A MOLECULAR SYSTEMATIC APPROACH TO EXPLORE DIVERSITY WITHIN THE SELLAPHORA PUPULA SPECIES COMPLEX (BACILLARIOPHYTA)1

The common and cosmopolitan freshwater benthic diatom Sellaphora pupula (Kütz.) Mereschk. is a model system for studying the nature of species in microalgae; the biological significance of morphological variation in this species complex has been widely demonstrated. The aim of this study was to establish a two‐gene phylogeny (18S rDNA and rbcL) for 23 Sellaphora taxa, including 19 S. pupula aggregate (agg.) demes or species, S. bacillum (Ehrenb.) D. G. Mann, and S. laevissima (Kütz.) D. G. Mann. A range of analyses on separate and combined data sets indicated that Sellaphora is a monophyletic group containing four major clades. Of the traditionally recognized species, S. bacillum and S. laevissima are natural groups, but S. pupula is paraphyletic or polyphyletic because S. bacillum groups with S. pupula“small lanceolate” and S. lanceolata. Thickened bars at the poles of valves within the core “pupula–bacillum” group may be a morphological synapomorphy; the fossil record suggests that this group is at least 12 million years old. Otherwise, there was no clear pattern in the distribution of different morphologies among the major clades; each clade was also heterogeneous with respect to mating system. More intensive and extensive sampling will doubtless uncover even greater diversity; the challenge lies in its interpretation. Our results demonstrate the limitations of paleoecological, ecological, and biogeographical research based on morphospecies.     

Ecological variation within Sellaphora species complexes (Bacillariophyceae): specialists or generalists?

The genus Sellaphora has become a model system for studies of the species concept, speciation and automated identification in diatoms. Three species, S. pupula, S. bacillum and S. laevissima, have proved to be complexes containing several or many species, which are difficult to distinguish morphologically but which are genetically differentiated and (where tested) reproductively isolated. Until now, however, there has been little information about the ecology of the species within this complex, except in relation to parasite sensitivity. In order to test whether semi- and pseudo-cryptic Sellaphora species are ecologically differentiated with respect to trophic status, we used tools recently developed in the UK in response to the EU Water Framework Directive (WFD). Diatom samples from three substrata (plants, rocks, sediment) were taken from 22 lakes in Scotland and England, covering a gradient from oligotrophic mountain lakes to eutrophic lowland ponds. The epilithic and epiphytic diatom assemblages were used to evaluate lake trophic status according to the UK WFD assessment system and showed that there was a strong environmental gradient in the dataset. Sellaphora species occurred in the sediment-derived epipelon and their distributions were analysed in relation to the trophic gradient. A total of 28 Sellaphora phenodemes (putative species) were found, and some of them differed in their environmental demands. Two groups were distinguished: (1) a group indicating rather oligotrophic conditions and containing several demes with linear valves and subcapitate or capitate poles (referred to here as S. [pupula] cap-A, cap-B and cap-C and (2) a group occurring in eutrophic lakes and containing the recently described species S. blackfordensis, S. capitata and S. obesa, as well as S. [pupula] Φ ‘small lanceolate’. The data obtained are also discussed with respect to Finlay’s hypothesis on microalgal cosmopolitanism. Handling editor: J. Padisak     

基于rDNA ITS序列分析莲塘湖萼花臂尾轮虫种群遗传结构的季节变化

对采自芜湖市莲塘湖水体中的萼花臂尾轮虫(Brachionus calyciflorus)夏季种群中的30个克隆(S1-S30)和秋季种群中的20个克隆(A1-A20)的rDNA ITS序列进行了分析;以十指臂尾轮虫B.patulus为外群,构建了50个克隆的NJ、MP和ME系统发生树。结果表明:三个系统树均支持将50个克隆分为两个支系;第一支系包括29个夏季采集的单元型(单元型S9除外)和1个秋季采集的单元型A2,第二个支系包括18个秋季采集的单元型(单元型A2除外)和1个夏季采集的单元型S9;两支系间ITS序列差异为5.1%-8.8%;它们应属于两个不同的姐妹种。夏季,莲塘湖水体中的萼花臂尾轮虫种复合体内以第一支系为主构成的姐妹种在相对丰度上占绝对优势,达29/30;而秋季则相反。上述结果表明:浅水湖泊中萼花臂尾轮虫种群密度的季节变动实际上主要在于各姐妹种相对丰度的季节变化;两姐妹种的种群遗传结构季节变化符合重叠模型。     

Genotyping of a Miso and Soy Sauce Fermentation Yeast,Zygosaccharomyces rouxii,Based on Sequence Analysis of the Partial 26S Ribosomal RNA Gene and Two Internal Transcribed Spacers

We analyzed sequences of the D1D2 domain of the 26S ribosomal RNA gene (26S rDNA sequence), the internal transcribed spacer 1, the 5.8S ribosomal RNA gene, and the internal transcribed spacer 2 (the ITS sequence) from 46 strains of miso and soy sauce fermentation yeast, Zygosaccharomyces rouxii and a closely related species, Z. mellis, for typing. Based on the 26S rDNA sequence analysis, the Z. rouxii strains were of two types, and the extent of sequence divergence between them was 2.6%. Based on the ITS sequence analysis, they were divided into seven types (I–VII). Between the type strain (type I) and type VI, in particular, a 12% difference was detected. The occurrence of these nine genotypes with a divergence of more than 1% in these two sequences suggests that Z. rouxii is a species complex including novel species and hybrids. Z. mellis strains were of two types (type α and type β) based on the ITS sequence. Z. rouxii could clearly be distinguished from Z. mellis by 26S rDNA and ITS sequence analyses, but not by the 16% NaCl tolerance, when used as the sole key characteristic for differentiation between the two species.     

Systematics of Cladophora spp. (Chlorophyta) from North Carolina,USA, based upon morphology and DNA sequence data with a description of Cladophora subtilissima sp. nov.

Identification of Cladophora species is challenging due to conservation of gross morphology, few discrete autapomorphies, and environmental influences on morphology. Twelve species of marine Cladophora were reported from North Carolina waters. Cladophora specimens were collected from inshore and offshore marine waters for DNA sequence and morphological analyses. The nuclear‐encoded rRNA internal transcribed spacer regions (ITS) were sequenced for 105 specimens and used in molecular assisted identification. The ITS1 and ITS2 region was highly variable, and sequences were sorted into ITS Sets of Alignable Sequences (SASs). Sequencing of short hyper‐variable ITS1 sections from Cladophora type specimens was used to positively identify species represented by SASs when the types were made available. Secondary structures for the ITS1 locus were also predicted for each specimen and compared to predicted structures from Cladophora sequences available in GenBank. Nine ITS SASs were identified and representative specimens chosen for phylogenetic analyses of 18S and 28S rRNA gene sequences to reveal relationships with other Cladophora species. Phylogenetic analyses indicated that marine Cladophorales were polyphyletic and separated into two clades, the Cladophora clade and the “Siphonocladales” clade. Morphological analyses were performed to assess the consistency of character states within species, and complement the DNA sequence analyses. These analyses revealed intra‐ and interspecific character state variation, and that combined molecular and morphological analyses were required for the identification of species. One new report, Cladophora dotyana, and one new species Cladophora subtilissima sp. nov., were revealed, and increased the biodiversity of North Carolina marine Cladophora to 14 species.     

MOLECULAR PHYLOGEOGRAPHY, RETICULATION, AND LINEAGE SORTING IN MEDITERRANEAN SENECIO SECT. SENECIO (ASTERACEAE)

Abstract The Mediterranean species complex of Senecio serves to illustrate evolutionary processes that are likely to confound phylogenetic inference, including rapid diversification, gene tree‐species tree discordance, reticulation, interlocus concerted evolution, and lack of complete lineage sorting. Phylogeographic patterns of chloroplast DNA (cpDNA) haplotype variation were studied by sampling 156 populations (502 individuals) across 18 species of the complex, and a species phylogeny was reconstructed based on sequences from the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. For a subset of species, randomly amplified polymorphic DNAs (RAPDs) provided reference points for comparison with the cpDNA and ITS datasets. Two classes of cpDNA haplotypes were identified, with each predominating in certain parts of the Mediterranean region. However, with the exception of S. gallicus, intraspecific phylogeographic structure is limited, and only a few haplotypes detected were species‐specific. Nuclear sequence divergence is low, and several unresolved phylogenetic groupings are suggestive of near simultaneous diversification. Two well‐supported ITS clades contain the majority of species, amongst which there is a pronounced sharing of cpDNA haplotypes. Our data are not capable of diagnosing the relative impact of reticulation versus insufficient lineage sorting for the entire complex. However, there is firm evidence that S. flavus subsp. breviflorus and S. rupestris have acquired cpDNA haplotypes and ITS sequences from co‐occurring species by reticulation. In contrast, insufficient lineage sorting is a viable hypothesis for cpDNA haplotypes shared between S. gallicus and its close relatives. We estimated the minimum coalescent times for these haplotypes by utilizing the inferred species phylogeny and associated divergence times. Our data suggest that ancestral cpDNA polymorphisms may have survived for ca. 0.4–1.0 million years, depending on molecular clock calibrations.     

A Phylogeographic Split in Buxus balearica (Buxaceae) as Evidenced by Nuclear Ribosomal Markers: When ITS Paralogues Are Welcome

Sequences from the ribosomal nuclear internal transcribed spacers (ITS) have been widely used to infer evolutionary hypotheses across a broad range of living organisms. Intraspecific sequence variation is assumed to be absent or negliable in most species, but few detailed studies have been conducted to assess the apportionment of ITS sequence variation within and between plant populations. Buxus balearica was chosen as a model species to assess the levels of infraspecific and intragenomic ITS variation in rare and endangered species occurring in disjunct populations around the Mediterranean basin. Intragenomic polymorphic sites were detected for western and eastern accessions of B. balearica and in two accessions of the sister species B. sempervirens. Overall, 19 different ribotypes were found in B. balearica after sequencing 48 clones, whereas 15 ribotypes were detected in 19 clones of B. sempervirens. The integrity and secondary structure stability of the ribosomal sequences suggest that they are not pseudogenes. The high number of ribotypes recovered through cloning suggested that some sequences could be chimeric or generated in vivo by partial homogenization through gene conversion or unequal crossing-over. Average sequence divergence among B. balearica clones was 0.768%, and the most divergent sequences differed by 1.62%. Available evidence does not suggest that B. balearica paralogues have been obtained from other extant Buxus species through interspecific hybridization. The presence of several ribosomal sequences in box implies that the molecular forces driving the concerted evolution of this multigene family are not fully operational in this genus. Phylogenetic analyses of cloned ITS sequences from B. balearica displayed very poor resolution and only two clades received moderate bootstrap support. Despite the marked intragenomic sequence divergence found, ribosomal data suggest a clear phylogeographic split in B. balearica between western and eastern accessions. The distinct, nonchimeric sequences that are postulated as being present in each biogeographic group suggest that box populations from Anatolia (eastern Mediterranean) are relict. [Reviewing Editor: Dr. Rafael Zardoya]     

Accelerated nrDNA evolution and profound AT bias in the medicinal fungus Cordyceps sinensis

Cordyceps sinensis is a reputed medicinal fungus growing parasitically on buried larvae of ghost moths in Asian high-altitude grassland ecosystems. We have analysed the intraspecific ITS nrDNA (ITS1, 5.8S gene, ITS2) variation among 71 sequences of C. sinensis available in EMBL/Genbank. The ITS sequences, submitted to Bayesian ML analyses, were distributed into five groups, referred to as A–E. Nine of the sequences (groups D and E) grouped with distantly related hypocrealean/clavicipitalean taxa and are interpreted as sequences erroneously accessioned under wrong taxon names. The remaining 62 sequences constituted three highly supported clades (groups A–C), that may represent cryptic (phylogenetic) species currently ascribed to C. sinensis. A remarkably high sequence divergence occurred in the 5.8S gene between the three groups. Sequences of groups B and C showed accelerated substitution rates and high AT nucleotide bias. We hypothesize that the accelerated evolution and AT bias have been caused by a shift in life historical attributes or ecology. We also suggest that the recorded differences in medicinal effects among C. sinensis populations may be attributed to the existence of genetically differentiated chemotypes in this morphotaxon.     

Molecular phylogeny reveals high diversity and endemism in the limestone karst-restricted land snail genus Sophina Benson, 1859 from Myanmar (Eupulmonata: Helicarionidae), with description of four new species

Sophina is a poorly known and neglected genus due to the inaccessibility of the Salween Basin, Southern Myanmar. Taxonomic status, distribution, and phylogeny are being revised based on an integrative analysis of genitalia, radula, and molecular data. Morphological variation in shells and genitalia, together with a phylogenetic tree from concatenated data of mitochondrial and nuclear genes, revealed the existence of ten species/subspecies. Penial morphology and genetic divergence were generally consistent and useful in delimiting species, while shell characters showed little overall taxonomic utility in some species. Taxonomic placement of the previous subspecies “bensoni” shows clear distinction in both genitalia and molecular evidence, and is currently recognized as a distinct species. The nominal species “S. schistostelis” and “S. calias” possess similar genitalia and shell morphology, and molecular evidence suggested that they are sister taxa representing geographically isolated populations. Four new species are additionally described herein as S. furfuracea n. sp., S. pisinna n. sp., S. salweenica n. sp., and S. tonywhitteni n. sp. based on both morphology and molecular evidence. Phylogenetic analyses supported monophyly of Sophina, and further a split into two principal clades. These two clades showed little difference in genitalia, but more clear differences in the umbilical area and allopatric distribution in upper and lower reaches of the Gyaing River. High genetic divergence was revealed and this coincided with remarkable degree of endemism and localization with a pattern of one outcrop for one lineage. These data highlight the importance of the Salween Basin's karst ecosystems as an evolutionary and endemic biodiversity hotspot, and indicate that a focus on conservation and management in this area is urgently required.     

Marked Intragenomic Heterogeneity and Geographical Differentiation of nrDNA ITS in <Emphasis Type="BoldItalic">Larix</Emphasis><Emphasis Type="BoldItalic">potaninii</Emphasis> (Pinaceae)

Nuclear ribosomal DNA (nrDNA) of gymnosperms, especially Pinaceae, is characterized by slow concerted evolution and exhibits substantial ITS-region length variation (975–3663 bp), in sharp contrast to the narrow range (565–700 bp) in angiosperms. Here we examined intra- and intergenomic heterogeneity of the nrDNA internal transcribed spacer (ITS) region in four varieties of Larix potaninii, a species from the mountainous areas of western China. Two clones with more than a 100-bp deletion in ITS1 were detected in L. potaninii var. chinensis and L. potaninii var. australis, respectively. The deletion resulted in the loss of most part, including the motif sequence, of subrepeat 1 (SR1). Sequence divergence and phylogenetic analyses showed that some clones would be pseudogenes given their low GC content, high substitution rates, unique positions in the phylogenetic trees, or significant length variation. These clones might represent orphons or paralogues at minor loci resulting from large-scale gene or chromosome reorganization. Some recombinants characterized by chimeric structure and discordant phylogenetic positions in partitioned sequence analyses indicate that unequal crossing-over plays an important role in the process of nrDNA concerted evolution. In addition, some varieties of L. potaninii might have experienced an nrDNA founder effect parallel to their geographical isolation.     

Observations on Copulation in Navicula pupula and Amphora ovalis in Relation to the Nature of Diatom Species

Within the same water body, morphological variants of the samediatom species often coexist. Observations of such phenodemesof Navicula pupula and Amphora ovalis show that, when sexual,they either fail to attract or fail to recognize each otherand do not interbreed. Since in both species the morphologicaland reproductive differences between demes appear to be reflectedalso in ecological differences, the present species conceptin diatoms needs re-evaluation. Amphora ovalis (Ktz.) Ktz., Navicula pupula Ktz., diatom microevolution, diatom systematics, reproductive isolation     

Intra-isolate heterogeneity of the ITS region of rDNA in Pythium helicoides

Heterogeneity of the rDNA ITS region in Pythium helicoides and the phylogenetic relationship between P. helicoides and closely related species were investigated. In PCR-RFLP analysis of the rDNA ITS region of six P. helicoides isolates investigated, including the type culture, intraspecific variation was found at the HhaI site. The total length of fragments was longer than before cutting, indicating sequence heterogeneity within isolates. Digestion of the cloned rDNA ITS region derived from seven isolates with HhaI revealed polymorphisms among and within single zoospore isolates, and variability of the region was also present among the clones derived from the same isolate. To test whether the rDNA ITS region of closely related species and other regions in the genome of P. helicoides are also variable, the rDNA ITS region of P. ultimum and the cytochrome oxydase II (cox II) gene encoded in mitochondria were sequenced. P. ultimum had little variation in the rDNA ITS region. The cox II gene sequences of both species revealed only a low intraspecific variability and no intra-isolate variation. In the phylogenic tree based on the rDNA ITS sequences, all clones of P. helicoides formed one large clade that was distinct from the clades comprising morphologically similar species, such as P. oedochilum and P. ostracodes, and was closely related to P. chamaehyphon rather than the other species.     

Analysis of nrDNA polymorphism in closely related diploid sexual, tetraploid sexual and polyploid agamospermous species

Nuclear sequences of ITS1-5.8S-ITS2 region of rDNA may be an important source of phylogenetically informative data provided that nrDNA is cloned and the character of sequence variation of clones is properly analyzed. nrDNA of selected Taraxacum sections was studied to show sequence variation differences among diploid sexual, tetraploid sexual and polyploid agamospermous species. We examined nucleotide characteristics, substitution pattern, secondary structure, and the phylogenetic utility of ITS1-5.8S-ITS2 from 301 clones of 32 species representing 11 sections. The most divergent sequences of ITS1&2 differed by 17.1% and in 5.8S only by 3.7%. The ITS1-5.8S-ITS2 characteristics, integrity and also stability of secondary structures confirmed that pseudogenes are not responsible for the above variation. The within-individual polymorphism of clones implies that the concerted evolution of ITS cistron of agamospermous polyploid Taraxacum is remarkably suppressed. Sequences of ITS clones proved to be a useful tool for mapping pathways of complex reticulation (polyploid hybridity) in agamospermous Taraxacum.     

STRUCTURE AND EVOLUTION OF THE RDNA INTERNAL TRANSCRIBED SPACER (ITS) REGION 2 IN THE SYMBIOTIC DINOFLAGELLATES (SYMBIODINIUM,DINOPHYTA)1

Internal transcribed spacer (ITS) regions of the eukaryotic rDNA operon are integral to the correct processing and maturation of rRNAs. To further understand the evolution of this region, we elucidated the secondary structure of ITS2 from representatives of the eight divergent clades of Symbiodinium Freud., a large genus of dinoflagellate endosymbionts occurring in association with zooxanthellate marine protists and invertebrates. Symbiodinium ITS2 molecules folded into one of two distinct conformations. One conformation, the “four‐fingered hand” model, has been described from a wide variety of eukaryotes, including free‐living dinoflagellates. A monophyletic assemblage comprising several Symbiodinium clades shared an unusual conformation, a five‐stem model previously known only from drosopholids, indicating that it arose in the common ancestor to this “superclade” of Symbiodinium. Several conserved features were identified in the ITS2 secondary structures, including a pyrimidine–pyrimidine bulge and a highly conserved 11 bp sequence motif, that correspond to known processing sites in other eukaryotes. Lastly, the ITS2 structural data are discussed in the context of Symbiodinium evolution, phylogenetics, and ecology.     

Nuclear ribosomal DNA sequence polymorphism and hybridization in checker mallows (Sidalcea, Malvaceae)

Checker mallows (Sidalcea, Malvaceae) constitute a western North American genus of annuals and perennials that have been regarded as taxonomically difficult because of complex patterns of morphological variation putatively stemming from hybridization and polyploidy. In recent molecular phylogenetic investigations extensive polymorphism was observed in the internal and external transcribed spacers (ITS and ETS) of 18S-26S nuclear ribosomal DNA for some Sidalcea samples. To resolve the evolutionary basis for this polymorphism and to readdress the evolutionary impact of hybridization in Sidalcea we cloned and sequenced the polymorphic DNAs and included the clones in phylogenetic analyses together with direct sequences of non-polymorphic samples. The positions of cloned spacer sequences in the phylogenetic trees suggest that S. reptans and two subspecies of S. malviflora may have been influenced by past hybridization with lineages of the "glaucescens" clade. Polymorphic sequence patterns in other taxa may be a result of extensive interbreeding within young clades, in keeping with the minimal sequence divergence, largely overlapping geographic distributions and morphology, and ploidy variation in these groups. Other possible explanations for polymorphic sequences in members of Sidalcea include slow concerted evolution relative to mutation rates, incomplete lineage sorting, and recent pseudogene formation.     

Pseudo-nitzschia pungens (Bacillariophyceae): A cosmopolitan diatom species?

Genetic, reproductive and morphological variation were studied in 193 global strains of the marine diatom species Pseudo-nitzschia pungens (Grunow ex Cleve) Hasle to assess potential intraspecific variation and biogeographic distribution patterns. Genetic differentiation between allo- and sympatric strains was investigated using the ITS1–5.8S–ITS2 rDNA region. Three ITS clades were found. Clones of opposite mating type were sexually compatible within clades I or II, and viable F1 hybrid offspring were produced in crosses between them. The molecular differences between these clades were correlated with slight but consistent morphological differences. At present, nothing can be said about morphology and mating behavior for clade III clones because only ITS data were available. The three ITS clades showed different geographic distributions. Clade II was restricted to the NE Pacific, whereas clones belonging to clade III originated from geographically widely separated areas (Vietnam, China and Mexico). ITS clade I was recovered in all locations studied: the North Sea (Belgium, The Netherlands, France), the eastern and western N Atlantic (Spain, Canada), the NW and S Pacific (Japan, New Zealand) and the NE Pacific (Washington State). Clade I thus appears to be globally distributed in temperate coastal areas and provides the first strong evidence to date for the global distribution of a biologically, genetically and morphologically defined diatom species.     

Transatlantic disjunction in fleshy fungi. I. The Sparassis crispa complex

Phylogenies based on ITS and LSU sequences show that the Sparassis crispa complex comprises several monophyletic clades, in some cases co\rresponding to named taxa (i.e. S. crispa, S. radicata), but others lacking names (i.e. eastern and southwestern North American S. “crispa”). In our study, morphological examination of numerous collections also distinguished subtle differences correlated with geographic distribution. Underlying these problems, several taxa lacked type specimens for taxonomic analysis. In this paper, appropriate epitypes are designated and names assigned. Extensive sexual compatibility experiments, described within, indicate that monokaryon, haploid isolates of collections from North America and Europe are consistently sexually compatible to some degree. Inherent in the study, different “species concepts” were tested, with the “biological species concept,” based on sexual compatibility, being the least restrictive. We propose two new taxa, S. americana and S. americana f. arizonica.     

Nuclear ribosomal DNA sequence polymorphism and hybridization in checker mallows (Sidalcea, Malvaceae)

Checker mallows (Sidalcea, Malvaceae) constitute a western North American genus of annuals and perennials that have been regarded as taxonomically difficult because of complex patterns of morphological variation putatively stemming from hybridization and polyploidy. In recent molecular phylogenetic investigations extensive polymorphism was observed in the internal and external transcribed spacers (ITS and ETS) of 18S–26S nuclear ribosomal DNA for some Sidalcea samples. To resolve the evolutionary basis for this polymorphism and to readdress the evolutionary impact of hybridization in Sidalcea we cloned and sequenced the polymorphic DNAs and included the clones in phylogenetic analyses together with direct sequences of non-polymorphic samples. The positions of cloned spacer sequences in the phylogenetic trees suggest that S. reptans and two subspecies of S. malviflora may have been influenced by past hybridization with lineages of the “glaucescens” clade. Polymorphic sequence patterns in other taxa may be a result of extensive interbreeding within young clades, in keeping with the minimal sequence divergence, largely overlapping geographic distributions and morphology, and ploidy variation in these groups. Other possible explanations for polymorphic sequences in members of Sidalcea include slow concerted evolution relative to mutation rates, incomplete lineage sorting, and recent pseudogene formation.     

Evolution of temperature responses in the Cladophora vagabunda complex and the C. albida/sericea complex (Chlorophyta)

Differentiation in temperature responses (survival and growth) was investigated among isolates of two tropical to temperate green algal lineages: the Cladophora vagabunda complex and the C. albida/sericea complex. The results were analysed in relation to published data on 18S rRNA and ITS sequence divergence, which have shown that the overall degree of genetic divergence is similar in the two lineages but that very different patterns of radiation have occurred. In the C. vagabunda complex, the two main clades in the well-resolved phylogenetic tree differed mainly in their tolerance to low temperatures. Within-clade variation was no stronger in the Atlantic/Pacific than in the all-Pacific clade. In the C. albida/sericea complex, six distinctive ITS types indicated early radiation. Although distinctive differences were found between some of these types, the thermal responses of others were very similar, indicating physiological stasis. In both lineages there was evidence for some adaptation to local temperature regimes but phylogenetic constraints were generally more important. Isolates with the same ITS sequences showed similar temperature responses even though collected from different climate zones. Evidence was found for a physiological trade-off between growth at high and at low temperatures in the C. albida/sericea complex, whereas, in the C. vagabunda complex, one clade showed more eurythermal growth responses than the other. In the C. vagabunda complex, which is the ancestral lineage of the C. albida/sericea complex, major differentiation was found in cold tolerance but not in heat tolerance, whereas the reverse pattern was found in the derived C. albida/sericea complex. These findings suggest that an acquisition of cold tolerance preceded the loss of heat tolerance during adaptation to colder climates.     

Determination of internal transcribed spacer regions (ITS) in Trichomonas vaginalis isolates and differentiation among Trichomonas species

The nucleotide sequence of the 5.8S rRNA gene and the flanked internal transcribed spacer (ITS) regions of six Trichomonas vaginalis isolates with different metronidazole sensitivity and geographic origin were genotyped. A multiple sequence alignment was performed with different sequences of other isolates available at the GenBank/EMBL/DDBJ databases, which revealed 5 different sequence patterns. Although a stable mutation in position 66 of the ITS1 (C66T) was observed in 26% (9/34) of the T. vaginalis sequences analyzed, there was 99.7% ITS nucleotide sequence identity among isolates for this sequence. The nucleotide sequence variation among other species of the genus Trichomonas ranged from 3.4% to 9.1%. Surprisingly, the % identity between T. vaginalis and Pentatrichomonas hominis was ~ 83%. There was > 40% divergence in the ITS sequence between T. vaginalis and Tritrichomonas spp., including Tritrichomonas augusta, Tritrichomonas muris, and Tritrichomonas nonconforma and with Tetratrichomonas prowazeki. Dendrograms grouped the trichomonadid sequences in robust clades according to their genera. The absence of nucleotide divergence in the hypervariable ITS regions between T. vaginalis isolates suggests the early divergence of the parasite. Importantly, these data show this ITS1-5.8S rRNA-ITS2 region suitable for inter-species differentiation.