Using DNA Sequences and Phylogenetic Trees as Tools for Teaching Entomology to Undergraduate Students: A Simple Approach

This technical note aims to show how any instructor teaching entomology can use the Basic Local Alignment Search Tool (BLAST) and the “one click” mode of Phylogeny.fr to teach undergraduate students about insect DNA similarity in a simple way. Teaching an entomology course requires the use of numerous tools to help students grasp different concepts. Knowing that there are more than one million described species of insects means that teaching students about insect identification and taxonomy can be challenging. However, here we present two easy exercises that could be used as classroom or take-home assignments to demonstrate various levels of DNA similarity among different insect taxa. Such exercises unlock students’ creativity and break the barrier of fear of bioinformatics. Moreover, they open up new ways for them to understand insect taxonomy through molecular biology and allow them to develop new skills that contribute to strengthening their scientific performance in the future, especially when they do research as graduate students. Finally, this note is an example of how to integrate simple bioinformatics tools into the teaching of entomology.

Distances Between Phylogenetic Trees: A Survey

Phylogenetic trees have been widely used in the study of evolutionary biology for representing the tree-like evolution of a collection of species. However, different data sets and different methods often lead to the construction of different phylogenetic trees for the same set of species. Therefore, comparing these trees to determine similarities or, equivalently, dissimilarities, becomes the fundamental issue. Typically, Tree Bisection and Reconnection（TBR）and Subtree Prune and Regraft（SPR） distances have been proposed to facilitate the comparison between different phylogenetic trees. In this paper, we give a survey on the aspects of computational complexity, fixed-parameter algorithms, and approximation algorithms for computing the TBR and SPR distances of phylogenetic trees.

Partial Fusion (F) Gene Analysis of Newcastle Disease Virus Detected in Pakistan during 2021-2022

Newcastle disease (ND) virus is a leading threat to commercial and domestic poultry in Pakistan. The virus infects and constitutes irreversible impairment to the nervous system, damages the respiratory system, and marks severe gastrointestinal lesions leading to heavy mortality in short-living birds and substantial losses in layers and breeders. The continuous emergence and evolution of the virus made it inclined to evade the humoral response and indirectly the circumvention of artificial active immunization. Newcastle disease is caused by the orthoavula genus of the paramyxoviridae family and has shown high genetic diversity even in their genotypes while information regarding enzootic trends of the virus is scanty in Pakistan. A total of 40 tracheal samples of NDV were collected from different commercial broiler farms and 11 isolates of NDV were identified. In the current study, we determined the genetic diversity of the Newcastle disease virus based on the partial sequencing of the fusion protein gene available in the NCBI database. Genetic analysis showed that seven isolates belonged to class I genotype VII and four belonged to class II genotype II. Interestingly, two isolates had epidemiological connections with vaccine-like class II genotype II. Our findings, concerning the recent outbreaks of class I genotype VII and class II genotype II of NDV in vaccinated commercial flocks, suggest possible potential partial mutations in the fusion protein gene. Genetic diversity and formation of the new cleavage site in an important neutralizing protein of wild strain are linked with the potency of artificial active immunization and a major cause of vaccine failure.

The Finding and Phylogenetic Evolution Analysis of Bovine Piroplasms in the Rasǒn Area of North Korea

The objective of this study was to investigate the epidemiology of bovine Piroplasms infections in the Rasǒn area of North Korea.The survey was carried out by light microscopic examination of Giemsa-stained blood smears,PCR,and phylogenetic evolution analysis of 128 blood samples collected from the Rasǒn area.The results showed that the infection rates of the small and large parasites were about 2.5 and 1.5% on average,respectively,in all Theileria sergenti and Babesia ovatapositive blood smears by microscopic examination of blood smears.The detection rate of T.sergenti Giemsa-stained smears was 43.75%,while that with PCR was 67.97%.The detection rate of B.ovata Giemsa-stained smears was 49.21%,while that with PCR was 71.88%.The sequence and phylogenetic analysis of DNA showed 98.84% homology between the 18S rRNA gene sequences of T.sergenti isolates from North Korean and that of Yanbian state from China,indicating the closest genetic relationship between both of them.Moreover,100% homology was shown between the 18S rRNA gene sequence of B.ovata isolates from North Korea and the published sequence AY081192 of GenBank,indicating the closest genetic relationship between both of them.This survey confirmed that Ras n is the endemic area of T.sergenti and B.ovata in North Korea.

Characterization of Avian Influenza A(H7N9) Virus Prevalence in Humans and Poultry in Huai′an,China：Molecular Epidemiology,Phylogenetic,and Dynamics Analyses

Objective To trace the source of human H7N9 cases in Huai＇an and elucidate the genetic characterization of Huai＇an strains associated with both humans and birds in live poultry market.Methods An enhanced surveillance was implemented when the first human H7N9 case was confirmed in Huai＇an.Clinical specimens,cloacal swabs,and fecal samples were collected and screened by real-time reverse transcription-polymerase chain reaction（RT-PCR） for H7N9 virus.The positive samples were subjected to further RT-PCR and genome sequencing.The phylodynamic patterns of H7N9 virus within and separated from Huai＇an and evolutionary dynamics of the virus were analyzed.Results Six patients with H7N9 infection were previously exposed to live poultry market and presented symptoms such as fever（〉38.0 °C） and headaches.Results of this study support the hypothesis that live poultry markets were the source of human H7N9 exposure.Phylogenetic analysis revealed that all novel H7N9 viruses,including Huai＇an strains,could be classified into two distinct clades,A and B.Additionally,the diversified H7N9 virus circulated in live poultry markets in Huai＇an.Interestingly,the common ancestors of the Huai＇an H7N9 virus existed in January 2012.The mean nucleotide substitution rates for each gene segment of the H7N9 virus were（3.09-7.26）×10-3 substitutions/site per year（95% HPD：1.72×10-3 to 1.16×10-2）.Conclusion Overall,the source of exposure of human H7N9 cases in Huai＇an was live poultry market,and our study highlights the presence of divergent genetic lineage of H7N9 virus in both humans and poultry specimens in Huai＇an.

The complete genome of hydrocarbon-degrading Pseudoalteromonas sp. NJ289 and its phylogenetic relationship

Genus Pseudoalteromonas belongs to Family Pseudoalteromonadaceae in Gammaproteobacteria. A cold-adapted gram-negative bacterium, hydrocarbon-degrading Pseudoalteromonas sp. NJ289, was isolated from sea-ice of the Antarctica region, and sequenced the whole genome through the next generation sequencing platform. The assembly yielded three contigs representing two chromosomes and one plasmid with the sizes of 3.2 Mb, 636 kb and 1.8 kb, respectively. The G＋C contents of genome were 40.83% and included 3 589 ORFs. Functional annotation indicated some potential roles in enzymatic activity and environmental adaptability. This study may help for understanding the population diverse, evolutionary ecology and the microbial interaction.

Phylogenetic analysis and biological characteristic tests of marine bacteria isolated from Southern Ocean(Indian sector)water

Fifty-seven bacteria were isolated from Southern Ocean （Indian sector） water samples which were collected from different latitude and longitude of the ocean. All the isolates were able to grow at 4℃, 20℃, 37℃ and tolerable NaCI concentration up to 13.5% （w/v）. 29 out of 57 isolates were identified using 16S rDNA amplification and the sequences were submitted to National Center for Biotechnology Information （NCBI）. All the isolates were classified by using Ribosomal Database Project （RDP） and found that isolates belongs to Proteobacteria and Bacteriodes. The average G＋C content was 56.4%. The isolates were screened for the presence of extracellular enzymes, viz. amylase, catalase, urease, esterase, lipase and protease. The disc diffusion method is used to screen antibiotic production by the isolates against four pathogenic bacteria, viz. Salmonella typhimurium （NCIM 2501）, Staphylococcus aureus （NCIM 2122）, Bacillus subtilis （NCIM 2193）, and Pseudomonas aeruginosa （NCIM 2036）. Nine out of 29 were found to be antibiotic producer.

Prevalence of cercarial infections in freshwater snails and morphological and molecular identification and phylogenetic trends of trematodes

Objective:To investigate the prevalence of cercarial infections in freshwater snails from several water sources in Nakhon Nayok,Nonthaburi,and Pathum Thani provinces of Central Thailand,and to reconstruct a phylogenetic tree for improved understanding of the relationships in the cercarial stage.Methods:The snail specimens were collected from 34 total sampling sites and investigated for cercarial infections using the crushing method.The cercarial specimens were classified and used for the phylogenetic tree analysis using the Internal Transcribed Spacer 2(ITS2).Results:A total of 1921 snail specimens were classified into five families and seven species.The results showed that four snail species were identified as intermediate hosts of the larval stages of trematodes,with an overall prevalence of infection of 2.45%(47/1921).The infected snail specimens included five groups of the cercarial type:cercariaeum cercariae,echinostome cercaria,megalurous cercaria,parapleurolophocercous cercaria,and xiphidiocercariae.This is particularly true of xiphidiocercariae,which was found to be the dominant type among cercarial infections in bithyniid snails by approximately 38.00%.With regard to molecular identification,the phylogenetic tree was reconstructed using the neighbor-joining method with 10000 bootstraps and separated the trematodes into three clades:Echinostomatoidea,Microphalloidea and Opisthorchioidea.Conclusions:The study reveals a high prevalence of cercarial infection for each cercarial type and maturation into a definite trematode genus and delineates morphological characteristics and evolutionary trends among each larval trematode in Nakhon Nayok,Nonthaburi and Pathum Thani provinces.In addition,the ITS2 sequence data of cercariae could be used to examine classification of these species at the family level.

Phylogenetic analysis of microRNA biomarkers for amyotrophic lateral sclerosis

Amnyotrophic lateral sderosis(ALS),also alled Lou Gchrig's disase,is an rreversible disase that is aused bythe degeneration and death of motor newons.Approximately 5-10%of cases are familial AIS(fALS),and the other cases are sporadic ALS(sALS).Gene mutations have been identifed both in fAIS and sALS patients.In this study,w disauss the four ALS-nelated genes,C9orf72,SODI,FUS,and TARDBP,and review the microRNAs(miRNAs)that are asociated with AlS and other neurological disordes from the litemature.A phylogentic analysis is used toexplore potential miRNAs that an be taken into acount when studying the dfference in pathology for AIS induced by the fowr genes and other ncurological discases such as frontotemporal dementia,spinal muscular atrophy,and narcolepsy.We found several miRNAs that an be taken into account to study the diference in pathology betwen AIS and other neurological disorders.

A simple way to visualize detailed phylogenetic tree of huge genome-wide SNP data constructed by SNPhylo

Phylogenetic trees based on genome-wide single nucleotide polymorphisms （SNPs） among diverse inbreds could provide valuable and intuitive information for breeding and germplasm management in crops. As a result of sequencing technology developments, a huge amount of whole genome SNP data have become available and affordable for breeders. However, it is a challenge to perform quick and reliable plotting based on the huge amount of SNP data. To meet this goal, a visualization pipeline was developed and demonstrated based on publicly available SNP data from the current important maize inbred lines, including temperate, tropical, sweetcorn, and popcorn. The detailed phylogenetic tree plotted by our pipeline revealed the authentic genetic diversity of these inbreds, which was consistent with several previous reports and indicated that this straightforward pipeline is reliable and could potentially speed up advances in crop breeding.

Phylogenetic analysis and in vitro culture of mosses fromthe Antarctic Fildes Peninsula

Molecular genetic techniques have proven very useful for initial analysis of the extent of genetic variation and dispersal in several Antarctic moss species. In the present study, the small subunit ribosomal RNA （SSU rDNA） and internal transcribed spacers of the nuclear ribosomal DNA （ITS rDNA） were sequenced in nine individuals of different mosses from the Fildes Peninsula of Antarctica. Sequence alignment showed that the extreme environment tended to increase the genetic diversity of Antarctic mosses. In addition, in our phylogenetic analysis, one previously unidentified Antarctic moss species was characterized by comparison with SSU and ITS rDNA sequences of known moss species. Moreover, the optimal culture medium and conditions for surface explant sterilization and protonemata induction in tissue culture of Pohlia nutans were investigated. The successful establishment of a tissue culture protocol together with the phylogenetic analysis of Antarctic mosses will provide technological support to establish an effective resource regeneration method for discovering new functional genes and gaining novel insights into the mechanisms of stress acclimation.

Study on Phylogenetic Relationships of Five Breeds of Pigs by Random Amplified Polymorphic DNA(RAPD)

RAPD was used to study the genetic divergency and phylogenetic relationships of five breeds of domestic pigs,including Min pig,Duroc,Yorkshired,Landrace and Junmu I pig.We selected fourteen primers from eighty random primers,caculated genetic distance index matrix and constructed phylogenetic tree with UPGMA methods.Genetic distance index matrix indicated that the genetic relationship between Junmu I pig and Landrace was the closest and the farthest between Duroc and min pig.

Phylogenetic Structure of Low Altitude Forest Communities in Baotianman Mountain

[Objectives]The paper was to reveal the phylogenetic structure of low altitude forest communities in Baotianman Mountain.[Methods]Ten 20 m×20 m plots were set at an altitude of 249-796 m in Baotianman Nature Reserve,in which 62 plant species were found.A phylogenetic tree was established on Phylomatic website,and the community related phylogenetic indexes at low altitude were calculated and analyzed,including net relatedness index(NRI),nearest taxon index(NTI),phylogenetic diversity Faith's PD index,phylogeneticβdiversity PhyloSor index and Dnn index.[Results]The NTI and NRI values of low altitude forest communities were generally greater than 0,showing phylogenetically clustered communities,and habitat filtration was the dominant factor in community construction.Combined with the species abundance matrix of plots,it was found that PD index had a significantly positive correlation with species richness.Phylogeneticβdiversity PhyloSor index was larger in the lower altitude area,which was exactly opposite to Dnn index.In other words,the more similar the community,the closer the distance between species,and the communities were phylogenetically clustered.[Conclusions]The study can provide a scientific basis for forest community management and restoration.

Phylogenomic analysis of transcriptomic sequences of mitochondria and chloroplasts of essential brown algae(Phaeophyceae)in China

The chloroplast and mitochondrion of brown algae （Class Phaeophyceae of Phylum Ochrophyta） may have originated from different endosymbiosis. In this study, we carried out phylogenomic analysis to distinguish their evolutionary lineages by using algal RNA-seq datasets of the 1 000 Plants （1KP） Project and publicly available complete genomes of mitochondria and chloroplasts of Kingdom Chromista. We have found that there is a split between Class Phaeophyceae of Phylum Ochrophyta and the others （Phylum Cryptophyta and Haptophyta） in Kingdom Chromista, and identified more diversity in chloroplast genes than mitochondrial ones in their phylogenetic trees. Taxonomy resolution for Class Phaeophyceae showed that it was divided into Laminariales-Ectocarpales clade and Fucales clade, and phylogenetic positions of Kjellmaniella crassi-folia, Hizikia fusifrome and Ishige okamurai were confirmed. Our analysis provided the basic phylogenetic relationships of Chromista algae, and demonstrated their potential ability to study endosymbiotic events.

Phylogenomic analysis of transcriptomic sequences of mitochondria and chloroplasts for marine red algae(Rhodophyta)in China

The chloroplast and mitochondrion of red algae （Phylum Rhodophyta） may have originated from different endosymbiosis. In this study, we carried out phylogenomic analysis to distinguish their evolutionary lin-eages by using red algal RNA-seq datasets of the 1 000 Plants （1KP） Project and publicly available complete genomes of mitochondria and chloroplasts of Rhodophyta. We have found that red algae were divided into three clades of orders, Florideophyceae, Bangiophyceae and Cyanidiophyceae. Taxonomy resolution for Class Florideophyceae showed that Order Gigartinales was close to Order Halymeniales, while Order Graci-lariales was in a clade of Order Ceramials. We confirmed Prionitis divaricata （Family Halymeniaceae） was closely related to the clade of Order Gracilariales, rather than to genus Grateloupia of Order Halymeniales as reported before. Furthermore, we found both mitochondrial and chloroplastic genes in Rhodophyta under negative selection （Ka/Ks〈1）, suggesting that red algae, as one primitive group of eukaryotic algae, might share joint evolutionary history with these two organelles for a long time, although we identified some dif-ferences in their phylogenetic trees. Our analysis provided the basic phylogenetic relationships of red algae, and demonstrated their potential ability to study endosymbiotic events.

Fifty years of character compatibility concepts at work

In the mid 19th century, systematic biologists realized that observable similarities and differences among a group of related species could be the basis for hypotheses about the evolutionary relationships among the species and their ancestors. Such hypotheses can be expressed as characters. A character is comprised of two or more character states of species considered to be similar with respect to a basis for comparison. The states of a character may also be arranged into a character state tree to hypothesize speciation events associated with changes from one character state to another. In the mid 20th century, some systematists realized that sometimes pairs of characters （or character state trees） could be incompatible as hypotheses, i.e., they could not both be true. Through the 1950s, ＇60s and ＇70s, tests for, and ways to resolve, incompatibilities were used to estimate an ancestor rela-tion based on mutually compatible characters. An estimate was often shown as a diagram connecting ancestors to their immediate descendants （not quite correctly） called a phylogenetic tree. More recently, other applications of compatibility concepts have been developed, including： identify characters that appear to be random in the context of their data set; combine estimates of ancestor relations for subsets of taxa in a larger collection into a single estimate （a so-called supertree） for the whole collection; and interpret geographic patterns in an evolutionary context.

Biological and molecular characterization of tomato brown rugose fruit virus and development of quadruplex RT-PCR detection

Tomato brown rugose fruit virus(ToBRFV) is a novel tobamovirus firstly reported in 2015 and poses a severe threat to the tomato industry. So far, it has spread to 10 countries in America, Asia, and Europe. In 2019, ToBRFV was identified in Shandong Province(ToBRFV-SD), China. In this study, it was shown that ToBRFV-SD induced mild to severe mosaic and blistering on leaves, necrosis on sepals and pedicles, and deformation, yellow spots, and brown rugose necrotic lesions on fruits. ToBRFV-SD induced distinct symptoms on plants of tomato, Capsicum annumm, and Nicotiana benthamiana, and caused latent infection on plants of Solanum tuberosum, Solanum melongena, and N. tabacum cv. Zhongyan 102. All the 50 tomato cultivars tested were highly sensitive to ToBRFV-SD. The complete genomic sequence of ToBRFV-SD shared the highest nucleotide and amino acid identities with isolate IL from Israel. In the phylogenetic tree constructed with the complete genomic sequence, all the ToBRFV isolates were clustered together and formed a sister branch with tobacco mosaic virus(TMV). Furthermore, a quadruplex RT-PCR system was developed that could differentiate ToBRFV from other economically important viruses affecting tomatoes, such as TMV, tomato mosaic virus, and tomato spotted wilt virus. The findings of this study enhance our understanding of the biological and molecular characteristics of ToBRFV and provide an efficient and effective detection method for multiple infections, which is helpful in the management of ToBRFV.

Sequence Analysis of mtDNA CO/Gene and Molecular Phylogeny of Different Geographical Populations of Bemisia tabaci (Gennadius)

Bemisia tabaci(Gennadius) is a serious pest in many cropping systems worldwide and occurs in different biotypes. ThemtDNA COI gene of the 12 Bemisia tabaci (Gennadius) populations from different regions and countries were analyzed.Based on mtDNA COI sequences, their biotypes were characterized and phylogenetic relationships among thesepopulations were established with the method of UPGMA. The results indicated the genetic similarity between thosepopulations from Beijing, Zhengzhou, Zaozhuang, Nanjing, Shanghai, Haikou, and the B-biotype populations fromCalifornia, Texas, Arizona reached 99.8-100%, which meant the nation-wide infested populations of B.tabaci in China inrecent years were B-biotypes. Another population collected from Kunming of Yunnan Province showed very high similaritywith Q-biotype B.tabaci from Spain and Morocco, which meant the Kunming population was Q-biotype. This is the firstreport on the invasion of Q-biotype into China.

Analysis and characterization of the Salix suchowensis chloroplast genome

By screening sequence reads from the Salix suchowensis chloroplast （cp） genome that were generated by next-generation sequencing platforms, we assembled a complete circular pseudomolecule for the cp genome. This pseudomolecule is 155,508 bp long and has a typical quadripartite structure that contains two single copy regions, a large single copy region （LSC, 84,385 bp）, and a small single copy region （SSC, 16,209 bp） separated by inverted repeat regions （IRs, 27,457 bp）. Gene annotation revealed that the S. suchowensis cp genome encoded 119 unique genes, including four ribosome RNA genes, 30 transfer RNA genes, 82 protein-coding genes, and three pseudogenes. Analysis of the repetitive sequences revealed 31 tandem repeats, 16 forward repeats, and five palindromic repeats. In addition, a total of 148 perfect microsatellites, which were characterized as A/T dominant in nucleotide composition, were detected. Significant shifting of the IR/SSC boundaries was revealed by comparing this cp genome with those of other rosid plants. We also constructed phylogenetic trees to demonstrate the phylogenetic position of S. suchowensis in Rosidae based on 66 orthologous protein-coding genes present in the cp genomes of 32 species. Sequencing 30 amplicons based on the pseudomolecule for experimental verification revealed 99.88% accuracy for the S. suchowensis cp genome assembly. Therefore, we assembled a high-quality pseudomolecule of the S. suchowensis cp genome, which is a useful resource for facilitating development of this shrub willow into a more productive bioenergy crop.

New geographic distribution and molecular diversity of Citrus chlorotic dwarf-associated virus in China

In 2009, an emerging citrus viral disease caused by Citrus chlorotic dwarf-associated virus(CCDaV) was discovered in Yunnan Province of China. However, the occurrence and spread of CCDaV in other citrus-growing provinces in China is unknown to date. To better understand the distribution and molecular diversity of CCDaV in China, a total of 1 772 citrus samples were collected from 11 major citrus-growing provinces and were tested for CCDaV by PCR. Among these, 134 citrus samples from Guangxi, Yunnan and Guangdong were tested positive for CCDaV, demonstrating that the occurrence and spread of CCDaV are increasing in China. The complete genome sequences of 17 CCDaV isolates from different provinces and hosts were sequenced. Comparisons of the whole-genome sequences of the 17 CCDaV isolates as well as the 15 isolates available in GenBank revealed that the sequence identity was about 99–100%, showing that the CCDaV isolates were highly conserved. Phylogenetic studies showed that the 32 CCDaV isolates belonged to four different groups based on geographical origins and host species, and that CCDaV isolates from China and Turkey were clustered into different groups. The results provide important information for clarifying the distribution and genetic diversity of CCDaV in China.