Development of polymorphic microsatellite markers for Dalbergia nigra (Papilionoideae), an endangered tree from the Brazilian Atlantic Forest

Dalbergia nigra is an endangered tree restricted to the Brazilian Atlantic Forest. Ten polymorphic microsatellite loci were isolated and characterized in 47 trees from two populations. The total number of alleles per locus ranged from three to 12 alleles. The levels of observed and expected heterozygosities ranged from 0.304 to 0.740 and from 0.278 to 0.872, respectively. Significant deviations from Hardy-Weinberg were detected for only three loci in each population. No pair of loci exhibited significant linkage disequilibrium. These microsatellites provide an efficient tool to investigate genetic structure in forest remnants with the purpose of conservation of this species.     

Diversity and genetic connectivity among populations of a threatened tree (<Emphasis Type="Italic">Dalbergia nigra</Emphasis>) in a recently fragmented landscape of the Brazilian Atlantic Forest

In this study we evaluated the influence of recent landscape fragmentation on the dynamics of remnant fragments from the Brazilian Atlantic Forest. This biome is one of the richest in the world and has been extensively deforested and fragmented. We sampled five populations of the threatened Dalbergia nigra, a tree endemic to the Brazilian Atlantic Forest, two located in a large reserve of continuous forest and three in fragments of different sizes and levels of disturbance. In order to assess historical changes, considering the longevity of the analyzed species, 119 adults and 116 saplings were genotyped for six microsatellite loci. Lower levels of genetic diversity were found in the most impacted fragments when compared to the most preserved population located inside the reserve, and there was significant genetic structure among the populations studied (pairwise F _ST = 0.031–0.152; pairwise D _EST = 0.039–0.301). However, genetic structure among saplings (F _ST = 0.056; D _EST = 0.231) was significantly lower than among adults (F _ST = 0.088; D _EST = 0.275). Estimates of contemporary gene flow based on assignment tests corroborated this result, suggesting that fragmentation led to an increase in gene flow. This connectivity among remnant fragments could mitigate the loss of genetic diversity through a metapopulation dynamic, but the high rate of habitat loss and the unknown long-term genetic effects add uncertainty. These results, taken together with the presence of private alleles in disturbed populations, highlight the importance of preserving the extant fragments.     

Isolation and characterization of microsatellite loci in the blue manakin,Chiroxiphia caudata (Aves,Pipridae)

We designed primers for amplifying 11 polymorphic microsatellite loci in the blue manakin, Chiroxiphia caudata, a neotropical passerine bird which inhabits a critically endangered tropical ecosystem, the Brazilian Atlantic forest. Based on genotypes from 24 individuals from a single population, we detected between four and 22 alleles per locus with observed heterozygosities ranging from 0.54 to 0.92. These highly variable loci will be useful for determining levels of population differentiation and assessing the impact of habitat fragmentation on levels of genetic variation in isolated populations of these birds.     

PERMANENT GENETIC RESOURCES: Characterization of eight microsatellite loci in the woolly mouse opossum, Micoureus paraguayanus, isolated from Micoureus demerarae

Eight novel microsatellite markers were isolated from the woolly mouse opossum from the Amazon Forest in Peru, Micoureus demerarae, using a partial genomic DNA library and an enrichment protocol. These loci were polymorphic in M. demerarae and Micoureus paraguayanus populations from the Atlantic Forest in Brazil with the number of alleles ranging from two to 23. Those eight loci plus another five already described for M. paraguayanus will allow for the evaluation of genetic diversity of populations from the 'Rio Doce' Park, one of the last Atlantic Forest fragments in Minas Gerais state, Brazil.     

Genetic assessment of the Atlantic Forest bristle porcupine, Chaetomys subspinosus (Rodentia: Erethizontidae), an endemic species threatened with extinction

The bristle-spined porcupine, Chaetomys subspinosus, an endemic rodent from Atlantic Forest, was considered to be abundant in the recent past, but population reductions due to habitat loss and expansion of human activities caused this species to be included in the "vulnerable" category of the World Conservation Union Red List. We performed the first genetic assessment in natural populations of this focal species along its geographical distribution. Thirty-five non-invasive samples (hair) were collected from three natural populations in the Brazilian States of Sergipe, Bahia and Espírito Santo. Genetic similarity obtained by Jaccard's index, based on dominant RAPD and ISSR markers, varied between 25 and 100%. Four clusters, mainly coincident with the geographical distribution of the populations, were observed. Analysis of molecular variance based on 47 polymorphic loci showed that there was 15.99% genetic variability among populations and 84.01% within populations. The estimated genetic structure among populations (Φ(ST)) was 0.16. The populations may have formed a continuum along the past distribution of the Atlantic rainforest but historical events of human occupation resulted in recent divergence among sampled populations.     

Isolation and characterization of microsatellite loci for Hymenaea courbaril and transferability to Hymenaea stigonocarpa, two tropical timber species

Hymenaea courbaril is a tropical timber species, intensely exploited and found in the Amazon, Atlantic Forest and Brazilian Cerrado biome. Nine highly polymorphic microsatellite loci were developed from a genomic library enriched for AG/TC repeats. In a total of 41 individuals, from two natural populations, seven to 13 alleles per locus were detected and expected heterozygosity ranged from 0.75 to 0.90. Seven loci were effectively transferred to Hymenaea stigonocarpa. High levels of polymorphism make the present primers useful for population genetic studies and are a powerful tool to investigate mating system, gene flow and spatial genetic structure.     

Development and characterization of microsatellite markers for Psychotria tenuinervis (Rubiaceae), a shrub species from the Atlantic forest, and primers transferability from Coffea

The shrub species Psychotria tenuinervis (Rubiaceae) is native to the Brazilian Atlantic forest and is largely found within natural and disturbed forest fragments. Aiming to develop studies on population genetic structure of forest fragment species, eigth microsatellite markers were developed for P. tenuinervis. Also, 15 loci already developed for Coffea (Rubiaceae) were tested for transferability to this species. We utilized 45 individuals from natural populations of three different fragments-anthropic edge, interior fragment and natural edge, within the Brazilian Atlantic forest. The average number of alleles per locus was 2.5 (two–four alleles/locus). These loci will be useful for future population genetic studies aiming to the conservation and management of this species.     

Phylogeography of the endangered rosewood Dalbergia nigra (Fabaceae): insights into the evolutionary history and conservation of the Brazilian Atlantic Forest

The Brazilian rosewood (Dalbergia nigra) is an endangered tree endemic to the central Brazilian Atlantic Forest, one of the world''s most threatened biomes. The population diversity, phylogeographic structure and demographic history of this species were investigated using the variation in the chloroplast DNA (cpDNA) sequences of 185 individuals from 19 populations along the geographical range of the species. Fifteen haplotypes were detected in the analysis of 1297 bp from two non-coding sequences, trnV-trnM and trnL. We identified a strong genetic structure (F_ST=0.62, P<0.0001), with a latitudinal separation into three phylogeographic groups. The two northernmost groups showed evidence of having maintained historically larger populations than the southernmost group. Estimates of divergence times between these groups pointed to vicariance events in the Middle Pleistocene (ca. 350 000–780 000 years ago). The recurrence of past climatic changes in the central part of the Atlantic forest, with cycles of forest expansion and contraction, may have led to repeated vicariance events, resulting in the genetic differentiation of these groups. Based on comparisons among the populations of large reserves and small, disturbed fragments of the same phylogeographic group, we also found evidence of recent anthropogenic effects on genetic diversity. The results were also analysed with the aim of contributing to the conservation of D. nigra. We suggest that the three phylogeographic groups could be considered as three distinct management units. Based on the genetic diversity and uniqueness of the populations, we also indicate priority areas for conservation.     

Maintenance of genetic differentiation across a transition zone in the sea: discordance between nuclear and cytoplasmic markers

To investigate the origin and maintenance of the genetic discontinuity between Atlantic and Mediterranean populations of the common sea bass (Dicentrarchus labrax) we analysed the genetic variation at a fragment of mitochondrial cytochrome b sequence for 18 population samples. The result were also compared with new or previously published microsatellite data. Seven mitochondrial haplotypes and an average nucleotidic divergence of 0.02 between Atlantic and Mediterranean populations that matches a Pleistocene allopatric isolation were found. The frequency variation at the cytochrome b locus was many times greater between Atlantic and Mediterranean populations (theta(C) = 0.67) than at microsatellite loci (theta(N)= 0.02). The examination of the different evolutionary forces at play suggests that a sex-biased hybrid breakdown is a likely explanation for part of the observed discrepancy between mitochondrial and nuclear loci. In addition, an analysis is made of the correlation between microsatellite loci points towards the possible existence of a hybrid zone in samples from the Alboran Sea.     

Contrasting levels of genetic diversity between the historically rare orchid Cypripedium japonicum and the historically common orchid Cypripedium macranthos in South Korea

Many terrestrial orchids are historically rare and occur in small, spatially isolated populations. Theory predicts that such species will harbour low levels of genetic variation within populations and will exhibit a high degree of population genetic divergence, primarily as a result of genetic drift. If the origin of the present‐day populations is relatively recent from the same genetically depauperate source population, a complete lack of genetic differentiation between conspecific populations is expected. If a terrestrial orchid was historically common with moderate or high levels of genetic diversity, but has experienced more recent anthropogenic disturbance as a result of over‐collection, it would still exhibit initial levels of genetic variation within populations and a low degree of genetic divergence between populations. To test these predictions, we examined the genetic diversity in six populations (N = 131) of the historically and currently rare Cypripedium japonicum and in four populations (N = 94) of the historically common but now rare C. macranthos from South Korea. Fourteen putative allozyme loci resolved from eight enzyme systems revealed no variation either within or among populations of C. japonicum, which supports the first prediction. In contrast, populations of C. macranthos harboured high levels of genetic variation (mean percentage of polymorphic loci %P = 46.7; mean expected heterozygosity H_e = 0.185) and exhibited a low degree of population genetic divergence (G_ST = 0.059), supporting the second prediction. The lack of genetic variation both within and among conspecific populations of C. japonicum may suggest that populations originated from the same genetically depauperate ancestral population. The high levels of genetic diversity maintained in populations of C. macranthos suggest that the collection‐mediated decrease in the number of individuals is still too recent for long‐term effects on genetic variation. Based on current demographic and genetic data, in situ and ex situ conservation strategies should be provided to preserve genetic variation and to ensure the long‐term survival of the two species in the Korean Peninsula. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160 , 119–129.     

Genetic differentiation of pedunculate oak Quercus robur L. in the European part of Russia based on RAPD markers

Using randomly amplified polymorphic DNA markers (RAPD), genetic variation and differentiation in four populations of pedunculate oak Quercus robur L. were examined. The populations occupy a large part of the Quercus robur range in the European Russia (Voronezh and Novgorod oblasts; Republics of Mordovia and Bashkortostan). With each of six random primers (A02, A09, A17, B01, B08, B11), 96 DNA samples were analyzed by PCR. In all, 48 putative polymorphic RAPD loci were detected. We failed to reveal population-specific DNA fragments for any primer although the frequencies of 14 fragments were significantly different among populations. The oak populations studied exhibited high variability: 73-90% of genes were polymorphic and the effective allele number was about 1.4. The total genetic variation varied from 0.202 (Vor) to 0.245 (Nov), which corresponded to the estimates for populations of this species from Central and Western Europe. The populations examined showed low among-population differentiation (GST = 0.098); gene flow Nem was 4.61. The proportion of among-population variation of the RAPD loci studied accounted for 7% of the total variability; more than 93% of the total variability was explained by individual and within--population variation.     

The historical bridge between the Amazon and the Atlantic Forest of Brazil: a study of molecular phylogeography with small mammals

Abstract Aim To examine how the genetic diversity of selected taxa of forest‐dwelling small mammals is distributed between and within the major rain forest domains of Amazonia and Atlantic Forest and the intervening interior forests of Brazil, as inferred by the relationships between gene genealogies and geography. I also addressed the historical importance of the central Brazilian forests in connecting Amazon and Atlantic Forest populations of rodents and marsupials. Methods I evaluated variation in the mitochondrial cytochrome b gene to estimate the levels of sequence divergence between those taxa occurring throughout the Amazon, Atlantic Forest, and forests in the Cerrado and Caatinga regions. I inferred the hierarchical relationships between haplotypes, populations and formal taxa using the cladistic approach of maximum parsimony. I compared areas and the clades identified by superimposing cladograms on the geographical distribution of samples. The degree of concordance both in phylogeny and the depth of the nodes in these phylogenies, in addition to patterns of geographical distribution of clades, permitted me to make inferences on how, when and where the taxa differentiated. Results Sequence similarity is often greater between samples from the Atlantic Forest and either Amazon or central Brazilian forests than it is within each of the two rain forest domains. The Atlantic Forest clades are either not reciprocally monophyletic or are the sister group to all the other clades. There is some indication of northern and southern components in the Atlantic Forest. Given the geographical distribution of clades and the relatively deep levels of divergence, the central Brazilian area does not behave as a separate region but is complementary to either Amazon or Atlantic Forest. Patterns of area relationships differ across taxa, suggesting that different processes and/or historic events affected the diversification within each lineage. Main conclusions The Amazon and the Atlantic forests are not exclusive in terms of their small mammal faunas; both overlap broadly with taxa occurring in gallery forests and dry forests in central Brazil. Central Brazilian forests are an integral part of the evolutionary scenario of lowland small mammals, playing an important role as present and past habitats for rain forest species. Therefore, representatives from this area should always be included in analyses of the evolutionary history of lowland rain forest faunas. The incongruence of branching patterns among areas is in agreement with recent results presented for Neotropical passerine birds and indicates that a single hypothesis of Neotropical area relationships is unlikely. These findings reinforce the idea that speciation in the Neotropics will not be explained by any single model of vicariance or climatic changes.     

Using dung beetles to evaluate the effects of urbanization on Atlantic Forest biodiversity

We used dung beetles to evaluate the impact of urbanization on insect biodiversity in three Atlantic Forest fragments in Londrina, Paraná, Brazil. This study provides the first empirical evidence of the impact of urbanization on richness, abundance, composition and guild structure of dung beetle communities from the Brazilian Atlantic Forest. We evaluated the community aspects (abundance, richness, composition and food guilds) of dung beetles in fragments with different degrees of immersion in the urban matrix using pitfall traps with four alternative baits (rotten meat, rotten fish, pig dung and decaying banana). A total of 1 719 individuals were collected, belonging to 29 species from 11 genera and six Scarabaeinae tribes. The most urban‐immersed fragment showed a higher species dominance and the beetle community captured on dung presented the greatest evenness. The beetle communities were distinct with respect to the fragments and feeding habits. Except for the dung beetle assemblage in the most urbanized forest fragment, all others exhibited contrasting differences in species composition attracted to each bait type. Our results clearly show that the degree of urbanization affects Atlantic Forest dung beetle communities and that the preservation of forest fragments inside the cities, even small ones, can provide refuges for Scarabaeinae.     

GEOGRAPHIC DISTRIBUTION OF MOLECULAR VARIANCE WITHIN THE BLUE MARLIN (MAKAIRA NIGRICANS): A HIERARCHICAL ANALYSIS OF ALLOZYME,SINGLE-COPY NUCLEAR DNA,AND MITOCHONDRIAL DNA MARKERS

This study presents a comparative hierarchical analysis of variance applied to three classes of molecular markers within the blue marlin (Makaira nigricans). Results are reported from analyses of four polymorphic allozyme loci, four polymorphic anonymously chosen single-copy nuclear DNA (scnDNA) loci, and previously reported restriction fragment length polymorphisms (RFLPs) of mitochondrial DNA (mtDNA). Samples were collected within and among the Atlantic and Pacific Oceans over a period of several years. Although moderate levels of genetic variation were detected at both polymorphic allozyme (H = 0.30) and scnDNA loci (H = 0.37), mtDNA markers were much more diverse (h = 0.85). Allele frequencies were significantly different between Atlantic and Pacific Ocean samples at three of four allozyme loci and three of four scnDNA loci. Estimates of allozyme genetic differentiation (θ_O) ranged from 0.00 to 0.15, with a mean of 0.08. The θ_O values for scnDNA loci were similar to those of allozymes, ranging from 0.00 to 0.12 with a mean of 0.09. MtDNA RFLP divergence between oceans (θ_O = 0.39) was significantly greater than divergence detected at nuclear loci (95% nuclear confidence interval = 0.04–0.11). The fourfold smaller effective population size of mtDNA and male-mediated gene flow may account for the difference observed between nuclear and mitochondrial divergence estimates.     

Concordance of genetic divergence among sockeye salmon populations at allozyme, nuclear DNA, and mitochondrial DNA markers

Abstract.— We examined genetic variation at 21 polymorphic allozyme loci, 15 nuclear DNA loci, and mitochondrial DNA in four spawning populations of sockeye salmon ( Oncorhynchus nerka ) from Cook Inlet, Alaska, to test for differences in the patterns of divergence among different types of markers. We were specifically interested in testing the suggestion that natural selection at allozyme loci compromises the effectiveness of these markers for describing the amount and patterns of gene flow among populations. We found concordance among markers in the amount of genetic variation within and among populations, with the striking exception of one allozyme locus ( sAH ), which exhibited more than three times the amount of among-population differentiation as other loci. A consideration of reports of discordance between allozymes and other loci indicates that these differences usually result from one or two exceptional loci. We conclude that it is important to examine many loci when estimating genetic differentiation to infer historical amounts of gene flow and patterns of genetic exchange among populations. It is less important whether those loci are allozymes or nuclear DNA markers.     

Allozyme,chloroplast DNA and RAPD markers for determining genetic relationships between Abies alba and the relic population of Abies nebrodensis

Allozyme, chloroplast (cpDNA) and random amplified polymorphic DNA (RAPD) markers have been used to estimate genetic and taxonomic relationships among different populations of Abies alba and the relic population of A. nebrodensis. Twelve isozyme gene loci, as well as restriction fragment length polymorphism (RFLP) at cpDNA spacer regions between t-RNA genes were analysed. Moreover, a set of 60 random sequence 10-mer primers were tested. Over all isozyme loci, evident differences in allele frequencies among A. nebrodensis and A. alba populations were found, particularly at 2 loci, phosphoglucose isomerase (Pgi-a) and shikimate dehydrogenase (Skd-a). More than 10% of the total genetic diversity was due to differences among populations. High values of genetic distances among populations were also found. Out of the 60 primers tested, 12 resulted in a polymorphic banding pattern both within and among populations. A total of 84 RAPD fragments were produced by the 12 selected primers. A phenogram of relationships among populations was constructed based on RAPD band sharing: the differentiation of the A. nebrodensis population was evident. The analysis of molecular variance (AMOVA) was used to apportion the variation among individuals within populations and among populations. There was considerable variation within each population: even so, genetic divergence was found among populations. This pattern of genetic variation was very different from that reported for inbred species. Identical cpDNA amplification and restriction patterns were observed among all the individuals sampled from the populations. Taken together, the results of allozyme and RAPDs show a clear differentiation among A. nebrodensis and A. alba populations and provide support for their classification into two different taxonomic groups.     

Genetic diversity within and among 11 juvenile populations of green alder (Alnus crispa) in Canada

Germinated seeds from 11 populations of green alder [ Alnus crispa (Ait.) Pursh] sampled in four Canadian provinces were analysed for electrophoretically demonstrable diversity of 10 enzymes encoded by 15 structural loci. Of these, nine were polymorphic, and on average, 52% of the loci per population were polymorphic. Assuming a diploid model of expression, average level of expected heterozygosity was 0.11 with nearly all populations in Hardy-Weinberg equilibrium for the set of polymorphic loci analysed. No significant inbreeding and associated subpopulation structuring were noted. Rates of gene flow appeared high within and among populations. Although little divergence was observed among populations, genetic and geographical distances between populations were related. Discriminant and cluster analyses revealed a pattern of genetic variation associated with geography. Populations from northern Quebec were poorly differentiated, whereas western populations from Alberta exhibited a larger degree of genetic differentiation. Introgresive hybridization with the sympatric species Alnus sinuata (Regel) Rydberg and partial isolation in the West are suggested as an explanation for this larger differentiation. The occurrence and significance of rare alleles is discussed in relation to the importance of geographical distance in the process of population differentiation in this species.     

Development and characterization of microsatellite markers for Echinopsis rhodotricha and cross-amplification in other species of Cactaceae

Located in central South America, the Chaco is a large subtropical dry forest characterized by plants with xeromorphic features, including numerous cacti. In the Brazilian Chaco at the northeasternmost part of the Chaquenian region, Echinopsis rhodotricha (Cactaceae, Trichocereeae) is one of the species threatened by intense deforestation caused by expanding pastureland. This study characterizes the microsatellite loci isolated from E. rhodotricha and cross-amplification in thirteen other cactus species. Twelve microsatellite loci were developed from an enriched genomic library. Eight of these were polymorphic and characterized in 48 individuals from three E. rhodotricha populations. The loci showed a mean of 3.2 alleles per locus and overall levels of expected and observed heterozygosities ranging from 0.00 to 0.83 and 0.00 to 0.66, respectively. Five loci showed significant departures from the Hardy–Weinberg equilibrium and also exhibited signs of null alleles. Cross-amplification in other Cactaceae species was successful, ranging from one (Ferocactus latispinus, Cacteae and Harrisia adscendens, Trichocereeae) to twelve loci (Echinopsis calochlora, Trichocereeae). The development of these microsatellite markers will contribute to investigations of population structure, genetic diversity, and gene flow in E. rhodotricha populations, as well as in other cactus species, providing information useful for the creation and delimitation of conservation areas in the Brazilian Chaco region.     

Informative microsatellites for genetic population studies of black-faced lion tamarins (Leontopithecus caissara)

Leontopithecus caissara is a critically endangered primate species from the Brazilian Atlantic Forest. Nineteen microsatellite loci, previously developed for congeneric species, were tested with 34 L. caissara individuals from Superagüi Island. Of the 19 loci, 17 (89.4%) produced robust alleles, nine (47.4%) of these proved to be polymorphic, with a total of 23 alleles and an average of 2.56 alleles per locus. Expected and observed heterozygosity averaged 0.483 and 0.561, respectively. The exclusion power for identifying the first parent of an arbitrary offspring was 0.315 over all loci. The results thus indicate both the usefulness and limitations of these nine microsatellite loci in the genetic analysis of L. caissara, as well as their potentiality for genetic investigation in other congeneric species.     

The Conservation Value of Small Fragments For Atlantic Forest Reptiles

Loss and fragmentation of natural habitats are key contributors to the decline of populations and impoverishment of biological communities. The response to these disturbances can vary substantially among taxa and depends on spatial metrics of habitat fragments and the surrounding landscape. Herein we test how fragment area, shape, isolation, and matrix quality affect reptile richness, abundance, and occurrence in Brazilian Atlantic Forest fragments, a biodiversity hotspot with a poorly studied reptile fauna. We used 23 forest fragments, ranging from 2 to 30 hectares, surrounded by different matrix types, including sugarcane crop fields, cattle ranching, subsistence farmlands and rural communities. Species richness, total reptile abundance, population abundance, and occurrence probability of many species decreased with fragment area. Model selection suggested that fragment area is the main predictor of both richness and abundance, but matrix quality as well as fragment shape are also important predictors. For population abundance and occurrence probability, fragment area and proximity were the most important predictors followed by fragment shape and matrix quality, but the strength and even the sign of predictors varied substantially among species. We highlight that the value of small fragments should not be neglected for the conservation of Atlantic Forest reptiles.