AFLP characterization of natural populations of Berberis ...

Plant Syst. Evol. 231: 133±142 (2002)

AFLP characterization of natural populations of Berberis (Berberidaceae) in Patagonia, Argentina M. C. J. Bottini1,3, A. De Bustos2, N. Jouve2, and L. Poggio1,3 1

Instituto FitoteÂcnico de Santa Catalina (FCAF, UNLP)- Centro de Investigaciones GeneÂticas (UNLP-CONICET-CIC), C. C. 4 (1836), Llavallol, Buenos Aires, Argentina 2 Department of Cell Biology and Genetics, University of AlcalaÂ (Madrid), Spain 3 Departamento de Ciencias BioloÂgicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina Received March 21, 2001 Accepted September 11, 2001

Abstract. AFLP markers were used to analyse the intra- and interspeci®c relationships among 22 natural populations of 13 Patagonian species of Berberis and the relationships among the taxa belonging to homoploid and polyploid complexes. Seven primer combinations gave rise to 231 AFLP bands, of which 199 were polymorphic. Correspondence between AFLP data, morphological traits and seed protein bands was also assessed. The dendrogram inferred from AFLP ®ngerprints showed that, in general, populations of the same species formed closely related groups with high coecients of similarity. Principal co-ordinates analysis showed two separate subgroups: (i) B. bidentata and their putative ancestors ± B. darwinii and B. linearifolia ± which form a homogamic group, and (ii) B. buxifolia, B. heterophylla and B. parodii ± which could form a polyploid hybrid complex. Key words: Berberis, Berberidaceae, michay, calafate, barberry, AFLP, molecular markers, Patagonia, Argentina.

The family Berberidaceae contains about 15 genera and 650 species mainly found in the Northern Hemisphere. A single genus, Berberis, is also found in temperate and Andean

South America (Landrum 1999). The members of this family are evergreen and semi-evergreen shrubs which grow in a wide range of ecological conditions (Orsi 1984). These species have sexual and clonal propagation, with longer rhizomes growing very far from the center of the plants (about 10 m). For this reason, populations show a big covering but are composed by a rather low number of individuals (Bottini 2000). The species of Berberis are used for medical (Shaer 1985) and food purposes (Martinez Crovetto 1980). The systematics of the genus is still uncertain, particularly with respect to the number of existent species in Argentine Patagonia (Orsi 1984, Landrum 1999). The Patagonian species of Berberis show two levels of ploidy: diploid (2n 28) and polyploid (2n 56) (Bottini et al. 1999a, 2000a). The diploid species include B. cabrerae Job., B. chillanensis Sprag. ex Sandwith., B. montana Gay, B. serrato-dentata Lechl., B. ilicifolia L., B. microphylla Frost., B. parodii Job., B. empetrifolia Lam., B. darwinii Hook., B. linearifolia Phil. and B. bidentata Lechl. The polyploid species are comprised of B. buxifolia Lam. and B. heterophylla Juss.

134

M. C. J. Bottini et al.: AFLP characterization of Berberis

Variation in genome size of all these species shows three levels of discontinuity (Bottini et al. 2000a). The DNA content ranges from 1.463 to 1.857 pg in the diploid species B. cabrerae, B. chillanensis, B. montana, B. serrato-dentata and B. bidentata. A second group of diploid species shows a DNA content which ranges from 2.875 to 3.806 pg includes B. linearifolia, B. darwinii, B. parodii and B. empetrifolia. The genome size of the polyploid species B. buxifolia and B. heterophylla ranges from 5.809 to 6.844 pg. The diploid species with lower DNA content grow at high altitudes with high rainfall and low water availability. Those with higher DNA content are common in medium-altitude forests with long vegetative periods, high water availability and moderate temperatures. The distribution of the polyploid species is considerably wider than the one shown by the diploid species, which are geographically and ecologically restricted to forest regions. This suggests that the C-value is associated with the ability of the species to adapt to dierent growing conditions (Bottini et al. 2000a). The existence of hybrid complexes in the genus was postulated on the basis of morphological, biochemical and cytological characterizations. These data suggest that Berberis bidentata, B. linearifolia and B. darwinii form a homogamic hybrid complex, while B. heterophylla, B. buxifolia ± and their putative progenitor B. parodii ± could form a polyploid complex (Bottini 2000; Bottini et al. 1999b, 2000a, b). DNA markers can be used to detect variation in the DNA level and have proved to be extremely eective in distinguishing between closely related genotypes (Ayele et al. 1999). A dominant DNA marker system based on two consecutive selective polymerase chain reactions called ampli®ed fragment length polymorphism (AFLP) (Vos et al. 1995), allows to detect relatively representative samples of genetic variation of a given genome. It is therefore a suitable tool for examining population genetics (Travis et al. 1996, Roa et al. 1997). AFLP markers have been used in the assessment of

genetic diversity in Camellia sinensis (Paul et al. 1997), Cocos nucifera (Perera et al. 1998), Euterpe edulis (Cardoso et al. 2000), Glycine max (Maughan et al. 1996), Hordeum spontaneum (Pakniyat et al. 1997), Lactuca (Hill et al. 1996), Lens (Sharma et al. 1996), Oryza sativa (Zhu et al. 1998), Oxalis tuberosa (Tosto and Hopp 2000), Phaseolus vulgaris (Tohme et al. 1996), Pinus sylvestris (Lerceteau and Szmidt 1999), Vitis vinifera (Cervera et al. 1998) and Zea mays (Ajmone et al. 1998). In this work, AFLPs markers were used to analyse the intra- and interspeci®c relationships among Patagonian species of the genus Berberis. Correspondence between AFLP data, morphological traits and seed protein bands was also investigated. The relationships among taxa that could constitute homoploid and polyploid complexes are discussed. Materials and methods Plant materials. Plants belonging to 22 natural populations of 13 species were collected in dierent localities of Argentine Patagonia. Seeds from dierent individuals were collected at each site and preserved at 41 °C. Previous studies (Bottini 2000) determined that, due to the clonal reproductive method of these species, the minimum distance between dierent individuals is about 10 m. For that reason, the collections were done taking in account these data to ensure an adequate evaluation of population genetic variability. Representative accessions were deposited in the herbaria of the Instituto de BotaÂnica Darwinion (SI) Argentina. Collector: Cecilia Bottini (CB). B. bidentata. ARGENTINA. Prov. NeuqueÂn. Dpto. Los Lagos: Highway 231, on the Brazo Huemul shoreline of Lake Nahuel Huapi, 845 m s.m., CB 109. Prov. RõÂo Negro. Dpto. Bariloche: Pto. Blest, on the way to Lago FrõÂ as, 764 m s.m., 30-I-1995, CB 59. B. buxifolia. ARGENTINA. Prov RõÂo Negro. Dpto Bariloche: C° Otto, 1000±1300 m s.m., 24-II1995, CB 42. Prov. Chubut. Dpto. Cushamen: National Highway 259, 9 km S of the intersection with Highway 40, 300 m s.m., 8-II-1995, CB 140. B. cabrerae. ARGENTINA. Prov. RõÂo Negro. Dpto. Bariloche: Picada a Laguna Los Clavos, 1200 m s.m., 19-II-1997, CB 446.

M. C. J. Bottini et al.: AFLP characterization of Berberis B. comberi Sprag. ex Sandwith. ARGENTINA. Prov. NeuqueÂn. Dpto. LoncopueÂ: National Highway 40, 9-XII-97, CB 506, 509. B. darwinii. ARGENTINA. Prov. NeuqueÂn. Dpto. Los Lagos: Pichi Traful, 800 m s.m., 2-II1995, CB 105. Prov. RõÂo Negro. Dpto. Bariloche: MallõÂ n Ahogado circuit, 10 km NE of El BolsoÂn, 465 m s.m., 9-II-1995, CB 160. B. empetrifolia. ARGENTINA. Prov. RõÂo Negro. Dpto. Bariloche: 5 km down National Highway 258, banks of RõÂ o Yoconto, 850 m s.m., 13-III-97, CB 484; La Veranada, track to Lake Steen, 890 m s.m., 13-III-97, CB 487. B. grevilleana Gillies ex Hook. ARGENTINA. Prov. Mendoza. Dpto. MalarguÈe. 5-II-98, CB 355. B. heterophylla. ARGENTINA. Prov. RõÂo Negro. Dpto. Bariloche: KaruÂ-KinkaÂ establishment, 15 km W of Colonia Paso Flores, 7-I-97, CB 494. Dpto. Pilcaniyeu: National Highway 23, 20 km SE of Pilcaniyeu del Limay, CanÄadoÂn Bonito, 700 m s.m., 12-II-1995, CB 183. B. ilicifolia. ARGENTINA. Prov. Tierra del Fuego. Dpto. Ushuaia: Ushuaia, 640 Houses, 5-II98, CB 545; Tierra del Fuego National Park, Hito XXIV, 4-II-98, CB 539. B. linearifolia. ARGENTINA. Prov. RõÂo Negro. Dpto. Bariloche: Pto. Blest, track to Lake FrõÂ as, 764 m s.m., 30-I-1995, CB 77. B. microphylla. ARGENTINA. Prov. Tierra del Fuego: Dpto. Ushuaia: Ushuaia, Municipal Campsite, 533 m s.m., 3-II-98, CB 527; Tierra del Fuego National Park, RõÂ o Pipo, 516 m s.m., 7-II98, CB 549. B. parodii. ARGENTINA. Prov. NeuqueÂn. Dpto Los Lagos: Pichi Traful, 800 m s.m., 2-II-1995, CB 98. Prov. RõÂo Negro. Dpto. Bariloche: Pto. Blest, track to Lake FrõÂ as, 764 m s.m., 30-I-1995, CB 67. B. serrato-dentata. ARGENTINA. Prov. RõÂo Negro. Dpto. Bariloche: C° Chal-Huaco, 1800 m s.m., 14-II-97, CB 425. DNA isolation. Total genomic DNA was isolated from 5±6 seeds taken from individual plants. The seeds were homogenised to a ®ne powder using a mortar at 25 °C. DNA was extracted using the QIAGEN DNeasy Plant Mini Kit, according to the manufacturer's instructions. To check and quantify the DNA preparation, an aliquot was resolved on a 0.8% agarose gel in 1X TAE buer (Sambrook et al. 1989) and compared with a standard of known mass (1 Kb Plus ADN LADDERä 1 lg/ll, Gibco BRL).

135

AFLP procedure. The AFLP protocol followed was the one developed by Vos et al. (1995) with minor modi®cations. Genomic DNA (350 ng) was digested with 2U each of EcoRI and MseI restriction enzymes. Two dierent adaptors, EcoRI and MseI were ligated to the ends of genomic restriction fragments. The EcoRI adaptor consisted of the combination of two primers: 5¢-CTCGTAGACTGCGTACC-3¢ and 3¢-CTGACGCATGGTTAA-5¢. Similarly, the MseI adaptor also consisted of the combination of two primers: 5¢-GACGATGAGTCCTGAG-3¢ and 3¢-TACTCAGGACTCAT-5¢. The digested and ligated template DNA was preampli®ed using EcoRI+1 (5¢-GACTGCGTACCAATTCA-3¢) and MseI+1 (5¢-GATGAGTCCTGAGTAAC-3¢) primers. AFLP ®ngerprints were generated using pairs of EcoRI+3 and MseI+3. Seven primer combinations were employed to detect AFLP polymorphism among the dierent genotypes: EcoRI+AAG/MseI+CAG, EcoRI+ AAG/MseI+CTG, EcoRI+AAG/MseI+CAA, EcoRI+AAG/MseI+CAT, EcoRI/AAC/MseI+ CAG, EcoRI+AAC/MseI+CTG and EcoRI+ AGC/MseI+CAT. The ampli®cation products produced were separated on 6% polyacrylamide gels. Electrophoresis was performed for 3±4 h in TBE 1X at 2150 v. Bands were visualised by silver nitrate staining (Cho et al. 1996). AFLP data analysis. All populations were scored to determine the presence (1) or absence (0) of AFLP polymorphic bands, and the data entered into a binary data matrix as discrete variables. Jaccard's coecient of similarity (Sneath and Sokal 1973) was calculated for all pairwise comparisons among populations, and a dendrogram was made through cluster analysis using the unweighted pair group method based on arithmetic averages (UPGMA). The correlation between the Jaccard's similarity and the cophenetic coecients for the clusters was calculated (Sneath and Sokal 1973). The dendrograms obtained from AFLP data were compared with morphological traits (Bottini et al. 1998) and seed protein data (Bottini et al. 2000b) using the Mantel correspondence analysis (Rohlf 1998). Mantel (1967) developed a test for the `goodness of ®t' of two matrices. Similarity matrices were also used in principal co-ordinates analysis (PCOORD) to resolve patterns of variation among and within species. The relative contribution signi®cance of AFLP bands in

136


species discrimination for the ®rst three co-ordinates was analysed. The NTSYS-pc 2.0f programme (Rohlf 1998) was used for all analyses.

Results The results obtained for the seven dierent primer combinations used in this study are shown in Table 1. A total of 231 AFLP bands were detected using seven combinations of primers. Out of them, 199 were polymorphic, representing 86% of the total number of bands. Band numbers varied among species (Table 2). Weak bands, or bands showing conspicuous dierences in their intensities were not considered in this study. The number of polymorphic products ranged from 11 for EcoRI+AGC/MseI+ CAT, to 34 for the primer combination EcoRI+AAG/MseI+CAT (Table 1). Among the polymorphic fragments, 16 bands (8%) were unique to diploid species. In the polyploid species, unique bands could not be detected (Table 3). The total number of AFLP bands for the diploid and polyploid species were not signif-

icantly dierent (P £ 0.05; mean 121 12 and 125 14, respectively). Grouping analysis. The dendrogram generated from AFLP data using the UPGMA method is shown in Fig. 1. The cophenetic correlation coecient was 0.97 (P £ 0.01), which indicates good agreement with the genetic similarity matrix. The dendrogram shows that the populations are divided into two main groups with a similarity coecient of 0.43. Group 1 contains the populations of B. buxifolia, B. parodii, B. heterophylla, B. cabrerae, B. empetrifolia, B. grevilleana and B. microphylla. The populations of B. buxifolia, B. parodii and B. heterophylla are closely grouped, and show a similarity coecient of 0.72. Berberis buxifolia and B. parodii are closely related with a similarity coecient of 0.78. Group 1 also includes B. cabrerae, B. empetrifolia, B. grevilleana and B. microphylla due to the high number of AFLP bands shared with B. parodii. Group 2 is formed by the populations of B. darwinii, B. bidentata, B. linearifolia, B. comberi, B. ilicifolia and B. serrato-dentata.

Table 1. Level of polymorphism and ®ngerprinting patterns of AFLP markers

1) 2) 3) 4) 5) 6) 7)

Primers

Total bands

Polymorphic bands

Polymorphism rate (%)

No. of unique bands

E-AAG M-CAG E-AAG M-CTG E-AAG M-CAA E-AAG M-CAT E-AAC M-CAG E-AAC M-CTG E-AGC M-CAT Sum Mean

36

32

89

4

34

32

94

5

41

33

81

4

36

34

92

1

33

30

91

1

31

27

87

1

20

11

55

0

231 33

199 28

84

16


137

Table 2. Distribution of total bands in species of Berberis Population

E-AAG M-CAG

E-AAG M-CTG

E-AAG M-CAA

E-AAG M-CAT

E-AAC M-CAG

E-AAC M-CTG

E-AGC M-CAT

Total bands

B. B. B. B. B. B. B. B. B. B. B. B. B.

14 15 14 15 16 20 10 12 22 15 17 24 9

18 17 21 16 16 18 14 12 23 13 15 16 13

22 22 19 23 24 23 17 16 21 17 19 23 22

17 18 15 19 17 19 11 21 21 14 16 16 14

19 19 18 19 18 16 17 12 21 18 15 20 18

19 16 18 18 18 21 21 22 20 16 12 16 20

17 17 17 15 15 15 18 18 18 17 14 16 14

126 124 122 125 124 132 108 113 146 110 108 131 110

buxifolia heterophylla parodii darwinii bidentata linearifolia cabrerae empetrifolia comberi grevilleana serrato-dentata ilicifolia microphylla

Table 3. AFLP unique bands and ploidy levels of Berberis Population

Ploidy level (2n)

No. of unique bands

B. buxifolia B. heterophylla B. parodii B. darwinii B. bidentata B. linearifolia B. cabrerae B. empetrifolia B. comberi B. grevilleana B. serrato-dentata B. ilicifolia B. microphylla Total

56 56 28 28 28 28 28 28 28 28 28 28 28

± ± ± ± ± ±

1 2

3 2 2 1 5 16

The populations of B. darwinii, B. bidentata, B. linearifolia are very closely grouped with a similarity coecient of 0.86. Principal co-ordinates analysis. The frequencies of the polymorphic bands of the 22 populations were used to generate a rectangular matrix to be employed as a contingency table in PCOORD. This statistical procedure determines the absolute contribution of each variable to the proportion of variance in each of the axes generated. Two groups of the populations of Berberis were clearly de®ned by

the ®rst and second principal coordinates, which represented 25.52% and 16.44% of the total variation respectively (Table 4, Fig. 2A). The ®rst principal coordinate was mainly related to bands 24 ()0.5109), 25 ()0.5109), 93 ()0.5109) and 94 ()0.5109). Group 1, as de®ned by cluster analysis, tended to distribute on the positive values of co-ordinate, while Group 2 showed negative values. The second principal coordinate was related to bands 105 ()0.5757), 121 ()0.5667), 160 ()0.5736) and 185 ()0.5667). The populations of B. darwinii, B. linearifolia and B. bidentata formed a sub-group since they are the only species that share bands 52, 57, 72, 87, 100, 120, 138, 140, 162 and 204. The third principal coordinate represented 12.27% of the variation (Table 4) and was related to bands 33 ()0.6324), 89 ()0.6091) and 122 ()0.6544) (Fig. 2B). The populations of B. buxifolia, B. parodii and B. heterophylla form a sub-group because they are the only species with bands 2, 30, 37, 46, 165 and 173. The two major groups (Groups 1 and 2) revealed by cluster analysis (Fig. 1A) were con®rmed by PCOORD (Fig. 2A and 2B). This analysis allowed discrimination among the populations and established the relevance of the bands to each principal coordinate. Comparison of AFLP, morphological and seed protein data. By means of the Mantel

138


Fig. 1. Dendrogram of twenty-two populations of Berberis species resulting from a UPGMA cluster analysis based on Jaccard estimates of similarity obtained from 231 AFLP bands. The herbarium number is indicated in brackets Table 4. PCOORD: eigenvalue, percentage of total variation and cumulative percentage of total contribution Principal co-ordinates

Eigenvalue

Percent total

Percent cumulative

1 2 3 4 5 6 7 8 9 10

2.545 1.639 1.223 0.885 0.577 0.460 0.436 0.358 0.339 0.330

25.525 16.438 12.273 8.876 5.789 4.617 4.381 3.594 3.404 3.040

25.525 41.963 54:236 63.111 68.900 73.517 77.897 81.491 84.895 87.935

correspondence test (Mantel 1967), the dendrogram was constructed using the AFLP data and was compared with others previously obtained using morphological traits (Bottini et al. 1998) and seed protein data (Bottini

et al. 2000b). The correlation between AFLP data and morphological traits is rather low, although signi®cant (r 0.55, P 0:01). In the case of seed proteins no signi®cant correlation was found at all. Discussion AFLP analysis proved to be an eective tool for providing quantitative estimates of genetic similarity among recognised Argentine Patagonian species of Berberis [i.e. those according to Orsi (1984)]. As shown in Fig. 1, most species clearly grouped into well-de®ned population clusters. The taxonomic treatment of species in the genus Berberis based on external morphology is still a matter of debate. Orsi (1984) recognized sixteen Patagonian species in Argentina, while Landrum (1999) argues for the existence of only nine. The results of the present work, particularly the topologies of the dendrogram


139

Fig. 2. A: Relationships among 22 populations of Berberis species by principal co-ordinate analysis using the Jaccard coecient. The ®rst (PC1) and second (PC2) principal co-ordinates accounted for 41.96% of the total variation. B: The ®rst (PC1) and third (PC3) principal co-ordinates accounted for 37.79% of the total variation. B. bidentata: Bi (109), Bi (52); B. buxifolia: Bu (140), Bu (42); B. cabrerae: Ca (373); B. comberi: Co (437), Co (439); B. darwinii: Da (105), Da (160); B. empetrifolia: Em (411), Em (414); B. grevilleana: Gre (355); B. heterophylla: He (183), He (421); B. ilicifolia: Ili (539), Ili (545); B. linearifolia: Li (77); B. microphylla: Mi (527), Mi (549); B. parodii: Pa (67), Pa (98); B. serrato-dentata: Sd (352)

140


and principal coordinate analyses using AFLP, numerical taxonomy of external morphology (Bottini et al. 1998) and seed proteins (Bottini et al. 2000b), are in agreement with Orsi's classi®cation. The AFLP markers show genetic variation in the DNA level and are free from environmental in¯uences against to morphological characters (Ayele et al. 1999). The signi®cant correlation between AFLP data and morphological traits and the absence of correlation between seed proteins and AFLP ®ngerprinting are not rare. There are several similar examples already published. The usual explanations are: low number of morphological traits imply incomplete genomic coverage, lack of independence of the variables, violation of certain assumptions made when computing estimates. Also, the erroneous scoring of non-homologous AFLP bands of similar mobility might lead to an overestimation of similarity in the AFLP matrix (Powell et al. 1996). Berberis buxifolia and B. heterophylla species are polyploid species (2n 56). Berberis parodii (2n 28) is one of their putative diploid ancestors (Bottini et al. 1999a). In Figs. 1 and 2B, these three species form a very clear, separate subgroup. A similar grouping is obtained when data for biochemical and morphological traits are analysed (Bottini et al. 1998, 2000b; Bottini 2000). Berberis buxifolia and B. heterophylla have no speci®c bands, although they share bands absent in their putative ancestor B. parodii, inferred through morphological and isoenzymatic data (Bottini et al. 1998, 1999b; Bottini 2000). The absence of speci®c bands in the polyploid species suggests scarce genomic divergence with respect to the putative ancestors, an evolutionary history involving very related species, and/or a recent origin. Moreover, the polyploid species do not show signi®cant increase of the amount of AFLP bands compared to the diploid species, which may support autopolyploidy. Additionally, the cytogenetic studies showed 1 to 3 quadrivalents in meiosis (Bottini et al. 1999a) and

isoenzymatic data showed similar interspeci®c genetic index and similar grade of isoenzymatic variability between polyploid species and the putative ancestor (Bottini 2000). These results, together with the morphological and biochemical data previously reported support the idea that polyploidy could have played an important role in the speciation of Berberis. B. buxifolia, B. heterophylla and B. parodii could form part of a polyploid complex according the hybrid complex concept of Grant (1989). Populations of other diploid species (B. cabrerae, B. microphylla, B. empetrifolia and B. grevilleana) are also present in Group 1 due to the high number of bands they share with B. parodii but they have not morphological or biochemical characters that resemble the polyploid species. There is morphological (Job 1942, Orsi 1984), cytological (Bottini et al. 1999a) and biochemical (Bottini et al. 1999b, Bottini 2000) evidence that B. bidentata could be a species of hybrid origin, with B. linearifolia and B. darwinii as their ancestors. Berberis bidentata (2n 28) has intermediate morphology between B. darwinii (2n 28) and B. linearifolia (2n 28). AFLP markers showed that populations of B. bidentata and B. darwinii are strongly associated, and that both species are associated with B. linearifolia. These three species form a well de®ned subgroup within Group 2 (Figs. 1 and 2A). The analysis of isozymes and seed proteins also shows close relationships among the three species (Bottini et al. 1999b, Bottini 2000), and strongly suggests that B. bidentata could have had a hybrid origin conforming an homogamic complex together with their ancestral progenitors B. linearifolia and B. darwinii. The authors thank the CICYT (ComisioÂn Asesora de Ciencia y TecnologõÂ a, Grant No. AGF97810) of Spain, Agencia Nacional de PromocioÂn CientõÂ ®ca y TecnoloÂgica (Grant No. 014443) Universidad de Buenos Aires and CONICET (Consejo Nacional de Investigaciones CientõÂ ®cas y TeÂcnicas) of Argentina for their ®nancial support to carry out this work, and Adrian Burton and MarõÂ a Laura for linguistic assistance.


References Ajmone Marsan P., Catiglioni P., Fusari F., Kuiper M., Maotto M. (1998) Genetic diversity and its relationships to hybrid performance in maize as revealed by RFLP and AFLP markers. Theor. Appl. Genet. 96: 219±227. Ayele M., Tefera H., Assefa K., Nguyen H. T. (1999) Genetic characterization of two Eragrostis species using AFLP and morphological traits. Hereditas 130: 33±40. Bottini M. C. J. (2000) Estudios multidisciplinarios en las especies patagoÂnicas argentinas del geÂnero Berberis L. (Berberidaceae). Tesis doctoral en Ciencias BioloÂgicas, Facultad de Ciencias Exactas y Naturales, UBA. 223 pp. Bottini M. C. J., Greizerstein E. J., Aulicino M. B., Poggio L. (2000a) Relationships among the genome size and environmental conditions and geographical distribution in natural populations of NW Patagonian species of Berberis L. (Berberidaceae). Ann. Bot. 86(3): 565±573. Bottini M. C. J., Greizerstein E. J., Poggio L. (1999a) Ploidy levels and their relationships with the rainfall in several populations of Patagonian species of Berberis L. Caryologia 52(1±2): 75±80. Bottini M. C. J., Greizerstein E. J., Poggio L. (2000b) Estudios electroforeÂticos de proteõÂ nas seminales (SDS-page) en las especies patagoÂnicas del geÂnero Berberis (Berberidaceae). Bol. Soc. Argent. Bot. 35(3±4): 245±258. Bottini M. C. J., Orsi M. C., Greizerstein E. J., Poggio L. (1998) Relaciones feneÂticas entre las especies de Berberis (Berberidaceae) del Noroeste de la regioÂn patagoÂnica. Darwiniana 35(1± 4): 115±129. Bottini M. C. J., Premoli A. C., Poggio L. (1999b) Hybrid speciation in Berberis L.: an isoenzymatic approach. XVI International Botanical Congress, St. Louis, Missouri, U.S.A. Bottini M. C. J., Greizerstein E. J., Poggio L. (1997) NuÂmeros cromosoÂmicos y contenido de ADN de cuatro especies patagoÂnicas del geÂnero Berberis (Berberidacae). BoletõÂ n de la Sociedad Argentina de BotaÂnica 32(3±4): 235±239. Cardoso S. R. S., Eloy N. B., Provan J., Cardoso M. A., Ferreira P. C. G. (2000) Genetic dierentiation of Euterpe edulis Mart. Populations estimated by AFLP analysis. Mol. Ecol. 9: 1753±1760. Cho Y. G., Blair M. W., Panaud O., McCouch S. R. (1996) Cloning and mapping of variety-speci®c rice genomic DNA sequences: ampli®ed fragment

141

length polymorphisms (AFLP) from silverstained polyacrylamide gels. Genome 39: 373± 378. Cervera M. T., Cabezas J. A., Sanch J. C., MartõÂ nez Detoda F., MartõÂ nez-Zapater J. M. (1998) Applications of AFLPs to the characterization of grapevine Vitis vinifera L. genetic resources. A case study with accessions from Rioja (Spain). Theor. Appl. Genet. 97: 51±59. Grant V. (1989) EspeciacioÂn vegetal, 2nd ed. Columbia Univ. Press, New York, 435 pp. Hill M., Witsenboer H., Zabeau M., Vos P., Kesseli R., Michelmore R. (1996) PCR-based ®ngerprinting using AFLPs as a tool for studying genetic relationships in Lactuca spp. Theor. Appl. Genet. 39: 1202±1210. Job M. M. (1942) Los Berberis de la RegioÂn del Nahuel Huapi. Revista Mus. La Plata, Secc. Bot. 5: 21±72. Landrum L. R. (1999) Revision of Berberis (Berberidaceae) in Chile and adjacent Southern Argentina. Ann. Missouri Bot. Gard. 86(4): 793±834. Lerceteau E., Szmidt A. E. (1999) Properties of AFLP markers in inheritance and genetic diversity studies of Pinus sylvestris L. Heredity 82: 252±260. Mantel N. A. (1967) The detection of disease clustering and a generalized regression approach. Cancer Res. 27: 209±220. MartõÂ nez Crovetto R. (1980) Apuntes sobre la vegetacioÂn de los alrededores del Lago Cholila. Univ. Nac. Del Noroeste Facultad de Cs. Agrarias. Corrientes. Maughan P. J., Saghai Maroof M. A., Buss G. R., Huestis G. M. (1996) Ampli®ed fragment length polymorphism (AFLP) in soybean: species diversity, inheritance, and near-isogenic line analysis. Theor. Appl. Genet. 93: 330±336. Orsi M. C. (1984) Berberidaceae. In: Correa M. N. (ed.) Flora PatagoÂnica Secc. 4a, pp. 325±348, INTA. Bs. As. Pakniyat H., Powell W., Baird E., Handley L. L., Robinson D., Scrimgeour C. M., Nevo E., Hackett C. A., Caligari P. D. S., Forster B. P. (1997) AFLP variation in wild barley (Hordeum spontaneum C. Koch) with reference to salt tolerance and associated site of origin ecogeography. Genome 40: 332±341. Paul S., Wachira F. N., Powell W., Waugh R. (1997) Diversity and genetic dierentiation among populations of Indian and Kenyan tea

142


(Camellia sinensis L.) revealed by AFLP markers. Theor. Appl. Genet. 94: 255±263. Perera L., Russell J. R., Provan J., McNicol J. W., Powell W. (1998) Evaluating genetic relationships between indigenous coconut (Cocos nucifera L.). Accessions from Sri Lanka by means of AFLP pro®ling. Theor. Appl. Genet. 96: 545± 550. Powell W., Morgante M., Chaz A., Manafey M., Vogel J., Tingey S., Rafalski A. (1996) The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germoplasm analysis. Molecular Breeding 2(3): 225±238. Roa A. C., Maya M. M., Duque M. C., Tohme J., Allem A. C., Bonierbale M. W. (1997) AFLP analysis of relationships among cassava and other Manihot species. Theor. Appl. Genet. 95: 741±750. Rohlf F. J. (1998) NTSYS-pc. Numerical taxonomy and multivariate analysis system (version 2.0f). Exeter Software Publishers Ltd., Setanket, New York. Sharma S. K., Knox M. R., Ellis T. H. N. (1996) AFLP analysis of the diversity and phylogeny of Lens and its comparison with RAPD analysis. Theor. Appl. Genet. 93: 751±758. Sambrook J., Fritsch E. F., Maniatis T. (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press. Shaer J. E. (1985) Inotropic chronotropic activity of Berberine on Isolated Guinea Pig Atria. J. Cardiovascular Pharmacology 7: 307±315. Sneath P. H. A., Sokal R. R. (1973) Numerical taxonomy: the principles and practice of numerical classi®cation (ed. Freeman), 359 pp. Tohme J., GonzaÂlez D. O., Beebs S., Duque M. C. (1996) AFLP analysis of gene pools of a wild bean core collection. Crop Science 36: 1375± 1384.

Tosto D. S., Hopp H. E. (2000) Suitability of AFLP markers for the study of genomic relationships within the Oxalis tuberosa alliance. Plant Syst. Evol. 223: 201±209. Travis S. E., Maschinski J., Keim P. (1996) An analysis of genetic variation in Astragalus cremnophylax, a critically endangered plant, using AFLP markers. Mol. Ecol. 5: 735±745. Vos P., Hogers R., Bleeker M., Reljans M., van de Lee T., Hornes M., Frijters A., Pot J., Peleman J., Kuiper M., Zabeau M. (1995) AFLP: a new technique for DNA ®ngerprinting. Nucl. Acids Res. 23: 4407±4414. Zhu J., Gale M. D., Quarrie S., Jackson M. T., Bryan G. J. (1998) AFLP markers for the study of rice biodiversity. Theor. Appl. Genet. 96: 602± 611.

Addresses of the authors: MarõÂ a Cecilia J. Bottini* and Lidia Poggio, Instituto FitoteÂcnico de Santa Catalina (FCAF, UNLP)- Centro de Investigaciones GeneÂticas (UNLP-CONICETCIC), C.C. 4, 1836 Llavallol, Buenos Aires and Departamento de Ciencias BioloÂgicas, Facultad de Ciencias Exactas y Naturales, Univ. de Buenos Aires, Argentina. Alfredo De Bustos and NicolaÂs Jouve, Department of Cell Biology and Genetics, Univ. of AlcalaÂ (Madrid), Spain. *Corresponding address: Instituto FitoteÂcnico de Santa Catalina (FCAF, UNLP)- Centro de Investigaciones GeneÂticas (UNLP-CONICET-CIC), C.C. 4, 1836 Llavallol, Buenos Aires (E-mail: [email protected]).