Weak prezygotic isolating mechanisms in threatened Caribbean Acropora corals

The Caribbean corals, Acropora palmata and A. cervicornis, recently have undergone drastic declines primarily as a result of disease. Previous molecular studies have demonstrated that these species form a hybrid (A. prolifera) that varies in abundance throughout the range of the parental distribution. There is variable unidirectional introgression across loci and sites of A. palmata genes flowing into A. cervicornis. Here we examine the efficacy of prezygotic reproductive isolating mechanisms within these corals including spawning times and choice and no-choice fertilization crosses. We show that these species have subtly different mean but overlapping spawning times, suggesting that temporal isolation is likely not an effective barrier to hybridization. We found species-specific differences in gametic incompatibilities. Acropora palmata eggs were relatively resistant to hybridization, especially when conspecific sperm are available to outcompete heterospecific sperm. Acropora cervicornis eggs demonstrated no evidence for gametic incompatibility and no evidence of reduced viability after aging four hours. This asymmetry in compatibility matches previous genetic data on unidirectional introgression.     

Phylogeny of Populus (Salicaceae) based on nucleotide sequences of chloroplast TRNT-TRNF region and nuclear rDNA

The species of the genus Populus, collectively known as poplars, are widely distributed over the northern hemisphere and well known for their ecological, economical, and evolutionary importance. The extensive interspecific hybridization and high morphological diversity in this group pose difficulties in identifying taxonomic units for comparative evolutionary studies and systematics. To understand the evolutionary relationships among poplars and to provide a framework for biosystematic classification, we reconstructed a phylogeny of the genus Populus based on nucleotide sequences of three noncoding regions of the chloroplast DNA (intron of trnL and intergenic regions of trnT-trnL and trnL-trnF) and ITS1 and ITS2 of the nuclear rDNA. The resulting phylogenetic trees showed polyphyletic relationships among species in the sections Tacamahaca and Aigeiros. Based on chloroplast DNA sequence data, P. nigra had a close affinity to species of section Populus, whereas nuclear DNA sequence data suggested a close relationship between P. nigra and species of the section Aigeiros, suggesting a possible hybrid origin for P. nigra. Similarly, the chloroplast DNA sequences of P. tristis and P. szechuanica were similar to that of the species of section Aigeiros, while the nuclear sequences revealed a close affinity to species of the section Tacamahaca, suggesting a hybrid origin for these two Asiatic balsam poplars. The incongruence between phylogenetic trees based on nuclear- and chloroplast-DNA sequence data suggests a reticulate evolution in the genus Populus.     

The origin of the C-genome and cytoplasm of <Emphasis Type="Italic">Avena</Emphasis> polyploids

The contribution of C-genome diploid species to the evolution of polyploid oats was studied using C-genome ITS-specific primers. SCAR analysis among Avena accessions confirmed the presence of C-genome ITS1-5.8S-ITS2 sequences in the genome of AACC and AACCDD polyploids. In situ hybridization and screening of more than a thousand rRNA clones in Avena polyploid species containing the C-genome revealed substantial C-genome rRNA sequence elimination. C-genome clones sequenced and Maximum Likelihood Parsimony analysis revealed close proximity to Avena ventricosa ITS1-5.8S-ITS2 sequences, providing strong evidence of the latter's active role in the evolution of tetraploid and hexaploid oats. In addition, cloning and sequencing of the chloroplastic trnL intron among the most representative Avena species verified the maternal origin of A-genome for the AACC interspecific hybrid formation, which was the genetic bridge for the establishment of cultivated hexaploid oats.     

ITS sequence variation and concerted evolution in the natural hybrid species Malus toringoides

The internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) is one of the most used molecular characters in plant systematics. Our previous studies based on morphological analysis and ITS sequence variation suggested that Malus toringoides (Rehd.) Hughes is derived from hybridization between M. transitoria (Batal.) Schneid. and M. kansuensis (Batal.) Schneid. To further understand the variation pattern of ITS sequences in M. toringoides, and to elucidate the evolutionary processes that affect ITS sequence variation after hybridization, we sampled 99 accessions from multiple populations of the hybrid and parental species, and then obtained totally 254 ITS sequences by cloning and sequencing. Our ITS variation data demonstrates three outcomes of ITS repeats after hybrid speciation. ～ 27–41% of M. toringoides have only M. transitoria type ITS sequence, ～ 40–70% have M. transitoria type ITS sequence plus one or two chimeric ITS sequences generated by recombination between parental ITS sequences, and six accessions retain both parental type ITS sequences. The plausible evolutionary processes that created the observed ITS variations were inferred to be the joint actions of recombination, concerted evolution, pseudogenization and backcrossing. Our study provides further understandings of the variation model of ITS repeats after hybridization as well as the evolution of M. toringoides after its hybrid speciation.     

A molecular approach to detect hybridization between bream Abramis brama, roach Rutlius rutilus and rudd Scardinius erythrophthalmus

Hybridization between roach Rutilus rutilus , bream Abramis brama and rudd Scardinius erythrophthalmus was investigated using morphological and genetic analysis employing both nuclear (ITS1) and mitochondrial (cytochrome b ) markers. Allele-specific amplification (ASA) reactions for both markers were developed and ITS1 sequence data for all three species are presented. Sequencing detected two ITS1 haplotypes within both roach and bream which most likely evolved in isolation and were subsequently brought together as a result of restocking by anglers. Analysis of cloned hybrid nuclear ribosomal DNA revealed evidence of recombination between parental ITS1 sequences. ASA proved to be an effective method for identifying hybrids and detected multiple ITS1 copies in fishes identified as purebred by morphological analysis. In addition this suggests post-F1 hybridization and introgression may be occurring between roach and bream, and rudd and bream, although some barriers appear to be suppressing backcrosses within the hybrid population. Analysis of the hybrid population demonstrated that hybridization has occurred in both directions.     

Spawning times,reproductive compatibilities and genetic structuring in the Acropora aspera group: evidence for natural hybridization and semi-permeable species boundaries in corals

Species boundaries among five sympatric coral species of the Indo-Pacific Acropora aspera group were examined by a combination of in vitro breeding trials, comparisons of spawning times and DNA sequence analysis of ribosomal DNA internal transcribed spacer (rDNA ITS) and 5.8S regions. The breeding trials showed that reproductive compatibility exists between at least some colonies of all the species pairs tested, suggesting a large potential for natural hybridization and introgression. The Acropora ITS regions exhibited extremely high levels of variability (up to approximately 62% for ITS1, approximately 11% for 5.8S and approximately 43% for ITS2), but most of the variation was shared among four of the five species, A. millepora, A. papillare, A. pulchra and A. spathulata, consistent with extensive introgression. Phylogenetic analyses did not resolve these four species as distinct clusters across a wide biogeographic region stretching from the southern Great Barrier Reef to Papua New Guinea. However, most colonies of the fifth species, A. aspera, constituted a distinct clade in phylogenetic analyses. This is consistent with our observations of a semi-permeable temporal barrier involving differences in spawning times between this and the other four species. Although the majority of colonies of all five species generally spawned within 90 min of each other, in two out of four years, gametes were absent prior to mass spawning episodes from at least some A. aspera colonies. Hence, our data suggest that transient reproductive barriers may be the result of year-to-year variation in the date of spawning and that this difference in spawning time contributes to the genetic structure detected among Acropora species in this group. Occasional leakage through the reproductive barrier was confirmed by the observation of A. aspera xA. pulchra F1 hybrids, identified based on additivity of ITS sequences.     

The identification of vahlkampfiid amoebae by ITS sequencing

We have determined the internal transcribed spacer (ITS) sequences (including the 5.8S ribosomal DNA) of 30 strains of 14 species belonging to eight vahlkampfiid genera. Each previously described species has a specific ITS sequence, except for Tetramitus aberdonicus, Tetramitus thorntoni, and Tetramitus jugosus, which have identical ITS sequences. The latter three may therefore constitute a single species despite their apparent phenotypic differences. The ITS sequence appears to be conserved within a species. The species Willaertia magna appears to be ubiquitous. The 5.8S rDNA sequences of Singhamoeba horticola and Learamoeba waccamwensis indicate that they do not represent different genera, but both belong to the genus Tetramitus. The ITS sequences of 16 undescribed vahlkampfiid isolates were determined. Based on these sequences, seven isolates were identified as belonging to described species, while nine probably represent seven new species. Five of these presumed new species belong to the genus Tetramitus, and one each to the genera Vahlkampfia and Paravahlkampfia.     

The internal transcribed spacers and 5.8S rRNA gene show extensive diversity among isolates of the Cryptococcus neoformans species complex

Sequences of the internal transcribed spacer (ITS) region including the 5.8S rRNA gene delineated seven genotypes within the three varieties of Cryptococcus neoformans via specific combinations of eight nucleotide differences located at positions 10, 11, 15, 19, 108 (ITS1), 221 (5.8S), 298 and 346 (ITS2). The ITS types correlated to polymerase chain reaction fingerprint/random amplification of polymorphic DNA (RAPD) molecular types: with ITS type 1 (ATACTAGC)=C. neoformans var. grubii, molecular types VNI+VNII and the serotype A allele of the AD hybrid, VNIIIA; ITS type 2 (ATATAGGC)=the serotype D allele of the AD hybrid, VNIIIB, and C. neoformans var. neoformans, VNIV; and ITS type 3 (GCGCTGGC) and ITS type 7 (ACGCTGGC)=VGI=RAPD type III, ITS type 4 (ACACTGAC)=VGII=RAPD type II, ITS type 5: (ACACTGGG)=VGIII=RAPD type I, ITS type 6 (ACACTGGC)=VGIV=RAPD type IV, all corresponding to C. neoformans var. gattii. Cloned sequences from serotype AD revealed that the hybrid serotype is diploid at the ITS1-5.8S-ITS2 locus carrying the ITS type 1 (ATACTAGC) and the ITS type 2 (ATATAGGC) alleles. ITS sequencing is a useful technique for genotyping the three C. neoformans varieties and for subtyping within C. neoformans var. gattii.     

Intrasporal variability of ribosomal sequences in the endomycorrhizal fungus Gigaspora margarita

The sequence variability of the ribosomal internal transcribed spacer (ITS) region, which comprises the 5.8 gene and the flanking regions ITS1 and ITS2, was investigated in the arbuscular mycorrhizal fungus Gigaspora margarita. DNA analysis of a multispore preparation and three single spores led to the identification of 11 slightly different sequences (three variants within a single spore), indicating substantial intersporal and intrasporal genetic variability (up to 9% sequence divergence). The sequence variations inside a single spore may be higher than that observed between spores. Even so, primers designed on the ITS1 and ITS2 regions identified Gi. margarita isolates and detected the endophyte during colonization.     

Using AFLP to resolve phylogenetic relationships in a morphologically diversified plant species complex when nuclear and chloroplast sequences fail to reveal variability

Inferring phylogenetic relationships among closely related plant species is often difficult due to the lack of molecular markers exhibiting enough nucleotide variability at this taxonomic level. Moreover, gene tree does not necessary represent the true species tree because of random sorting of polymorphic alleles in different lineages. A solution to these problems is to use many amplified fragment length polymorphisms (AFLP) distributed throughout the whole genome, to infer cladistic and phenetic among-species relationships. Phylogenetic relationships among interfertile species of Trollius L. (Ranunculaceae) were investigated using nuclear DNA (ITS1+5.8S rRNA+ITS2) and chloroplast DNA (trnL intron and trnL-trnF intergene spacer) sequences, and AFLP markers. ITS sequences were not informative at the intrageneric level, but confirmed the sister relationship between Trollius and Adonis genera, and provided new information on the phylogenetic relationships among five Ranunculaceae genera. Chloroplast DNA was more informative among Trollius species, but not consistent with the sections previously described. AFLP proved to be a powerful tool to resolve the complex genetic relationships between the morphological entities constituting the genus Trollius. Although as much as 76.1% of the total AFLP variability was found within a priori defined morphological groups, the remaining 23.9% variability differentiating groups was sufficient to generate congruent and robust cladistic and phenetic trees. Several morphological traits, independent from those used to define groups, were mapped onto the molecular phylogeny, and their evolution discussed in relation to the absence/presence of pollinator-seed parasite Chiastocheta flies.     

Restricted gene flow in the Caribbean staghorn coral Acropora cervicornis: implications for the recovery of endangered reefs

Coral reef conservation requires information about the distance over which healthy reefs can rescue damaged reefs through input of coral larvae. This information is desperately needed in the Caribbean where the 2 dominant shallow water corals Acropora cervicornis and Acropora palmata have suffered unprecedented declines. Here we compare the population genetic structure in the staghorn coral A. cervicornis across the greater Caribbean using DNA sequence data from 1 mitochondrial and 3 nuclear genes. Data from 160 individuals from 22 populations and 9 regions show that A. cervicornis exhibits significant population genetic structure across the greater Caribbean in both the mitochondrial (Phi(st) = 0.130) and nuclear data (Phi(st) = 0.067). The highest population structure was observed in the species' own, native mtDNA haplotypes (Phi(st) = 0.235). Introgressed alleles from A. palmata tempered higher population structure in A. cervicornis over regional scales but in some cases generated highly localized "introgression hot spots" and fine-scale genetic structure among reefs separated by as few as 2 km. These data show that larval dispersal over moderate or long distances (>500 km) is limited for this threatened species and in some cases locally limited as well. Thus, the endangered Caribbean staghorn corals require local source populations for their recovery and targeted conservation efforts over spatial scales much smaller than the hundreds to thousands of kilometers usually proposed for marine reserves.     

裸子植物DNA条形码ITS2高通用性引物的筛选

DNA条形码技术是利用标准DNA片段进行准确快速鉴定物种的一种方法,理想的DNA条形码片段应具有高通用性。虽然核糖体DNA内部转录间隔区II（ITS2）被建议作为种子植物有效的DNA条形码,但目前裸子植物还没有通用性高的引物可用。为获得高通用性的ITS2引物,本研究基于裸子植物55个属的5．8S基因的保守序列区设计了3个正向引物,与已有的ITS反向引物组合,组成了7对ITS2引物进行通用性的评价。选取了裸子植物8目、12科和40属的56个种用于本文的研究。引物组合5．8SR／ITS4、5．8SRa／ITS4和5．8SF2／S3R因为在科水平评价中通用性低或者产生的PCR产物有双带,因而排除在全部物种水平上进一步评价。其余4对引物（GYM-5．8SF1／ITS4、GYM-5．8SFl／S3R、GYM-5．8SF2／ITS4和S2F／S3R）在56个物种的PCR检测中,均有100％的扩增率。基于PCR产物的亮度、序列质量和正反向引物覆盖率的综合评价,建议引物GYM_5．8SF2／ITS4作为裸子植物条形码片段ITS2最好的通用引物。     

青藏高原黄绿蜜环菌纯培养菌种的分离培养及分子鉴定

首次从采自青藏高原、与高原牧草嵩草属Kobresia草本植物形成外生菌根的黄绿蜜环菌Armillarialuteo-virens子实体中分离获得一组织分离菌株,运用rDNA-ITS和rDNA-IGS-1测序技术对该组织分离菌株是否为黄绿蜜环菌的纯培养菌种进行分子鉴定,并基于黄绿蜜环菌的5.8S/ITS和IGS-1序列进行核酸序列数据库GenBank同源性检索比对、构建系统发育树。结果表明,本研究获得的黄绿蜜环菌子实体组织分离菌株即为其纯培养菌种。基于ITS的系统发育分析表明黄绿蜜环菌与口蘑科内其它属间物种的系统发育关系较远;基于IGS-1的系统发育分析表明黄绿蜜环菌与蜜环菌属内的其它种序列差异较大,系统发育关系较远,而与Lepiota属内的部分种具有较近的系统发育关系。本研究首次基于分子手段对我国青藏高原的黄绿蜜环菌种进行了分离培养、分子鉴定和系统发育分析,为黄绿蜜环菌的科学分类提供了分子依据。     

Determination of Trichuris skrjabini by sequencing of the ITS1-5.8S-ITS2 segment of the ribosomal DNA: comparative molecular study of different species of trichurids

Adults of Trichuris skrjahini have been isolated from the cecum of caprine hosts (Capra hircus), Trichuris ovis and Trichuris globulosa from Ovis aries (sheep) and C. hircus (goats), and Trichuris leporis from Lepus europaeus (rabbits) in Spain. Genomic DNA was isolated and the ITS1-5.8S-ITS2 segment from the ribosomal DNA (rDNA) was amplified and sequenced by polymerase chain reaction (PCR) techniques. The ITS1 of T. skrjabini, T. ovis, T. globulosa, and T. leporis was 495, 757, 757, and 536 nucleotides in length, respectively, and had G + C contents of 59.6, 58.7, 58.7, and 60.8%, respectively. Intraindividual variation was detected in the ITSI sequences of the 4 species. Furthermore, the 5.8S sequences of T. skrjabini, T. ovis, T. globulosa, and T. leporis were compared. A total of 157, 152, 153, and 157 nucleotides in length was observed in the 5.8S sequences of these 4 species, respectively. There were no sequence differences of ITS1 and 5.8S products between T. ovis and T. globulosa. Nevertheless, clear differences were detected between the ITS1 sequences of T. skrjabini, T. ovis, T. leporis, Trichuris muris, and T. arvicolae. The ITS2 fragment from the rDNA of T. skrjabini was sequenced. A comparative study of the ITS2 sequence of T. skrjabini with the previously published ITS2 sequence data of T. ovis, T. leporis, T. muris, and T. arvicolae suggested that the combined use of sequence data from both spacers would be useful in the molecular characterization of trichurid parasites.     

Phylogenetic Use of Noncoding Regions in the GenusGentianaL.: ChloroplasttrnL (UAA) Intron versus Nuclear Ribosomal Internal Transcribed Spacer Sequences

Sequence divergence was estimated within noncoding sequences of both chloroplast DNA (cpDNA)trnL (UAA) intron and nuclear ribosomal DNA (nrDNA) internal transcribed spacer sequences (ITS1 and ITS2) for 10 species of the genusGentianaL. (Gentianaceae). Comparisons of evolutionary rates among these sequences (cpDNA versus nrDNA, ITS1 versus ITS2) were performed. It appears that sequence divergence is on average two to three times higher in ITSs than in thetrnL intron sequences and higher in ITS1 than in ITS2. Both the cpDNA intron and ITSs of nrDNA give concordant phylogenetic trees. However, the ITS-based phylogeny displays higher bootstrap values. At the intrageneric level, at least inGentiana,ITSs (especially ITS2) sequences seem to be more appropriate in the assessment of plant phylogenies. Nevertheless, the cpDNAtrnL intron seems to be preferable at the intergeneric level.     

Genetic relatedness of Trichoderma sect. Pachybasium species based on molecular approaches

Molecular approaches including internal transcribed spacer (ITS) sequences of ribosomal DNA, universal primer polymerase chain reaction (UP-PCR) fingerprinting, and DNA-DNA hybridization were used to study the genetic relatedness of species within Trichoderma sect. Pachybasium. In the analysis of ITS and 5.8S sequences of ribosomal DNA, parsimony analysis demonstrated that forty-one strains were distributed into five main groups supported by high bootstrap values. The species of Trichoderma sect. Pachybasium were clustered into groups I, II, and IV, with the strains of Trichoderma fasculatum and Trichoderma strictipile forming a separate branch, an independent group V. Some species within each group showed nearly identical sequence differences (fewer than 1-3 bp). UP-PCR and DNA-DNA hybridization were further used to clarify the genetic relatedness of these species with highly similar ITS sequences. Highly similar or identical UP-PCR profiles and high values of DNA complementarity (>70%) were observed among some species, Trichoderma hamatum and Trichoderma pubescens; Trichoderma croceum, Trichoderma polysporum and Trichoderma album, Trichoderma crassum and Trichoderma flavofuscum; and Trichoderma strictipile and Trichoderma fasciculatum. Although every species can be differentiated morphologically, the species showed highly similar molecular characteristics in the above cases, indicating that they could be conspecific. However, in some cases (Trithoderma longipile, T. crassum and T. flavofuscum; Trichoderma fertile and Trichoderma minutisporum; Trichoderma tomentosum, Trichoderma inhamatum and Trichoderma harzianum) there were discriminative patterns of UP-PCR and (or) low levels (<50%) of DNA-DNA hybridization; even their ITS sequences were similar, suggesting a closely phylogenetic relationship.     

Nucleotide sequence diversity of rDNA internal transcribed spacers extracted from conidia and cleistothecia of several powdery mildew fungi

An improved protocol, including DNA extraction with Chelex, two amplifications with a nested primer set, and DNA purification by electrophoresis, made it possible to analyze nuclear rDNA sequences of powdery mildew fungi using at most several hundred conidia or 20 cleistothecia. Nucleotide sequence diversity of the nuclear rDNA region containing the two internal transcribed spacers (ITS1 and ITS2) and 5.8S rRNA gene derived from conidia and cleistothecia was investigated for four kinds of powdery mildew fungi including two isolates of the same species. The results showed that the nucleotide sequences of the nuclear rDNA region were highly conserved between the teleomorph and the anamorph. Thus, the nucleotide sequence data obtained from either developmental stage can be used for phylogenetic studies of powdery mildew fungi. The nucleotide sequences of the 5.8S rRNA genes of the four species were highly conserved, but those of their ITS regions were variable. This suggests that the nuclear rDNA region is not suitable for phylogenetic studies of distantly related powdery mildew fungi, because too much sequence diversity exists, within the ITS, and too little phylogenetic information is contained within the 5.8S rRNA gene. However, the ITS region will be useful for phylogenetic comparison of closely related species or intraspecies. Contribution No. 132 from the Laboratory of Plant Pathology, Mie University.     

Three divergent rDNA clusters predate the species divergence in Quercus petraea (Matt.) Liebl. and Quercus robur L

Quercus petraea and Quercus robur are two closely related oak species that frequently hybridize. We sequenced 70 clones containing the 5.8S and ITS2 regions of ribosomal DNA (rDNA) from these two species and did not detect a species-specific difference. Surprisingly, three divergent (up to 12.6%) rDNA families were identified in both species, indicating that they predate the speciation event. Despite a large between-rDNA-families divergence, rDNA sequences were very similar within families, suggesting ongoing concerted evolution. Expression analysis, relative-rate tests, and mutation spectrum analyses indicated that only a single rDNA family is functional. We propose that past hybridization events, combined with nucleolar dominance, were the evolutionary processes underlying the contemporary rDNA variability in Q. petraea and Q. robur.     

Molecular detection of Bartonella species infecting rodents in Slovenia

Rodents, collected in three zoogeographical regions across Slovenia, were tested for the presence of bartonellae using direct PCR-based amplification of 16S/23S rRNA gene intergenic spacer region (ITS) fragments from splenic DNA extracts. Bartonella DNA was detected in four species of rodents, Apodemus flavicollis, Apodemus sylvaticus, Apodemus agrarius and Clethrionomys glareolus, in all three zoogeographic regions at an overall prevalence of 40.4%. The prevalence of infection varied significantly between rodent species and zoogeographical regions. Comparison of ITS sequences obtained from bartonellae revealed six sequence variants. Four of these matched the ITS sequences of the previously recognized species, Bartonella taylorii, Bartonella grahamii, Bartonella doshiae and Bartonella birtlesii, but one was new. The identity of the bartonellae from which the novel ITS sequences was obtained were further assessed by sequence analysis of cell division protein-encoding gene (ftsZ) fragments. This analysis demonstrated that the strain is most likely a representative of possible new species within the genus.