Conserved chromatin and repetitive patterns reveal slow genome evolution in frogs.

Frogs are an ecologically diverse and phylogenetically ancient group of anuran amphibians that include important vertebrate cell and developmental model systems, notably the genus Xenopus. Here we report a high-quality reference genome sequence for the western clawed frog, Xenopus tropicalis, along with draft chromosome-scale sequences of three distantly related emerging model frog species, Eleutherodactylus coqui, Engystomops pustulosus, and Hymenochirus boettgeri. Frog chromosomes have remained remarkably stable since the Mesozoic Era, with limited Robertsonian (i.e., arm-preserving) translocations and end-to-end fusions found among the smaller chromosomes. Conservation of synteny includes conservation of centromere locations, marked by centromeric tandem repeats associated with Cenp-a binding surrounded by pericentromeric LINE/L1 elements. This work explores the structure of chromosomes across frogs, using a dense meiotic linkage map for X. tropicalis and chromatin conformation capture (Hi-C) data for all species. Abundant satellite repeats occupy the unusually long (~20 megabase) terminal regions of each chromosome that coincide with high rates of recombination. Both embryonic and differentiated cells show reproducible associations of centromeric chromatin and of telomeres, reflecting a Rabl-like configuration. Our comparative analyses reveal 13 conserved ancestral anuran chromosomes from which contemporary frog genomes were constructed.

The Arylabialis Muscle of the Túngara Frog (Engystomops pustulosus).

The current functional model of the anuran larynx includes four pairs of laryngeal muscles. Their contractions do not account, however, for the behavioral control of call complexity observed in male túngara frogs (Engystomops pustulosus), which optionally add a secondary note with distinct harmonic structure to their advertisement call. Examination of the túngara frog's laryngeal morphology through dissection and resin histology has revealed that the m. dilatator laryngis is divided into two separate bundles (superficial and deep). The superficial bundle closely matches the typical description of the m. dilatator laryngis and is well positioned to open the glottis. The deep bundle is exclusively innervated by the short laryngeal nerve and has an attachment to the fibrous mass, an internal laryngeal structure necessary for complex call production. This attachment indicates a separate role for the deep bundle in controlling the complexity of the call. Based on physical separation, exclusive attachments, distinct fiber orientation, exclusive innervation, and potential action, we recognize the deep bundle of the m. dilatator laryngis as a separate muscle. We also revalidate the name m. arylabialis which had been previously used to describe it. The split of the m. dilatator laryngis into two muscles results in a laryngeal innervation pattern that closely matches that of mammals. This study identified a novel laryngeal muscle in túngara frogs, a potential mechanism for the control of call complexity, and revealed new evidence of homologies between the laryngeal structures of amphibians and mammals. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:1966-1976, 2020. © 2019 American Association for Anatomy.

Laryngeal Demasculinization in Wild Cane Toads Varies with Land Use.

Anthropogenic factors, including the spread of endocrine-disrupting chemicals, have been linked to alterations in the reproductive physiology, morphology, and behavior of wildlife. Few studies of endocrine disruption, however, focus on secondary sexual traits that affect mating signals, despite their importance for reproductive success. The larynx of many anurans (frogs and toads), for example, is larger in males than in females and is crucial for producing mating calls. We aim to determine if wild populations of cane toads (Rhinella marina) near sugarcane fields in Florida have demasculinized larynges when compared to populations near urban areas. We find evidence of demasculinization in both primary and secondary sexual traits in male toads living near sugarcane. Relative to body size, the laryngeal mass, vocal cord length, and dilator muscle width are all reduced in males from sugarcane regions compared to their urban counterparts. Strong correlations between primary and secondary male sexual traits indicate that demasculinization occurs in concert both within and across diverse organs, including the testes, larynx, and skin. Our results show that anurans near sugarcane fields have demasculinized reproductive systems, that this disruption extends to secondary sexual traits like the larynx, and that it is likely due to anthropogenic causes.

Low temperature tolerance, cold hardening and acclimation in tadpoles of the neotropical túngara frog (Engystomops pustulosus).

Many frogs from temperate climates can tolerate low temperatures and increase their thermal tolerance through hardening and acclimation. Most tropical frogs, on the other hand, fail to acclimate to low temperatures. This lack of acclimation ability is potentially due to lack of selection pressure for acclimation because cold weather is less common in the tropics. We tested the generality of this pattern by characterizing the critical temperature minimum (CTMin), hardening, and acclimation responses of túngara frogs (Engystomops pustulosus). These frogs belong to a family with unknown thermal ecology. They are found in a tropical habitat with a highly constant temperature regime. The CTMin of the tadpoles was on average 12.5°C. Pre-metamorphic tadpoles hardened by 1.18°C, while metamorphic tadpoles hardened by 0.36°C. When raised at 21°C, tadpoles acclimated expanding their cold tolerance by 1.3°C in relation to larvae raised at 28°C. These results indicate that the túngara frog has a greatly reduced cold tolerance when compared to species from temperate climates, but it responds to cold temperatures with hardening and acclimation comparable to those of temperate-zone species. Cold tolerance increased with body length but cold hardening was more extensive in pre-metamorphic tadpoles than in metamorphic ones. This study shows that lack of acclimation ability is not general to the physiology of tropical anurans.

Reinterpreting features of the advertisement call of Dermatonotus muelleri (Boettger, 1885; Anura, Microhylidae).

The advertisement call of Dermatonotus muelleri was originally described by Nelson (1973) in a brief section of a review on the mating calls of the Microhylinae. He used two calls from São Leopoldo, state of Minas Gerais, in Brazil to determine that they have i) dominant frequency between 1.500-2.200 kHz (mean 1.854 + 0.216 kHz), and ii) harmonic intervals between 0.140 and 0.150 kHz (0.146 +/- 0.005 kHz). Nelson (1973) based his description on an audiospectrogram produced with high frequency resolution and did not quantify the pulse structure of the calls. More recently, Giaretta and colleagues (2013) expanded on the original description using a larger set of calls recorded from Gurinhat, state of Minas Gerais, in Brazil. They quantified the temporal structure of the call and confirmed that the dominant frequency is around 1.8 kHz. In addition, they identified a secondary low frequency band at 667 Hz.

Is the frequency content of the calls in north american treefrogs limited by their larynges?

A high diversity of mating calls is found among frogs. The calls of most species, however, are simple, in comparison to those of mammals and birds. In order to determine if the mechanics of the larynx could explain the simplicity of treefrog calls, the larynges of euthanized males were activated with airflow. Laryngeal airflow, sound frequency, and sound intensity showed a positive direct relationship with the driving air pressure. While the natural calls of the studied species exhibit minimal frequency modulation, their larynges produced about an octave of frequency modulation in response to varying pulmonary pressure. Natural advertisement calls are produced near the higher extreme of frequency obtained in the laboratory and at a slightly higher intensity (6 dB). Natural calls also exhibit fewer harmonics than artificial ones, because the larynges were activated with the mouth of the animal open. The results revealed that treefrog larynges allow them to produce calls spanning a much greater range of frequencies than observed in nature; therefore, the simplicity of the calls is not due to a limited frequency range of laryngeal output. Low frequencies are produced at low intensities, however, and this could explain why treefrogs concentrate their calling at the high frequencies.

The Response of Gray Treefrogs to Anesthesia by Tricaine Methanesulfonate (TMS or MS-222).

The design of anesthetic protocols for frogs is commonly hindered by lack of information. Results from fishes and rodents do not always apply to frogs, and the literature in anurans is concentrated on a few species. We report on the response of treefrogs (Hyla chrysoscelis and H. versicolor) to tricaine methanesulfonate. Body mass did not differ significantly between the species or between sexes. In the first exposure of a frog to TMS, variation in induction time was best explained by species (H. chrysoscelis resisted longer) and body mass (larger animals resisted longer). Multiple exposures revealed a strong effect of individual variation on induction time and a significant increase of induction time with number of previous anesthesia events within the same day. Recovery time was mostly explained by individual variation, but it increased with total time in anesthetic and decreased with induction time. It also increased with number of days since the last series of anesthesias and decreased with number of previous uses of the anesthetic bath. This is one of the first studies of anesthesia in hylids and also one of the first assessments of the factors that influence the variability of the response to anesthesia within a species.

Pure ultrasonic communication in an endemic Bornean frog.

Huia cavitympanum, an endemic Bornean frog, is the first amphibian species known to emit exclusively ultrasonic (i.e., >20 kHz) vocal signals. To test the hypothesis that these frogs use purely ultrasonic vocalizations for intraspecific communication, we performed playback experiments with male frogs in their natural calling sites. We found that the frogs respond with increased calling to broadcasts of conspecific calls containing only ultrasound. The field study was complemented by electrophysiological recordings from the auditory midbrain and by laser Doppler vibrometer measurements of the tympanic membrane's response to acoustic stimulation. These measurements revealed that the frog's auditory system is broadly tuned over high frequencies, with peak sensitivity occurring within the ultrasonic frequency range. Our results demonstrate that H. cavitympanum is the first non-mammalian vertebrate described to communicate with purely ultrasonic acoustic signals. These data suggest that further examination of the similarities and differences in the high-frequency/ultrasonic communication systems of H. cavitympanum and Odorrana tormota, an unrelated frog species that produces and detects ultrasound but does not emit exclusively ultrasonic calls, will afford new insights into the mechanisms underlying vertebrate high-frequency communication.

Environmental influences in the evolution of tetrapod hearing sensitivity and middle ear tuning.

Vertebrates inhabit and communicate acoustically in most natural environments. We review the influence of environmental factors on the hearing sensitivity of terrestrial vertebrates, and on the anatomy and mechanics of the middle ears. Evidence suggests that both biotic and abiotic environmental factors affect the evolution of bandwidth and frequency of peak sensitivity of the hearing spectrum. Relevant abiotic factors include medium type, temperature, and noise produced by nonliving sources. Biotic factors include heterospecific, conspecific, or self-produced sounds that animals are selected to recognize, and acoustic interference by sounds that other animals generate. Within each class of tetrapods, the size of the middle ear structures correlates directly to body size and inversely to frequency of peak sensitivity. Adaptation to the underwater medium in cetaceans involved reorganization of the middle ear for novel acoustic pathways, whereas adaptation to subterranean life in several mammals resulted in hypertrophy of the middle ear ossicles to enhance their inertial mass for detection of seismic vibrations. The comparative approach has revealed a number of generalities about the effect of environmental factors on hearing performance and middle ear structure across species. The current taxonomic sampling of the major tetrapod groups is still highly unbalanced and incomplete. Future expansion of the comparative evidence should continue to reveal general patterns and novel mechanisms.

Active control of ultrasonic hearing in frogs.

Vertebrates can modulate the sound levels entering their inner ears in the face of intense external sound or during their own vocalizations. Middle ear muscle contractions restrain the motion of the middle ear ossicles, attenuating the transmission of low-frequency sound and thereby protecting the hair cells in the inner ear. Here we show that the Chinese concave-eared torrent frog, Odorrana tormota, can tune its ears dynamically by closing its normally open Eustachian tubes. Contrary to the belief that the middle ear in frogs permanently communicates with the mouth, O. tormota can close this connection by contraction of the submaxillary and petrohyoid muscles, drastically reducing the air volume behind the eardrums. Mathematical modeling and laser Doppler vibrometry revealed that the reduction of this air volume increases the middle ear impedance, resulting in an up to 20 dB gain in eardrum vibration at high frequencies (10-32 kHz) and 26 dB attenuation at low frequencies (3-10 kHz). Eustachian tube closure was observed in the field during calling and swallowing. Besides a potential role in protecting the inner ear from intense low-frequency sound and high buccal air pressure during calling, this previously unrecognized vertebrate mechanism may unmask the high-frequency calls of this species from the low-frequency stream noise which dominates the environment. This mechanism also protects the thin tympanic membranes from injury during swallowing of live arthropod prey.

The structure of vocal sounds produced with the mouth closed or with the mouth open in treefrogs.

Frogs and toads mostly call with their mouths shut, unlike many other vertebrates. Sound is generated when air crosses the larynx, but there is no direct airflow to the external environment and radiation occurs at the skin. This study directly compares the acoustic output obtained from euthanized frogs with the mouth open against the output obtained with the mouth closed during activation of the larynx by airflow. With the mouth closed, the vocal sac was inflated and the acoustic energy was concentrated in the same harmonics as in the advertisement call, whereas with the mouth open, energy was spread in a wide range of harmonics. The acoustic output at the dominant frequency was more intense with the mouth closed than with the mouth open. More sound was radiated through the vocal sac and head than through the rest of the body. The spectral differences between open and closed mouth treatments matched the differences observed between natural advertisement calls, produced with the mouth closed, and distress calls, produced with the mouth open. By calling with the mouth closed, treefrogs can potentially produce advertisement calls with the energy concentrated in a narrower frequency range than with the mouth open.