MONOPHYLY OF ANEUPLOID ASTRAGALUS (FABACEAE): EVIDENCE FROM NUCLEAR RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER SEQUENCES

Evolutionary relationships within Astragalus L. (Fabaceae) were inferred from nucleotide sequence variation in nuclear ribosomal DNA of both New World and Old World species. The internal transcribed spacer regions (ITS) of 18S–26S nuclear ribosomal DNA from representatives of 26 species of Astragalus, three species of Oxytropis DC., and two outgroup taxa were analyzed by polymerase chain reaction amplification and direct DNA sequencing. The length of the ITS 1 region within these taxa varied from 221 to 231 bp, while ITS 2 varied in length from 207 to 217 bp. Of the aligned, unambiguous positions, approximately 34% were variable in each spacer region. In pairwise comparisons among Astragalus species and outgroup taxa, sequence divergence at these sites ranged from 0 to 18.8% in ITS 1 and from 0 to 21.7% in ITS 2. Parsimony analyses of these sequences resulted in a well-resolved phylogeny that is highly concordant with previous cytogenetic and chloroplast DNA evidence for a major phylogenetic division in the genus. These data suggest that the New World aneuploid species of Astragalus form a monophyletic but morphologically cryptic group derived from euploid species of Old World (Eurasian) origin, which are consequently paraphyletic.     

Polyploid speciation inHedera (Araliaceae): Phylogenetic and biogeographic insights based on chromosome counts and ITS sequences

Variation in chromosome number and internal transcribed sequences (ITS) of nrDNA is used to infer phylogenetic relationships of a wide range ofHedera species. Polyploidy was found to be frequent inHedera, with diploid, tetraploid, hexaploid and octoploid populations being detected. Nucleotide additivity occurs in the ITS sequences of one tetraploid (H. hibernica) and two hexaploid species (H. maderensis, H. pastuchovii), suggesting that all three species originated by allopolyploidisation. ITS sequence polymorphism and nucleotide characters may indicate the presence of an ancient genome persistent only in some allopolyploid species. Phylogenetic analyses of ITS sequence data reveal two lineages ofHedera: one containing all sequences belonging to extant diploids plus the tetraploidH. algeriensis, and a second that includes this ancient ITS type and others exclusive to several polyploid species. The origin of the polyploids is evaluated on the basis of morphology, chromosome counts, ITS sequence polymorphism, and phylogenetic analyses. Reconstruction of reticulate evolution inHedera agrees with two allopolyploid areas on both sides of the Mediterranean basin. Morphological, molecular and cytological evidence also suggests an active dispersal ofHedera populations that may account for three independent introductions in Macaronesia.     

Molecular phylogenetics of Hippophae L. (Elaeagnaceae) based on the internal transcribed spacer (ITS) sequences of nrDNA

 The genus Hippophae comprises 7 species and 8 subspecies according to the latest classification, and has shown enormous ecological, nutrient and medicinal values. Here we analyzed the phylogenetic relationships among 15 taxa of the genus by comparing sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA). ITS sequences in Hippophae varied in length from 651 bp to 666 bp. The aligned sequences were 690 bp in length and 269 (39.0%) were variable sites with 150 being parsimony-informative. The amount of polymorphism observed within a taxon was extremely low in most taxa except for two putative hybrid species. The aligned sequences were analyzed by maximum parsimony (MP) and neighbor-joining (NJ) methods. In the strict consensus trees of parsimony analysis, the monophyly of Hippophae was supported by 100% bootstrap value. H. tibetana was at the basal position of the genus, and the remaining taxa formed two clades with high bootstrap support. The first clade included subspecies of H.␣rhamnoides and the other one consisted of remaining species. Parsimony analysis also suggested that the species H. tibetana, H. neurocarpa and H.␣salicifolia were all distinct. Although the sequence divergence among subspecies of H. rhamnoides was also remarkably high, the molecular data supported the monophyly of H. rhamnoides when H. rhamnoides subsp. gyantsensis Rousi was excxluded. The NJ trees showed essentially the same topology. The taxonomical arrangement that divided the genus into two sections was not supported based on the ITS sequences. However, the hybrid origin of H. goniocarpa and H. litangensis proposed previously was supported by the present ITS data. Received January 7, 2002; accepted May 10, 2002 Published online: November 22, 2002 Addresses of the authors: Kun Sun, Xuelin Chen, Ruijun Ma, Qin Wang, Institute of Botany, Northwest Normal University, Lanzhou 730070, China. Changbao Li, Song Ge (e-mail: gesong@ns.ibcas.ac.cn or song_ge@hotmail.com), Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China.     

18S‐Based Taxonomy and Intraspecific Its Sequence Variation in the Marine Fungal Endosymbiont Haloguignardia Irritans Infecting Cystoseira Osmundacea Along the Californian Coast

The marine ascomycete genus Haloguignardia occurs endophytically in members of the marine brown algal family Sargassaceae globally. This example of endosymbiosis has been morphologically described: the fungal component internally infects the algal host resulting in prolific cell growth, forming galls composed chiefly of host algal cells but containing fungal reproductive structures and vegetative hyphae. H. irritans induces the formation of galls in the brown algae Cystoseira osmundacea and Halidrys dioica along the Pacific coast from Oregon to Baja California, Mexico. Using culture‐independent molecular techniques, I sequenced the 18S rDNA gene region for H. irritans and generated a 18S‐based taxonomy consistent with the current taxonomy for this morphological species. In order to study intraspecific genetic variation in H. irritans, I have sequenced the ITS rDNA (ITS 1, 2 and the 5.8 s) regions for five separate gall‐tissue samples from Santa Rosa Island in southern California and for five samples from Monterey and Carmel in central California. Intraspecific DNA sequence variation in the ITS regions of H. irritans reveals consistent sequence divergence between sites sampled. The fungal ITS regions for H. irritans total 613 bp in length and contain 40 synapomorphic characters for a total of 6.5% variation in informative loci between southern and central Californian sites. This value is similar to those found for the ITS and other gene regions previously used by researchers investigating species boundaries at the intraspecific level in symbiotic, terrestrial fungi. In addition to ITS 1, 2 and the 5.8 s gene regions, I am currently using the 5’ end of the EF1a coding region to construct intraspecific genealogies for H. irritans. By comparing these genealogies to each other and to the geographic distribution of samples, I aim to determine if more than one genetic species is present within the morphological species H. irritans.     

Genetic Diversity of Ostreopsis ovata (Dinophyceae) from Malaysia

The genus Ostreopsis is an important component of benthic and epiphytic dinoflagellate assemblages in coral reefs and seaweed beds of Malaysia. Members of the species may produce toxins that contribute to ciguatera fish poisoning. In this study, two species have been isolated and cultured, Ostreopsis ovata and Ostreopsis lenticularis. Analyses of the 5.8S subunit and internal transcribed spacer regions ITS1 and ITS2 of the ribosomal RNA gene sequences of these two species showed that they are separate species, consistent with morphological designations. The nucleotide sequences of the 5.8S subunit and ITS1 and ITS2 regions of the rRNA gene were also used to evaluate the interpopulation and intrapopulation genetic diversity of O. ovata found in Malaysian waters. Results showed a low level of sequence divergence within populations. At the interpopulation level, the rRNA gene sequence distinguished two groups of genetically distinct strains, representative of a Malacca Straits group (isolates from Port Dickson) and a South China Sea group (isolates from Pulau Redang and Kota Kinabalu). Part of the sequences in the ITS regions may be useful in the design of oligonucleotide probes specific for each group. Results from this study show that the ITS regions can be used as genetic markers for taxonomic, biogeographic, and fine-scale population studies of this species. Received September 15, 2000; accepted December 15, 2000     

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.     

Sequence analysis of the ribosomal internal transcribed spacers region in spider mites (Prostigmata: Tetranychidae) occurring in citrus orchards in Eastern Spain: use for species discrimination

Tetranychus urticae is a polyphagous mite which is an important pest of citrus worldwide. This mite can be found feeding on many plant species occurring in the citrus agrosystem moving from weeds to trees. Because field samples consist of a mixture of different Tetranychidae species, as a first step necessary to further implement population characterisation of T. urticae, species‐discriminating criteria based on molecular techniques are needed. In this study, the nucleotide variation of the internal transcribed spacers (ITS) 1 and 2 and the intergenic 5.8S fragment of nuclear rDNA of T. urticae, Tetranychus turkestani, Tetranychus evansi, Tetranychus ludeni and Panonychus citri have been determined. Results demonstrate that for these species, the rDNA ITS2 regions are much more conserved than the corresponding rDNA ITS1. The high homogeneity of the ITS2 sequence observed among the specimens of T. urticae obtained from the same ecoregion makes this DNA sequence an excellent tool for species discrimination. ITS sequences differentiate not only species but also specimens from different geographical origin. Furthermore, polymerase chain reaction–restriction fragment length polymorphism analysis of the ITS2 proved adequate for a quick screening of high numbers of field samples.     

IDENTIFICATION OF PFIESTERIA PISCICIDA (DINOPHYCEAE) AND PFIESTERIA‐LIKE ORGANISMS USING INTERNAL TRANSCRIBED SPACER‐SPECIFIC PCR ASSAYS1

The putative harmful algal bloom dinoflagellate, Pfiesteria piscicida (Steidinger et Burkholder), frequently co‐occurs with other morphologically similar species collectively known as Pfiesteria‐like organisms (PLOs). This study specifically evaluated whether unique sequences in the internal transcribed spacer (ITS) regions, ITS1 and ITS2, could be used to develop PCR assays capable of detecting PLOs in natural assemblages. ITS regions were selected because they are more variable than the flanking small subunit or large subunit rRNA genes and more likely to contain species‐specific sequences. Sequencing of the ITS regions revealed unique oligonucleotide primer binding sites for Pfiesteria piscicida, Pfiesteria shumwayae (Glasgow et Burkholder), Florida “Lucy” species, two cryptoperidiniopsoid species, “H/V14” and “PLO21,” and the estuarine mixotroph, Karlodinium micrum (Leadbetter et Dodge). These PCR assays had a minimum sensitivity of 100 cells in a 100‐mL sample (1 cell·mL^?1) and were successfully used to detect PLOs in the St. Johns River system in Florida, USA. DNA purification and aspects of PCR assay development, PCR optimization, PCR assay controls, and collection of field samples are discussed.     

Testing DNA barcodes in closely related species of Curcuma (Zingiberaceae) from Myanmar and China

The genus Curcuma L. is commonly used as spices, medicines, dyes and ornamentals. Owing to its economic significance and lack of clear‐cut morphological differences between species, this genus is an ideal case for developing DNA barcodes. In this study, four chloroplast DNA regions (matK, rbcL, trnH‐psbA and trnL‐F) and one nuclear region (ITS2) were generated for 44 Curcuma species and five species from closely related genera, represented by 96 samples. PCR amplification success rate, intra‐ and inter‐specific genetic distance variation and the correct identification percentage were taken into account to assess candidate barcode regions. PCR and sequence success rate were high in matK (89.7%), rbcL (100%), trnH‐psbA (100%), trnL‐F (95.7%) and ITS2 (82.6%) regions. The results further showed that four candidate chloroplast barcoding regions (matK, rbcL, trnH‐psbA and trnL‐F) yield no barcode gaps, indicating that the genus Curcuma represents a challenging group for DNA barcoding. The ITS2 region presented large interspecific variation and provided the highest correct identification rates (46.7%) based on BLASTClust method among the five regions. However, the ITS2 only provided 7.9% based on NJ tree method. An increase in discriminatory power needs the development of more variable markers.     

Morphological and molecular characterization of Anopheles (Anopheles) maculipennis Meigen, type species of the genus and nominotypical member of the Maculipennis Complex

Abstract. Adults and larvae of Anopheles maculipennis Meigen were collected in Greece in May and June 2001. Larvae and the progeny of wild‐caught females were individually reared to obtain samples of all life stages for integrated morphological and molecular study. Specimens were identified on the basis of egg morphology and correlation of their ITS2 rDNA sequences with those in GenBank. The adult, pupal, larval (fourth‐instar) and egg stages are described, and all stages except the egg are illustrated. Partial sequence data are provided for the mitochondrial cytochrome c oxidase I (COI) gene and complete sequences for the nuclear ITS2 region. Additionally, the bionomics and distribution of the species are reviewed, and its taxonomy and systematics are discussed. This study provides the first fully integrated morphological and molecular assessment of An. maculipennis, the nominotypical member of the Maculipennis Complex, and the type species of genus Anopheles, and serves as a foundation for further studies of the complex and subfamily Anophelinae.     

Molecular phylogenetics of the subtribeGentianinae (Gentianaceae) inferred from the sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA

The internal transcribed spacer (ITS) regions of 18S–25S nuclear ribosomal DNA from representatives of 23 species of the subtribeGentianinae and one outgroup species (Centaurium capitatum) were analyzed by polymerase chain reaction amplification and direct DNA sequencing. Within the taxa analyzed, the length of the ITS1 region varied from 221 to 233 bp, ITS2 from 226 to 234 bp. Of the aligned sequences of 497 positions, 151 sites involved gaps or nucleotide ambiguity, 133 were invariable and 213 showed divergence. In pairwise comparisons among the taxa of the subtribeGentianinae and the outgroup, sequence divergence ranged from 1.3% to 34.1% in ITS1, from 0 to 28.1% in ITS2 and from 0.6% to 27.5% in combined ITS1 and ITS2. Phylogenetic trees generated from ITS sequences were highly resolutive and principally concordant with morphological classifications for the major phylogenetic divisions in the subtribe. An ancient divergence leading to two evolutionary lines was suggested in the subtribe by both DNA sequence and morphological data. One line encompasses the generaGentiana, Crawfurdia andTripterospermum, morphologically characterized by their glands on the base of ovary and their plicate corolla, while the other line involves all other members of the subcribe surveyed, characterized by their epipetalous glands and simple corolla without plicae.Megacodon, with glands on the base of ovary but without plicae on its corolla, was revealed to be more related to the latter group than to the former.Comastoma, Gentianella andGentianopsis were shown to be well-defined monophyletic genera.Pterygocalyx showed much closer affinity toGentianopsis than to any other genus. Some conflictions were detected in the genusSwertia.     

Radiation of the endemic genus Dendroseris (Asteraceae) on the Juan Fernandez Islands: evidence from sequences of the its regions of nuclear ribosomal DNA

Phylogenetic relationships among nine of the 11 species of the endemic genus Dendroseris on the Juan Fernandez Islands were inferred from nucleotide sequences of the internal transcribed spacer regions (ITS) of the 18-26S nuclear ribosomal DNA. Sequences were determined for 15 populations of Dendroseris and one population for each of two outgroups from the genera Sonchus and Sventenia. Little length variation was detected in the ITS regions of Dendroseris, with ITS 1 253 or 254 bp long and ITS 2 224 or 225 bp. The sequence data provide strong support for the holophyly of Dendroseris despite the distinct morphological differences among the three subgenera. The molecular data also indicate that subg. Dendroseris and Phoenicoseris are holophyletic, but do not support holophyly of subg. Rea. The ITS sequences did not resolve relationships among subgenera, supporting the hypothesis of rapid adaptive radiation of Dendroseris on the islands. Relative rate tests indicate that rates of nucleotide substitutions in the ITS regions are not significantly different among the different lineages of Dendroseris following adaptive radiation. Comparisons of average pairwise sequence divergence of Dendroseris species in the ITS regions and chloroplast genome indicated that ITS sequences have evolved about 38 times faster than cpDNA in the genus. Rates of ITS sequence divergence of Dendroseris were estimated to be faster than (3.94 ± 0.10) × 10^-9 per site per year, and likely (6.06 ±0.15) × 10^-9 per site per year.     

Phenology of the rhodomelarian algae Neorhodomela aculeata and Ceramium kondoi and their survival strategies against herbivorous snails

The seasonal growth and reproductive phenology of Neorhodomela aculeata (Perestenko) Masuda and Ceramium kondoi Yendo, and the food preferences of herbivorous snails were examined to elucidate (i) why snails select the fronds of N. aculeata for their habitat; and (ii) the survival strategies of the two red algae under grazing pressures. The maximal lengths and weights of both algal species were recorded for each season over a 12‐month period beginning with the spring of 2003. C. kondoi grew in length at a faster rate than N. aculeate, whereas the turf alga N. aculeata produced new branches from the tips of broken branches. The reproductive period of C. kondoi was between the spring and summer but the reproductive organs of N. aculeata were observed throughout the year. The algal loss rate of fresh N. aculeata to snails was low but snails had a food preference for N. aculeata when compared to C. kondoi in an artificial food experiment. These results indicate that snails may adapt to chemical compounds characteristic of N. aculeata and that the alga further reduces predation damage by its structural resistance. In conclusion, the survival strategies of C. kondoi appear to be rapid growth, seasonal sexual reproduction, and a delicately branched frond morphology that reduces stable feeding patterns of its predators plus high tissue nitrogen content, whereas the survival strategy of N. aculeata includes regenerative growth responses, structural toughness and chemical defenses while under the grazing pressure of herbivorous snails.     

Evaluation of six candidate DNA barcoding loci in Ficus (Moraceae) of China

Ficus, with about 755 species, diverse habits and complicated co‐evolutionary history with fig wasps, is a notoriously difficult group in taxonomy. DNA barcoding is expected to bring light to the identification of Ficus but needs evaluation of candidate loci. Based on five plastid loci (rbcL, matK, trnH‐psbA, psbK‐psbI, atpF‐atpH) and a nuclear locus [internal transcribed spacer (ITS)], we calculated genetic distances and DNA barcoding gaps individually and in combination and constructed phylogenetic trees to test their ability to distinguish the species of the genus. A total of 228 samples representing 63 putative species in Ficus (Moraceae) of China were included in this study. The results demonstrated that ITS has the most variable sites, greater intra‐ and inter‐specific divergences, the highest species discrimination rate (72%) and higher primer universality among the single loci. It is followed by psbK‐psbI and trnH‐psbA with moderate variation and considerably lower species discrimination rates (about 19%), whereas matK, rbcL and atpF‐atpH could not effectively separate the species. Among the possible combinations of loci, ITS + trnH‐psbA performed best but only marginally improved species resolution over ITS alone (75% vs. 72%). Therefore, we recommend using ITS as a single DNA barcoding locus in Ficus.     

Factors affecting photoreactivation in UVB-irradiated herbivorous spider mite (Tetranychus urticae)

To investigate and identify the ticks prevalent in the North East part of India, scanning electron microscope (SEM) and DNA sequence of nuclear second internal transcribed spacer (ITS2) and mitochondrial 16S ribosomal DNA (rDNA) were used. Based on the morphological and molecular analysis, the ticks infesting cattle of North East India were found to be Rhipicephalus (Boophilus) microplus and Haemaphysalis bispinosa. ITS2 and 16S rDNA sequence from R. (B.) microplus and H. bispinosa were amplified using universal and gene specific primers, sequenced and analysed. The length of the amplified ITS2 sequence of R. (B.) microplus and H. bispinosa, were found to be approximately 1,500 and 1,700 bp, respectively. The length of the 16S rDNA sequences in both the ticks was found to be similar in size, but they differ in their base pair constitutions. This is the first report of the nucleotide sequences of ITS2 and 16S rDNA of H. bispinosa. Phylogenetic analysis revealed that H. bispinosa is a close relative of H. longicornis. A polymerase chain reaction–restriction fragment length polymorphism diagnostic tool was developed based on HindIII digestion of ITS2 in order to facilitate the identification of these two species which cannot be distinguished once it is fully-fed. Present study describes the use of SEM and 16S rDNA/ITS2 based molecular analysis in identification and differentiation of fully fed tick species.     

Lengths and nucleotide sequences of the internal spacers of nuclear ribosomal DNA in gymnosperms and pteridophytes

The nucleotide sequences of the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal DNA were analyzed in species belonging to gymnosperms and pteridophytes. The lengths of the ITSs of sixteen species of gymnosperms and seven species of pteridophytes were estimated. The gymnosperms have ITS1 regions larger than those observed in the pteridophytes and angiosperms (ca. 610–3100 bp versus 159–360 bp). On the other hand, the ITS2 regions appear to be of a conserved length (182–370 bp). We have determined the complete nucleotide sequences of ITS regions from four gymnosperm species and five pteridophyte species by cloning the PCR products. Sequence analysis showed the presence of three short tandem arranged subrepeats of about 70 bp in the 1112 bp ITS1 ofEphedra fragilis. Pyrimidine rich (about 90%) DNA segments of 40–50 bp were observed in the ITS1 ofGinkgo biloba. A highly conserved 16 bp long sequence known to be present in the ITS1 of the angiosperm species has been also found in the ITS1 ofCycas revoluta, Taxus baccata andEphedra fragilis. Dedicated to Prof.Emilio Battaglia.     

Species distinctions among closely related strains of Eustigmatophyceae (Stramenopiles) emphasizing ITS2 sequence-structure data: Eustigmatos and Vischeria

ABSTRACT

There is an increasing interest in the Eustigmatophyceae, a class of stramenopile microalgae, because they offer a variety of high-value health-beneficial compounds, e.g. polyunsaturated fatty acids (PUFAs), while concomitantly producing high biomass. Clarification of the taxonomy of these organisms at the species level is important in order to achieve reproducible results and constant yields of valuable compounds in their exploitation. Here the distinction of the, so far exclusively, morphologically defined species of the genera Eustigmatos and Vischeria was tested. Distinctions inferred from almost full 18S and ITS2 rRNA as well as plastid-encoded rbcL gene sequences were evaluated following a morphological investigation. The ITS2 secondary-structure-based phylogenies separated independent lineages (species) with long internal branches. This recommends ITS2 as a promising marker for a DNA metabarcoding approach (culture-independent biodiversity assessment). In contrast, the 18S V4 region which is commonly used in metabarcoding was almost invariant, whereas the almost full length sequences distinguished eight groups/types of strains. Monophyly of the species was supported by shared ITS2 secondary structure features, making them distinct from other eustigmatophyte lineages in concordance with phylogenetic analyses. No groups of strains were congruently supported by all three markers. Consequently, the previous distinction of two genera on the basis of morphology cannot be retained and the species should be accommodated in a single genus, Vischeria. Taxonomic changes among the species with the definition of epitypes, on the basis of cryopreserved strains, are recommended. Two findings point to a more complex evolutionary history of the species. The rbcL and nuclear markers resulted in disparate groupings of strains. In three species divergent intragenomic ITS2 paralogues were revealed. Therefore, a still broader taxon sampling, in conjunction with a deep sequencing approach, is needed for a more comprehensive understanding of the complex evolution of eustigmatophyte species.