Reinstatement of Ectocarpus crouaniorum Thuret in Le Jolis as a third common species of Ectocarpus (Ectocarpales, Phaeophyceae) in Western Europe, and its phenology at Roscoff, Brittany

Molecular phylogenetic is the branch of phylogeny that analyzes hereditary molecular diversity, mainly in DNA sequences, to increase data on an organism‘s evolutionary relationships. Due to the taxonomic levels of the study, various molecular markers are applied in molecular phylogeny. The selection of molecular instrument is of paramount matter to ensure that a proper level of variation is meliorated to respond the phylogenetic question. In this review, we have been trying to discuss about gene markers used in the plant phylogeny at various taxonomic levels. The current gene markers used in phylogeny include: the ribosomal nuclear genes, low copy nuclear genes and the extra-nuclear genome (mitochondrial and chloroplastic genomes). Conserved regions could be used at higher taxonomic levels in phylogenetics studies and regions with more changes could be applied between closely related taxa. One of the most common sequences for studying the phylogenetic relationships at the generic and infrageneric taxonomic levels in plants is the internal transcribed spacer (ITS) region of the 18S–5.8S–26S nuclear ribosomal cistron. Chloroplastic gene sequences have been used extensively at the family level and above but chloroplast non-coding sequences such as introns and intergenic spacers are used more frequently at lower taxonomic levels. Low-copy nuclear genes are most useful at the interspecific and intraspecific levels where cpDNA and/or nrDNA cannot provide adequate resolution. Evidence offers that for more strongly reconstruction of phylogeny, several discrete genes are needed. Now, uses of next generation sequencing (NGS) techniques are reported. Techniques for NGS are an alternative to prevalent methods that let access to hundreds of DNA regions.

Molecular phylogenetics of<i>Thlaspi</i>s.l. (Brassicaceae) based on chloroplast DNA restriction site variation and sequences of the internal transcribed spacers of nuclear ribosomal DNA

I studied the phylogeny and evolution of 25 species of ticks from the subfamily Rhipicephalinae with four molecular markers and morphology. I also studied 5 members of the subfamily Hyalomminae, which molecular analyses have shown arose within the Rhipicephalinae, and two members of Haemaphysalinae which were used as an outgroup. All taxa were studied with morphological characters and 12S rRNA gene sequences. Sequences for the other three genes, cytochrome oxidase c I, internal transcribed spacer 2 and 18S rRNA, were not available for all taxa. My initial analyses were with mitochondrial 12S rRNA gene sequences. I found that the subfamily Rhipicephalinae was paraphyletic with respect to the Hyalomminae (Hyalomma), but that the Hyalomma spp. studied were monophyletic. The species of Rhipicephalus and Boophilus studied were closely related but the monophyly of these genera could not be established.Because many parts of the 12S phylogeny were poorly resolved I combined mitochondrial cytochrome c oxidase I (COI) and 12S sequences. The phylogenetic resolution was markedly improved compared with separate analyses of each gene. My most controversial result was that the genus Rhipicephalus was apparently paraphyletic unless species of Boophilus were included in it. The species of Rhipicephalus most closely related to Boophilus spp. were ticks of the subgenus R. (Digineus) and members of the R. pravus species group.Next I sequenced the internal transcribed spacer 2 (ITS2) to obtain phylogenetic information from a nuclear gene. The phylogenies from ITS2 were mostly consistent with results from the mitochondrial genes. In addition, the ITS2 provided support for the monophyly of Boophilus spp., the monophyly of the ornate spp. of Rhipicephalus, and for Anocentor nitens arising within the genus Dermacentor. The genus Rhipicephalus was again found to be paraphyletic without the inclusion of Boophilus spp. A repeat sequence was found in almost all ticks studied. By mapping the presence of the second copy of the repeat onto the phylogeny from the ITS2 gene it appears that there have been many independent gains and losses of that copy. I inferred a putative secondary structure for the region comprising the repeat. It seems that each copy folds into a distinct and almost identical stem-loop complex. Gains and losses of one of the copies do not seem to impair the function of the ITS2 in these ticks.Phylogenetic inferences from the ITS2 and the mitochondrial genes, 12S rRNA and COI, were informative but no single set of characters produced a fully resolved tree. The three genes (12S, COI and ITS2) were then combined with 18S rRNA gene sequences and morphological characters and analysed separately and together in a total evidence analysis. The total evidence dataset had 33 taxa and 3303 characters. Analyses of this dataset led to robust hypotheses about the phylogeny of the Rhipicephalinae and Hyalomminae. I conclude that: (i) the genus Rhipicephalus is paraphyletic and should be revised to include Boophilus; (ii) the subgenus R. (Rhipicephalus) is paraphyletic without the inclusion of the subgenus Digineus and the genus Boophilus; (iii) Hyalomma should only comprise two sub-genera because the subgenus Hyalomma is paraphyletic without the inclusion of the sub genus Hyalommasta; (iv) A. nitens arose within the genus Dermacentor and thus should not retain generic status, and; (v) the subfamily Hyalomminae (Hyalomma) arose within the Rhipicephalinae, so the Rhipicephalinae should be revised to include Hyalomma. The phylogeny from my total evidence analysis was used to map life history and morphological features. It appears that the evolution of a truncated life cycle (one- or two- hosts) has occurred at least three times in the species studied here. Omateness (patterning on the scutum) appears to have evolved on three separate occasions (in N. monstrosum, in the R. pulchellus group and in the Dermacentor spp.) whereas there has been one reversal (in A. nitens). Mapping the geographic distribution of rhipicephaline and hyalommine taxa onto the phylogeny indicated that the Rhipicephalinae s.l. probably evolved at least 50 million years ago. The centre of diversity of most of the subfamily appears to have been Afrotropical, with secondary radiations of species to Palearctic, Nearctic, Oriental and Neotropical regions.Analyses of molecular and morphological characters together provided the greatest resolution of phylogeny in the Rhipicephalinae and Hyalomminae so far.

Thrixspermum (T.) is one of the genus in Orchidaceae that has small flowers. Among species in this genus has a high homology and also has many synonyms. Identification using morphological characters can be constrained since Thrixspermum flowering time occurs in a very short period. This study aimed to conduct molecular-based identification of Thrixspermum orchids using DNA barcoding . This method applied molecular-based species identification technique using DNA sequences from genomic fragments that are considered fast, accurate, and consistent. The molecular markers used were Internal Transcribed Spacer 2 (ITS2), while the samples used were T. centipeda, T. lucidum, and T. angustifolium. BLAST results show that T. centipeda has a close relationship with T. centipeda from Malaysia (KX679332) with 99.79% percent identity, T. lucidum has a high homology with T. linusii (KX679333; 97.30%), while T. angustifolium has a high homology with T. triangulare (KX679348; 99.38%). There is a unique sequence that only T. lucidum and T. angustifolium have that distinguishes the two from other species. ITS2 can be recommended as a molecular marker for determining the Thrixspermum orchid barcode . The benefit obtained from this research is the DNA barcode sequences ( ITS2 ) of Thrixspermum orchids would be very useful to enrich the plant barcodes database for further molecular taxonomy and biodiversity of orchid.Â

• The origin of the Ectocarpus strain used for genome sequencing (the 'genome strain') was Peru, where no Ectocarpus had been reported previously. To study the genetic diversity in the region and to increase the number of individuals from this area available for genetic experiments, 119 new Ectocarpus strains were isolated from eight localities along the 3000 km of coastline from central Peru to central Chile. • Internal transcribed spacer 1 (ITS1) genotyping revealed nine different genotypes, five of which were endemic to the area studied and three of which were previously unknown. • Individuals of the same genotype as the genome strain occurred from Peru to northernmost Chile, representing 61% of the samples in this area, from which five more genotypes were isolated. Further south, down to central Chile, most individuals belonged to Ectocarpus siliculosus, Ectocarpus fasciculatus and Ectocarpus crouaniorum. In sexual crosses, the genome strain and the new isolates of the same genotype were fully compatible. • Sequences from four nuclear and cytoplasmic genetic markers (ITS1, ITS2, Rubisco spacer and Cytochrome-c oxidase subunit 3 (cox3)) separated the genome strain from the known species of Ectocarpus. It may in future be recognized as a separate species.

The general purpose of the molecular systematic studies is to illuminate the ITS2 structure of the target populations, to determine its phylogenetic boundaries, and to clarify intra-species and inter-species relationships. Particularly, the internal transcribed spacer 2 (ITS2) region of nuclear ribosomal DNA is used in molecular systematics because of availability of conserved regions with its highly repeated in number in plant genomes. Addition to the primary sequences of ITS2, also secondary structure form of the region became a valuable feature in species divergence and became to use like a morphological character. In the current study to indicate the secondary structure form of the ITS2 region as a useful tool in systematics, different taxa from 22 genera were used. The DNA samples were collected in the field studies in 2021 and sequences were aligned using ClustalW and Kimura-2 parameter to calculate the genetic distances. Phylogenetic tree was also constructed with Maximum Likelihood method with the best suitable model at MEGA X software. The secondary structure predictions of species and ΔG (Gibbs) free energy calculations, the tools of both the ITS2 database and mFOLD web server were used. The results indicated that ITS2 secondary structure estimations represented the genetic differences visibly with its helices and motifs like a morphological character. Consequently, even if primary structure of the ITS2 region revealed as valuable marker in molecular systematic studies, also, all tested secondary structure forms of the region will be used as an ideal marker for taxonomic and phylogenetic reconstructions at all taxonomic levels.

Duohua huangjing (Polygonatum cyrtonema Hua) seedling basal stem rot caused by Fusarium redolens in China Tao Tang1, Fanfan Wang1, Jie Guo1, Xiaoliang Guo1, Yuanyuan Duan1,Jingmao You1* 1 Institute of Chinese Herbal Medicines, Hubei Academy of Agricultural Sciences, Enshi, 445000, China. Duohua huangjing (Polygonatum cyrtonema Hua), a herbal medicine, that is mostly planted in several provinces in China. In April 2020, severe diseases with about 40% seedling losse was found in the Huangjing seedling base in Shiyan city, Hubei province. The symptoms included softening and decay of the roots and stem bases, a progressive yellowing and wilting of leaves, and finally being completely rotted. Small pieces of symptomatic stems (0.5 cm in length) and leaves (0.5 × 0.5 cm in size) were surface sterilized with 75% ethanol for 30 s, followed by 0.1% HgCl2 for 1 min, rinsed three times with sterile water, and then dried with sterilized absorbent paper. The sections were placed on potato dextrose agar (PDA) medium containing 10 µg/ml of ampicillin and incubated at 25°C in the dark. After 3 days incubation, eight isolates with the same colony morphology were sub-cultured and purified by hyphal tip isolation. Macroconidia were sickle-shaped, 15.8 - 32.3 × 3.1 - 5.6 μm (n = 25), and three to five septate. Microconidia were oval or kidney-shaped, 5.2 - 11.4 × 2.0 - 3.2 μm (n = 25), and zero to one septate. To confirm the identity of the pathogen, molecular identification was performed with strain HJCD1. Following DNA extraction, PCR was performed using the TSINGKE 2×T5 Direct PCR Mix kit. Target areas of amplification were the internal transcribed spacer (ITS) and translation elongation factor 1α (TEF-1α) using ITS1/4 (White et al. 1990) , EF1/EF2 (Taylor et al. 2016), respectively. Following BLAST searches and phylogenetic reconstruction, the ITS region (GenBank MW485770.1) showed 99% identity with those of Fusarium redolens in GenBank (KU350713.1) and the TEF-1α (GenBank MW503930.1) showed 100% identity with F. redolens GenBank (MK922537.1). Pathogenicity tests were performed to fulfill Koch's postulates. Huangjing seedlings were rinsed with sterile water, wiped clean with sterile absorbent paper, and transferred to a tray covered with wet filter paper to maintain high humidity. The mycelial piugs of F. redolens HJCD1 were inoculated onto the surface of leaves and basal stems. Controls were inoculated with sterile PDA plugs. The inoculated seedlings were sealed with plastic wrap, and then cultivated in a 25 ℃ growth chamber with 16 h of light per day. The pathogen-inoculated plants exhibited etiolation and typical wilt symptoms after 4 days, whereas no symptoms were observed in the control plants. F. redolens was reisolated from the infected tissues, and colony morphology and ITS sequence of re-isolates were same as that of HJCD1. The pathogen has been reported previously in american ginseng in China (Fan et al. 2021), lentil in Pakistan (Rafique et al. 2020), and wild rocket in United Kingdom (Taylor et al. 2019). However, to the best of our knowledge, this is the first report of F. redolens causing seelding basal rot on Duohua huangjing in China.

Phylogenetic analysis of Eucalyptus subgenus Eudesmia is presented on the basis of the following three datasets: sequences of the internal transcribed spacer (ITS) and the external transcribed spacer (ETS) regions from nuclear rDNA, sequences of the psbA–trnH intergenic spacer region from chloroplast DNA, and morphological characters, including stamen bundling, operculum development, seeds and trichomes. Studies of floral development were essential for understanding the morphology of mature flowers and interpretation of synapomorphy and homoplasy. A summary phylogeny was constructed from a maximum parsimony analysis of those nodes coded as characters that had support in the molecular trees together with morphological characters. A revised infra-subgeneric classification is presented on the basis of the summary phylogeny, and compared with classifications of Hill and Johnson (1998) and Brooker (2000). Differences relate to relationships between clades and taxonomic rank (sections, series and subseries) and valid names of Brooker (2000) are conserved where possible. One main clade of 14 species (section Limbatae), many of mallee growth form, was found in all analyses; this clade is distributed in the South-West of Western Australia and adjacent Interzone and desert areas. A second main clade (section Complanatae) occurs in the northern and eastern tropical and subtropical regions of Australia, including Kimberley, Arnhem, Queensland and New South Wales. This section includes E. tetrodonta, previously treated as an isolated taxon in a monotypic section; however, this species is related to E. baileyana, E. similis, E. lirata and series Miniatae. The hypothesised phylogeny provides a framework for further analyses of biogeography and ecology, including functional traits.

Molecular markers from different genomic compartments reveal cryptic diversity within glaucophyte species

BackgroundSpecies in the genus Morus (Moraceae) are deciduous woody plants of great economic importance. The classification and phylogenetic relationships of Morus, especially the abundant mulberry resources in China, is still undetermined. Internal transcribed spacer (ITS) regions are among the most widely used molecular markers in phylogenetic analyses of angiosperms. However, according to the previous phylogenetic analyses of ITS sequences, most of the mulberry accessions collected in China were grouped into the largest clade lacking for phylogenetic resolution. Compared with functional ITS sequences, ITS pseudogenes show higher sequence diversity, so they can provide useful phylogenetic information.MethodsWe sequenced the ITS regions and the chloroplast DNA regions TrnL-TrnF and TrnT-TrnL from 33 mulberry accessions, and performed phylogenetic analyses to explore the evolution of mulberry.ResultsWe found ITS pseudogenes in 11 mulberry accessions. In the phylogenetic tree constructed from ITS sequences, clade B was separated into short-type sequence clades (clades 1 and 2), and a long-type sequence clade (clade 3). Pseudogene sequences were separately clustered into two pseudogroups, designated as pseudogroup 1 and pseudogroup 2. The phylogenetic tree generated from cpDNA sequences also separated clade B into two clades.ConclusionsTwo species were separated in clade B. The existence of three connection patterns and incongruent distribution patterns between the phylogenetic trees generated from cpDNA and ITS sequences suggested that the ITS pseudogene sequences connect with genetic information from the female progenitor. Hybridization has played important roles in the evolution of mulberry, resulting in low resolution of the phylogenetic analysis based on ITS sequences. An evolutionary pattern illustrating the evolution history of mulberry is proposed. These findings have significance for the conservation of local mulberry resources. Polyploidy, hybridization, and concerted evolution have all played the roles in the evolution of ITS sequences in mulberry. This study will expand our understanding of mulberry evolution.

Interspecific hybridization is one of the major factors leading to phylogenetic incongruence among loci, but the knowledge is still limited about the potential of each locus to introgress between species. By directly sequencing three DNA regions: chloroplast DNAs (matK gene and trnL-F noncoding region), the nuclear ribosomal external transcribed spacer (ETS) region, and internal transcribed spacer (ITS) regions, we construct three phylogenetic trees of Asian species of Mitella (Saxifragaceae), a genus of perennials in which natural hybrids are commonly observed. Within this genus, there is a significant topological conflict between chloroplast and nuclear phylogenies and also between the ETS and the ITS, which can be attributed to frequent hybridization within the lineage. Chloroplast DNAs show the most extensive introgression pattern, ITS regions show a moderate pattern, and the ETS region shows no evidence of introgression. Nonuniform concerted evolution best explains the difference in the introgression patterns between the ETS region and ITS regions, as the sequence heterogeneity of the ITS region within an individual genome is estimated to be twice that of an ETS in this lineage. Significant gene conversion patterns between two hybridizing taxa were observed in contiguous arrays of cloned ETS-ITS sequences, further confirming that only ITS regions have introgressed bidirectionally. The relatively slow concerted evolution in the ITS regions probably allows the coexistence of multiple alleles within a genome, whereas the strong concerted evolution in the ETS region rapidly eliminates heterogeneous alleles derived from other species, resulting in species delimitations highly concordant with those based on morphology. This finding indicates that the use of multiple molecular tools has the potential to reveal detailed organismal evolution processes involving interspecific hybridization, as an individual locus varies greatly in its potential to introgress between species.

The phylogenetic relationship of the Korean endemic genus,Hanabusaya, to other campanulaceous genera has been controversial since it was described by Nakai in 1911. Three genera of Campanuloideae,Symphyandra, Adenophora, andCampanula, have been considered closely related by various taxonomists on the basis of anther shape, gross morphology, and pollen characters, respectively. We have tested these competing taxonomic hypotheses using the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA from 12 taxa representing 7 genera of Campanulaceae. The molecular phylogeny indicates strongly thatHanabusaya is more closely allied toAdenophora than toCampanula orSymphyandra. The phylogenetic affinity ofHanabusaya andAdenophora is supported by a 100% bootstrap value and a high decay index (13). The average sequence divergence value (Kimura’s 2-parameter method) betweenHanabusaya and theAdenophora species is 2.58. The value is significantly (about ten times) lower than the ones observed betweenHanabusaya and the species ofCampanula (average of 23.52) and betweenHanabusaya andSymphyandra (24.95). The ITS sequence phylogeny suggests that some morphological characters, such as fused anthers and corolla shape, are homoplastic in the Campanulaceae genera.

Species diversity in the genus Ulva remains understudied worldwide. Using molecular analyses we investigated the species composition, diversity, distribution, and relative frequencies of the genus Ulva along the entire coast of Jeju Island, off the southern tip of Korea. Species identification was performed for 215 samples collected from 23 sites, based on comprehensive phylogenetic and model-based species delimitation analyses using the sequences of two molecular markers, chloroplast elongation factor Tu (tufA) and nuclear rDNA internal transcribed spacer (ITS). We identified 193 specimens as nine Ulva species, 14 specimens as Blidingia spp., and eight samples undetermined, based on the combined analysis of tufA and ITS phylogenies. Two model-based approaches generally supported nine groups of Ulva species. Previously documented species complex, such as U. ohnoi−U. spinulosa and U. procera−U. linza showed discordant relationships between the two phylogenies. The occurrence of U. torta on Jeju Island was first observed, despite its existence on the mainland previously reported. Ulva australis [16 of 23 sites; 34.4% (relative frequency)], U. ohnoi (16; 21.9%), and U. procera (11; 14%) were found to be the predominant species. Our study highlights that molecular analysis is critical for species delimitation in the genus Ulva and provides fundamental information for an understanding of green-tide assemblages on the “biological hotspot” coastal ecosystem, Jeju Island in Korea. This study will also help to monitor and manage local green tides at the areas that are currently encountering rapid climate changes.

The Rhizopus strains in the Universitas Indonesia Culture Collection (UICC) were isolated from tempeh from various regions in Indonesia. Five strains of Rhizopus (UICC 10, UICC 85, UICC 119, UICC 120, and UICC 135) were previously identified based on phenotypic characters (morphology and physiology) as R. oryzae. The aim of this study was to clarify the identities of five Rhizopus strains based on internal transcribed spacer (ITS) regions of ribosomal DNA sequence data. Molecular phylogeny was carried out to establish the identities of these five strains. Primer set ITS5 and ITS4 were used for amplification and sequencing of the ITS regions of rDNA. The sequence data was sent to genBank for homology search using basic local alignment search tool (BLAST) program. Construction of phylogenetic tree was conducted using Neighbor-Joining method with Kimura’s two-parameter model. The results on gel electrophoresis showed that sizes of the ITS regions of rDNA fragment lengths of all strains were approximately 650 bp. The BLAST homology search results of five strains showed 99.8% homology to R. oryzae CBS 112.07T type strain. The phylogenetic tree clearly indicated that these five strains were included in a monophyletic group with R. oryzae CBS 112.07T type strain, and it was supported by high bootstrap value (84%). Morphological observation of all strains showed the characters corresponded to R. oryzae. The ITS regions of rDNA sequences and morphological characters have confirmed the strains as R. oryzae.

Preliminary Analysis of the Molecular Phylogenetics of Toxocara canis (Nematoda: Ascaridoidea) Using Nuclear Ribosomal Second Internal Transcribed Spacer Sequences