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.     

Fine-scale spatial genetic structure of Dalbergia nigra (Fabaceae), a threatened and endemic tree of the Brazilian Atlantic Forest

The Atlantic Forest is one of the most diverse ecosystems in the world and considered a hotspot of biodiversity conservation. Dalbergia nigra (Fabaceae) is a tree endemic to the Brazilian Atlantic Forest, and has become threatened due to overexploitation of its valuable timber. In the present study, we analyzed the genetic diversity and fine-scale spatial genetic structure of D. nigra in an area of primary forest of a large reserve. All adult individuals (N = 112) were sampled in a 9.3 ha plot, and genotyped for microsatellite loci. Our results indicated high diversity with a mean of 8.6 alleles per locus, and expected heterozygosity equal to 0.74. The co-ancestry coefficients were significant for distances among trees up to 80 m. The Sp value was equal to 0.017 and indirect estimates of gene dispersal distances ranged from 89 to 144 m. No strong evidence of bottleneck or effects of human-disturbance was found. This study highlights that long-term efforts to protect a large area of Atlantic Forest have been effective towards maintaining the genetic diversity of D. nigra. The results of this study are important towards providing a guide for seed collection for ex-situ conservation and reforestation programmes of this threatened 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.     

Genetic differentiation among populations of an oceanic island: The case of Metrosideros boninensis, an endangered endemic tree species in the Bonin Islands

Conservation of endemic species on oceanic islands is an essential issue for biodiversity conservation. Metrosideros boninensis (Myrtaceae) is an endangered tree species endemic to the Bonin Islands of the western North Pacific Ocean. This species is considered to be extremely rare with less than 400 adult individuals, a number that has fluctuated between the 1880s and 1980s through human influence. We analyzed the genetic diversity and genetic structure of this species using amplified fragment length polymorphism markers and microsatellite markers. Genetic diversity of M. boninensis was extremely low compared to related taxa and similar endemic species from small islands. This low genetic diversity might be attributed to a stepwise colonization process with repeated founder bottlenecks in the dispersal pathway to the Bonin Islands. Populations of M. boninensis showed significant genetic differentiation and isolation by distance over a small geographical scale, despite the fact that this species should have extensive gene dispersal ability. This genetic differentiation might be caused by limited gene flow via pollen and seed among populations and genetic drift amid a small number of remnant individuals. Taken together, these findings suggest that the genetic diversity and connectivity of tree populations on islands are more vulnerable to habitat fragmentation than previously thought. We offer some recommendations for management to ameliorate habitat fragmentation and biological invasion.     

Rampant drift in artificially fragmented populations of the endangered tidewater goby (Eucyclogobius newberryi)

Habitat fragmentation and its genetic consequences are a critically important issue in evaluating the evolutionary penalties of human habitat modification. Here, we examine the genetic structure and diversity in naturally subdivided and artificially fragmented populations of the endangered tidewater goby (Eucyclogobius newberryi), a small fish restricted to discrete coastal lagoons and estuaries in California, USA. We use five naturally fragmented coastal populations from a 300‐ km spatial scale as a standard to assess migration and drift relative to eight artificially fragmented bay populations from a 30‐ km spatial scale. Using nine microsatellite loci in 621 individuals, and a 522‐base fragment of mitochondrial DNA control region from 103 individuals, we found striking differences in the relative influences of migration and drift on genetic variation at these two scales. Overall, the artificially fragmented populations exhibited a consistent pattern of higher genetic differentiation and significantly lower genetic diversity relative to the naturally fragmented populations. Thus, even in a species characterized by habitat isolation and subdivision, further artificial fragmentation appears to result in substantial population genetic consequences and may not be sustainable.     

Genetic structure and conservation of Mountain Lions in the South-Brazilian Atlantic Rain Forest

The Brazilian Atlantic Rain Forest, one of the most endangered ecosystems worldwide, is also among the most important hotspots as regards biodiversity. Through intensive logging, the initial area has been reduced to around 12% of its original size. In this study we investigated the genetic variability and structure of the mountain lion, Puma concolor. Using 18 microsatellite loci we analyzed evidence of allele dropout, null alleles and stuttering, calculated the number of allele/locus, PIC, observed and expected heterozygosity, linkage disequilibrium, Hardy-Weinberg equilibrium, F(IS), effective population size and genetic structure (MICROCHECKER, CERVUS, GENEPOP, FSTAT, ARLEQUIN, ONESAMP, LDNe, PCAGEN, GENECLASS software), we also determine whether there was evidence of a bottleneck (HYBRIDLAB, BOTTLENECK software) that might influence the future viability of the population in south Brazil. 106 alleles were identified, with the number of alleles/locus ranging from 2 to 11. Mean observed heterozygosity, mean number of alleles and polymorphism information content were 0.609, 5.89, and 0.6255, respectively. This population presented evidence of a recent bottleneck and loss of genetic variation. Persistent regional poaching constitutes an increasing in the extinction risk.     

High genetic differentiation among remnant populations of the endangered Caesalpinia echinata Lam. (Leguminosae–Caesalpinioideae)

Forest fragments along the Atlantic coastland of Brazil have been highly impacted by extensive human activities for the last 400 years. Caesalpinia echinata (Leguminosae– Caesalpinioideae), brazilwood, was overexploited during this period due to its economical importance as a dye. As a result, the species has become endangered and today its total population size is very restricted. We have assessed the distribution of genetic variation between five natural populations of brazilwood by means of RAPD (random amplified polymorphic DNA) markers. Of the total genetic variability, 28.5% was attributable to differences between two geographical groups, 29.6% to population differences within groups and 42.0% to individual differences within populations. The high level of population differentiation observed is in contrast to that expected for a primarily outcrossed woody perennial plant, and suggests that there may be a degree of inbreeding. Our results are in agreement with previous studies which postulated that C. echinata has always occurred in clumps, being common in some places but rare in between. From a conservation point of view, different populations representing different regions should be protected and, yet, plants with different origins should not be synthesized into populations in a recovery process at the risk of loss and dilution of genetic information. This study demonstrates that RAPD markers were effective in establishing a clear correlation between genetic and geographical distance and in identifying areas of maximum diversity, and may be used as an initial approach to assess the partitioning of genetic variation in this endangered species.     

Microsatellite analysis reveals interpopulation differentiation and gene flow in the endangered tree Changiostyrax dolichocarpa (Styracaceae) with fragmented distribution in central China

Polymorphic simple sequence repeat (SSR) markers were used to investigate the impact of habitat fragmentation on the population structure and gene flow of Changiostyrax dolichocarpa, a critically endangered tree in central China. Intrapopulation genetic diversity, population structure and gene flow in the five extant populations of this species were analysed by eight SSR markers. Intrapopulation genetic diversity results suggest that C. dolichocarpa remnants maintained a relatively high degree of genetic diversity despite severe fragmentation. Low genetic differentiation among populations was found based on Wright's F(ST) and amova analysis. Both the F(ST)-based estimate and private allele method revealed high historical gene flow among the remnant populations. Recent immigrants, detected by assignment tests, tend to decrease from the grandparent generation to the current generation. The potentially highly restricted current gene flow among fragments may render the fragmented populations of C. dolichocarpa at a higher risk of local extinction several generations after fragmentation. Both in situ and ex situ conservation management for the remnant populations of C. dolichocarpa are therefore urgently needed to rescue remaining genetic diversity.     

Genetic structure of red-handed howler monkey populations in the fragmented landscape of Eastern Brazilian Amazonia

We genotyped 15 microsatellite loci in order to evaluate the effects of habitat fragmentation, caused by flooding of the Tucuruí reservoir, on the genetic structure of Alouatta belzebul in eastern Amazonia. The analysis included two populations sampled in 1984, representing both margins of the Tocantins river, and three populations sampled 18 years later. Minimal differences in the diversity levels between present-day (Ho = 0.62-0.69 and A(R) = 6.07-7.21) and pre-flooding (Ho = 0.60-0.62 and A (R) = 6.27-6.77) populations indicated there was no significant loss of genetic variability, possibly because of successful management strategies applied during the flooding. The changes observed were limited to shifts in the composition of alleles, which presumably reflect the admixture of subpopulations during flooding. Given this, there were significant differences in the Rst values (p = 0.05) in all but one between-site comparison. Both present-day and original populations showed a deficit of heterozygotes, which suggests that this may be typical of the species, at least at a local level, perhaps because of specific ecological characteristics. The relatively large number of private alleles recorded in all populations may be a consequence of the Wahlund effect resulting from population admixture or a process of expansion rather than the loss of rare alleles through genetic drift. Additionally, the levels of genetic variability observed in this study were higher than those reported for other species of Neotropical primates, suggesting good fitness levels in these A. belzebul populations. Regular genetic monitoring of remnant populations, especially on islands, should nevertheless be an integral component of long-term management strategies.     

Phylogeography of the Bothrops jararaca complex (Serpentes: Viperidae): past fragmentation and island colonization in the Brazilian Atlantic Forest

The Brazilian Atlantic Forest is one of the world's major biodiversity hotspots and is threatened by a severe habitat loss. Yet little is known about the processes that originated its remarkable richness of endemic species. Here we present results of a large-scale survey of the genetic variation at the mitochondrial cytochrome b gene of the pitviper, jararaca lancehead (Bothrops jararaca), and two closely related insular species (Bothrops insularis and Bothrops alcatraz), endemic of this region. Phylogenetic and network analyses revealed the existence of two well-supported clades, exhibiting a southern and a northern distribution. The divergence time of these two phylogroups was estimated at 3.8 million years ago, in the Pliocene, a period of intense climatic changes and frequent fragmentation of the tropical rainforest. Our data also suggest that the two groups underwent a large size expansion between 50,000 and 100,000 years ago. However, the southern group showed a more marked signal of population size fluctuation than the northern group, corroborating evidences that southern forests may have suffered a more pronounced reduction in area in the late Pleistocene. The insular species B. alcatraz and B. insularis presented very low diversity, each one sharing haplotypes with mainland individuals placed in different subclades. Despite their marked morphological and behavioural uniqueness, these two insular species seem to have originated very recently and most likely from distinct costal B. jararaca populations, possibly associated with late Pleistocene or Holocene sea level fluctuations.     

Genetic heterogeneity among pelagic egg samples and variance in reproductive success in an endangered freshwater fish,Hybognathus amarus (Cyprinidae)

Synopsis A sweepstakes–mismatch process whereby reproduction is poorly coordinated with appropriate resources for larval development and recruitment can result in large variance in reproductive success among individuals and spawning aggregations. This process has been proposed to explain low ratio of genetic effective population size (N_e) to adult census size (N) ratios in marine species with high fecundity, pelagic spawning, and extensive mortality in early life stages. This process is also hypothesized to also account for very low N_e/N (≈ 0.001) observed in the federally endangered Rio Grande silvery minnow, Hybognathus amarus. This species is a freshwater fish that shares life-history features with marine pelagic spawners. We tested two key predictions of the sweepstakes–mismatch hypothesis using molecular data: (i) that temporally distinct samples of eggs differ in genetic composition and, (ii) that egg samples do not comprise a random subset of potential adult breeders. We present genetic data that supports both predictions and that are consistent with the hypothesis that high variance in reproductive success among adult breeders is an important factor that lowers N_e/N in H. amarus. This study highlights the importance of understanding the interaction of early life history and fragmentation in devising conservation plans for endangered aquatic organisms.     

Genetic structure and AFLP variation of remnant populations in the rare plant Pedicularis palustris (Scrophulariaceae) and its relation to population size and reproductive components

We investigated plant reproduction in relation to genetic structure, population size, and habitat quality in 13 populations of the rare biennial plant Pedicularis palustris with 3-28500 flowering individuals. We used AFLP (amplified fragment length polymorphism) profiles to analyze genetic similarities among 129 individuals (3-15 per population). In a cluster analysis of genetic similarities most individuals (67%) were arranged in population-specific clusters. Analysis of molecular variance indicated significant genetic differentiation among populations and among and within subpopulations (P < 0.001). Gene flow (N(e) m) was low (0.298). On average, plants produced 55 capsules, 17 seeds per fruit, and 42 seedlings in the following growing season. The number of seeds per capsule was independent of population size and of genetic variability. In contrast, the number of capsules per plant (P < 0.05) and the number of seedlings per plant (P < 0.05) were positively correlated with population size. The relation between population size and the number of seeds per plant was not significant (P = 0.075). The number of capsules and of seeds and seedlings per plant (P < 0.01) were positively correlated with genetic variability. Genetic variability was independent of actual population size, suggesting that historical population processes have to be taken into account, too. Stepwise multiple regressions revealed additional significant relationships of habitat parameters (soil pH, C:N ratio), vegetation composition, and standing crop on reproductive components. We conclude that populations of P. palustris are genetically isolated and that reproductive success most likely is influenced by population size, genetic variability, and habitat quality. Management strategies such as moderate grazing, mowing, and artificial gene flow should endeavor to increase population size as well as genetic variation.     

Fine-scale spatial genetic structure and gene flow in a small, fragmented population of Sinojackia rehderiana (Styracaceae), an endangered tree species endemic to China

Populations of Sinojackia rehderiana are highly threatened and have small and scattered distribution due to habitat fragmentation and human activities. Understanding changes in genetic diversity, the fine-scale spatial genetic structure (SGS) at different life stages and gene flow of S. rehderiana is critical for developing successful conservation strategies for fragmented populations of this endangered species. In this study, 208 adults, 114 juveniles and 136 seedlings in a 50 × 100-m transect within an old-growth forest were mapped and genotyped using eight microsatellite makers to investigate the genetic diversity and SGS of this species. No significant differences in genetic diversity among different life-history stages were found. However, a significant heterozygote deficiency in adults and seedlings may result from substantial biparental inbreeding. Significant fine-scale spatial structure was found in different life-history stages within 19 m, suggesting that seed dispersal mainly occurred near a mother tree. Both historical and contemporary estimates of gene flow (13.06 and 16.77 m) indicated short-distance gene dispersal in isolated populations of S. rehderiana. The consistent spatial structure revealed in different life stages is most likely the result of limited gene flow. Our results have important implications for conservation of extant populations of S. rehderiana. Measures for promoting pollen flow should be taken for in situ conservation. The presence of a SGS in fragmented populations implies that seeds for ex situ conservation should be collected from trees at least 19-m apart to reduce genetic similarity between neighbouring individuals.     

Phenological variation within and among populations of Plathymenia reticulata in Brazilian Cerrado, the Atlantic Forest and transitional sites

BACKGROUND AND AIMS: Plathymenia reticulata (Leguminosae) is a Brazilian tree that occurs in two biomes: Cerrado, a woody savanna vegetation, and the Atlantic Forest, a tropical forest. In this study, phenological patterns and their variability within and among populations located in these biomes and in transitional zones between them were assessed. METHODS: During a 15-month period, individuals from two populations in Cerrado, two in the Atlantic Forest, and six in transitional zones (three in a cerrado-like environment and three in forest fragments) were evaluated in Minas Gerais State, Brazil. The individuals were evaluated monthly according to the proportion of the canopy in each vegetative phenophase (leaf fall, leaf flush and mature leaves) and each reproductive phenophase (floral buds, flowers, immature fruits and mature fruit/seed dispersal). In order to assess the phenological variability within and among populations, habitats and biomes, the Shannon-Wiener diversity index, the Morisita-Horn similarity index and genetic population approach of partitioning diversity were used. KEY RESULTS: Populations of P. reticulata, in general, showed similar phenology; the main differences were related to leaf fall, a process that starts months earlier in the Cerrado than in transitional sites, and even later in forest areas. Considerable synchrony was observed for reproductive phenology among populations and between biomes. Most phenological diversity was due to differences among individuals within populations. CONCLUSION: In spite of environmental differences, P. reticulata from the Atlantic Forest and Cerrado showed similar phenological behavior with only about 10% of the total diversity being attributed to differences between biomes.     

Genetic diversity in Cytisus aeolicus Guss. (Leguminosae), a rare endemite of the Italian flora

ABSTRACT

Cytisus aeolicus Guss, is an endemic plant restricted to the isles of Vulcano, Stromboli and Alicudi in the Aeolian archipelago. All known populations were assayed for genetic variability using enzyme polymorphism. Allozyme variation at 16 loci coding for 10 enzyme systems was examined. The great majority of loci turned out to be monomorphic or fixed heterozygous. The observed genetic depauperation is indicative of historical factors, such as the bottleneck effect associated with migration, and the founder effect in population re-establishment. The low genetic diversity is largely partitioned within rather than among populations, indicating that extant populations have not been established long enough for divergence to have occurred. The genetic uniformity, combined with the scattered distribution of small populations, and the negative shift of population size point to a critically endangered species. Strategies for conservation are considered.     

Increased inbreeding and inter-species gene flow in remnant populations of the rare <Emphasis Type="Italic">Eucalyptus benthamii</Emphasis>

Eucalyptus benthamii Maiden & Cambage is a forest tree of interest for conservation and plantation forestry. It is vulnerable to extinction, occurring on the alluvial floodplains of the Nepean River and its tributaries, south-west of Sydney, Australia. These floodplains were largely cleared of native vegetation for agriculture by the mid-1800s. Flooding of the Cox Valley for Sydneys water supply further decreased the species distribution. The species is now confined to one population of approximately 6500 trees in the Kedumba valley and three remnant populations on the Nepean River at Bents Basin (about 300 trees), Wallacia (nine trees) and Camden (about 30 trees). Genetic analysis of the four populations using microsatellite markers revealed significant divergence among all populations, despite the Bents Basin, Wallacia and Camden remnants being separated by distances of only a few kilometres. Trees in these populations have been estimated to range from 35 to 200 years old, suggesting genetic divergence among populations occurred prior to land clearing. To investigate the impact of fragmentation on the next generation, outcrossing rates were estimated from 41 families. While no direct relationship was found between population size and outcrossing rates, fragmentation and the isolation of trees appears to have resulted in higher levels of selfing and biparental inbreeding in seed collected from the Camden and Wallacia remnants. There was also evidence from seedling morphology that inter-species gene flow increased with fragmentation since 20% of the progeny from Camden and 30% of the progeny from Wallacia were hybrids. Seed viability and germination rates were significantly lower in the remnant populations, reducing their value as seed sources for regeneration and plantation forestry. To maintain the genetic integrity of the remnant populations, germplasm should be sourced from the local area. Outcrossed, non-hybrid seed could be produced by controlled pollination in ex-situ conservation stands or by using seedling morphology and microsatellites to screen seedlings from the remnant populations.     

Population structure and genetic variation in the endangered Giant Kangaroo Rat (<Emphasis Type="Italic">Dipodomys ingens</Emphasis>)

Populations of the endangered giant kangaroo rat, Dipodomys ingens (Heteromyidae), have suffered increasing fragmentation and isolation over the recent past, and the distribution of this unique rodent has become restricted to 3% of its historical range. Such changes in population structure can significantly affect effective population size and dispersal, and ultimately increase the risk of extinction for endangered species. To assess the fine-scale population structure, gene flow, and genetic diversity of remnant populations of Dipodomys ingens, we examined variation at six microsatellite DNA loci in 95 animals from six populations. Genetic subdivision was significant for both the northern and southern part of the kangaroo rat’s range although there was considerable gene flow among southern populations. While regional gene diversity was relatively high for this endangered species, hierarchical F-statistics of northern populations in Fresno and San Benito counties suggested non-random mating and genetic drift within subpopulations. We conclude that effective dispersal, and therefore genetic distances between populations, is better predicted by ecological conditions and topography of the environment than linear geographic distance between populations. Our results are consistent with and complimentary to previous findings based on mtDNA variation of giant kangaroo rats. We suggest that management plans for this endangered rodent focus on protection of suitable habitat, maintenance of connectivity, and enhancement of effective dispersal between populations either through suitable dispersal corridors or translocations.     

Genetic variation in populations of the arctic perennial Pedicularis dasyantha (Scrophulariaceae), on Svalbard,Norway

Isozyme variability was examined in 13 geographically isolated populations of the endemic arctic hairy lousewort (Pedicularis dasyantha) in the Svalbard Archipelago, 80° N latitude, Norway. Of the 23 enzyme systems screened on five buffer systems 18 were interpretable. Of the 31 reliable loci, only 6-phosphogluconate dehydrogenase (6-Pgd), was polymorphic. However, no heterozygotes were detected. Frequencies for allele 1 among the populations varied from 1.00 in the north to 0.00 in the south and 0.53 in the central “overlap” region. At the species level the mean number of alleles per locus (A) was 1.03. Percentage of polymorphic loci (P) was 3%. Expected heterozygosity (H_e) was 0.016. At the population level the mean number of alleles per locus was 1.01, and 1.1% of the loci were polymorphic. H_e was 0.004. These values are low compared to endemic, widespread, selling, and outcrossed species. Flower color morphs were distinct. They varied within and among the 13 populations. The frequency of color morphs coincided with allele frequencies of 6-PGD: allele 1 was found in dark purple morphs, and allele 2 was found in light morphs. This species shows more isozyme genetic variability than the five other species reported in the genus but generally less variation than other species with limited regional distributions. Low-level genetic variation in this diploid species may be a result of colonization events coupled with genetic drift, founder effects, and strong natural selection. Additional factors include the self-compatible reproductive system and the long-lived perennial habit.