Population Genetic Structure of Neotropical Mangrove Species on the Colombian Pacific Coast: Avicennia germinans (Avicenniaceae)1

We investigated the genetic variation of Avicennia germinans using 172 AFLP (Amplified Fragment Length Polymorphism) bands of 45 plants from four localities on the Colombian Pacific coast: 11 from Virudó (Chocó), 10 from La Plata (Valle), 12 from Tumaco (Nariño), and 12 from Chontal (Nariño). AFLP variation among localities (16.2%) was highly significant (AMOVA; P < 0.0001). All the analyses showed that Tumaco was the most genetically distinct locality of the four under study. The other three localities, La Plata, Virudó, and Chontal, apparently form a large single subpopulation with high‐to‐moderate gene flow among localities. We also found the genetic diversity of A. germinans on the Colombian Pacific coast (H_E= 0.251) higher than that estimated by others over the broad geographic range of A. germinans. All these results together show that mangroves on the Colombian Pacific coast deserve a strong investigative effort to improve our ecological, evolutionary, and biogeographic knowledge of this important tropical forest type.     

Characterization of microsatellite markers for the mangrove tree Avicennia germinans L. (Avicenniaceae)

We describe 10 primers for amplification of microsatellite loci for the mangrove, Avicennia germinans. Eight loci were isolated from a DNA sample from the Pacific coast of Baja California, Mexico and two loci were isolated from a DNA sample from the Atlantic coast of Bermuda. Polymorphism was investigated in a population from the Mexican Pacific coast (n = 15) and in four samples scattered throughout the range of the species. Total number of alleles for the species ranged from two to 10. Observed heterozygosity in the Mexican Pacific coast population ranged from 0.27 to 0.60, with two loci having fixed alleles.     

Salinity effects on the leaf water relations components and ion accumulation patterns in Avicennia germinans (L.) L. seedlings

Physiological traits involved in leaf water relations were evaluated in Avicennia germinans (L.) L. seedlings growing at different salinities in the field. Analysis of pressure-volume (P-V) curves and sap osmometry were combined to evaluate osmotic adjustment and cell elasticity, and the contribution of accumulated inorganic ions to osmotic potential was estimated. Seedlings growing in soils with interstitial water salinity above that of normal sea water showed a modification of the relationship between water potential and relative water content. Thus, their leaf osmotic potential at maximum turgor (Ψ_{π( max )}) and at zero turgor (Ψ_π(0)) was 1.41 and 1.82 MPa lower respectively, than that of the seedlings from the low salinity site. Volumetric moduli of elasticity () were between 17 and 23 MPa. Thus, ɛ was about 6 MPa lower in high-salinity plants indicating that their cells were slightly more elastic. Ionic concentration analysis showed that Σ [anions] and Σ [cations] were higher in the high-salinity site (22–35%) while the water content per unit dry mass was only 12–17% lower. Reduction in water content was insufficient to explain the increase in ion concentration. Ion concentration explained 73 and 66% of the osmotic potential estimated by P-V curves for leaves from low- and high-salinity sites, respectively. In conclusion, this study provided evidence that leaves of A. germinans seedlings adapt to hypersaline soils by increasing solute concentration by 52% and cell elasticity by 26%. Both processes allow leaf water uptake and turgor maintenance over a large range of soil water potential. Received: 30 June 1997 / Accepted 26 November 1997     

Microsatellite analysis of genetic structure in the mangrove species Avicennia marina (Forsk.) Vierh. (Avicenniaceae)

The level of genetic variation throughout the entire worldwide range of the mangrove species Avicennia marina (Forsk.) Vierh. was examined using microsatellite markers. Three microsatellite loci detected high levels of allelic diversity (70 alleles in total), essential for an accurate estimation of population genetic parameters. The informativeness of the microsatellite loci tended to increase with increasing average number of repeats. The levels of heterozygosity detected for each population, over all loci, ranged from 0.0 to 0.8, with an average of 0.407, indicating that some populations had little or no genetic variation, whereas others had a large amount. Populations at the extremes of the distribution range showed reduced levels of heterozygosity, and significant levels of inbreeding. This is not unexpected as these populations may be subject to founder effects and environmental constraints. The presence of genetic structure was tested in A. marina populations using three models: (i) a single panmictic model; (ii) the discrete subpopulation model; and (iii) the isolation by distance model. The discrete subpopulations model was supported by the overall measures of population differentiation based on the infinite alleles model (F-statistics), and the stepwise mutation model (R statistics). In addition, an analysis of molecular variance (AMOVA), using both theoretical models, found that most of the variation was between populations (41-71%), and within individuals in the total population (31-49%). There was little variation among individuals within populations (0-10%). There was no significant isolation by distance. The high levels of genetic differentiation observed among populations of A. marina may be due to environmental and ecological factors, particularly past sea level and climatic changes.     

Genetic structure at range edge: low diversity and high inbreeding in Southeast Asian mangrove (Avicennia marina) populations

Understanding the genetic composition and mating systems of edge populations provides important insights into the environmental and demographic factors shaping species' distribution ranges. We analysed samples of the mangrove Avicennia marina from Vietnam, northern Philippines and Australia, with microsatellite markers. We compared genetic diversity and structure in edge (Southeast Asia, and Southern Australia) and core (North and Eastern Australia) populations, and also compared our results with previously published data from core and southern edge populations. Comparisons highlighted significantly reduced gene diversity and higher genetic structure in both margins compared to core populations, which can be attributed to very low effective population size, pollinator scarcity and high environmental pressure at distribution margins. The estimated level of inbreeding was significantly higher in northeastern populations compared to core and southern populations. This suggests that despite the high genetic load usually associated with inbreeding, inbreeding or even selfing may be advantageous in margin habitats due to the possible advantages of reproductive assurance, or local adaptation. The very high level of genetic structure and inbreeding show that populations of A. marina are functioning as independent evolutionary units more than as components of a metapopulation system connected by gene flow. The combinations of those characteristics make these peripheral populations likely to develop local adaptations and therefore to be of particular interest for conservation strategies as well as for adaptation to possible future environmental changes.     

Comparative analysis of genetic diversity in the mangrove species Avicennia marina (Forsk.) Vierh. (Avicenniaceae) detected by AFLPs and SSRs

Avicennia marina is an important mangrove species with a wide geographical and climatic distribution which suggests that large amounts of genetic diversity are available for conservation and breeding programs. In this study we compare the informativeness of AFLPs and SSRs for assessing genetic diversity within and among individuals, populations and subspecies of A. marina in Australia. Our comparison utilized three SSR loci and three AFLP primer sets that were known to be polymorphic, and could be run in a single analysis on a capillary electrophoresis system, using different- colored fluorescent dyes. A total of 120 individuals representing six populations and three subspecies were sampled. At the locus level, SSRs were considerably more variable than AFLPs, with a total of 52 alleles and an average heterozygosity of 0.78. Average heterozygosity for AFLPs was 0.193, but all of the 918 bands scored were polymorphic. Thus, AFLPs were considerably more efficient at revealing polymorphic loci than SSRs despite lower average heterozygosities. SSRs detected more genetic differentiation between populations (19 vs 9%) and subspecies (35 vs 11%) than AFLPs. Principal co-ordinate analysis revealed congruent patterns of genetic relationships at the individual, population and subspecific levels for both data sets. Mantel testing confirmed congruence between AFLP and SSR genetic distances among, but not within, population comparisons, indicating that the markers were segregating independently but that evolutionary groups (populations and subspecies) were similar. Three genetic criteria of importance for defining priorities for ex situ collections or in situ conservation programs (number of alleles, number of locally common alleles and number of private alleles) were correlated between the AFLP and SSR data sets. The congruence between AFLP and SSR data sets suggest that either method, or a combination, is applicable to expanded genetic studies of mangroves. The codominant nature of SSRs makes them ideal for further population-based investigations, such as mating-system analyses, for which the dominant AFLP markers are less well suited. AFLPs may be particularly useful for monitoring propagation programs and identifying duplicates within collections, since a single PCR assay can reveal many loci at once. Received: 3 October 2000 / Accepted: 19 February 2001     

Isolation and characterization of 10 new compound microsatellite markers for a mangrove tree species,Avicennia marina (Forsk.) Vierh. (Avicenniaceae)

Avicennia marina is an ecologically important mangrove tree species. We isolated 10 polymorphic microsatellite loci from this species using an improved technique. Our isolated loci provided compound microsatellite markers with polymorphism of two to six alleles per locus. The observed and expected heterozygosities ranged from 0.025 to 0.625 and from 0.096 to 0.767, respectively. These markers would be the useful tools for researching on the genetic diversity and population genetic structure of A. marina.     

Genetic diversity of Ranunculus acris L. (Ranunculaceae) populations in relation to species diversity and habitat type in grassland communities

Correlates between genetic diversity at intra- and interpopulation levels and the species diversity in plant communities are rarely investigated. Such correlates may give insights into the effect of local selective forces across different communities on the genetic diversity of local plant populations. This study has employed amplified fragment length polymorphism to assess the genetic diversity within and between 10 populations of Ranunculus acris in relation to the species diversity (richness and evenness) of grassland communities of two different habitat types, 'seminatural' and 'agriculturally improved', located in central Germany. Within-population genetic diversity estimated by Nei's unbiased gene diversity (HE) was high (0.258-0.334), and was not correlated with species richness (Pearson's r = -0.17; P = 0.64) or species evenness (Pearson's r = 0.15; P = 0.68) of the plant communities. However, the genetic differentiation between R. acris populations was significantly correlated with the difference in species evenness (Mantel's r = 0.62, P = 0.02), but not with difference in species richness of plant communities (r = -0.17, P = 0.22). Moreover, we also found that populations of R. acris from the 'seminatural' habitat were genetically different (amova, P < 0.05) from those in 'agriculturally improved' habitats, suggesting that gene flow between these habitat types is limited. The results reported in this study may indicate that habitat characteristics influence the genetic diversity of plant species.     

Genetic diversity and population structure of a cyprinid fish (Ancherythroculter nigrocauda) in a highly fragmented river

In this study, samples of Ancherythroculter nigrocauda, an endemic fish in the upper Yangtze River, were collected above and below dams in the Longxi River, a tributary of the upper Yangtze River, China, to investigate the genetic impacts of dams. The mitochondrial cytochrome b gene (cyt b) and 13 microsatellite (SSR) loci were used to analyze whether dams have resulted in loss of genetic diversity of the two fragmented populations or caused genetic differentiation between them. The results showed that the haplotype diversity (0.488; 0.486), nucleotide diversity (0.084%; 0.082%) and average expected heterozygosity (0.652; 0.676) of the two populations were all at a low level, and recent bottlenecks were detected. However, there was no genetic differentiation detected by the low genetic differentiation index (F_st, cyt b: ?0.1677, p = 0.99707; SSR: 0.00259, p = 0.81427). Besides, 11 pairs of half‐sibling relationship were found between the two populations indicating that there were individual movements and gene flow between them. This could be the larvae moving from upstream to downstream when water spilled over dams in flooding season. Therefore, our analysis showed that the dams have caused a loss of genetic diversity of the populations of A. nigrocauda in the Longxi River, blocked the active upstream movement but allowing passive downstream drift of larvae.     

Salt effects on membranes of the hypodermis and mesophyll cells of Avicennia germinans (Avicenniaceae): a freeze-fracture study

Freeze-fracture electron microscopy was used to investigate intramembranous particle (IMP) densities and particle distributions in the plasma membrane and tonoplast of the cells of secreting and nonsecreting leaves of Avicennia germinans (L.) Steam. Intramembranous particle densities of the protoplasmic (P) and exoplasmic (E) face of the plasma membrane and tonoplast were significantly higher in hypodermal cells of secreting leaves than of nonsecreting leaves. In contrast, no significant differences in the frequency of intramembranous particles were found in any membrane faces of secreting or nonsecreting mesophyll cells. However, particle densities were higher in the plasma membrane and tonoplast of the mesophyll cells, compared to the hypodermal cells, with the exception of the P-face of hypodermal plasma membranes of secreting tissue, which had the highest particle density measured. Particle distributions were dispersed and no discernible patterns such as paracrystalline arrays or other multi-IMP structures were observed. Results support the hypothesis that secretion is coupled to changes in membrane ultrastructure, and the possibility that salt secretion is an active process driven by integral membrane proteins such as the H⁺/ATPase. Additionally, the hypodermal cells of the leaf may function as storage reservoirs for salt as well as water, suggesting a regulatory role in salt secretion.     

A combination of molecular markers identifies evolutionarily significant units in Cedrela odorata L. (Meliaceae) in Costa Rica

The necessity for conservation of the geneticcomponent of biodiversity is now widelyrecognised. A broad genetic base is required tomaintain evolutionary potential and thepopulation erosion occurring in much of theworld's forests threatens the genetic integrityof many tree species. Spanish Cedar (Cedrela odorata L.) has been under severepressure for generations and is now the focusof a study aimed at assessing the levels anddistribution of genetic diversity in remainingpopulations. Ten Costa Rican populations wereanalysed using chloroplast and AFLP markers.The overall level of diversity was as expectedfor an outcrossing, long-lived, woody species(H _T = 0.27). However, this concealeda deep divergence within the species, forchloroplast and AFLP (_CT = 0.83)markers. Populations were differentiated in twogroups that exhibited contrasting habitatpreferences and two ecotypes, wet and dry, wereidentified. Within the ecotypes, all but onepopulation were fixed for a single chloroplasthaplotype and within populations, total genomicdiversity levels were low (H _S= 0.03–0.13). Populations possessing the dryecotype maintained significantly more diversitythan those from wet regions. Within the wetecotype group, pairwise genetic distancebetween populations fitted an isolation bydistance model. The group was stronglysubdivided and showed isolation by distancearound the southern edge of the centralmountain ranges. The genetic divergence of thetwo ecotypes, observed at both organellar andnuclear loci, identifies evolutionarilysignificant units that, taken together withprevious studies of the species, provide arational basis on which to build a conservationpolicy for the species.     

Genetic diversity and multilocus structure in sexual Otiorhynchus alpicola populations (Goleoptera: Gurculionidae)

Otiorhynchus alpicola Boh. (Coleoptera: Curculionidae) is distributed on high mountains of central, southern and south-eastern Europe. On the Balkan peninsula, this species is patchily distributed on mountain peaks at heights over 1800 m. To examine the organization of isoenzyme variation of the ten sexual (diploid) populations, starch gel electrophoresis of enzymes was used. The average heterozygosity over 14 gene loci was 0.11. For diploid O. alpicola, F-statistics were used to assess population heterogeneity and substructuring. The data indicate that, compared with other insects, this high-altitude weevil species is genetically very differentiated (the average F _ST= 0.35). In addition, gene-flow among populations was extremely low (the estimates of N_em from Wright's F _ST and Slatkin private-allele methods were 0.47 and 0.83, respectively). Pairwise linkage disequilibrium (LD) parameters were estimated from zygotic frequencies using Burrows' method. The average rate of significant LD in analysed populations of O. alpicola was about 27%. The pattern of LD over the diploid populations indicates that stochastic factors might be a primary cause of the observed multilocus associations.     

Genetic diversity and population structure of Rhododendron simsii (Ericaceae) as revealed by microsatellite markers

Rhododendron simsii Planch. (Ericaceae) is a valuable horticultural and medicinal plant species. In this study, the genetic diversity of eight wild R. simsii populations from the Dabie Mountains (central China) was investigated with 29 microsatellite markers. The results showed that R. simsii harbored a high level of genetic diversity (H_E: 0.64–0.79; H_O: 0.71–0.94; I = 1.917; h = 0.826), and 84.34% of this genetic variation was maintained within populations, while variation among populations only accounted for 15.66%. The number of alleles ranged from 6 to 11, with an average of 9.069. Heterozygote excess was found, with the mean F_IS and F_IT values of ?0.1739 and 0.0092, respectively. The average value of gene flow (Nm) was 1.3525. Within‐population, genetic diversity (I) ranged from 1.131 to 1.681. Cluster analyses divided the eight populations into two clades: the ‘Ltjiuzihe’ population formed its own cluster, while the other seven populations clustered together. There was a weak negative correlation between genetic and geographic distance. The results are highly relevant for the conservation and sustainable utilization of wild R. simsii germplasm resources in central China.     

Genetic diversity and structure of natural Pinus tabulaeformis populations in North China using amplified fragment length polymorphism (AFLP)

Chinese pine (Pinus tabulaeformis carr.), endemic to China, is a conifer species with extensive and fragmented distribution in North China. In this study, the genetic diversity and structure of 20 natural populations of this species were investigated using amplified fragment length polymorphism (AFLP) markers. A total of 445 fragments were revealed with 8 pairs of primers, 379 (85.17%) of which were polymorphic. A moderate level of genetic diversity was detected at the species level (Shannon's information index I = 0.356, Nei's gene diversity H_E = 0.271) and at the population level (I = 0.219, H_E = 0.206). Most of genetic variation was within populations while a considerable level of genetic differentiation was detected (G_ST = 0.352, Ф_ST = 0.304). The high differentiation could be attributed to the complex and fragmented habitats, and a limited gene flow among populations (N_m = 0.572). The Mantel test indicated that there was significant correlation (r = 0.455, P < 0.001) between Nei's genetic distance and geographical distance among all the populations. The results suggested that proper countermeasures should be taken to prevent the habitat further deterioration and maintain the genetic diversity of this species.     

The molecular population structure of the tall forb Cicerbita alpina (Asteraceae) supports the idea of cryptic glacial refugia in central Europe

There is an ongoing debate about the glacial history of non‐arctic species in central and northern Europe. The two main hypotheses are: (1) postglacial colonization from refugia outside this region; (2) glacial survival in microclimatically favourable sites within the periglacial areas. In order to clarify the glacial history of a boreo‐montane tall forb, we analysed AFLPs from populations of Cicerbita alpina through most of its range (Scandinavia, the mountains of central Europe, the Alps, the Pyrenees and the Balkan Peninsula). We found a major differentiation between the Pyrenean population and all others, supported by principal coordinate, neighbour joining and STRUCTURE analyses. Furthermore, three populations from the central and north‐eastern Alps were genetically distinct from the bulk of populations from Scandinavia, central Europe, the Alps and the Balkan Peninsula. Most populations, including those from central and northern Europe, had moderate to high levels of genetic diversity (mean Shannon index H_Sh = 0.292, mean percentage of polymorphic loci P = 54.1%, mean Nei's gene diversity H = 0.195). The results indicate separate glacial refugia in the Pyrenean region and the Italian Alps. Furthermore, they provide evidence of glacial persistence in cryptic refugia north of the Alps, from where Scandinavia and most of the Alps are likely to have been colonized following deglaciation. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 164 , 142–154.     

Genetic diversity and variance of Stentor coeruleus (Ciliophora: Heterotrichea) inferred from inter-simple sequence repeat (ISSR) fingerprinting

We used inter-simple sequence repeat fingerprinting to analyze the genetic structure of 16 populations of Stentor coeruleus from three lakes and three ponds in China. Using 14 polymorphic primers, a total of 99 discernible DNA fragments were detected, among which 76 (76.77%) were polymorphic, indicating median genetic diversity in these populations. Further, both Nei's gene diversity (h) and Shannon's information index (I) between the different populations revealed a median genetic diversity. At the same time, gene flow was interpreted to be low. The main factors responsible for the median level of diversity and low gene flow within populations are probably due to a low frequency of sexual recombinations. Analysis of molecular variance showed that there was high genetic differentiation among the five water bodies. Both cluster analysis and a nonmetric multidimensional scaling analysis suggested that genotypes isolated from the same locations displayed a higher genetic similarity than those from different ones, separating populations into subgroups according to their geographical locations. However, there is a weak positive correlation between the genetic distance and geographical distance.     

Characterisation and analysis of microsatellite loci in a mangrove species, Avicennia marina (Forsk.) Vierh. (Avicenniaceae)

An enriched microsatellite library of the mangrove species Avicennia marina was constructed, in which 85.8% of the clones contained microsatellite sequences. Of the microsatellite repeat sequences isolated, 55.0% were di-nucleotides, 34.2% were tri-nucleotides, 50.0% were perfect, 24.2% were imperfect, and 15.0% were compound. Four different di-nucleotide repeats were isolated with repeat lengths ranging from 5 to 33; ten different tri-nucleotide repeats were isolated with repeat lengths ranging from 3 to 25. The most common di-nucleotide was the AC/TG repeat; the most common tri-nucleotide was the CCG/GGC repeat. Sixteen microsatellite sequences were selected for primer design, and 6 primers were selected to investigate the polymorphism detected among 15 individuals of A. marina from three natural populations in Australia. A total of 40 alleles were detected at 6 microsatellite loci. The number of alleles per microsatellite locus ranged from 5 to 13. On average, 7 alleles were detected per locus. All microsatellite loci showed high levels of gene diversity (heterozygosity), with values ranging from 0.53 to 0.88; the mean value of gene diversity was 0.70. Microsatellite loci were also tested for conservation across Avicennia species. There was a decline in amplification success with increasing divergence between Avicennia species. The results indicate that microsatellites are abundant in the Avicennia genome and can be valuable genetic markers for assessing the effects of deforestation and forest fragmentation in mangrove communities, which is an important issue for mangrove conservation and afforestation schemes. Received: 8 June 1999 / Accepted: 21 September 1999