Replication banding and FISH analysis reveal the origin of the Hyla femoralis karyotype and XY/XX sex chromosomes

The pine woods treefrog, Hyla femoralis, is unique among North American hylid frogs in having a metacentric chromosome 6 and heteromorphic sex chromosomes of the XY/XX type. The X chromosome is distinguished by having a nucleolar organizing region (NOR) in the short arm. The Y chromosome does not possess an NOR. Until the present study, it was not known if the NOR was not present on the Y chromosome or inactive and therefore not detectable by conventional cytogenetic methods like silver staining. Exclusive of its unique features the karyotype of H. femoralis closely resembles those of North American frogs with karyotypes like H. chrysoscelis. We used replication banding and fluorescence in situ hybridization (FISH) with a DNA probe to the 18S + 28S ribosomal genes, which are located at the NOR, to characterize the H. femoralis karyotype. Our analysis revealed that the 18S + 28S ribosomal genes are not present on the Y chromosome, and that the karyotype of H. femoralis was derived from an H. chrysoscelis-like karyotype by relocation of the NOR to the X chromosome from chromosome 6 and either a concurrent or subsequent pericentric inversion of chromosome 6.     

Chromosomal differentiation of Hyla nana and Hyla sanborni (Anura,Hylidae) with a description of NOR polymorphism in H. nana

Specimens of Hyla nana and Hyla sanborni from a syntopic population were studied cytogenetically. These species are morphologically very similar and are frequently misidentified, confused with each other. Both species had a diploid chromosome number, 2n = 30. However, the karyotypes of H. nana and H. sanborni differed considerably from each other in the number of submetacentric and telocentric chromosomes. The two species also differed in their primary NOR-bearing chromosomes (metacentric pair 13 in H. nana and telocentric pair 12 in H. sanborni). Additional nucleolus organizer regions (NORs) were detected by Ag-NOR staining and FISH in chromosome pairs 1, 5, 6, 12, and 14 in seven specimens of H. nana. Thus, a total of six patterns of NOR were identified. These differences in karyotype and in NOR location allowed the unambiguous identification of syntopic individuals of the two species. However, the chromosomal morphology of both species differed from that reported for populations from other geographic regions, suggesting that a systematic reevaluation of this group of Hyla may be necessary.     

Advertisement-call preferences in diploid-tetraploid treefrogs (Hyla chrysoscelis and Hyla versicolor): implications for mate choice and the evolution of communication systems

Signals used for mate choice and receiver preferences are often assumed to coevolve in a lock-step fashion. However, sender-receiver coevolution can also be nonparallel: even if species differences in signals are mainly quantitative, females of some closely related species have qualitatively different preferences and underlying mechanisms. Two-alternative playback experiments using synthetic calls that differed in fine-scale temporal properties identified the receiver criteria in females of the treefrog Hyla chrysoscelis for comparison with female criteria in a cryptic tetraploid species (H. versicolor); detailed preference functions were also generated for both species based on natural patterns of variation in temporal properties. The species were similar in three respects: (1) pulses of constant frequency were as attractive as the frequency-modulated pulses typical of conspecific calls; (2) changes in preferences with temperature paralleled temperature-dependent changes in male calls; and (3) preference functions were unimodal, with weakly defined peaks estimated at values slightly higher than the estimated means in conspecific calls. There were also species differences: (1) preference function slopes were steeper in H. chrysoscelis than in H. versicolor; (2) preferences were more intensity independent in H. chrysoscelis than in H. versicolor; (3) a synergistic effect of differences in pulse rate and shape on preference strength occurred in H. versicolor but not in H. chrysoscelis; and (4) a preference for the pulse shape typical of conspecific calls was expressed at the species-typical pulse duration in H. versicolor but not in H. chrysoscelis. However, females of H. chrysoscelis did express a preference based on pulse shape when tested with longer-than-average pulses, suggesting a hypothesis that could account for some examples of nonparallel coevolution. Namely, preferences can be hidden or revealed depending on the direction of quantitative change in a signal property relative to the threshold for resolving differences in that property. The results of the experiments reported here also predict patterns of mate choice within and between contemporary populations. First, intraspecific mate choice in both species is expected to be strongly influenced by variation in temperature among calling males. Second, simultaneous differences in pulse rate and pulse shape are required for effective species discrimination by females of H. versicolor but not by females of H. chrysoscelis. Third, there is greater potential for sexual selection within populations and for discrimination against calls produced by males in other geographically remote populations in H. chrysoscelis than in H. versicolor.     

Conserved karyotypes in the Hyla pulchella species group (Anura, Hylidae)

Cytogenetic analyses were done on specimens of Hyla marginata and on three populations of H. semiguttata differing in morphology and in the physical parameters of their advertisement call, as well as in individuals of Hyla sp. (aff. semiguttata). All specimens had 2n=24 chromosomes with a morphology very similar to that of other 24-chromosome Hyla species. Hyla semiguttata and H. marginata showed the same C-banding pattern but were distinguished by the location of the NOR on pair 1 in H. semiguttata (in the three populations) and Hyla sp. (aff. semiguttata), and on pair 10 in H. marginata. The H. semiguttata populations did not differ cytogenetically, despite variations in their morphology and advertisement calls. Similarly, H. semiguttata and H. p. joaquini studied previously had identical C-banding patterns and NOR locations, suggesting that they are very closely related.     

Comparative cytochemical measurements in the diploid-tetraploid species pair of hylid frogs Hyla chrysoscelis and H. versicolor.

Hyla versicolor (2n = 48) is a tetraploid counterpart to H. chrysoscelis (2n = 24). Cytochemical measurements revealed that no cytological parameter of the two species conformed to the expected 2:1 ratio. Every cytological factor of the tetraploid appeared to have been regulated toward the diploid level. H. veriscolor-to-H. chrysoscelis mean ratios and their standard errors were: DNA 1.90 +/- 0.03; nuclear histone, 1.70 +/- 0.18; nuclear RNA, 1.63 +/- 0.19; total nuclear protein, 1.46 +/- 0.05; nuclear sizes, 1.42 +/- 0.08.     

Species specificity and temperature dependency of temporal processing by the auditory midbrain of two species of treefrogs

The mating (advertisement) calls of two sibling species of gray treefrogs, Hyla versicolor and Hyla chrysoscelis, are spectrally identical but differ in trill rate; being higher for H. chrysoscelis. Single-unit recordings were made from the torus semicircularis of both species to investigate the neural mechanisms by which this species-specific temporal feature is analyzed. Using sinusoidally amplitude-modulated (AM) white noise as a stimulus, the temporal selectivity of these midbrain auditory neurons could be described by five response categories: 'AM nonselective' (34%); 'AM high-pass' (7%); 'AM low-pass' (6%); 'AM band-suppression' (12%); 'AM tuned' (40%). The distributions of temporal tuning values (i.e., modulation rate at which each AM-tuned unit responds maximally) are broad; in both species, neurons were found which were tuned to modulation rates greater than those found in their advertisement calls. Nevertheless, the temporal tuning values for H. versicolor (median = 25 Hz) were significantly lower than those for H. chrysoscelis (median = 32.5 Hz). The temporal selectivities of AM band-suppression neurons were found to be temperature dependent. The modulation rate at which a response minimum was observed shifted to higher values as the temperature was elevated. These results extend our earlier findings of temperature-dependent temporal selectivity in the gray treefrog. The selectivity of band-suppression and AM-tuned neurons to various rates of amplitude modulation was largely, but not completely, independent of whether sinusoidal or natural forms of AM were used.     

Ribosomal RNA structure in the diploid and phylogenetically polyploid amphibian species Hyla and Odontophrynus

Ribosomal RNA of the diploid amphibian species Hyla chrysoscelis and Odontophrynus americanus is structurally modified by hidden breaks. Phylogenetically polyploid related species like the tetraploid Hyla versicolor, the tetraploid Odontophrynus americanus and the octoploid Ceratophrys ornata do not show hidden breaks in ribosomal RNA. Structural modifications of rRNA molecules in diploid amphibians has no detectable effect on the ribosomal activity in vitro.     

Climatic variation and the distribution of an amphibian polyploid complex

The establishment of polyploid populations involves the persistence and growth of the polyploid in the presence of the progenitor species. Although there have been a number of animal polyploid species documented, relatively few inquiries have been made into the large-scale mechanisms of polyploid establishment in animal groups. Herein we investigate the influence of regional climatic conditions on the distributional patterns of a diploid-tetraploid species pair of gray treefrogs, Hyla chrysoscelis and H. versicolor (Anura: Hylidae) in the mid-Atlantic region of eastern North America. Calling surveys at breeding sites were used to document the distribution of each species. Twelve climatic models and one elevation model were generated to predict climatic and elevation values for gray treefrog breeding sites. A canonical analysis of discriminants was used to describe relationships between climatic variables, elevation and the distribution of H. chrysoscelis and H. versicolor. There was a strong correlation between several climatic variables, elevation and the distribution of the gray treefrog complex. Specifically, the tetraploid species almost exclusively occupied areas of higher elevation, where climatic conditions were relatively severe (colder, drier, greater annual variation). In contrast, the diploid species was restricted to lower elevations, where climatic conditions were warmer, wetter and exhibited less annual variation. Clusters of syntopic sites were associated with areas of high variation in annual temperature and precipitation during the breeding season. Our data suggest that large-scale climatic conditions have played a role in the establishment of the polyploid H. versicolor in at least some portions of its range. The occurrence of the polyploid and absence of the progenitor in colder, drier and more varied environments suggests the polyploid may possess a tolerance of severe environmental conditions that is not possessed by the diploid progenitor. Our findings support the hypothesis that increased tolerance to severe environmental conditions is a plausible mechanism of polyploid establishment.     

Functional equivalence of non-lethal effects: generalized fish avoidance determines distribution of gray treefrog, Hyla chrysoscelis, larvae

How patterns of prey abundance are generated determines how predation influences population and community level dynamics. We investigated how a natural population of ovipositing treefrogs, Hyla chrysoscelis , partitioned their eggs amongst experimental ponds differing in the non-lethal presence of five fish predators, each representing a distinct family. Four fish predators were functionally equivalent in terms of the behavioral response they induced in H. chrysoscelis females and hence the distribution of larval H. chrysoscelis . Mean number of H. chrysoscelis eggs deposited was significantly decreased by the mere presence of these predators relative to controls, with three species eliciting a complete avoidance (e.g. no eggs deposited). One fish predator, Aphredoderus sayanus , was statistically indistinguishable from the control treatment. These data mimic patterns of species distribution observed in nature in response to the presence of fish, but suggest an alternative mechanism for generating patterns of prey abundance amongst habitats differing in their predator composition. Furthermore, our results expand the importance of predator induced non-lethal effects as a process that could dramatically affect population and community level dynamics.     

The thermal physiology of two sympatric treefrogs Hyla cinerea and Hyla chrysoscelis (Anura; Hylidae)

Metabolic rates, temperature acclimation, lipid deposition and temperature tolerance were investigated in two species of hylid treefrogs, the green treefrog (Hyla cinerea) and the coastal plain (Cope's) gray treefrog (Hyla chrysoscelis). The rate of oxygen consumption at rest differed between the two species only at 30 degrees C; there was no difference in respiratory metabolism at lower ambient temperatures. Hyla cinerea generally completed metabolic acclimation earlier than H. chrysoscelis, particularly at high temperatures; both species appeared to be fully acclimated in 6 days or less. The gray treefrog is less tolerant of high ambient temperatures than the green treefrog; mean upper lethal temperature was 41.5 degrees C for Hyla chrysoscelis and 43.7 degrees C for H. cinerea. Metabolized energy was higher at high ambient temperatures (i.e. 29 degrees C) for H. chrysoscelis than H. cinerea, while the reverse was true at 19 degrees C. The coefficient of utilization (100 X metabolized energy/gross energy intake) did not vary significantly between species or within species over the ambient temperature range of 19-24 degrees C; H. chrysoscelis had a significantly higher efficiency at 29 degrees C. Lipid reserves were generally similar in the two species throughout the summer. Differences in behavior, seasonal variation in activity and timing of reproduction are all related to thermal physiology and may play a role in determining the distributional limits of the two species.     

Temporal order and the evolution of complex acoustic signals

The evolution of complex signals may be favoured by hidden preferences or pre-existing sensory biases. Females of two species of grey treefrogs (Hyla chrysoscelis and Hyla versicolor) were tested with combinations of a conspecific advertisement call and acoustic appendages. Appendages consisted of aggressive calls and segments of advertisement calls from conspecific males and males of three other species and bursts of filtered noise. When a wide variety of these acoustic appendages followed the advertisement call, the resulting compound signal was often more attractive than the same advertisement call alone. When the same appendages led advertisement calls, however, the compound signal was never more attractive and sometimes less attractive. The order effect was especially strong in tests of H. versicolor in which advertisement-call duration was decreased. These results cannot be explained by a general pre-existing bias for extra stimulation per se. Rather, order and other effects may constrain the evolution and subsequent modification of complex and extravagant signals, examples of which have been reported for a wide range of taxa.     

Karyotype differentiation in Chromaphyosemion killifishes (Cyprinodontiformes, Nothobranchiidae). II: cytogenetic and mitochondrial DNA analyses demonstrate karyotype differentiation and its evolutionary direction in C. riggenbachi

African killifishes of the genus Chromaphyosemion show a high degree of phenotypic and karyotypic diversity. The latter is especially pronounced in C. riggenbachi, a morphologically defined species restricted to a small distribution area in Cameroon. This study presents a detailed reconstruction of karyotype differentiation within C. riggenbachi using conventional Giemsa staining and sequential chromosome banding as well as a phylogenetic analysis based on part of the mitochondrial (mt) cytochrome b gene from eleven populations. The cytogenetic analysis revealed differences in chromosome morphology, banding patterns and/or diploid chromosome number (2n) among all populations examined. Diploid number ranged from 2n = 20 to 2n = 36 and varied mainly among populations, while C-banding patterns and NOR phenotypes showed fixed differences among populations as well as some variability within populations. The mtDNA analysis disclosed five clearly differentiated haplotype groups. Mapping the karyotype data onto the mtDNA dendrogram revealed a decrease in 2n from the most basal to the most derived groups, thus demonstrating a reduction of 2n during their evolutionary history. Our results indicate that karyotype differentiation involved Robertsonian fusions as well as non-Robertsonian processes. Causes of the high karyotypic variability may include an elevated chromosomal mutation rate as well as certain features of the ecology and mating system that could facilitate the fixation of chromosomal rearrangements. The pattern of karyotype and haplotype differentiation and the results of previous crossing experiments suggest incipient speciation in C. riggenbachi.     

CALL ANALYSIS OF TRIPLOID HYBRIDS RESULTING FROM DIPLOID-TETRAPLOID SPECIES CROSSES OF HYLID TREE FROGS

ABSTRACT

Calls produced by hybrids resulting from laboratory crosses of tetraploid Hyla versicolor females and either diploid Hyla chrysoscelis (type I) or Hyla arborea (type II) males were induced through manipulation of environmental conditions. Type I hybrids produced trilled calls similar in note repetition to H. versicolor, but more similar in dominant frequency to H. chrysoscelis. Mean duration was shorter than in both parent calls. Type II hybrids produced calls which were longer in duration and lower in note repetition rate than H. versicolor, but shorter in duration and higher in note repetition rate than H. arborea. Dominant frequency of type II hybrids was lower than in H. arborea but not significantly different than in H. versicolor. Hybrid calls were not strictly intermediate, and may provide information regarding parental relationships.     

Non-parallel coevolution of sender and receiver in the acoustic communication system of treefrogs

Advertisement calls of closely related species often differ in quantitative features such as the repetition rate of signal units. These differences are important in species recognition. Current models of signal-receiver coevolution predict two possible patterns in the evolution of the mechanism used by receivers to recognize the call: (i) classical sexual selection models (Fisher process, good genes/indirect benefits, direct benefits models) predict that close relatives use qualitatively similar signal recognition mechanisms tuned to different values of a call parameter; and (ii) receiver bias models (hidden preference, pre-existing bias models) predict that if different signal recognition mechanisms are used by sibling species, evidence of an ancestral mechanism will persist in the derived species, and evidence of a pre-existing bias will be detectable in the ancestral species. We describe qualitatively different call recognition mechanisms in sibling species of treefrogs. Whereas Hyla chrysoscelis uses pulse rate to recognize male calls, Hyla versicolor uses absolute measurements of pulse duration and interval duration. We found no evidence of either hidden preferences or pre-existing biases. The results are compared with similar data from katydids (Tettigonia sp.). In both taxa, the data are not adequately explained by current models of signal-receiver coevolution.     

Parallel changes in mate-attracting calls and female preferences in autotriploid tree frogs

For polyploid species to persist, they must be reproductively isolated from their diploid parental species, which coexist at the same time and place at least initially. In a complex of biparentally reproducing tetraploid and diploid tree frogs in North America, selective phonotaxis--mediated by differences in the pulse-repetition (pulse rate) of their mate-attracting vocalizations--ensures assortative mating. We show that artificially produced autotriploid females of the diploid species (Hyla chrysoscelis) show a shift in pulse-rate preference in the direction of the pulse rate produced by males of the tetraploid species (Hyla versicolor). The estimated preference function is centred near the mean pulse rate of the calls of artificially produced male autotriploids. Such a parallel shift, which is caused by polyploidy per se and whose magnitude is expected to be greater in autotetraploids, may have facilitated sympatric speciation by promoting reproductive isolation of the initially formed polyploids from their diploid parental forms. This process also helps to explain why tetraploid lineages with different origins have similar advertisement calls and freely interbreed.     

Diploid and tetraploid grey treefrogs (Hyla chrysoscelis and Hyla versicolor) have similar metabolic rates

We measured oxygen consumption of the diploid frog Hyla chrysoscelis and its recently evolved tetraploid cryptic species Hyla versicolor at rest and during forced locomotory exercise. We also measured whole-body lactic acid concentrations of individuals of the two species at rest and following 4 min of exercise. Contrary to predictions based on tissue-culture experiments, rates of organismal metabolism are not different in this diploid/tetraploid species pair: the diploid and tetraploid species did not differ in rates of oxygen consumption at rest or during exercise. Furthermore, whole-body lactate concentrations of frogs at rest and following forced locomotion were the same in the diploid and tetraploid species. For these species, polyploidy does not appear to be associated with lower maintenance costs at the level of the organism. However, polyploidy also does not appear to have metabolic consequences that would limit the behavioral or ecological repertoire of an anuran.     

Polyploidy, B chromosomes, and heterochromatin characterization of Mimosa caesalpiniifolia Benth. (Fabaceae-Mimosoideae)

Eight populations of Mimosa caesalpiniifolia Benth. were investigated using a cytogenetic approach. Here, we describe for the first time details of the karyotype including chromosome morphology, physical mapping of chromomycin A3 (CMA) 4′,6-diamidino-2-phenylindole (DAPI) and silver staining of nucleolar organizer regions (Ag-NOR banding), as well as 45S rDNA sites. All populations studied showed karyotypes with 2n?=?2x?=?26 small metacentric and submetacentric chromosomes, although some individuals exhibited 2n?=?4x?=?52 chromosomes. Moreover, we observed putative additional B chromosomes in some populations. The CMA banding and fluorescent in situ hybridization techniques revealed NOR heteromorphism on the unique pair containing 45 rDNA site (chromosome 12) while the Ag-NOR banding indicated NORs on both cytotypes. Up to two and four nucleoli were observed, respectively, on individuals with 2n?=?2x?=?26 and 2n?=?4x?=?52 chromosomes and the differences in nucleolar size seems to be directly related to NOR heteromorphism in some individuals. The data present new and important information to understand karyotypic evolution of Mimosa and Fabaceae.     

Replication banding patterns in Atlantic salmon (Salmo salar).

Replication banding patterns have been obtained from in vivo treatment of Salmo salar using a modification of the 5-BrdU technique and in kidney cultures using the FPG staining method. Most of the chromosome pairs were identified in the karyotype based on the banding pattern, chromosome size, and centromere position. C-banding and replication banding patterns were compared.     

中国大麦属（Horseum）的核型研究

作者研究了我国野生二棱大麦(H. spontaneum)、栽培二棱大麦(H. distichon)、野生六棱大麦(H. agriocrithon)、野生瓶形大麦(H. lagunculiforme)、栽培六棱大麦(H. vulgare)和各种变种及过渡类型的染色体数目、核型和Giemsa N—带,结果表明,大麦种间核型差异甚微,带型则有一定区别,对大麦的进化途径进行了讨论。     

Standard karyotype of sea trout (Salmo trutta morpha trutta) based on replication banding patterns

Replication banding patterns have been obtained from in vivo treatment of Salmo trutta morpha trutta chromosomes using a modification of the 5-BrdU technique and in kidney cultures using the fluorochrome photolysis Giemsa (FPG) staining method. Each chromosome pair was identified in the karyotype based on the banding pattern, chromosome size, and centromere position. The standard karyotype of sea trout has been proposed. Similarities of replication, and restriction enzyme banding patterns of chromosomes 8 and 9, and of chromosomes 11 and 12, are discussed. Fluorescence in situ hybridization was used to determine the location of telomeric sequences.