Intergenic Spacer Research Articles

Stevia rebaudiana Is a New Host of Stagonosporopsis pogostemonis Associated with Leaf Spot Disease in China.

Stevia rebaudiana is a promising medicinal and edible plant, widely cultivated in China. In 2022-2023, a new leaf spot disease occurred in S. rebaudiana in Hongxing country (32°34'55″N, 118°2'12″E), Dingyuan city, Anhui province. Symptoms were observed on 10 to 15% of plants in three S. rebaudiana nursery beds (0.1 ha in total). The typical symptoms included dark brown spots on the leaves and foliar wilting, the development of brown stems with dieback of top buds, and occasional plant death (Fig. 1a). To identify the pathogen, twenty diseased leaves were collected, cut into small pieces, surface sterilized with 75% ethanol for 30 s, in 0.5% sodium hypochlorite for 2 min, washed three times in sterile water, placed on PDA, and incubated at 25℃ for 5 days. Pure cultures were prepared by subculturing hyphal tips. Twenty-five Stagonosporopsis-like isolates with similar morphology were obtained. After 7 days growth on PDA, colonies had a regular margin, were cottony, and formed concentric circles on the surface that were gray-green. The reverse side of the culture was dark brown with a creme-orange and white, margin. The growth rate was 9.5 mm/day on PDA. Pycnidia were mostly solitary, globose or subglobose, pale to dark brown, thin-walled, glabrous, ostiolate, 95.735~250.851×90.93~266.32 μm (n=50). Conidia were oblong, cylindrical to ellipsoidal, smooth-walled, aseptate, with rounded ends and two polar guttules and measured 3.41 to 5.83 × 1.78 to 3.07 μm (n = 50) (Fig.1 b-d). For molecular identification, the internal transcribed spacer (ITS) rDNA, large ribosomal subunit (LSU) gene, β-tubulin (TUB2) gene and RNA polymerase II (RPB2) gene sequences of two representative isolates (TYJ-SP1 and TYJ-SP2) were amplified by PCR (Woudenberg et al. 2009; Dong et al. 2021). The sequences were deposited in GenBank (accession nos.: OR506193 and OR506194 for ITS, OR533526 and OR533527 for LSU, OR545221and OR545222 for TUB; OR545223 and OR545224 for RPB2) and showed 99.60% to 99.2% similarity to ITS (502/504 bp and 507/511 bp; MZ156571), 100% similarity to LSU (857/857 bp and 857/857 bp; MZ191532), 98.67% to 99.3% similarity to TUB2 (296/300 bp and 298/300 bp; MZ203132) and 99.78% (888/890 bp and 868/870 bp; MZ203135) of S. pogostemonis strain ZHKUCC 21-0001. A maximum likelihood phylogenetic analysis based on the concatenated sequences of ITS, LSU, TUB2 and RPB2 using MEGA 11.0 showed the strains TYJ-SP1 and TYJ-SP2 formed a clade with S. pogostemonis (Fig. 2). Thus, the strains were identified as S. pogostemonis (Dong et al. 2021). To test pathogenicity, the strain TYJ-SP1 was inoculated onto 30-day-old S. rebaudiana seedlings which were surface sterilized with 70% alcohol and washed 3 times with water and air dried prior to inoculation. Ten seedlings were sprayed with a conidial suspension (105 conidia/mL) and ten seedlings were sprayed with sterile water to serve as the negative control. All seedlings were maintained in a growth chamber (25°C, 90% relative humidity) with a 16 h photoperiod. Brown spots were first observed on inoculated leaves 48 h after inoculation; typical symptoms appeared by 7 days post inoculation. All inoculated plants developed symptoms similar to naturally infected plants in the nursery beds, and the disease incidence reached 100% while control plants remained symptom free (Fig. 1 e-f). The same Stagonosporopsis isolates were reisolated from the inoculated plants and identified based on morphological and phylogenetic analyses. S. pogostemonis has been reported to cause leaf spot in Pogostemon cablin and Brassica oleracea var. botrytis (Dong et al. 2021; Habib et al. 2024). To our knowledge, this is the first report of S. pogostemonis causing leaf spot on S. rebaudiana in China. As a medicinal and economic plant, S. rebaudiana is widely planted in China and other Asian countries. The occurrence of this leaf spot disease seriously affects its medicinal and economic value. Therefore, it is crucial to establish and implement effective disease management practices to reduce the impact of the disease.

First Report of Rhizopus arrhizusi causing rot of Dictyophora rubrovolvata in China.

Dictyophora rubrovolvata, as an edible fungus with high medicinal value, is widely cultivated in several provinces in China (Hang et al. 2012). However, between December 2023 and March 2024, a rot disease occurred in the main production area in Fengxian District, Shanghai, China (N30°93', E121°49'). The disease incidence was 25% in the affected 1.33-ha growing area. High temperatures (>25℃) and poor ventilation provide favorable conditions for the spread of this disease. The disease mainly occurs at the stage of fruiting bodies formation of D. rubrovolvata. When the epidermis is damaged and broken, it becomes infested with mold, which then produces a layer of moldy rot with pus. The infected D. rubrovolvata tissues at the edge of the lesions were isolated, surface sterilized and cultured on potato dextrose agar (PDA) at 30 ℃ under dark conditions. Pure cultures were obtained by single-spore isolation. After 3 days, isolates were transferred to Czapek Yeast agar (CYA) (Samson et al, 2014). On CYA, the fungal colony consisted of white flocculent hyphae. Scanning electron microscopy analysis showed that the mycelium was white, and the internodes of the stolons formed characteristic pseudoroots, from which upwardly clustered erect, unbranched sporocarp peduncles expanded apically to form rounded sporocarp sacs, within which sporocarp spores were produced. (Hariprasath P, 2019). To confirm the identity of the pathogen, the genomic fragments for the internal transcribed spacer (ITS) and intergenic spacer (IGS) gene of the isolate were amplified by PCR (White et al. 1990; Liu XY. 2008). The resulting sequence was deposited in GenBank with accession PP951880 and PQ001670, respectively. PCR results and morphological observations indicated the isolated strain was a pure culture and the strain was designated as DIC01. Comparative results showed that the sequences with accession numbers MT603964.1 and DQ990323.1 showed high homology of 99.15% and 98.96% to the ITS and IGS sequences of Rhizopus arrhizusi DIC01, respectively. Phylogenetic analysis with ITS and IGS genes of the isolated strain and 7 Rhizopus spp. strains were performed using MEGAX with Neighbor-Joining (NJ) method. Based on the results of growth habits, morphological observations, and phylogenetic analysis, the pathogen was identified as R. arrhizusi. A spore suspension of the R. arrhizusi DIC01 (1 x107 conidia/mL) was inoculated back to healthy D. rubrovolvata. Five healthy fruit bodies of D. rubrovolvata were injected, and another five healthy morels were treated with potato dextrose broth (PDB) as controls. D. rubrovolvata was incubated at 25°C and 90% relative humidity without ventilation for 5 days. The pathogen successfully infected the D. rubrovolvata, which developed white moldy lesions similar to those of natural diseases. The controls remained healthy without any symptoms. The pathogen was reisolated from the affected lesions and identified as R. arrhizusi DIC01 based on its morphological characteristics and phylogenetic marker genes. R. arrhizusi has been reported to cause endothelial cell damage and mycelial invasion into blood vessels, leading to thrombosis and tissue necrosis. (Hariprasath P, 2019). To our knowledge, this is the first report of R. arrhizusi causing rot disease of D. rubrovolvata. This study confirmed that R. arrhizusi is the pathogenic fungus responsible for rotting disease in D. rubrovolvata farms in Fengxian, Shanghai.

Molecular and Morphological Characteristics of a Novel Cyst Nematode in the Rhizosphere of Artemisia lavandulaefolia DC. in Gansu Province, Northwest China.

Cyst nematodes are obligate parasitic nematodes found in the fields of many cultivated crops. These nematodes, which have great economic importance, pose a threat to food security, though they are frequently ignored or misdiagnosed as pests because of covert parasitism. A cyst nematode population parasitizing on Artemisia lavandulaefolia DC., one of the traditional Chinese medicines was collected in Gansu Province. The species was diagnosed using integrative taxonomy and molecular approaches. The cyst population is spherical or lemon-shaped, light brown or dark brown in color, with a long neck and a protruding vulval cone. The stylet of the second-stage juvenile is strong, and the front end of the ball at the base of the stylet is concave; the median bulb and excretory pore are prominent; the tail is blunt and circular, and the transparent tail is usually shorter than the stylet. A phylogenetic analysis was carried out using the internal transcribed spacer (ITS) and 28S genes of ribosomal DNA, which further confirmed the presence of Cactodera chenopodiae. According to our literature review, this is the first report on C. chenopodiae in Compositae. By following this research, we can better understand the challenges posed by A. lavandulaefolia DC. and develop effective strategies for managing its spread and impacts. This will help to protect vulnerable ecosystems and ensure the sustainability of agricultural and forestry activities in affected areas.

Complete chloroplast genome sequence of Artemisia littoricola (Asteraceae) from Dokdo Island Korea: genome structure, phylogenetic analysis, and biogeography study.

The Asteraceae family, particularly the Artemisia genus, presents taxonomic challenges due to limited morphological characteristics and frequent natural hybridization. Molecular tools, such as chloroplast genome analysis, offer solutions for accurate species identification. In this study, we sequenced and annotated the chloroplast genome of Artemisia littoricola sourced from Dokdo Island, employing comparative analyses across six diverse Artemisia species. Our findings reveal conserved genome structures with variations in repeat sequences and junction boundaries. Notably, the chloroplast genome of A. littoricola spans 150,985bp, consistent with other Artemisia species, and comprises 131 genes, including 86 protein-coding, 37 tRNA, and 8 rRNA genes. Among these genes, 16 possess a single intron, while clpP and ycf3 exhibit two introns each. Furthermore, 18 genes display duplicated copies within the IR regions. Moreover, the genome possesses 42 Simple Sequence Repeats (SSRs), predominantly abundant in A/T content and located within intergenic spacer regions. The analysis of codon usage revealed that the codons for leucine were the most frequent, with a preference for ending with A/U. While the chloroplast genome exhibited conservation overall, non-coding regions showed lower conservation compared to coding regions, with the Inverted Repeat (IR) region displaying higher conservation than single-copy regions. Phylogenetic analyses position A. littoricola within subgenus Dracunculus, indicating a close relationship with A. scoparia and A. desertorum. Additionally, biogeographic reconstructions suggest ancestral origins in East Asia, emphasizing Mongolia, China (North East and North Central and South Central China), and Korea. This study underscores the importance of chloroplast genomics in understanding Artemisia diversity and evolution, offering valuable insights into taxonomy, evolutionary patterns, and biogeographic history. These findings not only enhance our understanding of Artemisia's intricate biology but also contribute to conservation efforts and facilitate the development of molecular markers for further research and applications in medicine and agriculture.

Foliar blight, leaf spot, and stem blackening diseases caused by Stemphylium beticola, Alternaria alternata, and Alternaria atra on dragon's head (Lallemantia iberica) in Iran

Dragon's head, Lallemantia iberica, is an oilseed crop adapted to be grown in cold-dry areas. In the growing season of 2022–23 a severe foliar blight, leaf spot, and stem blacking symptoms were observed in commercial fields in northern and northwestern Iran, leading to more than 50 % yield loss. Infected samples were collected and transferred to the laboratory. Following the isolation of fungal agents, five representative isolates were selected for further studies. Morphological studies, based on conidial shape, conidiation, and other mycological characteristics, were carried out on a pure culture established from a single spore. Furthermore, multigene phylogenetic analyses were performed using sequences of translation elongation factor 1-α (TEF1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (RPB2), and Internal Transcribed Spacer (ITS) rDNA genes for Alternaria isolates, along with calmodulin (cmdA) for the Stemphylium isolate. The morphological and molecular studies identified the fungal isolates as Alternaria atra, Alternaria alternata, and Stemphyliium beticola. Pathogenicity tests on the commercial cultivar ‘Sara’ indicated that all isolates were pathogenic to dragon's head, but they differed significantly in aggressiveness. To the best of our knowledge, this is the first report of the association of these fungal species with an important foliar disease on this crop in Iran and worldwide.

First report of Colletotrichum perseae Causing Anthracnose Disease of Avocado (Persea americana) in Türkiye

Colletotrichum-induced anthracnose is a major disease of avocado (Persea americana), causing significant losses in production worldwide. In 2023, a survey of avocado fruits in markets within the Kızıltepe district of Mardin, Türkiye, revealed symptoms of anthracnose, characterized by brown-black lesions and internal necrosis. To identify the causal agent, 20 symptomatic avocado fruits were collected and fungal isolates resembling Colletotrichum species were obtained. Morphological characterization, multilocus sequence analysis of the internal transcribed spacer (ITS), actin (act), the apn2-Mat1-2 intergenic spacer (ApMat), and calmodulin (cal) loci, and pathogenicity testing were performed for only two representative isolates (Pa01 and Pa02). These analyses identified the pathogen as Colletotrichum perseae. The isolates readily formed ascomata on all culture media tested, suggesting a high capacity for sexual reproduction. Pathogenicity tests confirmed that C. perseae caused characteristic dark, sunken anthracnose lesions on avocado fruits. This is the first report of C. perseae causing anthracnose in avocados in Türkiye, expanding the known geographical distribution of this pathogen. The findings highlight the need for effective disease management strategies and further research to understand the pathogen’s lifecycle, environmental conditions favoring infection, and potential for seedborne transmission.

Characterization of the Complete Mitochondrial Genome of Pleurogenoides japonicus (Digenea, Pleurogenidae): Comparison With the Members of Microphalloidea and Phylogenetic Implications.

Pleurogenoides japonicus (Trematoda: Microphalloidea) is an important parasite in wood frogs with high infection rates and significant ecological, economic, and societal importance. The scarcity of molecular data for these parasites severely limits population genetics and phylogenetic studies. In the present study, for the first time, we determined and described the entire mitochondrial (mt) genome of P. japonicus as the first representative of the family Pleurogenidae. The entire mt genome of P. japonicus was circular, with 15,043 bp (GenBank accession number OR900118), containing 36 genes, comprising 12 protein-coding genes (cox1-3, nad1-6, nad4L, cytb, and atp6), two ribosomal RNA genes, 22 transfer RNA genes, and two non-coding regions. There were 23 intergenic spacers, ranging from 2 to 162 bp, and only one 40 bp overlap between nad4L and nad4 genes in the P. japonicus mt genome. The nucleotide composition of P. japonicus mt genome exhibited a strong AT bias with a 63.75% A + T content, while the AT- and GC-skews were - 0.435 and 0.407, respectively. Comparative analysis demonstrated that the P. japonicus mt genome shared the most common characteristics with Microphalloidea trematodes, and the cox1 gene was the longest and most conserved gene in Microphalloidea trematodes. The gene arrangements of Xiphidiata trematodes were of the same order based on protein-coding genes and rRNA genes, except for tRNA. More than two gene arrangement types exist in Echinostomata and Xiphidiata, and the gene rearrangement events mainly occurred in "trnE-trnG" and "trnG-trnE". Phylogenetic analysis suggested that trematodes of the family Pleurogenidae clustered more with Prosthogonimidae than Eucotylidae. The mt genome data of P. japonicus provide an accurate genetic marker for further studies of Xiphidiata trematodes.

Evaluating the effectiveness of DNA-based methods to detect mislabelling and adulteration of chestnut honey

Honey is a naturally sweet and viscous substance that is consumed worldwide for its specific flavour, taste and biological properties, which in turn depend on the type of plant from which bees collect nectar. The quality of honey and, in particular, the information on the product label determines consumer choice and market price. In this study, the true botanical composition of chestnut monofloral honeys produced in Calabria (South Italy) was determined by DNA barcoding, PCR-RFLP and LAMP approaches, all based on the chloroplast trnL-trnF intergenic spacer region. All three DNA-based techniques were reliable in unambiguously identifying the floral composition of the different honeys tested at species level. However, the LAMP assay has the advantage over DNA barcoding and PCR-RFLP of greatly simplifying and speeding up the identification of Castanea sativa as the dominant plant species of the Calabrian chestnut honeys. This may encourage the adoption of this method by the food industry for the rapid detection of intentionally or accidentally mislabelled ingredients in this valuable natural product, thus ensuring consumer safety and protection.

Acinetobacter baumannii clinical isolates from outbreaks in Erbil hospitals after the COVID-19 pandemic.

Acinetobacter baumannii is endemic in hospital environments, and since the coronavirus disease 2019 (COVID-19) pandemic, multidrug-resistant A. baumannii has become more potent. This potential evolution is driven by the undetectable numbers of gene resistances it has acquired. We evaluated the antibiotic-resistance genes in isolates from patients in Erbil hospitals. This is the first study to demonstrate the antimicrobial resistance epidemic in Erbil, Iraq. A total of 570 patients, including 100 COVID-19 patients were tested. Isolate identification, characterization, antibiotics susceptibility test, polymerase chain reaction (PCR) amplification of the antibiotic resistance genes in both bacterial chromosome and plasmid, 16S-23S rRNA gene intergenic spacer (ITS) sequencing using the Sanger DNA sequencing, and phylogenetic analysis were used in this study. Only 13% of A. baumannii isolates were from COVID-19 patients. All isolates were multi-drug resistant due because of 24 resistance genes located in both the bacterial chromosome or the plasmid. blaTEM gene was detected in the isolates; however, aadB was not detected in the isolated bacteria. New carbapenemase genes were identified by Sanger sequencing and resistance genes were acquired by plasmids. The study identified metabolic differences in the isolates; although all the strains used the coumarate pathway to survive. Several resistance genes were present in the isolates' plasmids and chromosome. There were no strong biofilm producers. The role of the plasmid in A. baumannii resistance development was described based on the results.

In Silico Evaluation of rbcL, matK, and psbA-trnH Regions on the Genus Spatholobus (Fabaceae)

Spatholobus is a genus that belongs to the Fabaceae that is known to contain various bioactive compounds and distributed across Asia, including Indonesia. However, exploration of Spatholobus in Indonesia is still rare. Therefore, DNA barcoding is used to support the exploration and conservation of Spatholobus in Indonesia. However, there is no universal marker that can be used across all plant species. In addition, there are still few studies related to DNA barcoding within the genus Spatholobus. The purpose of this study was to evaluate the rbcL, matK, and intergenic spacer psbA-trnH regions in silico that can be used as DNA barcodes for the genus Spatholobus. This study began with a sequence search on the NCBI database including the rbcL, matK, and intergenic spacer psbA-trnH genes in the genus Spatholobus and Phaseolus coccineus as the outgroup. Each sequence was then aligned with ClustalW. Then, a phylogenetic tree was constructed using the Maximum-Likelihood (ML) with 1000× bootstrap. As a result, the rbcL, matK, and psbA-trnH regions can be used as markers for DNA barcoding in the genus Spatholobus with different specifications. The rbcL and matK can be used to distinguish Spatholobus at the genus level, while the psbA-trnH can be used to distinguish Spatholobus at the species level.

Pseudoperonospora cubensis Causing Downy Mildew on Luffa aegyptiaca in Korea

In November 2021, sponge gourd (<i>Luffa aegyptiaca</i>) plants with typical downy mildew symptom were observed in Korea. Based on morphological characteristics of sporangiophores and sporangia, combined with molecular phylogenetic analysis of the internal transcribed spacer rDNA and <i>cox</i>2 mtDNA regions, the causal pathogen was identified as <i>Pseudoperonospora cubensis</i> (Oomycota). This is the first report of downy mildew caused by <i>P. cubensis</i> on <i>L. aegyptiaca</i> in Korea. Given that <i>P. cubensis</i> exhibits high disease incidence and severity on many Cucurbitaceae crops, it could pose a significant threat to sponge gourd cultivation.

Description of the complete rDNA repeat unit structure of Coturnixjaponica Temminck et Schlegel, 1849 (Aves).

Ribosomal RNA (18S, 5.8S, 28S) gene clusters in genomes form regions that consist of multiple tandem repeats. They are located on a single or several pairs of chromosomes and play an important role in the formation of the nucleolus responsible for the assembly of ribosome subunits. The rRNA gene cluster sequences are widely used for taxonomic studies, however at present, complete information on the avian rDNA repeat unit structure including intergenic spacer sequence is available only for the chicken (Gallusgallusdomesticus Linnaeus, 1758). The GC enrichment and high-order repeats peculiarities within the intergenic spacer described for the chicken rDNA cluster may be responsible for these failures. The karyotype of the Japanese quail (Coturnixjaponica Temminck et Schlegel, 1849) deserves close attention because, unlike most birds, it has three pairs of nucleolar organizer bearing chromosomes, two of which are microchromosomes enriched in repeating elements and heterochromatin that carry translocated terminal nucleolar organizers. Here we assembled and annotated the complete Japanese quail ribosomal gene cluster sequence of 21166 base pairs (GenBank under the registration tag BankIt2509210 CoturnixOK523374). This is the second deciphered avian rDNA cluster after the chicken. Despite the revealed high similarity with the chicken corresponding sequence, it has a number of specific features, which include a slightly lower degree of GC content and the presence of bendable elements in the content of both the transcribed spacer I and the non-transcribed intergenic spacer.

5s rDNA intergenic spacer of some species of Cruciata and Chondrophyllae sections and its application in taxonomy of genus Gentiana

Aim. The 5S rDNA intergenic spacer (IGS) region is a convenient tool for studying genome evolution and systematics of higher plants. The aim was to investigate the nucleotide sequence of 5S rDNA in some species of the sections Cruciata and Chondrophyllae, as well as to study the peculiarities of this region organisation by comparative analysis with other Gentiana species. Methods. Polymerase chain reaction, cloning, sequencing, bioinformatic analysis. Results. The cloned IGS regions of G. laciniata, G. cruciata and G. dahurica were sequenced and analysed. Typical regulatory elements were found in the regions of terminator and external promoter of 5S rRNA gene. Based on the comparative analysis, two groups of species were distinguished that differ significantly in the organisation of this region. One includes G. asclepiadea and species of the sections Gentiana, Ciminalis and Calathianae, and the other includes species of the sections Cruciata and Pneumonanthe. G. laciniata (sect. Chondrophyllae) differs in the IGS organization from species of both groups. Conclusions. The results of taxon grouping are generally consistent with the genus systematics and indicate that 5S rDNA IGS region can be a convenient tool for studying evolution and systematics of the genus.

Genetic diversity of populations of East Siberian ende

Based on nucleotide polymorphism of cpDNA intergenic spacers psbA-trnH, trnL-trnF, and trnS-trnG, we studied the genetic diversity and population structure of a mountain species Oxytropis vasskovskyi Jurtz., endemic to East Siberia. The populations were characterized by medium and high haplotype (0.524–1.000) and low nucleotide (0.0003–0.0008) diversity. Variability distribution analysis showed a moderate level of diff erentiation; the interpopulation component accounted for about 44% of the total genetic variation in O. vasskovskyi. We observed demographic expansion for three of fi ve O. vasskovskyi populations. The haplotypes of the fi ve studied populations form a single complex in the genealogical network, which is probably due to their common origin, relatively recent divergence and incomplete lineage sorting, as well as the existing gene exchange between populations. Thus, in four of the fi ve studied populations, a low level of nucleotide diversity and the absence of nucleotide substitutions were determined, which indicates the need to use certain measures to protect all known habitats, conduct regular monitoring of their state and search for new locations of the species.

Nothophoma spp. causing leaf blight of ancient Platycladus orientalis

Ancient Platycladus orientalis holds significant ecological, landscape, historical, and cultural value. In northern China, leaf blight has significantly impacted the growth and ornamental value of ancient P. orientalis. In this study, 26 blight leaf samples of ancient P. orientalis were collected in Beijing, China. Phylogenetic analysis based on the concatenated internal transcribed spacer (ITS), β-tubulin (tub2), and RNA polymerase second largest subunit (rpb2) DNA sequence data combined with fungal morphological characteristics revealed three taxa of Nothophoma, i.e. N. platycladus, N. spiraeae, and N. juglandis. Of which, N. platycladus is a species new to science. Koch’s postulates indicated that all these three species of Nothophoma could cause leaf blight of P. orientalis.

Extinct or Not? Confirming the “Extinct” Status of Hieracium tolstoii (Asteraceae) with Integrated Taxonomic Investigation

Hieracium is one of the largest flowering plant genera that has recently experienced a rapid evolutionary radiation. Due to the massive morphological variation among species, frequent hybridization, and polyploidization events, combined with apomictic reproduction in polyploids, the concept of species in Hieracium is widely debated, and species identification is incredibly challenging. Taxonomic investigation can lead to changes in conservation status or, in some cases, to de-extinctions of species. Taxa with doubtful classification are particularly sensitive to this kind of problem. Hieracium tolstoii was a narrow Italian endemic species that is nowadays considered extinct. Described as a morphological intermediate form between two other species, no further investigations were conducted after its first description. To clarify H. tolstoii’s origin and ensure the validity of its current conservation status, we conducted a taxonomic investigation on herbaria specimens of the early XX century. Specimens were studied using a morphometric analysis that compared H. tolstoii with other closely related taxa. Moreover, we performed phylogenetic investigations using three plastid intergenic spacers to evaluate the relationship between species. Plastid markers revealed the presence of indels and SNPs in H. tolstoii sequences that differ from sequences of the supposed parental species. Morphometry revealed differences among species that led us to confirm the validity of H. tolstoii as an independent apomictic species and, therefore, unfortunately, its extinction.

Origin and maintenance of large ribosomal RNA gene repeat size in mammals.

The genes encoding ribosomal RNA are highly conserved across life and in almost all eukaryotes are present in large tandem repeat arrays called the rDNA. rDNA repeat unit size is conserved across most eukaryotes but has expanded dramatically in mammals, principally through the expansion of the intergenic spacer region that separates adjacent rRNA coding regions. Here, we used long-read sequence data from representatives of the major amniote lineages to determine where in amniote evolution rDNA unit size increased. We find that amniote rDNA unit sizes fall into two narrow size classes: "normal" (∼11-20 kb) in all amniotes except monotreme, marsupial, and eutherian mammals, which have "large" (∼35-45 kb) sizes. We confirm that increases in intergenic spacer length explain much of this mammalian size increase. However, in stark contrast to the uniformity of mammalian rDNA unit size, mammalian intergenic spacers differ greatly in sequence. These results suggest a large increase in intergenic spacer size occurred in a mammalian ancestor and has been maintained despite substantial sequence changes over the course of mammalian evolution. This points to a previously unrecognized constraint on the length of the intergenic spacer, a region that was thought to be largely neutral. We finish by speculating on possible causes of this constraint.

Phlebotomine sand fly (Diptera: Psychodidae) fauna, blood meal source, and detection of Leishmania (Kinetoplastida: Trypanosomatidae) DNA in the Gurupi Biological Reserve, Eastern Amazon, Brazil.

This study was conducted in the Gurupi Biological Reserve (REBIO-Gurupi), the largest area of Amazon rainforest in Maranhão State, Brazil. The objectives were to survey the sand fly (Diptera: Psychodidae) fauna of REBIO-Gurupi, identify blood meal sources, and investigate the presence of Leishmania (Ross, 1903) (Kinetoplastida: Trypanosomatidae) DNA. Individuals were collected using Centers for Disease Control (CDC) light traps and black and white Shannon traps in May and Jun 2022 and Jan 2023. DNA was extracted from female sand flies and subjected to amplification and sequencing of cytochrome b molecular marker (CYTB) for identification of blood meal sources and the first internal transcribed spacer (ITS-1) of ribosomal DNA for Leishmania detection. A total of 514 sand flies individuals were sampled, of which 93 were identified at the genus or series level (9 taxa) and 421 were identified at the species level (24 taxa). Psychodopygus davisi (Root, 1934) (41.1%), Nyssomyia antunesi (Coutinho, 1939) (10.3%), and Psychodopygus (Mangabeira, 1941) Chagasi Series Barretto, 1962 (9.7%) were the most frequently collected. Human (Homo sapiens, Primates, Hominidae) and tapir (Tapirus terrestris, Perissodactyla, Tapiridae) DNA was detected in 10 female sand flies. Leishmania (Leishmania) infantum Cunha and Chagas, 1937 DNA was detected in 2 specimens of Ps. davisi. Given the presence of vectors of Leishmania in REBIO-Gurupi, it is imperative to conduct more comprehensive studies on the interactions among sand flies, Leishmania, and pathogen reservoirs in the area.

A resource of identified and annotated lincRNAs expressed during somatic embryogenesis development in Norway spruce.

Long non-coding RNAs (lncRNAs) have emerged as important regulators of many biological processes, although their regulatory roles remain poorly characterized in woody plants, especially in gymnosperms. A major challenge of working with lncRNAs is to assign functional annotations, since they have a low coding potential and low cross-species conservation. We utilised an existing RNA-Sequencing resource and performed short RNA sequencing of somatic embryogenesis developmental stages in Norway spruce (Picea abies L. Karst). We implemented a pipeline to identify lncRNAs located within the intergenic space (lincRNAs) and generated a co-expression network including protein coding, lincRNA and miRNA genes. To assign putative functional annotation, we employed a guilt-by-association approach using the co-expression network and integrated these results with annotation assigned using semantic similarity and co-expression. Moreover, we evaluated the relationship between lincRNAs and miRNAs, and identified which lincRNAs are conserved in other species. We identified lincRNAs with clear evidence of differential expression during somatic embryogenesis and used network connectivity to identify those with the greatest regulatory potential. This work provides the most comprehensive view of lincRNAs in Norway spruce and is the first study to perform global identification of lincRNAs during somatic embryogenesis in conifers. The data have been integrated into the expression visualisation tools at the PlantGenIE.org web resource to enable easy access to the community. This will facilitate the use of the data to address novel questions about the role of lincRNAs in the regulation of embryogenesis and facilitate future comparative genomics studies.

New insights on the phylogeny, evolutionary history, and ecological adaptation mechanism in cycle-cup oaks based on chloroplast genomes.

Cycle-cup oaks (Quercus section Cyclobalanopsis) are one of the principal components of forests in the tropical and subtropical climates of East and Southeast Asia. They have experienced relatively recent increases in the diversification rate, driven by changing climates and the Himalayan orogeny. However, the evolutionary history and adaptive mechanisms at the chloroplast genome level in cycle-cup oaks remain largely unknown. Therefore, we studied this problem by conducting chloroplast genomics on 50 of the ca. 90 species. Comparative genomics and other analyses showed that Quercus section Cyclobalanopsis had a highly conserved chloroplast genome structure. Highly divergent regions, such as the ndhF and ycf1 gene regions and the petN-psbM and rpoB-trnC-GCA intergenic spacer regions, provided potential molecular markers for subsequent analysis.The chloroplast phylogenomic tree indicated that Quercus section Cyclobalanopsis was not monophyletic, which mixed with the other two sections of subgenus Cerris. The reconstruction of ancestral aera inferred that Palaeotropics was the most likely ancestral range of Quercus section Cyclobalanopsis, and then dispersed to Sino-Japan and Sino-Himalaya. Positive selection analysis showed that the photosystem genes had the lowest ω values among the seven functional gene groups. And nine protein-coding genes containing sites for positive selection: ndhA, ndhD, ndhF, ndhH, rbcL, rpl32, accD, ycf1, and ycf2. This series of analyses together revealed the phylogeny, evolutionary history, and ecological adaptation mechanism of the chloroplast genome of Quercus section Cyclobalanopsis in the long river of earth history. These chloroplast genome data provide valuable information for deep insights into phylogenetic relationships and intraspecific diversity in Quercus.