Anatomy and mechanisms of vocal production in harvest mice.

Characterizing mechanisms of vocal production provides important insight into the ecology of acoustic divergence. In this study, we characterized production mechanisms of two types of vocalizations emitted by western harvest mice (Reithrodontomys megalotis), a species uniquely positioned to inform trait evolution because it is a sister taxon to peromyscines (Peromyscus and Onychomys spp.), which use vocal fold vibrations to produce long-distance calls, but more ecologically and acoustically similar to baiomyines (Baiomys and Scotinomys spp.), which employ a whistle mechanism. We found that long-distance calls (∼10 kHz) were produced by airflow-induced vocal fold vibrations, whereas high-frequency quavers used in close-distance social interactions (∼80 kHz) were generated by a whistle mechanism. Both production mechanisms were facilitated by a characteristic laryngeal morphology. Our findings indicate that the use of vocal fold vibrations for long-distance communication is widespread in reithrodontomyines (Onychomys, Peromyscus, Reithrodontomys spp.) despite overlap in frequency content that characterizes baiomyine whistled vocalizations. The results illustrate how different production mechanisms shape acoustic variation in rodents and contribute to ecologically relevant communication distances.

Evolutionary lability of food caching behaviour in mammals.

Food hoarding provides animals access to resources during periods of scarcity. Studies on mammalian caching indicate associations with brain size, seasonality and diet but are biased to a subset of rodents. Whether the behaviour is generalizable at other taxonomic scales and/or is influenced by other ecological factors is less understood. Population density may influence food caching due to food competition or pilferage, but this remains untested in a comparative framework. Using phylogenetic analyses, we assessed the role of morphology (body and brain size), climate, diet breadth and population density on food caching behaviour evolution at multiple taxonomic scales. We also used a long-term dataset on caching behaviour of red squirrels (Tamiasciurus fremonti) to test key factors (climate and population density) on hoarding intensity. Consistent with previous smaller scale studies, we found the mammalian ancestral state for food caching was larderhoarding, and scatterhoarding was derived. Caching strategy was strongly associated with brain size, population density and climate. Mammals with larger brains and hippocampal volumes were more likely to scatterhoard, and species living at higher population densities and in colder climates were more likely to larderhoard. Finer-scale analyses within families, sub-families and tribes indicated that the behaviour is evolutionary labile. Brain size in family Sciuridae and tribe Marmotini was larger in scatterhoarders, but not in other tribes. Scatterhoarding in tribe Marmotini was more likely in species with lower population densities while scatterhoarding in tribe Sciurini was associated with warmer climates. Red squirrel larderhoarding intensity was positively related to population density but not climate, implicating food competition or pilferage as an important mechanism mediating caching behaviour. Our results are consistent with previous smaller-scale studies on food caching and indicate the evolutionary patterns of mammalian food caching are broadly generalizable. Given the lability of caching behaviour as evidenced by the variability of our results at finer phylogenetic scales, comparative analyses must consider taxonomic scale. Applying our results to conservation could prove useful as changes in population density or climate may select for different food caching strategies and thus can inform management of threatened and endangered species and their habitats.

Variation in responses to conspecific and heterospecific advertisement vocalizations in sympatric grasshopper mice (Onychomys).

Advertisement vocalizations that function in mate acquisition and resource defense within species may also mediate behavioral interactions among species. While olfactory signals play an important role in mate choice and territoriality in rodents, less is known about the function of acoustic signals in influencing interspecific interactions. In this study, we used playback experiments in the laboratory to assess the function of long-distance vocalizations within and among three sympatric species of grasshopper mice. We found that, within each species, individuals of both sexes varied widely in spontaneous vocal behavior and response to playback. The largest species (Onychomys leucogaster) was most responsive to conspecifics, but smaller O. arenicola and O. torridus exhibited no clear pattern in their vocal behavior and were even responsive to the white noise controls. Our results indicate that grasshopper mice are broadly responsive to a range of sounds that resemble calls and that long-distance vocalizations function primarily as signals that facilitate localization for subsequent close-distance assessment by both sexes in various social contexts. Variation in vocal responses among species may depend on competitive dominance, degree of interaction, acoustic similarity, or behavioral changes resulting from captivity. Replicating playback experiments in the field will help validate whether the observed variation in the laboratory reflects ecologically relevant patterns in nature.

Mechanisms of sound production in deer mice (Peromyscus spp.).

Rodent diversification is associated with a large diversity of species-specific social vocalizations generated by two distinct laryngeal sound production mechanisms: whistling and airflow-induced vocal fold vibration. Understanding the relative importance of each modality to context-dependent acoustic interactions requires comparative analyses among closely related species. In this study, we used light gas experiments, acoustic analyses and laryngeal morphometrics to identify the distribution of the two mechanisms among six species of deer mice (Peromyscus spp.). We found that high frequency vocalizations (simple and complex sweeps) produced in close-distance contexts were generated by a whistle mechanism. In contrast, lower frequency sustained vocalizations (SVs) used in longer distance communication were produced by airflow-induced vocal fold vibrations. Pup isolation calls, which resemble adult SVs, were also produced by airflow-induced vocal fold vibrations. Nonlinear phenomena (NLP) were common in adult SVs and pup isolation calls, suggesting irregular vocal fold vibration characteristics. Both vocal production mechanisms were facilitated by a characteristic laryngeal morphology, including a two-layered vocal fold lamina propria, small vocal membrane-like extensions on the free edge of the vocal fold, and a singular ventral laryngeal air pocket known as the ventral pouch. The size and composition of vocal folds (rather than total laryngeal size) appears to contribute to species-specific acoustic properties. Our findings suggest that dual modes of sound production are more widespread among rodents than previously appreciated. Additionally, the common occurrence of NLP highlights the nonlinearity of the vocal apparatus, whereby small changes in anatomy or physiology trigger large changes in behavior. Finally, consistency in mechanisms of sound production used by neonates and adults underscores the importance of considering vocal ontogeny in the diversification of species-specific acoustic signals.

Pygmy mouse songs reveal anatomical innovations underlying acoustic signal elaboration in rodents.

Elaborate animal communication displays are often accompanied by morphological and physiological innovations. In rodents, acoustic signals used in reproductive contexts are produced by two distinct mechanisms, but the underlying anatomy that facilitates such divergence is poorly understood. 'Audible' vocalizations with spectral properties between 500 Hz and 16 kHz are thought to be produced by flow-induced vocal fold vibrations, whereas 'ultrasonic' vocalizations with fundamental frequencies above 19 kHz are produced by an aerodynamic whistle mechanism. Baiomyine mice (genus Baiomys and Scotinomys) produce complex frequency-modulated songs that span these traditional distinctions and represent important models to understand the evolution of signal elaboration. We combined acoustic analyses of spontaneously vocalizing northern pygmy mice (Baiomystaylori) in air and light gas atmosphere with morphometric analyses of their vocal apparatus to infer the mechanism of vocal production. Increased fundamental frequencies in heliox indicated that pygmy mouse songs are produced by an aerodynamic whistle mechanism supported by the presence of a ventral pouch and alar cartilage. Comparative analyses of the larynx and ventral pouch size among four additional ultrasonic whistle-producing rodents indicated that the unusually low 'ultrasonic' frequencies (relative to body size) of pygmy mice songs are associated with an enlarged ventral pouch. Additionally, mice produced shorter syllables while maintaining intersyllable interval duration, thereby increasing syllable repetition rates. We conclude that while laryngeal anatomy sets the foundation for vocal frequency range, variation and adjustment of central vocal motor control programs fine tunes spectral and temporal characters to promote acoustic diversity within and between species.

A broad filter between call frequency and peripheral auditory sensitivity in northern grasshopper mice (Onychomys leucogaster).

Acoustic communication is a fundamental component of mate and competitor recognition in a variety of taxa and requires animals to detect and differentiate among acoustic stimuli (Bradbury and Vehrencamp in Principles of animal communication, 2nd edn., Sinauer Associates, Sunderland, 2011). The matched filter hypothesis predicts a correspondence between peripheral auditory tuning of receivers and properties of species-specific acoustic signals, but few studies have assessed this relationship in rodents. We recorded vocalizations and measured auditory brainstem responses (ABRs) in northern grasshopper mice (Onychomys leucogaster), a species that produces long-distance calls to advertise their presence to rivals and potential mates. ABR data indicate the highest sensitivity (28.33 ± 9.07 dB SPL re: 20 µPa) at 10 kHz, roughly corresponding to the fundamental frequency (11.6 ± 0.63 kHz) of long-distance calls produced by conspecifics. However, the frequency range of peripheral auditory sensitivity was broad (8-24 kHz), indicating the potential to detect both the harmonics of conspecific calls and vocalizations of sympatric heterospecifics. Our findings provide support for the matched filter hypothesis extended to include other ecologically relevant stimuli. Our study contributes important baseline information about the sensory ecology of a unique rodent to the study of sound perception.

Grasshopper mice employ distinct vocal production mechanisms in different social contexts.

Functional changes in vocal organ morphology and motor control facilitate the evolution of acoustic signal diversity. Although many rodents produce vocalizations in a variety of social contexts, few studies have explored the underlying production mechanisms. Here, we describe mechanisms of audible and ultrasonic vocalizations (USVs) produced by grasshopper mice (genus Onychomys). Grasshopper mice are predatory rodents of the desert that produce both loud, long-distance advertisement calls and USVs in close-distance mating contexts. Using live-animal recording in normal air and heliox, laryngeal and vocal tract morphological investigations, and biomechanical modelling, we found that grasshopper mice employ two distinct vocal production mechanisms. In heliox, changes in higher-harmonic amplitudes of long-distance calls indicate an airflow-induced tissue vibration mechanism, whereas changes in fundamental frequency of USVs support a whistle mechanism. Vocal membranes and a thin lamina propria aid in the production of long-distance calls by increasing glottal efficiency and permitting high frequencies, respectively. In addition, tuning of fundamental frequency to the second resonance of a bell-shaped vocal tract increases call amplitude. Our findings indicate that grasshopper mice can dynamically adjust motor control to suit the social context and have novel morphological adaptations that facilitate long-distance communication.

Agonistic character displacement in social cognition of advertisement signals.

Interspecific aggression between sibling species may enhance discrimination of competitors when recognition errors are costly, but proximate mechanisms mediating increased discriminative ability are unclear. We studied behavioral and neural mechanisms underlying responses to conspecific and heterospecific vocalizations in Alston's singing mouse (Scotinomys teguina), a species in which males sing to repel rivals. We performed playback experiments using males in allopatry and sympatry with a dominant heterospecific (Scotinomys xerampelinus) and examined song-evoked induction of egr-1 in the auditory system to examine how neural tuning modulates species-specific responses. Heterospecific songs elicited stronger neural responses in sympatry than in allopatry, despite eliciting less singing in sympatry. Our results refute the traditional neuroethological concept of a matched filter and instead suggest expansion of sensory sensitivity to mediate competitor recognition in sympatry.

Asymmetric energetic costs in reciprocal-cross hybrids between carnivorous mice (Onychomys).

Aerobic respiration is a fundamental physiological trait dependent on coordinated interactions between gene products of the mitochondrial and nuclear genomes. Mitonuclear mismatch in interspecific hybrids may contribute to reproductive isolation by inducing reduced viability (or even complete inviability) due to increased metabolic costs. However, few studies have tested for effects of mitonuclear mismatch on respiration at the whole-organism level. We explored how hybridization affects metabolic rate in closely related species of grasshopper mice (genus Onychomys) to better understand the role of metabolic costs in reproductive isolation. We measured metabolic rate across a range of temperatures to calculate basal metabolic rate (BMR) and cold-induced metabolic rate (MRc) in O. leucogaster, O. torridus and O. arenicola, and in reciprocal F1 hybrids between the latter two species. Within the genus, we found a negative correlation between mass-specific BMR and body mass. Although O. arenicola was smaller than O. torridus, hybrids from both directions of the cross resembled O. arenicola in body mass. In contrast, hybrid BMR was strongly influenced by the direction of the cross: reciprocal F1 hybrids were different from each other but indistinguishable from the maternal species. In addition, MRc was not significantly different between hybrids and either parental species. These patterns indicate that metabolic costs are not increased in Onychomys F1 hybrids and, while exposure of incompatibilities in F2 hybrids cannot be ruled out, suggest that mitonuclear mismatch does not act as a primary barrier to gene flow. Maternal matching of BMR is suggestive of a strong effect of mitochondrial genotype on metabolism in hybrids. Together, our findings provide insight into the metabolic consequences of hybridization, a topic that is understudied in mammals.

Interspecific dominance via vocal interactions mediates altitudinal zonation in neotropical singing mice.

Interspecific aggression between ecologically similar species may influence geographic limits by mediating competitive exclusion at the range edge. Advertisement signals that mediate competitive interactions within species may also provide social information that contributes to behavioral dominance and spatial segregation among species. We studied the mechanisms underlying altitudinal range limits in Neotropical singing mice (Scotinomys), a genus of muroid rodent in which males vocalize to repel rivals and attract mates. We first delineated replacement zones and described temperature regimes on three mountains in Costa Rica and Panama where Chiriquí singing mice (S. xerampelinus) abruptly replace Alston's singing mice (S. teguina). Next, we conducted interspecific behavioral trials and reciprocal removal experiments to examine if interspecific aggression mediated species replacement. Finally, we performed reciprocal playback experiments to investigate whether response to song matched competitive interactions. Behavioral trials and removal experiments suggest that S. xerampelinus is behaviorally dominant and excludes S. teguina from higher, cooler altitudes. Playback experiments indicate that subordinate S. teguina is silenced and repelled by heterospecific song, whereas S. xerampelinus responded to heterospecifics with approach and song rates comparable to responses to conspecifics. Thus, interspecific communication reflects underlying dominance and suggests that acoustic signaling contributes to altitudinal zonation of ecologically similar congeners. Our findings implicate the use of social information in structuring spatial distributions of animal communities across landscapes and provide insight into how large-scale patterns are generated by individual interactions.

Vocal repertoire and auditory sensitivity of white-throated woodrats (Neotoma albigula).

Rodents produce a variety of acoustic signals to communicate different types of information such as identity, reproductive state, or danger. The degree to which hearing sensitivity matches particular frequencies of conspecific vocalizations may provide insight into the relative importance of different acoustic signals. In this experiment, we characterized vocal and footdrumming behaviors of white-throated woodrats (Neotoma albigula) and measured their hearing sensitivity using the auditory brainstem response. Adult and juvenile woodrats produced seven categories of vocalizations, with six categories containing frequencies that overlap their peak hearing sensitivity. In addition, woodrats produced low-frequency footdrumming signals in the presence of same- and opposite-sex social partners and in social isolation. Woodrats varied spectral and temporal characteristics of vocalizations based on social composition of the dyad. Woodrat audition spanned 1 to 42 kHz, with a broad range of best hearing sensitivity between 4 and 20 kHz. Compared to other rodents that primarily produce high-frequency vocalizations in social contexts, woodrat audition was more sensitive to low frequencies that typify their vocal repertoire. Our results suggest that the auditory system of white-throated woodrats is broadly tuned to detect behaviorally relevant acoustic signals. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Androgens modulate song effort and aggression in Neotropical singing mice.

Androgens are an important class of steroid hormones involved in modulating the expression and evolution of male secondary sex characters. Vocalizations used in the context of aggression and mate attraction are among the most elaborate and diverse androgen-dependent animal displays as reflected in a rich tradition of studies on bird song and anuran calls. Male Alston's singing mice (Scotinomys teguina) commonly emit trilled songs that appear to function in male-male aggression. In this study, we experimentally manipulated androgens in singing mice to assess their role in modulating aggression and song effort. Testosterone- and DHT-treated animals retained aggressive and song attributes similarly. However, castrated mice administered empty implants showed more subordinate behavior and sang fewer songs that were shorter, lower in power, higher in frequency, and less stereotyped. The extensive effects of androgens on a suite of phenotypes highlight their role in linking gonadal status with decisions about investment in reproductive behaviors.

Age-related and noise-induced hearing loss alters grasshopper mouse (Onychomys) vocalizations.

Age-related and noise-induced hearing loss disorders are among the most common pathologies affecting Americans across their lifespans. Loss of auditory feedback due to hearing disorders is correlated with changes in voice and speech-motor control in humans. Although rodents are increasingly used to model human age- and noise-induced hearing loss, few studies have assessed vocal changes after acoustic trauma. Northern grasshopper mice (Onychomys leucogaster) represent a candidate model because their hearing sensitivity is matched to the frequencies of long-distance vocalizations that are produced using vocal fold vibrations similar to human speech. In this study, we quantified changes in auditory brainstem responses (ABRs) and vocalizations related to aging and noise-induced acoustic trauma. Mice showed a progressive decrease in hearing sensitivity across 4-32 kHz, with males losing hearing more rapidly than females. In addition, noise-exposed mice had a 61.55 dB SPL decrease in ABR sensitivity following a noise exposure, with some individuals exhibiting a 21.25 dB recovery 300-330 days after noise exposure. We also found that older grasshopper mice produced calls with lower fundamental frequency. Sex differences were measured in duration of calls with females producing longer calls with age. Our findings indicate that grasshopper mice experience age- and noise- induced hearing loss and concomitant changes in vocal output, making them a promising model for hearing and communication disorders.

SNP variable selection by generalized graph domination.

BACKGROUND: High-throughput sequencing technology has revolutionized both medical and biological research by generating exceedingly large numbers of genetic variants. The resulting datasets share a number of common characteristics that might lead to poor generalization capacity. Concerns include noise accumulated due to the large number of predictors, sparse information regarding the p≫n problem, and overfitting and model mis-identification resulting from spurious collinearity. Additionally, complex correlation patterns are present among variables. As a consequence, reliable variable selection techniques play a pivotal role in predictive analysis, generalization capability, and robustness in clustering, as well as interpretability of the derived models. METHODS AND FINDINGS: K-dominating set, a parameterized graph-theoretic generalization model, was used to model SNP (single nucleotide polymorphism) data as a similarity network and searched for representative SNP variables. In particular, each SNP was represented as a vertex in the graph, (dis)similarity measures such as correlation coefficients or pairwise linkage disequilibrium were estimated to describe the relationship between each pair of SNPs; a pair of vertices are adjacent, i.e. joined by an edge, if the pairwise similarity measure exceeds a user-specified threshold. A minimum k-dominating set in the SNP graph was then made as the smallest subset such that every SNP that is excluded from the subset has at least k neighbors in the selected ones. The strength of k-dominating set selection in identifying independent variables, and in culling representative variables that are highly correlated with others, was demonstrated by a simulated dataset. The advantages of k-dominating set variable selection were also illustrated in two applications: pedigree reconstruction using SNP profiles of 1,372 Douglas-fir trees, and species delineation for 226 grasshopper mouse samples. A C++ source code that implements SNP-SELECT and uses Gurobi optimization solver for the k-dominating set variable selection is available (https://github.com/transgenomicsosu/SNP-SELECT).

Vocal divergence is concordant with genomic evidence for strong reproductive isolation in grasshopper mice (Onychomys).

Behavioral barriers to gene flow often evolve faster than intrinsic incompatibilities and can eliminate the opportunity for hybridization between interfertile species. While acoustic signal divergence is a common driver of premating isolation in birds and insects, its contribution to speciation in mammals is less studied. Here we characterize the incidence of, and potential barriers to, hybridization among three closely related species of grasshopper mice (genus Onychomys). All three species use long-distance acoustic signals to attract and localize mates; Onychomys arenicola and Onychomys torridus are acoustically similar and morphologically cryptic whereas Onychomys leucogaster is larger and acoustically distinct. We used genotyping-by-sequencing (GBS) to test for evidence of introgression in 227 mice from allopatric and sympatric localities in the western United States and northern Mexico. We conducted laboratory mating trials for all species pairs to assess reproductive compatibility, and recorded vocalizations from O. arenicola and O. torridus in sympatry and allopatry to test for evidence of acoustic character displacement. Hybridization was rare in nature and, contrary to prior evidence for O. torridus/O. arenicola hybrids, only involved O. leucogaster and O. arenicola. In contrast, laboratory crosses between O. torridus and O. arenicola produced litters whereas O. leucogaster and O. arenicola crosses did not. Call fundamental frequency in O. torridus and O. arenicola was indistinguishable in allopatry but significantly differentiated in sympatry, a pattern consistent with reproductive character displacement. These results suggest that assortative mating based on a long-distance signal is an important isolating mechanism between O. torridus and O. arenicola and highlight the importance of behavioral barriers in determining the permeability of species boundaries.

Laryngeal airway reconstruction indicates that rodent ultrasonic vocalizations are produced by an edge-tone mechanism.

Some rodents produce ultrasonic vocalizations (USVs) for social communication using an aerodynamic whistle, a unique vocal production mechanism not found in other animals. The functional anatomy and evolution of this sound production mechanism remains unclear. Using laryngeal airway reconstruction, we identified anatomical specializations critical for USV production. A robust laryngeal cartilaginous framework supports a narrow supraglottal airway. An intralaryngeal airsac-like cavity termed the ventral pouch was present in three muroid rodents (suborder Myomorpha), but was absent in a heteromyid rodent (suborder Castorimorpha) that produces a limited vocal repertoire and no documented USVs. Small lesions to the ventral pouch in laboratory rats caused dramatic changes in USV production, supporting the hypothesis that an interaction between a glottal exit jet and the alar edge generates ultrasonic signals in rodents. The resulting undulating airflow around the alar edge interacts with the resonance of the ventral pouch, which may function as a Helmholtz resonator. The proposed edge-tone mechanism requires control of intrinsic laryngeal muscles and sets the foundation for acoustic variation and diversification among rodents. Our work highlights the importance of anatomical innovations in the evolution of animal sound production mechanisms.

Comparing Phlebotomy by Tail Tip Amputation, Facial Vein Puncture, and Tail Vein Incision in C57BL/6 Mice by Using Physiologic and Behavioral Metrics of Pain and Distress.

Tail tip amputation with minimal restraint is not widely used for mouse phlebotomy. In part, this infrequency may reflect policies influenced by tail tip amputation procedures for genotyping, which involve greater handling and tissue removal. To assess tail tip amputation with minimal restraint as a phlebotomy technique, we compared it with 2 more common methods: scruffing with facial vein puncture and lateral tail vein incision with minimal restraint. Blood glucose levels, audible and ultrasonic vocalizations, postphlebotomy activity and grooming behavior, open field and elevated plus maze behaviors, nest-building scores, and histologic changes at the phlebotomy site were evaluated. Mice in the facial vein phlebotomy group produced more audible vocalizations, exhibited lower postphlebotomy activity in the open field, and had more severe histologic changes than did mice in the tail incision and tail tip amputation groups. Facial vein phlebotomy did not affect grooming behavior relative to sham groups, whereas tail vein incision-but not tail tip amputation-increased tail grooming compared with that in control mice. Blood glucose levels, nest-building scores, and elevated plus maze behavior did not differ between groups, and no mice in any group produced ultrasonic vocalizations. Tail tip amputation mice did not perform differently than sham mice in any metric analyzed, indicating that this technique is a potentially superior method of blood collection in mice in terms of animal wellbeing.

Cross-fostering alters advertisement vocalizations of grasshopper mice (Onychomys): Evidence for the developmental stress hypothesis.

Nutritional stress can have lasting impacts on the development of traits involved in vocal production. Cross-fostering experiments are often used to examine the propensity for vocal learning in a variety of taxa, but few studies assess the influence of malnourishment that can occur as a byproduct of this technique. In this study, we reciprocally cross-fostered sister taxa of voluble grasshopper mice (genus Onychomys) to explore their propensity for vocal learning. Vocalizations of Onychomys leucogaster did not differ between control and cross-fostered animals, but cross-fostered Onychomys arenicola produced vocalizations that were higher in frequency in a direction away from tutors. These same animals exhibited a transient reduction in body mass early in development, indicative of malnutrition. Our findings simultaneously refute vocal learning and support the developmental stress hypothesis to highlight the importance of early ontogeny on the production of vocalizations later in life.

Vocal ontogeny in neotropical singing mice (Scotinomys).

Isolation calls produced by dependent young are a fundamental form of communication. For species in which vocal signals remain important to adult communication, the function and social context of vocal behavior changes dramatically with the onset of sexual maturity. The ontogenetic relationship between these distinct forms of acoustic communication is surprisingly under-studied. We conducted a detailed analysis of vocal development in sister species of Neotropical singing mice, Scotinomys teguina and S. xerampelinus. Adult singing mice are remarkable for their advertisement songs, rapidly articulated trills used in long-distance communication; the vocal behavior of pups was previously undescribed. We recorded 30 S. teguina and 15 S. xerampelinus pups daily, from birth to weaning; 23 S. teguina and 11 S. xerampelinus were recorded until sexual maturity. Like other rodent species with poikilothermic young, singing mice were highly vocal during the first weeks of life and stopped vocalizing before weaning. Production of first advertisement songs coincided with the onset of sexual maturity after a silent period of ≧2 weeks. Species differences in vocal behavior emerged early in ontogeny and notes that comprise adult song were produced from birth. However, the organization and relative abundance of distinct note types was very different between pups and adults. Notably, the structure, note repetition rate, and intra-individual repeatability of pup vocalizations did not become more adult-like with age; the highly stereotyped structure of adult song appeared de novo in the first songs of young adults. We conclude that, while the basic elements of adult song are available from birth, distinct selection pressures during maternal dependency, dispersal, and territorial establishment favor major shifts in the structure and prevalence of acoustic signals. This study provides insight into how an evolutionarily conserved form of acoustic signaling provides the raw material for adult vocalizations that are highly species specific.

Experimentally induced metamorphosis in axolotls reduces regenerative rate and fidelity.

While most tetrapods are unable to regenerate severed body parts, amphibians display a remarkable ability to regenerate an array of structures. Frogs can regenerate appendages as larva, but they lose this ability around metamorphosis. In contrast, salamanders regenerate appendages as larva, juveniles, and adults. However, the extent to which fundamental traits (e.g., metamorphosis, body size, aging, etc.) restrict regenerative ability remains contentious. Here we utilize the ability of normally paedomorphic adult axolotls (Ambystoma mexicanum) to undergo induced metamorphosis by thyroxine exposure to test how metamorphosis and body size affects regeneration in age-matched paedomorphic and metamorphic individuals. We show that body size does not affect regeneration in adult axolotls, but metamorphosis causes a twofold reduction in regeneration rate, and lead to carpal and digit malformations. Furthermore, we find evidence that metamorphic blastemal cells may take longer to traverse the cell cycle and display a lower proliferative rate. This study identifies the axolotl as a powerful system to study how metamorphosis restricts regeneration independently of developmental stage, body size, and age; and more broadly how metamorphosis affects tissue-specific changes.