Modern morphological methods for tadpole studies. A comparison of micro-CT, and clearing and staining protocols modified for frog larvae.

Micro-computed tomography (micro-CT) scanning and three-dimensional reconstruction have revolutionized morphological studies. Whereas species descriptions and comparative studies formerly were based on external appearance and dissection, we now can visualize muscles, skeleton and viscera in intact animals. In most cases, visualization of internal structures depends on appropriate staining methods. Staining with iodine, phosphotungstic acid (PTA) and osmium tetroxide are established methods, but some problems remain. Agents like osmium tetroxide are toxic and the contrast of cartilage generally is unsatisfactory with osmium tetroxide, iodine or PTA. Furthermore, staining results vary for different animals and different developmental stages. We investigated critical point drying as an inexpensive, nontoxic and rapid alternative to staining for frog tadpoles. Critical point drying enables visualization of cartilage and its differentiation from muscle tissue. Shrinkage generally is acceptable. We also present a protocol for clearing and staining frog tadpoles.

Fossil evidence of the zygomycetous fungi.

Molecular clock data indicate that the first zygomycetous fungi occurred on Earth during the Precambrian, however, fossil evidence of these organisms has been slow to accumulate. In this paper, the fossil record of the zygomycetous fungi is compiled, with a focus on structurally preserved Carboniferous and Triassic fossils interpreted as zygosporangium-gametangia complexes and resembling those of modern Endogonales. Enigmatic microfossils from the Precambrian to Cenozoic that have variously been interpreted as, or compared to, zygomycetous fungi are also discussed. Among these, the spherical structures collectively termed 'sporocarps' are especially interesting because of their complex investments and abundance in certain Carboniferous and Triassic rocks. Circumstantial evidence suggests that at least some 'sporocarp' types represent mantled zygosporangia. Zygomycetous fungi probably were an important element in terrestrial paleoecosystems at least by the Carboniferous.

Perithecial ascomycetes from the 400 million year old Rhynie chert: an example of ancestral polymorphism.

We describe a perithecial, pleomorphic ascomycetous fungus from the Early Devonian (400 mya) Rhynie chert; the fungus occurs in the cortex just beneath the epidermis of aerial stems and rhizomes of the vascular plant Asteroxylon. Perithecia are nearly spherical with a short, ostiolate neck that extends into a substomatal chamber of the host plant; periphyses line the inner surface of the ostiole. The ascocarp wall is multilayered and formed of septate hyphae; extending from the inner surface are elongate asci interspersed with delicate paraphyses. Asci appear to be unitunicate and contain up to 16 smooth, uniseriate-biseriate ascospores. The method of ascospore liberation is unknown; however, the tip of the ascus is characterized by a narrow, slightly elevated circular collar. Ascospores appear 1-5 celled, and germination is from one end of the spore. Also present along the stems and interspersed among the perithecia are acervuli of conidiophores that are interpreted as the anamorph of the fungus. Conidiogenesis is thallic, basipetal and probably of the holoarthric-type; arthrospores are cube-shaped. Some perithecia contain mycoparasites in the form of hyphae and thick-walled spores of various sizes. The structure and morphology of the fossil fungus is compared with modern ascomycetes that produce perithecial ascocarps, and characters that define the fungus are considered in the context of ascomycete phylogeny.

An unusual, structurally preserved ovule from the Permian of Antarctica.

Anatomically preserved ovules are described from silicified peat of Late Permian age collected from Skaar Ridge in the central Transantarctic Mountains, Antarctica. The small ovules are significant in possessing fleshy apical appendages and a funnel-shaped micropylar extension formed by the sarcotestal layer of the integument, by which they differ from all other Permian ovules described to date. The apical modifications may have functioned in pollination and/or seed dispersal. Similarity with the apical organization of earlier Paleozoic ovules is shown to be superficial, since the analogous structures are developmentally derived from different tissues. Although the ovules occur in rocks in which glossopterids are the only gymnosperms represented, there is insufficient evidence to assign them to a taxonomic group. These ovules are of particular importance because there are so few anatomically preserved gymnosperm reproductive structures known from the Permian and thus provide new data on the diversity of late Paleozoic gymnosperms.

Cuticles of Mariopteris occidentalis White nov. emend. from the Middle Pennsylvanian of Oklahoma (USA), and a new type of climber hook for mariopteroid pteridosperms.

Cuticles of Mariopteris occidentalis are described from the Desmoinesian (Middle Pennsylvanian) of Oklahoma (USA). This species, like other mariopteroids, had a vine- to liana-like growth habit and climbed with specialized climber hooks. However, M. occidentalis is different from other mariopteroids in having small recurved hooks on the abaxial surfaces of the pinna axes. The diagnosis for M. occidentalis White 1899 is emended based on additional macroscopical observations and data on the epidermal anatomy; a lectotype is designated. M. occidentalis is compared with Pseudomariopteris cordato-ovata from the Stephanian and Autunian of Europe and North America, a taxon which is considered very similar and may be related. Although the two taxa indeed display similarities, significant evidence for a closer relationship could not be found. Finally, some features, e.g. marginal water pits and the stomatal structure, are considered with regard to their palaeoautecological significance where they are interpreted as adaptations to special physiological requirements of a vine- to liana-like life form.

Reconstruction of Pseudomariopteris busquetii, a vine-like Late Carboniferous-Early Permian pteridosperm.

The growth habit of the Late Carboniferous-Early Permian pteridosperm Pseudomariopteris busquetii is reconstructed based on compression material from the upper Stephanian of the Blanzy-Montceau and Commentry Basins (Massif Central, France), and the upper Rotliegend of the Saar-Nahe Basin (Nahe Group, N 4, Rheinland Pfalz, Germany). Pseudomariopteris busquetii was a medium-sized, vine- to liana-like plant with slender stems to which small bipartite fronds were attached. What is most interesting is that the species used at least two different strategies to both anchor and support the plant body. Most specimens possess specialized climber hooks developed from apical extensions of the pinna axes, indicating that the fronds were used to attach the plant. A few specimens suggest that the stem may also have had some capacity for attachment. In the absence of suitable supports, however, P. busquetii was apparently able to grow in dense stands or thickets in which the individual plants supported each other.

A filmy fern from the Upper Triassic of North Carolina (USA).

Modern cladistic analyses support earlier suggestions that the Hymenophyllaceae (the "filmy ferns") are basal filicaleans. However, the fossil record of the family is ambiguous. A new fossil fern, Hopetedia praetermissa gen. et sp. nov., from the Upper Triassic Pekin Formation of North Carolina (USA), is described and interpreted as the oldest unequivocal representative of the Hymenophyllaceae based primarily on general frond morphology, indirect evidence for a filmy (membranaceous) habit, and soral position and morphology. Particularly compelling as evidence for the hymenophyllaceous affinity of H. praetermissa is the funneliform structure of the indusium (involucre), which is similar to that found primarilly in the extant Trichomanes (sensu lato) clade. However, the receptacle in H. praetermissa is relatively short and, thus, more like most representatives of the Hymenophyllum (sensu lato) clade. The Triassic age of this fossil is consistent with the basal or near basal position of the Hymenophyllaceae in all recent phylogenetic analyses of the filicalean ferns. Hopetedia preatermissa is evaluated in relationship to several previous reports of fossil Hymenophyllaceae.

A contribution to the knowledge of the pteridosperm genera Pseudomariopteris Danzé-Corsin nov. emend. and Helenopteris nov. gen.

The macromorphology and epidermal anatomy of three Late Carboniferous-Early Permian pteridosperm species, conventionally assigned to the form-genus Pseudomariopteris Danzé-Corsin, are described from the Stephanian of the Blanzy-Montceau Basin (Central France). The generic diagnosis of Pseudomariopteris is emended, and P. busquetii is designated as the type species of the genus. The combination P. cordato-ovata (Weiss) Gillespie et al. is validated to replace the illegitimate name P. ribeyronii (Zeiller) Danzé-Corsin. The species diagnoses of P. busquetii and P. cordato-ovata are emended. On the basis of its epidermal anatomy, P. paleaui, another species orininally described from Central France, is transferred to the newly established genus Helenopteris Krings et Kerp, and the new combination Helenopteris paleaui is introduced. Lectotypes of P. busquetii and H. paleaui are selected. In addition, specimens from other basins are (re-)examined and illustrated. Seeds attached to a P. busquetii specimen suggest a callistophytalean relationship for Pseudomariopteris. Compression fossils and cuticles of Pseudomariopteris and Helenopteris are interpreted in terms of palaeoecology: Pseudomariopteris busquetii and P. cordato-ovata had climbing/scrambling growth habits; specialized climber hooks can be demonstrated for both species, either in compression or in cuticular preservation. Although the growth habit of H. paleaui is not yet entirely clear, the size of its fronds suggests that this taxon most probably was also a scrambler/climber. Finally, based on epidermal features, a tentative reconstruction of stages of the pinnule ontogeny of H. paleaui is provided.

Mitochondrial DNA sequences in prehistoric human remains from the Alps.

The spread of agriculture that started in the Near East about 10 000 years ago caused a dramatic change in the European archaeological record. It is still unclear if that change was caused mostly by movement of people or by cultural transformations. In particular, there is disagreement on what proportion of the current European gene pool is derived either from the pre-agricultural, paleolithic and mesolithic people, or from neolithic farmers immigrating from the south-east. To begin to characterise the mtDNA gene pool of prehistoric Europe we examined five human remains from the Eastern Italian Alps, dated between 14 000 and 3000 years ago. Three of them yielded sufficient amount of mtDNA for analysis. DNA extracts were prepared in two independent laboratories, and PCR products from the first hypervariable segment of the mtDNA control region were cloned and sequenced. Together with the 5200 year old 'ice man', these DNA sequences show that European mtDNA diversity was already high at the beginning of the neolithic period. All the neolithic sequences have been observed in contemporary Europeans, suggesting genealogical continuity between the neolithic and present-day European mtDNA gene pool. The mtDNA sequence from a 14 000 year-old specimen was not observed in any contemporary Europeans, raising the possibility of a lack of continuity between the mesolithic and present-day European gene pools.

The use of biological stains in the analysis of late Palaeozoic pteridosperm cuticles.

The use of biological stains in the cuticular analysis of late Palaeozoic pteridosperms based on specimens from the Stephanian Blanzy-Montceau Basin (Central France) is discussed. Bismarck Brown, Malachite Green G, Methylene Blue, Methyl Green, Neutral Red, Safranin T, and a double staining with Neutral Red and Malachite Green G, were tested. Bismarck Brown, Malachite Green G, Methylene Blue, and Neutral Red increase contrast and emphasize differences in cutinization. The double staining in Neutral Red and Malachite Green G enhances the three-dimensional morphology of complex epidermal structures. Safranin T increases contrast, emphasizes cutinization differences, and enhances the three-dimensional morphology of complex epidermal features. The colour photography of cuticles is normally not affected by the presence of stains, but some stains mask black-and-white half-tones.

DNA sequence of the mitochondrial hypervariable region II from the neandertal type specimen.

The DNA sequence of the second hypervariable region of the mitochondrial control region of the Neandertal type specimen, found in 1856 in central Europe, has been determined from 92 clones derived from eight overlapping amplifications performed from four independent extracts. When the reconstructed sequence is analyzed together with the previously determined DNA sequence from the first hypervariable region, the Neandertal mtDNA is found to fall outside a phylogenetic tree relating the mtDNAs of contemporary humans. The date of divergence between the mtDNAs of the Neandertal and contemporary humans is estimated to 465,000 years before the present, with confidence limits of 317,000 and 741,000 years. Taken together, the results support the concept that the Neandertal mtDNA evolved separately from that of modern humans for a substantial amount of time and lends no support to the idea that they contributed mtDNA to contemporary modern humans.

mtDNA analysis of Nile River Valley populations: A genetic corridor or a barrier to migration?

To assess the extent to which the Nile River Valley has been a corridor for human migrations between Egypt and sub-Saharan Africa, we analyzed mtDNA variation in 224 individuals from various locations along the river. Sequences of the first hypervariable segment (HV1) of the mtDNA control region and a polymorphic HpaI site at position 3592 allowed us to designate each mtDNA as being of "northern" or "southern" affiliation. Proportions of northern and southern mtDNA differed significantly between Egypt, Nubia, and the southern Sudan. At slowly evolving sites within HV1, northern-mtDNA diversity was highest in Egypt and lowest in the southern Sudan, and southern-mtDNA diversity was highest in the southern Sudan and lowest in Egypt, indicating that migrations had occurred bidirectionally along the Nile River Valley. Egypt and Nubia have low and similar amounts of divergence for both mtDNA types, which is consistent with historical evidence for long-term interactions between Egypt and Nubia. Spatial autocorrelation analysis demonstrates a smooth gradient of decreasing genetic similarity of mtDNA types as geographic distance between sampling localities increases, strongly suggesting gene flow along the Nile, with no evident barriers. We conclude that these migrations probably occurred within the past few hundred to few thousand years and that the migration from north to south was either earlier or lesser in the extent of gene flow than the migration from south to north.

Epidermal anatomy of Barthelopteris germarii from the Upper Carboniferous and Lower Permian of France and Germany.

The epidermal anatomy of Barthelopteris germarii, a late Paleozoic seed fern, is described on the basis of material from the Upper Stephanian of Central France. A number of features are described for the first time for this species. Some have never been reported for late Paleozoic pteridosperms. Although our material is in most respects very similar to previously described material of B. germarii from Germany, there are also some striking differences, especially the peltate glandular trichomes that are very common in the French but completely absent in the German material. Their presence is most probably an ecological adaptation as in many modern plants. This and other epidermal and gross-morphological features (e.g., the presence of papillae on subsidiary cells of the stomata, anastomosing venation) indicate that B. germarii was well adapted to stressed conditions. Of more general interest is the preservation of the material. There appears to exist a clear relationship between the preservation of anticlinal walls and the thickness of the parenchymatic mesophyll; anticlinal walls are very well preserved where parenchyma was thin. Therefore, the absence of anticlinal walls in fossil cuticles, which has often been used as a taxonomic character, is not necessarily a primary feature.

Neandertal DNA sequences and the origin of modern humans.

DNA was extracted from the Neandertal-type specimen found in 1856 in western Germany. By sequencing clones from short overlapping PCR products, a hitherto unknown mitochondrial (mt) DNA sequence was determined. Multiple controls indicate that this sequence is endogenous to the fossil. Sequence comparisons with human mtDNA sequences, as well as phylogenetic analyses, show that the Neandertal sequence falls outside the variation of modern humans. Furthermore, the age of the common ancestor of the Neandertal and modern human mtDNAs is estimated to be four times greater than that of the common ancestor of human mtDNAs. This suggests that Neandertals went extinct without contributing mtDNA to modern humans.

The retrieval of ancient human DNA sequences.

DNA was extracted from approximately 600-year-old human remains found at an archaeological site in the southwestern United States, and mtDNA fragments were amplified by PCR. When these fragments were sequenced directly, multiple sequences seemed to be present. From three representative individuals, DNA fragments of different lengths were quantified and short overlapping amplification products cloned. When amplifications started from <40 molecules, clones contained several different sequences. In contrast, when they were initiated by a few thousand molecules, unambiguous and reproducible results were achieved. These results show that more experimental work than is often applied is necessary to ensure that DNA sequences amplified from ancient human remains are authentic. In particular, quantitation of the numbers of amplifiable molecules is a useful tool to determine the role of contaminating contemporary molecules and PCR errors in amplifications from ancient DNA.

Global patterns of linkage disequilibrium at the CD4 locus and modern human origins.

Haplotypes consisting of alleles at a short tandem repeat polymorphism (STRP) and an Alu deletion polymorphism at the CD4 locus on chromosome 12 were analyzed in more than 1600 individuals sampled from 42 geographically dispersed populations (13 African, 2 Middle Eastern, 7 European, 9 Asian, 3 Pacific, and 8 Amerindian). Sub-Saharan African populations had more haplotypes and exhibited more variability in frequencies of haplotypes than the Northeast African or non-African populations. The Alu deletion was nearly always associated with a single STRP allele in non-African and Northeast African populations but was associated with a wide range of STRP alleles in the sub-Saharan African populations. This global pattern of haplotype variation and linkage disequilibrium suggests a common and recent African origin for all non-African human populations.

Phenobarbital-induced fibromyalgia as the cause of bilateral shoulder pain.

A female swimming instructor was seen with chronic bilateral shoulder pain and loss of range of motion. Intensive physical therapy significantly improved the range of motion but did not alleviate the pain. Osteopathic manipulative treatment produced no further improvement in pain or function. Results of laboratory tests were all within normal limits. Four months after the initial consultation, the patient, who was taking medication for tonic/clonic seizures, recalled that her symptoms began after her anticonvulsant medication was switched from hydantoin sodium to phenobarbital. Therefore, phenobarbital-induced fibromyalgia was diagnosed. In 4 months, pain had completely disappeared. The authors discuss several theories regarding the cause of fibromyalgia and the mechanism of action of phenobarbital, including its relationship to sleep disturbance, a probable contributor to pain and dysfunction in the patient described.

Ancient DNA: methodological challenges.

The study of ancient DNA offers the possibility of following genetic change over time. However, the field is plagued by a problem which is unique in molecular biology--the difficulty of verifying results by reproduction. Some of the reasons for this are technical and derive from the low copy number and damaged state of ancient DNA molecules. Other reasons are the unique nature of many of the objects from which DNA is extracted. We describe methodological approaches with which these problems can be alleviated in order to ensure that results are scientific in the sense that they can be reproduced by others.