Molecular evidence for the phylogenetic position ofHanabusaya asiatica Nakai (Campanulaceae), an endemic species in Korea

본 연구는 국내산 참취속 식물의 ITS(internal transcribed spacer) 염기서열을 결정하고 이들을 이용하여 <TEX>$Kalimeris$</TEX>, <TEX>$Gymnaster$</TEX>, <TEX>$Heteropappus$</TEX> 속 식물들간의 유연관계를 분석하고자 수행되었다. 섬쑥부쟁이(<TEX>$A.$</TEX> <TEX>$glehnii$</TEX>) 등을 포함한 11종의 참취속 식물의 ITS 염기서열을 결정하였으며 유전자 은행에 등록된 <TEX>$Kalimeris$</TEX> 속 식물의 ITS 염기서열을 포함하여 군집분석을 실시하였다. ITS1의 길이는 249-253bp로, ITS2의 길이는 181-217bp로 나타났으며 G + C 함량은 47-54%의 변이를 나타내었다. 본 연구에서 다룬 16종의 18개체간 염기 치환율은 한 site당 ITS1의 경우는 9%, ITS2는 10%로 나타나 ITS1과 2 부위간의 차이가 거의 없는 것으로 나타났다. 또한 협의의 참취속(<TEX>$Aster$</TEX> sensu strict) 식물과 <TEX>$Kalimeris$</TEX> 속 식물의 치환율을 비교한 결과 ITS1과 ITS2 지역 모두 <TEX>$Kalimeris$</TEX> 속 식물이 낮게 나타났다. 계통분석결과, 갯개미취는 군외군으로부터 가장 먼저 분지하였으며, 일본산 <TEX>$A.$</TEX> <TEX>$bellidiastrum$</TEX>이 나머지 분류군들을 위한 자매군으로 유집되었다. 참취는 다른 참취속 식물들과 분리되어 독립적인 분계조를 형성하였다. 갯쑥부쟁이(<TEX>$A.$</TEX> <TEX>$hispidus$</TEX>)를 제외한 <TEX>$Kalimeris$</TEX> 속 식물은 91%의 높은 지지율을 가지고 광의의 참취속내로 유집되었으며 <TEX>$Gymnaster$</TEX>와 <TEX>$Heteropappus$</TEX> 속 식물 역시 광의의 참취속으로 유집되었다. 이러한 연구결과는 <TEX>$Kalimeris$</TEX>, <TEX>$Gymnaster$</TEX> 및 <TEX>$Heteropappus$</TEX>로 세분되었던 참취속을 ITS 염기서열을 바탕으로 통합될 수 있을 것으로 판단된다. This study was carried out to decide ITS (internal transcribed spacer) sequence of some Korean native <TEX>$Aster$</TEX> species and to resolve their relationship among Korean native <TEX>$Aster$</TEX>, including <TEX>$Kalimeris$</TEX>, <TEX>$Gymnaster$</TEX>, <TEX>$Heteropappus$</TEX> genus separated from <TEX>$Aster$</TEX> in a previously study based on the pappus length. We registered 11 ITS sequences of <TEX>$Aster$</TEX> species including <TEX>$A.$</TEX> <TEX>$glehni$</TEX> to GenBank and those sequences were used for the cluster analysis with <TEX>$Kalimeris$</TEX> species. The size of ITS1 was varied from 248 to 256 bp, while ITS2 was varied from 220 to 222 bp. The G + C content of the ITS region ranged from 49.4 to 53.5%. Pairwise comparison results showed that the substitution rate of ITS1 and ITS2 region was 9% and 10%, respectively. <TEX>$Kalimeris$</TEX> sensu strict substitution rate was lower than that of <TEX>$Aster$</TEX> sensu strict species. The strict consensus parsimonious cluster analysis showed <TEX>$A.$</TEX> <TEX>$tripolium$</TEX> is the first branching from the clade and the next is <TEX>$A.$</TEX> <TEX>$scaber$</TEX>. The <TEX>$Kalimeris$</TEX> species except for the <TEX>$A.$</TEX> <TEX>$hispidus$</TEX> were grouped into the same clade with high bootstrap value (91%) within <TEX>$Aster$</TEX>. <TEX>$Gymnaster$</TEX> and <TEX>$Heteropappus$</TEX> that has been classified by morphological characters were also grouped into broad sense <TEX>$Aster$</TEX> clade. These results implied these three genera could be merged together into <TEX>$Aster$</TEX> based on the ITS sequences.

In order to resolve the relationships among species from Angelica s.s. and its allied genera in East Asia, ITS (Internal Transcribed Spacer) sequences of 44 taxa were used to analyze their sequence divergence and to construct three phylogenetic trees in this paper. Three taxa were used as the outgroup. Some conclusions could be achieved as follows. (1) Angelica s.s., Czernaevia, and Coelopleurum were closely related, but the relationship between Ostericum and them was ulterior. It was also supported by analyses on fruit anatomy and chemical constitutes. Ostericum was proposed as a relatively independent genus. (2) Results from ITS sequences supported the view that Angelica s.s. was monophyletic and could be divided into several sections. (3) Results from ITS sequences and chemical constitutes indicated that the relationship between Peucedanum and Angelica s.s. was very close. (4) Results from ITS sequences as well as from conformation and chemical constitutes showed that the divergence of A. sinensis from other taxa of Angelica s.s. was great. The taxonomic position of A. sinensis should be reconsidered. (5) Both analyses of ITS sequences and chemical constitutes revealed that Ligusticum was not a natural group.

The ascomycete fungal pathogen Ustilaginoidea virens (Cooke) Takahshi cause false smut in rice and considerable yield loss. In this study, we collected isolates of U. virens from the rice growing ecosystems of Karnataka and characterized for cultural, morphological and molecular characters. The isolates of the fungus on Potato Sucrose Agar media exhibited distinct colonies with colony growth ranging from 21.50 mm (Uv-20) to 70.00 mm (Uv-15). The colony colour appeared as whitish to yellowish with varied growth pattern from flat, raised flat to fluffy and raised fluffy colonies with sectoring in Uv-1, Uv-3, Uv-6 and Uv-9 isolates. The isolates of U. virens also showed variation in the morphology of spores, where the conidia were globose, irregularly round to elliptical and warty on the surface with spore radius ranging from 2.91 to 5.36 μm. The scanning electron microscopy revealed hyaline globose to irregularly rounded ornamented chlamydospores with prominent spines. Besides cultural and morphological characters, molecular identification of false smut isolates was confirmed through ITS sequencing which showed 91 to 99 per cent identity with U. virens in NCBI-BLAST analysis. Dendrogram constructed using ITS sequence data broadly separated the isolates into two major clusters with divergence among clusters. This ITS (internal transcribed spacer) sequencing of isolates should help better understanding of the phylogenetic relationships among these isolates.

Molecular Evolution and Phylogenetic Implications of Internal Transcribed Spacer Sequences of Nuclear Ribosomal DNA in the Phaseolus–Vigna Complex

Data processing can mask biology: towards better reporting of fungal barcoding data?

Basella alba (family Basellaceae) is a perennial vine that serves as an edible leaf vegetable in Malaysia. In May 2021, red spots were observed on leaf samples of B. alba in Lido, Sabah Province (5°56'39.1"N, 116°04'47.6"E). The disease severity was about 20% with 10% incidence. The spots enlarged and coalesced into larger necrotic spots. Small pieces (5 x 5 mm) of infected leaves were excised from three plants, and then surface disinfected based on Khoo et al. (2022). One fungal isolate (Lido01) was isolated and cultured on potato dextrose agar (PDA) at 25°C. A single isolate with cottony aerial mycelia and pink concentric rings was observed on the upper surface of the culture. Unicellular and multicellular chlamydospores were observed, and measured 7.1 to 14.3. × 17.8 to 74.5 μm. Conidia were unicellular, hyaline, oval, and measured 3.8 to 5.2 x 1.7 to 2.7 μm (n= 20). Pycnidia were spheroid, and measured 66.2 to 114.3 x 44.1 to 86.1 μm (n= 20). Genomic DNA was extracted from fresh mycelia according to the extraction method of Khoo et al. (2022a and 2022b). ITS1/ITS4, LR0R/LR7, ACT512F/ACT783R, and T10/Bt2b primers were used to amplify the internal transcribed spacer (ITS), large subunit (LSU), actin (ACT), and tubulin (TUB) genes, respectively (O'Donnell and Cigelnik, 1997; Chen et al. 2021). PCR products were Sanger sequenced by Apical Scientific Sdn. Bhd. (Serdang, Malaysia). Sequences of isolate Lido01 were deposited in GenBank as OM501130 (ITS), OM501128 (LSU), OM513916 (ACT) and OM513917 (TUB). Respective gene sequences of this isolate showed 100% homology to ITS sequence of isolate BPL01 (OM453926) (507/507 bp), LSU sequence of isolate BPL01 (OM453925) (1328/1328 bp), ACT sequence of isolate CZ01 (MN956831) (275/275 bp) and TUB sequence of isolate BJ-F1 (MF987525) (556/556 bp). The sequences of Lido01 established a supported clade (99% bootstrap value) to the related Epicoccum sorghinum type sequences, according to phylogenetic analysis using maximum likelihood based on the concatenated ITS, ACT, and TUB sequences. Morphological characters also matched the description of E. sorghinum (Li et al. 2020). Koch's postulates were tested as described by Chai et al. (2017) with modification by spray inoculation (106 spores/ml) on the leaves of three healthy one-month-old B. alba, while sterilized distilled water served as the control treatment. Monitoring and incubation were performed in a greenhouse based on Iftikhar et al. (2022). All inoculated leaves developed symptoms as described above by 8 days post-inoculation, whereas no symptoms occurred on controls, thus fulfilling Koch's postulates. The experiment was repeated twice. The reisolated pathogen was morphologically and genetically identical to E. sorghinum. E. sorghinum was reported causing leaf spot on Brassica parachinensis (Yu et al. 2019), Camellia sinensis (Bao et al. 2019), Myrica rubra (Li et al. 2020), Oryza sativa (Liu et al. 2020) and Zea mays (Chen et al. 2021). To our knowledge, this is the first report of E. sorghinum causing leaf spot on B. alba in Malaysia. Our findings have expanded the geographic range and host range of E. sorghinum in Malaysia, though the host range of this isolate is not known.

Phylogeny of Isatis (Brassicaceae) and allied genera based on ITS sequences of nuclear ribosomal DNA and morphological characters

한국산 담배풀속(Carpesium L.) 7분류군과 3개의 외군(Inula britannica L., Inula germanica L., Rhanteriopsis lannginosa(DC.) Rauschert)을 대상으로 유연관계를 파악하기 위하여 nuclear ribosomal DNA(nrDNA) 중 ITS(internal transcribed spacer) 지역의 계통분류학적 분석을 수행하였다. 계통분류학적 연구방법은 maximum parsimony, neighbor-joining와 maximum likelihood 방법을 사용하였다. 정렬된 계통분의 총 길이는 731 bp이며, ITS1, ITS2와 5.8S 부위의 길이는 각각 284~297 bp, 264~266 bp와 164 bp로 나타났다. 계통분류학 변이를 보이는 site는 111개로 확인 되었으며, 그 중 64개의 site가 계통학적으로 유효한 것으로 나타났고, ITS1 지역이 ITS2 지역보다 염기 변이가 다양하게 나타나는 것으로 확인되었다. 그 결과, 한국산 담배풀속은 단계통을 형성하였으며, 담배풀(C. abrotanoides L.)이 가장 기저부에 위치하였다. 여우오줌(C. macrocephalum Franch. & Sav.)와 두메담배풀(C. triste Maxim.)은 가까운 유연관계를 나타냈으며, 애기담배풀(C. rosulatum Miq.)와 천일담배풀(C. glossophyllum Maxim.) 그리고 좀담배풀(C. cernuum L.)와 긴담배풀(C. divaricatum Siebold & Zucc.)도 유연관계가 가깝게 나타났다. 이와 같은 결과로 담배풀속 nrDNA의 ITS 지역 염기서열에 기초한 분자 계통학적 연구는 계통분류를 이해하는데 유용한 방법으로 판단된다. Phylogenetic analyses were conducted to evaluate relationships of 7 taxa of Korean Carpesium including three outgroup (Inula britannica L., Inula germanica L., Rhanteriopsis lannginosa (DC.) Rauschert) by using ITS (internal transcribed spacer) sequences of nuclear ribosomal DNA. Phylogenetic studies used maximum parsimony, neighbor-joining and maximum likelihood methods analysis. The length of the ITS sequences was 731 bp, and the lengths of the ITS1, ITS2 and 5.8S regions were 284~297 bp, 264~266 bp and 164 bp, respectively. The total number of variable sites was 111 for the entire sequences, and a parsimony informative sites of 64 are valid. Base change appeared variously in ITS1 rather than in ITS2. As the result, Korean Carpesium were formed monophyletic group and C. abrotanoides situated as the most basal clade. The results show that C. macrocephalum is closely related with C. triste. C. rosulatum has the closest relationship with C. glossophyllum. C. cernuum is close to C. divaricatum. These results suggest that the ITS data used in this study could be useful for the phylogenetic analysis of Korean Carpesium.

Hydrangea macrophylla (Thunb.) Ser., an important genetic resource for the development of hydrangea cultivars, is endemic to a limited number of regions in Japan; however, there is a lack of comprehensive phytogeographical information essential for the conservation and effective utilization of this valuable genetic resource. In this study, we investigated the occurrence of natural hybridization between H. macrophylla and H. serrata within the distribution range of H. macrophylla by analyzing the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. Analysis of the H. macrophylla genome database revealed approximately 300 tandem repeats of ITS sequences in the genome. Two major ITS sequences, accounting for 55% to 59% of the total ITS sequences in the genome of H. macrophylla Aogashima-1 (the individual used for genome assembly), appeared to be inferable from electropherogram data obtained by direct sequencing of PCR products. From a total of 320 samples of H. macrophylla and H. serrata, 10 ITS types were identified. As a result of phylogenetic analysis, the ITS sequence types of H. macrophylla, H. serrata var. yesoensis, var. serrata, var. angustata, and some var. acuminata samples were grouped into a single cluster. Samples were collected from four of the Izu Islands, 16 populations on the Izu Peninsula, and 12 populations on the Boso Peninsula to estimate the occurrence of interspecific hybridization within the H. macrophylla distribution range. Direct sequencing results for PCR products of ITS sequences and morphological data showed that little interspecific hybridization had occurred in the Izu Islands; however, hybridization had occurred in many populations on the Boso and Izu Peninsulas. Furthermore, our findings suggest that habitat topography influences the frequency of interspecific hybridization among H. macrophylla populations. To conserve H. macrophylla genetic resources in the Boso and Izu Peninsulas, it is important to consider not only population decline, but also the impact of natural interspecific hybridization.

Genetic relationship of a newly discovered teosinte from Nicaragua, Zea nicaraguensis with waterlogging tolerance, was determined based on randomly amplified polymorphic DNA (RAPD) markers and the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA using 14 accessions from Zea species. RAPD analysis showed that a total of 5,303 fragments were produced by 136 random decamer primers, of which 84.86% bands were polymorphic. RAPD-based UPGMA analysis demonstrated that the genus Zea can be divided into section Luxuriantes including Zea diploperennis, Zea luxurians, Zea perennis and Zea nicaraguensis, and section Zea including Zea mays ssp. mexicana, Zea mays ssp. parviglumis, Zea mays ssp. huehuetenangensis and Zea mays ssp. mays. ITS sequence analysis showed the lengths of the entire ITS region of the 14 taxa in Zea varied from 597 to 605 bp. The average GC content was 67.8%. In addition to the insertion/deletions, 78 variable sites were recorded in the total ITS region with 47 in ITS1, 5 in 5.8S, and 26 in ITS2. Sequences of these taxa were analyzed with neighbor-joining (NJ) and maximum parsimony (MP) methods to construct the phylogenetic trees, selecting Tripsacum dactyloides L. as the outgroup. The phylogenetic relationships of Zea species inferred from the ITS sequences are highly concordant with the RAPD evidence that resolved two major subgenus clades. Both RAPD and ITS sequence analyses indicate that Zea nicaraguensis is more closely related to Zea luxurians than the other teosintes and cultivated maize, which should be regarded as a section Luxuriantes species.

A biogeographic study of Saxifraga section Saxifraga was performed based on phylogenetic analyses of ITS (internal transcribed spacer) sequences of nuclear ribosomal DNA. ITS sequences from 21 species and 31 populations were examined to identify colonization patterns for the two species of Saxifraga occurring in Macaronesia and for S. globulifera in the west Mediterranean basin. Phylogenetic analysis of the sequence data yield a single most parsimonious tree with many of the major clades well supported by bootstrap and decay values. The ITS tree provided resolution at specific and populational levels that points to two biogeographic patterns within the genus. In contrast to the molecular evidence provided by other authors for a Mediterranean origin of several Macaronesian genera of angiosperms, our results indicate that the Madeiran archipelago was colonized a single time by a species of Saxifraga originating from the Eurosiberian region. On the other hand, the molecular evidence also suggests that populations of S. globulifera from North Africa have been isolated for a long time from populations occurring in the Iberian Peninsula, and that the endemic S. reuteriana has evolved from the Iberian populations of S. globulifera. The Mediterranean Sea has probably been an effective isolating barrier for some plant groups that occur in Europe and North Africa.

In the present study, samples representing Toxascaris leonina and Tococara canis were collected from cougar (Panthera leo) and common wolf (Canis lupus ) from Harbin Northern Forest Zoo in Heilongjiang Province, China, and they were characterized by nuclear ribosomal DNA (rDNA) sequences of internal transcribed spacers (ITS). The ITS rDNA was amplified by polymerase chain reaction (PCR), then sequenced and compared with that of other members of ascaridida published in GenBankTM. Furthermore, the phylogenetic relationship was reconstructed using Maximum Parsimony and Bayesian analyses. For the T. leonina of cougar, the length of the ITS sequences was 698 bp (the ITS1 and ITS2 were 419 and 279 bp, respectively) and the corresponding sequences of T. canis from common wolf were 808 bp (the ITS1 and ITS2 were 485 and 323 bp, respectively). The identity of ITS sequences of T. leonina and T. canis from cougar and common wolf was 76%. The ITS sequences were analyzed by PCR-linked restriction fragment length polymorphism (PCR-RFLP) which was established for the unequivocal delineation of the T. leonina and T. canis with which were easily confused morphologically, using restriction endonuclease EcoRV and SalI. The valuable tool established in this study makes the molecular identification, the ecology and genetic structure studies of these two ascaridoids possible. Key words: Toxascaris leonina, Tococara canis, ribosomal DNA (rDNA), internal transcribed spacers (ITS), PCR-linked restriction fragment length polymorphism (PCR-RFLP)

Sequences of the internal transcribed spacers (ITS) of nuclear ribosomal DNA were analysed to precise their length (637–643 bp) and resolve phylogenetic relationships among eight Mediterranean species of the genus Hedysarum (Fabaceae). The infra-specific variability levels of the ITS sequences of spontaneous population of H. coronarium proved a lack of polymorphism both in the length and in the sequences examined in this species. Hence, a consensus ITS sequence characterising each Hedysarum species has been investigated for analysis of inter-specific polymorphisms. The level of variation of ITS sequence was high enough to make the ITS1 and ITS2 a useful tool for phylogenetic reconstruction. However, ITS2 seems to be relatively more polymorphic and more informative than ITS1 regarding length or GC percent. The phylogenic relationships in the genus Hedysarum based on ITS1 and ITS2 sequences taken independently or together, are discussed in the context of current work in molecular biosystematics. Results exhibited the distinctiveness of the two H. spinosissimum subspecies (i.e. H. spinosissimum ssp. capitatum and H. spinosissimum ssp. spinosissimum). In addition, the great similarity of the ITS sequences between H. coronarium (the only cultivated species of the genus) and H. carnosum suggests the usefulness of the latter in selection programmes to improve pastoral production in semi-arid areas.

The internal transcribed spacer (ITS) region of the 18 S–25 S nuclear ribosomal DNA repeat was sequenced from 19 populations of the tribeLactuceae, including all species of dwarf dandelion (Krigia) and five outgroup genera. The incidence of length changes and base substitutions was at least two times higher for ITS 1 than ITS 2. Interspecific sequence divergence withinKrigia averaged 9.62% (1.61%–15.19%) and 4.26% (0%–6.64%) in ITS 1 and ITS 2, respectively. Intergeneric sequence divergence ranged from 15.6% to 44.5% in ITS 1 and from 8.0% to 28.6% in ITS 2. High sequence divergence and homoplasy among genera of tribeLactuceae suggest that the phylogenetic utility of ITS sequence data is limited to interspecific studies or comparisons among closely related genera. Trees generated from ITS sequences are essentially identical to those from restriction site comparisons of the entire nuclear ribosomal (nr) DNA region. The degree of tree resolution differed depending on how gaps were treated in phylogenetic analyses. The ITS trees were congruent with the chloroplast DNA and morphological phylogenies in three major ways: 1) the sister group relationship betweenKrigia andPyrrhopappus; 2) the recognition of two monophyletic sections,Krigia andCymbia, in genusKrigia; and 3) the monophyly of theK. occidentalis-K. cespitosa clade in sect.Cymbia. However, the two nrDNA-based trees are not congruent with morphology/chloroplast DNA-based trees for the interspecific relationships in sect.Krigia. An average of 22.5% incongruence was observed among fourKrigia data sets. The relatively high degree of incongruence among data sets is due primarily to conflict between trees based on nrDNA and morphological/cpDNA data. The incongruence is probably due to the concerted evolution of nrDNA repeating units. The results fromKrigia and theLactuceae suggest that nrDNA data may have limited utility in phylogenetic studies of plants, especially in groups which exhibit high levels of sequence divergence. Our combined phylogenetic analysis as a total evidence shows the least conflict to each of the individual data sets.

Internal transcribed spacer sequence analysis of Angelica from different habitats