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Wild bees are vital for the pollination of native plants and crops, providing essential ecosystem services. Climate change is known to impact biodiversity and species distributions, but insects adapted to desert ecosystems may exhibit unique physiological, behavioral, and evolutionary responses. The desert pallid bee ( C. pallida ), a solitary bee native to the arid southwestern United States and northern Mexico, primarily forages on yellow palo verde ( P. microphylla ), blue palo verde ( P. florida ), and desert ironwood ( O. tesota ). This study used MaxEnt to estimate the current and projected geographical overlap of suitable habitats for C. pallida and its host plants. Here, we used MaxEnt to estimate the current and forecasted overlapping geographically suitable habitat of C. pallida with all three host plants. We forecasted potential environmentally suitable areas for each species to the year 2040 using the current distribution model and climate projections with moderate CO 2 levels. We found a continued spatial alignment in the suitable area of the bee and its host plants with a 70% increase in the range overlap area, though shifted to higher average altitudes and a slight northern expansion. These findings may provide insight to stakeholders on the conservation needs of desert-dwelling pollinators.

Butterfly populations are declining worldwide, reflecting our current global biodiversity crisis. Because butterflies are a popular and accurate indicator of insect populations, these declines reflect an even more widespread threat to insects and the food webs upon which they rely. As small ectotherms, insects have a narrow range of habitable conditions; hence, extreme fluctuations and shifts caused by climate change may increase insects' risk of extinction. We evaluated trends of butterfly richness and abundance and their relationship with relevant climate variables in Arizona, U.S.A., using the past 40 years of community science data. We focused on precipitation and temperature as they are known to be influential for insect survival, particularly in arid areas like southwestern U.S.A. We found that preceding winter precipitation is a driver of both spring and summer/fall butterfly richness and spring butterfly abundance. In contrast, summer/fall butterfly abundance was driven by summer monsoon precipitations. The statistically significant declines over the 40-year period were summer/fall butterfly abundance and spring butterfly richness. When controlling for the other variables in the model, there was an average annual 1.81% decline in summer/fall season butterfly abundance and an average annual decline of 2.13 species in the spring season. As climate change continues to negatively impact winter precipitation patterns in this arid region, we anticipate the loss of butterfly species in this region and must consider individual butterfly species trends and additional management and conservation needs.

Urban areas are proliferating quickly around the globe often with detrimental impacts on biodiversity. Insects, especially pollinators, have also seen record declines in recent decades, sometimes associated with land use change such as urbanization, but also associated with climate changes such as increased aridity. How these various factors play out in attracting and sustaining species richness in a complex urban matrix is poorly understood. Urban botanical gardens may serve as important refugia for insect pollinators in arid regions due to reliable water availability for both plants and insects. Here, we use community science data on butterfly observations to evaluate if botanical gardens can be hotspots of biodiversity in the arid urban landscapes of the southwest US. We found butterfly richness and diversity were proportionally overrepresented in botanical gardens compared with the urban landscape they were embedded in. We conclude that biodiversity-friendly botanical gardens in urban arid regions can make a valuable contribution to pollinator conservation, in particular, in face of the continued aridification due to climate change.

The global lockdown to mitigate COVID-19 pandemic health risks has altered human interactions with nature. Here, we report immediate impacts of changes in human activities on wildlife and environmental threats during the early lockdown months of 2020, based on 877 qualitative reports and 332 quantitative assessments from 89 different studies. Hundreds of reports of unusual species observations from around the world suggest that animals quickly responded to the reductions in human presence. However, negative effects of lockdown on conservation also emerged, as confinement resulted in some park officials being unable to perform conservation, restoration and enforcement tasks, resulting in local increases in illegal activities such as hunting. Overall, there is a complex mixture of positive and negative effects of the pandemic lockdown on nature, all of which have the potential to lead to cascading responses which in turn impact wildlife and nature conservation. While the net effect of the lockdown will need to be assessed over years as data becomes available and persistent effects emerge, immediate responses were detected across the world. Thus, initial qualitative and quantitative data arising from this serendipitous global quasi-experimental perturbation highlights the dual role that humans play in threatening and protecting species and ecosystems. Pathways to favorably tilt this delicate balance include reducing impacts and increasing conservation effectiveness., Competing Interests: Authors declare no competing interests., (© 2021 Published by Elsevier Ltd.)

Shutdowns associated with the COVID-19 pandemic have had extensive impacts on professional and volunteer-based biodiversity and conservation efforts. We evaluated the impact of the widespread pandemic-related closures in the spring of 2020 on participation patterns and rates on a national and a state-by-state basis in the United States in four biodiversity-themed community science programs: eBird, eButterfly, iNaturalist, and Nature's Notebook . We compared the number of participants, observations submitted, and proportion of observations collected in urban environments in spring 2020 to the expected values for these metrics based on activity in the previous five years (2015-2019), which in many cases exhibited underlying growth. At the national scale, eButterfly and Nature's Notebook exhibited declines in the number of participants and number of observations submitted during the spring of 2020 and iNaturalist and eBird showed growth in both measures. On a state-by-state basis, the patterns varied geographically and by program. The more popular programs - iNaturalist and eBird - exhibited increases in the Eastern U.S. in both the number of observations and participants and slight declines in the West. Further, there was a widespread increase in observations originating from urban areas, particularly in iNaturalist and eBird. Understanding the impacts of lockdowns on participation patterns in these programs is crucial for proper interpretation of the data. The data generated by these programs are highly valuable for documenting impacts of pandemic-related closures on wildlife and plants and may suggest patterns seen in other community science programs and in other countries., Competing Interests: The authors declare no conflicts of interest., (© 2021 Elsevier Ltd. All rights reserved.)

Uncertainty remains regarding the role of anthropogenic climate change in declining insect populations, partly because our understanding of biotic response to climate is often complicated by habitat loss and degradation among other compounding stressors. We addressed this challenge by integrating expert and community scientist datasets that include decades of monitoring across more than 70 locations spanning the western United States. We found a 1.6% annual reduction in the number of individual butterflies observed over the past four decades, associated in particular with warming during fall months. The pervasive declines that we report advance our understanding of climate change impacts and suggest that a new approach is needed for butterfly conservation in the region, focused on suites of species with shared habitat or host associations., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Agriculture has long been a part of the urban landscape, from gardens to small scale farms. In recent decades, interest in producing food in cities has grown dramatically, with an estimated 30% of the global urban population engaged in some form of food production. Identifying and managing the insect biodiversity found on city farms is a complex task often requiring years of study and specialization, especially in urban landscapes which have a complicated tapestry of fragmentation, diversity, pollution, and introduced species. Supporting urban growers with relevant data informs insect management decision-making for both growers and their neighbors, yet this information can be difficult to come by. In this study, we introduced several web-based citizen science programs that can connect growers with useful data products and people to help with the who, what, where, and when of urban insects. Combining the power of citizen science volunteers with the efforts of urban farmers can result in a clearer picture of the diversity and ecosystem services in play, limited insecticide use, and enhanced non-chemical alternatives. Connecting urban farming practices with citizen science programs also demonstrates the ecosystem value of urban agriculture and engages more citizens with the topics of food production, security, and justice in their communities.

Mimics should not exist without their models, yet often they do. In the system involving queen and viceroy butterflies, the viceroy is both mimic and co-model depending on the local abundance of the model, the queen. Here, we integrate population surveys, chemical analyses, and predator behavior assays to demonstrate how mimics may persist in locations with low-model abundance. As the queen becomes less locally abundant, the viceroy becomes more chemically defended and unpalatable to predators. However, the observed changes in viceroy chemical defense and palatability are not attributable to differing host plant chemical defense profiles. Our results suggest that mimetic viceroy populations are maintained at localities of low-model abundance through an increase in their toxicity. Sharing the burden of predator education in some places but not others may also lower the fitness cost of warning signals thereby supporting the origin and maintenance of aposematism., Competing Interests: The authors declare no competing interests.

By 2030, ten percent of earth's landmass will be occupied by cities. Urban environments can be home to many plants and animals, but surveying and estimating biodiversity in these spaces is complicated by a heterogeneous built environment where access and landscaping are highly variable due to human activity. Citizen science approaches may be the best way to assess urban biodiversity, but little is known about their relative effectiveness and efficiency. Here, we compare three techniques for acquiring data on butterfly (Lepidoptera: Rhopalocera) species richness: trained volunteer Pollard walks, Malaise trapping with expert identification, and crowd-sourced iNaturalist observations. A total of 30 butterfly species were observed; 27 (90%) were recorded by Pollard walk observers, 18 (60%) were found in Malaise traps, and 22 (73%) were reported by iNaturalist observers. Pollard walks reported the highest butterfly species richness, followed by iNaturalist and then Malaise traps during the four-month time period. Pollard walks also had significantly higher species diversity than Malaise traps.

Motivation: The BioTIME database contains raw data on species identities and abundances in ecological assemblages through time. These data enable users to calculate temporal trends in biodiversity within and amongst assemblages using a broad range of metrics. BioTIME is being developed as a community-led open-source database of biodiversity time series. Our goal is to accelerate and facilitate quantitative analysis of temporal patterns of biodiversity in the Anthropocene., Main Types of Variables Included: The database contains 8,777,413 species abundance records, from assemblages consistently sampled for a minimum of 2 years, which need not necessarily be consecutive. In addition, the database contains metadata relating to sampling methodology and contextual information about each record., Spatial Location and Grain: BioTIME is a global database of 547,161 unique sampling locations spanning the marine, freshwater and terrestrial realms. Grain size varies across datasets from 0.0000000158 km 2 (158 cm 2 ) to 100 km 2 (1,000,000,000,000 cm 2 )., Time Period and Grain: BioTIME records span from 1874 to 2016. The minimal temporal grain across all datasets in BioTIME is a year., Major Taxa and Level of Measurement: BioTIME includes data from 44,440 species across the plant and animal kingdoms, ranging from plants, plankton and terrestrial invertebrates to small and large vertebrates., Software Format: .csv and .SQL.

In contrast to the important role of hormones in the development of sexual traits in vertebrates (Cox RM, Stenquist DS, Calsbeek R. 2009. Testosterone, growth and the evolution of sexual size dimorphism. J Evol Biol. 22(8):1586-1598.), the differentiation of these traits in insects is attributed almost exclusively to cell-autonomous mechanisms controlled by members of the sex determination pathway (Verhulst EC, van de Zande L. 2015. Double nexus - doublesex is the connecting element in sex determination. Brief Funct Genomics 14(6):396-406.), such as doublesex. Although hormones can shape the development of sexual traits in insects, variation in hormone levels are not conclusively known to cause dimorphism in these traits (Prakash A, Monteiro A. 2016. Molecular mechanisms of secondary sexual trait development in insects. Curr Opin Insect Sci. 17:40-48.). Here, we show that butterflies use sex-specific differences in 20-hydroxyecdysone hormone titers to create sexually dimorphic wing ornaments. Females of the dry season (DS) form of Bicyclus anynana display a larger sexual ornament on their wings than males, whereas in the wet season form both sexes have similarly sized ornaments (Prudic KL, Jeon C, Cao H, Monteiro A. 2011. Developmental plasticity in sexual roles of butterfly species drives mutual sexual ornamentation. Science 331(6013):73-75.). High levels of circulating 20-hydroxyecdysone during larval development in DS females and wet season forms cause proliferation of the cells fated to give rise to this wing ornament, and results in sexual dimorphism in the DS forms. This study advances our understanding of how the environment regulates sex-specific patterns of plasticity of sexual ornaments and conclusively shows that hormones can play a role in the development of secondary sexual traits in insects, just like they do in vertebrates., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Data collection, storage, analysis, visualization, and dissemination are changing rapidly due to advances in new technologies driven by computer science and universal access to the internet. These technologies and web connections place human observers front and center in citizen science-driven research and are critical in generating new discoveries and innovation in such fields as astronomy, biodiversity, and meteorology. Research projects utilizing a citizen science approach address scientific problems at regional, continental, and even global scales otherwise impossible for a single lab or even a small collection of academic researchers. Here we describe eButterfly an integrative checklist-based butterfly monitoring and database web-platform that leverages the skills and knowledge of recreational butterfly enthusiasts to create a globally accessible unified database of butterfly observations across North America. Citizen scientists, conservationists, policy makers, and scientists are using eButterfly data to better understand the biological patterns of butterfly species diversity and how environmental conditions shape these patterns in space and time. eButterfly in collaboration with thousands of butterfly enthusiasts has created a near real-time butterfly data resource producing tens of thousands of observations per year open to all to share and explore., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; and in the decision to publish the results.

It is well established that steroid hormones regulate sexual behavior in vertebrates via organizational and activational effects. However, whether the organizational/activational paradigm applies more broadly to the sexual behavior of other animals such as insects is not well established. Here we describe the hormonal regulation of a sexual behavior in the seasonally polyphenic butterfly Bicyclus anynana is consistent with the characteristics of an organizational effect. By measuring hormone titer levels, quantifying hormone receptor gene expression in the brain, and performing hormone manipulations, we demonstrate steroid hormone signaling early in pupal development has a latent effect on adult male sexual behavior in B. anynana. These findings suggest the organizational/activational paradigm may be more highly conserved across animal taxa than previously thought.

Bodies are often made of repeated units, or serial homologs, that develop using the same core gene regulatory network. Local inputs and modifications to this network allow serial homologs to evolve different morphologies, but currently we do not understand which modifications allow these repeated traits to evolve different levels of phenotypic plasticity. Here we describe variation in phenotypic plasticity across serial homologous eyespots of the butterfly Bicyclus anynana, hypothesized to be under selection for similar or different functions in the wet and dry seasonal forms. Specifically, we document the presence of eyespot size and scale brightness plasticity in hindwing eyespots hypothesized to vary in function across seasons, and reduced size plasticity and absence of brightness plasticity in forewing eyespots hypothesized to have the same function across seasons. By exploring the molecular and physiological causes of this variation in plasticity across fore and hindwing serial homologs we discover that: 1) temperature experienced during the wandering stages of larval development alters titers of an ecdysteroid hormone, 20-hydroxyecdysone (20E), in the hemolymph of wet and dry seasonal forms at that stage; 2) the 20E receptor (EcR) is differentially expressed in the forewing and hindwing eyespot centers of both seasonal forms during this critical developmental stage; and 3) manipulations of EcR signaling disproportionately affected hindwing eyespots relative to forewing eyespots. We propose that differential EcR expression across forewing and hindwing eyespots at a critical stage of development explains the variation in levels of phenotypic plasticity across these serial homologues. This finding provides a novel signaling pathway, 20E, and a novel molecular candidate, EcR, for the regulation of levels of phenotypic plasticity across body parts or serial homologs.

Some eyespots are thought to deflect attack away from the vulnerable body, yet there is limited empirical evidence for this function and its adaptive advantage. Here, we demonstrate the conspicuous ventral hindwing eyespots found on Bicyclus anynana butterflies protect against invertebrate predators, specifically praying mantids. Wet season (WS) butterflies with larger, brighter eyespots were easier for mantids to detect, but more difficult to capture compared to dry season (DS) butterflies with small, dull eyespots. Mantids attacked the wing eyespots of WS butterflies more frequently resulting in greater butterfly survival and reproductive success. With a reciprocal eyespot transplant, we demonstrated the fitness benefits of eyespots were independent of butterfly behaviour. Regardless of whether the butterfly was WS or DS, large marginal eyespots pasted on the hindwings increased butterfly survival and successful oviposition during predation encounters. In previous studies, DS B. anynana experienced delayed detection by vertebrate predators, but both forms suffered low survival once detected. Our results suggest predator abundance, identity and phenology may all be important selective forces for B. anynana. Thus, reciprocal selection between invertebrate and vertebrate predators across seasons may contribute to the evolution of the B. anynana polyphenism.

Seasonal polyphenism demonstrates an organism's ability to respond to predictable environmental variation with alternative phenotypes, each presumably better suited to its respective environment. However, the molecular mechanisms linking environmental variation to alternative phenotypes via shifts in development remain relatively unknown. Here we investigate temporal gene expression variation in the seasonally polyphenic butterfly Bicyclus anynana. This species shows drastic changes in eyespot size depending on the temperature experienced during larval development. The wet season form (larvae reared over 24°C) has large ventral wing eyespots while the dry season form (larvae reared under 19°C) has much smaller eyespots. We compared the expression of three proteins, Notch, Engrailed, and Distal-less, in the future eyespot centers of the two forms to determine if eyespot size variation is associated with heterochronic shifts in the onset of their expression. For two of these proteins, Notch and Engrailed, expression in eyespot centers occurred earlier in dry season than in wet season larvae, while Distal-less showed no temporal difference between the two forms. These results suggest that differences between dry and wet season adult wings could be due to a delay in the onset of expression of these eyespot-associated genes. Early in eyespot development, Notch and Engrailed may be functioning as repressors rather than activators of the eyespot gene network. Alternatively, temporal variation in the onset of early expressed genes between forms may have no functional consequences to eyespot size regulation and may indicate the presence of an 'hourglass' model of development in butterfly eyespots.

We examined whether shared volatiles found in various heteropteran species and developmental stages function to repel predators. The nymphal dorsal abdominal gland secretions of Riptortus pedestris (Heteroptera: Alydidae) and Thasus acutangulus (Heteroptera: Coreidae), and the metathoracic scent gland secretion of Euschistus biformis (Heteroptera: Pentatomidae) adults were identified by gas chromatography/mass spectrometry (GC/MS). (E)-2-Hexenal, 4-oxo-(E)-2-hexenal (4-OHE), and (E)-2-octenal were found in all three species and deemed likely candidates for repelling predators. In addition to (E)-2-alkenals, the adult E. biformis secreted (E)-2-hexenyl acetate, (E)-2-octenyl acetate, and four hydrocarbons. We evaluated the potential predator repellent properties of these compounds and compound blends against a generalist, cosmopolitan insect predator, the Chinese praying mantid (Mantodea: Mantidae: Tenodera aridifolia sinensis). Mantids that experienced (E)-2-hexenal, (E)-2-octenal, and (E)-2-octenyl acetate moved away from the site of interaction, while 4-OHE and (E)-2-hexenyl acetate did not affect mantid behavior. The compound blends did not have additive or synergistic repellency effects on predator behavior. Compound repellency was not related to compound volatility. Instead, the repellent effect is likely related to predator olfaction, and the affinity of each compound to receptors on the antennae. Our results also suggest the repellents might intensify the visual defensive signals of aposematism (T. acutangulus nymphs) and mimicry (R. pedestris nymphs) in heteropteran bugs.

Current explanations for why sexual ornaments are found in both sexes include genetic correlation, same sex competition, and mutual mate choice. In this study, we report developmental plasticity in mating behavior as induced by temperature during development in the butterfly Bicyclus anynana. Males and females reciprocally change their sexual roles depending on their larval rearing temperatures. This switch is correlated with a change in mating benefits to females and costs to males. The discrete seasonal environments, wet season and dry season, are known to produce the two developmental forms and as a consequence impose alternating, symmetrical patterns of sexual selection, one season on male ornaments, the following season on female ornaments. Thus, reciprocal selection through time may result in mutual sexual ornamentation.

Background: The processes governing the origin and maintenance of mimetic phenotypes can only be understood in a phylogenetic framework. Phylogenetic estimates of evolutionary relationships can provide a context for analyses of character evolution; however, when phylogenetic estimates conflict, rigorous analyses of alternative evolutionary histories are necessary to determine the likelihood of a specific history giving rise to the observed pattern of diversity. The polyphenic butterfly Limenitis arthemis provides a case in point. This species is comprised of three lineages, two of which are mimetic and one of which is non-mimetic. Conflicting estimates of the relationships among these three lineages requires direct evaluation of the alternative hypotheses of mimicry evolution., Results: Using a coalescent framework, we found support for a sister-taxon relationship between the non-mimetic L. a. arthemis and the mimetic L. a. astyanax, congruent with the previous hypothesis that the non-mimetic form of L. a. arthemis was derived from a mimetic ancestor. We found no support for a mimetic clade (L. a. astyanax + L. a. arizonensis) despite analyzing numerous models of population structure., Conclusions: These results provide the foundation for future studies of mimicry, which should integrate phylogenetic and developmental analyses of wing pattern formation. We propose future analyses of character evolution accommodate conflicting phylogenetic estimates by explicitly testing alternative evolutionary hypotheses.

Heteropteran insects often protect themselves from predators with noxious or toxic compounds, especially when these insects occur in aggregations. The predators of heteropteran insects change from small insect predators to large avian predators over time. Thus, a chemical that is deterrent to one type of predator at one point in time may not be deterrent to another type of predator at another point in time. Additionally, these predator deterrent compounds may be used for other functions such as alarm signaling to other conspecifics. Defensive secretion compounds from the adult and the nymph giant mesquite bug (Thasus neocalifornicus: Coreidae) were isolated and identified by gas chromatography-mass spectrometry and NMR. The predominant compounds isolated from the nymph mesquite bugs during a simulated predator encounter were (E)-2-hexenal and 4-oxo-(E)-2-hexenal. In adults, the major compounds released during a simulated predator encounter were hexyl acetate, hexanal, and hexanol. Results from predator bioassays suggest the nymph compounds are more effective at deterring an insect predator than the adult compounds. By using behavioral bioassays, we determined the role of each individual compound in signaling to other mesquite bugs. The presence of the nymph secretion near a usually compact nymph aggregation caused nymph mesquite bugs to disperse but did not affect adults. Conversely, the presence of the adult secretion caused the usually loose adult aggregation to disperse, but it did not affect nymph aggregation. The compounds that elicited nymph behavioral responses were (E)-2-hexenal and 4-oxo-(E)-2-hexenal, while those that elicited adult behavioral responses were hexyl acetate and hexanal. The differences between the chemical composition of nymph and adult defensive secretions and alarm behavior are possibly due to differences in predator guilds.

Batesian mimics gain protection from predation through the evolution of physical similarities to a model species that possesses anti-predator defences. This protection should not be effective in the absence of the model since the predator does not identify the mimic as potentially dangerous and both the model and the mimic are highly conspicuous. Thus, Batesian mimics should probably encounter strong predation pressure outside the geographical range of the model species. There are several documented examples of Batesian mimics occurring in locations without their models, but the evolutionary responses remain largely unidentified. A mimetic species has four alternative evolutionary responses to the loss of model presence. If predation is weak, it could maintain its mimetic signal. If predation is intense, it is widely presumed the mimic will go extinct. However, the mimic could also evolve a new colour pattern to mimic another model species or it could revert back to its ancestral, less conspicuous phenotype. We used molecular phylogenetic approaches to reconstruct and test the evolution of mimicry in the North American admiral butterflies (Limenitis: Nymphalidae). We confirmed that the more cryptic white-banded form is the ancestral phenotype of North American admiral butterflies. However, one species, Limenitis arthemis, evolved the black pipevine swallowtail mimetic form but later reverted to the white-banded more cryptic ancestral form. This character reversion is strongly correlated with the geographical absence of the model species and its host plant, but not the host plant distribution of L. arthemis. Our results support the prediction that a Batesian mimic does not persist in locations without its model, but it does not go extinct either. The mimic can revert back to its ancestral, less conspicuous form and persist.

Aposematic coloration, or warning coloration, is a visual signal that acts to minimize contact between predator and unprofitable prey. The conditions favoring the evolution of aposematic coloration remain largely unidentified. Recent work suggests that diet specialization and resultant toxicity may play a role in facilitating the evolution and persistence of warning coloration. Using a phylogenetic approach, we investigated the evolution of larval warning coloration in the genus Papilio (Lepidoptera: Papilionidae). Our results indicate that there are at least four independent origins of aposematic larval coloration within Papilio. Controlling for phylogenetic relatedness among Papilio taxa, we found no evidence supporting the hypothesis that either diet specialization or chemical specialization facilitated the origin of aposematic larvae. However, there was a significant relationship between the signal environment and the evolution of aposematic larvae. Specifically, Papilio lineages feeding on herbaceous or narrow-leaved plants, regardless of the plants' taxonomic affiliation, were more likely to evolve aposematic larvae than were lineages feeding only on trees/shrubs or broad-leaved plants. These results demonstrate that factors other than diet specialization, such as the signal environment of predator-prey interactions, may play a large role in the initial evolution and persistence of aposematic coloration.

The viceroy-monarch and viceroy-queen butterfly associations are classic examples of mimicry. These relationships were originally classified as Batesian, or parasitic, but were later reclassified as Müllerian, or mutalistic, based on predator bioassays. The Müllerian reclassification implies that viceroy is unpalatable because it too is chemically defended like the queen and the monarch. However, unlike the queen and the monarch, the viceroy defensive chemistry has remained uncharacterized. We demonstrate that the viceroy butterfly (Limenitis archippus, Nymphalidae) not only sequesters nonvolatile defensive compounds from its larval host-plant, the Carolina willow (Salix caroliniana, Salicaceae), but also secretes volatile defensive compounds when disturbed. We developed liquid chromatography-mass spectrometry-mass spectrometry methods to identify a set of phenolic glycosides shared between the adult viceroy butterfly and the Carolina willow, and solid phase microextraction and gas chromatography-mass spectrometry methods to identify volatile phenolic compounds released from stressed viceroy butterflies. In both approaches, all structures were characterized based on their mass spectral fragmentation patterns and confirmed with authentic standards. The phenolics we found are known to deter predator attack in other prey systems, including other willow-feeding insect species. Because these compounds have a generalized defensive function at the concentrations we described, our results are consistent with the Müllerian reclassification put forth by other researchers based on bioassay results. It seems that the viceroy butterfly possesses chemical defenses different from its monarch and queen butterfly counterparts (phenolic glycosides vs. cardiac glycosides, respectively), an unusual phenomenon in mimicry warranting future study.

Butterflies and primates are interesting for comparative color vision studies, because both have evolved middle- (M) and long-wavelength- (L) sensitive photopigments with overlapping absorbance spectrum maxima (lambda(max) values). Although positive selection is important for the maintenance of spectral variation within the primate pigments, it remains an open question whether it contributes similarly to the diversification of butterfly pigments. To examine this issue, we performed epimicrospectrophotometry on the eyes of five Limenitis butterfly species and found a 31-nm range of variation in the lambda(max) values of the L-sensitive photopigments (514-545 nm). We cloned partial Limenitis L opsin gene sequences and found a significant excess of replacement substitutions relative to polymorphisms among species. Mapping of these L photopigment lambda(max) values onto a phylogeny revealed two instances within Lepidoptera of convergently evolved L photopigment lineages whose lambda(max) values were blue-shifted. A codon-based maximum-likelihood analysis indicated that, associated with the two blue spectral shifts, four amino acid sites (Ile17Met, Ala64Ser, Asn70Ser, and Ser137Ala) have evolved substitutions in parallel and exhibit significant d(N)/d(S) >1. Homology modeling of the full-length Limenitis arthemis astyanax L opsin placed all four substitutions within the chromophore-binding pocket. Strikingly, the Ser137Ala substitution is in the same position as a site that in primates is responsible for a 5- to 7-nm blue spectral shift. Our data show that some of the same amino acid sites are under positive selection in the photopigments of both butterflies and primates, spanning an evolutionary distance >500 million years.

This study examined the effects of increased leaf nitrogen in natural host-plants (Plantago spp.) on female oviposition preference, larval performance, and larval chemical defense of the butterfly Junonia coenia. Increased availability of soil nutrients caused the host-plant's foliar nitrogen to increase and its chemical defense to decrease. Larval performance did not correlate with increases in foliar nitrogen. Larval growth rate and survival were equivalent across host-plant treatments. However, larvae raised on fertilized host-plants showed concomitant decreases in chemical defense as compared to larvae reared on unfertilized host-plants. Since most butterfly larvae cannot move long distances during their first few instars and are forced to feed upon the plant on which they hatched, J. coenia larval chemical defense is determined, in large part, by female oviposition choice. Female butterflies preferred host-plants with high nitrogen over host-plants with low nitrogen; however, this preference was also mediated by plant chemical defense. Female butterflies preferred more chemically defended host-plants when foliar nitrogen was equivalent between host-plants. J. coenia larvae experience intense predation in the field, especially when larvae are not chemically well defended. Any qualitative or quantitative variation in plant allelochemical defense has fitness consequences on these larvae. Thus, these results indicate that females may be making sub-optimal oviposition decisions under a nutrient-enriched regime, when predators are present. Given the recent increase in fertilizer application and nitrogen deposition on the terrestrial landscape, these interactions between female preference, larval performance, and larval chemical defense may result in long-term changes in population dynamics and persistence of specialist insects.

Although much is known about the ecological significance of metamorphosis and metamorphic timing, few studies have examined the underlying genetic architecture of these traits, and no study has attempted to associate phenotypic variation to molecular variation in specific genes. Here we report on a candidate gene approach (CGA) to test specific loci for a statistical contribution to variation in metamorphic timing. Three segregating populations (SP1, SP2 and SP3) were constructed utilizing three species of paedomorphic Mexican ambystomatid salamander, including the axolotl, Ambystoma mexicanum. We used these replicated species to test the hypothesis that inheritance of alternate genotypes at two thyroid hormone receptor loci (TRalpha, TRbeta) affects metamorphic timing in ambystomatid salamanders. A significant TRalpha*SP effect indicated that variation in metamorphic timing may be influenced by TRalpha genotype, however, the effect was not a simple one, as both the magnitude and direction of the phenotypic effect depended upon the genetic background. These are the first data to implicate a specific gene in contributing to variation in metamorphic timing. In general, candidate gene approaches can be extended to any number of loci and to any organism where simple genetic crosses can be performed to create segregating populations. The approach is thus of particular value in ecological studies where target genes have been identified but the study organism is not one of the few well-characterized model systems that dominate genetic research.