James Kezer

Chromosome variation in the plethodontid salamander, Aneides ferreus

Karyotype variation in the plethodontid salamander, Aneides ferreus, has been analysed. 358 individuals from 14 populations, representing the major portion of the range of this salamander, have been karyologically examined. In A. ferreus, n=14. When the chromosomes are arranged in a decreasing relative length series, the karyotype is heteromorphic with respect to chromosome number 13, which may be either telocentric (T) or subtelocentric (ST). Variation in the heteromorphism over the range of the species is sex related, and probably also reflects relative population sizes. The heteromorphism in the isolated populations of A. ferreus on Vancouver Island, British Columbia, Canada, resembles a WZfemale/ZZmale sex chromosome dimorphism, suggesting the possibility that chromosome number 13 may be involved in sex determination in this population. The possibility that chromosome number 13 is involved in sex determination in all populations of A. ferreus is discussed. Our data suggest that the ancestral A. ferreus karyotype was homomorphic for T (T/T), and that the ST was derived from the T by a pericentric inversion. In peripheral populations, only the “W homologue” has been affected, whereas in central populations both the “W” and the “Z” chromosomes have been rearranged. Comparisons are made with other species of Aneides for which karyological information is available, and it is concluded that chromosome rearrangements have played an important role in the evolution of the genus. In C-banded chromosomes of A. ferreus, staining is most intense at the centromere regions of the larger chromosomes and is absent only in some of the smaller chromosomes. Implications of this C-banding pattern are discussed.

A fresh look at meiosis and centromeric heterochromatin in the red-backed salamander, Plethodon cinereus cinereus (Green)

Male meiosis, with special regard to the centromeric heterochromatin and to centromeric structure, has been studied in the salamander, Plethodon cinereus cinereus. In this salamander, n = 14. Early meiotic prophase proceeds as described by other authors. Pachytene is followed by a “diffuse” stage in which much of the chromosomal DNA becomes reorganized into fine lateral loops which spring from the bivalent axes. These loops can be seen along the bivalent axes as early as zygotene. Loops are maximally extended in the diffuse stage. The formation of diplotene bivalents involves a return of this extended DNA into the axes of the bivalents. — At leptotone, centromeric heterochromatin is in one or a few large masses. These masses break up during zygotene. At pachytene there is one mass of heterochromatin at the centromeric region of each bivalent. The heterochromatin remains condensed in the diffuse stage. During diplotene, centromeric heterochromatin becomes less conspicuous, and it is possible to see 4 centromere granules in each diplotene bivalent. These observations support the view that centromeres replicate at pre-meiotic S-phase when the associated hetero-chromatin is replicated. In the interphase before the 2nd division, the hetero-chromatin often forms a broken ring corresponding to the positions of the centromeres at the end of anaphase 1. There are 14 masses of heterochromatin in nuclei at prophase of the 2nd division. In spermatids, the heterochromatin appears as a single solid mass or a broken ring.

Cytogenetic evolution in the plethodontid salamander genus Aneides

Chromosomal variation in the plethodontid salamander genus Aneides was analyzed using C-banding, to visualize heterochromatin distribution, as well as silver and fluorochrome banding, to reveal nucleolus organizer regions. Evidence is presented that Aneides has undergone pronounced karyological divergence from a Plethodon-like ancestor. It is concluded that this divergence reflects accelerated rates of chromosomal rearrangements in Aneides relative to Plethodon. These rearrangements have involved mainly pericentric inversions that have occurred in one or more of the three smallest pairs of chromosomes in the karyotype. Karyological variation within and between species of Aneides was used to analyze phylogenetic relationships within the genus. This analysis revealed at least two karyologically distinct species that had not been previously recognized by morphological criteria.

Localization of 5S RNA genes on chromosomes of plethodontid salamanders

Ribosomal 5S RNA, labelled with 125 I, was annealled to denatured spermatocyte chromosomes of salamanders from 11 different genera of the family Plethodontidae. The salamanders studied have genomes with 1, 2 or 3 gene clusters. Eleven sites are located interstitially on short chromosome arms; 3 are found interstitially on long arms; 5 sites are at centromeric regions and one is telomeric. — Salamanders from five genera of Neotropical plethodontids carry a 5S gene cluster on the short arm of a large asymmetric chromosome, presumably a linkage group that has remained stable since the divergence of these genera in Tertiary time. In Lineatriton lineola this short arm is heterochromatic during pachytene and it shows a high incidence of chiasma failure at the first meiotic metaphase, contrasting with the situation found in two other species. The localization and number of 5S gene sites is consistent with the proposed phylogeny of these Neotropical genera by Wake and Lynch (1976).

Structural differentiation of the meiotic and mitotic chromosomes of the salamander, Ambystoma macrodactylum

The lampbrush chromosomes of the long-toed salamander, Ambystoma macrodactylum Baird, have been analysed and a map of the oocyte genome prepared. The location of C-bands and cold-induced-constrictions has been established in mitotic chromosomes and compared with the location of marker structures and chiasmata in several lampbrush bivalents. In the lampbrush chromosomes, C-bands are tentatively correlated with sphere-organizing loci and with regions of low chiasma frequency; cold-induced-constrictions are tentatively correlated with regions of high chiasma frequency. In general, in this salamander, C-bands do not coincide in position with cold-induced-constrictions. We have compared our results with those obtained by Callan (1966) in his investigation of chromosomes of the axolotl, Ambystoma mexicanum, and we present an analysis of the similarities and differences that are visible in the chromosome sets of these two ambystomatid species.

The meiotic structure and behavior of the strongly heteromorphic X/Y sex chromosomes of neotropical plethodontid salamanders of the genus Oedipina

Plethodontid salamanders in the genus Oedipina are characterized by a strongly heteromorphic sex-determining pair of X/Y chromosomes. The telocentric X chromosome and the subtelocentric Y chromosome are clearly distinguished from the autosomes and their behavior during meiosis can be sequentially followed in squash preparations of spermatocytes. In Oedipina the sex chromosomes are not obscured by an opaque “sex vesicle” during early meiotic stages, making it possible to observe details of sex bivalent structure and behavior not directly visible in other vertebrate groups. The sex chromosomes can first be distinguished from autosomal bivalents at the conclusion of zygotene, with X and Y synapsed only along a short segment at their non-centromeric ends, forming a bivalent that contrasts sharply with the completely synapsed autosomes. During pachytene, the XY bivalent becomes progressively shortened and more compact, disappearing as a visible structure when pachytene progresses into the diffuse stage of male meiosis. Diplotene bivalents gradually emerge from the diffuse nuclei, presumably by the return of the loops of chromatin into their respective chromomeres. During early diplotene, the X/Y bivalent is clearly visible with a single chiasma within the synapsed segment. This chiasma is terminalized by first meiotic metaphase with the X and Y appearing either in end-to-end synaptic contact or as univalents separated at opposite poles relative to the equatorially distributed autosomal bivalents. In C-banded preparations, the Y is entirely heterochromatic while the X contains a large centromeric C-band and another block of heterochromatin located at the telomeric end, in the region of synapsis with the Y. We find no cytological evidence of dosage compensation, such as differential staining of the X chromosomes or Barr bodies, in mitotic or interphase cells from female animals.

Supernumerary chromosome variation and heterochromatin distribution in the endemic New Zealand frog Leiopelma hochstetteri

Specimens of the endemic New Zealand frog Leiopelma hochstetteri from Tapu on North Island were found to have six, nine or ten supernumerary chromosomes in their karyotypes. In comparison with previously published data, these results further indicate probable geographic variation in supernumerary chromosome number between populations. Increased numbers of supernumeraries in these frogs is correlated with apparent decrease of centromeric heterochromatin in the five large metacentric chromosomes of the karyotype, as detected by C-banding. Meiosis was abnormal in a male with a high number of supernumeraries. In lampbrush preparations from a single female with one supernumerary univalent, the supernumerary often had a denser, beaded appearance in comparison with the regular bivalents. Evidence is consistent with the notion that these supernumerary chromosomes may have arisen from centromeric fragments.

Triploidy in Hochstetter's frog, Leiopelma hochstetteri, from New Zealand

Abstract Autotriploidy is described in a female of the endemic New Zealand frog Leiopelma hochstetteri. This frog was found to have 3n=33 chromosomes plus 2 supernumerary chromosomes. All the chromosomes in the karyotype of this species contained C-band heterochromatin at the centromeres. A prominent C-band was found to be associated with a secondary constriction on chromosome no. 7. The supernumerary chromosomes in this species appear to be mitotically stable and contain C-band heterochromatin at the centromeres. From the limited data presently available, the triploid individual may have resulted from the fertilisation of a diploid egg produced when the second meiotic division had been suppressed.

Loop size in newt lampbrush chromosomes

Lampbrush chromosome 11 from the newtTaricha granulosa was studied by scanning electron microscopy (SEM) to determine the size of all loops in this bivalent. Measurements with an XY digitizer revealed a mean loop length of 14.9 µm, with a large standard deviation and a skewed distribution toward higher values. The size of the loops at this stage of diplotene extension is similar to that reported in other eukaryotes studies with different approaches. We estimated, from the DNA content of chromosome 11, that between 0.4% and 2.2% of the DNA is found in the loops while the rest of the DNA must remain in the compact chromomeres.

Cytogenetics of the chinese giant salamander, Andrias davidianus (Blanchard): the evolutionary significance of cryptobranchoid karyotypes

Cytogenetic aspects of the cryptobranchid salamander Andrias davidianus of western China have been studied, including chromosome number and morphology, C-band patterns, meiosis, and the chromosomal localization of ribosomal 5S RNA genes. Our data regarding chromosome number (2n=60) and general chromosome morphology largely confirm the results of Morescalchi et al. (1977). The karyotype consists of 16 pairs of “macrochromosomes” that decrease gradually in relative length to 14 pairs of “microchromosomes”. Telocentric chromosomes are a conspicuous feature of the karyotype, representing more than half the genome. Differential staining reveals that all of the chromosomes, except four pairs of microchromosomes, have C-band heterochromatin in their centromeric regions, the amount varying irrespective of chromosome size. Faint bands of interstitial and telomeric C-band heterochromatin are found in mitotic chromosomes but are not seen in meiotic preparations. In C-banded mitotic preparations from a female, one of the smallest macrochromosome pairs is heteromorphic in respect to C-band heterochromatin and centromere position. In situ hybridization of an iodinated 5S RNA probe to meiotic chromosome preparations reveals that this repeated gene is clustered near the telomeric region of chromosome 7, a medium size telocentric, a location corresponding to a band of heterochromatin. Studies of spermatocytes indicate that the process of meiosis in A. davidianus closely resembles that of more advanced salamanders, and that the microchromosomes are meiotically stable. The significance of microchromosomes and chromosome morphology in the reorganization of salamander genomes during evolution is discussed on the basis of cytogenetic data available for A. davidianus and various other primitive and advanced salamanders.