Extensive phenotypic diversification coexists with little genetic divergence and a lack of population structure in the White Wagtail subspecies complex (Motacilla alba)

Geographically clustered phenotypes often demonstrate consistent patterns in molecular markers, particularly mitochondrial DNA (mtDNA) traditionally used in phylogeographic studies. However, distinct evolutionary trajectories among traits and markers can lead to their discordance. First, geographic structure in phenotypic traits and nuclear molecular markers can be co‐aligned but inconsistent with mtDNA (mito‐nuclear discordance). Alternatively, phenotypic variation can have little to do with patterns in neither mtDNA nor nuclear markers. Disentangling between these distinct patterns can provide insight into the role of selection, demography and gene flow in population divergence. Here, we examined a previously reported case of strong inconsistency between geographic structure in mtDNA and plumage traits in a widespread polytypic bird species, the White Wagtail (Motacilla alba). We tested whether this pattern is due to mito‐nuclear discordance or discrepancy between morphological evolution and both nuclear and mtDNA markers. We analysed population differentiation and structure across six out of nine commonly recognized subspecies using 17 microsatellite loci and a combination of microsatellites and plumage indices in a comprehensively sampled region of a contact between two subspecies. We did not find support for the mito‐nuclear discordance hypothesis: nuclear markers indicated a subtle signal of genetic clustering only partially consistent with plumage groups, similar to previous findings that relied on mtDNA. We discuss evolutionary factors that could have shaped the intricate patterns of phenotypic diversification in the White wagtail and the role that repeated selection on plumage ‘hotspots’ and hybridization may have played.     

Genetic linkage maps constructed by using an interspecific cross between Japanese and European pears

Genetic linkage maps of the European pear ( Pyrus communis L.) cultivar 'Bartlett' and the Japanese pear ( Pyrus pyrifolia Nakai) cultivar 'Housui' were constructed based on AFLPs, SSRs from pear, apple and Prunus, isozymes and phenotypic traits by using their F(1) progenies. The map of the female parent Bartlett consisted of 226 loci including 175 AFLPs, 49 SSRs, one isozyme and one S locus on 18 linkage groups over a total length of 949 cM, while that for 'Housui' contained 154 loci including 106 AFLPs, 42 SSRs, two phenotypic traits and the other four markers on 17 linkage groups encompassing a genetic distance of 926 cM. These maps were partially aligned using 20 codominant markers which showed segregating alleles in both parents. Compared with the reports of apple genetic maps, these pear maps were not saturated but were near saturation. Distorted segregation was observed in two and one regions of the genome of Bartlett and Housui, respectively. The position of 14 SSRs originating from apple could be successfully determined in pear maps, which enabled us to compare the two maps. Some SSRs developed from Prunus (peach, cherry) were also mapped. The relationships between pear and the other species belonging to the Rosaceae were discussed based on the position of SSRs.     

Low support for separate species within the redpoll complex (Carduelis flammea-hornemanni-cabaret) from analyses of mtDNA and microsatellite markers

The redpoll complex, consisting of three currently recognized species (Carduelis flammea, C. hornemanni and C. cabaret), is polytypic in biometry, morphology, physiology and behaviour. However, previous genetic work has not revealed any indications of genetic differentiation. We analysed sequence variation in the mtDNA control region, and allele frequencies of supposedly faster evolving microsatellites (n=10), in an attempt to detect molecular genetic support for the three species, as well as two subspecies of C. flammea (ssp. flammea and rostrata), within this complex. We used samples from two subspecies of the twite (Carduelis flavirostris, ssp. flavirostris and rufostrigata) as outgroup. We found no structure among redpoll individuals in mtDNA haplotypes or microsatellite allele frequencies, and only marginal differences between redpoll taxa in analyses of molecular variance (AMOVAs) of predefined groups. In contrast, the two twite subspecies constituted two well-supported monophyletic groups. Our study thus strengthens previous indications of low genetic support for current redpoll taxa. Two major alternative interpretations exist. Either redpolls form a single gene pool with geographical polymorphisms possibly explained by Bergmann's and Gloger's rules, or there are separate gene pools of recent origin but with too little time elapsed for genetic differentiation to have evolved in the investigated markers. Future studies should therefore examine whether reproductive isolation mechanisms and barriers to gene flow exist in areas with sympatric breeding.     

Linking porcine microsatellite markers to known genome regions by identifying their human orthologs.

Microsatellites, or tandem simple sequence repeats (SSRs), have become one of the most popular molecular markers in genome mapping because of their abundance across genomes and because of their high levels of polymorphism. However, information on which genes surround or flank them has remained very limited for most SSRs, especially in livestock species. In this study, an in silico comparative mapping approach was developed to link porcine SSRs to known genome regions by identifying their human orthologs. From a total of 1321 porcine microsatellites used in this study, 228 were found to have blocks in alignment with human genomic sequences. These 228 SSRs span about 1459 cM of the porcine genome, but with uneven distributions, ranging from 2 on SSC12 to 24 on SSC14. Linking these porcine SSRs to the known genome regions in the human genome also revealed 16 new putative synteny groups between these two species. Fifteen SSRs on SSC3 with identified human orthologs were typed on a pig-hamster radiation hybrid (RH) panel and used in a joint analysis with 80 known gene markers previously mapped on SSC3 using the same panel. The analysis revealed that they were all highly linked to either one or both adjacent markers. These results indicated that assigning the porcine SSRs to known genome regions by identifying their human orthologs is a reliable approach. The process will provide a foundation for positional cloning of causative genes for economically important traits.     

Comparative Analysis of SSR Markers Developed in Exon,Intron, and Intergenic Regions and Distributed in Regions Controlling Fruit Quality Traits in <Emphasis Type="Italic">Prunus</Emphasis> Species: Genetic Diversity and Association Studies

Simple sequence repeats (SSRs) are genome domains located in both coding and non-coding regions in eukaryotic genomes. Although SSRs are often characterized by low polymorphism, their DNA-flanking sequences could be a useful source of DNA markers, which could help in genetic studies and breeding because they are associated with genes that control traits of interest. In this study, 56 genotypes from different Prunus species were used, including peach, apricot, plum, and almond (already phenotyped for several agronomical traits, including self-compatibility, flowering and ripening time, fruit type, skin and flesh color, and shell hardness). These Prunus genotypes were molecularly characterized using 28 SSR markers developed in exons, introns, and intergenic regions. All these genes were located in specific regions where quantitative trait loci (QTLs) for certain fruit quality traits were also located, including flowering and ripening times and fruit flesh and skin color. A sum of 309 SSR alleles were identified in the whole panel of analyzed cultivars, with expected heterozygosity values of 0.61 (upstream SSRs), 0.17 (exonic SSRs), 0.65 (intronic SSRs), and 0.58 (downstream SSRs). These values prove the low level of polymorphism of the exonic (gene-coding regions) markers. Cluster and structural analysis based on SSR data clearly differentiated the genotypes according to either specie (for the four species) and pedigree (apricot) or geographic origin (Japanese plum). In addition, some SSR markers mainly developed in intergenic regions could be associated with genes that control traits of interest in breeding and could therefore help in marker-assisted breeding. These findings highlight the importance of using molecular markers able to discriminate between the functional roles of the gene allelic variants.     

Ecological exchangeability versus neutral molecular markers: the case of the great tit

Neutral genetic markers, such as mitochondrial DNA (mtDNA) sequences, are commonly used to discover independently evolving groups of populations in nature. These groups are often considered to be units of conservation because they preserve distinct organismal histories. However, because of the time lag between the isolation of populations and the evolution of diagnostic neutral markers, adaptive traits could be unrepresented by units defined by neutral markers. The concept of ecological exchangeability potentially provides a way to preserve populations possessing local adaptations that lack diagnostic neutral markers. Populations are surveyed for differences in fitness traits, either directly, or by examining morphological or genetic traits that could indirectly serve as proxies for them. The purpose of this paper is to compare the nature of units of conservation defined by neutral gene surveys and ecological exchangeability, using data recently available for the great tit Parus major , a wide-ranging Palearctic species. mtDNA surveys reveal a lack of differentiation across thousands of kilometer. In contrast, studies of body size and clutch size show locally adapted differences between populations separated by a few kilometer, meaning that these populations could be classified as ecologically in exchangeable. These two types of markers have dramatically different consequences for the geographic and evolutionary scale of conservation units. The concept of ecological exchangeability might be an inappropriate way to diagnose units of conservation in birds owing to the time required to document local adaptations and their potential ubiquity. Neutral genetic markers continue to provide a theoretically sound way of identifying units of conservation, and these units ought to be integrated into conservation plans without delay.     

AFLP-Based Genetic Diversity and Its Comparison with Diversity Based on SSR, SAMPL, and Phenotypic Traits in Bread Wheat

Data on AFLP (eight primer pairs) and 14 phenotypic traits, collected on 55 elite and exotic bread wheat genotypes, were utilized for estimations of genetic diversity. We earlier used these 55 genotypes for a similar study using SSRs and SAMPL. As many as 615 scorable AFLP bands visualized included 287 (46.6%) polymorphic bands. The phenotypic traits included yield and its component traits, as well as physiomorphological traits like flag leaf area. Dendrograms were prepared using cluster analysis based on Jaccard's similarity coefficients in case of AFLP and on squared Euclidean distances in case of phenotypic traits. PCA was conducted using AFLP data and a PCA plot was prepared, which was compared with clustering patterns in two dendrograms, one each for AFLP and phenotypic traits. The results were also compared with published results that included studies conducted elsewhere using entirely different wheat germplasm and our own SSR and SAMPL studies based on the same 55 genotypes used in the present study. It was shown that molecular markers are superior to phenotypic traits and that AFLP and SAMPL are superior to other molecular markers for estimation of genetic diversity. On the basis of AFLP analysis and keeping in view the yield performance and stability, a pair of genotypes (E3876 and E677) was recommended for hybridization in order to develop superior cultivars.     

Genetic and morphological divergence at different spatiotemporal scales in the grasshopper Mioscirtus wagneri (Orthoptera: Acrididae)

The study of the association between morphological and genetic divergence can provide important information on the factors determining population differentiation and gene flow at different spatiotemporal scales. In this study we analyze the congruence between morphological and genetic divergence in the Iberian populations of Mioscirtus wagneri, a specialized grasshopper exclusively inhabiting highly fragmented hypersaline low grounds. We have found strong morphological variation among the studied localities and among mtDNA- and microsatellite-based genetic clusters. However, we have detected some cases of morphological convergence between highly differentiated populations. By contrast, certain genetically homogeneous populations at both mtDNA and microsatellite markers showed significant morphological differentiation which may be explained by phenotypic plasticity or divergent selection pressures acting at different spatiotemporal scales. Mantel tests also revealed that morphological divergence was associated with microsatellite- but not with mtDNA-based genetic distances. Overall, this study suggests that morphological traits can provide additional information on the underlying population genetic structure when only data on scarcely variable mtDNA markers is available. Thus, morphology can retain useful information on genetic structure and has the benefit over molecular methods of being inexpensive, offering a preliminary/complementary useful criterion for the establishment of management units necessary to guide conservation policies.     

Nuclear genes (but not mitochondrial DNA barcodes) reveal real species: Evidence from the Brenthis fritillary butterflies (Lepidoptera,Nymphalidae)

Discordance between entities revealed by nuclear versus mitochondrial genes is a common phenomenon in evolutionary and taxonomic studies. However, little attention has been paid to analysis of how such discordant entities correspond to traditional species detected through investigation of their morphology, ecology, and distribution. Here, we used one mitochondrial (COI, DNA barcode fragment) and four nuclear (CAD, Ca‐ATPase, arginine kinase, wg) genes to analyze the genetic structure of the taxonomically well‐studied butterfly genus Brenthis (Lepidoptera, Nymphalidae). Analysis of COI revealed multiple diverged allopatric and sympatric mitochondrial lineages within the known Brenthis species hinting at possible presence of unrecognized cryptic species. However, these multiple‐species hypotheses were not supported by further studies of nuclear genes and phenotypic traits. The discovered mitochondrial lineages did not correspond to the clusters revealed by nuclear genes. Simultaneously, we found a complete congruence between (a) traditional species boundaries, (b) clusters recognized by nuclear genes, and (c) clusters identified via cladistic analysis of phenotypic traits (genitalia and wing pattern characters, ecological preferences, and chromosome numbers). We conclude that in case of the genus Brenthis, nuclear genes rather than mtDNA barcodes reveal real species boundaries. Additionally, we suggest to support each DNA barcode‐based taxonomic conclusion by analysis of phased alleles of nuclear genes, avoiding widely used practice of nuclear and mitochondrial genes concatenation without any examination of interaction of these different types of data.     

Conserving marine biodiversity: insights from life-history trait candidate genes in Atlantic cod (Gadus morhua)

Recent technological developments have facilitated an increased focus on identifying genomic regions underlying adaptive trait variation in natural populations, and it has been advocated that this information should be important for designating population units for conservation. In marine fishes, phenotypic studies have suggested adaptation through divergence of life-history traits among natural populations, but the distribution of adaptive genetic variation in these species is still relatively poorly known. In this study, we extract information about the geographical distribution of genetic variation for 33 single nucleotide polymorphisms (SNPs) associated with life-history trait candidate genes, and compare this to variation in 70 putatively neutral SNPs in Atlantic cod (Gadus morhua). We analyse samples covering the major population complexes in the eastern Atlantic and find strong evidence for non-neutral levels and patterns of population structuring for several of the candidate gene-associated markers, including two SNPs in the growth hormone 1 gene. Thus, this study aligns with findings from phenotypic studies, providing molecular data strongly suggesting that these or closely linked genes are under selection in natural populations of Atlantic cod. Furthermore, we find that patterns of variation in outlier markers do not align with those observed at selectively neutral markers, and that outlier markers identify conservation units on finer geographical scales than those revealed when analysing only neutral markers. Accordingly, results also suggest that information about adaptive genetic variation will be useful for targeted conservation and management in this and other marine species.     

Phylogeography of the endangered darkling beetle species of Pimelia endemic to Gran Canaria (Canary Islands)

Phylogenetic and geographical nested clade analysis (NCA) methods were applied to mitochondrial DNA sequences of Pimelia darkling beetles (Coleoptera, Tenebrionidae) endemic to Gran Canaria, an island in the Canary archipelago. The three species P. granulicollis, P. estevezi and P. sparsa occur on the island, the latter with three recognized subspecies. Another species, P. fernandezlopezi (endemic to the island of La Gomera) is a close relative of P. granulicollis based on partial Cytochrome Oxidase I mtDNA sequences obtained in a previous study. Some of these beetles are endangered, so phylogeographical structure within species and populations can help to define conservation priorities. A total of about 700 bp of Cytochrome Oxidase II were examined in 18 populations and up to 75 individuals excluding outgroups. Among them, 22 haplotypes were exclusive to P. granulicollis and P. estevezi and 31 were from P. sparsa. Phylogenetic analysis points to the paraphyly of Gran Canarian Pimelia, as the La Gomera P. fernandezlopezi haplotypes are included in them, and reciprocal monophyly of two species groups: one constituted by P. granulicollis, P. estevezi and P. fernandezlopezi (subgenus Aphanaspis), and the other by P. sparsa'sensu lato'. The two species groups show a remarkably high mtDNA divergence. Within P. sparsa, different analyses all reveal a common result, i.e. conflict between current subspecific taxonomic designations and evolutionary units, while P. estevezi and P. fernandezlopezi are very close to P. granulicollis measured at the mtDNA level. Geographical NCA identifies several cases of nonrandom associations between haplotypes and geography that may be caused by allopatric fragmentation of populations with some cases of restriction of gene flow or range expansion. Analyses of molecular variance and geographical NCA allow definition of evolutionary units for conservation purposes in both species-groups and suggest scenarios in which vicariance caused by geological history of the island may have shaped the pattern of the mitochondrial genetic diversity of these beetles.     

Relationships among durum wheat accessions. I. Comparative analysis of SSR, AFLP, and phenotypic data.

The determination of genetic relatedness among elite materials of crop species allows for more efficient management of breeding programs and for the protection of breeders' rights. Seventy simple sequence repeats (SSRs) and 234 amplified fragment length polymorphisms (AFLPs) were used to profile a collection of 58 durum wheat (Triticum durum Desf.) accessions, representing the most important extant breeding programs. In addition, 42 phenotypic traits, including the morphological characteristics recommended for the official distinctness, uniformity, and stability tests, were recorded. The correlation between the genetic similarities obtained with the 2 marker classes was high (r = 0.81), whereas lower values were observed between molecular and phenotypic data (r = 0.46 and 0.56 for AFLPs and SSRs, respectively). Morphological data, even if sampled in high numbers, largely failed to describe the pattern of genetic similarity, according to known pedigree data and the indications provided by molecular markers.     

Linkage mapping of AFLP and microsatellite DNA markers with the body color- and sex-determining loci in the guppy (Poecilia reticulata)

The guppy is an ornamental fish species that exhibits various phenotypic characteristics, such as body color and fin-shape. Although linkage relationships of a limited number of phenotypic traits have already been investigated, the association between phenotypic and molecular markers is still unknown. We constructed a total of 35 linkage groups for the guppy using 186 polymorphic loci of AFLP and microsatellite DNA. The locus related to the yellow body color was linked with ten markers and the sex-determination locus was linked with five markers.     

Contrasting patterns of genetic structure in two species of the coral trout Plectropomus (Serranidae) from east and west Australia: introgressive hybridisation or ancestral polymorphisms

Inter-specific genetic relationships among regional populations of two species of grouper (Plectropomus maculatus and Plectropomus leopardus) were examined using mitochondrial and nuclear markers. mtDNA revealed contrasting regional inter-specific patterns whilst nuclear markers revealed contrasting patterns among markers, irrespective of region. In eastern Australia (EA) the species form a single mtDNA lineage, but the two species are reciprocally monophyletic in Western Australia (WA). This supports previous evidence for hybridisation between these species on the east coast. WA P. leopardus forms a sister relationship with the EA P. leopardus-maculatus clade while WA P. maculatus is more basal and sister to the P. leopardus lineages, indicating mtDNA does not suffer from incomplete lineage sorting for these species. In contrast, one of three nuclear markers (locus 7-90TG) differentiated the species into two reciprocally monophyletic clades, with no evidence of hybridisation or ancestral polymorphism. The remaining two nuclear markers (2-22 and ETS-2) did not separate these two species, while distinguishing other plectropomid species, suggesting incomplete lineage sorting at these nuclear loci. These results together with coalescence analyses suggest that P. leopardus females have hybridised historically with P. maculatus males and that P. maculatus mitochondria were displaced through introgressive hybridisation and fixation in the P. maculatus founder population on the Great Barrier Reef. The contrasting regional patterns of mtDNA structure may be attributed to Quaternary sea-level changes and shelf width differences driving different reef configurations on each coast. These reef configurations have provided opportunities for local scale interaction and reproduction among species on the narrower EA continental shelves, but not on the broader WA continental shelves.     

Low, but Strongly Structured Mitochondrial DNA Diversity in Root Knot Nematodes (Meloidogyne)

Root-knot nematodes (genus Meloidogyne) have been the subject of recent and numerous studies of genetic variation because of the need to develop molecular diagnostics for the four globally distributed, parthenogenetic species that are significant agricultural pests. Our analysis of Meloidogyne mtDNA improves on previous studies: (i) by examining restriction site polymorphism among a large number of isolates also characterized for standard morphological, host range and allozyme phenotypes; (ii) by using higher resolution electrophoretic techniques; and (iii) by mapping variable restriction sites with reference to the complete nucleotide sequence. This revealed fivefold less sequence divergence (<0.6%) between variants than estimated in previous restriction fragment length polymorphism (RFLP) studies, but perfect correspondence between mtDNA haplotype and allozyme (esterase) phenotypes. The mtDNA variation, although limited, is strongly structured with as much divergence between two lineages of Meloidogyne arenaria as between either of these and Meloidogyne javanica. The low diversity of mtDNAs suggests that these parthenogenetic lineages arose from distinct but closely related sexual females, a pattern seen in other parthenogenetic complexes. In contrast to the concordance between mtDNA and allozyme markers, there were several discrepancies between the traditional methods of identification. We suggest that further studies of these nematodes should focus on well defined genetic groups, whether or not these coincide with existing taxonomic units.     

Genetic structure and ecogeographical adaptation in wild barley (<Emphasis Type="Italic">Hordeum chilense</Emphasis>Roemer et Schultes) as revealed by microsatellite markers

Background  

Multi-allelic microsatellite markers have become the markers of choice for the determination of genetic structure in plants. Synteny across cereals has allowed the cross-species and cross-genera transferability of SSR markers, which constitute a valuable and cost-effective tool for the genetic analysis and marker-assisted introgression of wild related species. Hordeum chilense is one of the wild relatives with a high potential for cereal breeding, due to its high crossability (both interspecies and intergenera) and polymorphism for adaptation traits. In order to analyze the genetic structure and ecogeographical adaptation of this wild species, it is necessary to increase the number of polymorphic markers currently available for the species. In this work, the possibility of using syntenic wheat SSRs as a new source of markers for this purpose has been explored.     

Microsatellite analysis supports mitochondrial phylogeography of the hellbender (Cryptobranchus alleganiensis)

We investigated genetic diversity of the hellbender (Cryptobranchus alleganiensis) throughout its range in the eastern US using nuclear markers and compared our results to a previously published mitochondrial analysis. A variety of nuclear markers, including protein-coding gene introns and microsatellites were tested but only microsatellites were variable enough for population level analysis. Microsatellite loci showed moderate among population sharing of alleles, in contrast to the reciprocal monophyly exhibited by mitochondrial DNA. However, analyses using F-statistics and Bayesian clustering algorithms showed considerable population subdivision and clustered hellbender populations into the same major groups as the mtDNA. The microsatellites combined with the mtDNA data suggest that gene flow is severely restricted or non-existent among eight major groups, and potentially among populations (rivers) within groups. The combined mtDNA and microsatellite data suggest that the currently recognized hellbender subspecies are paraphyletic. We suggest that the eight independent groups identified in our study should be managed as such, rather than basing conservation decisions on the two named subspecies of hellbender.     

Molecular characterization of relatedness among colour variants of Asian Arowana (Scleropages formosus)

Morphological identification of fish taxa can sometimes prove difficult because phenotypic variation is either being affected by environmental factors, phenotypic characters are highly conserved or marker selection has been inappropriate. DNA based markers especially neutral mitochondrial DNA (mtDNA) have been used widely in recent times to provide better resolution of systematic relationships among vertebrate taxa. The Asian Arowana (Scleropages formosus) is a high value ornamental fish belonging to the family Osteoglossidae with a number of different colour variants distributed geographically across different locations around Southeast Asia. Systematic relationships among colour variants still remain unresolved. Partial sequences of the Cytochrome B (Cyt B) and DNA barcoding gene, Cytochrome C Oxidase I (COI) were used here to assess genetic relationships among colour variants and as a tool for molecular identification for differentiating among colour variants in this species. Results of the study show that in general, colour pattern shows no relationship with extent of COI or Cyt B mtDNA differentiation and so cannot be used to identify taxa. Partial sequences of the mtDNA genes were sufficient however, to identify S. formosus from a closely related species within the order Osteoglossidae.     

Patterns of genetic and phenotypic variation in Iris haynei and I. atrofusca (Iris sect. Oncocyclus = the royal irises) along an ecogeographical gradient in Israel and the West Bank

Iris haynei and I. atrofusca are two closely related narrow endemics distributed vicariously along an ecogeographical north-south gradient in Israel and the West Bank. To obtain baseline information of the taxonomic status, conservation and population history of these taxa, we investigated patterns of phenotypic variation and the partitioning of genetic variation within and among populations using dominant random amplified polymorphic DNA (RAPD) markers. Multivariate (principal components analysis) and taxonomic distance analyses based on morphometric traits from eight populations revealed no unambiguous separation into two distinct groups. Results of genetic analyses for nine populations differed only slightly when either allele- or marker-based approaches were employed. Mean within-population diversity was high (0.258 for Nei's expected heterozygosity), but there was no significant relationship between genetic diversity and either population size or latitude. Although the range-wide estimate of GST ( approximately 0.20) revealed relatively high differentiation among populations this value was inflated because of a small, but significant, component of molecular variance among regions viz. taxa ( approximately 5%). Limited long-distance dispersal capabilities in conjunction with a linearized habitat distribution are proposed to contribute to the approximate isolation by distance pattern observed. It also appears that extant populations are currently deviating from equilibrium conditions because of primary divergence of a formerly more widespread ancestral population. Given the absence of deep genetic and phenotypic subdivision among northern (I. haynei) vs. central/southern (I. atrofusca) populations, we argue for a revision of their species status. Nonetheless, we recommend conservation attention to these geographically differentiated segments as separate management units, which can be seen as an instructive example of incipient species formation.