Genetic and genomic interactions of animals with different ploidy levels.

Polyploid animals have independently evolved from diploids in diverse taxa across the tree of life. We review a few polyploid animal species or biotypes where recently developed molecular and cytogenetic methods have significantly improved our understanding of their genetics, reproduction and evolution. Mitochondrial sequences that target the maternal ancestor of a polyploid show that polyploids may have single (e.g. unisexual salamanders in the genus Ambystoma) or multiple (e.g. parthenogenetic polyploid lizards in the genus Aspidoscelis) origins. Microsatellites are nuclear markers that can be used to analyze genetic recombinations, reproductive modes (e.g. Ambystoma) and recombination events (e.g. polyploid frogs such as Pelophylax esculentus). Hom(e)ologous chromosomes and rare intergenomic exchanges in allopolyploids have been distinguished by applying genome-specific fluorescent probes to chromosome spreads. Polyploids arise, and are maintained, through perturbations of the 'normal' meiotic program that would include pre-meiotic chromosome replication and genomic integrity of homologs. When possible, asexual, unisexual and bisexual polyploid species or biotypes interact with diploid relatives, and genes are passed from diploid to polyploid gene pools, which increase genetic diversity and ultimately evolutionary flexibility in the polyploid. When diploid relatives do not exist, polyploids can interact with another polyploid (e.g. species of African Clawed Frogs in the genus Xenopus). Some polyploid fish (e.g. salmonids) and frogs (Xenopus) represent independent lineages whose ancestors experienced whole genome duplication events. Some tetraploid frogs (P. esculentus) and fish (Squaliusalburnoides) may be in the process of becoming independent species, but diploid and triploid forms of these 'species' continue to genetically interact with the comparatively few tetraploid populations. Genetic and genomic interaction between polyploids and diploids is a complex and dynamic process that likely plays a crucial role for the evolution and persistence of polyploid animals. See also other articles in this themed issue.

Sex in unisexual salamanders: discovery of a new sperm donor with ancient affinities.

Although bisexual reproduction has considerable evolutionary benefits, several all-female vertebrates exist. Unisexual salamanders in the genus Ambystoma are common around the Great Lakes region in eastern North America. They originated from a hybridization event that involved a female that shared a common ancestor with Ambystoma barbouri 2.4 to 3.9 million years ago but, unexpectedly, A. barbouri nuclear genomes were unknown in unisexuals. Unisexual salamanders steal sperm from donors of normally bisexual species, so their reproductive mode is described as kleptogenesis. Most known unisexuals are polyploid and they all possess at least one A. laterale genome. One or more other genomes are taken from sperm donors that may include A. jeffersonianum, A. laterale, A. texanum and A. tigrinum. We examined unisexual adults and larvae in a southern Ohio pond where unisexual individuals coexist with male A. barbouri. This population provided an opportunity to test hypotheses pertaining to the role of A. barbouri in the evolution of the disparate cytoplasmic and nuclear genomes in unisexual salamanders. Microsatellite DNA loci, mitochondrial DNA sequences and genomic in situ hybridization were used to identify the genomic constitution of individuals. A. barbouri was found to be an acceptable sperm donor for unisexuals but only contributed genomes in ploidy-elevated individuals. In the absence of A. jeffersonianum, this Ohio population is likely experiencing a recent switch in sperm donors from A. jeffersonianum to A. barbouri and demonstrates the evolutionary flexibility and dynamics of kleptogenesis.

An examination of intergenomic exchanges in A. laterale-dependent unisexual salamanders in the genus Ambystoma.

The evolutionary longevity of unisexual salamanders in the genus Ambystoma may be attributed to their flexible reproductive system and meiotic intergenomic interactions. More than 20 different unisexual genomic combinations have been found and all the unisexuals live with at least one of the sexual species A. laterale, A. jeffersonianum, A. texanum, and A. tigrinum. Most unisexuals rely on A. laterale orA. jeffersonianum as sperm donors. Intergenomic exchanges were previously reported in A. jeffersonianum-dependent unisexual populations from southern Ontario and are believed to be an important meiotic mechanism that provides genetic diversity. The situations of intergenomic exchanges in many of A. laterale-dependent unisexual populations, however, remain unknown. In this study we collected specimens from populations where unisexuals use A. laterale as sperm donors, including mainly triploid A. 2 laterale--jeffersonianum (or LLJ), and employed genomic in situ hybridization (GISH) to examine the intergenomic exchanges. Five patterns of intergenomic exchanges were detected. Intergenomic exchanges are less frequent and lack association among populations in A. laterale-dependent than in A. jeffersonianum- dependent unisexual populations, but more recombined homeologues were observed in LLJ unisexuals. Our observations show that the patterns and frequencies of intergenomic exchanges are different when unisexuals use different sexual species as sperm donors. We propose a few possible mechanisms that may account for these different observations.

Two rare aneutriploids in the unisexual Ambystoma (Amphibia, Caudata) identified by GISH indicating two different types of meiotic errors.

We report two types of aneutriploids in unisexual salamanders Ambystomalaterale-2jeffersonianum (LJJ) and Ambystoma 2 laterale-jeffersonianum (LLJ). One karyotype has 3n = 42: L27 (L8-); J15 (J8p+), and we suggest that it was induced by homoeologous pairing after premeiotic endomitosis followed by an unequal L8;J8 segregation. The second karyotype has 3n = 43: L14 (L10q); J29 (J12+), which can be explained by meiotic nondisjunction followed by unbalanced segregation. These two rare aneutriploids demonstrate two different types of meiotic errors that might help to explain the high mortality observed in this complex. Case one also indicates that contemporary intergenomic exchanges and homoeologous recombinations may occur after a premeiotic chromosome doubling event. Our study provides additional evidence for the extremely flexible reproduction of unisexual Ambystoma.

Intergenomic translocations in unisexual salamanders of the genus Ambystoma (Amphibia, Caudata).

Intergenomic interactions that include homoeologous recombinations and intergenomic translocations are commonly observed in plant allopolyploids. Homoeologous recombinations have recently been documented in unisexual salamanders in the genus Ambystoma and revealed exchanged chromosomal segments between A. laterale and A.jeffersonianum genomes in individual unisexuals. We discovered intergenomic translocations in two widespread unisexual triploids A.laterale--2 jeffersonianum (or LJJ) and its tetraploid derivative A.laterale--3 jeffersonianum (or LJJJ) by genomic in situ hybridization (GISH). Two different types of intergenomic translocations were observed in two unisexual populations and one contained novel chromosomes generated by an intergenomic reciprocal translocation. We also observed chromosome deletions in several individuals and these chromosome fragmentations were all derived from the A. jeffersonianum genome. These observed intergenomic reciprocal translocations are believed to be caused by non-homologous pairing during meiosis followed by breakage-rejoining events. Genomes of unisexual Ambystoma undergo complicated structural changes that include various intergenomic exchanges that offer unisexuals genetic and phenotypic complexity to escape their evolutionary demise. Unisexual Ambystoma have persisted as natural nuclear genomic hybrids for about four million years. These unisexuals provide a vertebrate model system to examine the interaction of distinct genomes and to evaluate the corresponding genetic, developmental and evolutionary implications of intergenomic exchanges. Intergenomic translocations and homoeologous recombinations appear to be frequent chromosome reconstruction events among unisexual Ambystoma.

Identification of intergenomic recombinations in unisexual salamanders of the genus Ambystoma by genomic in situ hybridization (GISH).

Unisexual salamanders in the genus Ambystoma (Amphibia, Caudata) are endemic to eastern North America and are mostly all-female polyploids. Two to four of the bisexual species, A. laterale, A. jeffersonianum, A. texanum and A. tigrinum, contribute to the nuclear genome of unisexuals and more than 20 combinations that range from diploid to pentaploid have been identified in this complex. Because the karyotypes of the four bisexual species are similar, homologous and homoeologous chromosomes in the unisexuals can not be distinguished by conventional or banded karyotypes. We chose two widespread unisexual genomic combinations (A.laterale-2 jeffersonianum [or LJJ] and A. 2 laterale-jeffersonianum [or LLJ]) and employed genomic in situ hybridization (GISH) to identify the genomes in these unisexuals. Under optimum conditions, GISH reliably distinguishes the respective chromosomes attributed to both A.laterale and A. jeffersonianum. Of four populations examined, two were found to have independently evolved homoeologous recombinants that persist in both LJJ and LLJ individuals. Our results refute the previous hypothesis of clonal integrity and independent evolution of the genome combinations in these unisexuals. Our data provide evidence for intergenomic interactions between maternal chromosomes during meiosis in unisexuals and help to explain previously observed non-homologous bivalents and/or quadrivalents among lampbrush chromosomes that were possibly initiated by partial homosequential pairing among the homo(eo)logues. To explore the utility of GISH in other members of the complex, probes developed from A. laterale were also applied to unisexuals that contained A. tigrinum and A. texanum genomes. GISH is an effective tool that can be used to identify and to quantify genomic constituents and to investigate intergenomic interactions in unisexual salamanders. GISH also has potential application to examine possible genomic evolution in other unisexuals.

Perception and history: molecular phylogeny of a diverse group of neotropical frogs, the 30-chromosome hyla (anura: hylidae).

We used 16S rRNA, 12S rRNA, and cytochrome-b sequence to investigate the history of the "30-chromosome" Hyla, a diverse assemblage of neotropical treefrogs. Three aspects of these frogs were examined: (1) phylogenetic relationships among constituent species groups, among the species of one of these groups (Hyla leucophyllata group), and among populations of Hyla leucophyllata; (2) the apparent age of cladogenetic events; and (3) the phylogeography of H. leucophyllata. Mixed success in resolving the phylogeny is not because of a lack of character variation; levels of genetic divergence are high and suggest pre-Pleistocene diversification, even among populations. Close temporal proximity of ancient cladogenetic events might make resolution of the topology difficult using any character set. At the population level, current geographic proximity is a poor predictor of phylogenetic affinity. A long history of dispersal and colonization may complicate, or even preclude, the accurate recovery of the history of this species in the Amazon Basin. It remains to be seen whether the patterns found here will prove common among neotropical frogs.

Riverine barriers and the geographic distribution of Amazonian species.

Rivers have been suggested to have played an important role in shaping present-day patterns of ecological and genetic variation among Amazonian species and communities. Recent molecular studies have provided mixed support for the hypothesis that large lowland Amazonian rivers have functioned as significant impediments to gene flow among populations of neotropical species. To date, no study has systematically evaluated the impact that riverine barriers might have on structuring whole Amazonian communities. Our analyses of the phylogeography of frogs and small mammals indicate that a putative riverine barrier (the Juruá River) does not relate to present-day patterns of community similarity and species richness. Rather, our results imply a significant impact of the Andean orogenic axis and associated thrust-and-fold lowland dynamics in shaping patterns of biotic diversity along the Juruá. Combined results of this and other studies significantly weaken the postulated role of rivers as major drivers of Amazonian diversification.

Ridges and rivers: a test of competing hypotheses of Amazonian diversification using a dart-poison frog (Epipedobates femoralis).

Mitochondrial DNA cytochrome b sequence data from a dart-poison frog, Epipedobates femoralis, were used to test two hypotheses of Amazonian diversification: the riverine barrier and the ridge hypotheses. Samples were derived from sites located on both banks of the Rio Juruá and on both sides of the Iquitos Arch in western Amazonia. The phylogeographic structure was inconsistent with predictions of the riverine barrier hypothesis. Haplotypes from opposite river banks did not form monophyletic clades in any of our phylogenetic analyses, nor was the topology within major clades consistent with the riverine hypothesis. Further, the greatest differentiation between paired sites on opposite banks was not at the river mouth where the strongest barrier to gene flow was predicted to occur. The results instead were consistent with the hypothesis that ancient ridges (arches), no longer evident on the landscape, have shaped the phylogeographic relationships of Amazonian taxa. Two robustly supported clades map onto opposite sides of the Iquitos Arch. The mean haplotypic divergence between the two clades, in excess of 12%, suggests that this cladogenic event dates to between five and 15 million years ago. These estimates span a period of major orogenesis in western South America and presumably the formation of these ancient ridges.

Hybridization between tetraploid and diploid species of treefrogs (Genus hyla).

An indirect analysis of female meiotic mechanisms in the tetraploid (4n = 48) grey treefrog, Hyla versicolor, was performed by examining artificially produced hybrids. First generation hybrids between a H. versicolor female from Canada (4n = 48) and a H. arborea male from France (2n = 24) were all triploid and appeared to contain two sets of chromosomes from H. versicolor and one set from H. arborea. Males and females were produced in equal numbers but testes in general were more completely developed than ovaries. Electrophoretic analysis of selected allozyme loci suggested that gene products from the two parents were not equally expressed. Backcross hybrids were triploid, tetraploid, and pentaploid but did not appear to contain recognizable chromosomes from the H. arborea grandparent. Allozymes from these hybrids indicated that only H. versicolor alleles were expressed, as none of the distinctive H. arborea alleles present in the triploid male parent were present in the offspring. It was concluded that preferential pairing of chromosomes and gene regulatory biases may help to explain factors that relate to the ability of tetraploids to hybridize with even distantly related taxa and may be involved in the rediploidization process that usually follows polyploidization.

Comparative contractile dynamics of calling and locomotor muscles in three hylid frogs.

Isometric twitch and tetanus parameters, force-velocity curves, maximum shortening velocity (Vmax) and percentage relaxation between stimuli (%R) across a range of stimulus frequencies were determined for a muscle used during call production (the tensor chordarum) and a locomotor muscle (the sartorius) for three species of hylid frogs, Hyla chrysoscelis, H. versicolor and H. cinerea. The call of H. chrysoscelis has a note repetition rate (NRR) approximately twice as fast as the call of H. versicolor (28.3, 42.5 and 56.8 notes s-1 for H. chrysoscelis and 14.8, 21.1 and 27.4 notes s-1 for H. versicolor at 15, 20 and 25 degrees C, respectively). Hyla cinerea calls at a very slow NRR (Approximately 3 notes s-1 at 25 degrees C). Hyla versicolor evolved from H. chrysoscelis via autopolyploidy, so the mating call of H. chrysoscelis is presumably the ancestral mating call of H. versicolor. For the tensor chordarum of H. chrysoscelis, H. versicolor and H. cinerea at 25 degrees C, mean twitch duration (19.2, 30.0 and 52.9 ms, respectively), maximum isometric tension (P0; 55.0, 94.4 and 180.5 kN m-2, respectively), tetanic half-relaxation time (17.2, 28.7 and 60.6 ms, respectively) and Vmax (4.7, 5.2 and 2.1 lengths s-1, respectively) differed significantly (P < 0.05) among all three species. The average time of tetanic contraction to half-P0 did not differ significantly between H. chrysoscelis (14.5 ms) and H. versicolor (15.8 ms) but was significantly longer for H. cinerea (52.6 ms). At 25 degrees C, Vmax differed significantly among the sartorius muscles of H. chrysoscelis, H. versicolor and H. cinerea (5.2, 7.0 and 9.8 lengths s-1, respectively) but mean twitch duration (29.5, 32.2 and 38.7 ms, respectively), P0 (252.2, 240.7 and 285.1 kN m-2, respectively) and tetanic half-relaxation time (56.3, 59.5 and 60.7 ms, respectively) did not differ significantly. The average time of contraction to half-P0 did not differ significantly between H. chrysoscelis (23.7 ms) and H. versicolor (22.9 ms) but was significantly shorter for H. cinerea (15.6 ms). The only consistent contractile differences found in this study between the calling muscle and locomotor muscle of H. chrysoscelis, H. versicolor and H. cinerea were that the calling muscles generated less tension and their force-velocity relationship was much more linear. These differences may be attributable to ultrastructural differences between calling and locomotor muscles.(ABSTRACT TRUNCATED AT 400 WORDS)

The chromosomes of the living coelacanth and their remarkable similarity to those of one of the most ancient frogs.

The karyotype of the coelacanth is reported for the first time. Latimeria chalumnae has a 48-chromosome karyotype that contains metacentric, subtelocentric, and telocentric chromosomes as well as microchromosomes. This karyotype is unlike those found in lung-fishes but is very similar to the 46-chromosome karyotype of one of the ancient frogs, Ascaphus truei.

Cow's milk protein intolerance in infants under 1 year of age: a prospective epidemiological study.

Incidence and clinical manifestation of cow's milk protein intolerance (CMPI) were studied in 1158 unselected newborn infants followed prospectively from birth to 1 year of age. No food changes were required in 914 infants who were used as healthy controls. When CMPI was suspected (211 infants), diagnostic dietary interventions according to a standard protocol were performed. After exclusion of lactose intolerance, two positive cow's milk elimination/challenge tests were considered diagnostic of CMPI. Two hundred and eleven symptomatic infants were examined for possible CMPI. A large group of 80 infants improved on a lactose reduced formula. In 87/211 infants CMPI was excluded (sick controls). Finally CMPI was proven in 26 infants. The calculated incidence rate for CMPI was 2.8%. The principal symptoms in infants with CMPI were gastrointestinal, dermatological and respiratory in 50%, 31% and 19% respectively. A positive family history for atopy (first or second degree relatives) was more frequent in either CMPI infants (65%), or sick controls (63%) when compared to either healthy controls (35%) or infants improving on a low lactose formula (51%). Differences between patients with CMPI and sick controls were only found for the presence of atopy in at least 2 first degree relatives [(5/26 in CMPI infants and 4/87 in sick controls (P < 0.05)] and for multiorgan involvement [10/26 infants with CMPI as opposed to 12/87 in the sick control group (P < 0.02)]. These statistical differences are too weak to be of clinical value.

Ancestry of unisexual salamanders.

In eastern North America there are populations of all-female salamanders that incorporate the nuclear genomes of two or three of four sympatric bisexual species. The hybrids can be diploid, triploid, tetraploid or pentaploid, and 18 different combinations have been reported. All hybrids require sperm from a sympatric male of one of the bisexual species to reproduce, but the sperm may or may not be incorporated in the egg. Some of the hybrids are believed to represent separate, clonal species, but little is known of the origin of this hybrid complex. Vertebrate mitochondrial DNA is inherited maternally, allowing identification of the female parent that gave rise to hybrid lineages. A portion of the cytochrome b gene was sequenced from diploid and triploid hybrids that represent combinations of all four species. Nearly all hybrids had a similar mitochondrial genome sequence, independent of nuclear genome composition and ploidy, and the sequence was distinct from that of any of the four bisexual species. The hybrids maintain a mitochondrial lineage that has evolved independently of their nuclear genome and represent the most ancient known unisexual vertebrate lineage.

Temperature and sperm incorporation in polyploid salamanders.

Although most animals reproduce sexually, a number of all-female groups exist. Triploid hybrid salamanders appear to maintain themselves by using a male's sperm to activate their eggs, after which the sperm nucleus is eliminated (gynogenesis). The incidence of sperm nuclear incorporation in eggs of these salamanders depends on temperature. Triploid offspring derived gynogenetically are more frequent at lower temperature, whereas tetraploid offspring derived sexually are far more frequent at higher temperatures. Temperature-dependent variability in sperm nuclear incorporation helps explain the variability in reproductive modes reported for hybrid salamanders.

Reproduction and the origin of polyploids in hybrid salamanders of the genus Ambystoma.

Eggs and larvae produced by diploid, triploid, and tetraploid females collected from breeding ponds on Pelee Island in Lake Erie were studied to examine the reproductive mechanism. No instance of parthenogenesis was found as all examined females required sperm to produce viable progeny. Diploid females produced diploid and triploid larvae, triploid females produced triploid and tetraploid larvae, and tetraploid females produced triploid and tetraploid larvae. The majority of the eggs produced by hybrid females do not develop or do not complete embryogenesis. Electrophoretic examination of females and their offspring demonstrate that the male genome is being incorporated in reduced as well as unreduced eggs produced by all three ploidy classes of females. The elevation of ploidy among Pelee Island Ambystoma is attributed to sperm incorporation in unreduced eggs. Triploid as well as tetraploid individuals are constantly being produced. A critical examination of the literature on parthenogenetic or gynogenetic modes of reproduction in North America Ambystoma hybrids shows no conclusive evidence supporting these modes and it is suggested that the reproductive mechanism found among Pelee Island female hybrids may be more generally applied to other hybrid Ambystoma populations.