifi cation was carried out using the following PCR profi le: 2 min at 95ºC; RAPD analysis 2 cycles of 30 sec at 95 ºC, 1 min at 37ºC, 2 min at 72ºC; 2 cycles of 30 sec at 95ºC, Primer selection 1 min at 35ºC, 2 min at 72ºC; 41 cycles of 30 sec at 94ºC, 1 min at 35ºC, 2 min at From an initial screen of 54 10mer RAPD 72ºC; and 5 min at 72ºC, on a MJ Research primers (Operon RAPD kits OPC, OPG, PTC 200 DNA Engine. Negative controls OPP technologies, Alameda), were used to lacking template DNA were included in screen a subset of samples. Five primers each PCR run. were chosen that gave clear and repro- ducible banding pattern (OPC-06, OPG- PCR products were separated alongside 05,OPG-09,OPP-12 and OPP-14). All 60 1Kb ladder on 1.6% agarose gels, stained samples were screened for genetic variation with ethidium bromide (0.5 μg/ml) gel in

59 Núm. 30: 55-67 Septiembre 2010

TBE buffer and visualized using a Genes- D = 1 - (Sij/Tij) nap 4.0 (Syngene UK). where S is the total number of shared Data analysis present band positions and T is the total number of band positions shared between RAPD bands were scored visually as either the ith and the jth individuals. The genetic present (1) (all dominant homozygotes distance matrix generated was subjected (AA) and heterozygotes (Aa)) or absent to a Principal Coordinate Analysis (PCO) (0) (recessive homozygotes (aa)). A binary (R package, Legendre and Vaudor, 1991), matrix of band presence and absence was which produce a visual representation (prin- constructed and then used to measure the cipal coordinate plot (PCO)) of the genetic genetic variability and structure of species relationship among individuals in the sample and populations. Samples that failed to (Hollingsworth and Ennos, 2004). amplify for speciﬁ c primers were scored as missing data. Measures of genetic structures

Measures of genetic variation In order to analyse the population structure of the taxa, analysis of molecular varian- The Shannon diversity index (Lewontin, ce (AMOVA) was calculated from the 1972) was used to quantify levels of genetic pairwise genetic distance using Jaccard variation within each population. Estima- distance measure to examine variation tes of variation were calculated using the (i) between populations and (ii) within formula: populations. The Arlequin programme generates Ф statistics (Excoffier et al.,

S = -∑pi log2 pi 1992), which is analogous to Wright’s FST (Wright, 1951). This approach has been where pi is the frequency of presence or widely adopted in the analysis of RAPD absence of each RAPD band (RAPD phe- data (Allnut et al., 1999, Diaz et al., 2001, notypes). The Spop was calculated as the Bekessy et al., 2002, Newton et al., 2002, mean value of S over the entire population Nkongolo et al., 2002, Castro-Felix et al., sample. This analysis was calculated ma- 2008). This analysis was conducted using nually in an Excell spreadsheet. the Software program Arlequin (Schneider et al., 2000). The percent polymorphic RAPD loci (%P) were calculated for each population, as well RESULTS as the mean value for all populations. Genetic diversity in populations of Pinus Measures of genetic distance culminicola

Based on the phenotypic data, a pairwise The ﬁ ve primers tested for the analysis of genetic distance measure (Jaccard’s) was Pinus culminicola showed banding patterns calculated using the following formula with very high reproducibility and clear (Sneath and Sokal, 1973): band resolution. These ﬁ ve primers pro-

60 Favela Lara, S.: Population variation in the endemic Pinus culminicola detected by RAPD

Table 2. Shannon diversity index (S) and percent polymorphic RAPD loci (%P) of Pinus culminicola.

Population Shannon % Mean Mean % Index (S) Polymorphism polymorphism Spop Sierra La Viga 0.5692 57.8

Cerro El Potosí 0.5690 56.8 0.569 57.3

duced a total of 72 distinct bands, 52 were DISCUSION polymorphic across the whole sample. Genetic diversity between populations of The percentage of polymorphism found in Pinus culminicola P. culminicola was 57.3%. The degree of diversity in this taxon, measured by Shan- The percentage of polymorphism of RAPD non index was 56% (Table 2). loci was relatively low (57.3%) compared with the other species of Cembroides group Principal coordinate analysis (PCO) was analysed (Pinus remota 84.4% and P. cem- conducted to examine the relationship broides var. bicolor 69.9%, Favela, 2005) among the two populations of Pinus cul- and also slightly low when is compared minicola (Fig. 2). The two principal coor- with other pine and tree species. A reduc- dinates of RAPD distance described 7.78% tion in the proportion of polymorphic loci and 7.57% of the variation, respectively. has been previously reported for endemic Individual samples from each population species (Hamrick and Godt, 1996). Even plotted as a continuous scatter, with the though this value is reported as low it is two populations overlapping. not as low as has been reported in other isolated population of Mexican pine species The genetic variation between the two (P. chiapensis 24.5%, P. maximartinezii populations of Pinus culminicola was con- 30.3%, P. greggii 31.9% and P. rzedowskii ducted by AMOVA analysis (Table 3). 46.8%) (Newton et al., 2002, Ledig et al., 1999, Parraguirre et al., 2002, Delgado et The total variation found between the two al., 1999). populations was 5.98 % and this value was highly signiﬁ cant (P = 0.0001, tested using Diversity estimates indicated by Shannon 1000 replications). However most of the index (0.56) did not differ between popu- variation was found within populations lations of Pinus culminicola. This value is (94.02 %). comparable with other species of conifers

61 Núm. 30: 55-67 Septiembre 2010 2 of the variation). P. culminicola P. 1 and 7.57% for P. culminicola P. (describing 7.78% for rst two principal coordinates of pairwise distance derived from RAPD analysis populations ﬁ Pinus culminicola lot of the Fig. 2. P

62 Favela Lara, S.: Population variation in the endemic Pinus culminicola detected by RAPD

Table 3. AMOVA of RAPD variation among two populations of Pinus culminicola.

d.f. Sum of Variance % of total P value squares components variation

Among 1 20.7 0.45 5.98 0.0001 populations Within 58 414.3 7.14 94.02 populations

(0.53) (Araucaria, Fitzorya and Cedrela) when the genetic distance matrix was sub- survey by Bekessy et al. (2002), Allnut et jected to a PCO analysis (Fig. 2), the pattern al. (1999) and Gillies et al. (1997), respec- showed evidence of gene fl ow between po- tively. pulations due to pollen interchange. Howe- ver the genetic variation among populations Genetic diversity within populations of for long-lived species is strongly infl uenced Pinus culminicola by geographical distribution (Hamrick et al., 1992). Among the many species of Most of the variation in Pinus culminicola pines studied previously, some endemic or (94.02%) was recorded within populations, isolated scattered distributions were found a result consistent with those from most of to retain higher gene differentiation among the other pine species (Ledig, 1998) and populations (e.g. Pinus rzedowskii (0.17) woody plant species. Hamrick et al. (1992) (Delgado et al., 1999); P. pinceana (0.15) indicated that long-lived woody plant spe- (Ledig et al., 2001); P. lagunae (0.18), P. cies tend to maintain higher levels of allo- muricata (0.16) (Molina-Freaner et al., zyme variation within populations. This is 2001) and P. strobiformis, P. ayacahuite, also comparable with results obtained from P. lambertiana and P. chiapensis (0.5) RAPD data where most of the tree species (Castro-Felix et al., 2008)). In contrast pine examined showed high levels of variation species with widespread and continuous within populations (Gillies et al., 1997, distributions have retained lower gene Allnut et al., 1999, Bekessy et al., 2002, differentiation among populations (e.g. P. Newton et al., 2002). albicaulis (0.034) (Jorgensen and Hamrick, 1997); P. banksiana (0.03) (Dancik and Yeh, Genetic diversity among populations of 1983); P. ponderosa (0.015) (Hamrick et Pinus culminicola al., 1989) and P. oocarpa (0.0054) (Sáenz et al., 2003). The degree of genetic variation among populations found in Pinus culminicola was The low differentiation between the two small (5.9%), but signifi cant. For instance, populations of Pinus culminicola was par-

63 Núm. 30: 55-67 Septiembre 2010 ticularly unexpected, given the restricted even though populations of P. culminicola and isolated geographical range of dis- have a restricted and isolated distribution, tribution. However, Hamrick and Godt gene fl ow among populations has, at least (1996) counted 274 studies in Pinaceae presently, not lead to dramatic loss of alone. One of the emerging generalities genetic variation and population diffe- was that conifers had substantial genetic rentiation. Future work on the structure diversity within population and only low of population in pinyon pine species is levels of differentiation among popula- needed in order to have a better unders- tions. In a review of 195 isozyme studies tanding of the history of the species in the of long-lived perennial woody taxa, mountain range “Sierra Madre Oriental” specifi cally in gymnosperms, Hamrick et where these species grows. al. (1992) recorded an overall mean Gst value of 6.5% in the genetic variation LITERATURE CITED among populations. Such differentiation suggests that even though populations of Allnut, T.R., A.C. Newton, A. Lara, A. P. culminicola have restricted and isolated Premoli, J.J. Armesto, R. Vergara and distribution, gene fl ow among populations M. Gardner, 1999. “Genetic Variation has been suffi cient to prevent dramatic in Fitzroya cupressoides (alerce), a loss of genetic variation. Another point threatened South American conifer”. to consider is that these two populations Molecular Ecology, 8: 975-987. may have been in historical contact in the geologically recent time, and hence the Bekessy, S. A.,T.R.Allnut, A.C. Premoli, similarity may merely refl ect insuffi cient A. Lara, R.A. Ennos, M.A. Burgman, time for differentiation to occur. However, M. Cortes and A.C. Newton, 2002. there is no clear evidence to support this “Genetic variation in the vunerable last point due to the lack of studies related and endemic Monkey Puzzle tree, to the postglacial history of the species. detected using RAPDs”. Heredity, 88: Information does exist in Pinus banksiana 243-249. and Pinus contorta which can be used as the base for future studies in pinyon pine Castro-Felix, P., J.A. Pérez de la Rosa, G. species from the northeast of Mexico. Vargas Amado, S. Velázquez Magaña, A. Santerre, F. López-Dellamary To- CONCLUSION ral, A.R. Villalobos-Arámbula, 2008. “Genetic relationship among Mexican The genetic variation found within two white pines (Pinus, Pinaceae) based on populations of Pinus culminicola was RAPD markers”. Biochemical Syste- high and in concordance with previous matics and Ecology, 36: 523-530. studies where pine and tree species have been included. The degree of genetic di- Dancik, B.P. & F.C. Yeh, 1983. “Allozyme fferentiation found among populations of variability and evolution of lodgepole P. culminicola was low but consistent with pine (Pinus contorta var. latifolia) and values recorded for other conifers species. jack pine (Pinus banksiana) in Alber- The extent of differentiation suggests that ta”. Can. J. Genet. Cytol., 25: 57-64.

64 Favela Lara, S.: Population variation in the endemic Pinus culminicola detected by RAPD

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Recibido: 16 julio 2009. Aceptado: 6 mayo 2010.