Different Sex Allocations in Two Related Species: The Case of the Extant Hippopotamus

Social and reproductive systems remain among the main predictors affecting mammalian birth sex ratio. The two extant hippopotamus species differ in their social and reproductive systems. While common hippopotamus (Hippopotamus amphibius) form herds and tend to be polygynous, solitary living pygmy hippopotamus (Choeropsis liberiensis) are promiscuous. Although it is one of the most studied topics, only few empirical studies using large sample sizes have reported distorted birth sex ratio. We examined the birth sex ratio in both hippopotamus species using international studbooks including large data sets exceeding a thousand individuals (1138 for common hippopotamus and 1161 for pygmy ones). In both species, the birth sex ratio differed from 1:1. Whereas more males than females were recorded in common hippopotamus (53.9% males), the opposite was found in pygmy hippopotamus (41.5% males). We also found that the birth sex ratio was affected by individual dams in common hippopotamus, and by individual sires in pygmy hippopotamus. The most plausible explanation for differentially skewed birth sex ratios in both species may be related to differences in social and reproductive systems. Whereas the polygynous, sexually dimorphic common hippopotamus biased the birth sex ratio towards males, the promiscuous and sexually monomorphic pygmy hippopotamus skewed the sex ratio in favour of females. Our results are in line with recent studies showing that not only manipulation by the mother (in common hippopotamus), but also by the father (in pygmy hippopotamus), may be responsible for the birth sex ratio in different species.     

Genetic consequences of population expansions and contractions in the common hippopotamus (Hippopotamus amphibius) since the Late Pleistocene

Over the past two decades, an increasing amount of phylogeographic work has substantially improved our understanding of African biogeography, in particular the role played by Pleistocene pluvial–drought cycles on terrestrial vertebrates. However, still little is known on the evolutionary history of semi‐aquatic animals, which faced tremendous challenges imposed by unpredictable availability of water resources. In this study, we investigate the Late Pleistocene history of the common hippopotamus (Hippopotamus amphibius), using mitochondrial and nuclear DNA sequence variation and range‐wide sampling. We documented a global demographic and spatial expansion approximately 0.1–0.3 Myr ago, most likely associated with an episode of massive drainage overflow. These events presumably enabled a historical continent‐wide gene flow among hippopotamus populations, and hence, no clear continental‐scale genetic structuring remains. Nevertheless, present‐day hippopotamus populations are genetically disconnected, probably as a result of the mid‐Holocene aridification and contemporary anthropogenic pressures. This unique pattern contrasts with the biogeographic paradigms established for savannah‐adapted ungulate mammals and should be further investigated in other water‐associated taxa. Our study has important consequences for the conservation of the hippo, an emblematic but threatened species that requires specific protection to curtail its long‐term decline.     

Surgical castration of the male common hippopotamus (Hippopotamus amphibius)

In a prospective, clinical, surgery study we report here for the first time, in detail, on the surgical castration of 10 captive adult male common hippopotami (Hippopotamus amphibius). The successful procedures, a species-specific modification of standard equine castration techniques, provide valuable insight into the spatially dynamic nature of the common hippopotamus testis. The use of ultrasonography to locate the testis before and during the procedures and species-specific positioning during surgery greatly facilitated this distinctive procedure. Additionally, this surgical method provides an important additional tool for captive management of the common hippopotamus. Castration of individual males not only facilitates population control but can potentially also be employed to limit intermale aggression.     

A note on the structure of tail hairs from a pygmy hippopotamus (Choeropsis liberiensis)

The pygmy hippopotamus (Choeropsis liberiensis), like the Nile hippopotamus (Hippopotamus amphibius), defecates by backing into vertical objects while making a series of rapid, propellerlike tail movements that spread a mixture of urine and feces in a wide swath. Split hairs from the distal ventral surface of the pygmy hippopotamus tail were studied with the scanning electron microscope to determine whether the splitting was a normal character of the hair or was due to damage. The results suggest that splitting is a normal feature of the hair that may facilitate the dispersal of urine and feces.     

Hybrid cell lines Arabidopsis thaliana + Brassica campestris: No evidence for specific chromosome elimination

Summary Protoplasts from callus tissue of Arabidopsis thaliana and from leaves of Brassica campestris were fused using the PEG-high pH-high Ca⁺⁺-technique. Single dividing fusion products were isolated mechanically and cultured further in 1 l microdroplets. 31 cell lines each originating from a single fusion event have been obtained. Cytological analysis of 6 cell lines was conducted 4–7 months after isolation. All metaphases examined contained chromosomes of both species. Both Arabidopsis and Brassica specific chromosomes were still retained in cells of 7 month old cultures. Reconstituted di- and multiconstrictional chromosomes were also observed. Biochemical analysis of 12 cell lines was performed in 6–7-month old cultures. Electrophoresis on PAG and specific staining for esterase, lactate dehydrogenase, and peroxidase activities revealed isozymes of both parents to be present in all cell lines. The data obtained are interpreted as evidence for retention and functional activity in hybrid cells of specific chromosomes from each species for at least 7 months of culture.     

A SINE species from hippopotamus and its distribution among animal species

Thirty sequences of a short interspersed repetitive element (SINE) were isolated from genomic DNA of Hippopotamus amphibius (hippopotamus). RNA polymerase III split promoter sequence was observed in all of the 30 sequences; and poly(A)-like structure at 3′-end, as well as direct repeat flanking to the repetitive sequence in many of the 30 sequences. A comparison of the consensus sequence of the 30 sequences with sequences in a DNA database (DDBJ/GENBANK/EMBL) revealed 93% homology to the consensus sequence of a whale SINE, CHR-2, and 73% homology to mouse glutamic acid tRNA. Phylogenetic analysis of tRNA-related regions of the sequences with all of the mouse tRNAs revealed that glutamic acid tRNA was genetically closest to the hippopotamus SINE. In addition, the tRNA-related region of the consensus sequence was folded into a cloverleaf structure as with mouse glutamic acid tRNA. These findings led us to conclude that the SINE of hippopotamus was genetically related to a whale SINE, CHR-2 [the hippopotamus SINE was named CHR-2(hippo)] and was a retroposon derived from glutamic acid tRNA. Hipo53 and hipo95, which were the genetically most separated CHR-2(hippo) sequences in the present study, were used as a probe for dot-blot hybridization to examine the distribution of their homologous sequences among animal species. Although the distribution spectra of hipo53 and hipo95 homologous sequences in animal species differed to some extent, large amounts of both sequences were found in Hippopotamus amphibius and Globicephala macrorhynchus (whale); and small amounts in most of the animal species in Artiodactyla examined. These findings indicated that the hippopotamus and whale had more recently branched off from the clade that includes chevrotain and pecorans than the other animal species in the clade. The 30 CHR-2(hippo) sequences were aligned, and the substitution rates among the sequences were calculated with a different substitution rate model for transition and for transversion. The calculation combined with the mutation rate of the pseudogenes (r = 4.6 × 10⁹) indicated that CHR-2(hippo) sequences diversified at least 132 million years ago (Myr). Received: 1 December 1997 / Accepted: 4 March 1998     

Stephanofilaria thelazioides n. sp. (Nematoda: Filariidae) from a hippopotamus and its affinities with the species parasitic in the African black rhinoceros

Stephanofilaria thelazioides n. sp. (Filarioidea: Filariidae: Stephanofilariinae) is described from a hippopotamus Hippopotamus amphibius. This nematode is close to S. dinniki Round, 1964, a parasite of the black rhinoceros Diceros bicornis in Africa, but differs from it in the number of cuticular spines surrounding the mouth, the arrangement of the cloacal papillae and the measurements of the spicules, gubernaculum and microfilariae. Species of the genus Stephanofilaria possess spines on the head which have been derived by modification of the sensory papillae. S. thelazioides is the most primitive species of the genus and has the least modified arrangement of these papillae, with six bifid internal labial spines, four bifid external labial spines and four cephalic papillae. The genus appears to have diversified in various mammals which have in common a thick skin, such as rhinoceroses, elephants, buffaloes and now the hippopotamus. It appears to have become adapted secondarily to domestic bovines, initially in Asia and subsequently in North America.     

Clonal analysis of the intercellular variability in the functioning of human chromosomal nucleolus organizer regions

Intercellular variability of NOR activity detected with the aid of Ag-staining of human chromosomes was studied in mass and cloned fibroblast cultures obtained from 3 individuals. The intercellular variability was determined by different staining of one of 10 NORs. According to this trait the heterogeneity of the cell population was discovered in all cloned lines, with this heterogeneity being the same as in the parent cultures. That concerned the number of a variable chromosome and the percentage of the cells with Ag-stained and unstained chromosomes. It is suggested that genetic determination in the progenies of the somatic cells concerns the whole spectrum of potential variability observed in cell populations.     

Haploid and diploid cell cultures from a haplo-diploid insect

Summary

This is the first report of haploid and diploid cell culture from the haplo-diploid parasitoid wasp, Mormoniella vitripennis. Cells were cultured from haploid and diploid wasps by collecting populations of eggs from virgin females (unfertilized, haploid, parthenogenetic eggs) and mated females (mostly fertilized, diploid eggs). Eggs were surface sterilized in 70% ethanol, followed by 50% Chlorox, and rinsed in phosphate buffered saline; larvae were allowed to hatch in culture. Larval cells were dissociated and cultured at 28°C in the presence of Grace's medium supplemented with fetal bovine serum. Most cells in the HMV (predominantly haploid) and DMV (predominantly diploid) cell cultures grew in suspension in the first week, formed monolayers of fibroblasts and epithelial cells by the second week in culture, and continued to grow in monolayers and vesicle-like structures for up to three months. Chromosome analysis of HMV. cells demonstrated over 70% haploid cells, with five chromosomes (N=5). The remainder were aneuploid. No diploid cells (2N= 10) were found in the HMV cell culture. Chromosome analysis of DMV cultures revealed 62% diploid, with ten chromosomes; 13% were haploid, with five chromosomes; the remainder were aneuploid. These data confirm that haploid and diploid cells can be cultured from a haplo-diploid insect species. The HMV cells which are predominantly haploid, and DMV cells which are predominantly diploid may be valuable models for the study of cellular and gene activity in haploid and diploid genetic milieux.     

Digestion in the hippopotamus

The hippopotamus (Hippopotamus amphibius Linn.) has an enlarged stomach, divided into: (a) two anterior diverticula; (b) a large median chamber, and (c) a posterior chamber, secreting gastric juice. Fermentative digestion occurs in (a) and (b). The anatomy and function of the stomach are discussed. Two experiments were performed to compare digestive efficiency with that of ruminants. Exp. 1. In a total collection experiment, chopped grass wasfedtoahippopotamusand five ruminants. Overall digestibility was lower in the hippopotamus, but this may have been due to keeping the animal out of water. Exp. 2. A hippopotamus and two sheep were fed chopped elephant grass (Pennisetum purpureum), digestibility being measured by total collection (in the sheep) and the chromogen ratio method. Dry matter digestibility was similar in all three animals, while digestibility of crude protein was higher in the hippopotamus.     

First documentation of flehmen in a common hippopotamus (Hippopotamus amphibius)

A male common hippopotamus (Hippopotamus amphibius) was observed giving a flehmen grimace on 14 occasions in July and August of 1998 at the Denver Zoo. Twelve instances of flehmen occurred after anogenital investigation of the female as she was exiting the pool. The two other instances of flehmen occurred in the terrestrial portion of the enclosure after the male examined samples of the female's freshly voided urine. The frequency of flehmen increased as the female approached estrus. Flehmen in common hippos may be an example of an evolutionary remnant. Zoo Biol 18:415–420, 1999. © 1999 Wiley‐Liss, Inc.     

A molecular approach to the identification and individualization of human and animal cells in culture: Isozyme and allozyme genetic signatures

Summary The electrophoretic resolution of a group of geneticallymonomorphic gene-enzyme systems that are developmentally and biologically ubiquitous has been used to provide a species-specific and type-specific biochemical characterization of various cultured cells. The relative mobilities of gene-enzyme systems representing nine distinct gene products from cell cultures of 25 species fromDrosophils to man are presented. These isoenzymes effectively discriminate interspecies cell-to-cell contamination and almost invariably serve to identify the contaminating species. The resolution of eightpolymorphic gene-enzyme systems in human cell cultures provides a virtually unique allozyme genetic signature as a monitor of intraspecies cellular contamination. The genetic signatures of 47 commonly used human cells are presented. Included in the test were seven putative HeLa (human cervical carcinoma) contaminants each of which expressed a signature identical with that of HeLa. The probability that an unrelated human cell line will have a signature identical to a typed cell is computed for each line from the genotypic frequencies at each locus in a population of cultured human cells. The gene frequencies of this cell population are comparable to the same frequencies in natural human populations. The most common human signature has a frequency (and therefore a probability) of 0.02. The majority of the 17,010 possible signatures are far less probable. A calculation of the theoretical incidence of chance matching of signatures within test groups of two or more individuals is presented. The probability of a chance match between any two randomly selected individuals is 0.004 and among five randomly selected individuals is 0.034. The allozyme genetic signature represents a definitive monitor of cell identity and is presented as a standard of cell and tissue identification for a variety of biological studies. This work was supported in part by the Virus Cancer Program of the National Cancer Institute.     

CHROMOSOME NUMBERS OF SOME SACCHARUM SPECIES HYBRIDS AND THEIR CELL SUSPENSION CULTURES

Chromosome numbers of five Saccharum species hybrids and their cell suspension cultures were determined. The chromosome number was stable in four parental clones (2n = 122, 114, 114, and 112), but was variable in another (2n = 108-128). The cell suspension cultures have been maintained in a yeast extract-enriched nutrient medium for more than 6 years. These cultures were variable in chromosome number for all clones, with a partial aneuploid series at the haploid and/or polyploid level. Each clone had different chromosomal population modes after 6 years of culture. The loss of chromosomes over a period of time would have an effect on the genetic makeup of a cell population. This has implications in the use and interpretation of data from pathological, cytogenetical, biochemical, and physiological studies using cell cultures and is probably a partial explanation for the loss of totipotency in 6-year-old sugarcane tissue and suspension cultures.     

Establishment and characterization of two fetal fibroblast cell lines from the yak

The objective of this work was to not only establish two fetal fibroblast cell lines from yak lung and ear tissue using a primary explant technique and cell cryogenic preservation technology but also check for their quality and biological characteristics. The cells showed typical morphologic characteristics of fibrous and long spindle appearance. Outgrowth of fibroblast-like cells from the lung and ear explants was around 2 and 3 d, and reaching 90% confluence level was in the ninth day and the thirteenth day, respectively. Biological analysis showed that the average viability of the lung fibroblast cells (ear fibroblast cells) was 97.5% (95.0%) before freezing and 91.0% (89.5%) after thawing. Analysis of the growth of the fifth passage culture revealed an ??S??-shaped growth curve with the population doubling times of 30 h for lung fibroblast cell line and 35 h for ear fibroblast cell line. Karyotyping indicated the chromosome number of yak was 2n?=?60, comprising 29 pairs of autosomes and one pair of sex chromosomes (XY). All somatic chromosomes were telocentric autosomes except that the two sex chromosomes were submetacentric. Assays for bacteria, fungi, and mycoplasmas were negative. Immunocytochemical staining showed that the cells were positive for the expression of vimentin and negative for the expression of cytokeratin. In conclusion, two yak fetal fibroblast cell lines (YFLF and YFEF) from lung and ear explants are successfully established in culture. It will not only preserve the genetic resources of yaks at the cellular level but also provide valuable materials for somatic cell cloning and transgenic research.     

Genetic processes in intergeneric cell hybrids Atropa + Nicotiana

Summary The genetic constitution of the cell hybrids Atropa belladonna + Nicotiana chinensis, obtained by cloning of individual heteroplasmic protoplast fusion products (Gleba et al. 1982) and cultured in vitro for 12 months, has been studied. The study comprised 11 hybrid cell clones of independent origin and included analysis of a) chromosome number, size, morphology, and relative position in metaphase plates, b) multiple molecular forms of the enzymes esterase and amylase, and c) relative nuclear DNA content. The data obtained permit us to conclude that, after one year of unorganized growth in vitro, the cells of most (8) clones had retained chromosomes of both parents, while species-specific elimination of nearly all Atropa chromosomes had occurred in three clones. About half of the non-segregating clones possess 120–150 chromosomes including 50–70 of Atropa and 50–90 of Nicotiana. Other clones are polyploid and possess 200–250 chromosomes with a predominance of either Atropa or Nicotiana chromosome types. Only a few chromosomal changes (reconstituted chromosomes, ring chromosomes) have been detected. In some metaphase plates, chromosomes of the two parents tend to group separately, indicating non-random arrangement of chromosomes of the two parents within the hybrid nucleus. Cytophotometric studies of the relative nuclear DNA content showed that distribution histograms for cell clones were similar to those of non-hybrid cultured cells. Cell populations were relatively homogenous and do not indicate any genetic instability as a result of hybridization between remote plant species. Biochemical analysis of isoenzyme patterns confirmed that in most cell clones, species-specific multiple molecular forms of esterase and amylase from both parents were present, i.e. genetic material of both parental species was expressed in the cell hybrids.Dedicated to Professor G. Melchers with gratitude     

Cell culture and karyotype of Sakhalin sturgeon Acipenser mikadoi

A cell culture of rare and threatened species of Sakhalin sturgeon Acipenser mikadoi was established from a fragment of the pectoral fin and neighboring tissues. Initially, the culture consisted of different cell types including typical fibroblasts as well as cells of epithelial origin, myofibroblasts, etc. After approximately five passages, the culture largely consisted of cells with fibroblast morphology. Under normal culture conditions, these cells grew for more than one year at a constant rate and passed about 80 population doublings. In the absence of serum, cells entered the state of proliferative quiescence (G₀-state). When cultured without medium replacement for a long period of time, cells fused to form myofibers of about 1 cm in length. These myofibers could branch and acquired cross striation with time. About forty days after myofibers emerged, they degenerated, lost their shape, detached from the substrate, and finally died. The induction of adipogenic differentiation arrested cell proliferation and introduced lipophilic inclusions formed in a minor fraction of cells. The number of these inclusions was low, and cells with inclusions demonstrated various morphology distinct from typical adipocytes. The induction of osteogenic differentiation gave rise to cells that produce mineralized extracellular matrix and bone nodules. Chromosome analysis revealed a set of chromosomes typical for “high chromosome” sturgeon species. The variation in the chromosome number was very high (mean, 247 ± 33; modal value, 248). The analysis involving AT- and GC-specific fluorochromes has demonstrated that the telomeric and centromeric regions of all chromosomes are enriched in GC content. The distribution of AT- and GC-rich sequences along the chromosomes was heterogeneous. Long chromosomes were preferentially stained by the AT-specific dye, whereas small chromosomes demonstrated brighter fluorescence after 7-amino-actinomycin D staining; in particular, several small chromosomes fluoresced extremely brightly. This work is the first report of cell culture and karyotype analysis of Sakhalin sturgeon.     

Do Avian Mitochondria Recombine?

The dogma of strict maternal inheritance of mitochondria is now being tested with population genetics methods on sequence data from many species. In this study we investigated whether recombination occurs in the mitochondria of the blue tit (Parus caeruleus) by studying polymorphisms in the mitochondrial control region and in a recently identified (A)_n microsatellite on the W chromosome. The female heterogamety of avian sex chromosomes allows a test of whether mitochondrial recombination affects genealogical inference by comparison of mitochondrial and W-linked sequence variation. There is no discrepancy between mitochondrial and W-linked genealogies in blue tits, consistent with no recombination. We also analyzed mitochondrial sequence variation in both blue tits and peregrine falcons (Falco peregrinus) using a coalescent-based approach which accounts for recurrent mutation; in neither bird species did we find evidence of recombination. We conclude that it is unlikely that mitochondrial recombination has large effects on mitochondrial genetic variability in birds.     

Episodic Molecular Evolution of Pituitary Growth Hormone in Cetartiodactyla

The sequence of growth hormone (GH) is generally strongly conserved in mammals, but episodes of rapid change occurred during the evolution of primates and artiodactyls, when the rate of GH evolution apparently increased substantially. As a result the sequences of higher primate and ruminant GHs differ markedly from sequences of other mammalian GHs. In order to increase knowledge of GH evolution in Cetartiodactyla (Artiodactyla plus Cetacea) we have cloned and characterized GH genes from camel (Camelus dromedarius), hippopotamus (Hippopotamus amphibius), and giraffe (Giraffa camelopardalis), using genomic DNA and a polymerase chain reaction technique. As in other mammals, these GH genes comprise five exons and four introns. Two very similar GH gene sequences (encoding identical proteins) were found in each of hippopotamus and giraffe. The deduced sequence for the mature hippopotamus GH is identical to that of dolphin, in accord with current ideas of a close relationship between Cetacea and Hippopotamidae. The sequence of camel GH is identical to that reported previously for alpaca GH. The sequence of giraffe GH is very similar to that of other ruminants but differs from that of nonruminant cetartiodactyls at about 18 residues. The results demonstrate that the apparent burst of rapid evolution of GH occurred largely after the separation of the line leading to ruminants from other cetartiodactyls.     

Genetic structure,ecological versatility,and skull shape differentiation in Arvicola water voles (Rodentia,Cricetidae)

Water voles from the genus Arvicola display an amazing ecological versatility, with aquatic and fossorial populations. The Southern water vole (Arvicola sapidus) is largely accepted as a valid species, as well as the newly described Arvicola persicus. In contrast, the taxonomic status and evolutionary relationships within Arvicola amphibiussensu lato had caused a long-standing debate. The phylogenetic relationships among Arvicola were reconstructed using the mitochondrial cytochrome b gene. Four lineages within A. amphibiuss.l. were identified with good support: Western European, Eurasiatic, Italian, and Turkish lineages. Fossorial and aquatic forms were found together in all well-sampled lineages, evidencing that ecotypes do not correspond to distinct species. However, the Western European lineage mostly includes fossorial forms whereas the Eurasiatic lineage tends to include mostly aquatic forms. A morphometric analysis of skull shape evidenced a convergence of aquatic forms of the Eurasiatic lineage toward the typically aquatic shape of A. sapidus. The fossorial form of the Western European lineage, in contrast, displayed morphological adaptation to tooth-digging behavior, with expanded zygomatic arches and proodont incisors. Fossorial Eurasiatic forms displayed intermediate morphologies. This suggests a plastic component of skull shape variation, combined with a genetic component selected by the dominant ecology in each lineage. Integrating genetic distances and other biological data suggest that the Italian lineage may correspond to an incipient species (Arvicola italicus). The three other lineages most probably correspond to phylogeographic variations of a single species (A. amphibius), encompassing the former A. amphibius, Arvicola terrestris, Arvicola scherman, and Arvicola monticola.     

Patterns of morphological variation among populations of the widespread annual killifish Nothobranchius orthonotus are independent of genetic divergence and biogeography

Populations of annual killifish of the genus Nothobranchius occur in patchily distributed temporary pools in the East African savannah. Their fragmented distribution and low dispersal ability result in highly structured genetic clustering of their populations. In this study, we examined body shape variation in a widely distributed species, Nothobranchius orthonotus with known phylogeographic structure. We tested whether genetic divergence of major mitochondrial lineages forming two candidate species is congruent with phenotypic diversification, using linear and geometric morphometry analyses of body shape in 23 wild populations. We also conducted a common‐garden experiment with two wild‐derived populations to control for the effect of local environmental conditions on body shape. We identified different allometric trajectories for different mitochondrial lineages and candidate species in both sexes. However, in a principal components analysis of population‐level body shape, the separation among mitochondrial lineages was incomplete. Higher similarity of mitochondrial lineages belonging to different candidate species than that of same candidate species prevented distinction of the two candidate species on the basis of body shape. Analysis at the individual level demonstrated that N. orthonotus express high intrapopulation variability, with major overlap among individuals from all populations. In conclusion, we suggest that N. orthonotus be considered as a single species with an extensive geographic range, strong population genetic structure and high morphological variability.