Codon equilibrium II: Its use in estimating silent-substitution rates

Summary We study the equilibrium in the use of synonymous codons by eukaryotic organisms and find five equations involving substitution rates that we believe embody the important implications of equilibrium for the process of silent substitution. We then combine these five equations with additional criteria to determine sets of substitution rates applicable to eukaryotic organisms. One method employs the equilibrium equations and a principle of maximum entropy to find the most uniform set of rates consistent with equilibrium. In a second method we combine the equilibrium equations with data on the man-mouse divergence to determine that set of rates that is most neutral yet consistent with both types of data (i.e., equilibrium and divergence data). Simulations show this second method to be quite reliable in spite of significant saturation in the substitution process. We find that when divergence data are included in the calculation of rates, even though these rates are chosen to be as neutral as possible, the strength of selection inferred from the nonuniformity of the rates is approximately doubled. Both sets of rates are applied to estimate the human-mouse divergence time based on several independent subsets of the divergence data consisting of the quartet, C- or T-ending duet, and A- or G-ending duet codon sets. Both rate sets produce patterns of divergence times that are shortest for the quartet data, intermediate for the CT-ending duets, and longest for the AG-ending duets. This indicates that rates of transitions in the duet-codon sets are significantly higher than those in the quartet-codon sets; this effect is especially marked for AG, the rate of which in duets must be about double that in quartets.     

Codon equilibrium I: Testing for homogeneous equilibrium

Summary We present theoretical considerations that suggest that synonymous-codon usage might be expected to be close to an equilibrium distribution given a very homogeneous process of silent substitution. By homogeneous we mean that substitution depends only on the two bases involved, so that 12 base-substitution rates completely describe the silent substitution process. We have developed a method of statistically testing for such homogeneous equilibrium and applied it to reported data on the codon usages of different classes of organisms. Weakly expressed bacterial sequences and both mammalian and nonmammalian eukaryotic sequences deviate significantly from a random pattern of codon usage, in the direction of homogeneous equilibrium. On the other hand, highly expressed bacterial sequences do not exhibit homogeneous equilibrium, which may be correlated with recent experimental results showing that they are optimized to accept the most abundant tRNAs. To examine the effect of amino acid replacements on the homogeneous model of silent substitution, we divided the amino acids with degenerate codes into two classes, those with high mutabilities and those with low, and performed the same analysis on bacterial and eukaryotic data sets. The codon sets of the highly mutable class of amino acids are not further from homogeneous equilibrium than are the codon sets of the class with low mutabilities. We also found for the eukaryotic data that these independent classes of codon sets show very similar equilibrium patterns. The various results suggest a high level of uniformity in the process of silent fixation in the different synonymous-codon sets, especially in eukaryotes.     

Nonrandom frequency patterns of synonymous substitutions in homologous mammalian genes

All 69 homologous coding sequences that are currently available in four mammalian orders were aligned and the synonymous positions of quartet and duet (fourfold and twofold degenerate) codons were divided into three classes (that will be called conserved, intermediate, and variable) according to whether they show no change, one change, or more than one change, respectively. We observed (1) that the frequencies of conserved, intermediate, and variable positions of quartet and duet codons are different in different genes; (2) that the frequencies of the three classes are significantly different from expectations based on a random substitution process in the majority of genes (especially for GC-rich genes) for quartet codons and in a minority of genes for doublet codons; and (3) that the frequencies of the three classes of positions of quartet codons are correlated with those of duet codons, the conserved positions of quartet and duet codons being, in addition, correlated with the degree of amino acid conservation. Our main conclusions are that synonymous substitution frequencies: (1) are gene-specific; (2) are not simply the result of a stochastic process in which nucleotide substitutions accumulate at random, over time; and (3) are correlated in quartet and duet codons.     

Codon equilibrium I: Testing for homogeneous equilibrium

We present theoretical considerations that suggest that synonymous-codon usage might be expected to be close to an equilibrium distribution given a very homogeneous process of silent substitution. By homogeneous we mean that substitution depends only on the two bases involved, so that 12 base-substitution rates completely describe the silent substitution process. We have developed a method of statistically testing for such homogeneous equilibrium and applied it to reported data on the codon usages of different classes of organisms. Weakly expressed bacterial sequences and both mammalian and nonmammalian eukaryotic sequences deviate significantly from a random pattern of codon usage, in the direction of homogeneous equilibrium. On the other hand, highly expressed bacterial sequences do not exhibit homogeneous equilibrium, which may be correlated with recent experimental results showing that they are optimized to accept the most abundant tRNAs. To examine the effect of amino acid replacements on the homogeneous model of silent substitution, we divided the amino acids with degenerate codes into two classes, those with high mutabilities and those with low, and performed the same analysis on bacterial and eukaryotic data sets. The codon sets of the highly mutable class of amino acids are not further from homogeneous equilibrium than are the codon sets of the class with low mutabilities. We also found for the eukaryotic data that these independent classes of codon sets show very similar equilibrium patterns. The various results suggest a high level of uniformity in the process of silent fixation in the different synonymous-codon sets, especially in eukaryotes.     

Angry Birds Need Not Apply: A Perspective on the Flexible form and Multifunctionality of Avian Vocal Duets

Research into avian vocal duets has revealed much about the social interactions of mated pairs, including how communication signals mediate important components of fitness such as reproductive success and resource‐holding ability. Excellent reviews have been written on avian duets, but none have quantified the prevalence of different duet features and functions, or tested for potential links between structure and function. Here we have compiled a thorough list of studies at the species level and investigated whether there are trends in duet form, trends in duet function and matches between form and function. We have also provided recommendations for reporting of duet structure to help standardize future data collection and facilitate comparative analyses. We found that duet vocalizations are typically tightly coordinated (69% of 33 species), initiated by both partners (80% of 35 species), and have sex‐specific contributions (73% of 48 species). Duets vary substantially in structure both across and within species, and this flexibility in duet form may contribute to their use in multiple contexts. Among the well‐studied species (functions well supported by experimental evidence and studied post‐1983), duets are usually multifunctional, with functions including joint resource defence, signalling commitment, maintaining contact, ensuring reproductive synchrony and mate‐guarding. Levels of support for different hypotheses have changed over time as theory within the field developed, but overall, the best‐supported function of duets across avian species (81% of 26 species) has been joint resource defence. In all but one of these 26 species, duets are sometimes cooperative (meaning that duet participation benefits both duetting partners), and they often produce multiple benefits. We found associations between form and function, in that duets directed to non‐pair individuals have higher amplitude and are more likely to have sex‐specific notes than duets directed within the pair. We see the field moving away from labelling duets as representing ‘cooperation’ or ‘conflict’ and moving towards a general multifunctionality framework within which researchers test multiple hypotheses for duet function under the assumption that both duet form and function can vary moment to moment and that such flexibility can provide multiple benefits.     

Breeding season length predicts duet coordination and consistency in Neotropical wrens (Troglodytidae)

Many animals produce coordinated signals, but few are more striking than the elaborate male–female vocal duets produced by some tropical songbirds. Yet, little is known about the factors driving the extreme levels of vocal coordination between mated pairs in these taxa. We examined evolutionary patterns of duet coordination and their potential evolutionary drivers in Neotropical wrens (Troglodytidae), a songbird family well known for highly coordinated duets. Across 23 wren species, we show that the degree of coordination and precision with which pairs combine their songs into duets varies by species. This includes some species that alternate their song phrases with exceptional coordination to produce rapidly alternating duets that are highly consistent across renditions. These highly coordinated, consistent duets evolved independently in multiple wren species. Duet coordination and consistency are greatest in species with especially long breeding seasons, but neither duet coordination nor consistency are correlated with clutch size, conspecific abundance or vegetation density. These results suggest that tightly coordinated duets play an important role in mediating breeding behaviour, possibly by signalling commitment or coalition of the pair to mates and other conspecifics.     

Siberian crane duet as an individual signature of a pair: comparison of visual and statistical classification techniques

Vocal individuality varies between species and/or ontogenesis stages depending on needs in the vocal recognition, but also estimation of individual differences depends on the method of analysis. We studied pair-specific differences of duets elicited by mating pairs of Siberian crane Grus leucogeranus. We quantitatively described the duet structure and compared visual and statistical classification methods of pair identification by duet. Three methods were used: discriminant analysis, method of classification trees and visual classification of spectrogram. We found significant interpair differences. The pairs differ by duet structure that is by the ratio of male- and female-initiated duets and by the ratio of the number of male to female calls; temporal-frequency duet characteristics are pair-specific, too. All methods showed high interpair differences, which exceeded random values significantly. Discriminant analysis stepwise procedure based on 11 parameters resulted in 97.3% of correctly assigned duets. Human observers correctly assigned 80.7% of spectrograms. Our data provide a basis for remote monitoring of this endangered species with a wild population of only 3,000 birds.     

Cleavage in<Emphasis Type="Italic"> Nemertoderma westbladi</Emphasis> (Nemertodermatida) and its phylogenetic significance

Recent phylogenetic analyses of ribosomal and protein coding nuclear genes place the marine worms within the Nemertodermatida as one of the oldest lineages among the bilaterian animals. We studied the early embryonic cleavage in Nemertoderma westbladi to provide the first account of nemertodermatid early development. Live embryos were studied with interference microscopy and fixed embryos were either sectioned or studied with confocal laser scanning microscopy. Initially the divisions in the embryo are radial, but then micromeres are shifted clockwise generating a spiral pattern. The four-cell stage is characterized by duets of macromeres and micromeres and thus resembles the duet cleavage reported from members of the Acoela. However, subsequent stages differ from the acoel duet pattern and also from quartet spiral cleavage. The optimization of the cleavage pattern on current phylogenetic hypotheses with Nemertodermatida and Acoela as early bilaterian branches is discussed.     

The unique developmental program of the acoel flatworm, Neochildia fusca

Acoel embryos exhibit a unique form of development that some investigators argue is related to that found in polyclad turbellarians and coelomate spiralians, which display typical quartet spiral cleavage. We generated the first cell-lineage fate map for an acoel flatworm, Neochildia fusca, using modern intracellular lineage tracers to assess the degree of similarity between these distinct developmental programs. N. fusca develops via a "duet" cleavage pattern in which second cleavage occurs in a leiotropically oblique plane relative to the animal-vegetal axis. At the four-cell stage, the plane of first cleavage corresponds to the plane of bilateral symmetry. All remaining cleavages are symmetrical across the sagittal plane. No ectomesoderm is formed; the first three micromere duets generate only ectodermal derivatives. Endomesoderm, including the complex assemblage of circular, longitudinal, and oblique muscle fibers, as well as the peripheral and central parenchyma, is generated by both third duet macromeres. The cleavage pattern, fate map, and origins of mesoderm in N. fusca share little similarity to that exhibited by other spiralians, including the Platyhelminthes (e.g., polyclad turbellarians). These findings are considered in light of the possible evolutionary origins of the acoel duet cleavage program versus the more typical quartet spiral cleavage program. Finally, an understanding of the cell-lineage fate map allows us to interpret the results of earlier cell deletion studies examining the specification of cell fates within these embryos and reveals the existence of cell-cell inductive interactions in these embryos.     

Vocal negotiation over parental care? Acoustic communication at the nest predicts partners' incubation share

In species with biparental care, individuals adjust their workload to that of their partner to either compensate or match its investment. Communication within a pair might be crucial for achieving this adjustment. Zebra finches, Taeniopygia guttata, form life‐long monogamous pair bonds, in which partners are highly coordinated and both incubate the eggs. When relieving each other during incubation, partners perform a structured call duet at the nest. If this duet functions to coordinate incubation workload, disrupting the pair's usual nest‐relief pattern by delaying the male's return to the nest should affect the structure of the duet. Using domesticated birds breeding in a large aviary, we found that delaying the male's return induced shorter duets with higher call rates. In addition, we tracked the location of individuals with a transponder at the nest and the feeder, and showed that these accelerated duets were associated with an increased haste of the partners to take turns incubating and foraging. Females also spent less time incubating during their subsequent shift, and females' time off‐nest was best predicted by their mate's calling behaviour in the previous duet. Taken together, these results suggest that duets may function as ‘vocal negotiation’ over parental care.     

Functions of duetting in the tropical boubou, Laniarius aethiopicus: territorial defence and mutual mate guarding

Duetting, found in many animal taxa, is still a poorly understood form of signalling behaviour despite numerous hypotheses to explain its function. One contentious issue is whether duetting is a cooperative endeavour or signals conflict between the sexes. We studied the function of duetting in the tropical boubou, using interactive playback experiments. We staged encounters by presenting paired birds with four variants of each of four duet types (neighbour and stranger solos and neighbour and stranger duets). Multiple lines of evidence suggest that both joint territorial defence and mutual mate guarding are important functions of duetting, and that both cooperation and conflict between males and females have shaped duetting behaviour. Of a repertoire of 12 duet types, eight were used for joint territorial defence. Two of these eight duet types were probably also used for mutual mate guarding, suggesting that individual duets can have multiple functions. We found an unusual response for duetting birds in that females often synchronized notes with male solo playback to form precise duets. In turn, males attempted to jam the song of rivals when females joined the song of male solo playback, providing further evidence for the importance of acoustic mate guarding. Song jamming by males has not previously been described for duetting species. Finally, we suggest that the repertoire of duet types is used to fine-tune territorial encounters in a network environment of multiple interactants.     

The Responses of Duetting Antbirds to Stereo Duet Playback Provide Support for the Joint Territory Defence Hypothesis

In multiple animal taxa, including many birds and primates, members of mated pairs produce coordinated acoustic displays known as duets. By observing the behaviour of territorial animals as they respond to playback‐simulated duets of rivals, we can gain insight into the behavioural significance of vocal duets. Playback experiments, however, have been conducted across a very narrow range of duetting animals. Furthermore, many studies have been conducted with single‐speaker playback, whereas stereo‐speaker playback offers more spatially realistic simulation of duets. Moreover, by evaluating the reactions of animals to separate loudspeakers broadcasting male and female duet contributions, we can study the interactions of both males and females with same‐sex vs. opposite‐sex rivals. We used a paired experimental design to broadcast duet stimuli through a single‐speaker and a stereo‐speaker apparatus to 30 pairs of duetting barred antshrikes Thamnophilus doliatus in Costa Rica. Our goals were (1) to evaluate whether territorial antbirds respond more aggressively to male vs. female duet components and (2) to assess aggressive responses of antbirds towards single‐speaker vs. stereo‐speaker playback. Neither males nor females differentiated between the loudspeaker simulating the male vs. female duet contribution during stereo‐speaker playback trials. Barred antshrikes displayed significantly stronger responses to stereo‐speaker playback compared with single‐speaker playback. Males displayed stronger playback responses than females with closer, quicker and more vocal responses. These results provide evidence for a joint resource defence function of antbird duets given that pairs responded together with equivalent intensity to male and female simulated intruders. This is the first study to show that although duetting is an aggressive territorial signal, birds do not necessarily respond to sex‐specific components of duets. Our results support the idea that spatially realistic stereo presentation of duet stimuli is critical for experimental duet research.     

Context, Structural Variability and Distinctiveness of California Towhee (Pipilo crissalis) Vocal Duets

Avian vocal duets occur when paired birds produce temporally and structurally coordinated vocalizations. Duets are given by members of many species from taxonomically distinct lineages and show great variety in form that often reflects function. By describing the structure of vocal duets we can learn about the diversity of communication signals present in nature and also gain insight into the evolution and operation of those signals. This study quantified the usage patterns and acoustic structure of California towhee duet vocalizations, and tested the distinctiveness of duets among different pairs. California towhee duets consist of a 'squeal' vocalization that is highly unlike the species-typical call note or male advertizement song. California towhees duet an average of three times per hour during the breeding season, and all duets are accompanied by an approach response that brings duetting partners into close spatial proximity. Males and females produce duet contributions with the same syllabic structure. Individual birds produce highly variable squeal vocalizations that are distinctive enough to signal identity. California towhee duet characteristics indicate that the squeal vocalization has evolved separately from other vocal traits in this species and was promoted by natural selection as a duet-specific vocalization. Duet usage patterns and structure suggest that these communication signals function in a cooperative context.     

Song repertoire and duetting behaviour of the tropical boubou, Laniarius aethiopicus

Female song and the significance of duetting in birds remain puzzling, in part because of the limited number of species studied. We investigated duetting behaviour in the tropical boubou in West Africa. Birds produced a diverse song repertoire consisting of solos, duets and trios, with duets being the most conspicuous vocalization. We identified 12 distinct duet types in which tonal and broadband notes were combined by males and females in highly synchronized and temporally precise patterns. Molecular sexing and observations of colour-banded birds revealed that duets were initiated by both sexes, with strict sex-specific roles maintained within the duet. An equal number of duet types was initiated by males and females. Seven of 12 duets were terminated by the male. Male-initiated duets were also more common (89% of total) and were repeated more often, i.e. sung more frequently. Solo singing occurred when partners did not respond and was also used by unpaired birds. Trios were produced by subadult birds joining the duet of a resident pair. The wide variety of contexts in which duets were sung suggests that they serve several functions, including territorial defence and mutual mate guarding.     

Description of the acoustical interaction and synchronization between duetters of the Large-footed Finch (Pezopetes capitalis)

Duets are highly coordinated acoustical displays produced by two individuals. Studying the structure of duet songs and its causes is essential to understand the ecological role and evolution of this form of communication. Our goals in this study were to describe the structure of the duet of the Large-footed Finch, the temporal synchronization of each individual, and to test the effect of provoked (playback) duets. We recorded birds at Cerro de la Muerte, Costa Rica and simulated territory intrusions using playback trials. The duet of this species consisted of overlapping notes between both members of a mated pair, which were emitted either randomly or periodically. Temporal synchronization was similar between both individuals in a mated pair. Both partners adjust the silent intervals between notes in response to the duration of its partner’s note. The high frequency of the second part of the duet of this species decreased with time and the second individual, in duetting this part, not only synchronized its notes temporally with the first individual, but also synchronized the high frequency. The high frequency of the first note the second individual sung was a bit lower than the previous note sung by the first individual. Finally, birds responding to provoked duets produced duets with higher frequency and higher rate of notes compared to unprovoked duets. Our results provide information on the temporal and frequency synchronization in duets of an endemic emberizid and provide new information about a less reported vocal behaviour in bird duets, the acoustic frequency coordination.     

Effect of Divergence Time and Recombination Rate on Molecular Evolution of <Emphasis Type="Italic">Drosophila</Emphasis> INE-1 Transposable Elements and Other Candidates for Neutrally Evolving Sites

Interspecies divergence of orthologous transposable element remnants is often assumed to be simply due to genetic drift of neutral mutations that occurred after the divergence of the species. However, divergence may also be affected by other factors, such as variation in the mutation rate, ancestral polymorphisms, or selection. Here we attempt to determine the impact of these forces on divergence of three classes of sites that are often assumed to be selectively unconstrained (INE-1 TE remnants, sites within short introns, and fourfold degenerate sites) in two different pairwise comparisons of Drosophila (D. melanogaster vs. D. simulans and D. simulans vs. D. sechellia). We find that divergence of these three classes of sites is strongly influenced by the recombination environment in which they are located, and this is especially true for the closer D. simulans vs. D. sechellia comparison. We suggest that this is mainly a result of the contribution of ancestral polymorphisms in different recombination regions. We also find that intergenic INE-1 elements are significantly more diverged than intronic INE-1 in both pairwise comparisons, implying the presence of either negative selection or lower mutation rates in introns. Furthermore, we show that substitution rates in INE-1 elements are not associated with the length of the noncoding sequence in which they are located, suggesting that reduced divergence in long noncoding sequences is not due to reduced mutation rates in these regions. Finally, we show that GC content for each site within INE-1 sequences has evolved toward an equilibrium value (approximately 33%) since insertion.     

A review of acoustic playback techniques for studying avian vocal duets

ABSTRACT Playback experiments involve the broadcast of natural or synthetic sound stimuli and provide a powerful tool for studying acoustic communication in birds. Playback is a valuable technique for exploring vocal duetting behavior because it allows investigators to test predictions of the various hypotheses for duet function. Here, we adopt a methodological perspective by considering various challenges specific to studying duetting behavior, and highlighting the utility of different playback designs for testing duet function. Single‐speaker playback experiments allow investigators to determine how duetting birds react to different stimuli, but do not simulate duets in a spatially realistic manner. Multi‐speaker playback experiments are superior to single‐speaker designs because duet stimuli are broadcast with spatial realism and unique and additional predictions can be generated for testing duet function. In particular, multi‐speaker playback allows investigators to evaluate how birds respond to male versus female duet contributions separately, based on reactions to the different loudspeakers. Interactive playback allows investigators to ask questions about the time‐ and pattern‐specific singing behavior of birds, and to understand how singing strategies correspond to physical behavior during vocal interactions. Although logistically challenging, interactive playback provides a powerful tool for examining specific elements of duets (such as the degree of coordination) and may permit greater insight into their functions from an operational perspective. Interactive playback designs where the investigator simulates half of a duet may be used to describe and investigate the function of pair‐specific and population‐wide duet codes. Regardless of experimental design, all playback experiments should be based on a sound understanding of the natural duetting behavior of the species of interest, and should aim to produce realistic and carefully controlled duet simulations. Future studies that couple playback techniques with other experimental procedures, such as Acoustic Location System recordings for monitoring the position of birds in dense vegetation or multimodal techniques that combine acoustic with visual stimuli, are expected to provide an even better understanding of these highly complex vocal displays.     

Duet function in the yellow-naped amazon, Amazona auropalliata: evidence from playbacks of duets and solos

The question of why animals participate in duets is an intriguing one, as many such displays appear to be more costly to produce than individual signals. Mated pairs of yellow-naped amazons, Amazona auropalliata, give duets on their nesting territories. We investigated the function of those duets with a playback experiment. We tested two hypotheses for the function of those duets: the joint territory defense hypothesis and the mate-guarding hypothesis, by presenting territorial pairs with three types of playback treatments: duets, male solos, and female solos. The joint territory defense hypothesis suggests that individuals engage in duets because they appear more threatening than solos and are thus more effective for the establishment, maintenance and/or defense of territories. It predicts that pairs will be coordinated in their response (pair members approach speakers and vocalize together) and will either respond more strongly (more calls and/or more movement) to duet treatments than to solo treatments, or respond equally to all treatments. Alternatively, the mate-guarding hypothesis suggests that individuals participate in duets because they allow them to acoustically guard their mate, and predicts uncoordinated responses by pairs, with weak responses to duet treatments and stronger responses by individuals to solos produced by the same sex. Yellow-naped amazon pairs responded to all treatments in an equivalently aggressive and coordinated manner by rapidly approaching speakers and vocalizing more. These responses generally support the joint territory defense hypothesis and further suggest that all intruders are viewed as a threat by resident pairs.     

Duets in Yellow-Naped Amazons: Variation in Syntax, Note Composition and Phonology at Different Levels of Social Organization

Complex vocal signals composed of multiple notes are used by many species. Such signals may vary in a number of features such as the rules that govern note order and timing (syntax), the relative number and types of different notes (note composition), and the acoustic structure of notes (phonology). Previous research examining male song in songbirds typically has shown greater conservation of syntax than phonology. Here we investigated whether these patterns of variation are also found in the duets of parrots. We examined geographic variation in the pair duets of yellow-naped amazons, Amazona auropalliata , at five sites within one vocal dialect. We also examined variation at several levels of social organization, including within pairs, among pairs and among sites, to assess where variability was greatest. Variation was highest at the within-pair level, although variation was also present at all other levels and for all of the duet factors. We hypothesize that variation at the among-site level allows duets to indicate site membership, while variation at the within-pair level allows pairs to change their duet to match their current social or physical environment. Syntax and phonology are also more conserved than note composition for all levels of social organization. This pattern may result because note composition is less important in duet function than syntax or phonology, or because development of syntax and phonology is more heavily influenced by genetic templates.