Illuminating the Early Life of Salamanders: Exploring Biofluorescence During Development

Jennifer Y. Lamb, Holden Cooper, Alexander Seymour, Matthew P. Davis, and Lynne Beaty

Ichthyology & Herpetology 113: 104–116

Supplemental Appendix A. Publications documenting biofluorescence in tetrapods. This appendix compiles published resources detailing occurrences of biofluorescence in tetrapods up to January 2025. Each row represents a family in which authors found fluorescence. Orders agree with taxonomy and nomenclature from the NCBI Taxonomy Database (https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi). We note the excitation light sources used and whether authors quantified fluorescence excitation wavelengths in their publication. Resources that have not yet been published in a peer-reviewed journal (e.g., unpublished theses, pre-prints) are noted as “NA” in the Publication column. In some cases, authors did not categorize the colors fluoresced, or did not provide sufficient images and details for us to do so. These instances are labeled as “Not categorized” in the Color(s) fluoresced column. In other cases, studies did not provide excitation spectra, did not quantify emission spectra, or did not specify emission maxima if spectra were collected. These instances are labeled as “Not provided” in the Excitation wavelength(s) and Wavelength(s) fluoresced columns. It is important to note that studies may have tested multiple excitation sources but only reported emission data for some of those excitation lights.

Supplemental Table 1. Spectral shape data of biofluorescent emissions in response to blue excitation light across developmental stages of the salamanders studied. These data represent the average, raw peak intensity as count data (B3) and primary peak wavelengths (H1) of biofluorescent emissions in the species studied (measurements as described in Maia et al., 2013). Some species and life stages exhibit primary and less intense secondary peaks. ^ indicates that secondary peaks were calculated from measurements from a single individual, whereas ^^ that secondary peaks were calculated based on data from two individuals. Prior to calculating spectral shape data, spikes produced by instrument error (see Fig. 4B, D) in the spectral data collected from A. maculatum and N. viridescens viridescens were removed and replaced with the average count value for fluorescent intensity calculated from immediately adjacent wavelengths. Spectra were then smoothed in pavo (Maia et al., 2013) and the peakshape() function was used to quantify peak metrics for green and red light spectra.

Supplemental Figure 1. Early embryos of Blue-spotted Salamanders (Ambystoma laterale) do not fluoresce in response to blue excitation light. The early-stage embryos of this species are darkly pigmented (A; Shi and Boucaut, 1995). Although red fluorescence is visible along the outer edge of the egg capsule, and the egg jelly fluoresces green, the embryo itself does not appear fluorescent (B).

Supplemental Figure 2. Biofluorescence of Spotted Salamander (Ambystoma maculatum) eggs and embryos in response to blue excitation light. The eggs of A. maculatum primarily fluoresce red in response to blue excitation light, although there was faint green fluorescence from the jelly of the egg mass (A, B). This red fluorescence is likely from symbiotic algae that live within the eggs of A. maculatum. Embryos within the eggs fluoresce green in response to blue light with the ventral surfaces fluorescing more brightly than dorsal surfaces (B).

Supplemental Figure 3. Biofluorescence of Spotted Salamander (Ambystoma maculatum) larvae and Eastern Red-spotted Newt (Notophthalmus viridescens viridescens) larvae and efts to green light. Larval A. maculatum (A), which appear darker under white light, exhibit faint red fluorescence in response to green excitation light (D). Larval N. viridescens viridescens (B) and efts (C) fluoresce red and brighter in response to green excitation compared to larval A. maculatum (E, F). The dorsal red-orange spots on efts fluoresce a bright orange (F).