Molecular taxonomy of Hyrcanian Alnus using nuclear ribosomal ITS and chloroplast trnH-psbA DNA barcode markers

The taxonomy and phylogeny of Hyrcanian Alnus (eight taxa) were investigated using sequence data from the nuclear ribosomal ITS and the chloroplast trnH-psbA intergenic spacer. The mean nucleotide compositions of the ITS region were completely equal for two main Hyrcanian taxa, and the ITS1 region had fewer variable sites than the ITS2 region. Two relatively distinct types of ITS2 were identified for two main clades of Hyrcanian Alnus, the A. subcoradata complex and A. glutinosa. Two recently described species, A. dolichocarpa and A. djavanshirii, did not show any diagnostic sites and had a similar pattern with A. subcordata. Three recognized subspecies of A. glutinosa were distributed in the A. incana complex. In the analysis of trnH-psbA sequence data, the three subgenera of Alnus were poorly resolved relative to one another. Alnus glutinosa had minimal sequence divergence from A. incana and A. tenuifolia, and A. subcordata had a minimum distance from A. cordata and A. orientalis. A maximum pairwise distance also was observed between Hyrcanian species (A. glutinosa and A. subcordata) with A. pendula and A. sieboldiana, respectively. Ultimately, the molecular phylogeny of Alnus based on two DNA barcode markers was not congruent with recent morphological classifications, so additional DNA markers should be explored for identifying Alder taxa in the Hyrcanian forest.     

Sequence characteristics and phylogenetic implications of the nrDNA internal transcribed spacers (ITS) in the genus <Emphasis Type="Italic">Nymphaea</Emphasis> with focus on some Indian representatives

Nymphaea, an aquatic perennial herb with exceptionally beautiful flowers and floating leaves, is well represented globally. Out of ten species reported from India, the internal transcribed spacers (ITS) region of nrDNA was investigated in seven species of Nymphaea viz. N. alba var. rubra, N. caerulea, N. × marliacea, N. nouchali, N. pubescens, N. rubra and N. tetragona. Barring N. pubescens, whereby double peaks detected in the sequencing chromatograms may be due to random mutations occurring in some of the ITS paralogues, the additional signals detected for N. alba var. rubra and N. rubra are probably influenced by recent hybridization and introgression. Our study on sequence characteristics of ITS 1 and ITS 2 revealed high G + C content (ITS 1, 45.5–48.4%; ITS 2, 50.2–51.5%) and sequence divergence. Percentage of sequence divergence based on substitution and substitution plus indels is 44.15 and 57.19, respectively, for the ITS 1; 29.74 and 47.96% were recorded for the ITS 2. Although highly variable, conserved motifs within the ITS 1 and ITS 2 region of Nymphaea were identified and are found to be common throughout the order Nymphaeales. Sequence analysis of the ITS 1 and ITS 2 failed to detect any variation between two morphotypes of N. nouchali, namely N. nouchali JD 06 and N. nouchali JD 07, differing in flower color and found at the same geographical location. However, on comparison with another specimen of N. nouchali found at a different location, they showed considerable variation in nucleotide composition. Complemented by sequence data retrieved from GenBank, phylogenetic tree reconstruction of the genus Nymphaea based on neighbor-joining, maximum parsimony, maximum likelihood and Bayesian inference methods is presented and discussed.     

Molecular Systematic Study of <Emphasis Type="Italic">Chrysosplenium</Emphasis> Series <Emphasis Type="Italic">Pilosa</Emphasis> (Saxifragaceae) in Korea

To address systematic issues pertaining to series Pilosa of Korean Chrysosplenium, the phylogenetic relationships of the 35 accessions of Korean Chrysosplenium taxa were examined using the DNA sequence data obtained from the nuclear ribosomal internal transcribed spacer (ITS) regions and the psbK-I gene of the plastid genome. The results from both ITS and psbK-I sequence data indicated that Chrysosplenium flaviflorum formed a well-supported clade that merits species status of the taxon. The ITS phylogenetic trees detected two distinct lineages in Chrysosplenium pilosum var. fulvum. Geographically, one lineage was widely distributed throughout the country whereas the other was confined to Gyeonggi and Gangwon provinces. The plants sampled from Jeju Island, which have been recognized as C. pilosum var. sphaerospermum [C. hallaisanense] exhibited no DNA sequence feature segregating them from C. pilosum var. fulvum [C. barbatum] distributed in the mainland area of Korea. The plastid DNA sequence data was not fully in concordance with the nuclear ribosomal ITS data and the current taxa delimitation. These results suggest incomplete lineage sorting or that plastid genome capture may have occurred during the evolution of some taxa examined in the present study.     

Evolution of trichome types and its systematic significance in the genus Phlomoides (Lamioideae–Lamiaceae)

Despite a number of recent molecular phylogenetic studies on Phlomoides, in terms of trichome morphology the genus is still among the most poorly studied taxa in the family Lamiaceae. In order to test the utility of trichome characters for delimitation of sections, subsection and species of Phlomoides, we examined trichomes of 64 species representing all recognized sections and subsections using stereomicroscopy and scaning electron microscopy. Two basic types of trichomes could be identified: non‐glandular and glandular. Both trichome types can be simple or branched. The glandular trichomes were sessile, short stalked or long stalked. Different kinds of branched trichomes were observed in most species of P. sect. Phlomoides, i.e. symmetrically stellate, stellate with a central long branch, bi‐ or trifurcate. The species of P. sect. Filipendula were mostly covered by simple trichomes. Moreover, variation in trichome characters appears to have particular value, not only for the classification at sectional or subsectional rank, but also for delimitation of species within each section. For example, all studied species of P. subsect. Fulgentes are characterized by various kinds of stellate trichomes, while the trichome variability in P. subsect. Tetragonae was sufficiently high for species discrimination. An ancestral character state reconstruction was performed in order to investigate the evolution of trichome types and it revealed the following evolutionary trends in trichome characters of Phlomoides: 1) branched trichomes are primitive in Phlomoides as compared to simple ones, 2) long simple non‐glandular trichomes are derived as compared to short simple ones and 3) the presence of stalked glandular trichomes is advanced as compared to subsessile or sessile ones.     

Intraspecific invariability of the internal transcribed spacer region of rDNA of the truffleTerfezia terfezioides in Europe

ITS regions (internal transcribed spacers—ITS1 andITS2—with the 5.8S gene of the nuclear rDNA) of 25 fruit body samples ofTerfezia terfezioides, originating from Hungary and Italy, were compared. The amplification and sequencing of the ITS region was successful with both theITS1-ITS4 andITSIF-ITS4 primer pairs. No differences of the restriction fragment length polymorphism profiles were detected among 19 samples collected in one place at the same time. The sequences of the ITS region of 9 samples collected in different localities were highly invariable, differing in only two bases. Thus the intraspecific homogeneity of the ITS region seems to be an important species-specific characteristic ofT. terfezioides in contrast to otherTerfezia species. As the samples of the species were collected from different and distant localities of Europe, the ITS sequence ofT. terfezioides can be considered a very conservative, reliable molecular marker of the fungus. *** DIRECT SUPPORT *** A00EN076 00008     

Phylogeny of Bonatea (Orchidaceae: Habenariinae) based on molecular and morphological data

The genus Bonatea is widely distributed throughout southern and eastern Africa. Considerable debate surrounds the generic status of Bonatea, but there have been neither previous studies of evolutionary relationships among Bonatea species, nor any tests of the monophyly of the genus in relation to its close relative Habenaria. We investigated phylogenetic relationships between Bonatea and selected Habenaria species using morphology, as well as sequences of the chloroplast gene matK and the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. A fully resolved cladogram was obtained using morphological data, but neither the ITS, matK, nor combined data sets yielded well-resolved and well-supported phylogenetic structure for Bonatea. There is poor congruence between ITS and matK data for interspecific relationships in Bonatea, whilst the morphological results are largely congruent with the ITS analysis. Relative to Habenaria, there is little sequence variation between Bonatea species, which could indicate a recent and rapid radiation of Bonatea. Although the sampled Bonatea species form a distinct clade, more extensive sampling of Habenaria would be required to establish unambiguously whether or not Bonatea is monophyletic.     

Persistent soil seed banks in old-growth Hyrcanian Box tree (Buxus hyrcana) stands in northern Iran

Little is known about the soil seed bank and the influence of plant communities on the interaction between the seed bank and aboveground vegetation in the Hyrcanian temperate deciduous forest. We surveyed species composition and diversity of the persistent soil seed bank and the aboveground vegetation in six community types in old-growth Hyrcanian Box tree (Buxus hyrcana) stands in northern Iran. Fifty-two species with an average of 3,808 seeds/spores m⁻² germinated; forbs accounted for 64% of the seed bank flora. Thirty-four species in the aboveground vegetation were not presented in the seed bank, 32 species in the seed bank were not found in the vegetation, and 20 species were in both. The dominant tree species were Diospyros lotus and Alnus subcordata with an average of 17 and 4.6 seeds m⁻², respectively. Our results suggest that (1) vernal geophytes and shade-tolerant perennials are not incorporated in the seed bank, (2) early successional species are well represented in the seed bank, (3) plant community type has significant impacts on seed bank densities, and seed bank richness and diversity were significantly related to presence/absence of Box tree in the aboveground vegetation. The persistent seed bank contained species that potentially have a negative impact on the regeneration of forests, thus forest managers should retain old-growth Hyrcanian Box tree stands to conserve disturbance-sensitive indicator forest species.     

Structural investigation of the glandular trichomes of <Emphasis Type="Italic">Salvia argentea</Emphasis>

The morphology, anatomy and distribution of glandular trichomes on the aerial organs of Salvia argentea L. has been investigated. Two morphologically distinct types of glandular trichomes were determined. Capitate glandular trichomes forming a base 1–7 celled, a stalk 1–5 celled or no stalk and a head uni- or bicellular had various types. In capitate trichomes, the neck cell that has an important role especially for xeroformic plants, acting to prevent the backflow of secreted substance through the apoplast has been distinctively observed in the investigated species. The capitate trichomes were present abundantly on all aerial organs of S. argentea. Peltate glandular trichomes had a large secretory head forming 1–5, 8 central and 8–10, 12, 14 peripheral cells. Peltate trichomes are present on all aerial organs, except petiole, being the most abundant on calyx and corolla. Results were shown by tables and photographs.     

Inflorescence bracts of fossil and extant Tilia in North America,Europe, and Asia: patterns of morphologic divergence and biogeographic history

The enlarged inflorescence bract diagnostic of extant Tilia has an extensive Tertiary fossil record in the Northern Hemisphere. Diversity of bract morphology, and the extent of adnation between peduncle and bract, is reviewed for fossil and extant species of Tilia. An extinct type of bract with an orbicular outline and palmate venation is documented by the fossil species Tilia circularis (Chaney) comb. nov. from the early Oligocene of Oregon and is designated Type A. Living species of the genus have elongate bracts with predominately pinnate venation that are borne in two basic configurations: Type B, with the peduncle fused only to the extreme base of the bract lamina, as in extant Tilia endochrysea Hand.-Mzt. of southern China; and Type C with the peduncle fused medially along the basal one-third of the bract lamina, as in most extant species. Bracts of Type B were widely distributed in the Tertiary of western North America (late Eocene to Miocene) and Europe (early Miocene to Pliocene), while those of Type C are known in the fossil condition only from the middle and late Tertiary of Asia and Pliocene of Europe. The bracts of T. circularis, like those of type B, are borne on relatively long stalks and have the peduncle fused only at the extreme base. The fossil record supports recognition of the following characters as apomorphic in Tilia bract evolution: bracts sessile, peduncle adnate to the upper surface of the bract, and pinnate bract venation.     

Phylogenetic relationships among species of <Emphasis Type="Italic">Leotia</Emphasis> (Leotiales) based on ITS and RPB2 sequences

Thirty-three collections of Leotia were used to investigate inter-and infra-specific relationships in the genus. Collections were obtained from various parts of the world and represent the ascomatal color forms typical in species of the genus. The ITS rDNA and a variable region of the RNA polymerase II (RPB2) gene were sequenced and analyzed using parsimony and maximum likelihood methods. Although ITS and RPB2 tree topologies differed in regard to the position of two clades of L. lubrica and L. atrovirens, no significant conflict between ITS and RPB2 data or trees was found as determined by the partition homogeneity test. RPB2 sequences in general gave results comparable to ITS; the RPB2 sequences were more easily aligned. Phylogenetic analysis of the sequence data indicates that L. viscosa, L. lubrica and L. atrovirens are polyphyletic species. This suggests that ascomatal color in fresh specimens is not a reliable character alone for determining species in this group. Four major well-supported groups were found; these do not fully correspond to the commonly recognized species. Stipe color, in both fresh and dry condition, seems to correlate with the major recognized groups but features of the ascospores, asci and paraphyses prove too variable to be informative. The most basal group of Leotia species, identified as L. atrovirens, differ from all others by having stipes without gel tissue in their outer layers.     

Phylogenetic relationships within Pappophoreae s.l. (Poaceae: Chloridoideae): Additional evidence based on ITS and trnL-F sequence data

Historically, Pappophoreae included the genera Cottea, Enneapogon, Kaokochloa, Pappophorum and Schmidtia. Some authors consider this tribe as a well-supported monophyletic group; while other evidences reveals Pappophoreae as polyphyletic, with Pappophorum separated from the rest of the tribe. When the latter happens, it can form a clade with Tridens flavus. Molecular phylogenetic analyses of the subfamily Chloridoideae have included few species of Pappophoreae; therefore, further research involving more representatives of this tribe is needed. With the aim of providing new evidence to help clarify the phylogenetic position of Pappophorum and its relationships with other genera of the tribe and the subfamily Chloridoideae, eight new sequences of ITS and trnL-F regions of Pappophoreae species were generated. These sequences were analyzed together with other available sequence data obtained from GenBank, using maximum parsimony and Bayesian inference, for individual (trnL-F or ITS) or combined trnL-F/ITS data sets. All analyses reveal that Pappophoreae is polyphyletic, with Pappophorum separated from the rest of the tribe forming a well-supported clade sister to Tridens flavus.     

Comparative analysis of qualitative anatomical characters of <Emphasis Type="Italic">Trifolium</Emphasis> L. (Fabaceae) and their taxonomic implications: preliminary results

A comparative anatomical study of 20 Trifolium species was carried out using light and scanning electron microscopy in order to investigate the structure of their vegetative and reproductive organs and to evaluate the taxonomic significance of qualitative characters for distinguishing between the taxa. Characters were coded and analysed by multiple correspondence analysis. The combination of selected qualitative anatomical characters of the petiole, stipules, stem, peduncle and calyx (not previously studied in detail) were significant in delimiting sections of the genus Trifolium, but not for identification of species. The characters that contributed most to the separation of sections were peduncle cross-section shape, the presence of trichomes on the calyx tubes and the presence of trichomes and crystals in the stipules.     

Circumscription of Fulbrightiella gen. nov. and Sherwoodiella gen. nov., Two Novel Genera in the Calotrichaceae (Nostocales,Cyanobacteria)

Three novel strains in Calotrichaceae from tropical habitats were isolated and characterized with regard to their morphology, phylogenetic placement, and secondary structures of conserved domains in the 16S-23S internal transcribed spacer (ITS). The strains fell into two clades formerly identified as Calothrix from freshwater and brackish habitats. Based on both morphology and ecology, they differed from the type species of Calothrix, C. confervicola, which is marine, has wide trichomes with short cells, and narrows abruptly to a hyaline hair. The first clade grouped species with heteropolar filaments widened at the base and narrowed gradually toward the apex but not ending in a hair, with basal heterocytes that are formed in series as the apically placed heterocytes senesce; this clade is being named Fulbrightiella gen. nov., with two named species, F. bharadwajae sp. nov. and F. oahuensis sp. nov. The second clade was comprised of a single species with isopolar trichomes that are untapering as hormogonia, but which widen midfilament and taper toward both ends following growth. These trichomes develop pairs of heterocyte mid-filament, causing fragmentation into heteropolar trichomes with basal heterocytes and ends that taper, but not to a hair. This clade consists of a single species at present, Sherwoodiella mauiensis. With this action, four clades in the Calotrichaceae have been named: Macrochaete, Dulcicalothrix, Fulbrightiella, and Sherwoodiella. Calothrix sensu stricto is truly marine, morphologically distinct, and unsequenced; finding and sequencing the generitype for Calothrix remains as the most important and unfinished task in the revision of the Calotrichaceae.     

Morphological and phylogenetic analyses of <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> and <Emphasis Type="Italic">U. vignae</Emphasis> on legumes in Japan

Uromyces appendiculatus, inclusive of three varieties, is distinguished from U. vignae primarily by the position of urediniospore germ pores and putative host specificity. However, opinions concerning these morphological and physiological features as taxonomic characters have varied greatly, and distinction of these species has often been confused. To clarify the taxonomy of these two species, morphological features of urediniospores and teliospores of 225 rust fungus specimens on species of Phaseolus, Vigna, Apios, Lablab, and Dunbaria were examined by light microscopy and scanning electron microscopy. Forty-five specimens were subjected to molecular phylogenetic analyses. As a result, the position of germ pores in urediniospores and the teliospore-wall thickness were considered as good characters to separate three morphological groups. In molecular analyses, the specimens fell into two and three clades based on the nucleotide sequence at D1/D2 domain of LSU rDNA and ITS regions, respectively. One of the D1/D2 clades corresponded to one morphological group whereas another D1/D2 clade included two other morphological groups. In contrast, each of the three ITS clades corresponded to a separate morphological group. Neither morphological groups nor molecular clades were host limited. It is suggested that the three morphological groups that corresponded to three distinct ITS clades constitute distinct species.Contribution no. 186 from the Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

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.     

Morphology and ecology of the kalyptorhynchTyphlopolycystis rubra (Plathelminthes), an inmate of lugworm burrows in the Wadden Sea

Typhlopolycystis rubra, a new species of the taxon Polycystididae (Plathelminthes, Kalyptorhynchia), is described. The red species is characterized by copulatory hard structures which consist of a proximal girdle and 2 similar sized stylets.T. rubra occurs in intertidal sand near the island of Sylt in the North Sea. Here, it is virtually confined to the lowest parts of lugworm (Arenicola marina) burrows, where it aggregates in the coarse grained sand around the feeding pocket areas. This is an extremely narrow spatial niche within the sulfide layer of sediment. Population size over a period of 7 years is the most constant one among all species of Plathelminthes living on the tidal flat. The ability ofT. rubra to endure unsuitable conditions inside a cyst may contribute to this remarkably low population variability.     

Placozoa: at least two

It was shown that compensatory base changes (CBCs) in internal transcribed spacer 2 (ITS2) sequence-structure alignments can be used for distinguishing species. Using the ITS2 Database in combination with 4SALE — a tool for synchronous RNA sequence and secondary structure alignment and editing — in this study we present an in-depth CBC analysis for placozoan ITS2 sequences and their respective secondary structures. This analysis indicates at least two distinct species in Trichoplax (Placozoa) supporting a recently suggested hypothesis, that Placozoa is “no longer a phylum of one”. The first two authors contributed equally to this work.     

Complete Plastid Genome Sequencing of Four Tilia Species (Malvaceae): A Comparative Analysis and Phylogenetic Implications

Tilia is an ecologically and economically important genus in the family Malvaceae. However, there is no complete plastid genome of Tilia sequenced to date, and the taxonomy of Tilia is difficult owing to frequent hybridization and polyploidization. A well-supported interspecific relationships of this genus is not available due to limited informative sites from the commonly used molecular markers. We report here the complete plastid genome sequences of four Tilia species determined by the Illumina technology. The Tilia plastid genome is 162,653 bp to 162,796 bp in length, encoding 113 unique genes and a total number of 130 genes. The gene order and organization of the Tilia plastid genome exhibits the general structure of angiosperms and is very similar to other published plastid genomes of Malvaceae. As other long-lived tree genera, the sequence divergence among the four Tilia plastid genomes is very low. And we analyzed the nucleotide substitution patterns and the evolution of insertions and deletions in the Tilia plastid genomes. Finally, we build a phylogeny of the four sampled Tilia species with high supports using plastid phylogenomics, suggesting that it is an efficient way to resolve the phylogenetic relationships of this genus.     

Phylogeny of Weigela and Diervilla (Caprifoliaceae) Based on Nuclear rDNA ITS Sequences: Biogeographic and Taxonomic Implications

Weigela and three species of Diervilla were inferred from nucleotide sequence variation in the internal transcribed spacer (ITS) regions of 18-26S nuclear ribosomal DNA. Phylogenetic trees were obtained using parsimony, distance, and maximum likelihood methods. The ITS phylogenies did not support the monophyly of Weigela, which comprises three major clades. The first clade includes a single species, W. middendorffiana, which is more closely related with American genus, Diervilla. The second clade consisted of a single species, W. maximowiczii, but its relationship to other Weigela species is equivocal. The third one is a strongly supported core clade which contains the remaining Weigela species. Within the core group, W. hortensis exhibits the most distinct ITS sequence type. The hybrid origin of the species from the putative parents, W. florida and W. subsessilis, is not supported. The ITS sequence evidence favors the taxonomic hypothesis proposed by Nakai who recognized four distinct evolutionary lines (Diervilla, Macrodiervilla, Weigelastrum and Weigela). However, recognition of the two sections, Calysphyrum and Weigela was not supported in the ITS phylogenies. Substantial morphological differences between Diervilla and Weigela middendorffiana do not support the morphological stasis concept which has been considered as a general evolutionary mode among the Asian and North American disjunct taxa. Received 27 July 1998/ Accepted in revised form 7 July 1999     

Molecular markers reconstruct the invasion history of variable leaf watermilfoil (<Emphasis Type="Italic">Myriophyllum heterophyllum</Emphasis>) and distinguish it from closely related species

Genetic variation is increasingly recognized as an important factor influencing the establishment and spread of introduced species, and depends on both the introduction history and partitioning of genetic variation within and among potential source populations. We examine patterns of genetic variation in native and introduced populations of variable leaf watermilfoil, Myriophyllum heterophyllum, using chloroplast (trnL-F) and ribosomal (ITS) DNA sequences, as well as amplified fragment length polymorphisms (AFLPs). We identify a strong phylogeographic break distinguishing populations located on the Atlantic Coastal Plain (ACP) versus other (“Continental”) portions of the native range. Within these distinct biogeographic regions, we also find genetic variation to be strongly partitioned among populations as analysis of molecular variance (AMOVA) partitioned 91 and 75% of cpDNA and ITS diversity among populations, respectively. We demonstrate that the introduced ranges of variable leaf watermilfoil (northeastern and western US) result from multiple independent introductions from a variety of source populations, including lineages from both the ACP and Continental portions of the native range. In addition, we used our molecular markers to demonstrate that variable leaf watermilfoil is genetically distinct from three closely-related species that it is morphologically similar to. In particular, we demonstrate that M. heterophyllum is clearly distinct from a morphologically similar native species in the western US, M. hippuroides—whose distinctiveness from M. heterophyllum has been questioned—and therefore confirm the introduction of M. heterophyllum in the western US. Furthermore, we provide the first evidence for hybridization between these two species. Finally, our molecular markers identify previously unrecognized genetic variation in these four species, and therefore demonstrate the need for further taxonomic investigation.