A total of 95 isolates, belonging to 33 species of five dermatophyte genera, i.e. Arthroderma (15 species), Chrysosporium (two), Epidermophyton (one), Microsporum (three) and Trichophyton (12), were studied using internal transcribed spacer 2 (ITS2)-PCR-RFLP analysis (ITS2-RFLP), consisting of amplification of the ITS2 region, restriction digestion with BstUI (CG/CG) and restriction fragment length determination by capillary electrophoresis. ITS2-RFLP analysis proved to be most useful for identification of species of the genera Arthroderma, Chrysosporium and Epidermophyton, but could not distinguish between several Trichophyton species. The identification results are in agreement with established and recent taxonomical insights into the dermatophytes; for example, highly related species also had closely related and sometimes difficult-to-discriminate ITS2-RFLP patterns. In some cases, several ITS2-RFLP groups could be distinguished within species, again mostly in agreement with the taxonomic delineations of subspecies and/or genomovars, confirming the relevance of ITS2-RFLP analysis as an identification technique and as a useful taxonomic approach.

An isolate of Arthroderma simii was successfully mated with a tester strain of A. vanbreuseghemii cultured on the plate of simple agar with some hair on it at 27 degrees C. Confirmation of sexual reproduction was made by the detection of hybrids of two parental genotypes. The implications of this result are discussed from the viewpoint of a reevaluation of the species boundaries of dermatophytes.

To determine the relationships among Trichophyton species we constructed three phylogenies, based on the nucleotide sequences of the actin, rRNA and DNA topoisomerase II genes. These phylogenies showed several conflicting branch points. For example, strains of T. verrucosum,T. concentricum and T. mentagrophytes var. erinacei were mingled with strains of Arthroderma benhamiae and could not be separated into their own phylogenic groups. In addition, strains of A. vanbreuseghemii, T. tonsurans, T. mentagrophytes var. interdigitale and T. mentagrophytes var. quinckeanum were mingled with strains of A. simii and could not be separated into their own phylogenic groups. T. rubrum and T. violaceum made up a clade, which was phylogenetically related to the A. benhamiae clade or A. simii clade, depending on the gene examined. These findings indicate the need to reevaluate the boundaries among Trichophyton species using an alternative to morphological or molecular biological methods.

Taxonomy is an artificial system to classify living organisms. For this purpose, different concepts of how to define a species can be used. In recent years, DNA analysis has especially helped to better understand the phylogenetic relations of dermatophytes and thus influenced the distinction of dermatophyte species. In some cases species previously considered separate were made synonymous. It may be necessary to discriminate different varieties within a particular species if this is required by their typical morphologic, physiologic or epidemiological characteristics. Trichophyton eboreum with its perfect state Arthroderma olidum was recently described as a new species. The taxonomic order of dermatophytes must continuously be updated by integrating new findings; at the same time, meaningful and proven criteria should be retained.

Fungal infections of the skin and nails are a common global problem. The high prevalence of superficial mycotic infections shows that 20-25% of the world's population has skin mycoses, making these one of the most frequent forms of infection. Pathogens responsible for skin mycoses are primarily anthropophilic and zoophilic dermatophytes from the genera Trichophyton (T.), Microsporum (M.) and Epidermophyton (E.). There appears to be considerable inter- and intra-continental variability in the global incidence of these fungal infections. Trichophyton rubrum, T. interdigitale (mentagrophytes var. interdigitale), M. canis, M. audouinii, T. tonsurans and T. verrucosum are the most common, but the attack rates and incidence of specific mycoses can vary widely. Local socio-economic conditions and cultural practices can also influence the prevalence of a particular infection in a given area. For example, tinea pedis (athlete's foot) is more prevalent in developed countries than in emerging economies and is likely to be caused by the anthropophilic germ T. rubrum. In poorer countries, scalp infections (tinea capitis) caused by T. soudanense or M. audouinii are more prevalent. This review summarises current epidemiological trends for fungal infections and focuses on dermatomycosis of glabrous skin on different continents.

In cases of highly inflammatory dermatophytosis in humans, it is important to identify the possible source of animal transmission in order to prevent recurrence, family outbreaks or rapidly progressing epidemics. A survey of dermatophytes in pets during a 14-month period in Switzerland revealed, in addition to Microsporum canis, two different species of the Trichophyton mentagrophytes complex, Arthroderma benhamiae and Arthroderma vanbreuseghemii, all causing inflammatory dermatophytoses. Arthroderma benhamiae was only and frequently isolated from guinea pigs. Arthroderma vanbreuseghemii was isolated mainly from European short hair cats, but also from dogs and in one case from a pure-bred cat. Ninety-three percent of the cats carrying A. vanbreuseghemii were hunters and all had skin lesions. In contrast, cats with skin lesions that were strictly indoors were found to be almost exclusively infected by M. canis. Therefore, it can be suspected that infection with A. vanbreuseghemii occurred during hunting and that the natural source of this dermatophyte is either soil or an animal other than the cat, most probably a rodent.

An intra-familial transmission of Arthroderma benhamiae in Canadian porcupines (Erethizon dorsatum) housed in a Japanese zoo was studied. The family consisted of an adult couple and two offspring (a male and a female). The porcupettes, born in Japan, showed severe hair loss while the parent animals, imported from the USA.(male) and Canada (female), showed mild symptoms or were asymptomatic. Morphologically identical Tricophyton spp. isolates were recovered within seven days from quills of all animals on chloramphenicol-supplemented potato dextrose agar plates incubated at 37 degrees C. Two representative colonies from each animal were identified as Arthroderma benhamiae Americano-European race based on mating type (+) and the internal transcribed spacer (ITS) 1-5.5S-ITS 2 region of the rRNA gene sequences (AB236404-AB236408). The present cases constituted the second isolation of dermatophytes from porcupines. There were two different ITS types, i.e., the predominant one isolated from all animals and a secondary one recovered from only the mother porcupine. The sequences have never been recorded in Japan or in the GenBank database to the best of our knowledge. In addition, they were located at a cluster involving the type strain and mating strains of A. benhamiae Americano-European race and its F1 progeny. In contrast, 28 rodents (eight species) and three insectivora (1 species) exhibited in the petting zoo were negative for any dermatophytes as determined by culture.

Identification of dermatophytes is currently performed based on morphological criteria and is increasingly supported by genomic sequence comparison. The present study evaluates an alternative based on the analysis of clinical fungal isolates by mass spectrometry. Samples originating from skin and nail were characterized morphologically and by sequencing the internal transcribed spacer 1 (ITS1), ITS2 and the 5.8S rDNA regions of the rDNA clusters. In a blind comparative study, samples were analyzed by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF MS). The mass spectra were compared to a database comprising of the spectral data of reference strains by applying the saramis software package. All fungal isolates belonging to the taxa Trichophyton rubrum, T. interdigitale, T. tonsurans, Arthroderma benhamiae and Microsporum canis were correctly identified, irrespective of host origin and pathology. To test the robustness of the approach, four isolates were grown on five different media and analyzed. Although the resulting mass spectra varied in detail, a sufficient number of signals were conserved resulting in data sets exploitable for unequivocal species identification. Taken together, the usually widespread dermatophytes can be identified rapidly and reliably by mass spectrometry. Starting from pure cultures, MALDI-TOF MS analysis uses very simple sample preparation procedures, and a single analysis is performed within minutes. Costs for consumables as well as preparation time are considerably lower than for PCR analysis.

Dermatophytes are keratinophilic fungi able to infect keratinized tissues of human or animal origin. Among them, Trichophyton mentagrophytes is known to be a species complex composed of several species or variants, which occur in both human and animals. Since the T. mentagrophytes complex includes both anthropophilic and zoophilic pathogens, accurate molecular identification is a critical issue for comprehensive understanding of the clinical and epidemiological implications of the genetic heterogeneity of this complex. Here, 41 T. mentagrophytes isolates from either human patients (14 isolates) or animals (27 isolates) with dermatophytosis were prospectively isolated by culture and identified on morphological bases at the University Hospital Centres of Lille and Poitiers, and the Veterinary School of Alfort, respectively. The isolates were differentiated by DNA sequencing of the variable internal transcribed spacer (ITS) regions flanking the 5.8S rDNA, and of the housekeeping gene encoding the manganese-containing superoxide dismutase (MnSOD), an enzyme which is involved in defence against oxidative stress and has previously provided interesting insight into both fungal taxonomy and phylogeny. ITS1-ITS2 regions and MnSOD sequences successfully differentiate between members of the T. mentagrophytes complex and the related species Trichophyton rubrum. Whatever the phylogenetic marker used, members of this complex were classified into two major clades exhibiting a similar topology, with a higher variability when the ITS marker was used. Relationships between ITS/MnSOD sequences and host origin, clinical pattern and phenotypic characteristics (macroscopic and microscopic morphologies) were analysed.

Altogether 285 dermatophyte isolates of 21 different species - including both Trichophyton rubrum and T. interdigitale, but also eight additional Trichophyton species, Microsporum canis and seven other Microsporum species, as well as Epidermophyton floccosum and Arthroderma spp.- were analyzed using Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and the AnagnosTec 'SARAMIS'(Spectral Archiving and Microbial Identification System) software. In addition, sequence analysis of the internal transcribed spacer (ITS) of the ribosomal DNA was performed for a high number of the tested strains. Sufficient agreement was found between the results obtained with standard identification methods and those with the MALDI-TOF MS for species identification of dermatophytes. A mass spectra database was constructed which contained the species identifications of all 285 isolates. The results were confirmed for 164 of the isolates by sequence analysis of the internal transcribed spacer (ITS) of the ribosomal DNA. Statistical analysis of all 285 dermatophyte strains showed that conventional identification matched the results of MALDI-TOF MS for 78.2% of the isolates tested. In the case of the 164 isolates for which the identifications were confirmed by PCR, the results of their conventional diagnosis and MALDI-TOF MS were in agreement for only 68.9 %(113 of 164 strains) of the test isolates. In contrast, there was agreement of 99.3 % or 98.8 % in the identifications obtained with PCR and MALDI-TOF MS techniques (283/285 or 162/164). The two exceptions were isolates that proved to be T. violaceum which could not be identified by the MALDI-TOF MS technique. In conclusion, the MALDI-TOF mass spectroscopy represents a fast and very specific method for species differentiation of dermatophytes grown in culture.

The localisation of a dermatophytosis on the face is rare. Differential diagnoses include a broad range of dermatological disorders, e. g. contact dermatitis, psoriasis vulgaris, seborrhoeic dermatitis, demodicosis, and polymorphic photo eruptions. Two patients suffering from tinea faciei incognito caused by Trichophyton rubrum are presented. Diagnosis was based on mycological diagnostics of skin scrapings using Calcofluor preparation and cultivation of the causative dermatophyte. Both patients were suffering from tinea pedis and tinea unguium caused by the same dermatophyte species. An infection caused by Trichophyton rubrum of the face always occurs following autoinoculation from a pre-existing tinea pedis and tinea unguium of feet and toenails, sometimes of the fingernails.

The prevalence of onychomycosis is increasing steadily, sevenfold alone in the US within the last twenty years. An important aspect in this development is the demographic development of the human population of the industrial countries like Germany. A fast and accurate laboratory diagnosis is essential for successful treatment because 50% of the cases are misdiagnosed when relying on the clinical appearance only. The current diagnosis of dermatophytosis, based on direct microscopy and culture of the clinical specimen, is problematic given the lacking specificity of the former and the length of time needed for the latter. Molecular techniques can help to solve these problems. In recent years, a number of in-house PCR assays have been developed to identify dermatophytes directly from clinical specimens. Based on the "Mikrobiologisch-infektiologischen Qualitätsstandards (MIQ) für Nukleinsäure-Amplifikationstechniken" and the MIQE guideline (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) 11 studies are reviewed which were published between 2007 and 2010. The present article evaluates the quality of the PCR assays regarding false positive and false negative results due to contamination, PCR format, statistical analysis, and diagnostic performance of the studies. It shows that we are only at the beginning of providing high quality PCR diagnosis of dermatophytes.

Some members of the genus Candida are among the most common human fungal pathogens and cause serious diseases especially in immunocompromised people. A yeast was isolated from a blood culture from an immunocompromised cancer patient who suffered from acute pneumonia. The growth characteristics of the yeast on CHROMagar Candida were similar to those of Candida tropicalis, whereas the API ID 32C system identified the yeast as Candida silvicola. On the basis of the nucleotide divergence in the D1/D2 domain of the 26S nuclear rRNA (nrRNA) gene, as well as the internal transcribed spacer (ITS) domain of the nrRNA gene region, a new species, Candida pseudoaaseri sp. nov. with type strain VK065094 (CBS 11170(T)), which was found to be closely related to Candida aaseri, is proposed. While C. aaseri strains were susceptible to all tested antifungals, the new species is resistant to flucytosine and may also be distinguished from C. aaseri by its ability to assimilate l-rhamnose, whereas its colony morphology on CHROMagar Candida may be helpful for differentiation.

Species identification of yeasts is based on biochemical (e.g. API ID 32 C(®), bioMérieux) and molecular biological approaches. As an alternative to DNA-dependent methods, mass spectral analysis based identification of micro-organisms has become increasingly recognized. In a number of studies, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been applied for the rapid classification and identification of micro-organisms. In this study, the applicability of MALDI-TOF MS for identifying yeasts isolated from dermatological patients was analysed and compared with the results from the API ID 32 C(®) system. Furthermore, sequencing the internal transcribed spacer (ITS) regions of the ribosomal DNA was employed as reference method. Candida (C.) albicans was isolated in 41.9% of all cases, C. parapsilosis in 20.3%, C. glabrata in 10.8%, and C. krusei in 6, 8.1%. Rarely isolated yeasts were Candida colliculosa, famata, guilliermondii, lusitaniae, and tropicalis as well as Geotrichum candidum, Rhodotorula mucilaginosa and Trichosporon mucoides. The MALDI TOF results were equal to the results gained by ITS sequence analysis in 94%, whereas API ID 32 C(®) provided the correct diagnosis in 84.3%(of all cases). This lower identification rate is mostly referable to frequent misidentifications of C. krusei as C. inconspicua/norvegensis,Candida tropicalis, or Geotrichum capitatum. In contrast, all C. krusei strains were correctly identified by MALDI TOF MS. In conclusion, species identification by MALDI-TOF MS was proven to be consistent with ITS sequence analysis; the technique has a resolving power comparatively as high as ITS sequence analysis.

A novel dermatophyte species is described in the Microsporum cookei clade. It differs significantly from known taxa in the two molecular markers analyzed, i.e., ITS and partial β-tubulin (BT2). Morphologically the species was characterized by smooth- or only slightly rough-walled conidia, but isolates rapidly became pleomorphic with sparse, smooth- and thick-walled macroconidia in addition to microconidia. A teleomorph was found after mating.

Literature data analysis, providing an exact explanation of the lichen planus pathogenesis, as well as its transition into other rare forms such as Keratosis lichenoides chronica or Graham Lassueur Piccardi Little Syndrome are scant, or totally missing. The chronological course of the disease, known in the literature as lichen planus, varies. Some patients develop Lichen planus or lichen nitidus and there is no logical explanation why. It is also not clear why single patients initially develop ulcerative lesions in the area of the mucosa and only in a few of them these lesions affect the skin. Antigen Mimicry and Epitope Spreading could be the possible pathogenic inductor in cases of lichenoid dermatoses, as well as the cause for their transition into ulcerative, exanthematous or other rare forms. The Epitope Spreading is probably not the leading pathogenetic factor in lichen planus but a phenomenon which occurs later. This manuscript analyzes some basic pathogenic aspects and presents some possible medical hypotheses regarding the heterogenic clinical picture and pathogenesis of lichen planus and lichenoid like pathologies of the skin which, in the near future should be analyzed in details in order to clarify several dilemmas the clinical dermatologist has to face.

Trichophyton mentagrophytes is epidemiologically divided into two distinct forms, zoophilic and anthropophilic. The zoophilic isolates of T.mentagrophytes have generally been identified by morphological and biochemical examination as well as through mating experiments. The confirmed teleomorphs of the zoophilic isolates of the T.mentagrophytes complex are Arthroderma benhamiae, A. simii and A. vanbreuseghemii. On the other hand, no teleomorph has been identified in an anthropophilic isolate of T. mentagrophytes, such as T. mentagrophytes var. interdigitale (T. interdigitale) or in the other anthropophilic strains. In the present study, the mating type (MAT)(－)-specific gene of the MAT1-1 (alpha-box) and the MAT (＋)-specific gene of the MAT1-2 (high-mobility-group : HMG) DNA binding domain were confirmed in zoophilic dermatophytes of A. benhamiae, A. simii and A. vanbreuseghemii. The sequence of the MAT1-1 was about 1.3kbp, containing 2 exons in the A. benhamiae, A. simii and A. vanbreuseghemii (－) mating type strain. The sequence of the MAT1-2 was 1.9 kbp, containing 2 exons in the A. benhamiae, A. simii and A. vanbreuseghemii (＋) mating type strain. Of 15 animal isolates and 72 human isolates examined, the MAT1-1 was detected in 5 of the animal isolates and in none of the human isolates, while the MAT1-2 was detected in the other 10 of the animal isolates and in all of the human isolates. These results indicate that anthropophilic T. mentagrophytes evolved from the A. vanbreuseghemii (＋) mating strain.

BACKGROUND Trichophyton mentagrophytes var. erinacei is a zoophilic dermatophyte transmitted by hedgehogs which human infections manifest as highly inflammatory and pruritic eruptions. METHODS We report a 21-year-old woman who presented with a two-week history of a pruritic scaly erythematous plaque on the nose. The patient had kept hedgehogs as pets, and one had bitten her on the tip of the nose two months prior to the appearance of the nasal lesion. RESULTS Fungal culture from the scales on the tip of the patient's nose was compatible with T. mentagrophytes var. erinacei. The strains isolated from cultures made from samples taken from the noses and ears of the pet hedgehogs were morphologically similar to the strain isolated from the patient. Morphological identification was confirmed by sequencing the internal transcribed spacer. The lesion was diagnosed as tinea faciei, and therapy was initiated with topical and systemic terbinafine 250mg/day for eight weeks, which resulted in complete improvement. ConcLUSIONS: Trichophyton mentagrophytes var. erinacei is the most common dermatophyte isolated in hedgehogs. Usually T. mentagrophytes var. erinacei infection manifests as an extremely inflammatory and pruritic eruption that is confined to the site of contact with the hedgehog. Although the identification of T. mentagrophytes by traditional methods is possible, identification by ITS region analysis is a fast, simple, and increasingly available method. The increasing frequency of the practice of keeping exotic animals as pets has resulted in the emergence of several zoonotic diseases that can potentially be transmitted to humans.

A newly proposed taxonomy of Trichophyton mentagrophytes and related species was introduced and verified with the grounds for the new classification, phylogenetic analysis, and Templeton's cohesive species concept. So-called asexual species were shown to retain sexual ability and different host preferences were shown not to be comparable to different ecological niches. We showed that genealogical concordance phylogenetic species recognition (GCPSR) can be applied to so-called asexual Trichophyton species.The results of GCPSR analysis and mating experiments demonstrated that Arthroderma simii, A. vanbreuseghemii, T. mentagrophytes var. interdigitale, and T. tonsurans may be considered as single phylogenetic species, and that A. benhamiae, T. concentricum, T. verrucosum, and T. mentagrophytes var. erinacei may also be considered as single phylogenetic species. In addition, the conspecificity of three Arthroderma species may also be demonstrated in future.In conclusion, we should not hastily revise the nomenclature based on the phylogenetic tree from only one gene, because different genes can yield different phylogenetic trees. Therefore, we recommend retaining the previous taxonomy until an acceptable taxonomy is proposed.

Thirty-nine Arthroderma vanbreuseghemii isolates identified by mating behavior were assayed to determine their ribosomal RNA gene internal transcribed spacer(ITS)region genotypes. Twenty-two isolates of mating type(+)and 4 of(-)showed the genotype of Trichophyton mentagrophytes var. interdigitale, 9 isolates of mating type(+)and 4 of(-)showed the genotype of A. vanbreuseghemii. Nine of 14 isolates with granular or asteroid colony morphology showed the genotype of T. mentagrophytes var. interdigitale. There was no relation between ITS genotype and morphology or mating type of the isolates. Isolates of T. mentagrophytes var. interdigitale genotype were shown to include non-sexually degenerate zoophilic strains with granular colony morphology as well as sexually and morphologically degenerate anthropophilic strains and may be just one genotype of a heterogeneous species, A. vanbreuseghemii.

Isolates of the Trichophyton mentagrophytes complex vary phenotypically. Whether the closely related zoophilic and anthropophilic anamorphs currently associated with Arthroderma vanbreuseghemii have to be considered as members of the same biological species remains an open question. In order to better delineate species in the T. mentagrophytes complex, we performed a mating analysis of freshly collected isolates from humans and animals with A. benhamiae and A. vanbreuseghemii reference strains, in comparison to internal transcribed spacer (ITS) and 28S rDNA sequencing. Mating experiments as well as ITS and 28S sequencing unambiguously allowed the distinction of A. benhamiae and A. vanbreuseghemii. We have also shown that all the isolates from tinea pedis and tinea unguium identified as T. interdigitale based on ITS sequences mated with A. vanbreuseghemii tester strains, but had lost their ability to give fertile cleistothecia. Therefore, T. interdigitale has to be considered as a humanized species derived from the sexual relative A. vanbreuseghemii.

Fungi of the Trichophyton mentagrophytes complex are important pathogenic dermatophytes capable of causing various human skin infections. There are many species and varieties involved in this complex. Due to morphological similarity and existing intermediate forms and variants, unequivocally separating these dermatophytes is not always straightforward, and sampling appropriate isolates for research is often troublesome. Communicating epidemiological statistics of these dermatophytes is greatly impeded because taxonomic schemes adopted by different research groups are inconsistent and hardly comparable. To analyse the molecular types existing within isolates of the T. mentagrophytes complex in Taiwan and evaluate the applicability of the current taxonomy on these isolates. Forty-eight isolates of the T. mentagrophytes complex were obtained from humans and animals (rabbit, guinea pig and hedgehog) in Taiwan. Sequences of ITS1-5.8S-ITS2 and b-tubulin gene regions of each isolate were used for molecular typing. Among the 48 isolates, 43 belonged to the T. interdigitale clade, including 21 of the anthropophilic strain and 22 of the zoophilic strain, and five belonged to the T. erinacei clade. No isolates of the T. mentagrophytes genotype were found. The isolates previously identified as T. mentagrophytes should be relabelled as T. interdigitale according to current taxonomy. Because the taxonomy is quite different from what it used to be, confusion in the nomenclature of the T. mentagrophytes complex is to be expected. The applicability of the current taxonomic concept may require further evaluation.

Background: Dermatophytes are usually identified on the basis of macroscopic characteristics and microscopic examination of the cultures. Identification of dermatophytes often remains difficult or uncertain because there are variations from one isolate to another and overlapping characteristics between species. Objective: To identify dermatophyte species producing numerous microconidia and resembling Trichophyton mentagrophytes by DNA sequence analysis. Methods: The complete ITS1 + 5.6s + ITS2 rDNA region of various dermatophytes isolated in culture was amplified by PCR and sequenced. Results: Nine isolates of a fast-growing dermatophyte species were identified as Arthroderma benhamiae by DNA sequencing. Retrospective investigations revealed that the isolates were from 8 children and 1 adult suffering from inflammatory dermatophytosis. Eight of the 9 patients had had previous contact with rodents, mostly guinea pigs. Conclusion: It is the first time that A. benhamiae is reported in Switzerland. In cases of dermatophytosis attributed to A. benhamiae, a rodent is the most likely cause of infection.

Cypripedium calceolus was found in 1980 in Rebun Island off the north coast of Hokkaido, Japan, but the origin of this plant has been a controversial issue. In this study, we have made a comparative study by chloroplast DNA sequencing analysis among C. calceolus which occurs in Rebun Island and populations of C. calceolus from western Europe, China and far eastern Russia (Nakhodka), and also as references, C. macranthos in Japan and other Cypripedium species in North America. A Cypripedium cf."calceolus", found recently in eastern Hokkaido, was also included in this analysis. The C. calceolus samples analyzed were categorized into three groups, i.e., those from Western Europe, from China and far eastern Russia, and from Rebun Island. The C. calceolus in Rebun Island was clearly different from the others in terms of DNA sequence and morphological features. The C. cf. calceolus from eastern Hokkaido and one sample from Nadhodka, Russia, were also classified into the same group as those from Rebun Island, although some differences in their morphological features were observed. It is concluded that the C. calceolus found in Rebun Island is not identical with those growing in Europe and China. In addition, it was found that it may be possible to classify C. macranthos into two groups, namely groups which include or do not include var. rebunense. An unidentified Cypripedium species found in Rebun Island falls into the same group as var. rebunense.

It has been suggested in the literature that primates of the genus Cacajao have been restricted to flooded-forest habitats of western Amazonia since their split from the Chiropotes line in the Tertiary. It has been proposed further that the differentiation of the two species of this genus, Cacajao melanocephalus and Cacajao calvus, occurred during the Pleistocene period as a result of the fragmentation of the Amazon forest and the isolation of populations in these forest fragments or refuges. However, recent evidence has shown that at least C. melanocephalus is not dependent on flooded-forest habitats, and molecular analysis of mitochondrial DNA shows that the two species had already differentiated during the Pliocene, thus Pleistocene glaciations do not explain the speciation in Cacajao. Considering that C. melanocephalus and its closest relative, Chiropotes, inhabit terra firme forests, it is suggested that preference for flooded-forest habitats may be an apomorphy in C. calvus.

The group of anopheline mosquitoes referred to as 'Anopheles maculipennis complex' includes the most important malaria vectors of the Palearctic Western region. The species belonging to this complex, however, are difficult or impossible to distinguish by morphological characters. To differentiate sibling palearctic species belonging to this complex, interspecific differences in the ITS2 sequences were used to set up a rapid and sensitive diagnostic tool based on heteroduplex analysis. The relative heteroduplex mobility allowed the following seven species to be readily distinguished: An. atroparvus, An. labranchiae, An. maculipennis s.s., An. martinius, An melanoon, An. messeae and An. sacharovi.