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Background Haemophilus influenzae is an opportunistic bacterial pathogen that can cause severe respiratory tract and invasive infections. The emergence of β-lactamase-negative ampicillin-resistant (BLNAR) strains and unclear correlations between genotypic (i.e., gBLNAR) and phenotypic resistance are challenging empirical treatments and patient management. Thus, we sought to revisit molecular resistance mechanisms and to identify new resistance determinants of H. influenzae . Methods We performed a systematic meta-analysis of H. influenzae isolates ( n  = 291) to quantify the association of phenotypic ampicillin and cefotaxime resistance with previously defined resistance groups, i.e., specific substitution patterns of the penicillin binding protein PBP3, encoded by ftsI . Using phylogenomics and a genome-wide association study (GWAS), we investigated evolutionary trajectories and novel resistance determinants in a public global cohort ( n  = 555) and a new clinical cohort from three European centers ( n  = 298), respectively. Results Our meta-analysis confirmed that PBP3 group II- and group III-related isolates were significantly associated with phenotypic resistance to ampicillin ( p  < 0.001), while only group III-related isolates were associated with resistance to cefotaxime ( p  = 0.02). The vast majority of H. influenzae isolates not classified into a PBP3 resistance group were ampicillin and cefotaxime susceptible. However, particularly group II isolates had low specificities (< 16%) to rule in ampicillin resistance due to clinical breakpoints classifying many of them as phenotypically susceptible. We found indications for positive selection of multiple PBP3 substitutions, which evolved independently and often step-wise in different phylogenetic clades. Beyond ftsI , other possible candidate genes (e.g., oppA, ridA, and ompP2 ) were moderately associated with ampicillin resistance in the GWAS. The PBP3 substitutions M377I, A502V, N526K, V547I, and N569S were most strongly related to ampicillin resistance and occurred in combination in the most prevalent resistant haplotype H1 in our clinical cohort. Conclusions Gradient agar diffusion strips and broth microdilution assays do not consistently classify isolates from PBP3 groups as phenotypically resistant . Consequently, when the minimum inhibitory concentration is close to the clinical breakpoints, and genotypic data is available, PBP3 resistance groups should be prioritized over susceptible phenotypic results for ampicillin. The implications on treatment outcome and bacterial fitness of other extended PBP3 substitution patterns and novel candidate genes need to be determined.

Background and objectives: In America, of the 44 species of Anopheles, nine are main vectors of malaria and, of these, genetic information exists for seven. Hence, this study sought to know the gene flow and diversity of the seven principal vectors of malaria at the Americas level. Methods: For the seven species and the sequences of the mitochondrial cytochrome c oxidase I (COI) gene obtained from the GenBank and Bold System, genetic analyzes of populations and genetic structure were performed and haplotype networks and phylogenetic trees were obtained. Results: For the seven species, 1440 sequences were analyzed and 519 haplotypes were detected. The Hd and π values were higher within a continental context than by countries. Neutrality tests indicated positive and negative values with most of these being significant (p < 0.05). Phylogenetic analyses for all the species recovered three clades with no geographic pattern among them. Interpretation & conclusion: Studies suggest that native species of Anopheles from the Americas have greater haplotype diversity and low genetic differentiation due to the lack of physical barriers to impede gene flow among these populations. Moreover, all the species are interconnected by roadways. This scenario complicates the epidemiological picture of malaria in the Americas.

We report Microhyla ninhthuanensis Hoang, Nguyen, Ninh, Luong, Pham, Nguyen, Orlov, Chen, Wang, Ziegler & Jiang, 2021 for the first time in China, from Zhuhai and Zhongshan, Guangdong, based on the molecular and morphological evidence. This extends its geographic range northward and eastward by over 550 km and 500 km, respectively. The hypothesis that this species may have been recently introduced into China through human activities is also discussed based on the haplotype network analysis.

We investigated diverse genomic selections using high-density single nucleotide polymorphism data of five distinct cattle breeds. Based on allele frequency differences, we detected hundreds of candidate regions under positive selection across Holstein, Angus, Charolais, Brahman, and N'Dama. In addition to well-known genes such as KIT, MC1R, ASIP, GHR, LCORL, NCAPG, WIF1, and ABCA12, we found evidence for a variety of novel and less-known genes under selection in cattle, such as LAP3, SAR1B, LRIG3, FGF5, and NUDCD3. Selective sweeps near LAP3 were then validated by next-generation sequencing. Genome-wide association analysis involving 26,362 Holsteins confirmed that LAP3 and SAR1B were related to milk production traits, suggesting that our candidate regions were likely functional. In addition, haplotype network analyses further revealed distinct selective pressures and evolution patterns across these five cattle breeds. Our results provided a glimpse into diverse genomic selection during cattle domestication, breed formation, and recent genetic improvement. These findings will facilitate genome-assisted breeding to improve animal production and health.

ABSTRACT Melting snowfields in polar and alpine regions often exhibit a red and orange colouration caused by microalgae. The diversity of these organisms is still poorly understood. We applied a polyphasic approach using three molecular markers and light and electron microscopy to investigate spherical cysts sampled from alpine mountains in Europe, North America and South America as well as from both polar regions. Molecular analyses revealed the presence of a single independent lineage within the Chlamydomonadales. The genus Sanguina is described, with Sanguina nivaloides as its type. It is distinguishable from other red cysts forming alga by the number of cell wall layers, cell size, cell surface morphology and habitat preference. Sanguina nivaloides is a diverse species containing a total of 18 haplotypes according to nuclear ribosomal DNA internal transcribed spacer 2, with low nucleotide divergence (≤3.5%). Based on molecular data we demonstrate that it has a cosmopolitan distribution with an absence of geographical structuring, indicating an effective dispersal strategy with the cysts being transported all around the globe, including trans-equatorially. Additionally, Sanguina aurantia is described, with small spherical orange cysts often clustered by means of mucilaginous sheaths, and causing orange blooms in snow in subarctic and Arctic regions. Red and orange spherical cysts causing snow colouration across several continents were investigated with regards to their geographic distribution, ecology, ultrastructure and phylogeny; the cosmopolitan distribution of a new independent lineage Sanguina within the Chlamydomonadales was molecularly confirmed.

Background Haplotype networks are useful for investigating the genealogical relationships among haplotypes and have been extensively used in molecular evolution and population genetic studies. However, existing tools for visualizing haplotype networks lack comprehensive functionalities for data exploration and layout adjustments. Therefore, there is a need for a useful tool capable of constructing and visualizing haplotype networks, facilitating the exploration of data, particularly with datasets with large sample sizes. Results We present HapNetworkView, a user-friendly tool that facilitates the construction and interactive visualization of haplotype networks, enabling users to explore haplotypes and mutation information effectively. HapNetworkView offers both automatic optimization and manual adjustment for haplotype network layouts. Additionally, HapNetworkView supports various input and output formats, as well as color customization. Conclusions These comprehensive functionalities collectively satisfy various needs of users in haplotype network analysis, making HapNetworkView a valuable tool.

Background The Aedes aegypti  mosquito is the primary vector for dengue, chikungunya, yellow fever and Zika viruses worldwide. The first record of Ae. aegypti in southwestern Saudi Arabia was in 1956. However, the first outbreak and cases of dengue fever were reported in 1994, and cases have increased in recent years. Vector control for Ae. aegypti mainly uses pyrethroid insecticides in outdoor and indoor space spraying. The constant use of pyrethroids has exerted intense selection pressure for developing target-site mutations in the voltage-gated sodium channel ( vgsc ) gene in Ae. Aegypti against pyrethroids—mutations that have led to knockdown resistance ( kdr ). Methods Aedes aegypti  field populations from five regions (Jazan, Sahil, Makkah, Jeddah and Madinah) of southwestern Saudi Arabia were genotyped for known  kdr  mutations in domains IIS6 and IIIS6 of the vgsc gene using polymerase chain reaction (PCR) amplification and sequencing. We estimated the frequency of kdr mutations and genotypes from Saudi Arabia as well as from other countries, Thailand, Myanmar (Southeast Asia) and Uganda (East Africa). We constructed haplotype networks to infer the evolutionary relationships of these gene regions. Results The three known kdr mutations, S989P, V1016G (IIS6) and F1534C (IIIS6), were detected in all five regions of Saudi Arabia. Interestingly, the triple homozygous wild genotype was reported for the first time in two individuals from the highlands of the Jazan region and one from the Al-Quoz, Sahil region. Overall, nine genotypes comprising four haplotypes were observed in southwestern Saudi Arabia. The median-joining haplotype networks of eight populations from Saudi Arabia, Southeast Asia and East Africa for both the IIS6 and IIIS6 domains revealed that haplotype diversity was highest in Uganda and in the Jazan and Sahil regions of Saudi Arabia, whereas haplotype diversity was low in the Jeddah, Makkah and Madinah regions. Median-joining haplotype networks of both domains indicated selection acting on the kdr -mutation containing haplotypes in Saudi Arabia. Conclusions The presence of wild type haplotypes without any of the three kdr mutations, i.e. that are fully susceptible, in Saudi Arabia indicates that further consideration should be given to insecticide resistance management strategies that could restore pyrethroid sensitivity to the populations of Ae. aegypti in Saudi Arabia as part of an integrative vector control strategy. Graphical Abstract

Metabarcoding is by now a well‐established method for biodiversity assessment in terrestrial, freshwater, and marine environments. Metabarcoding data sets are usually used for α‐ and β‐diversity estimates, that is, interspecies (or inter‐MOTU [molecular operational taxonomic unit]) patterns. However, the use of hypervariable metabarcoding markers may provide an enormous amount of intraspecies (intra‐MOTU) information mostly untapped so far. The use of cytochrome oxidase (COI) amplicons is gaining momentum in metabarcoding studies targeting eukaryote richness. COI has been for a long time the marker of choice in population genetics and phylogeographic studies. Therefore, COI metabarcoding data sets may be used to study intraspecies patterns and phylogeographic features for hundreds of species simultaneously, opening a new field that we suggest to name metaphylogeography. The main challenge for the implementation of this approach is the separation of erroneous sequences from true intra‐MOTU variation. Here, we develop a cleaning protocol based on changes in entropy of the different codon positions of the COI sequence, together with co‐occurrence patterns of sequences. Using a data set of community DNA from several benthic littoral communities in the Mediterranean and Atlantic seas, we first tested by simulation on a subset of sequences a two‐step cleaning approach consisting of a denoising step followed by a minimal abundance filtering. The procedure was then applied to the whole data set. We obtained a total of 563 MOTUs that were usable for phylogeographic inference. We used semiquantitative rank data instead of read abundances to perform AMOVAs and haplotype networks. Genetic variability was mainly concentrated within samples, but with an important between seas component as well. There were intergroup differences in the amount of variability between and within communities in each sea. For two species, the results could be compared with traditional Sanger sequence data available for the same zones, giving similar patterns. Our study shows that metabarcoding data can be used to infer intra‐ and interpopulation genetic variability of many species at a time, providing a new method with great potential for basic biogeography, connectivity and dispersal studies, and for the more applied fields of conservation genetics, invasion genetics, and design of protected areas.

Flowering time of crops is critical for maximizing yield, quality and regional adaptability under target environments. In rice, Heading date 3a (Hd3a) and Rice Flowering Locus T 1 (RFT1) proteins are the key factors of flowering time regulation in the florigen activation complex. However, effects of hd3a and rft1 mutations on the flowering time under long-day conditions are not clear. In this study, we generated single and double mutants of Hd3a and RFT1 in japonica varieties Shen Nong 9816 (SN9816) background, termed hd3a-SNs, rft1-SNs and doub-SNs , by using CRISPR/Cas9 system. The flowering time of hd3a-SN mutants was similar to wild-type (WT); but mutations of RFT1 delayed flowering time by 11 to 14 days comparing to WT under nature long day (NLD) conditions. Double mutants of Hd3a and RFT1 ( doub-SNs ) exhibited an extremely late flowering phenotype, and they did not flower up to 365 days after regeneration under either NLD or short-day conditions. The leaf emergency rate of RFT1 mutants was similar to WT, suggesting that the late flowering phenotype of rft1-SNs was caused by delayed floral transition, but not by retarded growth. The expression of the key floral organ identity genes was significantly decreased in rft1-SN and doub-SN mutants, resulting in late flowering phenotypes. The expression profiles, evolution and haplotypes of Hd3a and RFT1 was also analyzed. Our studies provide insight into the potential utilization of Hd3a and RFT1 gene resources to improving growth period and regional adaptability in rice breeding.