FY2014 Annual Report

Ecology and Evolution Unit
Assistant Professor Alexander Mikheyev


In FY2014 projects have began to come to fruition. For example, after years of research and development, we finally applied our degraded DNA sequencing techniques to a biological problem – how honey bees respond to novel diseases. We also published collaborative work on the evolution of castes in social insects, and on snake venom evolution.

Lab visitors

This year we two rotation students, who worked on snake venom evolution and fly transgenerationa inheritance, respectively:

  • Jigyasa Arora
  • Dorothy Immaculate

Tsai-Ming worked on annotating genes involved in meiosis in the little fire ant ( Wasmannia auropunctata ), while Shikha worked on snake venom proteomics in two Okinawan habus. We expect that both studies will be submitted for publication in FY2014.

1. Lab members

  • Dr. Alexander Mikheyev, Asistant Professor (PI)
  • Dr. Yutaka Watanabe, Researcher
  • Dr. Misato Okamoto, Researcher
  • Mandy Tin, Technical Staff
  • Miguel Grau Lopez, Technical Staff
  • Carmen Emborski, Special Student
  • Hitomi Shinzato, Research Administrator
  • Yoko Fujitomi, Research Administrator

2. Ongoing joint research collaborations (in order of duration)

2.1. Molecular signatures of co-evolution between fungus-gardening


2.2. Evolutionary ecology of host-associated differentiation in Ephydryas butterflies


2.3. Evolution and composition of snake venoms

  • Steven Aird, OIST

2.4. Using museum collections to understand the evolution of feral honeybees in the United States


2.5. Evolution of social parasitism in harvester ants


2.6. Genetic control of reproductive division of labor in a queenless Diacamma ants


2.7. Genetic and epigenetic control of termite caste regulation

  • Kenji Matsuura , Kyoto University
  • Kazuya Kobayashi, Kyoto University

2.8. Genetics of caste differentiation


3. Published activities and findings

3.1 How do honey bees respond to novel parasites? ( Mikheyev et. al. 2015, Nature Communications )

Understanding genetic changes caused by novel pathogens and parasites can reveal mechanisms of adaptation and genetic robustness. Using whole-genome sequencing of museum and modern specimens, we describe the genomic changes in a wild population of honey bees in North America following the introduction of the ectoparasitic mite, Varroa destructor. Even though colony density in the study population is the same today as in the past, a major loss of haplotypic diversity occurred, indicative of a drastic mitochondrial bottleneck, caused by massive colony mortality. In contrast, nuclear genetic diversity did not change, though hundreds of genes show signs of selection. The genetic diversity within each bee colony, particularly as a consequence of polyandry by queens, may enable preservation of genetic diversity even during population bottlenecks. These findings suggest that genetically diverse honey bee populations can recover from introduced diseases by evolving rapid tolerance, while maintaining much of the standing genetic variation.

Figure 1. Preparing the libraries. (a) DNA fragments are denatured, and (b) a controlled number of riboguanidines are added to the 3′ end.(c) This tail was then used to ligate a full-length Illumina sequencing primer, (d) which can then be used to synthesize the second strand. (e) The other sequencing adaptor can then be ligated to the double-stranded product. This protocol permits direct sequencing of nanogram-scale input templates, without biases and contamination introduced by PCR.

Figure 2. The vast majority of mitochondrial genetic diversity in the old population (blue) has been lost in the modern population (red). Coalescent analysis estimated the 95% highest posterior density of the mitochondrial Ne between 2 × 10 5 and 2 × 10 6 fifty years ago versus between ~300 and 1,800 today. These data suggest that the arrival of V. destructor was associated with massive colony mortality and intense selection acting on the bees. A major mitochondrial clade appears to have completely disappeared. The most common haplotype present in many of the modern bees and one of the old bees, is identical to the A. mellifera ligustica (Italian) mitochondrial haplotype.

3.2 How do organisms create novel phenotypes? ( Smith et al. , 2015, Molecular Biology & Evolution )

A central goal of biology is to uncover the genetic basis for the origin of new phenotypes. A particularly effective approach is to examine the genomic architecture of species that have secondarily lost a phenotype with respect to their close relatives. In the eusocial Hymenoptera, queens and workers have divergent phenotypes that may be produced via either expression of alternative sets of caste-specific genes and pathways or differences in expression patterns of a shared set of multifunctional genes. To distinguish between these two hypotheses, we investigated how secondary loss of the worker phenotype in workerless ant social parasites impacted genome evolution across two independent origins of social parasitism in the ant genera Pogonomyrmex and Vollenhovia. We sequenced the genomes of three social parasites and their most-closely related eusocial host species and compared gene losses in social parasites with gene expression differences between host queens and workers. Virtually all annotated genes were expressed to some degree in both castes of the host, with most shifting in queen-worker bias across developmental stages. As a result, despite >1 My of divergence from the last common ancestor that had workers, the social parasites showed strikingly little evidence of gene loss, damaging mutations, or shifts in selection regime resulting from loss of the worker caste. This suggests that regulatory changes within a multifunctional genome, rather than sequence differences, have played a predominant role in the evolution of social parasitism, and perhaps also in the many gains and losses of phenotypes in the social insects.

Figure 3. The evolutionary relationships, and divergence dates (with 95% CI), for the two monophyletic groups of host and parasites in the subgenus Myrmicinae which are included in this study. The images represent the known castes present in each species; social parasites are those lacking an image for the worker caste. Images from antweb.org.

3.3 What selective forces are acting on snake venoms? ( Aird et. al. 2015, BMC Genomics )

Venomous snakes employ well-integrated systems of proteins and organic constituents to immobilize prey. While many studies have shown snake venom proteins evolve rapidly, how selection acts on them remains poorly understood. In this study we looked at the interaction between ecology, expression level, and evolutionary rate in snake venom evolution. We examined microevolution in two Okinawan pitvipers, allopatrically separated for at least 1.6 million years. Although transcriptomes generally similar compositions in regard to protein families, but for a given protein family, the homologs present and concentrations thereof sometimes differed dramatically. Interestingly, expression levels (i.e., concentrations in the final venom) had dramatic effects on the evolutionary rates of the venoms. Namely, protein evolutionary rates were positively correlated with transcriptomic and proteomic abundances, and the most abundant proteins showed positive selection. This pattern appears to be general and we found it in other published crotaline transcriptomes, from two more genera, and also in the king cobra genome, suggesting that rapid evolution of abundant proteins may be generally true for snake venoms.

Figure 4.
This study started out as an investigation of what happens when two different species hybridize, but moved on to a more general finding. The two parental species were (a) Protobothrops flavoviridis (the Okinawa habu). (b) P. flavoviridis x P. elegan s hybrid. (c) Protobothrops elegans (the Sakishima habu). The Sakishima habu is invasive in Okinawa, and the two snakes occasionally hybridize, producing, most likely, F 1 hybrids.

Figure 5. Secreted proteins evolve rapidly, and more abundant venom toxins evolve most rapidly of all. (a) Violin plot of evolutionary rates of secreted proteins (those detected by mass spectrometry in the venom) vs. the rest of the transcriptome. Secreted proteins evolve significantly faster than the rest of the transcriptome, suggesting that they are subject to atypically strong selection within the genome. (b) Relationship between abundance and nucleic acid evolutionary rate for secreted proteins. See Figure 6 of Aird et al. , 2015 for more details.

4. Publications

4.1. Journals

  • C.R. Smith S. Helms Cahan, C. Kemena, S.G. Brady, W. Yang, E. Bronberg-Bauer, T. Eriksson, J. Gadau, M. Helmkampf, D. Gotzek, M. O. Miyakawa, A.V. Suarez and A.S. Mikheyev, How do genomes create novel phenotypes? Insights from the loss of the worker caste in ant social parasites, Molecular Biology and Evolution, 32: 2919-2931, 2015

  • A.S. Mikheyev, M.M.Y. Tin, J. Arora and T.D. Seeley, Museum samples reveal rapid evolution by wild honey bees exposed to a novel parasite, Nature Communications, 6: 7991, 2015

  • S.D. Aird, S. Aggarwal, A. Villar-Briones, M.M.Y. Tin, K. Terada, and A. S. Mikheyev, Snake venoms are integrated systems, but abundant venom proteins evolve more rapidly. BMC Genomics, 16:647, 2015

  • C. Parmesan, A. Williams-Anderson, M. Moskwik, A.S. Mikheyev and M.C. Singer. Endangered Quino checkerspot butterfly: A climate change success story? Journal of Insect Conservation. 19:185-204, 2015.

  • M.O. Miyakawa and A.S. Mikheyev. Males are here to stay: fertilization enhances viable egg production by clonal queens of the little fire ant ( Wasmannia auropunctata ). The Science of Nature. 102:15, 2015.

  • A.S. Mikheyev and T. Linksvayer. Genes associated with ant social behavior show distinct transcriptional and evolutionary patterns. eLife 2015;10.7554/eLife.04775, 2015.

4.2 Oral and Poster Presentations

  • Kzan’ Federal University, Kazan’, Russia: “Working with ancient DNA: tools and insights”
  • CSIRO, Canberra, Australia: “Working with degraded DNA: laboratory and bioinformatic approaches”
  • Australian National University, Canberra, Australia: “Whole genome re-sequencing of museum specimens revels resilience to disease in a feral population of European honey bees”
  • OIST-NTU Invasive Ant Symposium , Okinawa, Japan: “Molecular signatures of ancient mutualistic coevolution in attine ants and their fungal cultivars”
  • Experimental Evolution Discussion Group, Wageningen University, Wageningen, Netherlands: “Molecular signatures of ancient mutualistic coevolution in attine ants and their fungal cultivars”
  • University of Southern California, Los Angeles: “Whole genome re-sequencing of museum specimens revels resilience to disease in a feral population of European honey bees”
  • University of Southern California, Los Angeles: “Working with degraded DNA: laboratory and bioinformatic approaches”