Whole genome sequencing of the black grouse (Tetrao tetrix): reference guided assembly suggests faster-Z and MHC evolution

Background

The different regions of a genome do not evolve at the same rate. For example, comparative genomic studies have suggested that the sex chromosomes and the regions harbouring the immune defence genes in the Major Histocompatability Complex (MHC) may evolve faster than other genomic regions. The advent of the next generation sequencing technologies has made it possible to study which genomic regions are evolutionary liable to change and which are static, as well as enabling an increasing number of genome studies of non-model species. However, de novo sequencing of the whole genome of an organism remains non-trivial. In this study, we present the draft genome of the black grouse, which was developed using a reference-guided assembly strategy.

Results

We generated 133 Gbp of sequence data from one black grouse individual by the SOLiD platform and used a combination of de novo assembly and chicken reference genome mapping to assemble the reads into 4572 scaffolds with a total length of 1022 Mb. The draft genome well covers the main chicken chromosomes 1 ~ 28 and Z which have a total length of 1001 Mb. The draft genome is fragmented, but has a good coverage of the homologous chicken genes. Especially, 33.0% of the coding regions of the homologous genes have more than 90% proportion of their sequences covered. In addition, we identified ~1 M SNPs from the genome and identified 106 genomic regions which had a high nucleotide divergence between black grouse and chicken or between black grouse and turkey.

Conclusions

Our results support the hypothesis that the chromosome X (Z) evolves faster than the autosomes and our data are consistent with the MHC regions being more liable to change than the genome average. Our study demonstrates how a moderate sequencing effort can be combined with existing genome references to generate a draft genome for a non-model species.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-180) contains supplementary material, which is available to authorized users.     

The Mhc class II of the Black grouse (Tetrao tetrix) consists of low numbers of B and Y genes with variable diversity and expression

We found that the Black grouse (Tetrao tetrix) possess low numbers of Mhc class II B (BLB) and Y (YLB) genes with variable diversity and expression. We have therefore shown, for the first time, that another bird species (in this case, a wild lek-breeding galliform) shares several features of the simple Mhc of the domestic chicken (Gallus gallus). The Black grouse BLB genes showed the same level of polymorphism that has been reported in chicken, and we also found indications of balancing selection in the peptide-binding regions. The YLB genes were less variable than the BLB genes, also in accordance with earlier studies in chicken, although their functional significance still remains obscure. We hypothesize that the YLB genes could have been under purifying selection, just as the mammal Mhc-E gene cluster.     

Sex and age influence intestinal parasite burden in three boreal grouse species

Parasite infections are often more common in male vertebrates than in females. Sexual selection leading to dimorphism can cause sexual differences in immune defence, behaviour or body size. Possible proximate explanations for male bias in parasitism are the immunosuppressive effect of male sex hormones and the large body size of males which increases the likelihood of being parasitized. To evaluate these hypotheses, we studied the prevalence and abundance of intestinal helminth parasites in three boreal grouse species, the capercaillie Tetrao urogallus , the black grouse Tetrao tetrix , and the hazel grouse Bonasa bonasia . The first two are sexually dimorphic polygynous species while the latter species is sexually monomorphic and monogamous. We found a male-bias in the prevalence and abundance of the nematode Ascaridia compar . The bias was most pronounced in the polygynous black grouse and capercaillie. In the monogamous hazel grouse, there was a slight male-bias in occurrence of ascarids, but no bias in abundance. In juvenile grouse, the male-bias was larger than in adult grouse. No sexual bias was found in regard to the cestodes ( Skrjabinia cesticillus , Paroniella urogalli and Hymenolepis spp.). However, age was a factor in cestode prevalence: juvenile grouse were more commonly infected than adults. Differences in growth rates and body size are potential factors that may lead to male-biased parasitism in these grouse species, and their impact requires further studies.     

Genetic variation in the acorn barnacle from allozymes to population genomics

Understanding the patterns of genetic variation within and among populations is a central problem in population and evolutionary genetics. We examine this question in the acorn barnacle, Semibalanus balanoides, in which the allozyme loci Mpi and Gpi have been implicated in balancing selection due to varying selective pressures at different spatial scales. We review the patterns of genetic variation at the Mpi locus, compare this to levels of population differentiation at mtDNA and microsatellites, and place these data in the context of genome-wide variation from high-throughput sequencing of population samples spanning the North Atlantic. Despite considerable geographic variation in the patterns of selection at the Mpi allozyme, this locus shows rather low levels of population differentiation at ecological and trans-oceanic scales (F(ST)?～?5%). Pooled population sequencing was performed on samples from Rhode Island (RI), Maine (ME), and Southwold, England (UK). Analysis of more than 650 million reads identified approximately 335,000 high-quality SNPs in 19 million base pairs of the S. balanoides genome. Much variation is shared across the Atlantic, but there are significant examples of strong population differentiation among samples from RI, ME, and UK. An F(ST) outlier screen of more than 22,000 contigs provided a genome-wide context for interpretation of earlier studies on allozymes, mtDNA, and microsatellites. F(ST) values for allozymes, mtDNA and microsatellites are close to the genome-wide average for random SNPs, with the exception of the trans-Atlantic F(ST) for mtDNA. The majority of F(ST) outliers were unique between individual pairs of populations, but some genes show shared patterns of excess differentiation. These data indicate that gene flow is high, that selection is strong on a subset of genes, and that a variety of genes are experiencing diversifying selection at large spatial scales. This survey of polymorphism in S. balanoides provides a number of genomic tools that promise to make this a powerful model for ecological genomics of the rocky intertidal.     

Establishing genomic tools and resources for Guizotia abyssinica (L.f.) Cass.-the development of a library of expressed sequence tags, microsatellite loci, and the sequencing of its chloroplast genome

We present an EST library, chloroplast genome sequence, and nuclear microsatellite markers that were developed for the semi-domesticated oilseed crop noug (Guizotia abyssinica) from Ethiopia. The EST library consists of 25 711 Sanger reads, assembled into 17 538 contigs and singletons, of which 4781 were functionally annotated using the Arabidopsis Information Resource (TAIR). The age distribution of duplicated genes in the EST library shows evidence of two paleopolyploidizations—a pattern that noug shares with several other species in the Heliantheae tribe (Compositae family). From the EST library, we selected 43 microsatellites and then designed and tested primers for their amplification. The number of microsatellite alleles varied between 2 and 10 (average 4.67), and the average observed and expected heterozygosities were 0.49 and 0.54, respectively. The chloroplast genome was sequenced de novo using Illumina’s sequencing technology and completed with traditional Sanger sequencing. No large re-arrangements were found between the noug and sunflower chloroplast genomes, but 1.4% of sites have indels and 1.8% show sequence divergence between the two species. We identified 34 tRNAs, 4 rRNA sequences, and 80 coding sequences, including one region (trnH-psbA) with 15% sequence divergence between noug and sunflower that may be particularly useful for phylogeographic studies in noug and its wild relatives.     

Guardian or threat: does golden eagle predation risk have cascading effects on forest grouse?

Previous studies on intraguild predation have mainly focused on within-class assemblages, even though avian top predators may also influence mammalian mesopredator prey. By using nation-wide long-term data from Finland, northern Europe, we examined the impacts of golden eagles (Aquila chrysaetos) together with red foxes (Vulpes vulpes) and pine martens (Martes martes) on forest-dwelling herbivores, black grouse (Tetrao tetrix) and hazel grouse (Tetrastes bonasia). We hypothesized that eagles may alleviate the overall predation pressure on grouse by imposing intraguild predation risk on mesopredators. The predation impact of eagle was modelled using eagle density estimates and distance to eagle nest. Wildlife triangle counts were used as predation impact proxies of mammalian mesopredators and as measures of response in grouse. Our results show that eagle density correlated negatively with black grouse abundance indices while being positively associated with the proportion of juveniles in both grouse species, irrespective of the abundance of mesopredators. Yet, foxes and martens alone had a negative effect on the abundance indices and the proportion of young in the two grouse species. This suggests that the possible cascading effects of eagles are not mediated by decreased mesopredator numbers, but instead by fear effects. Alternatively, they may be mediated by other species than fox or marten studied here. In conclusion, we found support for the hypothesis that eagles provide protection for juvenile black and hazel grouse, whereas they are a threat for adult grouse. This important information helps us to better understand the role of avian top predators in terrestrial ecosystems.     

Development of genomic resources for Nothofagus species using next‐generation sequencing data

Using next‐generation sequencing, we developed the first whole‐genome resources for two hybridizing Nothofagus species of the Patagonian forests that crucially lack genomic data, despite their ecological and industrial value. A de novo assembly strategy combining base quality control and optimization of the putative chloroplast gene map yielded ~32 000 contigs from 43% of the reads produced. With 12.5% of assembled reads, we covered ~96% of the chloroplast genome and ~70% of the mitochondrial gene content, providing functional and structural annotations for 112 and 52 genes, respectively. Functional annotation was possible on 15% of the contigs, with ~1750 potentially novel nuclear genes identified for Nothofagus species. We estimated that the new resources (13.41 Mb in total) included ~4000 gene regions representing ~6.5% of the expected genic partition of the genome, the remaining contigs potentially being nongenic DNA. A high‐quality single nucleotide polymorphisms resource was developed by comparing various filtering methods, and preliminary results indicate a strong conservation of cpDNA genomes in contrast to numerous exclusive nuclear polymorphisms in both species. Finally, we characterized 2274 potential simple sequence repeat (SSR) loci, designed primers for 769 of them and validated nine of 29 loci in 42 individuals per species. Nothofagus obliqua had more alleles (4.89) on average than N. nervosa (2.89), 8 SSRs were efficient to discriminate species, and three were successfully transferred in three other Nothofagus species. These resources will greatly help for future inferences of demographic, adaptive and hybridizing events in Nothofagus species, and for conserving and managing natural populations.     

Temporal dynamics of grouse populations at the southern edge of their distribution

Studies of grouse conducted at northern latitudes have shown that tetraonids frequently exhibit cyclic fluctuations in abundance but little is known about the dynamics of grouse species at the southerly edge of their range. Hunting statistics from four species of grouse based on 30 yr of data collected from 210 hunting areas were examined from the Dolomitic Alps in the province of Trentino. These data were summed to represent 18 time series from discrete mountain groups. Analyses identified cycles of ca 5 yr in the minority of rock ptarmigan Lagopus mutus and hazel grouse Bonasa bonasia populations. These cycles only showed significant negative autocorrelation at half the cycle period and were classified as phase-forgetting quasi-cycles. Cycles were not found in time series of black grouse Tetrao terix or capereaillie Tetrao urogallus. Correcting time series for hunting effort or hunting restrictions tended to increase the proportion of populations that exhibited cycles but no difference in the strength of second order density dependence, A linear first order density-dependent autoregressive model described the dynamics of most of the populations with the exception of a proportion of rock ptarmigan and black grouse populations where a non linear first order model provided the best tit. We compare the findings with studies conducted in Finland and suggest possible reasons for the reduced tendency to cycle in the populations of southern Europe.     

Drift and selection influence geographic variation at immune loci of prairie-chickens

Previous studies of immunity in wild populations have focused primarily on genes of the major histocompatibility complex (MHC); however, studies of model species have identified additional immune-related genes that also affect fitness. In this study, we sequenced five non-MHC immune genes in six greater prairie-chicken (Tympanuchus cupido) populations that have experienced varying degrees of genetic drift as a consequence of population bottlenecks and fragmentation. We compared patterns of geographic variation at the immune genes with six neutral microsatellite markers to investigate the relative effects of selection and genetic drift. Global F(ST) outlier tests identified positive selection on just one of five immune genes (IAP-1) in one population. In contrast, at other immune genes, standardized G'(ST) values were lower than those at microsatellites for a majority of pairwise population comparisons, consistent with balancing selection or with species-wide positive or purifying selection resulting in similar haplotype frequencies across populations. The effects of genetic drift were also evident as summary statistics (e.g., Tajima's D) did not differ from neutrality for the majority of cases, and immune gene diversity (number of haplotypes per gene) was correlated positively with population size. In summary, we found that both genetic drift and selection shaped variation at the five immune genes, and the strength and type of selection varied among genes. Our results caution that neutral forces, such as drift, can make it difficult to detect current selection on genes.     

The endangered Attwater's prairie chicken and an analysis of prairie grouse helminthic endoparasitism

Although managers have speculated that parasitism might have contributed to the endangerment of the Attwater's prairie chicken Tympanuchus cupido attwateri , no data are available to evaluate this hypothesis. Because time is constraining, I attempted to determine whether Attwater's prairie chicken are likely to harbor helminthic endoparasites known to cause disease in other grouse so that a more informed decision regarding whether to commit time and other resources to field studies and experimental trials can be made. Using polar ordination, 1 found a gradient among 10 surveys of helminthic endoparasites of prairie grouse collected from several midcontinental ecoregions of the USA (1931 -1977). A regression model including variables representing 1) normal annual precipitation and 2) normal winter (Dec.-Feb.) temperature and the proportion of normal annual precipitation received during the winter could account for this gradient. When meteorological data from areas inhabited by the remaining 3 Attwater's prairie chicken populations were incorporated, the models predicted that Attwater's prairie chicken might be expected to harbor helminthic endoparasite communities similar to those of greater prairie chicken T. c. pinnatus populations surveyed in Illinois, Kansas, and Missouri. Two parasite species associated with disease in North American grouse Dispharynx nasuta, Heterakis gallinarum were found during these studies. Additionally, Attwater's prairie chicken might be expected to maintain parasites not found during any of the surveys I evaluated. This study suggests that it would be worthwhile to survey free-living Attwater's prairie chicken to determine whether individuals harbor helminthic endoparasites shown pathogenic to other grouse species, even if the methodologies employed must be somewhat constrained.     

The genetic network of greater sage‐grouse: Range‐wide identification of keystone hubs of connectivity

Genetic networks can characterize complex genetic relationships among groups of individuals, which can be used to rank nodes most important to the overall connectivity of the system. Ranking allows scarce resources to be guided toward nodes integral to connectivity. The greater sage‐grouse (Centrocercus urophasianus) is a species of conservation concern that breeds on spatially discrete leks that must remain connected by genetic exchange for population persistence. We genotyped 5,950 individuals from 1,200 greater sage‐grouse leks distributed across the entire species’ geographic range. We found a small‐world network composed of 458 nodes connected by 14,481 edges. This network was composed of hubs—that is, nodes facilitating gene flow across the network—and spokes—that is, nodes where connectivity is served by hubs. It is within these hubs that the greatest genetic diversity was housed. Using indices of network centrality, we identified hub nodes of greatest conservation importance. We also identified keystone nodes with elevated centrality despite low local population size. Hub and keystone nodes were found across the entire species’ contiguous range, although nodes with elevated importance to network‐wide connectivity were found more central: especially in northeastern, central, and southwestern Wyoming and eastern Idaho. Nodes among which genes are most readily exchanged were mostly located in Montana and northern Wyoming, as well as Utah and eastern Nevada. The loss of hub or keystone nodes could lead to the disintegration of the network into smaller, isolated subnetworks. Protecting both hub nodes and keystone nodes will conserve genetic diversity and should maintain network connections to ensure a resilient and viable population over time. Our analysis shows that network models can be used to model gene flow, offering insights into its pattern and process, with application to prioritizing landscapes for conservation.