DOI: 10.5061/dryad.wdbrv162t
title: Evolutionary constraints shape the diversity of microinsects' wing morphology

description: Miniaturisation profoundly alters animal morphology, particularly
 locomotory structures like insect wings. Larger insects possess membranous
 wings optimised for flight dominated by inertial forces, while
 microinsects have highly derived bristled wings with a reduced membrane,
 adapted to viscous interactions. Distantly related microinsects share
 striking similarities in some aspects of wing architecture, such as
 elongated bristles or narrowed wing blades, while features such as
 venation or proportion of bristled wing area vary widely. The basis of
 these differences remains unknown. Although insect wing morphology is
 largely shaped by evolutionary history, the role of evolutionary
 constraints in macro-to-micro wing transition has not been examined. For
 the first time, we combined morphological analyses with evolutionary
 modelling to explore how selection for wing optimisation during
 miniaturisation is constrained by evolutionary inertia in key wing
 features. Analysing 39 bark beetle species, ranging greatly in size, we
 found that some modifications, like bristle elongation or wing narrowing,
 exhibit very low evolutionary constraints, enabling rapid adaptation to
 miniaturisation. In contrast, traits like venation development or bristled
 area proportion were highly constrained, requiring longer evolutionary
 timescales to adapt. Our findings provide novel insights into the origins
 of wing‑architecture diversity in microinsects, emphasising the role of
 evolutionary constraints in modulating the transition from macro‑ to
 micro‑wings.
description: <strong>Studied taxa</strong> We
 analysed 39 species of bark beetles and pinhole borers (Curculionidae:
 Scolytinae, Platypodinae), representing a broad range of body sizes, from
 8.7 mm to 1.2 mm in length. Whenever possible, both wings from at least
 two males and two females per species were examined. However, the count of
 marginal bristles along the wing edge was based on only two specimens per
 species. Specimens were randomly selected from the insect collection at
 the Department of Forest Ecosystem Protection, Faculty of Forestry,
 University of Agriculture in Krakow.
 <strong>Sample preparation and morphological
 measurements</strong> Specimens were identified
 to species and sex [19–21] before analysis. Each individual was
 photographed using the Keyence VHX-7000 4K high-accuracy digital
 microscope (Keyence, Japan). Wings were carefully removed from each
 specimen using microsurgical tools, washed in pure ethanol, placed on a
 microscope slide, gently straightened in a drop of ethanol using a soft
 microbrush, and preparations were made for imaging. Wing preparations were
 subsequently photographed using the VHX-7000 microscope. Body length (from
 the top of the pronotum to the apex of the elytra), wing length, and wing
 width (see Fig. 1a for details) were measured based on the obtained body
 and wing images. Subsequently, seven parameters of wing architecture
 previously considered to be strongly affected by miniaturisation were
 analysed:  1)     Bristled area – The percentage of the
 bristled area relative to the total wing area (see <strong>Fig.
 1b</strong>). 2)     Bristle length – The mean
 length of marginal bristles at the central region of the wing (zones 2/4
 and 3/4, see <strong>Fig. 1b</strong>). Although bristle
 length was measured in four wing zones (see Fig. 1b), zones 2/4 and 3/4
 were chosen for analysis as they were the most representative for the
 analyzed groups. Bristles occur in this region even in larger species,
 allowing for the observation of gradual changes with decreasing
 size. 3)     Bristle number – The total number of
 bristles divided by the wing length (to obtain a size-independent relative
 variable). 4)     Bristled region percentage – The
 percentage of the bottom wing edge (trailing edge) covered with bristles
 relative to the total length of the bottom wing edge (see
 <strong>Fig. 1b</strong>). 5)     Wing
 aspect ratio – The ratio of wing length to wing width (see
 <strong>Fig. 1a</strong>), see [22]. 6)    
 Veins divergence – Measured as the distance between the medial posterior
 and cubital veins (including the cubitoanal strut), calculated by
 subtracting distance A from distance B (see <strong>Fig.
 1a</strong>). Both distances A and B were divided by wing length to
 obtain a size-independent relative variable. 7 Veins
 length – Measured as the relative sum of vein length (sum of vein length
 divided by wing length), see <strong>Fig.
 1a</strong>. Measurements were conducted using
 Digimizer v. 6.3.0 software (MedCalc Software Ltd, Belgium).
 <strong>Phylogenetic
 reconstruction</strong> We started with a
 genus-level phylogenetic topology from the literature [23], which was
 based on five genes (COI, EF-1a, 28S, CAD, ArgK). All genera not
 represented in our data set were pruned from this phylogeny before missing
 genera were coded in based on other published phylogenies [24]. Genera
 with only one representative species in our data were assigned the
 appropriate species names. Genera* *with two species representatives were
 added as sister species; while the relationship between species from
 genera with more than two species representatives was obtained from the
 literature [25–29], before coding these manually into our
 phylogeny. The node heights of the phylogeny were
 inferred by analysing COI sequences downloaded from BOLD in BEAST v2.7.6
 [30]. <em>Cryphalus intermedius</em> sequences, of which none
 were published in BOLD, were obtained from the authors of the publication
 of genus <em>Cryphalus</em> taxonomy [27]. Only the node
 heights were sampled during the BEAST analysis – the topology, which was
 obtained from the literature, remained fixed. Based on analysis of our
 alignment in ModelTest-NG [31], we used a GTR site model, with gamma
 category count set to four, and without estimation of invariant sites. The
 analysis was performed with a Yule tree prior and a relaxed molecular
 clock. Phylogenetic comparative analyses
 For our phylogenetic comparative analysis of wing traits, we
 applied the <strong>SLOUCH</strong> (Stochastic Linear
 Ornstein-Uhlenbeck Models for Comparative Hypotheses) method [32],
 implemented in R (4.4.1) [33]. SLOUCH generates two models:
 <strong>The null model (intercept-only
 model):</strong> This model does not include any predictor variables
 and assesses the phylogenetic effect on a trait. It estimates how long it
 takes for a trait to lose half of its correlations with the ancestral
 trait value. If the <strong>intercept-only half-life</strong>
 (measured in tree length units) is zero, the trait is not influenced by
 ancestral conditions. An intercept-only half-life greater than zero
 indicates that ancestral influences persist, with the strength of this
 effect proportional to the intercept-only half-life value. For example, a
 half-life of 0.5 implies that half of the evolutionary history of the
 group is needed for the trait to lose half of the correlation with the
 ancestral trait value. If the intercept-only half-life exceeds 30 times
 the phylogeny length, the phylogenetic effect is so strong that the model
 approximates Brownian motion (diffusion with negligible pull towards an
 optimum). <strong>The regression
 model:</strong> This model includes a predictor variable (selection
 driver), assuming the trait (response variable) evolves toward an
 <strong>adaptive optimum</strong> under selection. The
 adaptive optimum is the state where the trait achieves perfect adaptation
 to the predictor (selection driver; in our case, it is the body size
 regime), ancestral constraints have disappeared, and selection pressure
 from this particular driver drops to zero. In our specific case, the
 <strong>adaptive optimum</strong> for a given wing parameter
 (e.g., bristle length) is the state in which that parameter is perfectly
 adapted (i.e., bristles have the optimal length) to the prevailing
 body-size regime (with body size acting as the selection driver), so that
 no further selection for optimisation of bristle length to body size
 occurs. Any delay in reaching this adaptive optimum is measured by the
 phylogenetic half-life, indicating how long it takes for the trait to
 evolve halfway toward adaptation. A phylogenetic half-life of zero means
 the trait adjusts instantly to the given predictor. A phylogenetic
 half-life of 1 implies that the full evolutionary history of the group is
 required for halfway adaptation to the adaptive optimum. The larger the
 half-life estimate, the stronger the phylogenetic inertia. The regression
 model produces an evolutionary and optimal regression slope. The
 evolutionary regression slope shows the observed relationship between the
 predictor and response variables, while the optimal slope shows the
 expected relationship in a scenario with no constraints on adaptation,
 i.e., the optimal adaptive values in the response variable as a function
 of the predictor. Since the half-life parameter in
 SLOUCH is expressed as a proportion of phylogeny age, we set the root
 depth of our phylogeny to 1. This means that all estimates of half-life
 can be read as a percentage of the group’s evolutionary history. We also
 supply half-life parameters in Myr; the root age of our phylogeny was set
 to 105 Myr, which is based on the best available estimate for the split
 between Scolytinae and Platypodinae [34]. The root age used corresponds
 closely with a previously published phylogenetic study, which suggests
 ~100 Myr [35]. The null model and the regression model,
 for each of the seven wing traits (with body size [body length] as the
 singular predictor variable, i.e., the selection driver), were compared
 using <strong>δAICc</strong> (differences in the Akaike
 Information Criterion corrected for small sample size). When a regression
 model had an AICc score &gt;2 points smaller than the corresponding
 null model, the regression model was considered statistically significant
 [36].
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description: # Evolutionary constraints shape the diversity of microinsects' wing
 morphology Dataset DOI:
 [10.5061/dryad.wdbrv162t](https://doi.org/10.5061/dryad.wdbrv162t) ##
 Description of the data and file structure The *.csv file contains body
 and hind-wing measurements for **39 species** of bark beetles
 (Curculionidae: Scolytinae) and pinhole borers (Curculionidae:
 Platypodinae). Each row is a **species mean** computed from several
 specimens (males and/or females; counts in columns D–E). The *.r file
 contains a complete R software pipeline used for SLOUCH analysis of data.
 ### Files and variables #### File: dryad_script.r Description: a complete
 R software pipeline used for SLOUCH analysis. Models: each model fits
 response ~ Body length with OU regression (slouch.fit) and compares
 against a null (no covariate) via ΔAICc. The tree is internally rescaled
 to height = 1. ##### **Input:** * dryad_tree.tree — phylogeny in NEXUS
 format; tip labels must exactly match `Species_name`. * dryad_data.csv —
 CSV with species-level means (header present). Columns used by the script:
 **Models specification:** 1\. Bristled area (Column Z) ~ Body length
 (Column H) 2\. Vein length (column AB) ~ body length (column H) 3\.
 Bristle length (Column AA) ~ Body length (Column H) 4\. Vein divergence
 (column AU) ~ body length (column H) 5\. Bristled region percentage
 (column AF) ~ body length (column H) 6\. Number of bristles (column AO) ~
 body length (column H) 7\. Wing aspect ratio (Column AG) ~ Body length
 (Column H) #### File: dryad_data.csv **Description:** body and hind-wing
 measurements for **39 species** of bark beetles (Curculionidae:
 Scolytinae) and pinhole borers (Curculionidae: Platypodinae). #####
 Variables: * Order: taxa order * Species abbreviation: abbreviations of
 scientific species names * Species name: scientific name of taxa * No. of
 analysed females: number of analysed females * No. of analysed males:
 number of analysed males * Subfamily: subfamily affiliation * Tribe: tribe
 affiliation * Body length [mm]: body lengt: adults, pronotum apex to
 elytral apex. * Bristles length zone 0 (wing base) [mm]: marginal bristle
 length, zone 0 (base) * Bristles length zone 1/4 [mm]: marginal bristle
 length, zone 1/4 (1/4 of wing length) * Bristles length zone 2/4 [mm]:
 marginal bristle length, zone 2/4 (2/4 of wing length) * Bristles length
 zone 3/4 [mm]: marginal bristle length, zone 3/4 (3/4 of wing length) *
 Bristles length zone 4/4 (wing tip) [mm]: marginal bristle length, zone
 4/4 (wing tip) * Wing length [mm]: wing length from base to tip * Wing
 width [mm]: wing with masured at marginal joint perpendicular to the
 horizontal axis of the wing * Length: AA3+4 [mm]: length of vein AA3+4 *
 Length: Cu + CAS [mm]: length of vein Cu + CAS * Length: MP1+2 [mm]:
 length of vein Cu + CAS * Length: MSP [mm]: length of vein MSP * Length:
 RB [mm]: length of vein RB * Length: RP3+4 [mm]: length of vein RP3+4 *
 Length: RP 1+2 [mm]: length of vein RP 1+2 * Total wing area (solid area +
 bristled area) [mm2]: sum of solid area + bristled area * Solid
 (membranous) wing area [mm2]: Solid (membranous) wing area * Bristled area
 [mm2]: area formed by bristles * Bristled area proportion [%]: proportion
 of the bristled area to the total wing area * Mean bristles length (zones
 2,3) [mm]: mean of bristle lenght (zone 2 and 3) * Relative vein length
 [mm]: sum of wing vein divided by wing length * Number of bristles [pcs]:
 number of marginal bristles * Length of bristled part of the bottom wing
 edge [mm]: length of botttom wing edge part bearing marginal bristles *
 Length of bottom wing edge [mm]: length of whole botttom wing edge *
 Bristled region percentage [%]: percentage of bootom wing edge bearing
 marginal bristles * Wing aspect ratio: wing wing to length ratio *
 Relative length: AA3+4: length of AA3+4 vein divided by wing length *
 Relative length: Cu + CAS: length of Cu + CAS vein divided by wing length
 * Relative length: MP1+2: length of MP1+2 vein divided by wing length *
 Relative length: MSP: length of MSP vein divided by wing length * Relative
 length: RB: length of RB vein divided by wing length * Relative length:
 RP3+4: length of RP3+4 vein divided by wing length * Relative length: RP
 1+2: length of RP 1+2 vein divided by wing length * Relative number of
 bristles [pcs/mm]: relative number of bristles (per mm of wing length). *
 Mean gap between bristles [mm]: mean gap between * Distance MP-CAS
 -(Distance B, half): distance between MP1+2 vein and CAS+Cu vein measured
 in the half od CAS+Cu vein length * Distance MP-CAS V (Distance A, end):
 distance between MP1+2 vein and CAS+Cu vein measured at the end of CAS+Cu
 vein * Relative distance MP-CAS -(Distance B, half): relative distance
 between MP1+2 vein and CAS+Cu vein measured in the half od CAS+Cu vein
 length * Relative distance MP-CAS V (Distance A, end): relative distance
 between MP1+2 vein and CAS+Cu vein measured at the end of CAS+Cu vein *
 Delta end - half distance MP-CAS: Delta distance between MP1+2 vein and
 CAS+Cu vein obtained by subtracting relative distance A from relative
 distance BFile: #### File: dryad_tree.tree **Description:** Phylogeny (39
 taxa, branch lengths included) used in the SLOUCH analyses. Tip labels are
 species names and are intended to match the `Species_name` column in the
 data file. Format: NEXUS. ## Code/software *         R software (≥ 4.x) *
         Packages: `ape`, `phytools`, `slouch`