Appendix A from Macroevolutionary patterning of woodpecker drums reveals how sexual selection elaborates signals under constraint

Supplementary methods and information regarding the use of quantile regression in behavioral analyses

Supplementary Data File from Macroevolutionary patterning of woodpecker drums reveals how sexual selection elaborates signals under constraint

Drum and morphological dat

Supplementary Recording List from Macroevolutionary patterning of woodpecker drums reveals how sexual selection elaborates signals under constraint

Sexual selection drives elaboration in animal displays used for competition and courtship, but this process is opposed by morphological constraints on signal design. How do interactions between selection and constraint shape display evolution? One possibility is that sexual selection continues exaggeration under constraint by operating differentially on each signal component in complex, modular displays. This is seldom studied on a phylogenetic scale, but we address the issue herein by studying macroevolutionary patterning of woodpecker drum displays. These territorial displays are produced when an individual rapidly hits its bill on a hard surface, and drums vary across species in the number of beats included (length) and the rate of drumbeat production (speed). We report that species body size limits drum speed, but not drum length. As a result of this biomechanical constraint, there is less standing variation in speed than length. We also uncover a positive relationship between sexual size dimorphism and the unconstrained trait (length), but with no effect on speed. This suggests that when morphology limits the exaggeration of one component, sexual selection instead exaggerates the unconstrained trait. Modular displays therefore provide the basis for selection to find novel routes to phenotypic elaboration after previous ones are closed

Sex differences in neuromuscular androgen receptor expression and sociosexual behavior in a sex changing fish

<div><p>Androgen signaling, via receptor binding, is critical for regulating the physiological and morphological foundations of male-typical reproductive behavior in vertebrates. Muscles essential for male courtship behavior and copulation are highly sensitive to androgens. Differences in the distribution and density of the androgen receptor (AR) are important for maintaining dimorphic musculature and thus may provide for anatomical identification of sexually selected traits. In <i>Lythrypnus dalli</i>, a bi-directional hermaphroditic teleost fish, both sexes produce agonistic approach displays, but reproductive behavior is sexually dimorphic. The male-specific courtship behavior is characterized by rapid jerky movements (involving dorsal fin erection) towards a female or around their nest. Activation of the supracarinalis muscle is involved in dorsal fin contributions to both agonistic and sociosexual behavior in other fishes, suggesting that differences in goby sexual behavior may be reflected in sexual dimorphism in AR signaling in this muscle. We examined sex differences in the local distribution of AR in supracarinalis muscle and spinal cord. Our results demonstrate that males do express more AR in the supracarinalis muscle relative to females, but there was no sex difference in the number of spinal motoneurons expressing AR. Interestingly, AR expression in the supracarinalis muscle was also related to rates of sociosexual behavior in males, providing evidence that sexual selection may influence muscle androgenic sensitivity to enhance display vigor. Sex differences in the distribution and number of cells expressing AR in the supracarinalis muscle may underlie the expression of dimorphic behaviors in <i>L</i>. <i>dalli</i>.</p></div

Immunolocalization of spinal cord androgen receptor (AR) expression between the sexes and differences in expression between the sexes in the ventral and dorsal horn of the spinal cord.

<p>Representative AR staining in the dorsal and ventral horn of male (n = 12) (<b>A</b>) and female (n = 8) (<b>B</b>) <i>L</i>. <i>dalli</i>. Mean (±SEM) AR staining intensity within the dorsal and ventral horns of the spinal cord in males and females (<b>C</b>). DH = dorsal horn, VH = ventral horns.</p

Immunolocalization of androgen receptor (AR) expression between sexes in different muscle types.

<p>Representative AR staining within the supracarinalis muscle of males (<b>A</b>) and females (<b>B</b>), and the epaxial and hypaxial muscles of males (n = 12) (<b>C/E</b>) and females (n = 8) (<b>D/F</b>) respectively. The mean (±SEM) number of AR positive cells within the supracarinalis, epaxial, and hypaxial muscles in males and females (<b>G</b>). All images were captured under a 40x objective. Scale bar = 50 μm *** denotes a significant difference at p < 0.001.</p

Location and size of the supracarinalis muscle in <i>L</i>. <i>dalli</i>.

<p>Illustration of where the supracarinalis muscle is located (shown in red) and relative area sectioned in this study to assess receptor expression (<b>A</b>). Hemotoxylin and eosin stained cross section showing the exact location of the supracarinalis muscle relative to other muscles and peripheral tissues (<b>B</b>). Mean (±SEM) relative supracarinalis muscles size (muscle area (μm²) / standard length (mm) in male (n = 10) and female (n = 7) <i>L</i>. <i>dalli</i> (<b>C</b>).</p

Identification of forebrain structures with specialized <i>parvalbumin</i> (<i>PV</i>) mRNA expression.

(A-F) Representative radioactive in situ hybridization microscope images of PV mRNA in species representing 8 different avian orders (see S1 Fig for penguin and emu). PV-rich forebrain nuclei were present in only (E) vocal learning hummingbirds (positive control) and (F) downy woodpeckers. (Ei-iii) High magnification of 3 telencephalic “song control” nuclei in hummingbirds. (Fi) High magnification of the woodpecker drumming nucleus of the anterior nidopallium (dAN); and (Fii) drumming nucleus of arcopallium (dA). Each scale bar is equal to 2 mm. Neuroanatomical markers shown in “A” are as follows: Hyper, hyperpallium; Meso, mesopallium; Nido, nidopallium; GP, globus pallidus; T, Thalamus; Ot, optic tectum; St, striatum; Arco, arcopallium; Ento, entopallium. White dashed lines in high-magnification images (Ei-iii and Fi-ii) indicate boundaries for different telencephalic regions (e.g., boundary between nidopallium and mesopallium), whereas blue dashed lines indicate specialized PV regions identified in Anna’s hummingbirds and downy woodpeckers. Image credits: flamingo from Wilfredo Rodríguez; turaco from Edelmauswaldgeist; duck from Orso della campagna e Papera dello stagno; hawk from Cheva; hummingbird from Stickpen, and downy woodpecker from Greg Schechter. All image licenses: CC Public Domain via WikiMedia.</p

Parvalbumin cell count data.

(CSV)</p

Activity induced <i>Arc</i> expression in the dorsal anterior nidopallium (dAN) of downy woodpeckers positively correlates with drumming behavior.

(A-B) Representative low-magnification in situ hybridization microscope images of Arc expression (white), with fast red as a counterstain (grey) in downy woodpeckers that were either passively caught (A) or (B) after drumming during a simulated territorial encounter (STI). (C-F) Representative high-magnification examples of Arc mRNA image in (C) passively caught birds (n = 3) or birds that listened to STI playback (see Materials and methods) but either (D) did not produce vocalizations or drums (silent, n = 3), (E) produced only vocalizations and no drums (n = 2), or (F) produced drums (n = 10). Dash blue outline represents the PV-rich dAN area on each section determined from PV mRNA on an adjacent section. (G) Violin plots (horizontal line denotes median) illustrating differences in Arc gene expression in the PV-rich dAN in male downy woodpeckers caught after producing different behaviors. Arc mRNA expression significantly differed in the dAN (F3,14 = 21.14, p Arc expression than all other groups (all relevant comparisons: p Arc mRNA expression in the dAN nucleus was positively correlated with the number of drums (p = 0.01). (I) No correlation was detected with the total number of aggressive vocalizations. Yet, (J) there was a marginally significant negative correlation with woodpeckers that flew less having greater Arc mRNA expression in dAN. Significant correlations denoted by solid lines (p PV regions identified in downy woodpeckers. Data for G-J can be found in S2 Data.</p