Studies on the speciation of Japanese freshwater planarian Polycelis auriculata based on the analysis of its karyotypes and constitutive proteins

Various kinds of chromosomal polymorphisms or karyotypic variations are found in the Japanese freshwater planarian Polycelis auriculata. Within this species, there are found worms whose chromosome numbers are 2n = 6, 10, 11, 12 and others, and 3x = 6 and 9. There are some which have cells with triploidy and tetraploidy complements (3x = 6 & 4x = 8), and others which have cells with triploidy and hexaploidy complements (3x = 6 & 6x = 12). These worms with such varied karyotypes are usually found in separate habitats, though occasionally they occur together. Electrophoretic analysis of the proteins extracted from the karyotipically different worms which belong to three different local populations shows some dissimilarity in the constitutive proteins according to their karyotypic differences. The results obtained suggest that this species is still in the process of speciation or chromosomal evolution.     

Geographical distribution of Polycelis (Seidlia) auriculata in Japan

Polycelis (Seidlia) auriculata is endemic to mountain districts of Japan, from the central part of Honshû to the area of the Daisetsu Mts of Hokkaidô. In northern Japan, it sometimes occurs in cold-water biotopes of lowland areas. The progenitor of P. auriculata appears to have been the oldest immigrant into northern Japan among the Japanese Polycelis species, entering through a northern route as a preglacial faunal element. P. auriculata now shows a discontinious distribution in northern Japan. By virtue of its geographical and vertical distribution, ecological niche, variation in anatomy of the copulatory apparatus, and cytodemes, this species appears to be in the process of transformation.     

Relationship between variations of cAMP,neuromediators and the stimulation of nucleic acid synthesis during planarian (Polycelis tenuis) regeneration

Regenerating planarians of the species Polycelis tenuis have been studied with respect to the two distinct phases which occur during regeneration, viz., the first 24 h involving cellular activation, and the following days when differentiation of the blastema occurs after a period of cellular proliferation. We have studied particularly the biochemical events that control regeneration with respect to the nature of the signals which induce cellular activation, the membrane receptors of these signals, and the consequences of these signals for the cellular metabolism of DNA, RNA and proteins. The roles of neurohormones such as serotonin, dopamine, and noradrenaline are assessed, and a provisional model of the process of cellular activation is proposed which takes account of all the information that is now available concerning planarian regeneration.     

An electron microscope study of primary epidermis formation in freshwater planarian embryos

Early development in freshwater planarians is generally considered to be highly modified to the point of being unique. A careful examination by TEM, however, suggests that the primary epidermis (Skaer, 1965) is formed in a rather regular manner but is partially inverted with respect to the definitive body axes. After formation of the yolk cell syncytium, the blastomeres enclosed within it increase in number in the central area. Some of these blastomeres then move peripherally as a group and fuse to form another syncytium, the primordium of the primary epidermis. This primordium contacts the surface of the yolk-cell syncytium at the place where the primordium will subsequently flow out. The primordium spreads to the opposite pole through the spaces among the syncytial and non-syncytial yolk cell masses.     

Long-term frequency shifts in the chromosomal polymorphisms of Drosophila robusta in the Great Smoky Mountains

We resurveyed an elevational transect in the Great Smoky Mountains National Park first sampled in 1947 for chromosomal polymorphisms in populations of Drosophila robusta . Combining these results with those from previous surveys, unpublished data, and long-term meteorological data from this region up to 2003, we found that these chromosomal polymorphisms had continued to shift in frequency consistent with long-term temperature changes, yet had maintained elevational clines. Intensity of linkage disequilibrium for X-chromosome gene arrangements had shifted up and down the transect over the 56-year sampling period, suggesting shifting patterns of adaptation. Chromosomal frequency changes through the 1980s clearly demonstrated concerted directional evolution in response to cooler temperatures, but over the 20 years until 2003, frequency changes in most high-elevation populations reversed for many of the most temperature-sensitive gene arrangements.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 88 , 131–141.     

Comparative morphological studies on the visual systems in a binocular and a multi-ocular species of freshwater planarian

The visual systems of Bdellocephala brunnea Ijima & Kaburaki, a species with two eyes, and Polycelis sapporo (Ijima & Kaburaki), a species with multiple eyes, were investigated using light and electron microscopy. The eye of the binocular species consisted of 40–50 photoreceptor cells and 6–12 pigmented eyecup cells. The eye of the multi-ocular species was smaller and consisted in most specimens of one photoreceptor cell and one pigmented eyecup cell. The ultrastructure of the photoreceptor cells and of the pigmented cells was similar in the two species. Despite differences in numbers of constitutive cells, the arrangement of functional elements in the ocelli of these planarians is the same.     

Studies on the speciation of the European freshwater planarians Polycelis nigra and Polycelis tenuis based on the analysis of enzyme variation by means of iso-electric focusing

Polycelis nigra Ehrenberg and Polycelis tenuis Ijima differ morphologically and karyologically. No difference, however, was found in the isozyme pattern of malate dehydrogenase and tetrazolium oxidase, indicating a close relationship. Most sibling species differ at half of the loci.It could be deduced that the reproductive behaviour of a single population of Polycelis nigra in a Dutch pond was not panmictic. Two genetically different strains retained their identities during two years of observation. If pseudogamy occurs in this diploid planarian, the presence of heterozygous specimens indicates the absence of a true meiosis.The iso-electrofocusing technique by which these population-genetical studies were carried out, also lends itself to a comparison of overall protein banding patterns. The membrane proteins especially are conservative. The sodium dodecyl sulphate extracted proteins of Polycelis nigra-tenuis, Planaria torva and Phagocata vitta were very similar, while their water soluble proteins were not. This technique may be of great help in taxonomic studies of the higher taxa.     

Karyology of a marine population of Gyratrix hermaphroditus (Turbellaria,Rhabdocoela) and chromosomal evolution in this species complex

Karyology and reproductive biology of a marine population of the species complex Gyratrix hermaphroditus, from Roscoff (Brittany, France), have been investigated. A diploid complement of six chromosomes was determined from spermatogonial mitotic figures. One chromosome pair is metacentric, the second is intermediate between meta- and submetacentric, and the third is subtelocentric.In this population, regular meiosis occurs in both female and male germ lines, and the animals reproduce only by means of amphimictic eggs. Certain specimens of the population showed the elimination of one of the three bivalents during the first meiotic division in spermatogenesis. It seems that such animals produce normal and aneuploid sperm simultaneously; the aneuploid sperm are not capable of fertilization.The Roscoff population differs in its karyotype (2n = 6) from freshwater populations, which are either diploid (2n = 4) or polyploid (3n = 6, 4n = 8). These results suggest that aneuploidy played a role in the differentiation of freshwater populations from an originally marine species complex.     

The geographical distribution of Polycelis (Polycelis) sapporo and Seidlia schmidti in the Far East

Polycelis (Polycelis) sapporo (Ijima et Kaburaki, 1916) is a non-stenothermal species, common in Hokkaidô (including all of the adjacent islands), Northern Japan, except in the alpine region. It is also distributed in Aomori Prefecture in the northernmost part of Honshû. To the south, its range is bounded by a line from the base of the Tsugaru Peninsula, over Mt. Iwaki, Mt. Shirakami, Mt. Tashiro, Lake Towada, Mt. Hakkôda, the Natsudomari Peninsula, to the west of Mutsu and Ôhata in the Shimokita Peninsula. In Russia, the species is recorded only from the southern part of Sakhalin. Seidlia schmidti (Zabusov, 1916) is a stenothermal species found in cold-water biotops. It occurs in the North and East of Hokkaidô including the Shiretoko Peninsula and Rishiri Island. In Russia, the species is recorded from the southern part of Sakhalin, Primorskiy, Habarovsk including the base of the Chukotskiy Peninsula, and the Kamchatka Peninsula (and Bering Island).     

GABA in the nervous system of the planarian Polycelis nigra

Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in vertebrates and it has a similar inhibitory role in several invertebrate taxa. The transmitters serotonin, octopamine, catecholamines and histamine are present in flatworms while evidence for GABA is still lacking. Therefore, we have studied the occurrence of GABA-like immunoreactivity (IR) in the planarian nervous system. Specimens of Polycelis nigra were fixed in 4% 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide with 2% paraformaldehyde. The GABA-antiserum was raised in rabbits against GABA conjugated to keyhole limpet hemocyanin. Preabsorption with GABA-ovalbumin conjugate abolished all IR. The results were further confirmed with an monoclonal antibody and high pressure liquid chromatography (HPLC). In P. nigra GABA-like IR was seen as long, often varicose, sparsely distributed fibers in the ventral longitudinal nerve cords. IR was also located in a few cell somata in the brain and in the neuropil of the brain. The IR was restricted to the central nervous system and was absent in peripheral nerves and plexuses. The HPLC analysis supported the presence of GABA.Our results suggest that GABA is an interneuronal transmitter in P. nigra. The results also suggest a phylogenetically old origin of GABAergic neurotransmission.     

Chromosomal variability in natural populations of Chironomus cingulatus meigen (Diptera, Chironomidae)

Chromosomal polymorphism has been studied in seven natural populations of Chironomus cingulatus from Western Europe, Western Siberia, and the Republic of Sakha (Yakutia). The banding sequences pool of the species includes 15 banding sequences. Chromosomal polymorphism was revealed in five out of seven chromosomal arms. Arm B is the most polymorphic with four banding sequences. There are three banding sequences in arm A. Arms D, E, and G have two banding sequences. None of the chromosome rearrangements were revealed in arms C and F. The populations of C. cingulatus differ clearly in their number and frequency of banding sequences, which indicates that different gene sequences are adaptive in different populations.     

Proposed chromosomal phylogeny for the South American primates of the Callitrichidae family (Platyrrhini)

Cytogenetic and cytotaxonomic studies (G, C, sequential G/C, and NOR banding) were performed on 110 specimens representing the four genera of South American primates of the family Callitrichidae: Cebuella (C. pygmaea), Callithrix, groups argentata (C. argentata, C. emiliae, C. chrysoleuca, C. humeralifera, C. mauesi), and jacchus (C. aurita, C. geoffroyi, C. jacchus, C. kuhli, C. penicillata), Leontopithecus (L. chrysomelas, L. rosalia), and Saguinus (S. midas midas, S. m. niger). Mitotic chromosomes are characterized, and the rearrangements distinguishing the karyotypes of the taxa are inferred from arm homologies. The results were then converted into numerical data and submitted to cladistic analysis. The following conclusions were achieved: 1) Five karyotypic classes were observed, which correspond to the five taxa studied. Differences between them are as follows: a) Cebuella (2n = 44, 10 acrocentrics, A + 32 bi‐armed autosomes, bi) and the argentata group (2n = 44, 10A + 32bi) are different from each other due to a reciprocal translocation; b) both can be distinguished from the jacchus group (2n = 46, 14A + 30bi) by a centric fusion/fission rearrangement and a paracentric inversion; c) Leontopithecus (2n = 46, 14A + 30bi) and Saguinus (2n = 46, 14A + 30bi) differ from the jacchus group by a reciprocal translocation and three paracentric inversions; and d) Saguinus is different from the others by one paracentric inversion and pericentric inversions in at least four pairs of acrocentric autosomes. 2) The cladistic analysis separates Cebus (used as an outgroup) from the Callitrichidae groups, which forms a clade. Among the Callitrichidae, marmosets (Cebuella and Callithrix) form a sub‐clade, Cebuella and the argentata group being more closely related to each other than both are to the jacchus group. Tamarins (Leontopithecus and Saguinus) are also quite close, so that if one was not derived from the other, they with the marmosets share a common ancestor. Among the tamarins, Leontopithecus is karyotypically closest to the marmosets, specifically to the jacchus group. 3) Based on the chromosome information and considering the possible direction of the evolutionary changes (primitivity or phyletic dwarfism hypothesis, previously advanced by other authors), it was possible to propose the ancestral karyotypes and to develop two alternatives for the origin, differentiation and dispersion of the callitrichid. Both proposals are plausible, but when the geographical distribution is considered, the phyletic dwarfism hypothesis seems to be the most probable. Am. J. Primatol. 49:133–152, 1999. © 1999 Wiley‐Liss, Inc.     

An electron-microscopic study of syncytium formation during early embryonic development of the freshwater planarian Bdellocephala brunnea

To substantiate the assumption that the egg cell and blastomeres in planarian embryos influence surrounding yolk cells to form a syncytium, embryos at 1- to 8-cell stages were examined by electron microscopy. Within special areas of the endoplasmic reticulum both in the egg cell and in the blastomeres, a large number of vacuoles of various sizes formed and then disappeared at least four times over the period from egg-laying through the 8-cell stage as if their contents were being secreted. These activities diminished markedly at the 8-cell stage. Yolk cells surrounding the egg cell and blastomeres were aggregated in close contact with one another in a small clump shortly after egg-laying, and then, late in the 4-cell stage, became fused, forming a syncytium. The correlation between release of vacuoles by the egg cell and blastomeres and the formation of a syncytium by the yolk cells indicate that the cell fusion could be induced by a factor contained in the vacuoles.     

The freshwater planarian Dugesia (G.) tigrina contains a great diversity of homeobox genes

To identify potential pattern control and cell determination and/or differentiation genes in the freshwater planarian Dugesial (G.) tigrina, we searched for homeobox genes of different types in the genome of this primitive metazoan. We applied two basic approaches: 1) Screening the cDNA library with degenerate oligonucleotides corresponding to the most conserved amino acid sequence from helix-3 of the homeodomain of each family; and 2) PCR amplification of genomic DNA or cDNA, using two sets of degenerated oligonucleotides corresponding to helices 1 and 3 of the homeodomain or two specific domains of the POU family. Using the first strategy we have identified and characterized two tissue-specific cell determination and/or differentiation NK-type homeobox genes. Using the second strategy we have identified several homeobox genes that belong to the HOM/Hox, paired (prd) or POU families.     

Analysis of ploidy in a planarian by flow cytometry

Flow cytometry (flow microfluorimetry) provides a quick means for analysis of ploidy in planarians. Nuclei from homogenized tissues of the freshwater planarian Dugesia japonica japonica Ichikawa et Kawakatsu were stained with propidium iodide and measured with an argon-laser flow cytometer to produce histograms of DNA content. Tissues from sexually mature individuals produced histograms with a 1n (haploid) peak but no 3n peak (triploid peak), whereas those from asexual individuals showed a 2n peak or a 3n peak or both, but no 1n peak. Thus, the 1n peak distinguished sexual individuals. Mixoploid individuals, i.e., mosaics with both diploid and triploid tissues, were identified by the presence of both a 2n peak and a 3n peak. The ratios of the heights of the 2n and 3n peaks from tissues in different parts of a single mixoploid individual were similar, suggesting that the diploid and triploid cells are homogeneously distributed.     

The distribution of oxidizable mucosubstances and polysaccharides in the planarian Polycelis tenuis iijima

A chromic acid oxidation-silver technique was used to localize polysaccharide material in Polycelis tenuis at the electron microscope level. In the epithelium, staining was observed within apical vacuoles and on the free surfaces of the cells. A similar staining was observed in relation to the glycocalyx of the pharyngeal epithelia and that of the flame cells. Silver was deposited in the basement membrane. In the parenchyma, the major components giving a positive reaction were the cyanophil and mucous gland cells. Particularly strong silver staining (confirmed by X-ray microanalysis) was observed in the granules and Golgi apparatus of the cyanophil cells. IDPase activity was also found in relation to the Golgi apparatus and its secretory products. The overall distribution of mucopolysaccharide material was confirmed with the PAS and Alcian blue techniques. The fine structural localization of the Alcian blue was also determined using electron microscopy and X-ray microanalysis.     

Sex chromosome variability outlines the pathway to the chromosomal evolution in Microtus thomasi (Rodentia, Arvicolinae)

The underground vole Microtus thomasi , a Balkan endemic, displays remarkable variability in sex chromosome size and morphology. In the present study, we demonstrate this variability in two of its chromosomal races with 2 n  = 44 (i.e. 'thomasi' and 'atticus') with the use of C-banding on a sample of 189 individuals from 50 localities of Greece. In 'thomasi', five different, acrocentric X chromosome variants (X₀–X₄) are described, which differ significantly in size, due to heterochromatin addition. Also, three Y chromosome variants are described (Y₀–Y₂), ranging in size from very small (Y₀) to large (Y₂). The 'atticus' race displays three subtelocentric variants of the X chromosome (X_st0–X_st2), which differ in arm length ratio and heterochromatin content. In Peloponnesus, males of this race exhibit Y₀ and Y₁, whereas, in Attiki (south-east Sterea Ellada), males carry the small metacentric, Y_m. Overall, there is a trend towards sex chromosome size increase in a south to north direction. We propose that the last glaciation must have restricted M. thomasi to a refugium in southern Peloponnesus. During post-glacial colonization, limited northward expansion of its distribution area must have been accompanied by consecutive heterochromatin addition, which is proven today by comparatively larger sex chromosomes in the northern populations of 'thomasi' and 'atticus' in Greece than in their southern populations.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 685–695.     

Ultrastructural features of the epidermis of the planarian Artioposthia triangulata (Dendy)

The epidermis of the land planarian Arthioposthia triangulatawas examined by scanning and transmission electron microscopy. Thisinvestigation revealed that the flatworm was covered entirely withcilia and was especially densely populated on the ventral surface.In all regions the epidermis consisted of a one-layered columnarepithelium resting on a prominent basement membrane, but lacking aterminal web. Various secretions were found in the epidermistogether with epidermal rhabdoids. Below the basement membraneother secretory material was visible and this included thecytoplasmic lamellated granules and adenal rhabdites. The basementmembrane consisted of fibrils with a beaded appearance and thesewere arranged parallel to the epidermal layer but did not displaycross-banding. The secretory cells above and below the basementmembrane were compared and their products characterized on thebasis of shape, size and location. Their possible function isdiscussed.     

Conventional and ultrastructural analyses of the chromosomes of Discocyrtus pectinifemur (Opiliones, Laniatores, Gonyleptidae)

Among the Opiliones, species of the suborders Cyphophthalmi, Eupnoi, Dyspnoi and Laniatores have shown very diverse diploid chromosome numbers. However, only certain Eupnoi species exhibit XY/XX and ZZ/ZW sex chromosome systems. Considering the scarcity of karyotypical information and the absence of structurally identifiable sex chromosomes in the suborder Laniatores, we decided to analyse the chromosomes and bivalents of Discocyrtus pectinifemur (Gonyleptidae) to identify possible sex differences. Testicular cells examined under light microscopy showed a high diploid number, 2 n  = 88, meta/submetacentric chromosome morphology and a nucleolar organizer region on pair 35. Prophase I microspreading observed in transmission electron microscopy exhibited 44 synaptonemal complexes with similar electron density and thickness. The total and regular synapsis between the chromosomes of the bivalents was also noted in pachytene nuclei. Male mitotic and meiotic chromosomes revealed no distinct characteristic that could be related to the occurrence of heteromorphic sex chromosomes. Evolutionary trends of chromosome differentiation in the four suborders of Opiliones are discussed here.