Behavioral Evidence for Host Transitions in Plant, Plant Parasite, and Insect Interactions

Behavioral Evidence for Host Transitions in Plant, Plant Parasite, and Insect Interactions Abstract Specialized herbivorous insects have the ability to transition between host plant taxa, and considering the co-evolutionary history between plants and the organisms utilizing them is important to understanding plant insect interactions. We investigated the role of a pine tree parasite, dwarf mistletoe (Arceuthobium spp.) M. Bieb. Santalales: Viscaceae, in mediating interactions between Neophasia (Lepidoptera: Pieridae) butterflies and pine trees, the butterflies’ larval hosts. Mistletoe is considered the butterflies’ ancestral host, and the evolutionary transition to pine may have occurred recently. In Arizona, United States, we studied six sites in pine forest habitats: three in Neophasia menapia (Felder and R. Felder, 1859) habitat and three in Neophasia terlooii Behr, 1869 habitat. Each site contained six stands of trees that varied in mistletoe infection severity. Butterfly behavior was observed and ranked at each stand. Volatile compounds were collected from trees at each site and analyzed using gas chromatography-mass spectroscopy. Female butterflies landed on or patrolled around pine trees (i.e., interacted) more than males, and N. terlooii interacted more with pine trees than N. menapia. Both butterfly species interacted more with tree stands harboring greater mistletoe infection, and N. terlooii interacted more with heavily infected tree stands than did N. menapia. The influence of mistletoe on Neophasia behavior may be mediated by differences in tree volatiles resulting from mistletoe infection. Volatile profiles significantly differed between infected and uninfected pine trees. The role of mistletoe in mediating butterfly interactions with pines has implications for conservation biology and forest management, and highlights the importance of understanding an organism’s niche in an evolutionary context. butterfly, evolution, mistletoe, pine, volatile compound Specialized herbivorous insects can transition to different host plant taxa through rapid genetic adaptation (Singer et al. 1993) or via more gradual evolutionary processes (Dobler et al. 1996). Understanding the co-evolutionary history between plants and insects is key to understanding contemporary plant–insect interactions, especially when considering environmental stressors. Plant–insect interactions are critical to ecosystem function and developing successful habitat management strategies (Raffa et al. 2008, Soler et al. 2012). Other organisms can influence these interactions and may contribute to host transitioning. For example, plant pathogens can influence the attractiveness of the plants to herbivorous insects (McLeod et al. 2005, Mauck et al. 2010, Mann et al. 2012). Dwarf mistletoes, Arceuthobium spp. (M. Bieb. Santalales: Viscaceae), are flowering plants and parasites of conifers, and affect associated biological communities and silviculture (Stevens and Hawksworth 1970, Drummond 1982, Conklin 2000, Hoffman 2004). The ponderosa pine ecosystem occupies a significant portion of western North America, ranging from extreme southwestern Canada to central Mexico (Little 1971) and the pines host dwarf mistletoes (Hawksworth and Wiens 1996). Here, we investigate a tri-trophic interaction and a hypothesized host switch involving an insect herbivore, its contemporary conifer host, and the mistletoe parasite in a ponderosa-pine dominated ecosystem. Pine butterflies in the genus Neophasia Behr, 1869 (Lepidoptera: Pieridae) belong to the subtribe Aporiina and, in the New World, members of Aporiina are primarily mistletoe (Santalales) feeders and are restricted to South America (Braby and Nishida 2010). However, Neophasia and Eucheira Westwood, 1834 are two North American aporiine genera that feed on conifers and madrone (Arbutus) (L. Ericales: Ericaceae) trees, respectively. This break in phylogenetic conservatism via the exploitation of new larval host plants may have facilitated the northward expansion of these two genera, along with adaptation to colder climates. When combined, the geographic ranges of Neophasia menapia (Felder and R. Felder, 1859) and Neophasia terlooii Behr, 1869 span 30 degrees of latitude in western North America, which overlap considerably with the ponderosa pine ecosystem (for butterfly distributions, see: Scott 1986, Bailowitz and Brock 1991, Lotts and Naberhaus 2015). Central Arizona marks the southern range limit of N. menapia and the northern range limit of N. terlooii (Fig. 1). We focused on habitats for N. menapia and N. terlooii in Arizona at the southern and northern range boundaries of each butterfly species, respectively. Fig. 1. View largeDownload slide Geographic distribution of the six sites in Arizona, each containing six tree stands. The blue shaded area indicates the distribution of Neophasia menapia at the county level. The red shaded area indicates the distribution of N. terlooii at the county level. The area in purple indicates the county where both species can be found, with the river marking the dividing line. The inset map shows the entire range of N. menapia in blue and that of N. terlooii in red. Arizona is purple because it contains both butterfly species. With the exception of a small area in SW Texas, inset distribution data are at the state or province scale. Distribution data were obtained from Lotts and Naberhaus (2015). NJ = North Jacob Lake, SC = Schultz Pass, MO = Mormon Lake, BF = Barfoot Park, SW = Sawmill Canyon, and CR = Carr Canyon. Fig. 1. View largeDownload slide Geographic distribution of the six sites in Arizona, each containing six tree stands. The blue shaded area indicates the distribution of Neophasia menapia at the county level. The red shaded area indicates the distribution of N. terlooii at the county level. The area in purple indicates the county where both species can be found, with the river marking the dividing line. The inset map shows the entire range of N. menapia in blue and that of N. terlooii in red. Arizona is purple because it contains both butterfly species. With the exception of a small area in SW Texas, inset distribution data are at the state or province scale. Distribution data were obtained from Lotts and Naberhaus (2015). NJ = North Jacob Lake, SC = Schultz Pass, MO = Mormon Lake, BF = Barfoot Park, SW = Sawmill Canyon, and CR = Carr Canyon. From July through mid-August, adult N. menapia spend sunny days patrolling pine trees in search of mates and trees suitable for oviposition. In northern Arizona, we observed N. menapia ovipositing on Rocky Mountain ponderosa pine (ponderosa pine, henceforth), Pinus ponderosa ssp. scopulorum (Engelmann) (Pinales: Pinaceae), but a considerably greater diversity of conifers is utilized in the rest of its range (see Discussion). N. terlooii has two flights per year, one in late spring/early summer and the primary flight, which is in October. Like its congener, N. terlooii patrols pine trees in search of mates and trees suitable for oviposition. In the Arizona sky islands, we observed N. terlooii ovipositing on Apache pine, Pinus englemannii Carrière, Arizona pine, P. ponderosa ssp. arizonica (Engelmann), ponderosa pine, and southwestern white pine, Pinus strobiformis Engelmann. Southwestern dwarf mistletoe, Arceuthobium vaginatum subsp. eryptopodium (Engelmann) occurs throughout Arizona and parasitizes ponderosa pine, Apache pine, and Arizona pine (Olsen 2003). Dwarf mistletoes induce a stress response that causes trees to alter their biochemistry (Nebeker et al. 1995). Stressed trees produce volatile chemicals such as monoterpenes, which have been shown to attract wood-boring insects (Costello et al. 2008). Four primary chemical isolates are emitted by infected Pinus contorta (Douglas ex Loudon) trees: α-pinene, β-pinene, α-terpineol, and tricyclene (Nebeker et al. 1995). Butterflies have odor receptors on their antennae (Hansson 1995, Mercader et al. 2008), but the ability of Neophasia to detect airborne chemical cues has never been tested, nor have the airborne volatiles been described for the specific pine hosts of Neophasia in Arizona. We explored the role of a plant parasite in influencing plant–insect interactions, specifically how dwarf mistletoe mediates the interactions between butterflies and dwarf mistletoe-infected and uninfected pine trees. We hypothesized that the presence of parasitism and its severity would alter the volatile compound signatures of pine trees. If this was the case, we expected differential behavior between butterflies flying among infected and uninfected trees. Butterflies were predicted to interact more with infected trees and be more abundant in areas with greater dwarf mistletoe presence, which, in an evolutionary context, could reflect their origin from a mistletoe-feeding ancestor and subsequent switch to pine. Materials and Methods Field Sites and Tree Stands Three field sites per Neophasia spp. were selected to observe the interactions between the butterflies and stands of their larval host trees harboring various levels of dwarf mistletoe infection. For N. menapia, one site on the Kaibab Plateau (South Jacob Lake) and two sites on the Mogollon Rim (Schultz Pass and Mormon Lake) (Fig. 1) were visited. For N. terlooii, one site in the Chiricahua Mountains (Barfoot Park) and two sites in the Huachuca Mountains (Sawmill and Carr Canyons) (Fig. 1) were visited. Sites comprised roughly rectangular patches of pine forest, ranging from, 335- to 715-m long and 130- to170-m wide. The latitude and longitude for each site are listed in Table 1. Table 1. Locations of the field sites for the behavioral observations and volatile samples taken from pine trees Habitat for  Site  Latitude  Longitude  Neophasia menapia  North Jacob Lake  36°42′14.88″N  112°15′49.08″W  Schultz Pass  35°15′15.95″N  111°40′5.52″W  Mormon Lake  34°59′22.03″N  111°30′28.32″W  Neophasia terlooii  Barfoot Park  31°54′59.04″N  109°16′48.66″W  Sawmill Canyon  31°26′41.86″N  110°22′8.76″W  Carr Canyon  31°25′45.17″N  110°18′14.84″W  Habitat for  Site  Latitude  Longitude  Neophasia menapia  North Jacob Lake  36°42′14.88″N  112°15′49.08″W  Schultz Pass  35°15′15.95″N  111°40′5.52″W  Mormon Lake  34°59′22.03″N  111°30′28.32″W  Neophasia terlooii  Barfoot Park  31°54′59.04″N  109°16′48.66″W  Sawmill Canyon  31°26′41.86″N  110°22′8.76″W  Carr Canyon  31°25′45.17″N  110°18′14.84″W  GPS coordinates indicate the approximate center of each site. View Large Table 1. Locations of the field sites for the behavioral observations and volatile samples taken from pine trees Habitat for  Site  Latitude  Longitude  Neophasia menapia  North Jacob Lake  36°42′14.88″N  112°15′49.08″W  Schultz Pass  35°15′15.95″N  111°40′5.52″W  Mormon Lake  34°59′22.03″N  111°30′28.32″W  Neophasia terlooii  Barfoot Park  31°54′59.04″N  109°16′48.66″W  Sawmill Canyon  31°26′41.86″N  110°22′8.76″W  Carr Canyon  31°25′45.17″N  110°18′14.84″W  Habitat for  Site  Latitude  Longitude  Neophasia menapia  North Jacob Lake  36°42′14.88″N  112°15′49.08″W  Schultz Pass  35°15′15.95″N  111°40′5.52″W  Mormon Lake  34°59′22.03″N  111°30′28.32″W  Neophasia terlooii  Barfoot Park  31°54′59.04″N  109°16′48.66″W  Sawmill Canyon  31°26′41.86″N  110°22′8.76″W  Carr Canyon  31°25′45.17″N  110°18′14.84″W  GPS coordinates indicate the approximate center of each site. View Large Within each site, six stands of trees were selected such that each stand was at least 50 m from another stand running roughly lengthwise through the center of each site. Each stand consisted of two to six trees, where the two most distant trees in a stand were at most 15 m apart from each other. Each stand was set apart from the surrounding forest such that the tree crowns within the stands were not touching the tree crowns of the surrounding forest matrix. The tree crowns within each stand were in contact. Each site contained three stands that had visible dwarf mistletoe infection and three stands with little to no visible infection. Stands were scored for infection on a scale from 0 to 6, with 0 being uninfected and 6 being heavily infected. Scoring methods followed those of Hawksworth (1977), where the live crown of each tree in a stand was divided into thirds, each third was visually scored as either 0 (no visible infections), 1 (50% or less of the branches infected), or 2 (greater than 50% of branches infected), and the sum of the thirds was the score for the tree. We summed the scores of each tree in a stand and took the average as the score for the stand. To investigate the potential for dwarf mistletoe infection in the surrounding forest to influence butterfly abundance at each of the six stands at each of the N. menapia sites, all ponderosa pine trees within a 25-m radius of the centroid of each stand were scored for dwarf mistletoe infection. The 25-m-radius circle was divided into eight slices, starting with the slice containing trees between 0° (N) and 45° (NE). Slice perimeters were marked with irrigation flags and trees were marked with chalk after they had been scored. This was repeated in a clockwise fashion for the remaining seven slices. The scores of all trees in a 25-m radius circle were averaged. The average infection scores were compared to butterfly abundance at each tree stand, i.e., the total number of butterflies seen per stand during each visit. Butterfly Abundance and Behavior N. menapia were observed from 22 July 2015 through 9 August 2015, and N. terlooii were observed from 18 October 2015 through 28 October 2015. Each site was visited between one and five times during a field season between 9:00 a.m. and 3:00 p.m. Butterfly observations at each of the six tree stands at each site were completed in a random order, and each observation lasted for 30 min with at least 15 min of full sun. If there was not at least 15 min of full sun, the stand was re-visited after the others were completed, time permitting (i.e., the last 30-min observation had to start by 2:30 pm). Butterfly behavior was ranked in a qualitative manner, from weak to strong interactions. If a butterfly passed by a stand and did not significantly alter its flight direction, it was classified as ‘flyby’ (weakest interaction). If a butterfly encountered a stand and patrolled the canopy by circling the treetops, zigzagging up and down the canopy, or both, it was classified as ‘patrol’. If a butterfly landed on a tree branch, it was classified as ‘land’ (strongest interaction for males. If a butterfly oviposited on a pine needle, it was classified as ‘oviposit’ (strongest interaction for females). Sex was determined by wing color: males of both species are white with black markings, female N. terlooii are orange with black markings, and female N. menapia are pale yellow with heavier black markings than males, and have bright red dots along their outer hind wing margins. Collection and Analysis of Tree Volatiles Airborne volatile compounds were collected from infected and uninfected trees within the habitats of both species of Neophasia. Each infected tree had two samples taken from it: one branch that had dwarf mistletoe on it and one that did not have dwarf mistletoe on it. Branches from ponderosa pines were sampled from sites in N. menapia habitat, and ponderosa pine, Apache pine, and Arizona pine branches were sampled from sites in N. terlooii habitat. Volatile samples were collected by placing a living branch roughly 45-cm long into a 48.2-cm × 59.6-cm oven bag (Reynold’s Oven Bags, Manufacturer # 1001090000510), which had been pre-baked for 3.5 h at 121°C to remove unwanted volatiles. Bag openings were gathered together and sealed around the basal stem of the branch with a plastic zip tie. Care was taken to ensure the bags were sheltered from direct sunlight by suspending black plastic sheets in branches above the bags to provide shade. One control bag of air from each site was collected. There was a total of 25 volatile samples collected between the two habitats. Branches were bagged at the start of a site visit for the behavioral observations and branches were then cut off the tree in the afternoon, leaving about 15 cm of stem outside the bag. Approximately 12 h after a branch was bagged, the bag was affixed to a 1.5-gallon shop vacuum (Stanley, Part # SL18125P1) calibrated using a rheostat (GE, Model # 18019) that was used to draw air out of the bag through an adsorbent 30-mg HayeSep Q filter (Volatile Assay Systems, VAS) at 160 ml/min for 1 h. Clean filtered air was allowed into the bag through a glass pipette filled with activated charcoal and attached to an opposite corner of the bag. This allowed air to continue flowing over the pine samples while minimizing external contaminants. Filters containing volatiles were stored in airtight glass vials and kept in a freezer (−15°C), later shipped on ice, and stored in a freezer until analysis. Gas chromatography-mass spectroscopy (GC-MS) conducted at the Citrus Research and Education Center, University of Florida, Lake Alfred, FL was used to identify the volatile compounds in our samples. Each sample was eluted from its filter into a 200-µl glass vial insert with two 75-µl rinses of methylene chloride. Following elution, 5 μl of 1.5 ng/μl nonyl acetate were added as an internal standard. One-μl aliquots of each sample were then run on a 30-m × 0.25-mm-ID DB-5 capillary column in a Clarus 500 GC-MS (PerkinElmer, Waltham, MA). The column was held at 35°C for 3 min after injection and then increased 10°C/min until reaching 260°C, where it remained for an additional 5 min. Helium was used as a carrier gas at a flow rate of 2 ml/min. Electron ionization spectra were compared with references found in the National Institute of Standards and Technology database and then confirmed with available standards. Retention times, peak heights, peak areas, and the start and end times for each peak were recorded and downloaded for analysis. Statistical Analyses External factors (i.e., surrounding forest condition) influencing N. menapia abundance (i.e., total number of butterflies observed at a stand) were investigated with negative binomial regression using generalized linear models with a log link function to account for overdispersion in the abundance data. These models were better fits than Poisson models based on comparisons of log-likelihood values, goodness of fit tests, and visual examination of the conditional mean-variance relationship. All factors (i.e., mean dwarf mistletoe infection severity of trees surrounding a stand, number of trees surrounding a stand, date of observation, site, stand, and percentage of surrounding trees infected) as well as two- and three-level interactions were evaluated for inclusion in the models. Model factors were selected for inclusion based on analysis of deviance, chi-square tests of log-likelihood values, pseudo-R2 values, goodness of fit, and examination of residual diagnostic plots. Internal factors (i.e., stand conditions) influencing N. menapia and N. terlooii behavior at each tree stand were examined using cumulative link models (sometimes referred to as ordinal regression) with a logit link function. All factors (i.e., butterfly species, sex, and tree stand dwarf mistletoe infection severity) as well as two-factor interactions were evaluated for inclusion in the models. Model factors were selected for inclusion based on analysis of deviance, chi-square tests of log-likelihood values, goodness of fit and examination of residual diagnostic plots. Tukey’s HSD tests were used to make post-hoc comparisons. To investigate differences in tree volatile profiles potentially influenced by dwarf mistletoe infection, variable importance measures from random forests models were first used to identify compounds of interest. Differences in abundances of those compounds corresponding to dwarf mistletoe infection status were then modeled using multivariate analysis of variance (MANOVA) after assuring adherence to assumptions of normality and homoscedasticity. Canonical discriminant analysis was then used to identify relative influences of each compound. Data were collated in Microsoft Excel and analyzed using R version 3.3.1 in the RStudio version 0.99.902 development environment (R Core Team 2015). The following packages facilitated analysis: dplyr (Wickham and Francois 2015) and tidy (Wickham 2014) for data formatting, xlsx (Dragulescu 2014) for the R-Excel interface, car (Fox and Weisberg 2011) and lsmeans (Lenth 2016) for regression analysis, ordinal (Christensen 2015) for cumulative link models, candisc (Friendly and Fox 2016) for canonical discriminant analysis, MASS (Venables and Ripley 2002) for negative binomial regression, randomForest (Liaw and Wiener 2002) for random forests models, and ggplot2 (Wickham 2009) and ggmap (Kahle and Wickham 2013) for graphics. Raw data and R scripts are available upon request. Results Butterfly Abundance Greater numbers of trees surrounding each stand corresponded to greater numbers of N. menapia seen at each stand (χ2 = 14.1; df = 1; P < 0.001) (Fig. 2). The number of trees surrounding each stand ranged from 37 to 335, with the highest numbers indicating many small saplings and lower numbers indicating fewer, but larger, mature trees. Holding the sampling date constant, for every unit increase in the number of trees surrounding each stand, there was a 0.6 % (95% CI: 0.3%, 0.9%) increase in numbers of butterflies seen at each stand. Butterfly abundance was affected by the observation date, with greatest numbers seen at the beginning and end of the summer sampling period (χ2 = 7.55; df = 1; P = 0.06). Dwarf mistletoe infection of the trees surrounding each stand did not influence butterfly abundance at each stand (χ2 = 1.04; df = 1; P = 0.31). The model incorporating these factors was better than the null model (LR3 = 30.2; P < 0.0001), did not demonstrate a lack of fit (χ2 = 34.9; df = 30; P = 0.25), and explained 58.1% of the observed variance. Fig. 2. View largeDownload slide Tree and butterfly abundance were positively correlated. The number of butterflies indicates the total number of butterflies observed at each tree stand. The line and shaded area indicate the model fit and 95% confidence intervals, respectively. Results were averaged across sampling date at each stand. Fig. 2. View largeDownload slide Tree and butterfly abundance were positively correlated. The number of butterflies indicates the total number of butterflies observed at each tree stand. The line and shaded area indicate the model fit and 95% confidence intervals, respectively. Results were averaged across sampling date at each stand. Butterfly Behavior Butterfly species, average dwarf mistletoe infection severity of each tree stand, and the interaction between butterfly sex and dwarf mistletoe infection severity influenced butterfly behavior (Table 2). Females interacted significantly more with trees than did males (P < 0.001; Fig. 3). Interestingly, N. terlooii interacted more with trees than did N. menapia (P = 0.0002) (Fig. 3). An increase in dwarf mistletoe infection severity increased butterfly interactions with trees for both males (P = 0.012) and females (P = 0.048) (Fig. 4). Table 2. Analysis of deviance for models of Neophasia behavior Source  χ2  df  P  Speciesa  21.0  1  <0.0001  Sex  0.3  1  0.567  Hawksworthb  13.5  1  0.0002  Sex:Hawksworth  14.0  1  0.0002  Total  56.5  7  <0.0001  Residual  1.1  3  0.777  Source  χ2  df  P  Speciesa  21.0  1  <0.0001  Sex  0.3  1  0.567  Hawksworthb  13.5  1  0.0002  Sex:Hawksworth  14.0  1  0.0002  Total  56.5  7  <0.0001  Residual  1.1  3  0.777  aSpecies are N. menapia and N. terlooii. bHawksworth is the index of mistletoe infection severity. View Large Table 2. Analysis of deviance for models of Neophasia behavior Source  χ2  df  P  Speciesa  21.0  1  <0.0001  Sex  0.3  1  0.567  Hawksworthb  13.5  1  0.0002  Sex:Hawksworth  14.0  1  0.0002  Total  56.5  7  <0.0001  Residual  1.1  3  0.777  Source  χ2  df  P  Speciesa  21.0  1  <0.0001  Sex  0.3  1  0.567  Hawksworthb  13.5  1  0.0002  Sex:Hawksworth  14.0  1  0.0002  Total  56.5  7  <0.0001  Residual  1.1  3  0.777  aSpecies are N. menapia and N. terlooii. bHawksworth is the index of mistletoe infection severity. View Large Fig. 3. View largeDownload slide Neophasia behavior with respect to sex and species. Flyby, Patrol, Landing, and Oviposition indicate potential interactions with host trees irrespective of infection status. A Flyby is a weak interaction while Oviposition is a strong interaction. Points and error bars denote mean behavior and 95% confidence intervals from cumulative link models of Neophasia behavior, respectively. Letters A, B, C, and D indicate significant differences (P < 0.05) based on Tukey’s HSD test. Fig. 3. View largeDownload slide Neophasia behavior with respect to sex and species. Flyby, Patrol, Landing, and Oviposition indicate potential interactions with host trees irrespective of infection status. A Flyby is a weak interaction while Oviposition is a strong interaction. Points and error bars denote mean behavior and 95% confidence intervals from cumulative link models of Neophasia behavior, respectively. Letters A, B, C, and D indicate significant differences (P < 0.05) based on Tukey’s HSD test. Fig. 4. View largeDownload slide Effect of dwarf mistletoe infection severity on Neophasia behavior. The Hawksworth Score ranges from 0 (uninfected) to 6 (heavily infected). A Flyby is a weak interaction while Oviposition is a strong interaction. Lines and shaded areas denote mean behavior and 95% confidence intervals from cumulative link models of Neophasia behavior, respectively. Fig. 4. View largeDownload slide Effect of dwarf mistletoe infection severity on Neophasia behavior. The Hawksworth Score ranges from 0 (uninfected) to 6 (heavily infected). A Flyby is a weak interaction while Oviposition is a strong interaction. Lines and shaded areas denote mean behavior and 95% confidence intervals from cumulative link models of Neophasia behavior, respectively. Volatiles Variable importance measures from random forests models identified seven primary volatile compounds that differed based on dwarf mistletoe infection status (F = 3.73; df = 21,42; P < 0.0001). Volatile profiles of branches from infected trees with dwarf mistletoe present on the branches were different from the profiles of branches from uninfected trees or uninfected branches from infected trees (t1 = −6.765; df = 18; P < 0.0001) (Fig. 5a). Volatile profiles of branches of infected trees without dwarf mistletoe present on the branches were marginally different from those of branches from uninfected trees (t = −2.38; df = 18; P = 0.05) (Fig. 5a). Germacrene-D and δ-cadinene were the primary contributors to the volatile index that provided resolution to detect differences in infection status (Fig. 5b). Fig. 5. View largeDownload slide The dwarf mistletoe infection status of a tree influences its volatile chemical profile. (a) Differences in volatile profiles based on infection status. Black points and error bars denote mean and 95% bootstrapped confidence intervals, respectively. Grey points denote observed values. (b) Relative weighting of each compound used to construct the volatile index. The magnitude of arrows denotes relative weighting while direction denotes the effect of contributions (i.e., Germecrene-D had higher levels in volatile profiles from uninfected branches, while d-longifolene had higher levels in profiles from infected branches). Fig. 5. View largeDownload slide The dwarf mistletoe infection status of a tree influences its volatile chemical profile. (a) Differences in volatile profiles based on infection status. Black points and error bars denote mean and 95% bootstrapped confidence intervals, respectively. Grey points denote observed values. (b) Relative weighting of each compound used to construct the volatile index. The magnitude of arrows denotes relative weighting while direction denotes the effect of contributions (i.e., Germecrene-D had higher levels in volatile profiles from uninfected branches, while d-longifolene had higher levels in profiles from infected branches). Discussion We are potentially witnessing an evolutionary transition of host plant utilization in Neophasia. Shifts between conifers as larval hosts and conifer parasites like dwarf mistletoe as larval hosts may be fairly common in Lepidoptera because the mistletoes are growing on the trees very close to the pine needles on which caterpillars feed (Mooney 2003). Braby and Trueman (2006) hypothesized that this close tree-to-parasite proximity could be responsible for the numerous shifts between caterpillar host preference of trees and their associated mistletoes observed in Aporiina. Our behavioral evidence suggests that Neophasia has an affinity for pine trees infected with dwarf mistletoe. It is possible that some element of mistletoe host-seeking behavior has been retained in extant Neophasia as an evolutionary relic. N. terlooii showed a stronger preference for infected trees than N. menapia, suggesting that N. terlooii may have retained more of its hypothesized ancestral traits. From a biogeographic context, the distribution of N. terlooii is closer geographically to the distributions of remaining New World Aporiina, which are predominantly Neotropical (Braby et al. 2007), while the distribution of N. menapia has shifted farther north. A host plant shift away from mistletoe opens a new niche for Neophasia, as conifers are more abundant than mistletoes, in terms of biomass, as larval food sources. N. menapia is fairly polyphagous within the Pinaceae, feeding on Pinus, Pseudotsuga Carrière, Abies Mill., Tsuga Carrière, and Picea Mill. (Evenden 1926, Cole 1971, Scott 1986, Robinson et al. 2002). Less is known about N. terlooii, but they may be more limited in host plant options and have thus far been documented on only two genera: Pinus and Picea (Arizona Game and Fish Department 2001). Dwarf mistletoes can be found on all aforementioned tree genera (Hawksworth and Wiens 1996). Assuming the latter host-parasite relationships have persisted, and that ancestral Neophasia specialized on a more limited number of dwarf mistletoe species, this could explain why N. terlooii is more restricted in its pine hosts. N. terlooii has a greater affinity for pines that are parasitized by specific dwarf mistletoe species, while N. menapia is more likely to utilize other pines over a greater geographic range that are parasitized by more species of dwarf mistletoe. Dwarf mistletoe infection has a significant effect on Neophasia behavior. Neophasia interacted more with trees that were heavily infected. Male behavior predominantly consisted of patrolling, which is typical mate-seeking behavior (Scott 1986, Lotts and Naberhaus 2015). Despite the low abundance of females and greater variability compared to those of males, females still interacted significantly more with infected trees, tending to land and oviposit on them more often as compared with uninfected counterparts. Although butterfly behavior was affected by dwarf mistletoe infection in the tree stands from where the butterflies were observed, butterfly abundance at these stands was influenced more by the number of trees surrounding the stands rather than infection severity. There is evidence to suggest N. menapia on the Kaibab Plateau formed a population distinct from conspecifics found on the Mogollon Rim (D. A. Halbritter, unpublished data), suggesting the latter two regions would comprise the geographic scale necessary to make inferences of how forest structure affects population dynamics. While dwarf mistletoe infection may influence butterfly behavior within a tree stand, overall abundances of butterflies seem to be affected primarily by differences in other attributes of available host resources (i.e., the number and size of trees in the area). However, replicated population-scale surveys of dwarf mistletoe infection, tree sizes and abundance, and butterfly population size estimates are needed before any conclusions can be drawn about how spatial characteristics of forests and dwarf mistletoe infections affect Neophasia populations. The success of Neophasia larvae is at least partially dependent on the nutritional content of the pine needles onto which they were placed as ova. In a related species of tree, Pinus contorta, infected trees were found to have significantly lower starch, total nitrogen, and free amino-nitrogen composition in their phloem (Nebeker et al. 1995). Phloem samples in the latter study were taken from tree trunks at breast height, but dwarf mistletoe infections can have more localized effects, causing infected branches to become nutrient sinks at the expense of the tree as a whole (Hawksworth and Wiens 1996). Eventually tree health declines and the tree can die from an infection. Butterflies in our study seemed to randomly explore the forest matrix, but then spent more time searching the canopies of specific trees. Within close proximity to those trees, butterflies may select branches on infected trees that have higher nutritional content, which may be reflected in the different volatile profiles detected at close range. In addition to signaling nutritional quality, tree volatiles released as a result of mistletoe infection and other stressors may be cues to Neophasia indicating compromised tree defenses. We did not document feeding damage from other insects or infections from other pathogens, the latter of which are also known to alter terpene blends in pines (Nebeker et al. 1995). Pathogen-infected host plants are often more attractive to the insect vectors of these pathogens than uninfected counterparts (Mauck et al. 2010, Mann et al. 2012). For example, the bark beetle, Hylurgopinus rufipes (Eichhoff 1868), is preferentially attracted to elm trees infected with the Dutch elm disease pathogen (Ophiostoma novo-ulmi) compared with uninfected elms (McLeod et al. 2005). Although not vectors of mistletoe, ancestral Neophasia spp. likely fed on mistletoe and therefore would have had a direct dependence on it and would later retain an association with infected trees, potentially responding to kairomones in the pine forest matrix that signal compromised trees. Pine branches infected with mistletoe were characterized by an overall greater quantitative release of volatiles, and also qualitatively released a blend of volatiles characterized by specific terpenes, such as germacrene-D and δ-cadinene, as compared with uninfected branches. The mistletoe itself may have contributed to the volatile profile of branches with mistletoe on them as well. Additionally, individual volatile compounds can be emitted by non-host tree species, thereby adding to the complex milieu of forest volatiles. For these reasons, host recognition is likely dependent on a particular blend of volatiles (Witzgall et al. 2005). Additional behavioral assays with butterflies in the laboratory and field will be necessary to determine the specific volatile signals that influence host-seeking in Neophasia. As a result of mistletoe infection, it may be an overall quantitative increase in volatile production, a specific blend of volatiles, or both, that influence host selection. Neophasia may be drawn to volatiles from the mistletoe itself, or the butterflies may be attracted to trees afflicted with other diseases or insect damage. Conclusions Dwarf mistletoe and Neophasia butterflies are two conspicuous members of the ponderosa pine community that utilize the pine trees as hosts and have been shown here to interact with each other, comprising a multitrophic ecological interaction. Pine forests in the western United States are facing threats from fires exacerbated by climate change (Allen and Breshears 1998), insect damage (Kenaley et al. 2006), synergistic effects of fire and insects (McHugh et al. 2003), and from interactions between insects and tree parasites (Wagner and Mathiasen 1985). Neophasia are generally not detrimental to pine forests, but there have been occasional, localized population irruptions that result in defoliation (Ciesla 1974, Young 1986). Neophasia are abundant and easily detectable as adults, making them effective representatives of the ponderosa pine community. Changes in Neophasia population dynamics could reflect changes in dwarf mistletoe populations and consequently changes in forest health. Presence of butterflies may be indicative of new or ongoing mistletoe infestations. Understanding the ecology of Neophasia in a community context will therefore be important to inform forest management and conservation practices. Studies such as ours, which integrate understanding of evolutionary histories and field studies on multitrophic ecological interactions, will be important for the improvement of conservation science. Acknowledgments We thank Alan Yanahan, the Merriam-Powell Research Station, and the University of Arizona Entomology Department for providing lodging and laboratory space in Arizona, and Matthew Standridge for handling volatile sample shipments. We extend our gratitude to Fort Huachuca and the National Forest Service and for their collaboration. Funds from the William C. and Bertha M. Cornett Fellowship, the Florida Museum of Natural History Travel Award, and an Entomology and Nematology Department Scholarship, the latter awarded to author J.M.G., through the University of Florida were used to cover travel and lodging expenses. References Cited Allen, C. D., and D. D. Breshears. 1998. Drought-induced shift of a forest-woodland ecotone: rapid landscape response to climate variation. Proc. Natl. Acad. Sci. USA  95: 14839– 14842. Google Scholar CrossRef Search ADS   Arizona Game and Fish Department. 2001. Neophasia terlootii . Unpublished abstract compiled and edited by the Heritage Data Management System, Arizona Game and Fish Department, Phoenix, AZ. Bailowitz, R. A., and J. P. Brock. 1991. Butterflies of Southeastern Arizona . Sonoran Arthropod Studies, Inc, Tucson, AZ. Braby, M. F., and K. Nishida. 2010. The immature stages, larval food plants and biology of Neotropical mistletoe butterflies (Lepidoptera: Pieridae). II. The Catasticta group (Pierini: Aporiina). J. 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Behavioral Evidence for Host Transitions in Plant, Plant Parasite, and Insect Interactions

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Entomological Society of America
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© The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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0046-225X
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1938-2936
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10.1093/ee/nvy033
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Abstract

Abstract Specialized herbivorous insects have the ability to transition between host plant taxa, and considering the co-evolutionary history between plants and the organisms utilizing them is important to understanding plant insect interactions. We investigated the role of a pine tree parasite, dwarf mistletoe (Arceuthobium spp.) M. Bieb. Santalales: Viscaceae, in mediating interactions between Neophasia (Lepidoptera: Pieridae) butterflies and pine trees, the butterflies’ larval hosts. Mistletoe is considered the butterflies’ ancestral host, and the evolutionary transition to pine may have occurred recently. In Arizona, United States, we studied six sites in pine forest habitats: three in Neophasia menapia (Felder and R. Felder, 1859) habitat and three in Neophasia terlooii Behr, 1869 habitat. Each site contained six stands of trees that varied in mistletoe infection severity. Butterfly behavior was observed and ranked at each stand. Volatile compounds were collected from trees at each site and analyzed using gas chromatography-mass spectroscopy. Female butterflies landed on or patrolled around pine trees (i.e., interacted) more than males, and N. terlooii interacted more with pine trees than N. menapia. Both butterfly species interacted more with tree stands harboring greater mistletoe infection, and N. terlooii interacted more with heavily infected tree stands than did N. menapia. The influence of mistletoe on Neophasia behavior may be mediated by differences in tree volatiles resulting from mistletoe infection. Volatile profiles significantly differed between infected and uninfected pine trees. The role of mistletoe in mediating butterfly interactions with pines has implications for conservation biology and forest management, and highlights the importance of understanding an organism’s niche in an evolutionary context. butterfly, evolution, mistletoe, pine, volatile compound Specialized herbivorous insects can transition to different host plant taxa through rapid genetic adaptation (Singer et al. 1993) or via more gradual evolutionary processes (Dobler et al. 1996). Understanding the co-evolutionary history between plants and insects is key to understanding contemporary plant–insect interactions, especially when considering environmental stressors. Plant–insect interactions are critical to ecosystem function and developing successful habitat management strategies (Raffa et al. 2008, Soler et al. 2012). Other organisms can influence these interactions and may contribute to host transitioning. For example, plant pathogens can influence the attractiveness of the plants to herbivorous insects (McLeod et al. 2005, Mauck et al. 2010, Mann et al. 2012). Dwarf mistletoes, Arceuthobium spp. (M. Bieb. Santalales: Viscaceae), are flowering plants and parasites of conifers, and affect associated biological communities and silviculture (Stevens and Hawksworth 1970, Drummond 1982, Conklin 2000, Hoffman 2004). The ponderosa pine ecosystem occupies a significant portion of western North America, ranging from extreme southwestern Canada to central Mexico (Little 1971) and the pines host dwarf mistletoes (Hawksworth and Wiens 1996). Here, we investigate a tri-trophic interaction and a hypothesized host switch involving an insect herbivore, its contemporary conifer host, and the mistletoe parasite in a ponderosa-pine dominated ecosystem. Pine butterflies in the genus Neophasia Behr, 1869 (Lepidoptera: Pieridae) belong to the subtribe Aporiina and, in the New World, members of Aporiina are primarily mistletoe (Santalales) feeders and are restricted to South America (Braby and Nishida 2010). However, Neophasia and Eucheira Westwood, 1834 are two North American aporiine genera that feed on conifers and madrone (Arbutus) (L. Ericales: Ericaceae) trees, respectively. This break in phylogenetic conservatism via the exploitation of new larval host plants may have facilitated the northward expansion of these two genera, along with adaptation to colder climates. When combined, the geographic ranges of Neophasia menapia (Felder and R. Felder, 1859) and Neophasia terlooii Behr, 1869 span 30 degrees of latitude in western North America, which overlap considerably with the ponderosa pine ecosystem (for butterfly distributions, see: Scott 1986, Bailowitz and Brock 1991, Lotts and Naberhaus 2015). Central Arizona marks the southern range limit of N. menapia and the northern range limit of N. terlooii (Fig. 1). We focused on habitats for N. menapia and N. terlooii in Arizona at the southern and northern range boundaries of each butterfly species, respectively. Fig. 1. View largeDownload slide Geographic distribution of the six sites in Arizona, each containing six tree stands. The blue shaded area indicates the distribution of Neophasia menapia at the county level. The red shaded area indicates the distribution of N. terlooii at the county level. The area in purple indicates the county where both species can be found, with the river marking the dividing line. The inset map shows the entire range of N. menapia in blue and that of N. terlooii in red. Arizona is purple because it contains both butterfly species. With the exception of a small area in SW Texas, inset distribution data are at the state or province scale. Distribution data were obtained from Lotts and Naberhaus (2015). NJ = North Jacob Lake, SC = Schultz Pass, MO = Mormon Lake, BF = Barfoot Park, SW = Sawmill Canyon, and CR = Carr Canyon. Fig. 1. View largeDownload slide Geographic distribution of the six sites in Arizona, each containing six tree stands. The blue shaded area indicates the distribution of Neophasia menapia at the county level. The red shaded area indicates the distribution of N. terlooii at the county level. The area in purple indicates the county where both species can be found, with the river marking the dividing line. The inset map shows the entire range of N. menapia in blue and that of N. terlooii in red. Arizona is purple because it contains both butterfly species. With the exception of a small area in SW Texas, inset distribution data are at the state or province scale. Distribution data were obtained from Lotts and Naberhaus (2015). NJ = North Jacob Lake, SC = Schultz Pass, MO = Mormon Lake, BF = Barfoot Park, SW = Sawmill Canyon, and CR = Carr Canyon. From July through mid-August, adult N. menapia spend sunny days patrolling pine trees in search of mates and trees suitable for oviposition. In northern Arizona, we observed N. menapia ovipositing on Rocky Mountain ponderosa pine (ponderosa pine, henceforth), Pinus ponderosa ssp. scopulorum (Engelmann) (Pinales: Pinaceae), but a considerably greater diversity of conifers is utilized in the rest of its range (see Discussion). N. terlooii has two flights per year, one in late spring/early summer and the primary flight, which is in October. Like its congener, N. terlooii patrols pine trees in search of mates and trees suitable for oviposition. In the Arizona sky islands, we observed N. terlooii ovipositing on Apache pine, Pinus englemannii Carrière, Arizona pine, P. ponderosa ssp. arizonica (Engelmann), ponderosa pine, and southwestern white pine, Pinus strobiformis Engelmann. Southwestern dwarf mistletoe, Arceuthobium vaginatum subsp. eryptopodium (Engelmann) occurs throughout Arizona and parasitizes ponderosa pine, Apache pine, and Arizona pine (Olsen 2003). Dwarf mistletoes induce a stress response that causes trees to alter their biochemistry (Nebeker et al. 1995). Stressed trees produce volatile chemicals such as monoterpenes, which have been shown to attract wood-boring insects (Costello et al. 2008). Four primary chemical isolates are emitted by infected Pinus contorta (Douglas ex Loudon) trees: α-pinene, β-pinene, α-terpineol, and tricyclene (Nebeker et al. 1995). Butterflies have odor receptors on their antennae (Hansson 1995, Mercader et al. 2008), but the ability of Neophasia to detect airborne chemical cues has never been tested, nor have the airborne volatiles been described for the specific pine hosts of Neophasia in Arizona. We explored the role of a plant parasite in influencing plant–insect interactions, specifically how dwarf mistletoe mediates the interactions between butterflies and dwarf mistletoe-infected and uninfected pine trees. We hypothesized that the presence of parasitism and its severity would alter the volatile compound signatures of pine trees. If this was the case, we expected differential behavior between butterflies flying among infected and uninfected trees. Butterflies were predicted to interact more with infected trees and be more abundant in areas with greater dwarf mistletoe presence, which, in an evolutionary context, could reflect their origin from a mistletoe-feeding ancestor and subsequent switch to pine. Materials and Methods Field Sites and Tree Stands Three field sites per Neophasia spp. were selected to observe the interactions between the butterflies and stands of their larval host trees harboring various levels of dwarf mistletoe infection. For N. menapia, one site on the Kaibab Plateau (South Jacob Lake) and two sites on the Mogollon Rim (Schultz Pass and Mormon Lake) (Fig. 1) were visited. For N. terlooii, one site in the Chiricahua Mountains (Barfoot Park) and two sites in the Huachuca Mountains (Sawmill and Carr Canyons) (Fig. 1) were visited. Sites comprised roughly rectangular patches of pine forest, ranging from, 335- to 715-m long and 130- to170-m wide. The latitude and longitude for each site are listed in Table 1. Table 1. Locations of the field sites for the behavioral observations and volatile samples taken from pine trees Habitat for  Site  Latitude  Longitude  Neophasia menapia  North Jacob Lake  36°42′14.88″N  112°15′49.08″W  Schultz Pass  35°15′15.95″N  111°40′5.52″W  Mormon Lake  34°59′22.03″N  111°30′28.32″W  Neophasia terlooii  Barfoot Park  31°54′59.04″N  109°16′48.66″W  Sawmill Canyon  31°26′41.86″N  110°22′8.76″W  Carr Canyon  31°25′45.17″N  110°18′14.84″W  Habitat for  Site  Latitude  Longitude  Neophasia menapia  North Jacob Lake  36°42′14.88″N  112°15′49.08″W  Schultz Pass  35°15′15.95″N  111°40′5.52″W  Mormon Lake  34°59′22.03″N  111°30′28.32″W  Neophasia terlooii  Barfoot Park  31°54′59.04″N  109°16′48.66″W  Sawmill Canyon  31°26′41.86″N  110°22′8.76″W  Carr Canyon  31°25′45.17″N  110°18′14.84″W  GPS coordinates indicate the approximate center of each site. View Large Table 1. Locations of the field sites for the behavioral observations and volatile samples taken from pine trees Habitat for  Site  Latitude  Longitude  Neophasia menapia  North Jacob Lake  36°42′14.88″N  112°15′49.08″W  Schultz Pass  35°15′15.95″N  111°40′5.52″W  Mormon Lake  34°59′22.03″N  111°30′28.32″W  Neophasia terlooii  Barfoot Park  31°54′59.04″N  109°16′48.66″W  Sawmill Canyon  31°26′41.86″N  110°22′8.76″W  Carr Canyon  31°25′45.17″N  110°18′14.84″W  Habitat for  Site  Latitude  Longitude  Neophasia menapia  North Jacob Lake  36°42′14.88″N  112°15′49.08″W  Schultz Pass  35°15′15.95″N  111°40′5.52″W  Mormon Lake  34°59′22.03″N  111°30′28.32″W  Neophasia terlooii  Barfoot Park  31°54′59.04″N  109°16′48.66″W  Sawmill Canyon  31°26′41.86″N  110°22′8.76″W  Carr Canyon  31°25′45.17″N  110°18′14.84″W  GPS coordinates indicate the approximate center of each site. View Large Within each site, six stands of trees were selected such that each stand was at least 50 m from another stand running roughly lengthwise through the center of each site. Each stand consisted of two to six trees, where the two most distant trees in a stand were at most 15 m apart from each other. Each stand was set apart from the surrounding forest such that the tree crowns within the stands were not touching the tree crowns of the surrounding forest matrix. The tree crowns within each stand were in contact. Each site contained three stands that had visible dwarf mistletoe infection and three stands with little to no visible infection. Stands were scored for infection on a scale from 0 to 6, with 0 being uninfected and 6 being heavily infected. Scoring methods followed those of Hawksworth (1977), where the live crown of each tree in a stand was divided into thirds, each third was visually scored as either 0 (no visible infections), 1 (50% or less of the branches infected), or 2 (greater than 50% of branches infected), and the sum of the thirds was the score for the tree. We summed the scores of each tree in a stand and took the average as the score for the stand. To investigate the potential for dwarf mistletoe infection in the surrounding forest to influence butterfly abundance at each of the six stands at each of the N. menapia sites, all ponderosa pine trees within a 25-m radius of the centroid of each stand were scored for dwarf mistletoe infection. The 25-m-radius circle was divided into eight slices, starting with the slice containing trees between 0° (N) and 45° (NE). Slice perimeters were marked with irrigation flags and trees were marked with chalk after they had been scored. This was repeated in a clockwise fashion for the remaining seven slices. The scores of all trees in a 25-m radius circle were averaged. The average infection scores were compared to butterfly abundance at each tree stand, i.e., the total number of butterflies seen per stand during each visit. Butterfly Abundance and Behavior N. menapia were observed from 22 July 2015 through 9 August 2015, and N. terlooii were observed from 18 October 2015 through 28 October 2015. Each site was visited between one and five times during a field season between 9:00 a.m. and 3:00 p.m. Butterfly observations at each of the six tree stands at each site were completed in a random order, and each observation lasted for 30 min with at least 15 min of full sun. If there was not at least 15 min of full sun, the stand was re-visited after the others were completed, time permitting (i.e., the last 30-min observation had to start by 2:30 pm). Butterfly behavior was ranked in a qualitative manner, from weak to strong interactions. If a butterfly passed by a stand and did not significantly alter its flight direction, it was classified as ‘flyby’ (weakest interaction). If a butterfly encountered a stand and patrolled the canopy by circling the treetops, zigzagging up and down the canopy, or both, it was classified as ‘patrol’. If a butterfly landed on a tree branch, it was classified as ‘land’ (strongest interaction for males. If a butterfly oviposited on a pine needle, it was classified as ‘oviposit’ (strongest interaction for females). Sex was determined by wing color: males of both species are white with black markings, female N. terlooii are orange with black markings, and female N. menapia are pale yellow with heavier black markings than males, and have bright red dots along their outer hind wing margins. Collection and Analysis of Tree Volatiles Airborne volatile compounds were collected from infected and uninfected trees within the habitats of both species of Neophasia. Each infected tree had two samples taken from it: one branch that had dwarf mistletoe on it and one that did not have dwarf mistletoe on it. Branches from ponderosa pines were sampled from sites in N. menapia habitat, and ponderosa pine, Apache pine, and Arizona pine branches were sampled from sites in N. terlooii habitat. Volatile samples were collected by placing a living branch roughly 45-cm long into a 48.2-cm × 59.6-cm oven bag (Reynold’s Oven Bags, Manufacturer # 1001090000510), which had been pre-baked for 3.5 h at 121°C to remove unwanted volatiles. Bag openings were gathered together and sealed around the basal stem of the branch with a plastic zip tie. Care was taken to ensure the bags were sheltered from direct sunlight by suspending black plastic sheets in branches above the bags to provide shade. One control bag of air from each site was collected. There was a total of 25 volatile samples collected between the two habitats. Branches were bagged at the start of a site visit for the behavioral observations and branches were then cut off the tree in the afternoon, leaving about 15 cm of stem outside the bag. Approximately 12 h after a branch was bagged, the bag was affixed to a 1.5-gallon shop vacuum (Stanley, Part # SL18125P1) calibrated using a rheostat (GE, Model # 18019) that was used to draw air out of the bag through an adsorbent 30-mg HayeSep Q filter (Volatile Assay Systems, VAS) at 160 ml/min for 1 h. Clean filtered air was allowed into the bag through a glass pipette filled with activated charcoal and attached to an opposite corner of the bag. This allowed air to continue flowing over the pine samples while minimizing external contaminants. Filters containing volatiles were stored in airtight glass vials and kept in a freezer (−15°C), later shipped on ice, and stored in a freezer until analysis. Gas chromatography-mass spectroscopy (GC-MS) conducted at the Citrus Research and Education Center, University of Florida, Lake Alfred, FL was used to identify the volatile compounds in our samples. Each sample was eluted from its filter into a 200-µl glass vial insert with two 75-µl rinses of methylene chloride. Following elution, 5 μl of 1.5 ng/μl nonyl acetate were added as an internal standard. One-μl aliquots of each sample were then run on a 30-m × 0.25-mm-ID DB-5 capillary column in a Clarus 500 GC-MS (PerkinElmer, Waltham, MA). The column was held at 35°C for 3 min after injection and then increased 10°C/min until reaching 260°C, where it remained for an additional 5 min. Helium was used as a carrier gas at a flow rate of 2 ml/min. Electron ionization spectra were compared with references found in the National Institute of Standards and Technology database and then confirmed with available standards. Retention times, peak heights, peak areas, and the start and end times for each peak were recorded and downloaded for analysis. Statistical Analyses External factors (i.e., surrounding forest condition) influencing N. menapia abundance (i.e., total number of butterflies observed at a stand) were investigated with negative binomial regression using generalized linear models with a log link function to account for overdispersion in the abundance data. These models were better fits than Poisson models based on comparisons of log-likelihood values, goodness of fit tests, and visual examination of the conditional mean-variance relationship. All factors (i.e., mean dwarf mistletoe infection severity of trees surrounding a stand, number of trees surrounding a stand, date of observation, site, stand, and percentage of surrounding trees infected) as well as two- and three-level interactions were evaluated for inclusion in the models. Model factors were selected for inclusion based on analysis of deviance, chi-square tests of log-likelihood values, pseudo-R2 values, goodness of fit, and examination of residual diagnostic plots. Internal factors (i.e., stand conditions) influencing N. menapia and N. terlooii behavior at each tree stand were examined using cumulative link models (sometimes referred to as ordinal regression) with a logit link function. All factors (i.e., butterfly species, sex, and tree stand dwarf mistletoe infection severity) as well as two-factor interactions were evaluated for inclusion in the models. Model factors were selected for inclusion based on analysis of deviance, chi-square tests of log-likelihood values, goodness of fit and examination of residual diagnostic plots. Tukey’s HSD tests were used to make post-hoc comparisons. To investigate differences in tree volatile profiles potentially influenced by dwarf mistletoe infection, variable importance measures from random forests models were first used to identify compounds of interest. Differences in abundances of those compounds corresponding to dwarf mistletoe infection status were then modeled using multivariate analysis of variance (MANOVA) after assuring adherence to assumptions of normality and homoscedasticity. Canonical discriminant analysis was then used to identify relative influences of each compound. Data were collated in Microsoft Excel and analyzed using R version 3.3.1 in the RStudio version 0.99.902 development environment (R Core Team 2015). The following packages facilitated analysis: dplyr (Wickham and Francois 2015) and tidy (Wickham 2014) for data formatting, xlsx (Dragulescu 2014) for the R-Excel interface, car (Fox and Weisberg 2011) and lsmeans (Lenth 2016) for regression analysis, ordinal (Christensen 2015) for cumulative link models, candisc (Friendly and Fox 2016) for canonical discriminant analysis, MASS (Venables and Ripley 2002) for negative binomial regression, randomForest (Liaw and Wiener 2002) for random forests models, and ggplot2 (Wickham 2009) and ggmap (Kahle and Wickham 2013) for graphics. Raw data and R scripts are available upon request. Results Butterfly Abundance Greater numbers of trees surrounding each stand corresponded to greater numbers of N. menapia seen at each stand (χ2 = 14.1; df = 1; P < 0.001) (Fig. 2). The number of trees surrounding each stand ranged from 37 to 335, with the highest numbers indicating many small saplings and lower numbers indicating fewer, but larger, mature trees. Holding the sampling date constant, for every unit increase in the number of trees surrounding each stand, there was a 0.6 % (95% CI: 0.3%, 0.9%) increase in numbers of butterflies seen at each stand. Butterfly abundance was affected by the observation date, with greatest numbers seen at the beginning and end of the summer sampling period (χ2 = 7.55; df = 1; P = 0.06). Dwarf mistletoe infection of the trees surrounding each stand did not influence butterfly abundance at each stand (χ2 = 1.04; df = 1; P = 0.31). The model incorporating these factors was better than the null model (LR3 = 30.2; P < 0.0001), did not demonstrate a lack of fit (χ2 = 34.9; df = 30; P = 0.25), and explained 58.1% of the observed variance. Fig. 2. View largeDownload slide Tree and butterfly abundance were positively correlated. The number of butterflies indicates the total number of butterflies observed at each tree stand. The line and shaded area indicate the model fit and 95% confidence intervals, respectively. Results were averaged across sampling date at each stand. Fig. 2. View largeDownload slide Tree and butterfly abundance were positively correlated. The number of butterflies indicates the total number of butterflies observed at each tree stand. The line and shaded area indicate the model fit and 95% confidence intervals, respectively. Results were averaged across sampling date at each stand. Butterfly Behavior Butterfly species, average dwarf mistletoe infection severity of each tree stand, and the interaction between butterfly sex and dwarf mistletoe infection severity influenced butterfly behavior (Table 2). Females interacted significantly more with trees than did males (P < 0.001; Fig. 3). Interestingly, N. terlooii interacted more with trees than did N. menapia (P = 0.0002) (Fig. 3). An increase in dwarf mistletoe infection severity increased butterfly interactions with trees for both males (P = 0.012) and females (P = 0.048) (Fig. 4). Table 2. Analysis of deviance for models of Neophasia behavior Source  χ2  df  P  Speciesa  21.0  1  <0.0001  Sex  0.3  1  0.567  Hawksworthb  13.5  1  0.0002  Sex:Hawksworth  14.0  1  0.0002  Total  56.5  7  <0.0001  Residual  1.1  3  0.777  Source  χ2  df  P  Speciesa  21.0  1  <0.0001  Sex  0.3  1  0.567  Hawksworthb  13.5  1  0.0002  Sex:Hawksworth  14.0  1  0.0002  Total  56.5  7  <0.0001  Residual  1.1  3  0.777  aSpecies are N. menapia and N. terlooii. bHawksworth is the index of mistletoe infection severity. View Large Table 2. Analysis of deviance for models of Neophasia behavior Source  χ2  df  P  Speciesa  21.0  1  <0.0001  Sex  0.3  1  0.567  Hawksworthb  13.5  1  0.0002  Sex:Hawksworth  14.0  1  0.0002  Total  56.5  7  <0.0001  Residual  1.1  3  0.777  Source  χ2  df  P  Speciesa  21.0  1  <0.0001  Sex  0.3  1  0.567  Hawksworthb  13.5  1  0.0002  Sex:Hawksworth  14.0  1  0.0002  Total  56.5  7  <0.0001  Residual  1.1  3  0.777  aSpecies are N. menapia and N. terlooii. bHawksworth is the index of mistletoe infection severity. View Large Fig. 3. View largeDownload slide Neophasia behavior with respect to sex and species. Flyby, Patrol, Landing, and Oviposition indicate potential interactions with host trees irrespective of infection status. A Flyby is a weak interaction while Oviposition is a strong interaction. Points and error bars denote mean behavior and 95% confidence intervals from cumulative link models of Neophasia behavior, respectively. Letters A, B, C, and D indicate significant differences (P < 0.05) based on Tukey’s HSD test. Fig. 3. View largeDownload slide Neophasia behavior with respect to sex and species. Flyby, Patrol, Landing, and Oviposition indicate potential interactions with host trees irrespective of infection status. A Flyby is a weak interaction while Oviposition is a strong interaction. Points and error bars denote mean behavior and 95% confidence intervals from cumulative link models of Neophasia behavior, respectively. Letters A, B, C, and D indicate significant differences (P < 0.05) based on Tukey’s HSD test. Fig. 4. View largeDownload slide Effect of dwarf mistletoe infection severity on Neophasia behavior. The Hawksworth Score ranges from 0 (uninfected) to 6 (heavily infected). A Flyby is a weak interaction while Oviposition is a strong interaction. Lines and shaded areas denote mean behavior and 95% confidence intervals from cumulative link models of Neophasia behavior, respectively. Fig. 4. View largeDownload slide Effect of dwarf mistletoe infection severity on Neophasia behavior. The Hawksworth Score ranges from 0 (uninfected) to 6 (heavily infected). A Flyby is a weak interaction while Oviposition is a strong interaction. Lines and shaded areas denote mean behavior and 95% confidence intervals from cumulative link models of Neophasia behavior, respectively. Volatiles Variable importance measures from random forests models identified seven primary volatile compounds that differed based on dwarf mistletoe infection status (F = 3.73; df = 21,42; P < 0.0001). Volatile profiles of branches from infected trees with dwarf mistletoe present on the branches were different from the profiles of branches from uninfected trees or uninfected branches from infected trees (t1 = −6.765; df = 18; P < 0.0001) (Fig. 5a). Volatile profiles of branches of infected trees without dwarf mistletoe present on the branches were marginally different from those of branches from uninfected trees (t = −2.38; df = 18; P = 0.05) (Fig. 5a). Germacrene-D and δ-cadinene were the primary contributors to the volatile index that provided resolution to detect differences in infection status (Fig. 5b). Fig. 5. View largeDownload slide The dwarf mistletoe infection status of a tree influences its volatile chemical profile. (a) Differences in volatile profiles based on infection status. Black points and error bars denote mean and 95% bootstrapped confidence intervals, respectively. Grey points denote observed values. (b) Relative weighting of each compound used to construct the volatile index. The magnitude of arrows denotes relative weighting while direction denotes the effect of contributions (i.e., Germecrene-D had higher levels in volatile profiles from uninfected branches, while d-longifolene had higher levels in profiles from infected branches). Fig. 5. View largeDownload slide The dwarf mistletoe infection status of a tree influences its volatile chemical profile. (a) Differences in volatile profiles based on infection status. Black points and error bars denote mean and 95% bootstrapped confidence intervals, respectively. Grey points denote observed values. (b) Relative weighting of each compound used to construct the volatile index. The magnitude of arrows denotes relative weighting while direction denotes the effect of contributions (i.e., Germecrene-D had higher levels in volatile profiles from uninfected branches, while d-longifolene had higher levels in profiles from infected branches). Discussion We are potentially witnessing an evolutionary transition of host plant utilization in Neophasia. Shifts between conifers as larval hosts and conifer parasites like dwarf mistletoe as larval hosts may be fairly common in Lepidoptera because the mistletoes are growing on the trees very close to the pine needles on which caterpillars feed (Mooney 2003). Braby and Trueman (2006) hypothesized that this close tree-to-parasite proximity could be responsible for the numerous shifts between caterpillar host preference of trees and their associated mistletoes observed in Aporiina. Our behavioral evidence suggests that Neophasia has an affinity for pine trees infected with dwarf mistletoe. It is possible that some element of mistletoe host-seeking behavior has been retained in extant Neophasia as an evolutionary relic. N. terlooii showed a stronger preference for infected trees than N. menapia, suggesting that N. terlooii may have retained more of its hypothesized ancestral traits. From a biogeographic context, the distribution of N. terlooii is closer geographically to the distributions of remaining New World Aporiina, which are predominantly Neotropical (Braby et al. 2007), while the distribution of N. menapia has shifted farther north. A host plant shift away from mistletoe opens a new niche for Neophasia, as conifers are more abundant than mistletoes, in terms of biomass, as larval food sources. N. menapia is fairly polyphagous within the Pinaceae, feeding on Pinus, Pseudotsuga Carrière, Abies Mill., Tsuga Carrière, and Picea Mill. (Evenden 1926, Cole 1971, Scott 1986, Robinson et al. 2002). Less is known about N. terlooii, but they may be more limited in host plant options and have thus far been documented on only two genera: Pinus and Picea (Arizona Game and Fish Department 2001). Dwarf mistletoes can be found on all aforementioned tree genera (Hawksworth and Wiens 1996). Assuming the latter host-parasite relationships have persisted, and that ancestral Neophasia specialized on a more limited number of dwarf mistletoe species, this could explain why N. terlooii is more restricted in its pine hosts. N. terlooii has a greater affinity for pines that are parasitized by specific dwarf mistletoe species, while N. menapia is more likely to utilize other pines over a greater geographic range that are parasitized by more species of dwarf mistletoe. Dwarf mistletoe infection has a significant effect on Neophasia behavior. Neophasia interacted more with trees that were heavily infected. Male behavior predominantly consisted of patrolling, which is typical mate-seeking behavior (Scott 1986, Lotts and Naberhaus 2015). Despite the low abundance of females and greater variability compared to those of males, females still interacted significantly more with infected trees, tending to land and oviposit on them more often as compared with uninfected counterparts. Although butterfly behavior was affected by dwarf mistletoe infection in the tree stands from where the butterflies were observed, butterfly abundance at these stands was influenced more by the number of trees surrounding the stands rather than infection severity. There is evidence to suggest N. menapia on the Kaibab Plateau formed a population distinct from conspecifics found on the Mogollon Rim (D. A. Halbritter, unpublished data), suggesting the latter two regions would comprise the geographic scale necessary to make inferences of how forest structure affects population dynamics. While dwarf mistletoe infection may influence butterfly behavior within a tree stand, overall abundances of butterflies seem to be affected primarily by differences in other attributes of available host resources (i.e., the number and size of trees in the area). However, replicated population-scale surveys of dwarf mistletoe infection, tree sizes and abundance, and butterfly population size estimates are needed before any conclusions can be drawn about how spatial characteristics of forests and dwarf mistletoe infections affect Neophasia populations. The success of Neophasia larvae is at least partially dependent on the nutritional content of the pine needles onto which they were placed as ova. In a related species of tree, Pinus contorta, infected trees were found to have significantly lower starch, total nitrogen, and free amino-nitrogen composition in their phloem (Nebeker et al. 1995). Phloem samples in the latter study were taken from tree trunks at breast height, but dwarf mistletoe infections can have more localized effects, causing infected branches to become nutrient sinks at the expense of the tree as a whole (Hawksworth and Wiens 1996). Eventually tree health declines and the tree can die from an infection. Butterflies in our study seemed to randomly explore the forest matrix, but then spent more time searching the canopies of specific trees. Within close proximity to those trees, butterflies may select branches on infected trees that have higher nutritional content, which may be reflected in the different volatile profiles detected at close range. In addition to signaling nutritional quality, tree volatiles released as a result of mistletoe infection and other stressors may be cues to Neophasia indicating compromised tree defenses. We did not document feeding damage from other insects or infections from other pathogens, the latter of which are also known to alter terpene blends in pines (Nebeker et al. 1995). Pathogen-infected host plants are often more attractive to the insect vectors of these pathogens than uninfected counterparts (Mauck et al. 2010, Mann et al. 2012). For example, the bark beetle, Hylurgopinus rufipes (Eichhoff 1868), is preferentially attracted to elm trees infected with the Dutch elm disease pathogen (Ophiostoma novo-ulmi) compared with uninfected elms (McLeod et al. 2005). Although not vectors of mistletoe, ancestral Neophasia spp. likely fed on mistletoe and therefore would have had a direct dependence on it and would later retain an association with infected trees, potentially responding to kairomones in the pine forest matrix that signal compromised trees. Pine branches infected with mistletoe were characterized by an overall greater quantitative release of volatiles, and also qualitatively released a blend of volatiles characterized by specific terpenes, such as germacrene-D and δ-cadinene, as compared with uninfected branches. The mistletoe itself may have contributed to the volatile profile of branches with mistletoe on them as well. Additionally, individual volatile compounds can be emitted by non-host tree species, thereby adding to the complex milieu of forest volatiles. For these reasons, host recognition is likely dependent on a particular blend of volatiles (Witzgall et al. 2005). Additional behavioral assays with butterflies in the laboratory and field will be necessary to determine the specific volatile signals that influence host-seeking in Neophasia. As a result of mistletoe infection, it may be an overall quantitative increase in volatile production, a specific blend of volatiles, or both, that influence host selection. Neophasia may be drawn to volatiles from the mistletoe itself, or the butterflies may be attracted to trees afflicted with other diseases or insect damage. Conclusions Dwarf mistletoe and Neophasia butterflies are two conspicuous members of the ponderosa pine community that utilize the pine trees as hosts and have been shown here to interact with each other, comprising a multitrophic ecological interaction. Pine forests in the western United States are facing threats from fires exacerbated by climate change (Allen and Breshears 1998), insect damage (Kenaley et al. 2006), synergistic effects of fire and insects (McHugh et al. 2003), and from interactions between insects and tree parasites (Wagner and Mathiasen 1985). Neophasia are generally not detrimental to pine forests, but there have been occasional, localized population irruptions that result in defoliation (Ciesla 1974, Young 1986). Neophasia are abundant and easily detectable as adults, making them effective representatives of the ponderosa pine community. Changes in Neophasia population dynamics could reflect changes in dwarf mistletoe populations and consequently changes in forest health. Presence of butterflies may be indicative of new or ongoing mistletoe infestations. Understanding the ecology of Neophasia in a community context will therefore be important to inform forest management and conservation practices. Studies such as ours, which integrate understanding of evolutionary histories and field studies on multitrophic ecological interactions, will be important for the improvement of conservation science. Acknowledgments We thank Alan Yanahan, the Merriam-Powell Research Station, and the University of Arizona Entomology Department for providing lodging and laboratory space in Arizona, and Matthew Standridge for handling volatile sample shipments. We extend our gratitude to Fort Huachuca and the National Forest Service and for their collaboration. Funds from the William C. and Bertha M. 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Environmental EntomologyOxford University Press

Published: Mar 31, 2018

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