DOI: 10.5061/dryad.qz612jmpw
title: Selection drives divergence of eye morphology in sympatric Heliconius butterflies

description: When populations experience different sensory conditions, natural
 selection may favor sensory system divergence, affecting peripheral
 structures and/or downstream neural pathways. We characterized the outer
 eye morphology of sympatric Heliconius species from different forest types
 and their first-generation reciprocal hybrids to test for adaptive visual
 system divergence and hybrid disruption. In Panama, Heliconius cydno
 occurs in closed forests, whereas Heliconius melpomene resides at the
 forest edge. Among wild individuals, H. cydno has larger eyes than H.
 melpomene, and there are heritable, habitat-associated differences in the
 visual brain structures that exceed neutral divergence expectations.
 Notably, hybrids have intermediate neural phenotypes, suggesting
 disruption. To test for similar effects in the visual periphery, we reared
 both species and their hybrids in common garden conditions. We confirm
 that H. cydno has larger eyes and provide new evidence that this is driven
 by selection. Hybrid eye morphology is more H. melpomene-like despite body
 size being intermediate, contrasting with neural trait intermediacy.
 Overall, our results suggest that eye morphology differences between H.
 cydno and H. melpomene are adaptive, and that hybrids may suffer fitness
 costs due to a mismatch between the peripheral visual structures and
 previously described neural traits that could affect visual performance.
description: Butterfly specimens We collated samples from
 outbred stocks of <em>Heliconius cydno chioneus</em> (C) and
 <em>Heliconius melpomene rosina</em> (M), established from
 wild butterflies caught in Gamboa and the nearby Soberanía National Park,
 Panama. Most butterflies were sampled in 2015-2017 (9 <em>H.
 cydno</em> and 20 <em>H. melpomene</em>) or 2018-2019
 (16 <em>H. cydno</em> and 10 <em>H.
 melpomene</em>) versus fewer samples in 2008-2009 (9 <em>H.
 cydno</em> and 3 <em>H. melpomene</em>). We also
 exploited previously sampled reciprocal F1 hybrids between <em>H.
 cydno</em> and <em>H. melpomene</em>, generated by
 crossing a <em>H. cydno</em> female with a <em>H.
 melpomene</em> male (CxM) or a <em>H. melpomene</em>
 female with a <em>H. cydno</em> male (MxC). All hybrids were
 generated in 2017-2019, involving four CxM crosses and six MxC crosses
 (unique parents for each cross; see Table S1, Fig. S2). All butterflies
 were reared under common garden conditions in the Smithsonian Tropical
 Research Institute insectaries in Gamboa, and all specimens were preserved
 in DMSO/EDTA/NaCl and stored at -80° C as described in Merrill et al.
 (2019). Sample preparation Samples
 were prepared following previously published methods (Seymoure et al.,
 2015; Wright et al., 2023). In brief, we thawed specimens at room
 temperature, imaged the entire head (minus antennae and proboscis), and
 dissected out both eyes and the hind legs. The legs were immediately
 imaged (see below), while the eyes were placed in 20% sodium hydroxide
 (NaOH) for 18-24 hours to loosen the tissues behind the cuticular cornea.
 The following day, we cleaned each eye cuticle of excess tissue and
 mounted it on a microscope slide in Euparal (Carl Roth GmbH). The sample
 was left to dry overnight before imaging. Image
 analysis We used ImageJ/Fiji (Schindelin et al., 2012)
 to analyze each mounted cornea for the total number of facets and total
 corneal area. All slides were imaged at 7.5x on a Leica M80
 stereomicroscope fitted with a Leica Flexacam C1 camera and the
 <em>Leica Application Suite X</em> (LAS X) software. Each
 image contained a 1mm scale bar for calibration. Facet counts were
 measured via image thresholding and the <em>Analyze
 particles</em> function, and corneal surface area was measured with
 the <em>Freehand selection</em> and <em>Measure
 </em>options (full protocol provided as supplementary methods). This
 semi-automated method differs slightly from the approach used by Seymoure
 et al. (2015) but gives quantitatively similar results (Fig. S1). To
 account for differences in head and/or body size, we measured the distance
 between each eye cuticle (inter-ocular width, e.g., (Gaspar et al., 2020;
 Posnien et al., 2012; Wainwright et al., 2023)) and hind tibia length
 using the <em>Straight line</em> and
 <em>Measure</em> options. Finally, we also measured facet
 diameter as in Seymoure et al. (2015): within each anatomical region of
 the eye, we measured across ten facets in a row in two separate locations
 at least ten facets apart (using the <em>Straight line</em>
 and <em>Measure</em> options). Per facet diameter was
 calculated by dividing each line segment by ten, and facet diameter per
 eye region was calculated as the mean of the two locations.
description: # GENERAL INFORMATION 1\. Title of Dataset: Selection drives divergence of
 eye morphology in sympatric *Heliconius* butterflies 2\. Author
 Information: 1. Daniel Shane Wright (Division of Evolutionary Biology,
 Faculty of Biology, LMU Munich, Planegg-Martinsried, Germany) 2. Juliana
 Rodriguez-Fuentes (Division of Evolutionary Biology, Faculty of Biology,
 LMU Munich, Planegg-Martinsried, Germany) 3. Lisa Ammer (Division of
 Evolutionary Biology, Faculty of Biology, LMU Munich, Planegg-Martinsried,
 Germany) 4. Kathy Darragh (Department of Zoology, University of Cambridge,
 Cambridge, United Kingdom; Smithsonian Tropical Research Institute,
 Panama) 5. Chi-Yun Kuo (Division of Evolutionary Biology, Faculty of
 Biology, LMU Munich, Planegg-Martinsried, Germany) 6. W. Owen McMillan
 (Smithsonian Tropical Research Institute, Panama) 7. Chris D. Jiggins
 (Department of Zoology, University of Cambridge, Cambridge, United
 Kingdom; Smithsonian Tropical Research Institute, Panama) 8. Stephen H.
 Montgomery (School of Biological Science, University of Bristol, Bristol,
 United Kingdom; Smithsonian Tropical Research Institute, Panama) 9.
 Richard M. Merrill (Division of Evolutionary Biology, Faculty of Biology,
 LMU Munich, Planegg-Martinsried, Germany; Smithsonian Tropical Research
 Institute, Panama) 3\. Date of data collection (single date, range,
 approximate date): 2008-2019 4\. Geographic location of data collection:
 Panama ### SHARING/ACCESS INFORMATION 1\. Links to publications that cite
 or use the data:
 [https://doi.org/10.1101/2023.10.26.564160](https://doi.org/10.1101/2023.10.26.564160) 2\. Links to publicly accessible locations of the data: [https://github.com/SpeciationBehaviour/Adaptive_divergence_Heliconius_eyes](https://github.com/SpeciationBehaviour/Adaptive_divergence_Heliconius_eyes) 3\. Links/relationships to ancillary data sets: None 4\. Was data derived from another source? No ### DATA & FILE OVERVIEW 1\. File List: 1. eye_morph_selection_analyses.R 2. eye_morphology_cp_mp_f1.csv 3. legs.csv 4. method_comp.csv 5. fst_martin2013.csv 6. supplementary_analyses.R 2\. Relationship between files, if important: None 3\. Additional related data collected that was not included in the current data package: None 4\. Are there multiple versions of the dataset? No --- **FILE-SPECIFIC INFORMATION FOR**: eye_morph_selection_analyses.R Annotated R-scripts for all related analyses and figures --- **DATA-SPECIFIC INFORMATION FOR**: eye_morphology_cp_mp_f1.csv Dataset for the outer eye morphology of *H. cydno*, *H. melpomene*, and their reciprocal F1 hybrids. 1. Number of variables: 22 2. Number of cases/rows: 131 3. Variable List: * ID: sample number for each specimen * date_sampled: date when the specimen was preserved (dd.mm.yyyy) * year_sampled: year when the specimen was preserved (yyyy) * collection: sample period for the specimens (PAN_2007, PAN_2015, PAN_2017) * sex: F = female; M= male * type: species (CP: *Heliconius cydno* from Panama; MP: *Heliconius melpomene* from Panama; CxM: F1 cross of CP mother x MP father; MxC: F1 cross of MP mother x CP father) * brood: family identity of the crosses (stock = outbred stock populations) * wild_insectary: butterfly origin (wild or insectary-reared) * raised: location where the butterfly was reared (Gamboa = Panama) * observer: researcher that processed the eye sample * inter-eye width: distance (mm) between the eye cuticles (a.k.a inter-ocular width) * tibia_length: length of the hind tibia (mm) * L_area: corneal area of the left eye (mm^2) * L_whole_count: number of facets in the left eye * R_area: corneal area of the right eye (mm^2) * R_whole_count: number of facets in the right eye * lateral: mean facet diameter for the lateral region of the eye (um) * dorsal: mean facet diameter for the dorsal region of the eye (um) * ventral: mean facet diameter for the ventral region of the eye (um) * posterior: mean facet diameter for the posterior region of the eye (um) * anterior: mean facet diameter for the anterior region of the eye (um) * anterioventral: mean facet diameter for the anterioventral region of the eye (um) 4. Missing data codes: NA (data not available) 5. Specialized formats or other abbreviations used: None --- **DATA-SPECIFIC INFORMATION FOR**: legs.csv The dataset of leg vs. right hind tibia lengths from sampled butterflies. 1. Number of variables: 3 2. Number of cases/rows: 103 3. Variable List: * id: individual identification of each butterfly * L_length: length (mm) of the left hind tibia * R_length: length (mm) of the right hind tibia 4. Missing data codes: None 5. Specialized formats or other abbreviations used: None --- **DATA-SPECIFIC INFORMATION FOR**: method_comp.csv Dataset of extracted values from Figure 7 in Seymoure et al. (2015): *Peripheral eye dimensions in Longwing (Heliconius) butterflies vary with body size and sex but not light environment nor mimicry ring.* 1. Number of variables: 4 2. Number of cases/rows: 7 3. Variable List * type: species (CP: *Heliconius cydno* from Panama; MP: *Heliconius melpomene* from Panama) * sex: F = female; M= male * area: corneal area of the eye (mm^2) * whole_count: number of facets in the eye 4. Missing data codes: None 5. Specialized formats or other abbreviations used: None --- **DATA-SPECIFIC INFORMATION FOR**: fst_martin2013.csv Genome-wide Fst values between H. c. chioneus and H. m. rosina from: *Martin, Simon H. et al. (2013). Data from: Genome-wide evidence for speciation with gene flow in Heliconius butterflies [Dataset]. Dryad. [https://doi.org/10.5061/dryad.dk712](https://doi.org/10.5061/dryad.dk712)* 1. Number of variables: 7 2. Number of cases/rows: 15147 3. Variable List: * scaffold: * position: * start: * end: * midpoint: * sites: * chi_rosFst: Fst values between *H. c. chioneus* and *H. m. rosina* 4. Missing data codes: NA (data not available) 5. Specialized formats or other abbreviations used: None --- **FILE-SPECIFIC INFORMATION FOR**: supplementary_analyses.R Annotated R-scripts for supplementary analyses and figures