DOI: 10.5061/dryad.3n5tb2rp4
title: Vocal range and ultraviolet-induced photoluminescence in gliding mammals and their relatives

description: Gliding is only present in six extant groups of mammals – interestingly,
 despite divergent evolutionary histories, all mammalian gliders are
 strictly nocturnal. Gliding mammals also seem to have relatively high
 rates of ultrasound use and ultraviolet-induced photoluminescence (UVP) in
 contrast with their close relatives. Therefore, we hypothesized that,
 despite diverging lineages, gliding mammals use similar modes of cryptic
 communication compared to their non-gliding counterparts. We developed two
 datasets containing the vocal range (minimum-maximum of the dominant
 harmonic; kHz) and UVP of 73 and 82 species, respectively; we report five
 novel vocal repertoires and 57 novel observations of the presence or
 absence of UVP. We complemented these datasets with information about body
 size, diel activity patterns, habitat openness, and sociality to explore
 possible covariates related to vocal production and UVP. We found that the
 maximum of the dominant harmonic was significant higher in gliding mammals
 when vocalizing than their non-gliding relatives. Additionally, we found
 that nocturnality was the only significant predictor of UVP, consistent
 with the previous hypothesis that luminophores primarily drive UVP in
 mammal fur. In contrast, however, we did not find UVP ubiquitous in
 nocturnal mammals, suggesting that some unknown process may contribute to
 variation in this trait.
description: <strong>Vocalizations</strong> We
 developed a database beginning with a list of publications describing
 gliding mammal vocalizations (summarized in Table S1). The minimum
 requirement for each publication was describing at least one call with
 either a spectrographic analysis or numerical data. However, most
 publications described multiple call types per species or multiple species
 per publication (7 gliding mammals represented across 9 publications,
 summarized in Table S1). The databases used to search for these
 publications were Google Scholar, JSTOR, Web of Science, and Wiley Online
 Library. We used the keywords acoustics, acoustic repertoire, calls,
 frequency, Hz, vocalizations, and ultrasound paired with an exhaustive
 list of currently valid and invalid genera (the most updated nomenclature
 was taken from the Integrated Taxonomic Information System). Across all
 published calls, we took the absolute minimum and maximum frequencies
 (kHz) of the dominant harmonic for the final analyses (this often
 corresponded to the fundamental harmonic, if multiple harmonics were
 present [1]). For noisy calls, such as broadband calls, where the
 harmonics are not well defined, we estimated the minimum and maximum of
 the loudest parts of the call. We did not include calls produced by
 neonates or juveniles as there is evidence of some frequencies and calls
 being different in younger individuals [2,3]. To
 compare gliding mammals to closely related species, we systematically
 searched for vocalization data using the same methodology described above
 (Fig. 1). Flying squirrels are unique amongst the gliders as there are
 many extant species that occupy the same family (Sciuridae); therefore, we
 kept all relatives from the same subfamily (Sciurinae) and a random subset
 of squirrels from the other subfamilies (26 squirrels across 62
 publications). Other gliders have few extant relatives and we
 strategically chose taxa that shared similar evolutionary histories and
 traits. For the scaly-tailed gliders, we selected springhares
 (<em>Pedetes capensisi</em>), the only other extant taxa of
 the Anomaluromorpha suborder, and a variety of small-bodied rodents (12
 species across 16 publications) exhibiting a range of vocal frequencies
 (maximum dominant frequency range: 9.86 (<em>Sicista subtilis
 </em>[4]) - 109.8kHz (<em>Mus musculus </em>[5]).For
 colugos, the only extant members of the order Dermoptera, we selected tree
 shrews (<em>Tupaia belangeri</em>) which form a sister clade
 with Dermoptera [6] and similarly sized taxa from the order Primates (19
 primates across 27 publications), which are the next closest sister taxa
 [7]. For marsupial gliders, we expanded our search to include similarly
 sized taxa of the order Diprotodontia as there were few records of
 marsupial vocalizations (5 marsupials across 6 publications). The
 vocalization data for two marsupial gliders (<em>Petaurus
 breviceps</em>, and <em>P. norfolcensis</em>) and two
 glider relatives (<em>Pedetes capensis</em> and
 <em>Pseudocheirus peregrinus</em>; Fig. 1) were not available
 in the literature, and we worked with co-authors and collaborators to
 develop novel call descriptions  for our study (methods in Article S1). We
 also provide vocalization data from free-ranging <em>Petaurus
 australis </em>(methods in Articles S1) to opportunistically
 contrast our recordings with previously reported calls in the literature
 [8,9]<em>. </em>In the literature, four species were
 represented by a single subspecies only: <em>Otolemur garnettii
 lasiotis</em>, <em>Petaurista alborufus lena</em>,
 <em>Sciurus aberti kaibabensis</em>, and <em>Sciurus
 niger rufiventer</em>.
 <strong>Ultraviolet-Induced
 Photoluminescence</strong> To expand on our
 vocalization dataset, we assessed the UVP of pelage for 83 species.
 Previous literature accounted for 19 species in our dataset; we sampled an
 additional 64 species from the mammal collections at the Canadian Museum
 of Nature and the Royal Ontario Museum (one mounted specimen
 (<em>Sicista subtilis</em>), otherwise all dry-preserved
 pelts; specimen and museum information provided on Dryad). We sampled
 species from the vocalization dataset preferentially. However, we
 opportunistically added ten species (bold type in Table S1) to increase
 the sample size of luminescing species. We followed the same vocalization
 protocol detailed above for both opportunistic and previously published
 UVP species; we found vocalization data for eight additional species (four
 opportunistic and four from previous UVP literature).
 We used a Vansky UV flashlight (395nm wavelength) to illuminate
 museum specimens (held 75 cm above the individual) and a Huawei P30 Pro
 phone (held directly beside the light) to capture any luminescence. A
 yellow gel filter was held in front of the camera lens to reduce the input
 of purple-blue light [10,11]. To minimize the additional yellow hue
 created by the filter, we manually color-corrected the photos in Photoshop
 (details in Info. S1). We took pictures of each specimen’s ventral and
 dorsal sides under white-light conditions, UV-light only, UV-light +
 filter, and UV-light + filter + correction (example provided in Fig. 2;
 complete photoset available on Dryad). We additionally photographed a few
 live <em>Glaucomys </em>individuals trapped in the Kawartha
 Highlands, Ontario, following the same protocol (animals studied under
 Trent University animal care protocol 27909). In our
 investigation, some species expressed visible photoluminescence in white
 pelage or in some cases, the white ends of guard hairs. While “white” UVP
 has been noted in some species, this “white” coloration has been reported
 as a bluish-white (as seen in the striped possum (<em>Dactylopsila
 trivirgata</em>) and some marsupial gliders [13]). The underlying
 cause of UVP expressed as distinct colors have been linked to porphyrins
 (red or pink) or tryptophan metabolites (cyan, blue, lavender [13]).
 However, the expression of exclusively “white” coloration is not commonly
 reported, nor has a clear explanation been proposed for producing UVP
 without a dominant color. Furthermore, white human hair may emit a bluish
 hue similar to the pelage of minks, rabbits and goats and sheep, which
 have been described as being photoluminescent due to the presence of
 tryptophan metabolites [14]. Given that we could not photograph museum
 specimens in complete darkness, the available visible light may have
 excited white hairs that would otherwise not express UVP. Therefore, to
 remove the potential bias of visible light, we removed individuals that
 only expressed “white” photoluminescence (but model outputs for all
 species, including those with “white” UVP, are included in Table S2).
 While UVP varied dramatically in color (e.g., pink, blue/green),
 placement, and patterning across museum specimens and published
 literature, we reduced variability to absence/presence to increase the
 sample size in each category. <strong>Dataset
 Assembly</strong> Once we had assembled our
 database of vocalizing mammals with UVP records, we searched for the body
 mass (g), diel activity pattern (diurnal or nocturnal), social complexity,
 and habitat openness of the dominant habitat (open or closed) of each
 species. We preferentially took these data from the relevant vocalization
 or UVP papers, though this information was rarely provided; therefore,
 other resources, including articles and online databases such as Mammalian
 Species accounts and the Animal Diversity Web [15], were reviewed to
 complete our dataset. If a range was provided for the body mass, we took
 the mean of the given values; we took a mean of male and female body
 masses as we were not capturing the effect of sex on vocalization
 frequencies or UVP. Social variability was reduced to social or solitary
 living to reduce model parameters; species that exhibit dynamic social
 structures, where adult individuals will seasonally or cyclically shift
 between solitary and social living (e.g., flying squirrels engaging in
 social nesting during the winter only [16]), were treated as socially
 living.
 <strong>Phylogeny</strong> While
 multiple subspecies were present in the vocalization dataset, we
 calculated the vocalization maxima at the species level for the final
 dataset and analyses (Fig. 1; subspecies-specific information noted in
 Table S1). Only one subspecies was excluded from analyses
 (<em>Peromyscus maniculatus bairdii</em>) due to a binary
 variable inconsistency with the parent species – this subspecies only
 occurs in open habitats [17], while the parent species is most commonly
 found in closed habitats. In addition, Masters et al. [18] recently
 proposed that the <em>Paragalago </em>genus is a distinct
 clade from the <em>Galagoides </em>genus to which the
 <em>Paragalago </em>species had been previously assigned; we
 reassigned these species accordingly. For the final
 species dataset (n = 93), we pruned 1000 node-dated completed trees from
 the mammalian supertree on VertLife, an online database used to produce
 pruned random distribution trees of vertebrate species [19]. The nexus
 outputs were compiled into a consensus tree using the <em>phytools
 </em>[20]<em> </em>package in R [21] (Fig. 1).
 <em>Petaurus notatus </em>is a recently described species
 (previously incorporated within <em>P. breviceps</em>), and
 therefore, it was the only species not available on Vertlife; we
 incorporated this species into the final consensus tree by splitting the
 <em>P. breviceps </em>lineage at a divergence time of 1Ma
 [22]. <strong>Analyses</strong>
 We built phylogenetic generalized least square (PGLS) models to
 account for variation in the vocal range that could be explained by
 phylogenetic relatedness. PGLS models estimate phylogenetic relatedness as
 lambda (<em>λ</em>), which<em> </em>varies between
 0 (no phylogenetic trace) and 1 (absolute Brownian motion) [23,24]. Full
 models were built for each frequency limit (β<sub>0</sub> +
 body mass (β<sub>Mass</sub>) + diel activity pattern
 (β<sub>Diel</sub>) + sociality
 (β<sub>Sociality</sub>) + habitat openness
 (β<sub>Open</sub>) + UVP (β<sub>UVP</sub>)) using
 the <em>caper </em>[25]<em> </em>package in R
 [21]. We reported the regression coefficient estimates
 (<em>x̄</em> ± <em>SE</em>) to evaluate
 significance and effect size (<em>F-</em>statistic,
 <em>P-</em>value, and adjusted
 <em>R</em><sup>2</sup>). We
 also built a phylogenetic generalized linear mixed (PGLM) model for binary
 data using the <em>ape </em>[26]<em> </em>package
 in R to assess the presence of UVP. This binary PGLM model accounted for
 variation in UVP while dealing with the bimodal distribution that violates
 other tests [27]. The same independent variables were used
 (β<sub>0</sub> + β<sub>Mass</sub> +
 β<sub>Diel</sub> + β<sub>Sociality</sub> +
 β<sub>Open</sub>); β<sub>Mass</sub> was
 standardized to have a mean of 0 and variance of 1, while the categorical
 variables were reconstructed into dummy variables (2 categories = 0, 1)
 for the PGLM model. We standardized the variables to improve the
 interpretation of regression coefficients as they more accurately
 represent the effect size of the independent variables (Ives &
 Garland, 2010). The PGLM model represents the phylogenetic signal
 (<em>s<sup>2</sup></em>)<em> </em>as
 the scalar magnitude of the phylogenetic variance across all species
 comparisons (estimated from the phylogenetic variance-covariance matrix
 [27]). References [1] Newar SL, Bowman J. 2020. Think
 Before They Squeak: Vocalizations of the Squirrel Family.
 <em>Frontiers in Ecology and Evolution</em> 8:1–15.
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 Analysis of The Alarm Call Frequency in Different Age Rodent Groups (in
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 </em>86:499–504.<br>[3] Schneiderová I,
 Schnitzerová P, Uhlíková J, Brandl P, Zouhar J, Matějů J. 2015.
 Differences in alarm calls of juvenile and adult European ground squirrels
 (<em>Spermophilus citellus</em>): findings on permanently
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 AA, Abramov A V., Ilchenko OG, Volodina E V. 2019. Ultrasonic bouts of a
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 Tanomtong A, Volobouev V, Ferguson-Smith MA, Yang F. 2008. Flying lemurs -
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 DOI: 10.1186/1741-7007-6-18.<br>[7] Beard KC. 1993. Phylogenetic
 systematics of the Primatomorpha, with special reference to Dermoptera.
 In: Szalay FS, Novecaek MJ, McKenna MC eds. <em>Mammal Phylogeny:
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 10.1007/978-1-4613-9246-0_10.<br>[8] Kavanagh RP, Rohan-Jones WG.
 1982. Calling behaviour of the yellow-bellied glider, <em>Petaurus
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 DA, McKinnon F, Lefoe M, Rendall AR. 2021. Passive acoustic monitoring for
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 <em>PLOS ONE </em>16:e0252092. DOI:
 10.1371/journal.pone.0252092.<br>[10]Kohler AM, Olson ER, Martin JG,
 Anich PS. 2019 Ultraviolet fluorescence discovered in New World flying
 squirrels (<em>Glaucomys</em>). <em>Journal of
 Mammalogy</em> 100:21–30. (doi:10.1093/jmammal/gyy177)<br>[11] Nummert G, Ritson K, Nemvalts K. 2023 Photoluminescence in the Garden dormouse (<em>Eliomys quercinus</em>). <em>Zoology</em> 157. (doi:10.1016/j.zool.2023.126075)<br>[12] Reinhold L. 2021 Mammals with fluorescent fur: Observations from the Wet Tropics. <em>North Queensland Naturalist </em>51:1–8.<br>[13] Reinhold LM, Rymer TL, Helgen KM, Wilson DT. 2023. Photoluminescence in mammal fur: 111 years of research. <em>Journal of Mammalogy</em>. DOI: 10.1093/jmammal/gyad027.<br>[14] Millington KR. 2020 Anomalous fluorescence of white hair compared to other unpigmented keratin fibres. <em>International Journal of Cosmetic Science</em> 42:289–301. (doi:10.1111/ics.12614)<br>[15] Myers P, Espinosa R, Parr C, Jones T, Hammond G, Dewey T. 2023. The Animal Diversity Web (online). <em>Available at </em><em>https://animaldiversity.org</em><em> </em>(accessed 18 September 2023).<br>[16] Garroway CJ, Bowman J, Wilson PJ. 2013 Complex social structure of southern flying squirrels is related to spatial proximity but not kinship. <em>Behavioural Ecology and Sociobiology</em> 67:113–122. (doi:10.1007/s00265-012-1431-3)<br>[17] Wecker SC. 1963 The Role of Early Experience in Habitat Selection by the Prairie Deer Mouse, <em>Peromyscus maniculatus bairdi. Ecological Monographs</em> 33:307–325.<br>[18] Masters JC, Génin F, Couette S, Groves CP, Nash SD, Delpero M, Pozzi L. 2017 A new genus for the eastern dwarf galagos (Primates: Galagidae). <em>Zoologial Journal of the Linnean Society</em> 181:229–241.<br>[19] Upham N, Esselstyn J, Jetz W. 2019 Inferring the mammal tree: species-level sets of phylogenies for questions in ecology, evolution, and conservation. <em>PLOS Biology</em> 17:e3000494.<br>[20] Revell LJ. 2012 phytools: an R package for phylogenetic comparative biology (and other things). <em>Methods of Ecology and Evolution</em> 3:217–223. DOI: 10.1111/j.2041-210X.2011.00169.x.<br>[21] R Core Team. 2022 R: A Language and Environment for Statistical Computing. <br>[22] Cremona T, Baker AM, Cooper SJB, Montague-Drake R, Stobo-Wilson AM, Carthew SM. 2021 Integrative taxonomic investigation of Petaurus breviceps (Marsupialia: Petauridae) reveals three distinct species. <em>Zoological Journal of the Linnean Society </em>191:503–527.<br>[23] Freckleton RP, Harvey PH, Pagel M. 2002 Phylogenetic analysis and comparative data: A test and review of evidence. <em>American Naturalist</em> 160:712–726. DOI: 10.1086/343873.<br>[24] Martin K, Tucker MA, Rogers TL. 2016 Does size matter? Examining the drivers of mammalian vocalizations. <em>Evolution </em>71:249–260. DOI: 10.1111/evo.13128.<br>[25] Orme D, Freckleton R, Thomas G, Petzoldt T, Fritz S, Isaac N, Pease W. 2018 caper: Comparative Analyses of Phylogenetics and Evolution in R. <br>[26] Paradis E, Schliep K. 2019 ape 5.0: an environment for modern phylogenetics and evolutionary analyses in R. <em>Bioinformatics</em> 35:526–528. DOI: 10.1093/bioinformatics/bty633.<br>[27] Ives AR, Garland T. 2010 Phylogenetic Logistic Regression for Binary Dependent Variables. <em>Systematic Biology</em> 59:9–26. DOI:10.1093/sysbio/syp074.<br>
description: <strong>R</strong> is required to open
 <em>UltrasoundandUltraviolet_Script.R</em> and
 <em>UltrasoundandUltraviolet</em><em>_Trees.nex</em> <br><strong>Microsoft Excel </strong>can be used to view <em>UltrasoundandUltraviolet</em><em>_Data.csv </em>and <em>UltrasoundandUltraviolet</em><em>_MuseumSpecimens.csv <br><strong>Any PDF viewer</strong></em><strong> </strong>can be used to view <em>UltrasoundandUltraviolet</em><em>_UVP.pdf</em><em><br></em>
description: # Ultrasound and Ultraviolet: Open Source Data --- We have submitted our
 raw data (**UltrasoundandUltraviolet_Data.csv**), museum specimen
 information (**UltrasoundandUltraviolet_MuseumSpecimens.csv**), raw
 trimmed phylogenetic trees (**UltrasoundandUltraviolet_Trees.nex**), R
 script (**UltrasoundandUltraviolet_Script.R**), and photo deck of all
 ultraviolet-induced photoluminescence samples (including all species with
 no UVP; **UltrasoundandUltraviolet_UVP.pdf**). ## Descriptions ###
 **UltrasoundandUltraviolet**\_Data * *Name*: Currently valid scientific
 name * *Mass_g_Females*: Average mass of adult females (g) *
 *Mass_g_Males*: Average mass of adult males (g) * *Temporality*: The main
 temporal activity patten (Nocturnal or Diurnal) * *Gliding*: Whether or
 not this species can glide as a form of locomotion (Y/N) * *Openness*:
 Openness of the dominant habitat * Closed = closed canopy, tall vegetation
 * Open = open canopy, short or minimal vegetation * *Sociality*: Social
 interactions of adult individuals * Social = Stable or cyclical social
 groups * Solitary = Stable solitary living * *UV_Lit*: The dominant
 colour, if any, of ultraviolet-induced photoluminescence reported by other
 researchers in the literature; NA indicates that we could not find a
 record of ultraviolet-induced photoluminescence in the current literature.
 * *UV_Pub*: The dominant colour, if any, of ultraviolet-induced
 photoluminescence reported in this publication (photos available in
 **UltrasoundandUltraviolet_UVP**); NA indicates that we did not test for
 ultraviolet-induced photoluminescence in this publication. * *Lowest_kHz*:
 The absolute lowest frequency (kHz) of the dominant harmonic reported in
 literature (a list of all references and vocal ranges is provided in the
 supplemental information of the main article (Supplemental Information S1
 and Supplemental References); NA indicates that we could not find vocal
 frequency range in the current literature. * *Highest_kHz*: The absolute
 highest frequency (kHz) of the dominant harmonic reported in literature (a
 list of all references and vocal ranges is provided in the supplemental
 information of the main article (Supplemental Information S1 and
 Supplemental References); NA indicates that we could not find vocal
 frequency range in the current literature. ###
 UltrasoundandUltraviolet\_MuseumSpecimens * *Name*: Currently
 valid scientific name (note that this may not reflect the name used in the
 collections, we recommend using the ID to locate specimens in museum
 databases) * *Collection*: The museum that the specimen was stored in *
 *ID*: The catalogue ID for the specimen (this is the most reliable way to
 relocate specimens in both collections) * *Location of Origin*: The
 location that the specimen was obtained from (if available from the
 collection, NA indicates that location of origin was not catalogued by the
 collection). The city and province/state/territory were provided if
 possible, though some specimens were only listed with a country. * *Year
 of Origin*: The year that the specimen was obtained (if available from the
 collection, NA indicates that year of origin was not catalogued by the
 collection) * *Specimen Type*: The majority of specimens were pelts (which
 were sometimes stuffed), but we also note mounted specimens which were
 posed with additional features like glass eyes. Mounts are more likely to
 produce false positives as they are often treated with additional
 chemicals to achieve a desired look. * *Dorsal/Ventral_UV*: The
 conclusions reached by two independent observers as to the presence of
 ultraviolet-induced photoluminescence and the colour, if present. These
 conclusions are also present on each page of
 **UltrasoundUltraviolet_UVP**. ### **UltrasoundandUltraviolet**\_Trees
 1000 node-dated trees were generated from the species list in
 **UltrasoundandUltraviolet_Data**. Some species had nomenclature
 inconsistent with our dataset: * *Lagothrix lagothricha*, Vertlife =
 *Lagothrix lagotricha* * *Micoureous demerarae*, Vertlife = *Marmosa
 demerarae* * *Paragalago* spp., Vertlife = *Galagoides* spp. *
 *Plecturocebus moloch*, Vertlife = *Callicebus moloch* Additionally,
 *Petaurus notatus* is a recently described species that was not available
 at Vertlife at the time of publication; this species was added at 1MYA to
 the *Petaurus breviceps* tip using **UltrasoundUltraviolet_Script**. ###
 **UltrasoundandUltraviolet**\_UVP Museum specimens photographed under
 normal conditions (white light), ultraviolet light, ultraviolet light +
 yellow gel filter (to reduce the amount of excess purple-blue light), and
 the filtered photograph with a slight edit to reduce the excess of yellow
 (details provided in the supplemental information of the main article
 (SupplementalInformationS1)). The ventral and dorsal sides were
 photographed for all specimens; museum collection indicated in the
 bottom-right corner. ## Key Information Sources Body Mass, Habitat
 Openness, Sociality, Temporality, and Gliding were derived from the
 following sources: * Animal Diversity Web * Integrative Taxonomic
 Information System * IUCN Red List Phylogenetic trees were trimmed from
 the Mammalian Super Tree: * Vertlife ## Code/Software R is required to run
 *Nocturnal Gliders_Script*; the script was created using version 4.2.3.
 Annotations are provided throughout the script through 1) library loading,
 2) dataset loading and cleaning, 3) analyses, and 4) figure creation.