the african butterfly bicyclus anynana: a model for evolutionary genetics and evolutionary...

doi: 10.1101/pdb.emo122Cold Spring Harb Protoc; Paul M. Brakefield, Patrícia Beldade and Bas J. Zwaan Evolutionary Developmental Biology

A Model for Evolutionary Genetics andBicyclus anynana:The African Butterfly

ServiceEmail Alerting click here.Receive free email alerts when new articles cite this article -

CategoriesSubject Cold Spring Harbor Protocols.Browse articles on similar topics from

(880 articles)Laboratory Organisms, general (322 articles)Genetics, general

(284 articles)Emerging Model Organisms (582 articles)Developmental Biology

http://cshprotocols.cshlp.org/subscriptions go to: Cold Spring Harbor Protocols To subscribe to

Cold Spring Harbor Laboratory Press at UNIVERSITE LAVAL on May 31, 2014 - Published by http://cshprotocols.cshlp.org/Downloaded from


http://cshprotocols.cshlp.org/cgi/alerts/ctalert?alertType=citedby&addAlert=cited_by&saveAlert=no&cited_by_criteria_resid=protocols;10.1101/pdb.emo122&return_type=article&return_url=http://cshprotocols.cshlp.org/content/10.1101/pdb.emo122.full.pdf

http://cshprotocols.cshlp.org/cgi/collection/developmental_biology

http://cshprotocols.cshlp.org/cgi/collection/emerging_model_organisms

http://cshprotocols.cshlp.org/cgi/collection/genetics_general

http://cshprotocols.cshlp.org/cgi/collection/laboratory_organisms_general

http://cshprotocols.cshlp.org/cgi/subscriptions

http://cshprotocols.cshlp.org/

http://www.cshlpress.com



The African Butterfly Bicyclus anynana: A Model for EvolutionaryGenetics and Evolutionary Developmental Biology

Paul M. Brakefield,1 Patrícia Beldade, and Bas J. Zwaan

Institute of Biology, Leiden University, 2300 RA Leiden, The Netherlands

INTRODUCTION

The butterfly model based on laboratory stocks of the African species Bicyclus anynana provides a spe-cial system for several reasons. First, a range of phenotypes has proven to be amenable to examina-tion in this system. These include wing color patterns (including eyespots), seasonal forms, maleandroconia (secondary sexual traits), and a range of life-history traits (relevant to aging research).These phenotypes have a clear ecological relevance that is associated with dramatic differences in eco-logical environments represented by the dry and wet seasons in East Africa. Second, the Bicyclus genusand closely related genera from independent radiations in Asia and Madagascar are highly speciose,thereby providing opportunities to explore diversity among species for wing patterning, life histories,and male secondary sexual traits. There are also rich opportunities for examining interactions amongall of these phenotypes and both natural and sexual selection. Moreover, the size of the organismsprovides important practical advantages. B. anynana individuals are small enough to be readily rearedin large numbers, but big enough to allow marking and tracking and also to facilitate such manipu-lations as microsurgical procedures on developing wing discs and the noninvasive sampling ofhemolymph. Here, we explore the characteristics of B. anynana that enable integrative research link-ing variations among genotypes via development and physiology to variations in phenotypes and vari-ations in adaptation to natural environments.

BACKGROUND INFORMATION

Natural History of Bicyclus Butterflies

The tropical butterfly B. anynana (Butler 1879) was first established as a laboratory stock in Leiden in1988. The stock was founded with more than 80 gravid females collected by Paul Brakefield in theunderstory of a rubber plantation adjacent to primary forest near Nkhata Bay on the western shore ofLake Malawi in Malawi, Africa. B. anynana is a small, brown butterfly of the nymphalid tribe Satyrinae(Lepidoptera; Nymphalidae). Condamin (1973) published an important monograph on the genusBicyclus (Kirby 1871). This species-rich genus includes approximately 80 species distributed through-out sub-Saharan Africa (excluding Madagascar). Most species are forest or woodland dwellers, nor-mally flying at or close to the ground. The larvae feed on different species of grasses. Adults feed onfallen fruit and can be collected readily with handheld nets or fruit-baited traps (Windig et al. 1994;Molleman et al. 2006).

The male secondary sexual traits represented by wing androconia are a crucial taxonomic char-acteristic together with features of the wing venation and the genitalia. The androconia distribute sexpheromones over the female antennae during courtship (Robertson and Monteiro 2005; Nieberdinget al. 2008), and they vary in number, position, and morphology among species. The wing patternsare also diverse (Roskam and Brakefield 1996) and include marginal eyespots that can function indeflecting the attacks of birds away from the vulnerable body (Lyytinen et al. 2004). Species range

© 2009 Cold Spring Harbor Laboratory Press 1 Vol. 4, Issue 5, May 2009

1Corresponding author ([email protected])This article is also available in Emerging Model Organisms: A LaboratoryManual, Vol. 1. CSHL Press, Cold Spring Harbor, NY, USA, 2009.Cite as: Cold Spring Harb Protoc; 2009; doi:10.1101/pdb.emo122 www.cshprotocols.org

Emerging Model Organisms




www.cshprotocols.org 2 Cold Spring Harbor Protocols

from those with a wide distribution to narrowly distributed endemics found in a single forest. Manyspecies occur in wet equatorial forests, whereas others inhabit highly seasonal environments (Roskamand Brakefield 1996). A molecular phylogeny for about two-thirds of the species, based on both mito-chondrial and nuclear gene sequences, has been produced by Monteiro and Pierce (2001).

The Story of B. anynana

As usual, the story of why the research community initiated studies using Bicyclus is a mixture of sci-entific logic and serendipity. Paul Brakefield had studied the ecological genetics of the marginal wingspots of the meadow brown butterfly in Europe. Presenting this work at the first conference on but-terfly biology in London in 1981, Torben Larsen suggested that the tropical satyrids that express sea-sonal polyphenism would provide far better opportunities for analyzing how natural selection workson butterfly eyespots (Brakefield and Larsen 1984). A Kenyan field trip and some pilot studies in Cardiffwith Bicyclus safitza (which proved less suitable for laboratory culture) led to collection of the originalstock of B. anynana in Malawi. Then, following Fred Nijhout’s classic microsurgical studies on eyespotformation in Junonia (Precis) coenia, Vernon French in Edinburgh helped to set up the B. anynanaresearch program in evolutionary developmental biology that became based in Leiden. Sean Carroll’steam at the University of Wisconsin at Madison analyzed the expression of key developmental path-ways from Drosophila wings in the eyespot organizer in J. coenia; this analysis stimulated the develop-ment of molecular approaches in B. anynana. More recently, genomic and transgenesis tools havebrought the system up to be a front-player in modern research.

B. anynana as the Butterfly “Lab Rat”

The B. anynana system was first established for studies in ecological and evolutionary genetics. Anoverall aim has been to link genetic variation via developmental and physiological mechanisms to thevariation in phenotype that is screened by natural selection (Beldade and Brakefield 2002). Researchon the processes that generate phenotypic variation in this species can be done in the context of thefunctional significance of the variation in natural environments. This effort is beginning to explore howthose processes, together with natural selection, influence the directions taken in evolution and thepatterns of diversity observed within lineages (Beldade et al. 2002b; Zijlstra et al. 2004; Allen et al.2008). This work on adaptive evolution is now being extended to examining the processes of specia-tion in the Bicyclus lineage, especially those involving diversification in the wing androconia and theassociated male sex pheromone system (Nieberding et al. 2008).

B. anynana Stock Center

Stocks kept in laboratories reflect different “flavors” of variation, including phenotypic plasticity, muta-tions of large effect, and segregating quantitative variant traits. More than 30 spontaneous mutationsidentified through their discrete effects on morphology, usually in wing pattern, have been isolated inLeiden and used to establish genetic stocks (Fig. 1). X-ray mutagenesis has been applied to adult malesto screen for phenotypic variants in their offspring, but it proved impossible to establish any variantsas breeding stocks (Monteiro et al. 2003). In addition, artificial selection has provided a major toolused to explore patterns of existing genetic variation for morphological and life-history traits (dis-cussed below).

SOURCES AND HUSBANDRY

The laboratory stock of B. anynana has been maintained in Leiden with several hundred adults in each(non-overlapping) generation. The stock is now also kept in several other laboratories in Europe andNorth America. Effective population sizes are about two-thirds of the census number under our stan-dard culturing conditions in the laboratory (Brakefield et al. 2001).

Larvae are typically fed and maintained on pot-grown maize plants (Zea mays). Different-sizedgroups of larvae are raised in net sleeves or in larger cages. Prepupae and pupae can be readilyremoved from the foliage and will eclose (emerge from the pupal stage) successfully when layered inPetri dishes. For further details on the care and propagation of B. anynana, see Culture andPropagation of Laboratory Populations of the African Butterfly Bicyclus anynana (Brakefield et al.2009a).




RELATED SPECIES

B. anynana is a member of the subtribe Mycalesini that is distributed from northeast Australia throughAsia and into sub-Saharan Africa. Mycalesina butterflies have shown three of the most spectacular radi-ations of butterflies in the Old World tropics: in mainland Africa, on Madagascar, and in Asia, respec-tively. These expansions appear to have occurred in concert with those of the Poaceae (the plants theirlarvae eat), some 30-25 million years ago in the Oligocene epoch (Peña and Wahlberg 2008).

Several other species of Bicyclus have been brought to the laboratory in Leiden, but none havebeen maintained successfully for more than a few generations. The species from tropical wet foresthabitats have comparatively long larval developmental times and are very difficult to culture. B. safitzaand some other species from highly wet-dry seasonal environments may eventually prove possible toculture continuously in the laboratory. In addition, we have recently successfully established a sisterspecies from the radiation on Madagascar, Heteropsis iboina, for comparative studies with Bicyclus.

STUDIES IN EVOLUTION AND DEVELOPMENT

Adaptive Phenotypic Plasticity

Research on B. anynana concentrated initially on the evolution of developmental plasticity, which isexpressed as a seasonal polyphenism in the adult stage. An analysis of the daily trap captures of five


FIGURE 1. Mutant stocks illustrating variation in B. anynana color patterns. At the center, the “wild-type” phenotypeof the ventral surface of the adult (top) forewing and (bottom) hindwing shows the series of marginal eyespots charac-teristic of B. anynana adults. The surrounding images are examples of spontaneous mutants with large effects on dif-ferent aspects of adult wing pattern (variations in eyespot number and morphology, in wing venation coupled witheyespot pattern, and in overall pigmentation) and preadult pigmentation. (The Chocolate mutant with dark larvalintegument is shown next to a typical wild-type final instar larva.) (For color figure, see doi: 10.1101/pdb.emo122online at www.cshprotocols.org.)





species of Bicyclus, including B. anynana, in a forest-edge environment in Malawi provided baselineinformation on this phenomenon (Windig et al. 1994). A wet-season form with eyespots and a medialband flies during the hot rainy season. This form alternates with a brown, uniformly colored, crypticform that survives throughout the long, cool dry season (Fig. 2). The ways in which this polyphenismis influenced by natural selection, differences in resting backgrounds, and predation have been inves-tigated by use of capture-recapture experiments (Brakefield and Frankino 2009) and laboratory stud-ies with potential predators (Lyytinen et al. 2004). The phenotypic plasticity is induced by ambienttemperature in the late larval period and is mediated by the ecdysteroid hormones (Brakefield et al.1998; Kooi and Brakefield 1999; see further below). Declining temperatures in the early dry seasonact as a cue for larvae that will develop into the dry-season-form generation (Windig et al. 1994). Theseasonal polyphenism provides an adaptive response to the alternating seasonal environments thatdiffer in mortality factors and reproductive opportunities. Lines produced by artificial selection nowyield only one or the other of the alternative seasonal forms across the whole range of rearing tem-peratures (Brakefield et al. 1996). A set of crosses between these lines has suggested that from five to10 genes are involved in producing the highly divergent phenotypes (Wijngaarden and Brakefield2000). Work on the wing patterns of the seasonal forms has also been extended to cover differencesin life-history traits (see below).

Eyespot Evolutionary Developmental Biology

Following earlier work by Fred Nijhout (for review, see Nijhout 1991), the evolution and developmentof wing eyespot formation became an important research focus (for reviews, see Beldade andBrakefield 2002; McMillan et al. 2002). Each eyespot is made up of concentric rings of color formedby specialized epithelial cells (scale cells) that have synthesized a particular color pigment shortlybefore adult eclosion. The decisions that specify pigment identity are made earlier in development,shortly before and immediately after pupation. Each eyespot is formed around an inductive organizerknown as an eyespot focus. Some of the genetic pathways and developmental mechanisms involvedin pattern determination have been identified using a combination of microsurgical manipulations and

FIGURE 2. Phenotypic plasticity in Bicyclus. (Top row) Photographs of the habitat of the butterflies at the end of the wetseason (shown online in color as a green background in a) and in the early dry season (shown online in color as brown,dry leaves in b). Photographs were taken at exactly the same spot near Zomba, Malawi. (Bottom row, c) A mating pairof the wet-season form (B. safitza); (d) an individual of the dry-season form of Bicyclus cottrelli that has just alighted onleaf litter, thus hiding the ventral eyespot; (e) both forms of B. safitza. Photographs c and d were taken in the wild atZomba, whereas e is of two sisters raised in the laboratory. In e, the wet-season-form individual was reared throughoutdevelopment at 27°C, whereas the late larva of the dry-season-form butterfly was switched to a lower temperature(Reproduced from Brakefield and Frankino 2009, with permission from Science Publishers © 2009). (For color figure,see doi: 10.1101/pdb.emo122 online at www.cshprotocols.org.)




gene-expression studies (see, e.g., Brakefield et al. 1996; Brunetti et al. 2001; Reed and Serfas 2004;Saenko et al. 2008). An eyespot focus can be transplanted to a different site in very early pupae, anexperiment that results in an ectopic eyespot being formed around the grafted tissue. A series of genesfrom well-known signaling pathways has been shown to be expressed at different stages in the eye-spot focus and, later, in the surrounding epithelial cells. An early study has mapped a component ofphenotypic variation in eyespot size to the Distal-less locus (Beldade et al. 2002a). A modelingapproach has also begun to be applied to eyespot pattern determination in Bicyclus (Dilão and Sainhas2004; Marcus and Evans 2008). The Bicyclus community as a whole is now developing the toolsrequired for gene mapping, genomics, transgenics (Marcus et al. 2004; Beldade et al. 2006, Beldadeet al. 2008; Ramos et al. 2006), and studies of embryonic development, which has been shown to beassociated with eyespot development (Saenko et al. 2008).

STUDIES IN BEHAVIOR AND LIFE-HISTORY EVOLUTION

Behavioral Studies

The two wing androconia of male B. anynana have been found to produce three sex pheromone com-ponents that enhance male mating success via receptors on female antenna (Nieberding et al. 2008).Six consecutive steps have been detected by video analysis in a typical successful courtship: location,orientation, flickering, thrust, attempting, and copulation. The flickering phase, during which the maleinitiates rapid flapping wing movements in front of the female, causes the androconia to fan out andpresumably spread the pheromone on to the female. We have developed a useful tool for assayingmale mating success and studying sexual selection (Joron and Brakefield 2003). Males painted with afluorescent dust on their genitalia transfer the dust during copulation. Thus, males with different phe-notypes can be marked with a group-specific color of dust and released in a flight cage or greenhouse.Receptive females released into the same greenhouse will mate during a period of 24-48 h and canthen be recaptured and inspected under UV light to identify their mating partner by group. This tech-nique was first used to demonstrate a mating advantage for outbred over inbred males under free-flying conditions. The genetic load of B. anynana is higher than that of Drosophila melanogaster and isinherited mainly through the paternal line (Saccheri et al. 2005).

Life-History Evolution

Research studies have also focused on conservation genetics and inbreeding depression (Saccheriet al. 2005), on fluctuating asymmetry (Breuker and Brakefield 2003), and, especially, on life-historyevolution, including the basis of variation in rates of aging (http://www.lifespannetwork.nl). The lat-ter area of interest in evolutionary medicine has followed from the recognition that although adults ofthe wet-season form are short-lived and reproduce rapidly in a favorable environment, those of thedry-season form are long-lived and allocate more resources to survival in a stressful environment. Thedry-season form individuals must survive as active adults for several months of reproductive dormancyin a cool climate before they can reproduce with the rains and increased temperatures of the early wetseason. These differences in life histories have been the focus for work on the evolutionary genetics ofkey life-history traits, including egg size, developmental time, size at maturity, life span, and starvationresistance (e.g., Zijlstra et al. 2004; Fischer et al. 2007; Pijpe et al. 2008).

Physiology, Plasticity, and Life Histories

The roles of ecdysteroid hormones in regulating the phenotypic plasticity of the wing pattern and inpre-adult developmental time have been investigated (Brakefield et al. 1998; Zijlstra et al. 2004).Microinjections or infusions of 20-OH-ecdysone into early pupae fated to develop into the dry-seasonform yield adults with wing patterns more characteristic of the wet-season form. By studying theecdysone receptor using immunological staining against the receptor protein, PB Koch and PMBrakefield (unpubl.) showed that (1) the location of ecdysone receptors coincides with the location ofthe developing eyespot; (2) upon injection of ecdysone, the number of ecdysone receptors increases;and (3) butterflies artificially selected for smaller eyespots on the ventral wing surface have fewerecdysone receptors at the position of the developing eyespot.

Correlated responses are regularly observed in artificial experiments on phenotypic plasticity orlife-history traits. These can be explained in part by the involvement of ecdysone in the regulation ofthe different traits (Zijlstra et al. 2004). For example, lines that have been selected for short or long






pre-adult developmental time show corresponding changes toward larger or smaller ventral eyespots,respectively. Fast-developing butterflies have higher levels of ecdysone shortly after pupation than doslow-developing butterflies. In addition, the slow-selected butterflies show a much smaller responseto ecdysone injection in the pupal stage (increase in pupal developmental time and increase in ven-tral eyespot size) than do the fast-selected butterflies. In general, measuring ecdysone levels in larval,pupae, or adult butterflies will provide information on ecdysone production, whereas the responses toecdysone injection will provide information on the sensitivity of the target tissues to ecdysone (likelyto be at least partly caused by differences in the number of ecdysone receptors). We have also begunto explore the involvement of juvenile hormone (JH). We have used several JH forms and analogs(JH-III, methoprene, and periproxyfen) to study the influence of this hormone on several life-historytraits, such as egg size and life span. Generally, the effects of these manipulations are very small(BJ Zwaan, J Pijpe, L Doorduin, ANM Bot, and FMHN Kesbeke, unpubl.), probably because we lack thebasic knowledge of JH titer dynamics in the life history of Bicyclus. Only very recently werewe able to measure the titers of JH, because of the very low levels of JH normally present in insects.

GENETIC RESOURCES: POPULATIONS

Artificial selection is routinely applied for a series of morphological and life-history traits in B. anynana.The following are among the traits that we have targeted (usually in upward and downward direc-tions) and for which we have retained selected lines in the laboratory:

1. Seasonal polyphenism: A dry-season-form line and a wet-season-form line (Brakefield et al. 1996);the elevation and shape of the norm of reaction for eyespot size against rearing temperature(Brakefield and Frankino 2009).

2. Eyespot patterns: The size, color composition, shape, and position of individual eyespots(McMillan et al. 2002) and the pattern of relative size and color composition among particular eye-spots (Beldade et al. 2002b; Allen et al. 2008).

3. Allometric growth patterns: Wing size relative to body size and forewing size relative to hindwingsize (Frankino et al. 2005, Frankino et al. 2007).

4. Life-history traits: Egg size, developmental time (both as a single trait and in combination with eye-spot plasticity), pupal weight, protandry, adult starvation resistance, and life span (e.g., Zijlstra etal. 2004; Fischer et al. 2007; Pijpe et al. 2008).

Mutant stocks are available that carry alleles arising from spontaneous mutations and yielding adiscrete change in morphology. These stocks include the following, as grouped by the principalphenotypic change:

1. Eyespot patterns (see Fig. 1): Eyespot size, color, or shape and the presence or absence of specificsubsets of eyespots. One of these mutants also has vestigial development of the wing androconia,although such pleiotropic effects on other wing or adult traits are the exception. Five of more than20 of the stocks are, however, homozygous lethal in embryonic development (Saenko et al. 2008).

2. Medial band: The pale band across the ventral wings has a dent in it on both wings rather thanbeing nearly straight.

3. Venation: Wing pattern mutants such as Cyclops (Brakefield et al. 1996). Individual wing veins ortrachea are either vestigial or additional to the normal pattern, or the whole venation system isweakly formed (Saenko et al. 2008).

4. Eye color: The pearl mutant with cream-colored eyes (the ultrastructure of the eye and the opsinshave been studied by DG Stavenga, K Arikawa, and PM Brakefield (unpubl.).

5. Pigmentation: Several wing color mutants make the brown background color either darker orpaler, or yield a distal wing portion that is a pale cream color (Brakefield et al. 2001). In addition,spontaneous mutations have also been isolated that affect other tissues and/or developmentalstages, including a larval mutant with a much darker brown color in the final instar (Chocolate) anda yellow pupal mutant whose color is due to loss of synthesis of a blue, pterobilin pigment (yellowpigments are grass-derived carotenoids).

6. Size: Pygmy-sized adults with an extended larval developmental time.




GENETIC RESOURCES: GENOMICS AND TRANSGENESIS

Recent and ongoing efforts to develop genomic and transgenic tools for B. anynana are opening upa new generation of questions concerning linking genotypes to phenotypes and to fitness (Beldade etal. 2008). The complex repetitive genome of B. anynana is now subject to analysis by newly devel-oped resources and tools including (1) sequence information, (2) a linkage map, (3) microarrays, and(4) germline transformation. A recent project that involved sequencing approximately 10,000expressed sequence tags (ESTs) from B. anynana developing wings has identified more than 4000 newgenes expressed in these tissues (Beldade et al. 2006). We continue to sequence ESTs and expect toadd to the increasing number of genes expressed in developing wings and other tissues. We havecreated cDNA libraries from diverse tissues in order to sequence those ESTs, and we have also selectedbacterial artificial chromosome (BAC) clones containing candidate genes of interest, enablingsequence analysis of potential regulatory non-coding regions of those genes. Thus far, we havesequenced 11 BAC clones and more than 100,000 ESTs (in collaboration with the Department ofEnergy Joint Genome Institute; see http://jgi.doe.gov/sequencing/why/3112.html), which wereassembled into more than 17,000 UniGenes. Various types of DNA sequence polymorphisms havebeen identified (including microsatellites, amplified-fragment-length polymorphisms [AFLPs], and sin-gle-nucleotide polymorphisms) in expressed genes that can be used in gene mapping. A linkage mapcovering all 28 chromosome pairs of B. anynana is now available, and the microsatellite and AFLPmarkers (Van’t Hof et al. 2005) are currently being enriched with markers in genes expressed duringwing development (Beldade et al. 2009). Moreover, all approximately 17,000 B. anynana UniGenesidentified to date from ESTs have been used to design the first generation of B. anynana microarrays(using NimbleGen-Roche technology), which are currently being tested (P Beldade and AD Long,unpubl.). The use of these microarrays will enable high-throughput expression profiling analysis,which complements the single-gene approach possible with quantitative real-time polymerase chainreaction. Finally, B. anynana is the first (and, to date, the only published) butterfly for which germlinetransformation has been developed (Marcus et al. 2004). Such techniques, coupled with an elegantlaser-mediated method that enables precise transcriptional activation of transgenes in developingpupal wings (Ramos et al. 2006), will enable fine-scale functional analysis of candidate genes. Thesestudies hold great promise for furthering our understanding of the mechanisms of morphologicalevolution, beautifully represented in butterfly wing patterns.

TECHNICAL APPROACHES

Protocols are available that describe the preparation and manipulation of Bicyclus embryos and devel-oping wing discs for studies in gene expression and the development of wing patterns, includingCulture and Propagation of Laboratory Populations of the African Butterfly Bicyclus anynana(Brakefield et al. 2009a), Surgical Manipulations on Pupal Wings from the African ButterflyBicyclus anynana: Damage and Cauteries (Brakefield et al. 2009b), Surgical Manipulations onPupal Wings from the African Butterfly Bicyclus anynana: Grafts (Brakefield et al. 2009c), Fixationand Dissection of Embryos from the African Butterfly Bicyclus anynana (Brakefield et al. 2009d),Dissection of Larval and Pupal Wings from the African Butterfly Bicyclus anynana (Brakefield et al.2009e), and In Situ Hybridization of Embryos and Larval and Pupal Wings from the AfricanButterfly Bicyclus anynana (Brakefield et al. 2009f). There are also protocols that describe stainingtechniques for the detection of proteins in developing embryos and wings, such asImmunohistochemistry Staining of Embryos from the African Butterfly Bicyclus anynana(Brakefield et al. 2009g) and Immunohistochemistry Staining of Wing Discs from the AfricanButterfly Bicyclus anynana (Brakefield et al. 2009h). The protocol Extraction and GasChromatography Analysis of Adult Pheromones from the African Butterfly Bicyclus anynana(Brakefield et al. 2009i) describes methods used to isolate male sex pheromones. Other protocolsdescribe methods used in insect physiology, such as Fresh Weight, Dry Weight, and Fat Content ofAdult African Butterflies (Bicyclus anynana) (Brakefield et al. 2009j), Constant VolumeRespirometry in the African Butterfly Bicyclus anynana (Brakefield et al. 2009k), HemolymphExtraction from Various Developmental Stages of the African Butterfly Bicyclus anynana(Brakefield et al. 2009l), and Injection of Chemicals into Pupae of the African Butterfly Bicyclusanynana (Brakefield et al. 2009m).





Allen CE, Beldade P, Zwaan B, Brakefield PM. 2008. Developmentexplains differences in evolvability of serially repeated color pat-tern elements on butterfly wings. BMC Evol Biol 8: 94. doi:10.1186/1471-2148-8-94.

Beldade P, Brakefield PM. 2002. The genetics and evo-devo of but-terfly wing patterns. Nat Rev Genet 3: 442–452.

Beldade P, Brakefield PM, Long AD. 2002a. Contribution of Distal-lessto quantitative variation in butterfly eyespots. Nature 415: 315–318.

Beldade P, Koops K, Brakefield PM. 2002b. Developmental con-straints versus flexibility in morphological evolution. Nature 416:844–847.

Beldade P, Rudd S, Gruber JD, Long AD. 2006. A wing expressedsequence tag resource for Bicyclus anynana butterflies, an evo-devomodel. BMC Genomics 7: 130. doi: 10.1186/1471-2164-7-130.

Beldade P, McMillan WO, Papanicolaou A. 2008. Butterfly genomicseclosing. Heredity 100: 150–157.

Beldade P, Saenko SV, Pul N, Long AD. 2009. A gene-based linkagemap for Bicyclus anynana butterflies allows for a comprehensiveanalysis of synteny with the lepidopteran references genome.PLoS Genet 5: e1000366. doi: 10.1371/journal.pgen.1000366.

Brakefield PM, Frankino WA. 2009. Polyphenisms in Lepidoptera:Multidisciplinary approaches to studies of evolution. In Phenotypicplasticity of insects: Mechanisms and consequences (eds. DWWhitman and TN Ananthakrishnan), pp. 121–152. SciencePublishers, Plymouth, UK.

Brakefield PM, Larsen TB. 1984. The evolutionary significance of dryand wet season forms in some tropical butterflies. Biol J Linn Soc22: 1–12.

Brakefield PM, Gates J, Keys D, Kesbeke F, Wijngaarden PJ, MonteiroA, French V, Carroll SB. 1996. Development, plasticity and evolu-tion of butterfly eyespot patterns. Nature 384: 236–242.

Brakefield PM, Kesbeke F, Koch PB. 1998. The regulation of pheno-typic plasticity of eyespots in the butterfly. Bicyclus anynana AmNat 152: 853–860.

Brakefield PM, El Jilali E, van der Laan R, Breuker C, Saccheri I, ZwaanBJ. 2001. Effective population size, reproductive success andsperm precedence in the butterfly, Bicyclus anynana, in captivity.J Evol Biol 14: 148–156.

Brakefield PM, Beldade P, Zwaan BJ. 2009a. Culture and propagationof laboratory populations of the African butterfly Bicyclus any-nana. Cold Spring Harb Protoc (this issue). doi: 10.1101/pdb.prot5203.

Brakefield PM, Beldade P, Zwaan BJ. 2009b. Surgical manipulationson pupal wings from the African butterfly Bicyclus anynana:Damage and cauteries. Cold Spring Harb Protoc (this issue). doi:10.1101/pdb.prot5204.

Brakefield PM, Beldade P, Zwaan BJ. 2009c. Surgical manipulationson pupal wings from the African butterfly Bicyclus anynana:Grafts. Cold Spring Harb Protoc (this issue). doi: 10.1101/pdb.prot5205.

Brakefield PM, Beldade P, Zwaan BJ. 2009d. Fixation and dissectionof embryos from the African butterfly Bicyclus anynana. ColdSpring Harb Protoc (this issue). doi: 10.1101/pdb.prot5206.

Brakefield PM, Beldade P, Zwaan BJ. 2009e. Dissection of larval andpupal wings from the African butterfly Bicyclus anynana. ColdSpring Harb Protoc (this issue). doi: 10.1101/pdb.prot5207.

Brakefield PM, Beldade P, Zwaan BJ. 2009f. In situ hybridization ofembryos and larval and pupal wings from the African butterflyBicyclus anynana. Cold Spring Harb Protoc (this issue). doi:10.1101/pdb.prot5208.

Brakefield PM, Beldade P, Zwaan BJ. 2009g. Immunohistochemistrystaining of embryos from the African butterfly Bicyclus anynana.Cold Spring Harb Protoc (this issue). doi: 10.1101/pdb.prot5209.

Brakefield PM, Beldade P, Zwaan BJ. 2009h. Immunohistochemistrystaining of wing discs from the African butterfly Bicyclus anynana.Cold Spring Harb Protoc (this issue). doi: 10.1101/pdb.prot5210.

Brakefield PM, Beldade P, Zwaan BJ. 2009i. Extraction and gas chro-matography analysis of adult pheromones from the African but-terfly Bicyclus anynana. Cold Spring Harb Protoc (this issue). doi:10.1101/pdb.prot5211.

Brakefield PM, Beldade P, Zwaan BJ. 2009j. Fresh weight, dry weight,and fat content of adult African butterflies (Bicyclus anynana).Cold Spring Harb Protoc (this issue). doi: 10.1101/pdb.prot5212.

Brakefield PM, Beldade P, Zwaan BJ. 2009k. Constant volumerespirometry in the African butterfly Bicyclus anynana. Cold SpringHarb Protoc (this issue). doi: 10.1101/pdb.prot5213.

Brakefield PM, Beldade P, Zwaan BJ. 2009l. Hemolymph extractionfrom various developmental stages of the African butterfly Bicyclusanynana. Cold Spring Harb Protoc (this issue). doi: 10.1101/pdb.prot5214.

Brakefield PM, Beldade P, Zwaan BJ. 2009m. Injection of chemicalsinto pupae of the African butterfly Bicyclus anynana. Cold SpringHarb Protoc (this issue). doi: 10.1101/pdb.prot5215.

Breuker CJ, Brakefield PM. 2003. Lack of response to selection forlower fluctuating asymmetry of mutant eyespots in the butterflyBicyclus anynana. Heredity 91: 17–27.

Brunetti CR, Selegue JE, Monteiro A, French V, Brakefield PM, CarrollSB. 2001. The generation and diversification of butterfly eyespotcolor patterns. Curr Biol 11: 1578–1585.

Butler AG. 1879. Mycalesis anynana. Ann Mag Nat Hist 5: 187.Condamin M. 1973. Monographie du genre Bicyclus (Lepidoptera:

Satyridae). Mem Inst Fond Afr Noire 88: 1–324.Dilão R, Sainhas J. 2004. Modelling butterfly wing eyespot patterns.

Proc R Soc B 271: 1565–1569.Fischer K, Zwaan BJ, Brakefield PM. 2007. Realized correlated

responses to artificial selection on pre-adult life-history traits in abutterfly. Heredity 98: 157–164.

Frankino WA, Zwaan BJ, Stern DL, Brakefield PM. 2005. Natural selec-tion and developmental constraints in the evolution of allome-tries. Science 307: 718–720.

Frankino WA, Zwaan BJ, Stern DL, Brakefield PM. 2007. Internal and


ACKNOWLEDGMENTS

We are indebted to all who have worked with B. anynana, from research staff and students to labora-tory technicians and maize growers. Torben Larsen was the initial catalyst to begin work with thegenus, and Vernon French supplied the expertise in developmental biology that was crucial to shap-ing the evo-devo work (following a chance teatime conversation with Linda Partridge). Cornell Dudleyand John Wilson were most helpful in Malawi. Sean Carroll, Antónia Monteiro, and Tony Long havebeen especially influential in the modern genomics era, and Bernd Koch and Klaus Fischer in the areasof physiology and life histories. We thank all of the funding bodies for their contributions, especiallythe Human Frontiers Science Program, NWO, Leiden University, NSF, and JGI. We thank SuzanneSaenko and Martin Brittijn for the photos and artwork and Nicolien Pul for the organization of all pro-tocols. We are also grateful to the people of Malawi for the early support of their research authoritiesand for providing access to their exciting biodiversity.

REFERENCES




external constraints in the evolution of morphological allometriesin a butterfly. Evolution 61: 2958–2970.

Joron M, Brakefield PM. 2003. Captivity masks inbreeding effects onmale mating success in butterflies. Nature 424: 191–194.

Kirby WF. 1871. A synonymic catalogue of the diurnal Lepidoptera. VanVorst, London.

Kooi RE, Brakefield PM. 1999. The critical period for wing patterninduction in the polyphenic tropical butterfly Bicyclus anynana(Satyrinae). J Insect Physiol 45: 201–212.

Lyytinen A, Brakefield PM, Lindstrom L, Mappes J. 2004. Does pre-dation maintain eyespot plasticity in Bicyclus anynana? Proc R SocLond B Biol Sci 271: 279–283.

Marcus JM, Evans TM. 2008. A simulation study of mutations in thegenetic regulatory hierarchy for butterfly eyespot focus determi-nation. Biosystems 93: 250–255.

Marcus JM, Ramos DM, Monteiro A. 2004. Germline transformationof the butterfly Bicyclus anynana. Proc R Soc Lond B Biol Sci(Suppl 5) 271: S263–S265.

McMillan WO, Monteiro A, Kapan DD. 2002. Development andevolution on the wing. Trends Ecol Evol 17: 125–133.

Molleman F, Kop A, Brakefield PM, De Vries PJ, Zwaan BJ. 2006.Vertical and temporal patterns of biodiversity of fruit-feedingbutterflies in a tropical forest in Uganda. Biodivers Conserv 15:107–121.

Monteiro A, Pierce NE. 2001. Phylogeny of Bicyclus (Lepidoptera:Nymphalidae) inferred from COI, COII, and EF-1α genesequences. Mol Phylogenet Evol 18: 264–281.

Monteiro A, Prijs J, Bax M, Hakkaart T, Brakefield PM. 2003. Mutantshighlight the modular control of butterfly eyespot patterns. EvolDev 5: 180–187.

Nieberding CM, de Vos H, Schneider MV, Lassance J-M, Estramil N,Andersson J, Bång J, Hedenström E, Löfstedt C, Brakefield PM.2008. The male sex pheromone of the butterfly Bicyclus anynana:Towards an evolutionary analysis. PLoS ONE 3: e2751. doi:10.1371/journal.pone.0002751.

Nijhout HF. 1991. The development and evolution of butterfly wingpatterns. Smithsonian Institution Press, Washington, DC.

Peña C, Wahlberg N. 2008. Prehistorical climate change increased

diversification of a group of butterflies. Biol Lett 4: 274–278.Pijpe J, Brakefield PM, Zwaan BJ. 2008. Increased lifespan in a

polyphenic butterfly artificially selected for starvation resistance.Am Nat 171: 81–90.

Ramos DM, Kamal F, Wimmer EA, Cartwright AN, Monteiro A. 2006.Temporal and spatial control of transgene expression using laserinduction of the hsp70 promoter. BMC Dev Biol 6: 55. doi:10.1186/1471-213X-6-55.

Reed RD, Serfas MS. 2004. Butterfly wing pattern evolution is associ-ated with changes in a Notch/Distal-less temporal pattern forma-tion process. Curr Biol 14: 1159–1166.

Robertson KA, Monteiro A. 2005. Female Bicyclus anynana butterflieschoose males on the basis of their dorsal UV-reflective eyespotpupils. Proc R Soc B Biol Sci 272: 1541–1546.

Roskam JC, Brakefield PM. 1996. A comparison of temperature-induced polyphenism in African Bicyclus butterflies from a sea-sonal savannah-rainforest ecotone. Evolution 50: 2360–2372.

Saccheri IJ, Lloyd HD, Helyar SJ, Brakefield PM. 2005. Inbreedinguncovers fundamental differences in the genetic load affectingmale and female fertility in a butterfly. Proc R Soc Lond B Biol Sci272: 39–46.

Saenko SV, French V, Brakefield PM, Beldade P. 2008. Conserveddevelopmental processes and the formation of evolutionary nov-elties: Examples from butterfly wings. Philos Trans R Soc Lond B BiolSci 363: 1549–1555.

Van’t Hof AE, Zwaan BJ, Saccheri IJ, Daly D, Bot ANM, Brakefield PM.2005. Characterization of 28 microsatellite loci for the butterflyBicyclus anynana. Mol Ecol Notes 5: 169–172.

Wijngaarden PJ, Brakefield PM. 2000. The genetic basis of eyespotsize in the butterfly Bicyclus anynana: An analysis of line crosses.Heredity 85: 471–479.

Windig JJ, Brakefield PM, Reitsma N, Wilson JGM. 1994. Seasonalpolyphenism in the wild: Survey of wing patterns in five speciesof Bicyclus butterflies in Malawi. Ecol Entomol 19: 285–298.

Zijlstra WG, Steigenga MJ, Koch PB, Zwaan BJ, Brakefield PM. 2004.Butterfly selected lines explore the hormonal basis of interactionsbetween life histories and morphology. Am Nat 163: E76–E87.





the african butterfly bicyclus anynana: a model for evolutionary genetics and evolutionary...

Documents