Application of Genetic Bottleneck Testing to the Investigation of Amphibian Declines: a Case Study with Natterjack Toads

Abstract: Declines of amphibians are causing widespread concern and in some cases have been explained by factors such as climate change and the spread of microbial pathogens. A problem remains, however, in the unequivocal demonstration of decline in situations where populations may be undergoing natural fluctuations in abundance. We describe the application of a genetic test for bottlenecks ( Cornuet & Luikart 1996 ) that should distinguish between natural oscillations and true population declines. British natterjack toads (   Bufo calamita  ) provide examples of populations that have not declined in recent decades, populations that have declined, and populations in which trends are unclear. Microsatellite allele frequency data from these populations were tested for heterozygote excess and shifts in allele frequency distributions, and inferences from these computations about bottlenecks (i.e., persistently smaller population sizes than the recent means) were compared with demographic information. The genetic test correctly identified bottlenecks and should therefore prove useful in demonstrating whether amphibian declines have occurred where long-term demographic time series are not available.     

产生全雄子代罗非鱼亲本的分子标记及其遗传多样性分析

通过1对1的配对实验,选取产生全雄性杂交子一代的奥利亚罗非鱼和尼罗罗非鱼亲本,对其进行RAPD分析.在40个引物中筛选出22个重复性好的引物用于两个亲本群体的分子标记和遗传多样性研究,其中5个引物(S236、S328、S471、Opz6、Opz8)扩增出的特异性DNA片段,可以作为区分两个亲本群体的分子标记.在奥利亚罗非鱼群体内,共检出133个位点,多态位点数15,多态位点比例为11.28%,在奥利亚罗非鱼群体内,共检出134个位点,多态位点数26,多态位点比例为19.40%.两个亲本群体内较高的遗传相似指数(S分别为0.9787、0.9462),说明两个群体内的遗传变异较小,纯度高;奥利亚罗非鱼和尼罗罗非鱼的种间遗传距离较大(D=0.2595),表明有产生较强杂种优势的可能     

Field and Genetic Survey of the Endangered Butte County Meadowfoam—Limnanthes floccosa subsp. californica (Limnanthaceae)

The Butte County meadowfoam ( Limnanthes floccosa subsp, californica ), an endangered annual endemic to vernal pools in Butte County, California, is of agronomic interest as a sperm whale oil substitute. Because it is threatened by rapid development of the community of Chico, a field and genetic survey was required to guide a conservation program. Eight new populations were discovered, bringing the total known to eleven. Population sizes varied from 220 to 45,689 plants; mean seed set per flower, a bioassay of site quality, ranged from 0.82 to 2.56 among populations. The array of associated taxa, an indicator of appropriate habitat was fairly uniform across populations. Based on allozyme and morphometric data no introgression between L. floccosa subsp, californica and L. alba subsp, alba at sympatric sites was found. Populations were remarkably monomorphic, probably due to past population bottlenecks in conjunction with high selfing rates. Electrophoretic analysis of 28 isozyme loci revealed that 96% of total genetic diversity was distributed among populations. The migration rate, Nm , calculated from the mean frequency of private alleles, ( P (1)), and from the proportion of genetic variation distributed between populations, Gst , estimated exchange between local populations of one diploid individual every 50 to 100 generations, respectively. Mean genetic identity among populations was 0.91 ± 0.068. Grouping of populations on the basis of genetic distance identified two distinct populations and three clusters of populations that deserve high priority for preservation because they are likely to have high frequencies of locally adapted alleles. The low migration rates and substantial differentiation among populations suggest a conservation plan that emphasizes preservation of as many populations as possible, at possible expense of numbers of individuals.     

Genetic evidence for the occurrence of a cryptic species with the littoral nemerteans Lineus ruber and L. viridis (Nemertea: Anopla)

Lineus ruber and L. viridis are common intertidal heteronemerteans found on the coasts of northern Europe and the east coast of North America. The two species resemble each other morphologically and were synonymised for many years prior to being separated on the basis of larval development. In this study, specimens of L. ruber and L. viridis were collected from sites along the west and southwest coast of Britain, northern France and the east coast of North America. The external morphology and allele frequencies of isozymes of the two species were compared from all sites. The external morphology of L. ruber and L. viridis was similar but they could generally be separated by colour. Allele frequencies for up to 13 enzyme loci between sympatric populations of L. ruber and L. viridis indicated that these two species are genetically very different (Nei's genetic identity=0.090 to 0.083). Allele-frequency data also indicated the presence of a third genetic type occurring sympatrically with populations of both L. ruber and L. viridis on the coasts of Britain and France but not on the coast of North America. Fixed differences in allele frequencies between populations of the third genetic type and sympatric populations of L. ruber and L. viridis were observed across multiple loci. Genetic identity between the third genetic type and sympatric populations of L. ruber and L. viridis were extremely low (Nei's genetic identity =<0.078). Such large genetic differences between populations indicate a barrier to gene flow and reproductive isolation. The aberrant type, which exhibits sufficient morphological variation to prevent individuals being distinguished from those of both L. ruber and L. viridis, therefore represents a separate species. The low genetic identities found in intrageneric comparisons of species found in this study have been found in other studies on nemerteans. They may indicate systematic problems within these groups or other phenomena such as morphological stasis.     

Population biology of hyperbenthic crustaceans in a cold water environment (Conception Bay,Newfoundland). II.<Emphasis Type="Italic"> Acanthostepheia malmgreni</Emphasis> (Amphipoda)

Seasonal population dynamics of the gammarid Acanthostepheia malmgreni Goës in Conception Bay, Newfoundland, were examined from October 1998 to November 2000. This species exhibited a 2.5-year life span, with the reproductive cycle correlating with seasonal phytoplankton flux. Females were semelparous and died following a 5-month brooding period and the subsequent release of juveniles in April and May. The biennial life cycle of this population should result in the presence of two cohorts in the hyperbenthos at any given time. However, the cohorts alternated in strength from year to year, which affected annual density, biomass and production during the study period. Densities were 64±87 ind. per 100 m³ in 1999 and 491±492 ind. per 100 m³ (mean±SD) in 2000. Secondary production was estimated at 18–44 mg C m^–2 in 1999 and 180–311 mg C m^–2 in 2000. The annual P/B ratios were 0.89 and 2.27 in 1999 and 2000, respectively. Growth varied both among and within cohorts, with different life-history stages exhibiting variable growth rates ranging from 0 to 12 mg dry mass month^–1.Communicated by J.P. Grassle, New Brunswick     

Population biology of hyperbenthic crustaceans in a cold water environment (Conception Bay,Newfoundland). I.<Emphasis Type="Italic"> Mysis mixta</Emphasis> (Mysidacea)

Seasonal population dynamics of Mysis mixta Lilljeborg were studied from December 1998 to November 2000 at a 240 m deep site in Conception Bay, Newfoundland. At this depth, temperature was <0°C and salinity between 32.0 and 34.0 psu year-round. The spring phytoplankton bloom began in early or late March and reached a maximum in late April to mid-May. M. mixta exhibited a highly synchronised life cycle, with spawning and mating occurring in October to November, embryos brooded for ~5 months, and juveniles released during spring bloom sedimentation in April and May. Females were semelparous and died at age 2.5 years, following release of juveniles in spring, whereas the majority of mature males died at age 2 years, following mating in November. The biennial life cycle of this population resulted in the presence of two cohorts in the hyperbenthos at any given time. Variation in density and biomass was low among cohorts but high within cohorts, the latter probably due to the high motility of mysids. Densities in 1999 and 2000 were 242±379 and 544±987 ind. per 100 m³ (mean±SD), respectively. Although growth rates were similar between years, rates measured from changes in dry mass differed both seasonally and among life-history stages (range from –4 to 7 mg month^–1). Annual secondary production was estimated at 29–73 mg C m^–2 in 1999 and 53–205 mg C m^–2 in 2000. The annual P/B ratios were 1.62 and 1.19 in 1999 and 2000, respectively.Communicated by J.P. Grassle, New Brunswick     

Population ecology of the mud crab <Emphasis Type="Italic">Scylla paramamosain</Emphasis> (Estampador) in an estuarine mangrove system; a mark-recapture study

Mud crabs of the genus Scylla are commercially important mangrove residents that are extensively fished throughout their range in the Indo-West Pacific. Despite this high level of exploitation very little is known about the population dynamics of any of the Scylla species. The present study concentrated on an exploited population of Scylla paramamosain in a natural estuarine mangrove on Can Coc Island in the mouth of the Hau River, Mekong Delta, Vietnam. A total of 6,114 juvenile crabs captured on the seaward mangrove fringe were internally tagged by injection of coded microwire tags and released over a period of 29 days. Recaptures were monitored over the following 144 days. There was little migratory movement within the estuary; of 285 recaptured crabs, 93% were recovered within the island mangrove study area and only two individuals were recovered from mangroves lining the opposite mainland riverbanks. A von Bertalanffy growth function was fitted to growth increment data from recaptured crabs, with L _max = 150 mm to derive a growth constant, k = 2.39 year⁻¹. The theoretical age at which carapace width is zero (t ₀) was derived from known size at recruitment at instar 1, giving a value of −0.0095 years. Previous studies of the same population have shown that female S. paramamosain reach maturity at a mean size of 102 mm carapace width. The present study indicates that they attain this size at around 160 days from first settlement in the mangrove fringe. Abundance of juvenile crabs in the study area was estimated by the Petersen method as 1,101,500 (95% CI 4,17,300–1,785,800, representing 1,102 crabs ha⁻¹ of mangrove. Estimation of mortality from tag returns and from the age-catch curves during a period of constant recruitment were comparable (Z = 1.11 and 1.04 month⁻¹, respectively). Fishing only accounted for 14% of total mortality, suggesting that at the time exploitation was not at a critical level despite the apparent high level of fishing activity.     

Population structure of the common sole (<Emphasis Type="Italic">Solea solea</Emphasis>) in the Northeastern Atlantic and the Mediterranean Sea: revisiting the divide with EPIC markers

Spatial and temporal population genetic structures of the common sole, Solea solea, were studied in Northeastern Atlantic and Mediterranean Sea populations, using three polymorphic exon-primed intron-crossing (EPIC) markers. Results demonstrated significant multilocus differentiation among Eastern Mediterranean and a group composed by Western Mediterranean and Atlantic populations (θ = 0.150, P < 0.001), but also suggested unrecorded genetic differentiation of the Adriatic Sea population. No pattern of isolation-by-distance was recorded across the range covered by sampling, from the Kattegat to the Aegean Sea. Conversely to genetically structured Mediterranean populations, Atlantic populations ranging from Denmark to Portugal could be considered as representative of the same panmictic unit (θ = 0.009, not significant). Results further demonstrated stability of multilocus genetic structure among temporarily replicated cohort samples [0+, 1+, subadults] from several coastal and estuarine locations from Bay of Biscay, excepted for the amylase locus Am2B3-2 at one location (Pertuis d’Antioche). Despite coherence of such observed patterns of multilocus differentiation with previous allozymic surveys in sole, and with patterns generally obtained for other marine fish species, single-locus results from EPICs indicated divergent coalescence schemes supporting a complex response to ecology and history of sole’s populations. Results stress the use of nuclear genes such as EPIC markers to investigate population structure, but also historical, demographic, and possibly selective processes in marine fishes.     

Population structure of Brachidontes pharaonis (P. Fisher, 1870) (Bivalvia, Mytilidae) in the Mediterranean Sea, and evolution of a novel mtDNA polymorphism

Brachidontes pharaonis (Fisher P, 1870) is an Indo-Pacific mussel that has colonized the Mediterranean Sea via the Suez Canal. Mussels may have migrated by natural dispersal of pelagic larvae, or they may have been transported on the hulls of ships, or in ballast water, or by some combination of these. Mitochondrial COI sequences (618 bp) from 101 mussels from six localities in the central and eastern Mediterranean Sea and from one site in the Red Sea were used to describe population structure. Analysis of molecular variance indicated that frequencies differed among populations, and that 92% of the variation resided within populations. The majority of haplotypes were private alleles. No simple pattern of longitudinal variation was detected for haplotype frequencies, haplotype diversity, or nucleotide diversity. A variety of tree-building algorithms (NJ, MP, ME) gave consistent results, showing two sister groups. One clade had a leucine (L-form) and the other one a methionine (M-form) at the 30th amino acid. These sympatric forms were detected within all localities and they had similar frequencies in males and females. The M- and L-form were separated by a 7.3% sequence divergence, indicating that this is an ancient polymorphism. Closely related species of mussels had exclusively the L-form, so we infer that the M-form evolved from the L-form lineage of B. pharaonis, probably in the northern portion of the Red Sea. Neutrality tests detected an excess of rare mutations, a pattern of variation expected in expanding populations. Patterns of variation detected by Tajima’s D suggest that some form of balancing selection has maintained this polymorphism.     

Genetic diversity of Nymphon (Arthropoda: Pycnogonida: Nymphonidae) along the Antarctic Peninsula with a focus on Nymphon australe Hodgson 1902

Sea spiders are conspicuous, and often abundant, members of the Antarctic benthic community. Nymphonidae (Pycnogonida) in Southern Ocean waters comprise over 240 species which are often difficult to assign due to their intraspecific ‘highly variable’ morphology. In particular, Nymphon australe, the numerically dominant species in Antarctic waters is known to have a high level of phenotypic variation in external morphology and is also reported to have a circumpolar distribution. Circumpolarity seems contradictory to the pycnogonid’s brooding lifestyle and presumably limited dispersal. Here we examine the genetic diversity of several Nymphon species collected in the Antarctic Peninsular region. Concomitantly, we assess the genetic structure of N. australe to gain insight into Nymphon dispersal capacity. Cytochrome c oxidase subunit I (COI) and 16S ribosomal gene data suggest a recent common history and/or recent gene-flow of N. australe populations across nearly 800 km of the Antarctic Peninsula. Furthermore, these data support that the Antarctic Peninsula region may hold two previously unrecognized species of Nymphon. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Population dynamics and secondary production of the wedge clam <Emphasis Type="Italic">Donax</Emphasis><Emphasis Type="Italic">hanleyanus</Emphasis> (Bivalvia: Donacidae) on a high-energy,subtropical beach of Brazil

The population biology of Donax hanleyanus (Philippi, 1845) (Bivalvia: Donacidae) was studied by monthly sampling from June 1998 through June 2000, at Restinga da Marambaia Beach, Brazil. Two transects were established and divided into ten strata parallel to the waterline, and five replicates were taken with a 0.04 m² sampler in each stratum. The highest densities of D. hanleyanus were recorded in winter (September 1998, July 1999) and autumn (April 2000). A stratified distribution was observed: recruits were found mainly in the middle swash zone, while juveniles and adults occurred across the tidal gradient up to the retention zone. Mortality rates did not differ significantly between years. Annual production (in ash-free dry mass) ranged from 0.76 g AFDM m^-2 year^-1 (1998) to 3.67 g AFDM m^-2 year^-1 (1999), while the production-to-biomass (P/B) ratio varied from 1.45 to 1.59. Life span was ca. 17 months. Of all variables tested, only two, one biological and one physical, seem to have influenced the population dynamics of this species. A significant negative correlation between the densities of the suspension-feeders D. hanleyanus and Emerita brasiliensis (Crustacea: Decapoda) indicated possible intraguild competition, and there was a significant exponential correlation between beach slope and the mean across-shore position of D. hanleyanus. Data compiled from literature suggests a latitudinal gradient in population parameters, with mortality and renewal rate (P/B) of different species of Donax increasing and life span decreasing from temperate to tropical regions. Other factors influencing population dynamics, such as food availability, and the contributions of individual and community biomass to energy and nutrient cycling are also discussed.     

Population structure of <Emphasis Type="Italic">Symbiodinium</Emphasis> sp. associated with the common sea fan, <Emphasis Type="Italic">Gorgonia ventalina</Emphasis>, in the Florida Keys across distance,depth, and time

Numerous marine invertebrates form endosymbiotic relationships with dinoflagellates in the genus Symbiodinium. However, few studies have examined the fine-scale population structure of these symbionts. Here, we describe the genetic structure of Symbiodinium type “B1/B184” inhabiting the gorgonian Gorgonia ventalina along the Florida Keys. Six polymorphic microsatellite loci were utilized to examine 16 populations along the Upper, Middle, and Lower Keys spanning a range of ~200 km. Multiple statistical tests detected significant differentiation in 54–92% of the 120 possible pairwise comparisons between localities, suggesting low levels of gene flow in these dinoflagellates. In general, populations clustered by geographic region and/or reefs in close proximity. Some of the sharpest population differentiation was detected between Symbiodinium from deep and shallow sites on the same reef. In spite of the high degree of population structure, alleles and genotypes were shared among localities, indicating some connectivity between Symbiodinium populations associated with G. ventalina. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic diversity in two barnacle species, <Emphasis Type="Italic">Chthamalus stellatus</Emphasis> and <Emphasis Type="Italic">Tesseropora atlantica</Emphasis> (Crustacea,Cirripedia), with different larval dispersal modes in the archipelago of the Azores

ISSRs (Inter Simple Sequence Repeats) were employed to compare the genetic structure of Chthamalus stellatus and Tesseropora atlantica in the Azores. The two barnacles differ as regards life cycle: the former conducting pelagic life for about 22 days, the latter for only 24 h. Thirty individuals of each species were analysed from four locations of three Azorean islands. Five ISSR primers produced, respectively, 117 and 79 polymorphic bands in C. stellatus and T. atlantica. Estimates of among-samples genetic diversity suggested extensive connectivity in the former species and isolation in the latter. AMOVA further supported these results by attributing 2% of genetic variance to the among-islands component of C. stellatus and 30% to T. atlantica. Results are consistent with expectations based on species life history and corroborate the importance of pelagic stages in determining the degree of genetic structuring in benthic marine invertebrates.     

Identifying key life-traits for the dynamics and gene flow in a weedy crop relative: Sensitivity analysis of the GeneSys simulation model for weed beet (Beta vulgaris ssp. vulgaris)

The benefits of genetically modified herbicide-tolerant (GMHT) sugar beet (Beta vulgaris) varieties stem from their presumed ability to improve weed control and reduce its cost, particularly targeting weed beet, a harmful annual weedy form of the genus Beta (i.e. B. vulgaris ssp. vulgaris) frequent in sugar beet fields. As weed beet is totally interfertile with sugar beet, it is thus likely to inherit the herbicide-tolerance transgene through pollen-mediated gene flow. Hence, the foreseeable advent of HT weed beet populations is a serious threat to the sustainability of GM sugar beet cropping systems. For studying and quantifying the long-term effects of cropping system components (crop succession and cultivation techniques) on weed beet population dynamics and gene flow, we developed a biophysical process-based model called GeneSys-Beet in a previous study. In the present paper, the model was employed to identify and rank the weed life-traits as function of their effect on weed beet densities and genotypes, using a global sensitivity analysis to model parameters. Monte Carlo simulations with simultaneous randomization of all life-trait parameters were carried out in three cropping systems contrasting for their risk for infestation by HT weed beets. Simulated weed plants and bolters (i.e. beet plants with flowering and seed-producing stems) were then analysed with regression models as a function of model parameters to rank processes and life-traits and quantify their effects. Key parameters were those determining the timing and success of growth, development, seed maturation and the physiological end of seed production. Timing parameters were usually more important than success parameters, showing for instance that optimal timing of weed management operations is more important than its exact efficacy. The ranking of life-traits though depended on the cropping system and, to a lesser extent, on the target variable (i.e. GM weeds vs. total weed population). For instance, post-emergence parameters were crucial in rotations with frequent sugar beet crops whereas pre-emergence parameters were most important when sugar beet was rare. In the rotations with frequent sugar beet and insufficient weed control, interactions between traits were small, indicating diverse populations with contrasted traits could prosper. Conversely, when sugar beet was rare and weed control optimal, traits had little impact individually, indicating that a small number of optimal combinations of traits would be successful. Based on the analysis of sugar beet parameters and genetic traits, advice for the future selection of sugar beet varieties was also given. In climatic conditions similar to those used here, the priority should be given to limiting the presence of hybrid seeds in seed lots rather than decreasing varietal sensitivity to vernalization.     

Paternity and performance of golden-winged warblers (<Emphasis Type="Italic">Vermivora chrysoptera</Emphasis>) and golden-winged X blue-winged warbler (<Emphasis Type="Italic">V. pinus</Emphasis>) hybrids at the leading edge of a hybrid zone

We examined two components of reproduction in a population of golden-winged warblers in the initial stages of hybridization with blue-winged warblers. First, we used genetic analyses of mate choice to determine whether copulations outside the social pairbond (extra-pair copulations; EPCs) occur in these species and their hybrids. Second, we compared several aspects of reproduction (pairing success, clutch size, hatching success, fledging success, and loss of paternity) in nests raised by phenotypic golden-winged warblers to those raised by hybrid individuals. Together, these data provide us with the first quantitative analysis of reproduction within this hybrid system. Our data suggest several reasons why the level of hybridization between these species is likely being underestimated. First, many birds in our population showed only subtle phenotypic signs of introgression indicating that hybrid status can only be determined by close examination. Second, high rates of extra-pair paternity indicate that we cannot base our estimates of hybridization on pairbond data alone. More than 30% of nestlings were the result of EPCs, occurring in 55% of all nests. Third, there was no difference in the number of hybrid or “pure” golden-winged warbler males chosen as social mates, relative to their abundance. Indeed, based on several components of nesting success, it appears as though hybrids are having as great realized reproductive success as are phenotypic golden-winged warblers in our population. Accordingly, we argue that hybrid reproductive success and extra-pair fertilizations are likely playing a major role in the ongoing hybridization between blue-winged and golden-winged warblers. Studies such as this one are important to our understanding of the process and implications of hybridization, and findings may be applicable to other hybrid zones.