wFleaBase: the <Emphasis Type="Italic">Daphnia</Emphasis> genome database

Background  

wFleaBase is a database with the necessary infrastructure to curate, archive and share genetic, molecular and functional genomic data and protocols for an emerging model organism, the microcrustacean Daphnia. Commonly known as the water-flea, Daphnia's ecological merit is unequaled among metazoans, largely because of its sentinel role within freshwater ecosystems and over 200 years of biological investigations. By consequence, the Daphnia Genomics Consortium (DGC) has launched an interdisciplinary research program to create the resources needed to study genes that affect ecological and evolutionary success in natural environments.     

The systematics of Australian Daphnia and Daphniopsis (Crustacea: Cladocera): a shared phylogenetic history transformed by habitat-specific rates of evolution

This study examines the molecular‐genetic divergence and evolution of Australian aquatic micro‐Crustacea Daphnia and Daphniopsis. The results indicate that species of Daphniopsis are accommodated within the genus Daphnia. Although their phyletic integrity is no longer supported, all Daphniopsis species (save one from North America) form a monophyletic group and may warrant subgeneric recognition pending further systematic investigations. A total of five lineages are shown to occupy Australian inland waters, including an endemic subgenus (Australodaphnia) and representatives of the subgenus Ctenodaphnia. The subgenera (Daphnia and Hyalodaphnia) that dominate the North American fauna are absent in Australia. The large extent of sequence divergence among major groups suggests that continental isolation has helped shape the early evolution of daphniids. More recent speciation is also evident, particularly by the Daphnia carinata species complex, whose numbers have grown to 13 members by the addition of a species previously assigned to the nominal subgenus and species yet to be formally described. The molecular data provide more evidence that the colonization of distinct habitats and ecological settings is a key factor in spurring diversification in the genus, while also modulating the pace of molecular evolution. This study attributes habitat‐specific molecular clocks to the intense ultraviolet (UV) exposure in both saline and transparent oligohaline waters. Adaptations to these harsh environments by at least four independent lineages include the convergent acquisition of a melanic carapace. Yet some lineages, clearly under mutational duress, lack this commonly acquired protective trait. There are numerous adaptive lines of defense against UV damage, including the complex regulatory mechanisms required to initiate a cellular response to guard and repair DNA. Functional molecular studies may soon challenge a notion built on morphology that convergence is the general directive to Daphnia’s ecological and evolutionary success. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89 , 469–488.     

Linking genes to communities and ecosystems: Daphnia as an ecogenomic model

How do genetic variation and evolutionary change in critical species affect the composition and functioning of populations, communities and ecosystems? Illuminating the links in the causal chain from genes up to ecosystems is a particularly exciting prospect now that the feedbacks between ecological and evolutionary changes are known to be bidirectional. Yet to fully explore phenomena that span multiple levels of the biological hierarchy requires model organisms and systems that feature a comprehensive triad of strong ecological interactions in nature, experimental tractability in diverse contexts and accessibility to modern genomic tools. The water flea Daphnia satisfies these criteria, and genomic approaches capitalizing on the pivotal role Daphnia plays in the functioning of pelagic freshwater food webs will enable investigations of eco-evolutionary dynamics in unprecedented detail. Because its ecology is profoundly influenced by both genetic polymorphism and phenotypic plasticity, Daphnia represents a model system with tremendous potential for developing a mechanistic understanding of the relationship between traits at the genetic, organismal and population levels, and consequences for community and ecosystem dynamics. Here, we highlight the combination of traits and ecological interactions that make Daphnia a definitive model system, focusing on the additional power and capabilities enabled by recent molecular and genomic advances.     

A CHLOROPLAST-DNA PHYLOGENY OF THE WILD PERENNIAL RELATIVES OF SOYBEAN (GLYCINE SUBGENUS GLYCINE): CONGRUENCE WITH MORPHOLOGICAL AND CROSSING GROUPS

Hypotheses of evolutionary relationships among the Australian wild perennial relatives of soybean (Glycine subgenus Glycine) are based largely on patterns of meiotic pairing in intra- and interspecific experimental hybrids. This evidence has indicated a number of genome groupings within the subgenus but has not resolved most phylogenetic relationships. Restriction-endonuclease site variation of chloroplast DNA (cpDNA) within the perennial subgenus is reported here, representing a sampling of approximately 3% of the approximately 150-kilobase plastome. Seven hundred twenty-one unique restriction sites were compared within Glycine using 29 restriction endonucleases; 157 sites varied within the genus. Distance and parsimony methods using these data yielded congruent results, recognizing the existence of three major groups within subgenus Glycine: the species-rich and geographically diverse A clade consisting of G. canescens and related taxa; the B clade, which includes the stoloniferous species; and the C group, containing two species with distinctive curved pods. These results are in general agreement with hypotheses based on genome analysis; inconsistencies involve the inclusion of genetically divergent taxa such as G. falcata in well-supported plastome clades comprised of otherwise interfertile species. Such findings are not unexpected if crossing barriers are considered to be unique features of such anomalous species, paralleling their often numerous morphological and cpDNA autapomorphies. Consideration of cpDNA divergence within the three major clades of subgenus Glycine indicates that the rate of plastome evolution is uncoupled from rates of morphological or ecological diversification.     

Comparative mapping in Pinus: sugar pine (Pinus lambertiana Dougl.) and loblolly pine (Pinus taeda L.)

The majority of genomic research in conifers has been conducted in the Pinus subgenus Pinus mostly due to the high economic importance of the species within this taxon. Genetic maps have been constructed for several of these pines and comparative mapping analyses have consistently revealed notable synteny. In contrast, little genomic research has been conducted on the Pinus subgenus Strobus, even though these pines have strong ecological relevance. We report a consensus genetic linkage map for sugar pine (Pinus lambertiana Dougl.) constructed with 399 single nucleotide polymorphisms markers derived from annotated genes. The map is 1,231 cM in length and organized into 19 linkage groups. Two of the mapping populations were derived from trees that were segregating for the major gene of resistance (Cr1) to Cronartium ribicola, the fungal pathogen responsible for white pine blister rust. The third mapping population was derived from a full-sib cross segregating for partial resistance to white pine blister rust. In addition, we report the first comparative mapping study between subgenera Strobus and Pinus. Sixty mapped markers were found in common between sugar pine and the loblolly pine reference map with 56 of them (93%) showing collinearity. All 19 linkage groups of the sugar pine consensus map coaligned to the 12 linkage groups of the loblolly pine reference map. The syntenic relationship observed between these two clades of pines provides a foundation for advancing genomic research and genetic resources in subgenus Strobus.     

Biodiversity assessment using markers for ecologically important traits

Most studies of genetic variation within species to date are based on random markers. However, how well this correlates with quantitative variation is contentious. Yet, functional, or'ecotypic' variation in quantitative traits determines the ecological niche of a species, its future evolutionary potential, and, for livestock, crops and their wild relatives, their usefulness as a genetic resource for breeding. But nowadays we can also assess genetic diversity using markers directly targeted at specific genes or gene families. Such gene-targeted, multilocus profiles of markers can contribute to ex-situ management of genetic resources, ecological studies of diversity, and conservation of endangered species.     

Variable-Number-of-Tandem-Repeats Analysis of Genetic Diversity in <Emphasis Type="Italic">Pasteuria ramosa</Emphasis>

Variable-number-of-tandem-repeats (VNTR) markers are increasingly being used in population genetic studies of bacteria. They were recently developed for Pasteuria ramosa, an endobacterium that infects Daphnia species. In the present study, we genotyped P. ramosa in 18 infected hosts from the United Kingdom, Belgium, and two lakes in the United States using seven VNTR markers. Two Daphnia species were collected: D. magna and D. dentifera. Six loci showed length polymorphism, with as many as five alleles identified for a single locus. Similarity coefficient calculations showed that the extent of genetic variation between pairs of isolates within populations differed according to the population, but it was always less than the genetic distances among populations. Analysis of the genetic distances performed using principal component analysis revealed strong clustering by location of origin, but not by host Daphnia species. Our study demonstrated that the VNTR markers available for P. ramosa are informative in revealing genetic differences within and among populations and may therefore become an important tool for providing detailed analysis of population genetics and epidemiology.     

Plant DNA‐barcode library and community phylogeny for a semi‐arid East African savanna

Applications of DNA barcoding include identifying species, inferring ecological and evolutionary relationships between species, and DNA metabarcoding. These applications require reference libraries that are not yet available for many taxa and geographic regions. We collected, identified, and vouchered plant specimens from Mpala Research Center in Laikipia, Kenya, to develop an extensive DNA‐barcode library for a savanna ecosystem in equatorial East Africa. We amassed up to five DNA barcode markers (rbcL, matK, trnL‐F, trnH–psbA, and ITS) for 1,781 specimens representing up to 460 species (~92% of the known flora), increasing the number of plant DNA barcode records for Africa by ~9%. We evaluated the ability of these markers, singly and in combination, to delimit species by calculating intra‐ and interspecific genetic distances. We further estimated a plant community phylogeny and demonstrated its utility by testing if evolutionary relatedness could predict the tendency of members of the Mpala plant community to have or lack “barcode gaps”, defined as disparities between the maximum intra‐ and minimum interspecific genetic distances. We found barcode gaps for 72%–89% of taxa depending on the marker or markers used. With the exception of the markers rbcL and ITS, we found that evolutionary relatedness was an important predictor of barcode‐gap presence or absence for all of the markers in combination and for matK, trnL‐F, and trnH–psbA individually. This plant DNA barcode library and community phylogeny will be a valuable resource for future investigations.     

Characterization of microsatellite markers in peach [Prunus persica (L.) Batsch]

Microsatellites have emerged as an important system of molecular markers. We evaluated the potential of microsatellites for use in genetic studies of peach [Prunus persica (L.) Batsch]. Microsatellite loci in peach were identified by screening a pUC8 genomic library, a λZAPII leaf cDNA library, as well as through database searches. Primer sequences for the microsatellite loci were tested from the related Rosaceae species apple (Malus×domestica) and sour cherry (Prunus cerasus L.). The genomic library was screened for CT, CA and AGG repeats, while the cDNA library was screened for (CT)_n- and (CA)_n-containing clones. Estimates of microsatellite frequencies were determined from the genomic library screening, and indicate that CT repeats occur every 100 kb, CA repeats every 420 kb, and AGG repeats every 700 kb in the peach genome. Microsatellite- containing clones were sequenced, and specific PCR primers were designed to amplify the microsatellite- containing regions from genomic DNA. The level of microsatellite polymorphism was evaluated among 28 scion peach cultivars which displayed one to four alleles per primer pair. Five microsatellites were found to segregate in intraspecific peach-mapping crosses. In addition, these microsatellite markers were tested for their utility in cross-species amplification for use in comparative mapping both within the Rosaceae, and with the un- related species Arabidopsis thaliana L. Received: 18 June 1999 / Accepted: 6 December 1999     

Genetic analysis of Pinus strobus and Pinus monticola populations from Canada using ISSR and RAPD markers: development of genome-specific SCAR markers

Pinus is the largest genus of conifers, containing over 100 species and is also the most widespread genus in the Northern Hemisphere. Pinus monticola and P. strobus are two closely related and economically important species in Canada. Morphological and allometric characteristics have been used to assess genetic variation within these two species but these markers are not reliable due to ecological variations. The purpose of the present study was to determine the level of genetic diversity within and among Canadian populations from the two species using molecular markers and to identify and characterize genome-specific inter-simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) markers. The level of genetic variation among populations was much lower for P. monticola than P. strobus. For both species, the among population variation values were smaller than within population variation. The populations from P. monticola were more closely genetically related than populations from P. strobus based on ISSR and RAPD analyses. Six ISSR and four RAPD markers specific to either P. monticola or P. strobus were cloned and sequenced. Primer pairs flanking these specific sequences were designed and genome specific SCAR markers for P. monticola and P. strobus were developed and characterized.     

Genetic variation and biogeography of the disjunct Vitis subg. Vitis (Vitaceae)

Aim Vitis subg. Vitis provides an example of a plant disjunction occurring in the Northern Hemisphere. It shows broad morphological variation but is assumed to be a species complex with limited genetic differentiation. Based on a comprehensive sampling of taxa and polymorphism in both chloroplast and nuclear DNA, we assessed genetic variation within this subgenus. Our aims were to clarify the relationships among species and to examine their historical biogeography. Location Asia, Europe, North America. Methods We analysed a total of 30 species and putative hybrids from subgenus Vitis and examined the infra‐specific variation in some species. Polymorphism in chloroplast DNA was assessed in trnL and trnH–psbA–trnK sequences (c. 2170 bp) and in 15 microsatellite loci. We also obtained nuclear data for size variation at 24 microsatellite loci. Phylogenetic inference was performed with Bayesian analyses. A maximum parsimony network was constructed to depict the evolutionary relationships among haplotypes, and microsatellite data were also subjected to hierarchical clustering analysis using the Ward distance. In addition, we assessed size homoplasy by sequencing both chloroplast and nuclear microsatellite loci. Results Chloroplast polymorphisms resolved subgenus Vitis as a monophyletic group with limited genetic variation. The ancestral haplotypes were found in Eurasia. American taxa all harboured derived haplotypes. Most of them formed a monophyletic group that did not include Vitis californica. The four main haplotypes in Vitis vinifera corresponded to two different origins. Nuclear microsatellites indicated that genetic variation was especially large in North America. Asian species exhibited a lower level of nuclear divergence and the European V. vinifera corresponded to a differentiated nuclear lineage. Main conclusions We obtained some evidence that subgenus Vitis has an Asian origin and then dispersed to Europe and North America. Geographic separation was followed by diversification, presumably during the Pleistocene, resulting in phylogeographic patterns similar to other biota. In contrast to chloroplast DNA, nuclear DNA shows a larger than expected genetic variation. Our molecular data also highlight the need to re‐examine certain aspects of the current subgeneric classification.     

A linkage disequilibrium perspective on the genetic mosaic of speciation in two hybridizing Mediterranean white oaks

We analyzed the genetic mosaic of speciation in two hybridizing Mediterranean white oaks from the Iberian Peninsula (Quercus faginea Lamb. and Quercus pyrenaica Willd.). The two species show ecological divergence in flowering phenology, leaf morphology and composition, and in their basic or acidic soil preferences. Ninety expressed sequence tag-simple sequence repeats (EST-SSRs) and eight nuclear SSRs were genotyped in 96 trees from each species. Genotyping was designed in two steps. First, we used 69 markers evenly distributed over the 12 linkage groups (LGs) of the oak linkage map to confirm the species genetic identity of the sampled genotypes, and searched for differentiation outliers. Then, we genotyped 29 additional markers from the chromosome bins containing the outliers and repeated the multilocus scans. We found one or two additional outliers within four saturated bins, thus confirming that outliers are organized into clusters. Linkage disequilibrium (LD) was extensive; even for loosely linked and for independent markers. Consequently, score tests for association between two-marker haplotypes and the ‘species trait'' showed a broad genomic divergence, although substantial variation across the genome and within LGs was also observed. We discuss the influence of several confounding effects on neutrality tests and review the evolutionary processes leading to extensive LD. Finally, we examine how LD analyses within regions that contain outlier clusters and quantitative trait loci can help to identify regions of divergence and/or genomic hitchhiking in the light of predictions from ecological speciation theory.     

Genetic Identification of Hyalodaphnia Species and Interspecific Hybrids

Species of the genus Daphnia, in particular the subgenus Hyalodaphnia, represent a taxonomically problematic group due to their phenotypic plasticity, local races and the formation of interspecific hybrids and backcrosses. In this study, we present a genetic approach utilising nuclear DNA to unequivocally identify species and interspecific hybrids. Several nuclear loci (ITS1-ITS2, CA14 and GA13) were amplified by PCR and products were subjected to diagnostic restriction enzymes (restriction fragment length polymorphism; RFLP). The application of this approach to several populations across Europe revealed that the markers are highly consistent and reproducible. In addition, we illustrate with a number of examples how this approach contributed to unravel previously unrecognised taxa, increased the sensitivity of biodiversity studies or contributed to the analysis of resting egg banks. Advantages and disadvantages of this approach compared to existing techniques are discussed and several empirical studies and their results are summarised.     

Hybridization and polyploidy as drivers of continuing evolution and speciation in Sorbus

Interspecific hybridization and polyploidy are pivotal processes in plant evolution and speciation. The fate of new hybrid and polyploid taxa is determined by their ability to reproduce either sexually or asexually. Hybrids and allopolyploids with odd chromosome numbers are frequently sterile but some establish themselves through asexual reproduction (vegetative or apomixis). This allows novel genotypes to become established by isolating them from gene flow and leads to complex patterns of variation. The genus Sorbus is a good example of taxonomic complexity arising from the combined effects of hybridization, polyploidy and apomixis. The Avon Gorge in South‐west Britain contains the greatest diversity of Sorbus in Europe, with three endemic species and four putative endemic novel hybrids among its 15 native Sorbus taxa. We used a combination of nuclear microsatellite and chloroplast DNA markers to investigate the evolutionary relationships among these Sorbus taxa within the Avon Gorge. We confirm the genetic identity of putative novel taxa and show that hybridization involving sexual diploid species, primarily S. aria and S. torminalis and polyploid facultative apomictic species from subgenus Aria, has been responsible for generating this biodiversity. Importantly our data show that this creative evolutionary process is ongoing within the Avon Gorge. Conservation strategies for the rare endemic Sorbus taxa should therefore consider all Sorbus taxa within the Gorge and must strive to preserve this evolutionary process rather than simply the individual rare taxa that it produces.     

Genome skimming and plastid microsatellite profiling of alder trees (<Emphasis Type="Italic">Alnus</Emphasis> spp., Betulaceae): phylogenetic and phylogeographical prospects

Alders (Alnus spp.) represent keystone species trees of riparian and mountainous habitats of the northern hemisphere. Previous genetic studies have suggested a complex intrageneric diversification with numerous events of interspecific hybridization and polyploidization. Here, we first aim to test the present taxonomical treatment of Alnus by generating phylogenetic hypotheses based on plastid and nuclear data obtained from species belonging to the three main alder subgenera (Alnus, Alnobetula, and Clethropsis). A genome-skimming strategy was used to assemble the complete plastome and the nuclear ribosomal DNA cluster of 22 Eurasian and American alder individuals. Phylogenies based on these data strongly support an early diverging subgenus Alnobetula, while members of the subgenus Clethropsis do not constitute a monophyletic clade and are embedded within the subgenus Alnus. Incongruent topologies also sustain reticulate evolution within this group. Our results thus suggest considering the subgenera Clethropsis and Alnus within the same taxonomical unit. Our second aim is to test for the utility of highly variable plastid markers (microsatellites) to investigate the phylogeographic patterns of Eurasian alder species. Fifty-two polymorphic plastid microsatellite markers were developed and tested on 33 populations of the subgenus Alnus in western Eurasia. On average, 4.3 alleles per locus were revealed in 131 individuals of Alnus glutinosa, allowing the identification of 30 chlorotypes (multiloci profiles). Strong phylogeographic signals and recurrent cytoplasmic captures between co-occurring species are revealed, demonstrating that our plastid microsatellite profiling method is suitable for tracing the post-glacial spread of maternal lineages among alder species. All these results finally support the use of nuclear genomic regions for species identification and of plastid markers for phylogeographic aspects and origin certification in genetic resource management.     

Genetic diversity in European chestnut populations by means of genomic and genic microsatellite markers

Microsatellite or simple sequence repeats (SSRs) are one of the most used markers in population genetic studies. SSR markers developed from expressed sequence tags (EST) have proved useful to examine functional diversity in relation to adaptive variation. The information provided by both genomic and genic microsatellite markers could offer more accurate indication on the distribution of the genetic diversity among and within populations assuming different evolutionary drivers. This is the first study on chestnut (Castanea sativa Mill.) in which the genetic diversity was evaluated by means of genomic (SSRs) and genic (EST-SSRs) microsatellite markers. We genotyped nine natural European chestnut populations distributed throughout representative areas of contrasting climatic conditions in the Mediterranean basin. Genomic SSRs showed significantly higher levels of diversity in terms of number of alleles, effective number of alleles, expected heterozygosity and level of polymorphism. Furthermore, there were significant differences in the level of differentiation among populations. The UPGMA analysis revealed different clustering pattern between populations, being the grouping according to geographic distances in the case of genomic SSRs and two differentiated groups based on the northern–southern distribution of the populations for EST-SSRs. Furthermore, the EST-SSR transferability among related Castanea and Quercus species was stated. Our results confirm that combining genomic SSRs and EST-SSRs is a useful tool to give complementary information to explain the genetic and adaptive diversity in chestnut.     

Characterising functionally important and ecologically meaningful genetic diversity using a candidate gene approach

Over the past two decades the fields of molecular ecology and population genetics have been dominated by the use of putatively neutral DNA markers, primarily to resolve spatio-temporal patterns of genetic variation to inform our understanding of population structure, gene flow and pedigree. Recent emphasis in comparative functional genomics, however, has fuelled a resurgence of interest in functionally important genetic variation that underpins phenotypic traits of adaptive or ecological significance. It may prove a major challenge to transfer genomics information from classical model species to examine functional diversity in non-model species in natural populations, but already multiple gene-targeted candidate loci with major effect on phenotype and fitness have been identified. Here we briefly describe some of the research strategies used for isolating and characterising functional genetic diversity at candidate gene-targeted loci, and illustrate the efficacy of some of these approaches using our own studies on red grouse (Lagopus lagopus scoticus). We then review how candidate gene markers have been used to: (1) quantify genetic diversity among populations to identify those depauperate in genetic diversity and requiring specific management action; (2) identify the strength and mode of selection operating on individuals within natural populations; and (3) understand direct mechanistic links between allelic variation at single genes and variance in individual fitness.     

Confirmation of clonal reproduction of Fagus crenata Blume from Sado Island,Niigata Prefecture

While clonal growth is important in the East Asian Fagus subgenus Engleriana and the North American Fagus grandifolia (subgenus Fagus), for other subgenus Fagus species the vast majority of regeneration involves sexual reproduction with clonal growth only rarely observed. Here we aim to confirm using nuclear microsatellite markers whether clonal growth occurs in the Japanese endemic species Fagus crenata (subgenus Fagus) by investigating the origin of multi-stemmed clumps found within a high-elevation dwarf beech forest on Sado Island, Niigata Prefecture. We found that all stems collected from three separate clumps belonged to the same clump-specific multi-locus genotypes. The maximum size of clones was 3–4 m in diameter, comparable in size to those seen in the predominantly asexually regenerating Japanese species Fagus japonica (subgenus Engleriana). The species capacity for clonal growth is likely to underlie its ability to persist at high-elevation exposed sites at the limits of its ecological range.