Subspecies Level Research Articles

Identification of the Bartonella autotransporter CFA as a protective antigen and hypervariable target of neutralizing antibodies in mice

The bacterial genus Bartonella comprises numerous emerging pathogens that cause a broad spectrum of disease manifestations in humans. The targets and mechanisms of the anti-Bartonella immune defense are ill-defined and bacterial immune evasion strategies remain elusive. We found that experimentally infected mice resolved Bartonella infection by mounting antibody responses that neutralized the bacteria, preventing their attachment to erythrocytes and suppressing bacteremia independent of complement or Fc receptors. Bartonella-neutralizing antibody responses were rapidly induced and depended on CD40 signaling but not on affinity maturation. We cloned neutralizing monoclonal antibodies (mAbs) and by mass spectrometry identified the bacterial autotransporter CFA (CAMP-like factor autotransporter) as a neutralizing antibody target. Vaccination against CFA suppressed Bartonella bacteremia, validating CFA as a protective antigen. We mapped Bartonella-neutralizing mAb binding to a domain in CFA that we found is hypervariable in both human and mouse pathogenic strains, indicating mutational antibody evasion at the Bartonella subspecies level. These insights into Bartonella immunity and immune evasion provide a conceptual framework for vaccine development, identifying important challenges in this endeavor.

Landmark-Based Geometric Morphometric of Apis dorsata and A. d. binghami Wing Venation in Indonesian Archipelagos

The giant honey bee Apis dorsata has a vast distribution in Asia, including in the Indonesian archipelagos, whereas A. d. binghami is endemic to Sulawesi Island. This research aimed to analyze the variations of landmarks in wing venation of A. dorsata from Sumatra, Belitung, Java, Sumbawa, Moluccas islands, and A. d. binghami in Sulawesi, based on geometric morphometric analysis. Nineteen landmarks from 200 wing venations were analyzed using Thin Plate Spline software. Our study found landmarks 16 and 17, known as Cubital Index in traditional morphometrics, contributed to the wing shape of A. dorsata and A. d. binghami Among all samples, these two landmarks show high displacement in the wing shape of A. d. binghami in Sulawesi and A. dorsata in the Moluccas. On the contrary, we found that the Sumatra, Belitung, and Java Apis dorsata revealed low displacement in the deformation grid; resulting in their shapes being more similar to the reference landmark. The variations of wing shape separate A. d. binghami from Sulawesi and Sumbawa in the Principle Component Analysis and agreement with the Neighbor-joining tree. Therefore, the geometric morphometric based on landmarks of wing venation is a powerful tool to discriminate the subspecies level of A. dorsata.

Does Living in Human-Altered Environments Affect Life-History and Personality of Wild Mice?

In urban habitats, animals are faced with different and often challenging environmental conditions compared to their native habitats. Behavior is the fastest response to environmental change and therefore a very important component to adjust to human-altered environments. Behaviors such as novelty responses and innovativeness which allow animals to cope with novel stimuli are often altered in urban populations. The mechanisms producing such adaptations are currently not well understood. In this study, we investigate whether urban living has an impact on the microevolution of mouse behavioral and life-history traits including boldness, stress-coping, growth, longevity, and emphasis on reproduction. We hypothesized that animals living together with humans for longer show increased novelty-seeking and boldness characteristics at the species and subspecies level. We, therefore, compared behavior and life history characteristics among Mus musculus, a commensal rodent, Mus spicilegus as a synanthropic but not commensal, and Apodemus uralensis as a strictly rural species. In addition, we compared three subspecies of M. musculus (in total six populations) that differ in the time living together with humans. Behavioral and life history differences are stronger between populations even of the same subspecies rather than showing a structural trend with the time animals have spent with humans. In addition, species differ in behavior and life history, albeit not in a pattern that suggests an evolutionary adaptation to living in human-altered habitats. We, therefore, suggest that behavioral adaptations of wild mice are geared toward environmental differences such as geographic origin or habitat specifics but not necessarily directly evolve by living together with humans.

Dental macrowear reveals ecological diversity of Gorilla spp.

Size and shape variation of molar crowns in primates plays an important role in understanding how species adapted to their environment. Gorillas are commonly considered to be folivorous primates because they possess sharp cusped molars which are adapted to process fibrous leafy foods. However, the proportion of fruit in their diet can vary significantly depending on their habitats. While tooth morphology can tell us what a tooth is capable of processing, tooth wear can help us to understand how teeth have been used during mastication. The objective of this study is to explore if differences in diet at the subspecies level can be detected by the analysis of molar macrowear. We analysed a large sample of second lower molars of Grauer’s, mountain and western lowland gorilla by combining the Occlusal Fingerprint Analysis method with other dental measurements. We found that Grauer’s and western lowland gorillas are characterised by a macrowear pattern indicating a larger intake of fruit in their diet, while mountain gorilla’s macrowear is associated with the consumption of more folivorous foods. We also found that the consumption of herbaceous foods is generally associated with an increase in dentine and enamel wear, confirming the results of previous studies.

Microbial Community Analysis and Food Safety Practice Survey-Based Hazard Identification and Risk Assessment for Controlled Environment Hydroponic/Aquaponic Farming Systems.

Hydroponic and aquaponic farming is becoming increasingly popular as a solution to address global food security. Plants in hydroponic systems are grown hydroponically under controlled environments and are considered to have fewer food safety concerns than traditional field farming. However, hydroponics and aquaponics might have very different sources of microbial food safety risks that remain under-examined. In this study, we investigated the microbiomes, microbial hazards, and potential bacterial transmission routes inside two commercial hydroponic and aquaponic farming systems using 16S-ITS-23S rRNA sequencing and a hydroponic food safety practice survey. The hydroponic farming system microbiome was analyzed from the fresh produce, nutrient solution, tools, and farmworkers. Proteobacteria, Actinobacteria, Cyanobacteria, Bacteroidetes, and Firmicutes were the main components of hydroponic/aquaponic farming systems, with Pseudomonas being the most abundant genus in fresh produce samples. We further identified the presence of multiple spoilage bacteria and potential human, plant, and fish pathogens at the subspecies level. Spoilage Pseudomonas spp. and spoilage Clostridium spp. were abundant in the hydroponic microgreen farm and aquaponic lettuce farm, respectively. Moreover, we demonstrated the mapping of Escherichia coli 16s-ITS-23s rRNA sequence reads (∼2,500 bp) to small or large subunit rRNA databases and whole-genome databases to confirm pathogenicity and showed the potential of using 16s-ITS-23s rRNA sequencing for pathogen identification. With the SourceTracker and overlapping amplicon sequence variants, we predicted the bidirectional transmission route between plants and the surrounding environment and constructed the bacteria transmission map, which can be implemented in future food safety risk control plans.

Taxonomic analysis of certain taxa of Epipactis in Slovenia

The predominantly allogamous Broad-leaved Helleborines (Epipactis helleborine) and the predominantly autogamous Narrow-lipped Helleborines (Epipactis leptochila) represent two taxonomically complex groups with great morphological diversity and close genetic relationships at the subspecies level. They are influenced by environment, clinal variability, hybridization between allogamous taxa, and in particularly by speciation through transitions from allogamy to autogamy. We analysed floral traits, genome size and performed molecular analyses (Internal transcribed spacer region, ITS; Amplified Fragment Length Polymorphism, AFLP) of several individuals of E. helleborine and E. leptochila aggregate from different geographical regions in Slovenia, focusing on intermediate traits of E. helleborine subsp. moratoria, E. helleborine subsp. leutei and E. leptochila subsp. neglecta. In order to successfully distinguish studied taxa, the main attention should be paid to the shape of the epichil and the ovary, as well as to the size ratio of sepals and petals. Among the microfloral characters, the shape and size of the lower margin of the stigma and staminodes are found to be supportive. E. neglecta clearly shows intermediate floral characteristics between E. leptochila and E. leutei, suggesting that the transition to autogamy evolved E. neglecta as an intermediate stage in the speciation process, coinciding with the colonisation of forest sites with lower light intensity. Analysis of genome size revealed significant differences, with E. neglecta having the greatest value. Using molecular markers, we were able to determine two main ribotype clusters within the E. helleborine group, which were also determined by AFLP analyses. E. leutei and E. neglecta formed two clearly defined subclusters within the E. leptochila lineage. The results of the presented study suggest that combining molecular markers and genome size with information on how variation in floral morphology is distributed, using multivariate analyses, is the most reliable way to gain improved insight into the relationships among the taxa analysed.

Identification workflow of endotoxins by pyrolysis–gas chromatography–mass spectrometry based on a database and chemometrics

Single Shot Pyrolysis-Gas Chromatography-Mass Spectrometry (SS Py-GC-MS) has been previously demonstrated to directly evaluate the endotoxins of Gram-negative bacteria with minimal sample preparation. The analytical method was quick and sensitive, but data processing was time-consuming, especially when comparing two sets of data that were quite similar. In this research article, the data handling workflow was optimized, mainly using two approaches for comparing critical compound quantities to distinguish endotoxins from different sources. Firstly, a library of endotoxin biomarkers for bacteria profiling was constructed and the identification workflow was automated. Bacterial endotoxins could be fingerprinted down to the subspecies level. The approach was then tested on environmental, food, and water samples subjected to minimal sample preparation, and the results were highly promising. In the second approach, Chemometrics was utilized to create a similar but more encompassing database with predictive capability. The results supported the library technique, indicating that distinct bacteria species and subspecies produce endotoxins with varied amounts of each critical compound that could be identified using Py-GC-MS. A Hotelling T2 range plot was successful in differentiating Gram-negative bacterial endotoxins from Fungal mycotoxins and Cyanobacterial endotoxins. This illustrates the potential for the second approach for endotoxins which might not have been discovered. Additionally, the correlation between the molecular structure of lipopolysaccharides (LPS) and the endotoxin's virulence was observed for the first time, using a quantitative model of supervised Multivariate Analysis (MVA).

Strong migratory connectivity indicates Willets need subspecies-specific conservation strategies

AbstractBy combining all available banding and tracking data, we found that Willets (Tringa semipalmata) have a strong migratory connectivity between breeding and nonbreeding locations at the range-wide and subspecies levels, exposing two subspecies to varying threats such as hunting for the eastern subspecies (Tringa semipalmata semipalmata) and climatically-altered coastal habitats for both subspecies. We found that western Willets (Tringa semipalmata inornata) primarily used nonbreeding habitats along the Pacific Coast of the United States, although their reported nonbreeding range extends to the US Atlantic and Gulf Coasts and the Pacific Coast of Central and South America. Eastern Willets wintered in Central and South America, which covers much of the subspecies’ known nonbreeding range. By quantifying migratory connectivity within and between two subspecies, we could suggest subspecies-specific threats and potential limiting factors in the breeding and nonbreeding periods of the annual cycle of a declining migratory shorebird. Effective management of the species will likely require a range of conservation strategies across the diverse nonbreeding regions the two subspecies occupy within the United States, Central America, and South America. However, more data are needed from Willets breeding in mid-continental North America to understand the complete extent of overlap of the two subspecies throughout the annual cycle. The strong migratory connectivity documented here highlights the need to manage Willets by subspecies and protect a diversity of breeding and nonbreeding habitats, which will benefit the conservation of other shorebird species that overlap with Willets throughout the annual cycle.

The Use of Comparative Genomic Analysis for the Development of Subspecies-Specific PCR Assays for Mycobacterium abscessus.

Mycobacterium abscessus complex (MABC) is an important pathogen of immunocompromised patients. Accurate and rapid determination of MABC at the subspecies level is vital for optimal antibiotic therapy. Here we have used comparative genomics to design MABC subspecies-specific PCR assays. Analysis of single nucleotide polymorphisms and core genome multilocus sequence typing showed clustering of genomes into three distinct clusters representing the MABC subspecies M. abscessus, M. bolletii and M. massiliense. Pangenome analysis of 318 MABC genomes from the three subspecies allowed for the identification of 15 MABC subspecies-specific genes. In silico testing of primer sets against 1,663 publicly available MABC genomes and 66 other closely related Mycobacterium genomes showed that all assays had >97% sensitivity and >98% specificity. Subsequent experimental validation of two subspecies-specific genes each showed the PCR assays worked well in individual and multiplex format with no false-positivity with 5 other mycobacteria of clinical importance. In conclusion, we have developed a rapid, accurate, multiplex PCR-assay for discriminating MABC subspecies that could improve their detection, diagnosis and inform correct treatment choice.

Rapid and Accurate Differentiation of Mycobacteroides abscessus Complex Species by Liquid Chromatography-Ultra-High-Resolution Orbitrap™ Mass Spectrometry.

In this study, a Liquid Chromatography-Mass Spectrometry (LC-MS) method for the identification of clinically relevant Mycobacteroides abscessus (Mabs) complex organisms is tested using a set of microbial Type strains. This methodology is based on profiling proteins derived from Mycobacteroides abscessus complex isolates. These protein profiles are then used as markers of species differentiation. To test the resolving power, speed, and accuracy of this assay four ATCC type strains and 32 recent clinical isolates of closely related Mabs species collected at ARUP laboratories (10 clinical isolate strains of M. abscessus subsp. abscessus, 10 M. abscessus subsp. massiliense, 2 M. abscessus subsp. bolletii and 10 M. chelonae) were subjected to this approach. Using multiple deconvolution algorithms, we identified hundreds of individual proteins, with subpopulations of these used as species-specific markers. This assay identified 150, 130, 140 and 110 proteoforms with isocratic elution and 230, 180, 200 and 190 proteoforms with gradient elution for M. abscessus (ATCC 19977), M. massiliense (DSM 45103), M. bolletii (DSM 45149) and M. chelonae (ATCC 35752) respectively. Taxonomic species were identified correctly down to the species level with 100% accuracy. The ability to differentiate Mycobacteroides abscessus complex at sub-species level can in-turn be helpful for patient management. Data analysis showed ~7-17 proteoforms potentially able to differentiate between subspecies. Here, we present a proof-of-principle study employing a rapid mass spectrometry-based method to identify the clinically most common species within the Mabs species complex.

Comparative mitochondrial genomes of the Rhus gall aphid Kaburagia rhusicola subspecies with variable gall shapes

Rhus gall aphids (Hemiptera: Aphididae: Eriosomatinae) stimulate the formation of galls on their primary host plants (sumacs: Rhus spp., Anacardiaceae). The shapes of galls are often used as an extended phenotype to identify the aphid species and subspecies. We collected four Rhus galls with conspicuously different shapes formed by Kaburagia rhusicola aphids, whose sequences of the complete mitochondrial genomes (mitogenomes) were obtained by high-throughput sequencing. Each mitogenome was assembled into a circular molecule containing 13 protein-coding genes, two rRNAs, 22 tRNAs and one control region. All the protein-coding genes had a typical ATN initiation codon and TAA termination codon except for cox1 and nad4, which had a single T as stop codon. All the tRNAs could be folded as a typical clover-leaf secondary structure, except for trnS1 lacking a dihydrouridine (DHU) arm. The relative synonymous codon usage and ratio of nonsynonymous to synonymous substitution rates showed that the four K. rhusicola samples were highly similar to the subspecies K. r. ovogallis. The phylogenetic analyses grouped these samples with K. r. ovogallis in a clade sister to K. r. rhusicola. All these molecular analyses demonstrated that our current samples represented one subspecies of Kaburagia rhusicola, i.e., K. r. ovogallis, and the gall shape was variable even at the subspecies level in Kaburagia gall aphids.

Protein FT-IR amide bands are beneficial to bacterial typing

Bacterial FT-IR signals are extremely specific and highly reproducible, making FT-IR an efficient tool for bacterial typing at the subspecies level. The polysaccharide and nucleic acid FT-IR regions (1200–900 cm−1) are recommended as a precise and reproducible pattern for bacterial typing. However, proteins are the major macromolecules present in bacteria, and the FT-IR spectral region of proteins (1800–1300 cm−1) is conceivably an important factor in bacterial typing. In this study, we investigated the influence of water on bacterial protein amide bands by comparing spectra obtained with and without FT-IR system dehydration. Eight Escherichia coli, ten Klebsiella pneumoniae, and eleven Staphylococcus aureus strains were typed by FT-IR under different conditions in a blinded experimental setup. Hierarchical clustering analysis (HCA) showed that, when protein signals were included (1800–900 cm−1), the typing accuracies for select E. coli, K. pn and S. aureus strains without system dehydration were 50%, 30% and 18.2%, respectively. However, the accuracies greatly improved to 100%, 90% and 90.9% when the FT-IR system was dehydrated. These results indicate that the FT-IR signals of protein amide bands are beneficial for bacterial typing.

Applicability of rpoB Gene for PCR-RFLP based Discrimination of Bifidobacterial Species Isolated from Human and Animal Sources

Bifidobacteria are widely used as probiotics for their application in the development of functional food and prophylactic therapy. This has necessitated the development of a molecular approach for the genera to be widely identified up to species and subspecies level. In the current study, PCR-RFLP of the partial RNA polymerase β-subunit (rpoB) gene fragment was evaluated for differential identification of Bifidobacterium species. The rpoB gene partial sequences of 575 bp were amplified from 93 previously identified isolates collected from various sources of human and animal origin along with 12 standard reference strains. The PCR amplified products were digested with three restriction endonucleases HhaI, HinfI and BanI separately. Dendrograms constructed from the patterns of HhaI, were found to be more discriminatory and successfully differentiated all the twelve species and also at sub-species level in between B. longum subsp. longum and B. longum subsp. infantis. However, B. adolescentis and B. pseudocatenulatum group clusters were not separated and represented by one group. The groups were further discriminated by HinfI restriction digestion. A separate combination thereof may be used for inferring the classification of bifidobacterial species targeted on rpoB PCR-RFLP analysis. To our knowledge, this work is the first report based on use of rpoB PCR-RFLP for discrimination of the isolates of genus Bifidobacterium and also provides insights into specific advantages of this method over hsp60 PCR-RFLP in differentiating B. longum subsp. longum and B. longum subsp. infantis.

Guide to the genus Rubus L. (Rosaceae) in South Africa – Disentangling a taxonomic Gordian knot with the help of ploidy and reproductive data

Rubus is notorious for its taxonomic complexity. Although some light has been recently shed on the taxonomy of the species from the Greater Cape Floristic Region in South Africa, many questions remain unanswered. This contribution is based on extensive field and herbarium studies across South Africa and aims at clarifying Rubus taxonomy in the region with the help of DNA-ploidy estimation and assessment of reproductive mode. We distinguish a total of 18 taxa from the region, of which six are native. Furthermore, the two native species R. pinnatus and R. ludwigii exhibit geographic variation that can be treated at the level of subspecies, and the new combination R. pinnatus subsp. pappei is provided. We do not recognise any infraspecific taxa in the widely distributed sexual hexaploid R. rigidus. All of the taxa are described and are photographically documented. Several other taxa were found only as solitary inadequate specimens and need further study. Moreover, 12 primary hybrids were identified, of which seven were derived from hybridization between native and alien species. Contrary to previous reports, each of the species exhibited a single ploidy level, and hybrids were, with one or two exceptions, intermediate between the parents in their ploidy. Whereas native taxa are strictly sexual, most polyploid aliens reproduce by facultative apomixis and hybrids are mostly sterile or sexual. However, apomixis was detected in R. sect. Arguti × R. pinnatus and R. bergii × R. thaumasius.

Contribution to the knowledge of the non-calcareous grasslands of the Monti Sibillini National Park (central Italy): coenological structure, syntaxonomy, ecology, and floristic aspects

Abstract This study provides the first contribution to the knowledge of the non-calcareous grasslands of the arenaceous sector of the Monti Sibillini National Park (central Italy). We conducted 55 relevés using the Braun-Blanquet phytosociological method and analysed the ecology of plant communities by Redundancy analysis using topographic variables and Ellenberg Indicator Values as predictors. We identified nine plant communities, belonging to four classes. Communities of Calluno-Ulicetea and Nardetea strictae were found on acidic soils at higher elevations, those of Festuco-Brometea were mainly associated with steeper south-facing slopes, while Molinio-Arrhenatheretea communities were associated with low altitudes and gentle slopes, semi-flat lands, and high soil nutrient and moisture values. We recorded a total of 410 taxa at the species and subspecies level, representing about 20% of the flora of the Monti Sibillini National Park. Some of these are of high conservation interest, e.g. Calluna vulgaris, Genista sagittalis, Juncus capitatus, and Ophioglossum vulgatum. Eight species are new to the flora of Sibillini National Park.

Illumina Short-Read Sequencing of the Mitogenomes of Novel Scarites subterraneus Isolates Allows for Taxonomic Refinement of the Genus Scarites Fabricius 1775, within the Carabidae Family.

Simple SummaryGround beetles (Carabidae) have important ecological functions and serve as food, biological pest control, and models in biological research. Although there are over 40,000 ground beetle species worldwide, only a small fraction of those has genomic information currently available, which limits their classification and understanding of diversity. Since next-generation genome sequencing has become more mainstream, we used Illumina short-read sequencing to obtain complete mitogenomes from two Scarites beetles that we collected ourselves in Nebraska and Arkansas. Scarites are large ground beetles that resemble tropical beetles with a big head and large mandibles, and their role as predator and prey helps maintain sustainability in local ecosystems. This straightforward sequencing and analysis was found to be accurate and sufficient to help classify these isolates to the subspecies level. This is the first report of mitogenomes for Scarites subterraneus and only the second in that genus. This method is easily applicable to more beetle species and can be used to increase our understanding of beetles worldwide.We sequenced the complete mitogenomes, 18S and 28S rRNA of two new Scarites isolates, collected in Eastern Nebraska and Northern Arkansas (US). Based on molecular sequence data comparison and morphological characteristics, the new isolates were identified as a subspecies of Scarites subterraneus Fabricius 1775, for which we propose the subspecies names ‘nebraskensis’ and ‘arkansensis’. The new 18S and 28S rRNA sequences were found to be 99% and 98% identical to Scarites subterraneus. There are no other Scarites 18S or 28S rRNA sequences in the Genbank database, however, phylogenetic analysis of the Cox1 genes showed S. vicinus Chaudoir, 1843, and S. aterrimus Morawitz, 1863, as the closest relatives. This is the first report of a mitogenome for S. subterraneus, and only the second mitogenome for that genus. The nucleotide sequence identity between the mitogenomes of the two isolates is 98.8%, while the earlier sequenced S. buparius Forster 1771 mitogenome is more distantly related, with only 90% (to ssp. nebraskensis) and 89% (to ssp. arkansensis) overall nucleotide sequence identity. These new mitogenomes, and their phylogenetic analysis, firmly establish the position of Scarites on the Carabidae family tree and further refine the genus. In addition to the molecular data provided for the Scarites species, this approach also allowed us to identify bacterial and viral signatures for Providencia, Myroides, Spiroplasma, and a giant Nucleocytoviricota virus, associated with the Scarites species. We hereby present a simple and efficient protocol for identification and phylogenetic analysis of Scarites, that is applicable to other Coleoptera, based on total DNA extraction and Illumina short-read Next-Gen sequencing.

Development of a CAPS Marker and a LAMP Assay for Rapid Detection of Xylella fastidiosa Subsp. multiplex and Differentiation from X. fastidiosa Subsp. fastidiosa on Blueberry.

Bacterial leaf scorch (BLS), caused by Xylella fastidiosa (Xf), is a prevalent disease of blueberries in the southeastern United States. Initially, this disease was reported to be caused by X. fastidiosa subsp. multiplex (Xfm). However, a recent survey revealed the presence of another subspecies, X. fastidiosa subsp. fastidiosa (Xff), within naturally infected blueberry plantings in Georgia. Since knowledge regarding the origins of isolates causing Xf outbreaks can impact management recommendations, a routine method for identifying the pathogen at the subspecies level can be beneficial. Several detection strategies are available to identify Xf infection at the subspecies level. However, none of these have been developed for the routine and rapid differentiation of the blueberry-infecting Xf subspecies. Here, we developed two separate straightforward and rapid detection techniques, a cleaved amplified polymorphic sequence (CAPS) marker, and a loop-mediated isothermal amplification (LAMP) assay, targeting the RNA polymerase sigma-70 factor (rpoD) gene sequence of Xfm to discriminate between the two Xf subspecies infecting blueberry. With the CAPS marker, specific detection of Xfm isolates was possible from pure cultures, inoculated greenhouse-grown plant samples, and field infected blueberry samples by restriction digestion of the rpoD gene PCR product (amplified with primers RST31 and RST33) using the BtsI enzyme. The LAMP assay allowed for specific real-time amplification of a 204-bp portion of the Xfm rpoD gene from both pure bacterial cultures and infected plant material using the Genie® III system, a result further affirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. These detection strategies have the potential to greatly aid existing diagnostic methods for determining the distribution and prevalence of these Xf subspecies causing bacterial leaf scorch (BLS) in blueberries in the southeastern United States.

Molecular phylogeny, systematics and generic classification of the butterfly subfamily Trapezitinae (Lepidoptera: Papilionoidea: Hesperiidae)

Abstract Trapezitinae skippers are restricted to Australia and New Guinea. Despite decades of taxonomic work, their systematics and phylogeny remain little understood. To resolve the composition of genera and determine their evolutionary relationships, we inferred a comprehensive multilocus molecular phylogeny of Trapezitinae. Our results recover a monophyletic Trapezitinae as sister to Barcinae, with a poorly resolved backbone possibly indicating an early rapid radiation. We recover two main clades comprising all currently described genera, including the previously contentious Prada Evans, 1949 but excluding Tiacellia Evans, 1949. Several genera are recovered as paraphyletic or polyphyletic, rendering the classification of Trapezitinae incompatible with the new phylogeny. Therefore, we synonymize Motasingha Watson, 1893 syn. nov. and Oreisplanus Waterhouse & Lyell, 1914 syn. nov. with Hesperilla Hewitson, 1868 and Neohesperilla Waterhouse & Lyell, 1914 syn. nov. with Toxidia Mabille, 1891. To reflect the placement of Anisynta dominula outside of Anisynta, we describe the new genus Atkinsia Braby & Toussaint gen. nov., which includes the sole species Atkinsia dominula (Plötz, 1884) comb. nov. Two species of Toxidia and two species of Signeta Waterhouse & Lyell, 1914 are placed into Timoconia Strand, 1909, yielding four new combinations: Timoconia melania (Waterhouse, 1903) comb. nov., Timoconia peron (Latreille, 1824) comb. nov., Timoconia flammeata (Butler, 1882) comb. nov. and Timoconia tymbophora (Meyrick & Lower, 1902) comb. nov. Thus, SignetaWaterhouse & Lyell, 1914 syn. nov. is now regarded as a junior synonym of Timoconia. Overall, our phylogeny has provided the basis for 20 nomenclatural changes at the species and subspecies level, including 14 new combinations.

Highly Discriminative Genotyping of Mycobacterium abscessus Complex Using a Set of Variable Number Tandem Repeats in China.

In this study, our aims were to comparatively analyze the power of variable number tandem repeat (VNTR) typing to discriminate isolates within subspecies and to identify a potential genetic marker for better molecular typing of Mycobacterium abscessus complex (MABC) strains. A total of 103 clinical MABC isolates were collected from a nationwide cross-sectional study in China. Eighteen VNTR loci were chosen to genotype the MABC isolates. Of the 103 clinical MABC isolates, there were 76 (73.8%) M. abscessus subsp. abscessus (MAA) and 27 (26.2%) M. abscessus subsp. massiliense (MAM) isolates. Among the patients with MAA lung diseases, the percentage of patients older than 45 years (67.1%) was significantly higher than that of patients with MAM lung diseases [33.3%, adjusted odds ratio (aOR) = 0.36, 95% CI = 0.13–0.98, p = 0.046]. Fifteen VNTR loci were designated as being “highly discriminant” in our sample, except for TR109. The total of 103 MABC isolates were fully discriminated into 103 unique patterns by an 18-locus VNTR set [Hunter–Gaston Discriminatory Index (HGDI) = 1.000], of which the inclusion of the top 12 loci yielded a comparative HGDI value (HGDI = 0.9998). Remarkably, the order of the diversity of the VNTR loci showed significant difference between the MAA and MAM isolates. TR137 and TR2, two loci with high diversity indices for the MAA isolates, only yielded poor discriminatory power for the MAM isolates; the allelic diversity (h) values were 0.0000 and 0.2621, respectively. A detailed analysis of TR137 in combination with the other 17 VNTR loci showed that the combination of TR137–TR2 could fully distinguish MAA from MAM isolates. In conclusion, our data revealed that MAA is more prone to affect elderly patients. Additionally, the population structure of the MABC isolates circulating in China has high diversity. The combined use of the TR137 and TR2 loci provides a simple criterion for the precise identification of MABC to the subspecies level.

African or Asiatic origin? Genome analyses solved the mystery of the hybrid origin of the rescued lion cub

Species assignment of any seized material using DNA analysis has been a routine and widely accepted standard procedure in providing scientific advisory for the legal prosecution of wildlife cases. Scientific advancements and rigorous application of genetic tools have led to the development of a variety of molecular markers with their defined efficacy in wildlife forensics. However, in a few unusual cases where a hybrid needs to be identified or assignment need to be made at sub-species level, mitochondrial markers often fail or else provide biased results, which can affect the overall judgment in the court of law. Here, we report one such challenging case of lion cub rescued by the law enforcement from illegal trafficking. Phylogenetic assessment based on complete mitogenome assigned rescued lion cub with African lion (Panthera leo leo). However, the TSPY gene of the Y chromosome established that the lion cub shared its paternal lineage from Asiatic lion (Panthera leo persica). With the use of maternally and paternally inherited markers, we conclude a hybrid origin of the rescued lion cub which shared ancestry from both Asiatic as well as African lion. The present study exhibits the application of genome sequencing in thinking beyond routine identification and contributes to the operating procedures of wildlife forensics.