Genetic diversity in natural populations of Jacaranda decurrens Cham. determined using RAPD and AFLP markers

Jacaranda decurrens (Bignoniaceae) is an endemic species of the Cerrado with validated antitumoral activity. The genetic diversity of six populations of J. decurrens located in the State of São Paulo was determined in this study by using molecular markers for randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). Following optimization of the amplification reaction, 10 selected primers generated 78 reproducible RAPD fragments that were mostly (69.2%) polymorphic. Two hundred and five reproducible AFLP fragments were generated by using four selected primer combinations; 46.3% of these fragments were polymorphic, indicating a considerable level of genetic diversity. Analysis of molecular variance (AMOVA) using these two groups of markers indicated that variability was strongly structured amongst populations. The unweighted pair group method with arithmatic mean (UPGMA) and Pearson''s correlation coefficient (RAPD -0.16, p = 0.2082; AFLP 0.37, p = 0.1006) between genetic matrices and geographic distances suggested that the population structure followed an island model in which a single population of infinite size gave rise to the current populations of J. decurrens, independently of their spatial position. The results of this study indicate that RAPD and AFLP markers were similarly efficient in measuring the genetic variability amongst natural populations of J. decurrens. These data may be useful for developing strategies for the preservation of this medicinal species in the Cerrado.     

Life history variation between species of the relictual genus Borderea (Dioscoreaceae): phylogeography, genetic diversity, and population genetic structure assessed by RAPD markers

The genus Borderea consists of two species, B. pyrenaica and B. chouardii, taxa which have been previously considered as conspecific due to their overall close morphology. These two sole species of the rare genus of Dioscoreaceae are endemic to the Pyrenees (Spain, France). This mountain range likely operated as a refugium for these plants during the last glaciations. B. chouardii is only known from a single population in the Spanish Prepyrenees and has been classified as at risk of extinction in the Red List of Endangered Species (IUCN); B. pyrenaica shows a narrow distribution range in the central Pyrenees and Prepyrenees. We analysed genetic variation, population structure and differentiation in these two taxa using RAPD markers. Our study was conducted on the same seven populations for which very low levels of genetic differentiation were detected previously through allozyme analysis. By contrast, high levels of genetic variability were detected through the RAPD hypervariable markers. Twelve RAPD primers produced 112 distinct bands in the 397 surveyed individuals, totalling 395 different RAPD phenotypes. Only four bands were monomorphic across all samples of Borderea, whereas 21 of the polymorphic bands were species‐specific (20 for B. chouardii, and one for B. pyrenaica). The largest genetic distances were those between the B. chouardii and the B. pyrenaica phenotypes. An analysis of molecular variance showed greater variance between groups (B. chouardii vs. B. pyrenaica, 76.08%) than within groups (3.60%). RAPD band specificity, phenotypic distances, and the partitioning of variance all support the taxonomic separation of the two species. Statistical evaluation of within‐ and among‐population RAPD genetic variability in B. pyrenaica showed that genetic variability was higher within populations (>80%) than among them. No clear pattern of RAPD differentiation could be observed among the six studied populations of this taxon though slight differences in genetic diversity could be observed in the more isolated Prepyrenean populations compared with the more widespread Pyrenean ones. These results suggest a recent postglacial origin of the present B. pyrenaica populations. © 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80 , 483–498.     

Genetic variability in the endemic <Emphasis Type="Italic">Leucojum valentinum</Emphasis>

The genetic variability of Leucojum valentinum Pau (Amaryllidaceae), a vulnerable endemic species restricted to a small area in the region of Valencia (Eastern Spain), has been studied using random amplified polymorphic DNA (RAPD) markers. A total of 197 individuals from eleven populations were studied using 13 RAPD primers. Our results show high variability for the species, low differentiation among populations and uncorrelated levels of genetic variability and population size. Four groups in which three populations (SAG, PUG and COL) are separated from all the others were found, but without connection to geographical location.     

RAPD Profiling in Detecting Genetic Variation in Endemic Coelonema (Brassicaceae) of Qinghai-Tibet Plateau of China

Random amplified polymorphic DNA (RAPD) markers were used to measure genetic diversity of Coelonema draboides (Brassicaceae), a genus endemic to the Qilian Mountains of the Qinghai-Tibet Plateau. We sampled 90 individuals in 30 populations of Coelonema draboides from Datong and Huzhu counties of Qinghai Province in P.R. China. A total of 186 amplified bands were scored from the 14 RAPD primers, with a mean of 13.3 amplified bands per primer, and 87% (161 bands) polymorphic bands (PPB) was found. Analysis of molecular variance (AMOVA) shows that a large proportion of genetic variation (84.2%) resides among individuals within populations, while only 15.8% resides among populations. The species shows higher genetic diversity between individuals than other endemic and endangered plants. The RAPDs provide a useful tool for assessing genetic diversity of rare, endemic species and for resolving relationships among populations. The results show that the genetic diversity of this species is high, possibly allowing it to adapt more easily to environmental variations. The main factor responsible for the high level of differentiation within populations and the low level of diversity among populations is probably the outcrossing and long-lived nature of this species. Some long-distance dispersal, even among far separated populations, is also a crucial determinant for the pattern of genetic variation in the species. This distributive pattern of genetic variation of C. draboides populations provides important baseline data for conservation and collection strategies for the species. It is suggested that only populations in different habitats should be studied and protected, not all populations, so as to retain as much genetic diversity as possible.     

Conservation genetics of the rare and endangered Leucopogon obtectus (Ericaceae)

Leucopogon obtectus Benth. is a declared rare species found in the kwongan vegetation in Western Australia. Plants on a mineral sand mine and the rehabilitation area are subject to disturbance. Genetic diversity was examined within and among all known populations using random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphisms (AFLPs) for conservation. Both molecular markers revealed a high percentage (> 89%) of polymorphic markers and a high mean genetic distance among individuals (D = 0.3). Analysis of molecular variance showed that 86.7% (RAPD) and 89.7% (AFLP) of variability was partitioned among individuals within populations. Exact tests showed no significant population differentiation. The analyses indicated that L. obtectus exhibits high levels of genetic diversity despite small population sizes. The high levels of variability among individuals and the lack of clear population differentiation suggest that this species comprises a single, genetically diverse group. Conservation and management of L. obtectus should concentrate on maintaining the high levels of genetic variability through mixing genotypes and promoting outcrossing.     

Population genetic structure of two rare tree species (Colubrina oppositifolia and Alphitonia ponderosa,Rhamnaceae) from Hawaiian dry and mesic forests using random amplified polymorphic DNA markers

Hawaiian dry and mesic forests contain an increasingly rare assemblage of species due to habitat destruction, invasive alien weeds and exotic pests. Two rare Rhamnaceae species in these ecosystems, Colubrina oppositifolia and Alphitonia ponderosa, were examined using random amplified polymorphic DNA (RAPD) markers to determine the genetic structure of the populations and the amount of variation relative to other native Hawaiian species. Relative variation is lower than with other Hawaiian species, although this is probably not a consequence of genetic bottleneck. Larger populations of both species contain the highest levels of genetic diversity and smaller populations generally the least as determined by number of polymorphic loci, estimated heterozygosity, and Shannon's index of genetic diversity. Populations on separate islands were readily discernible for both species as were two populations of C. oppositifolia on Hawai'i island (North and South Kona populations). Substructure among Kaua'i subpopulations of A. ponderosa that were ecologically separated was also evident. Although population diversity is thought to have remained at predisturbance levels, population size continues to decline as recruitment is either absent or does not keep pace with senescence of mature plants. Recovery efforts must focus on control of alien species if these and other endemic dry and mesic forest species are to persist.     

Genetic diversity across natural populations of <Emphasis Type="Italic">Dendrobium officinale</Emphasis>, the endangered medicinal herb endemic to China,revealed by ISSR and RAPD markers

Dendrobium officinale is a rare and endangered herb with special habitats and endemic to China. Genetic diversity was examined within and among nine natural populations using inter-simple sequence repeat (ISSR) and random amplified polymorphic (RAPD) for conservation. Both molecular markers revealed a high percentage (>89%) of polymorphic bands and ISSR markers detected more diversity than RAPD markers. Analysis of molecular variance (AMOVA) revealed that 78.84% (ISSR) and 78.88% (RAPD) of variability was partitioned among individuals within populations. This genetic structure was probably due to severe genetic drift resulting from habitat fragmentation and human overexploitation since 1950s. Moreover, there is a lack of significant association between genetic and geographic distances (r = 0.276; p > 0.05) in the populations of D. officinale. From the conservation point of view, populations GL, GS and GSD with higher genetic diversity should be protected firstly to maintain the species potential for evolutionary change and population YG with lower diversity but representing a novel evolutionary unit should also be paid more attention to during D. officinale conservation practice. Published in Russian in Genetika, 2009, Vol. 45, No. 3, pp. 375–382. The article is published in the original.     

Genetic diversity analysis of Rhododendron aureum Georgi (Ericaceae) located on Changbai Mountain using ISSR and RAPD markers

Rhododendron aureum Georgi (Ericaceae) is a perennial alpine shrub endemic to Changbai Mountain in China. We used ISSR and RAPD markers to describe the diversity and genetic structure within and among four natural populations located at different altitudes. DNA from 66 individuals was amplified with ten ISSR markers and seven RAPD markers. High genetic diversity was observed by these two techniques at the species level. The genetic diversity of populations increased with altitudinal gradients from low to high. The coefficient of gene differentiation (G_ST 0.3652 in ISSR and 0.2511 in RAPD) and AMOVA analysis revealed that most genetic diversity was distributed within populations (61.96% in ISSR and 70.23% in RAPD). The estimate of gene flow based on G_ST was 0.8690 in ISSR and 1.4910 in RAPD. The UPGMA clustering results using ISSR and RAPD showed that all individuals from the same altitude were gathered together, and the two populations (TYD2a and YHLa) from middle altitudes always clustered together. Compared with populations from different altitudes, similar genetic diversity and low genetic differentiation were obtained from populations at the same altitudes, as revealed by ISSR markers. In addition to the reproductive strategy of R. aureum, these data highlight that local environmental conditions may play an important role in shaping the diversity and genetic structure of this species.     

Genetic diversity of the endangered Chinese endemic herb Primulina tabacum (Gesneriaceae) revealed by amplified fragment length polymorphism (AFLP)

Primulina tabacum Hance, is a critically endangered perennial endemic to limestone area in South China. Genetic variability within and among four extant populations of this species was assessed using AFLP markers. We expected a low genetic diversity level of this narrowly distributed species, but our results revealed that a high level of genetic diversity remains, both at population level (55.5% of markers polymorphic, H _E = 0.220, I _S = 0.321), and at species level (P = 85.6% of markers polymorphic, H _E = 0.339, I _S = 0.495), probably resulting from its refugial history and/or breeding system. High levels of genetic differentiation among populations was apparent based on Nei’s genetic diversity analysis (G _st=0.350). The restricted gene flow between populations is a potential reason for the high genetic differentiation. The population genetic diversity of P. tabacum revealed here has clear implications for conservation and management. To maintain present levels of genetic diversity, in situ conservation of all populations is necessary.     

Genetic diversity in the tetraploid sand dune endemic Deschampsia mackenzieana and its widespread iploid progenitor D. Cespitosa (Poaceae)

Electrophoretic variation was examined in 14 populations of tetraploid Deschampsia mackenzieana, an endemic of the Athabasca sand dunes in northern Saskatchewan, Canada, and 20 populations of its geographically widespread diploid progenitor, D. cespitosa. Three of the D. cespitosa populations were sympatric with the endemic on the Athabasca sand dunes. Populations of the endemic were found to have fewer alleles per locus (1.22 vs. 1.52), fewer alleles per polymorphic locus (2.17 vs. 2.70), lower percent polymorphic loci (18.9 vs. 30.5), and lower heterozygosity (0.062 vs. 0.119) than progenitor populations. Species level genetic diversity parameters also indicated that D. mackenzieana was genetically depauperate relative to its progenitor D. cespitosa. Deschampsia mackenzieana had no novel alleles but did share one allele with sympatric progenitor populations that did not occur in populations of D. cespitosa from other habitats. Although both species were found to partition most of their genetic diversity within populations, D. mackenzieana did have more of its limited genetic diversity partitioned among populations than D. cespitosa. The close genetic relationship between D. mackenzieana and sympatric populations of D. cespitosa may suggest the endemic tetraploid evolved from the sympatric diploid gene pool in the Athabasca sand dune region. The low levels of genetic diversity in D. mackenzieana suggest a restricted origin with limited gene flow from the progenitor since speciation.     

Genetic diversity of three endangered species of <Emphasis Type="Italic">Echium</Emphasis> L. (Boraginaceae) endemic to Cape Verde Islands

Echium hypertropicum, E. stenosiphon and E. vulcanorum are the three endemics representative of the genus Echium (Boraginaceae) in Cape Verde archipelago. The aim of this study is to provide a first attempt at estimating genetic diversity among natural populations of these endangered Echium species based on RAPD, so as to provide data available for future appropriate strategies for their conservation. PCO and UPGMA of RAPD analysis suggests a close genetic proximity between the Southern endemic species (E. hypertropicum and E. vulcanorum) and shows that the levels of polymorphism strongly differ between these two Echium species (27 and 29% respectively) and E. stenosiphon (74%), the Northern endemic species. Mantel test also corroborates a close genetic proximity between genetic and geographic data. Population genetic analysis of E. stenosiphon revealed low levels of gene flow between islands (Nm = 0.32) being S. Nicolau the most isolated as evident in PCO. Furthermore the differentiation between groups of individuals belonging to putative subspecies was tested by AMOVA. According to our results there is no genetic basis to consider the two subspecies of E. stenosiphon namely ssp. stenosiphon and ssp. lindbergii. Our results enable us to suggest that E. stenosiphon must be ranked as a Threatened species. Measures aiming at conservation of E. hypertropicum and E. vulcanorum must be implemented at short-term taking into account the small number of existing plants and its low genetic variability.     

Molecular variation and population structure in Elymus trachycaulus and comparison with its morphologically similar E. alaskanus

Random amplified polymorphic DNA (RAPD) markers were used to determine the levels and pattern of molecular variation in four populations of Elymus trachycaulus, and to estimate genetic similarity among different populations of E. trachycaulus from British Columbia and the Northwest Territories and one population of Elymus alaskanus from the Northwest Territories. Based on 124 RAPD bands (loci), mean percent polymorphic loci for E. trachycaulus (P_P) was 67.4% (a range 41.2% to 86.3%), and mean gene diversity (H_e) for E. trachycaulus species was 0.23 (range 0.18 to 0.27). The total genetic diversity was 0.32. Differentiation among populations was 31% (F_ST = 0.31) with most of the genetic variation found within populations (69%). This pattern of genetic variation was different from that reported for inbred species in general.The authors are very grateful to Michael Bond for excellent Laboratory assistance, to Dr. Mary Barkworth for her encouragement. This study was supported by a Natural Science and Engineering Research Council (NSERC) discovery grant and by a Saint Marys University Internal grant to G.S.     

Genetic diversity of Mexican brook lamprey <Emphasis Type="Italic">Lampetra (Tetrapleurodon) geminis</Emphasis> (Alvarez del Villar, 1966)

Lampreys are the only surviving representatives of the oldest known vertebrates. The Mexican lamprey L. geminis (nonparasitic), is particularly interesting, because it is an endemic, biogeographical relict, and a threatened species. RAPD markers were used to describe genetic diversity in L. geminis A total of 77 specimens were collected from five populations, three in the R'o Grande de Morelia-Cuitzeo basin and two in the R'o Duero-Lerma-Chapala basin, Mexico. Eighty-eight RAPD markers were obtained from eight primers. Genetic diversity within each population was estimated using Shannon's index (S), heterozygosity (H) and gene diversity (h). These estimates revealed significant variation within populations, although a variance homogeneity test (HOMOVA) showed no significant differences among populations or between basins. Nei genetic distance values indicate a low genetic differentiation among populations. Analysis of molecular variance (AMOVA) indicates that most of the genetic diversity occurs within populations (91.4%), but that a statistically significant amount is found among populations (P0.001). Principal coordinates and cluster analyses of RAPD phenotypes show that specimens are not grouped by geographical origin. The genetic diversity found within L. geminispopulations may be explained by its breeding system and an overlapping of generations. The scarce genetic differentiation among populations is likely to the low rate of DNA change that characterizes the lamprey group.     

The distribution of genetic variability in Baccharis concinna (Asteraceae), an endemic, dioecious and threatened shrub of rupestrian fields of Brazil

Distribution of genetic variability may becorrelated with life history traits, such asreproductive system, pollination, seeddispersal mode, geographical distribution, andlocal abundance. We used RAPD markers to studythe distribution of genetic variability of sixpopulations of Baccharis concinna(Asteraceae), a rare, dioecious and threatenedshrub, endemic to Serra do Cipó,southeastern Brazil, along an altitudinalgradient, ranging from 950 to 1300 m above sealevel. From 113 RAPD markers, 35 wererestricted to one of the six populations.Genetic variability distribution in B.concinna populations was weakly related to thealtitudinal gradient. The largest proportion ofthe genetic variability was due to variationwithin populations (82.82%, P < 0.001), while17.18% (P < 0.001) was due to variation amongpopulations from different elevations.Similarity among populations along thealtitudinal gradient was high, ranging from 65to 74%, suggesting an intensive gene flowamong them or a recent fragmentation of anancient population into many small populations.Dioecy and other life history traits such aswind pollination and seed dispersal mayinfluence the distribution of the geneticvariability in this species. In addition, fireand habitat fragmentation represent otherimportant factors that influence thesurvivorship of this rare and threatenedspecies, and hence the distribution of itsgenetic variability.     

Comparative population structure of three snook species (Teleostei: Centropomidae) from the eastern central Pacific

Three snook species, Centropomus viridis, Centropomus medius, and Centropomus robalito, from the eastern central Pacific, representing three of the four proposed phyletic lineages in the genus, were analyzed for genetic variability by means of allozyme and RAPD to evaluate the divergence between populations at different levels of dispersal ability and to evaluate the importance of barriers to dispersal in the population subdivision and genetic diversity. Levels of genetic diversity among species estimated by allozymes were similar and consistent with the observed levels of differentiation in marine fish species. Mean heterozygosity ranged from 0.089 for C. viridis to 0.10 for C. robalito. Genetic diversity for the snook species studied was slightly higher than the mean estimation reported in allozymes for 106 marine fish (0.055) and for anadromous fish species (0.043 to 0.057). Multilocus allele frequency homogeneity tests and population-subdivision estimates for both allozyme and RAPD markers revealed the existence of population structure in C. viridis and C. medius, in coincidence with geographic separation of samples, whereas no divergence was detected in C. robalito. This finding may be attributed to the greater population size of C. robalito, which originated by a recent population range expansion, and hence the potential for dispersal is mediated by larval drift. Fluctuations in population size and population range expansion are used to explain discrepancies between levels of genetic diversity and population structure in the studied species. Supplementary material to this paper is available in electronic format at     

Geographical diversity and genetic relationships among Cedrus species estimated by AFLP

Genetic diversity was described in 17 cedar populations covering the geographical range of the four species of the genus Cedrus. The study was conducted using amplified fragment length polymorphism (AFLP) on haploid tissues (megagametophytes). Eleven selective AFLP primer pairs generated a total of 107 polymorphic amplification products. Correspondence and genetic distance analyses indicated that Cedrus deodara constitutes a separate gene pool from the Mediterranean cedars. Within Mediterranean cedars, we distinguished two groups: the first one is made of Cedrus atlantica, while the second one is made of Cedrus libani and Cedrus brevifolia, these latter two species being genetically similar despite important divergence previously observed for morphological and physiological traits. The lowest intrapopulation variability was found in the two C. deodara populations analyzed. Surprisingly, C. brevifolia, the endemic taxon from the island of Cyprus that is found in small and fragmented populations, showed one of the highest levels of diversity. This unexpected pattern of diversity and differentiation observed for C. brevifolia suggests a recent divergence rather than a relictual, declining population. Patterns of diversity within- and among-populations were used to test divergence and fragmentation hypotheses and to draw conclusions for the conservation of Cedrus gene pools.     

Conservation genetics of the endangered endemic Sambucus palmensis Link (Sambucaceae) from the Canary Islands

Five polymorphic microsatellites (simple sequence repeat; SSR) markers were used to estimate the levels of genetic variation within and among natural populations from different islands of the endangered endemic from the Canary Islands Sambucus palmensis Link (Sambucaceae). Genetic data were used to infer potential evolutionary processes that could have led to present genetic differentiation among islands. The levels of genetic variability of S. palmensis were considerably high; proportion of polymorphic loci (P = 100%), mean number of alleles per locus (A = 6.8), average expected heterozygosity (He = 0.499). In spite of its small population size and endemic character, 58 different multilocus genotypes were detected within the 165 individuals analyzed. All samples located in different islands always presented different multilocus genotypes. Principal Coordinates Analysis, genetic differentiation analysis (F _ST and G _ST ′) and Bayesian Cluster Analysis revealed significant genetic differences among populations located in different islands. However, this genetic differentiation was not recorded among Tenerife and La Gomera populations, possibly revealing the uncontrolled transfer of material between both islands. AMOVA analysis attributed 77% of the variance to differences within populations, whereas 8% was distributed between islands. The levels of genetic differentiation observed among populations, and the genetic diversity distribution within populations in S. palmensis, indicate that management should aim to conserve as many of the small populations as possible. Concentrating conservation efforts only on the few large populations would result in the likelihood of loss of genetic variability for the species.     

Use of RAPD analysis in devising conservation strategies for the rare and endangered Grevillea scapigera (Proteaceae)

Extensive human impact in south western Australia has resulted in a high incidence of rarity throughout the highly endemic flora of the region. Grevillea scapigera (Proteaceae) is a typical example, with 27 plants (represented by four extant populations) remaining in the wild. In order to devise an appropriate strategy for the conservation of this species, its population genetics were studied using RAPD analysis, which enabled the discrimination of individual plants and the detection of a relatively high amount of variability (V = 0.32) within G. scapigera. This variability was found to be evenly distributed within the plants analysed despite the clear distinction between most populations (87% of the variability being attributable to single plant difference and 13% to population difference). Finally, RAPD analysis was used to select a small group of plants that captured maximum genetic variability to be used in the recovery program of the species. Because of the low genetic difference between populations, the mixing of these selected plants during the recovery process should not create genetic imbalances. The methods used in this study provide a useful model for future projects involving the recovery of rare flora.     

Molecular and morphological variation of rare endemic oncocyclus irises (Iridaceae) of Lebanon

Oncocyclus irises endemic to Lebanon form a complex of three closely related taxa replacing each other over short geographical distances in a linear habitat. In order to characterize the appropriate taxonomic levels and to assess their conservation status, we investigated patterns of phenotypic variability and the partitioning of genetic variation within and among populations using random amplified polymorphic DNA (RAPD) markers. Multivariate analysis (principal components analysis and multiple correspondence analysis), based on 16 quantitative and six qualitative characters, revealed no separation between populations or taxa. Moreover, no morphological character could be used to define clear boundaries between populations/taxa. The genetic characterization revealed high levels of polymorphism and diversity (H_s). Principal components analysis showed population delimitations, but no groupings reflecting the currently defined taxa could be identified. Both morphological and genetic data showed that Lebanese oncocyclus irises could not be fitted into clear taxonomic boundaries. Consequences for conservation are discussed. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 123–135.     

Isolated populations or isolated taxa? A case study in narrowly-distributed snapdragons (Antirrhinum sect. Sempervirentia) using RAPD markers

RAPD fingerprinting was used to study species boundaries in narrowly distributed endemic species in Antirrhinum section Sempervirentia. Based on RAPD data, similarity values within species were relatively high but pair-wise similarity values among species were low. Partitioning of the overall RAPD variation using AMOVA showed that most of the variation was found among species (58.06%), whereas the remaining phenotypic diversity was distributed among populations (25.18%) and among individuals within populations (16.76%). Comparison of the matrices of geographical distances and phenetic distances (1-Dice index) among populations using the Mantel test showed a moderate, but statistically significant correlation (r=0.588, P < 0.01), suggesting that isolation by distance is responsible for the distribution of genetic variation among Antirrhinum populations. Phenetic relationships among Antirrhinum samples based on a Dice similarity matrix showed a clear taxonomic pattern, confirming the grouping of individuals within their own populations and the clustering of populations within species. Individuals of A. charidemi, A. valentinum and A. subbaeticum, from subsection Valentina, made up a discrete group, whereas the samples belonging to subsection Sempervirentia (A. petegasii, A. sempervirens, A. microphyllum, A. pulverulentum) clustered together. RAPD data are entirely congruent with the subsection classification scheme proposed by Fernández Casas (1997) in section Sempervirentia. However, A. subbaeticum, treated as a synonym of A. valentinum by Fernández-Casas (1997), showed an unique RAPD profile characterized by the highest number of fixed species-specific markers found in section Sempervirentia. Thus, although A. valentinum appeared the most closely related species to A. subbaeticum, molecular data suggested that this species merits taxonomic distinction.