Diploid genome assembly of the Malbec grapevine cultivar enables haplotype-aware analysis of transcriptomic differences underlying clonal phenotypic variation.

To preserve their varietal attributes, established grapevine cultivars (Vitis vinifera L. ssp. vinifera) must be clonally propagated, due to their highly heterozygous genomes. Malbec is a France-originated cultivar appreciated for producing high-quality wines and is the offspring of cultivars Prunelard and Magdeleine Noire des Charentes. Here, we have built a diploid genome assembly of Malbec, after trio binning of PacBio long reads into the two haploid complements inherited from either parent. After haplotype-aware deduplication and corrections, complete assemblies for the two haplophases were obtained with a very low haplotype switch-error rate (<0.025). The haplophase alignment identified > 25% of polymorphic regions. Gene annotation including RNA-seq transcriptome assembly and ab initio prediction evidence resulted in similar gene model numbers for both haplophases. The annotated diploid assembly was exploited in the transcriptomic comparison of four clonal accessions of Malbec that exhibited variation in berry composition traits. Analysis of the ripening pericarp transcriptome using either haplophases as a reference yielded similar results, although some differences were observed. Particularly, among the differentially expressed genes identified only with the Magdeleine-inherited haplotype as reference, we observed an over-representation of hypothetically hemizygous genes. The higher berry anthocyanin content of clonal accession 595 was associated with increased abscisic acid responses, possibly leading to the observed overexpression of phenylpropanoid metabolism genes and deregulation of genes associated with abiotic stress response. Overall, the results highlight the importance of producing diploid assemblies to fully represent the genomic diversity of highly heterozygous woody crop cultivars and unveil the molecular bases of clonal phenotypic variation.

'Nebbiolo' genome assembly allows surveying the occurrence and functional implications of genomic structural variations in grapevines (Vitis vinifera L.).

BACKGROUND: 'Nebbiolo' is a grapevine cultivar typical of north-western Italy, appreciated for producing high-quality red wines. Grapevine cultivars are characterized by possessing highly heterozygous genomes, including a great incidence of genomic rearrangements larger than 50 bp, so called structural variations (SVs). Even though abundant, SVs are an under-explored source of genetic variation mainly due to methodological limitations at their detection. RESULTS: We employed a multiple platform approach to produce long-range genomic data for two different 'Nebbiolo' clones, namely: optical mapping, long-reads and linked-reads. We performed a haplotype-resolved de novo assembly for cultivar 'Nebbiolo' (clone CVT 71) and used an ab-initio strategy to annotate it. The annotated assembly enhanced our ability to detect SVs, enabling the study of genomic regions not present in the grapevines' reference genome and accounting for their functional implications. We performed variant calling analyses at three different organizational levels: i) between haplotypes of clone CVT 71 (primary assembly vs haplotigs), ii) between 'Nebbiolo' and 'Cabernet Sauvignon' assemblies and iii) between clones CVT 71 and CVT 185, representing different 'Nebbiolo' biotypes. The cumulative size of non-redundant merged SVs indicated a total of 79.6 Mbp for the first comparison and 136.1 Mbp for the second one, while no SVs were detected for the third comparison. Interestingly, SVs differentiating cultivars and haplotypes affected similar numbers of coding genes. CONCLUSIONS: Our results suggest that SVs accumulation rate and their functional implications in 'Nebbiolo' genome are highly-dependent on the organizational level under study. SVs are abundant when comparing 'Nebbiolo' to a different cultivar or the two haplotypes of the same individual, while they turned absent between the two analysed clones.

Independent evolution of intermediate bill widths in a seabird clade.

Interspecific introgression can occur between species that evolve rapidly within an adaptive radiation. Pachyptila petrels differ in bill size and are characterised by incomplete reproductive isolation, leading to interspecific gene flow. Salvin's prion (Pachyptila salvini), whose bill width is intermediate between broad-billed (P. vittata) and Antarctic (P. desolata) prions, evolved through homoploid hybrid speciation. MacGillivray's prion (P. macgillivrayi), known from a single population on St Paul (Indian Ocean), has a bill width intermediate between salvini and vittata and could also be the product of interspecies introgression or hybrid speciation. Recently, another prion population phenotypically similar to macgillivrayi was discovered on Gough (Atlantic Ocean), where it breeds 3 months later than vittata. The similarity in bill width between the medium-billed birds on Gough and macgillivrayi suggest that they could be closely related. In this study, we used genetic and morphological data to infer the phylogenetic position and evolutionary history of P. macgillivrayi and the Gough medium-billed prion relative other Pachyptila taxa, to determine whether species with medium bill widths evolved through common ancestry or convergence. We found that Gough medium-billed prions belong to the same evolutionary lineage as macgillivrayi, representing a new population of MacGillivray's prion that originated through a colonisation event from St Paul. We show that macgillivrayi's medium bill width evolved through divergence (genetic drift) and independently from that of salvini, which evolved through hybridisation (gene flow). This represents the independent convergence towards a similarly medium-billed phenotype. The newly discovered MacGillivray's prion population on Gough is of utmost conservation relevance, as the relict macgillivrayi population in the Indian Ocean is very small.

Whole genome resequencing and custom genotyping unveil clonal lineages in 'Malbec' grapevines (Vitis vinifera L.).

Grapevine cultivars are clonally propagated to preserve their varietal attributes. However, genetic variations accumulate due to the occurrence of somatic mutations. This process is anthropically influenced through plant transportation, clonal propagation and selection. Malbec is a cultivar that is well-appreciated for the elaboration of red wine. It originated in Southwestern France and was introduced in Argentina during the 1850s. In order to study the clonal genetic diversity of Malbec grapevines, we generated whole-genome resequencing data for four accessions with different clonal propagation records. A stringent variant calling procedure was established to identify reliable polymorphisms among the analyzed accessions. The latter procedure retrieved 941 single nucleotide variants (SNVs). A reduced set of the detected SNVs was corroborated through Sanger sequencing, and employed to custom-design a genotyping experiment. We successfully genotyped 214 Malbec accessions using 41 SNVs, and identified 14 genotypes that clustered in two genetically divergent clonal lineages. These lineages were associated with the time span of clonal propagation of the analyzed accessions in Argentina and Europe. Our results show the usefulness of this approach for the study of the scarce intra-cultivar genetic diversity in grapevines. We also provide evidence on how human actions might have driven the accumulation of different somatic mutations, ultimately shaping the Malbec genetic diversity pattern.

Untangling the Hypogeococcus pungens species complex (Hemiptera: Pseudococcidae) for Argentina, Australia, and Puerto Rico based on host plant associations and genetic evidence.

Hypogeococcus pungens, a mealybug native of southern South America, is devastating native cacti in Puerto Rico and threatening cactus diversity in the Caribbean, and potentially in Central and North America. The taxonomic status of H. pungens is controversial since it has been reported feeding not only on Cactaceae but also on other plant families throughout its distribution range. However, in Australia, where the species had been exported from Argentina to control weedy American cacti, it was never found on host plants other than Cactaceae. These conflicting pieces of evidence not only cast doubt on the species identity that invaded Puerto Rico, but also have a negative impact on the search for natural enemies to be used in biological control programs against this pest. Here we present reproductive incompatibility and phylogenetic evidences that give support to the hypothesis that H. pungens is a species complex in which divergence appears to be driven by the host plants. The nuclear EF1α and 18S and the mitochondrial COI genes were used as markers to evaluate the phylogenetic relationships among H. pungens populations collected in Argentina, Australia and Puerto Rico feeding on Cactaceae and/or Amaranthaceae. Additionally, we conducted reciprocal crosses between mealybugs from both hosts. Species delimitation analysis revealed two well-supported putative species within H. pungens, one including mealybugs feeding on Amaranthaceae (H. pungens sensu stricto), and a new undescribed species using Cactaceae as hosts. Additionally, we found asymmetric reproductive incompatibility between these putative species suggesting recent reproductive isolation. The Bayesian species delimitation also suggested that the Australian mealybug population may derive from another undescribed species. Overall, the patterns of genetic differentiation may be interpreted as the result of recent speciation events prompted by host plant shifts. Finally, the finding of a single haplotype in the Puerto Rico population suggests only one invasive event. We still need to identify the geographical origin of the pest in order to enable the use of biological control to reduce the threat to cacti diversity in the Caribbean.

Additive Traits Lead to Feeding Advantage and Reproductive Isolation, Promoting Homoploid Hybrid Speciation.

Speciation through homoploid hybridization (HHS) is considered extremely rare in animals. This is mainly because the establishment of reproductive isolation as a product of hybridization is uncommon. Additionally, many traits are underpinned by polygeny and/or incomplete dominance, where the hybrid phenotype is an additive blend of parental characteristics. Phenotypically intermediate hybrids are usually at a fitness disadvantage compared with parental species and tend to vanish through backcrossing with parental population(s). It is therefore unknown whether the additive nature of hybrid traits in itself could lead successfully to HHS. Using a multi-marker genetic data set and a meta-analysis of diet and morphology, we investigated a potential case of HHS in the prions (Pachyptila spp.), seabirds distinguished by their bills, prey choice, and timing of breeding. Using approximate Bayesian computation, we show that the medium-billed Salvin's prion (Pachyptila salvini) could be a hybrid between the narrow-billed Antarctic prion (Pachyptila desolata) and broad-billed prion (Pachyptila vittata). Remarkably, P. salvini's intermediate bill width has given it a feeding advantage with respect to the other Pachyptila species, allowing it to consume a broader range of prey, potentially increasing its fitness. Available metadata showed that P. salvini is also intermediate in breeding phenology and, with no overlap in breeding times, it is effectively reproductively isolated from either parental species through allochrony. These results provide evidence for a case of HHS in nature, and show for the first time that additivity of divergent parental traits alone can lead directly to increased hybrid fitness and reproductive isolation.

Forest corridors between the central Andes and the southern Atlantic Forest enabled dispersal and peripatric diversification without niche divergence in a passerine.

The central Andean rainforests and the Atlantic Forest are separated by the Chaco and the Cerrado domains. Despite this isolation, diverse evidence suggests that these rainforests have been connected in the past. However, little is known about the timing and geographic positions of these connections, as well as their effects on diversification of species. In this study, we used the Black-goggled Tanager (Trichothraupis melanops, Thraupidae) as a model to study whether the Andean and the Atlantic forests have acted as a refugia system, and to evaluate biogeographic hypotheses of diversification and connection between these rainforests. We compared alternative biogeographic scenarios by using Approximate Bayesian Computation (ABC), modeled range shifts across time, and assessed niche divergence between regions. The results indicated that the major phylogeographic gap within T. melanops is located between these rainforests. The ABC analysis supported peripatric diversification, with initial dispersal from the Atlantic Forest to the Andes during the Mid-Pleistocene. Also, the results supported an Andean-Atlantic forests connection through the current Cerrado-Chaco transition, linking the southern Atlantic Forest with the central Andes. Our findings, taken together with other studies, support that the connection between these biomes has been recurrent, and that has occurred mostly through the Cerrado and/or the Cerrado-Chaco transition. The data also support that the connection dynamic has played an important role in the biological diversification, by promoting peripatric divergence in some forest taxa restricted to both biomes.

Can the intake of antiparasitic secondary metabolites explain the low prevalence of hemoparasites among wild Psittaciformes?

BACKGROUND: Parasites can exert selection pressure on their hosts through effects on survival, on reproductive success, on sexually selected ornament, with important ecological and evolutionary consequences, such as changes in population viability. Consequently, hemoparasites have become the focus of recent avian studies. Infection varies significantly among taxa. Various factors might explain the differences in infection among taxa, including habitat, climate, host density, the presence of vectors, life history and immune defence. Feeding behaviour can also be relevant both through increased exposure to vectors and consumption of secondary metabolites with preventative or therapeutic effects that can reduce parasite load. However, the latter has been little investigated. Psittaciformes (parrots and cockatoos) are a good model to investigate these topics, as they are known to use biological control against ectoparasites and to feed on toxic food. We investigated the presence of avian malaria parasites (Plasmodium), intracellular haemosporidians (Haemoproteus, Leucocytozoon), unicellular flagellate protozoans (Trypanosoma) and microfilariae in 19 Psittaciformes species from a range of habitats in the Indo-Malayan, Australasian and Neotropical regions. We gathered additional data on hemoparasites in wild Psittaciformes from the literature. We considered factors that may control the presence of hemoparasites in the Psittaciformes, compiling information on diet, habitat, and climate. Furthermore, we investigated the role of diet in providing antiparasitic secondary metabolites that could be used as self-medication to reduce parasite load. RESULTS: We found hemoparasites in only two of 19 species sampled. Among them, all species that consume at least one food item known for its secondary metabolites with antimalarial, trypanocidal or general antiparasitic properties, were free from hemoparasites. In contrast, the infected parrots do not consume food items with antimalarial or even general antiparasitic properties. We found that the two infected species in this study consumed omnivorous diets. When we combined our data with data from studies previously investigating blood parasites in wild parrots, the positive relationship between omnivorous diets and hemoparasite infestation was confirmed. Individuals from open habitats were less infected than those from forests. CONCLUSIONS: The consumption of food items known for their secondary metabolites with antimalarial, trypanocidal or general antiparasitic properties, as well as the higher proportion of infected species among omnivorous parrots, could explain the low prevalence of hemoparasites reported in many vertebrates.

Does genetic structure reflect differences in non-breeding movements? A case study in small, highly mobile seabirds.

BACKGROUND: In seabirds, the extent of population genetic and phylogeographic structure varies extensively among species. Genetic structure is lacking in some species, but present in others despite the absence of obvious physical barriers (landmarks), suggesting that other mechanisms restrict gene flow. It has been proposed that the extent of genetic structure in seabirds is best explained by relative overlap in non-breeding distributions of birds from different populations. We used results from the analysis of microsatellite DNA variation and geolocation (tracking) data to test this hypothesis. We studied three small (130-200 g), very abundant, zooplanktivorous petrels (Procellariiformes, Aves), each sampled at two breeding populations that were widely separated (Atlantic and Indian Ocean sectors of the Southern Ocean) but differed in the degree of overlap in non-breeding distributions; the wintering areas of the two Antarctic prion (Pachyptila desolata) populations are separated by over 5000 km, whereas those of the blue petrels (Halobaena caerulea) and thin-billed prions (P. belcheri) show considerable overlap. Therefore, we expected the breeding populations of blue petrels and thin-billed prions to show high connectivity despite their geographical distance, and those of Antarctic prions to be genetically differentiated. RESULTS: Microsatellite (at 18 loci) and cytochrome b sequence data suggested a lack of genetic structure in all three species. We thus found no relationship between genetic and spatial structure (relative overlap in non-breeding distributions) in these pelagic seabirds. CONCLUSIONS: In line with other Southern Ocean taxa, geographic distance did not lead to genetic differences between widely spaced populations of Southern Ocean petrel species.

High prevalence of Trichomonas gallinae in wild columbids across western and southern Europe.

BACKGROUND: Avian trichomonosis is known as a widespread disease in columbids and passerines, and recent findings have highlighted the pathogenic character of some lineages found in wild birds. Trichomonosis can affect wild bird populations including endangered species, as has been shown for Mauritian pink pigeons Nesoenas mayeri in Mauritius and suggested for European turtle doves Streptopelia turtur in the UK. However, the disease trichomonosis is caused only by pathogenic lineages of the parasite Trichomonas gallinae. Therefore, understanding the prevalence and distribution of both potentially pathogenic and non-pathogenic T. gallinae lineages in turtle doves and other columbids across Europe is relevant to estimate the potential impact of the disease on a continental scale. RESULTS: We examined 281 samples from four wild columbid species for Trichomonas infection and determined the genetic lineages. The overall prevalence was 74%. There were significant differences between the species (P = 0.007). The highest prevalence was found in stock doves Columba oenas (86%, n = 79) followed by wood pigeons Columba palumbus (70%, n = 61) and turtle doves (67%, n = 65), while three of five collared doves Streptopelia decaocto (60%) were infected. We found seven different lineages, including four lineages present in columbids in the UK, one lineage already described from Spain and three new lineages, one of those found in a single turtle dove migrating through Italy and another one found in a breeding stock dove. Stock doves from Germany and collared doves from Malta were infected with a potentially pathogenic lineage (lineage A/B), which is known to cause lesions and mortality in columbids, raptors and finches. CONCLUSIONS: Generally, turtle doves showed high prevalence of Trichomonas infection. Furthermore, the potentially pathogenic lineage A/B (or genotype B according to previous literature) was found in a recovering stock dove population. Both findings are worrying for these columbid species due to the occasional epidemic character of trichomonosis, which can have severe negative effects on populations.

Genomic evidence of demographic fluctuations and lack of genetic structure across flyways in a long distance migrant, the European turtle dove.

BACKGROUND: Understanding how past climatic oscillations have affected organismic evolution will help predict the impact that current climate change has on living organisms. The European turtle dove, Streptopelia turtur, is a warm-temperature adapted species and a long distance migrant that uses multiple flyways to move between Europe and Africa. Despite being abundant, it is categorized as vulnerable because of a long-term demographic decline. We studied the demographic history and population genetic structure of the European turtle dove using genomic data and mitochondrial DNA sequences from individuals sampled across Europe, and performing paleoclimatic niche modelling simulations. RESULTS: Overall our data suggest that this species is panmictic across Europe, and is not genetically structured across flyways. We found the genetic signatures of demographic fluctuations, inferring an effective population size (Ne) expansion that occurred between the late Pleistocene and early Holocene, followed by a decrease in the Ne that started between the mid Holocene and the present. Our niche modelling analyses suggest that the variations in the Ne are coincident with recent changes in the availability of suitable habitat. CONCLUSIONS: We argue that the European turtle dove is prone to undergo demographic fluctuations, a trait that makes it sensitive to anthropogenic impacts, especially when its numbers are decreasing. Also, considering the lack of genetic structure, we suggest all populations across Europe are equally relevant for conservation.

Evolutionary factors affecting the cross-species utility of newly developed microsatellite markers in seabirds.

Microsatellite loci are ideal for testing hypotheses relating to genetic segregation at fine spatio-temporal scales. They are also conserved among closely related species, making them potentially useful for clarifying interspecific relationships between recently diverged taxa. However, mutations at primer binding sites may lead to increased nonamplification, or disruptions that may result in decreased polymorphism in nontarget species. Furthermore, high mutation rates and constraints on allele size may also with evolutionary time, promote an increase in convergently evolved allele size classes, biasing measures of interspecific genetic differentiation. Here, we used next-generation sequencing to develop microsatellite markers from a shotgun genome sequence of the sub-Antarctic seabird, the thin-billed prion (Pachyptila belcheri), that we tested for cross-species amplification in other Pachyptila and related sub-Antarctic species. We found that heterozygosity decreased and the proportion of nonamplifying loci increased with phylogenetic distance from the target species. Surprisingly, we found that species trees estimated from interspecific FST provided better approximations of mtDNA relationships among the studied species than those estimated using DC , even though FST was more affected by null alleles. We observed a significantly nonlinear second order polynomial relationship between microsatellite and mtDNA distances. We propose that the loss of linearity with increasing mtDNA distance stems from an increasing proportion of homoplastic allele size classes that are identical in state, but not identical by descent. Therefore, despite high cross-species amplification success and high polymorphism among the closely related Pachyptila species, we caution against the use of microsatellites in phylogenetic inference among distantly related taxa.

Phylogeography and genetic structure of two Patagonian shag species (Aves: Phalacrocoracidae).

We compared the phylogeographic and genetic structure of two sympatric shag species, Phalacrocorax magellanicus (rock shag) and Phalacrocorax atriceps (imperial shag), from Patagonia (southern South America). We used multilocus genotypes of nuclear DNA (microsatellite loci) from 324 individuals and mitochondrial DNA sequences (ATPase) from 177 individuals, to evaluate hypotheses related to the effect of physical and non-physical barriers on seabird evolution. Despite sharing many ecological traits, the focal species strongly differ in two key aspects: P. magellanicus has a strong tendency to remain at/near their breeding colonies during foraging trips and the non-breeding season, while P. atriceps exhibits the converse pattern. Both species showed similar mtDNA genetic structure, where colonies from the Atlantic Coast, Pacific Coast and Fuegian region were genetically divergent. We also found similarities in the results of Bayesian clustering analysis of microsatellites, with both species having four clusters. However population differentiation (e.g. Fst, Φst) was higher in P. magellanicus compared to P. atriceps, and average membership probabilities of individuals to specific clusters (Q-values) were also higher in the former. Phalacrocorax magellanicus has strong phylogeographic structure, consistent with the impact of Pleistocene glaciations, with diagnostic haplotypes associated with each of the three mentioned regions. The same pattern was not as evident for P. atriceps. Migration rate estimators were higher for P. atriceps than for P. magellanicus; however both species followed an n-island-like model of gene flow, this implies that dispersal occurs across the continental land mass that separates Atlantic and Pacific Oceans. Our results supported the hypothesis that non-physical barriers are important drivers of the genetic and phylogeographic structure in seabirds, and also that physical barriers constitute effective but not absolute impediments to gene flow.