Where do I come from? Using student’s mitochondrial DNA to teach about phylogeny, molecular clocks, and population genetics

Phylogenetic reconstruction, divergence times, and population genetics are critical concepts for a complete understanding of evolution. Unfortunately, students generally lack “tree-thinking” skills and are often unmotivated to explore these concepts using typical classroom exercises that feature taxa unknown to students or simulated datasets. To generate greater student interest, we have developed an affordable practical lab ($16 dollars per student) where students extract and sequence their own mtDNA and use it for exercises involving phylogenetic reconstruction (placement of own DNA into the world tree), divergence (speciation) time (comparing current student population with chimps, gorillas, and Neanderthal), and population genetics (demographic change calculation based on student’s sample). In contrast to traditional labs, we found that students were highly motivated and enthusiastic throughout the four-week activity. Students had a 100% rate of success in obtaining DNA sequences and their evaluations report high satisfaction with the learning outcome. Here we provide all details and datasets needed to run the lab and discuss a series of assessments and possible exercises

Characterisation and expression of microRNAs in developing wings of the neotropical butterfly Heliconius melpomene.

BACKGROUND: Heliconius butterflies are an excellent system for studies of adaptive convergent and divergent phenotypic traits. Wing colour patterns are used as signals to both predators and potential mates and are inherited in a Mendelian manner. The underlying genetic mechanisms of pattern formation have been studied for many years and shed light on broad issues, such as the repeatability of evolution. In Heliconius melpomene, the yellow hindwing bar is controlled by the HmYb locus. MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that have key roles in many biological processes, including development. miRNAs could act as regulators of genes involved in wing development, patterning and pigmentation. For this reason we characterised miRNAs in developing butterfly wings and examined differences in their expression between colour pattern races. RESULTS: We sequenced small RNA libraries from two colour pattern races and detected 142 Heliconius miRNAs with homology to others found in miRBase. Several highly abundant miRNAs were differentially represented in the libraries between colour pattern races. These candidates were tested further using Northern blots, showing that differences in expression were primarily due to developmental stage rather than colour pattern. Assembly of sequenced reads to the HmYb region identified hme-miR-193 and hme-miR-2788; located 2380 bp apart in an intergenic region. These two miRNAs are expressed in wings and show an upregulation between 24 and 72 hours post-pupation, indicating a potential role in butterfly wing development. A search for miRNAs in all available H. melpomene BAC sequences (~2.5 Mb) did not reveal any other miRNAs and no novel miRNAs were predicted. CONCLUSIONS: Here we describe the first butterfly miRNAs and characterise their expression in developing wings. Some show differences in expression across developing pupal stages and may have important functions in butterfly wing development. Two miRNAs were located in the HmYb region and were expressed in developing pupal wings. Future work will examine the expression of these miRNAs in different colour pattern races and identify miRNA targets among wing patterning genes.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Geographic variation in phenotypic divergence between two hybridizing field cricket species

Patterns of morphological divergence across species' ranges can provide insight into local adaptation and speciation. In this study, we compared phenotypic divergence among 4,221 crickets from 337 populations of two closely related species of field cricket, Gryllus firmus and G. pennsylvanicus, and their hybrids. We found that these species differ across their geographic range in key morphological traits, such as body size and ovipositor length, and we directly compared phenotype with genotype for a subset of crickets to demonstrate nuclear genetic introgression, phenotypic intermediacy of hybrids, and essentially unidirectional mitochondrial introgression. We discuss how these morphological traits relate to life history differences between the two species. Our comparisons across geographic areas support prior research suggesting that cryptic variation within G. firmus may represent different species. Our study highlights how variable morphology can be across wide-ranging species and the importance of studying reproductive barriers in more than one or two transects of a hybrid zone

Major improvements to the Heliconius melpomene genome assembly used to confirm 10 chromosome fusion events in 6 million years of butterfly evolution

The Heliconius butterflies are a widely studied adaptive radiation of 46 species spread across Central and South America, several of which are known to hybridize in the wild. Here, we present a substantially improved assembly of the Heliconius melpomene genome, developed using novel methods that should be applicable to improving other genome assemblies produced using short read sequencing. First, we whole-genome-sequenced a pedigree to produce a linkage map incorporating 99% of the genome. Second, we incorporated haplotype scaffolds extensively to produce a more complete haploid version of the draft genome. Third, we incorporated ~20x coverage of Pacific Biosciences sequencing, and scaffolded the haploid genome using an assembly of this long-read sequence. These improvements result in a genome of 795 scaffolds, 275 Mb in length, with an N50 length of 2.1 Mb, an N50 number of 34, and with 99% of the genome placed, and 84% anchored on chromosomes. We use the new genome assembly to confirm that the Heliconius genome underwent 10 chromosome fusions since the split with its sister genus Eueides, over a period of about 6 million yr

Los orígenes de un nuevo gen de diseño de alas de mariposa dentro de una familia de reguladores conservados del ciclo celular

A major challenge in evolutionary biology is to understand the origins of novel structures. The wing patterns of butterflies and moths are derived phenotypes unique to the Lepidoptera. Here we identify a gene that we name poikilomousa (poik), which regulates colour pattern switches in the mimetic Heliconius butterflies. Strong associations between phenotypic variation and DNA sequence variation are seen in three different Heliconius species, in addition to associations between gene expression and colour pattern. Colour pattern variants are also associated with differences in splicing of poik transcripts. poik is a member of the conserved fizzy family of cell cycle regulators. It belongs to a faster evolving subfamily, the closest functionally characterised orthologue being the cortex gene in Drosophila, a female germ-line specific protein involved in meiosis. poik appears to have adopted a novel function in the Lepidoptera and become a major target for natural selection acting on colour and pattern variation in this group

Genomic Hotspots for Adaptation: The Population Genetics of Müllerian Mimicry in the Heliconius melpomene Clade

Wing patterning in Heliconius butterflies is a longstanding example of both Müllerian mimicry and phenotypic radiation under strong natural selection. The loci controlling such patterns are “hotspots” for adaptive evolution with great allelic diversity across different species in the genus. We characterise nucleotide variation, genotype-by-phenotype associations, linkage disequilibrium, and candidate gene expression at two loci and across multiple hybrid zones in Heliconius melpomene and relatives. Alleles at HmB control the presence or absence of the red forewing band, while alleles at HmYb control the yellow hindwing bar. Across HmYb two regions, separated by ∼100 kb, show significant genotype-by-phenotype associations that are replicated across independent hybrid zones. In contrast, at HmB a single peak of association indicates the likely position of functional sites at three genes, encoding a kinesin, a G-protein coupled receptor, and an mRNA splicing factor. At both HmYb and HmB there is evidence for enhanced linkage disequilibrium (LD) between associated sites separated by up to 14 kb, suggesting that multiple sites are under selection. However, there was no evidence for reduced variation or deviations from neutrality that might indicate a recent selective sweep, consistent with these alleles being relatively old. Of the three genes showing an association with the HmB locus, the kinesin shows differences in wing disc expression between races that are replicated in the co-mimic, Heliconius erato, providing striking evidence for parallel changes in gene expression between Müllerian co-mimics. Wing patterning loci in Heliconius melpomene therefore show a haplotype structure maintained by selection, but no evidence for a recent selective sweep. The complex genetic pattern contrasts with the simple genetic basis of many adaptive traits studied previously, but may provide a better model for most adaptation in natural populations that has arisen over millions rather than tens of years

BARRIERS TO GENE EXCHANGE IN A FIELD CRICKET HYBRID ZONE

Understanding how speciation influences patterns of molecular evolution and how molecular changes drive speciation are central questions in evolutionary biology. In this dissertation I address questions of behavioral barriers to gene exchange, cytoplasmic incompatibility, molecular evolution, population history, gene flow and species boundaries in the hybridizing field crickets Gryllus firmus and G. pennsylvanicus. I examine the role of behavioral barriers to gene exchange in the context of previous studies that documented temporal and ecological isolation and a one-way post-mating incompatibility. My results reveal strong behavioral premating barriers, but no apparent fecundity or fertility costs for G. firmus females when they mate with conspecific and heterospecific males. I also document a failure of heterospecific males to induce normal oviposition in G. firmus females, a previously unknown post-mating, pre-zygotic barrier. Gryllus firmus and G. pennsylvanicus exhibit a very clear unidirectional incompatibility and have been cited as a possible example of Wolbachia-induced cytoplasmic incompatibility. Wolbachia are cytoplasmically inherited alpha-proteobacteria that can cause cytoplasmic incompatibility in insects. I conduct curing experiments, intra- and interspecific crosses, cytological examination of Wolbachia in testes, and Wolbachia quantifications via Real-Time PCR. All of the data strongly suggest that Wolbachia are not involved in the reproductive incompatibility between G. firmus and G. pennsylvanicus. Finally I analyze DNA sequence divergence for seminal protein loci, housekeeping loci, and mtDNA, using a combination of analytical approaches and extensive sampling. In recently diverged species, such as G. firmus and G. pennsylvanicus, ancestral polymorphism and introgression can cause incongruence between gene trees and species trees. In the face of hybridization only genomic regions that cannot cross the species boundaries will show reciprocal monophyly. These regions, usually evolving rapidly under selection, are essential for the maintenance of species identity. I report discordant genealogical patterns and differential introgression rates across the genome. The most dramatic outliers, showing near zero introgression and more structured species trees, are also the only two seminal protein loci under selection. These are candidate barrier genes with possible reproductive functions. I also use the genealogical data to examine the demographic history and the current structure of the hybrid zone

High-Throughput Microsatellite Marker Development for the Distylous Herb <i>Primula mistassinica</i> (Primulaceae)

Premise of the study: Twelve microsatellite markers were developed for Primula mistassinica, a distylous, diploid arctic-alpine plant. The markers will be used to investigate the landscape genetics of a disjunct population on Isle Royale, Michigan, and the phylogeographic patterns of the species. Methods and Results: We used Roche/454 high-throughput technology to sequence microsatellite-enriched regions in the P. mistassinica genome. We developed 12 polymorphic microsatellite primer sets. These loci contained di-, tri-, and tetranucleotide repeats with two to nine alleles per locus when assessed in 23 individuals. Conclusions: Understanding the historical movements of P. mistassinica will provide insight to the survival prospects of current Arctic plant populations, which face the pressures of global, anthropogenic climate change

Development and Characterization of 10 Microsatellite Markers in <i>Sagina nodosa</i> (Caryophyllaceae)

Premise of the study: We developed 10 novel microsatellite loci for Sagina nodosa, a diploid perennial arctic-alpine herb. To our knowledge, these are the first microsatellite loci for a Sagina species. Methods and Results: We performed a low-coverage 454 next-generation sequencing of enriched genomic fragments derived from one individual to generate a massive library of contigs containing potential polymorphic microsatellites. We present data for 10 novel polymorphic microsatellite loci containing di-, tri-, tetra-, and hexanucleotide repeats with two to nine alleles per locus assessed in 29 individuals. Conclusions: These polymorphic microsatellite loci in S. nodosa will provide insights on the population structure and life history of S. nodosa in Isle Royale and other North American populations