Comparative Phylogenetic Analysis Research Articles

Genomic Comparative Analysis of Two Multi-Drug Resistance (MDR) Acinetobacter baumannii Clinical Strains Assigned to International Clonal Lineage II Recovered Pre- and Post-COVID-19 Pandemic.

After the emergence of COVID-19, numerous cases of A. baumannii/SARS-CoV-2 co-infection were reported. Whether the co-infecting A. baumannii strains have distinctive characteristics remains unknown. A. baumannii AMA_NO was isolated in 2021 from a patient with COVID-19. AMA166 was isolated from a mini-BAL used on a patient with pneumonia in 2016. Both genomes were similar, but they possessed 337 (AMA_NO) and 93 (AMA166) unique genes that were associated with biofilm formation, flagellar assembly, antibiotic resistance, secretion systems, and other functions. The antibiotic resistance genes were found within mobile genetic elements. While both strains harbored the carbapenemase-coding gene blaOXA-23, only the strain AMA_NO carried blaNDM-1. Representative functions coded for by virulence genes are the synthesis of the outer core of lipooligosaccharide (OCL5), biosynthesis and export of the capsular polysaccharide (KL2 cluster), high-efficiency iron uptake systems (acinetobactin and baumannoferrin), adherence, and quorum sensing. A comparative phylogenetic analysis including 239 additional sequence type (ST) 2 representative genomes showed high similarity to A. baumannii ABBL141. Since the degree of similarity that was observed between A. baumannii AMA_NO and AMA166 is higher than that found among other ST2 strains, we propose that they derive from a unique background based on core-genome phylogeny and comparative genome analysis. Acquisition or shedding of specific genes could increase the ability of A. baumannii to infect patients with COVID-19.

Genome-wide analysis of R2R3-MYB genes in cultivated peanut (Arachis hypogaea L.): Gene duplications, functional conservation, and diversification.

The cultivated Peanut (Arachis hypogaea L.), an important oilseed and edible legume, are widely grown worldwide. The R2R3-MYB transcription factor, one of the largest gene families in plants, is involved in various plant developmental processes and responds to multiple stresses. In this study we identified 196 typical R2R3-MYB genes in the genome of cultivated peanut. Comparative phylogenetic analysis with Arabidopsis divided them into 48 subgroups. The motif composition and gene structure independently supported the subgroup delineation. Collinearity analysis indicated polyploidization, tandem, and segmental duplication were the main driver of the R2R3-MYB gene amplification in peanut. Homologous gene pairs between the two subgroups showed tissue specific biased expression. In addition, a total of 90 R2R3-MYB genes showed significant differential expression levels in response to waterlogging stress. Furthermore, we identified an SNP located in the third exon region of AdMYB03-18 (AhMYB033) by association analysis, and the three haplotypes of the SNP were significantly correlated with total branch number (TBN), pod length (PL) and root-shoot ratio (RS ratio), respectively, revealing the potential function of AdMYB03-18 (AhMYB033) in improving peanut yield. Together, these studies provide evidence for functional diversity in the R2R3-MYB genes and will contribute to understanding the function of R2R3-MYB genes in peanut.

Genome size evolution in grasshoppers (Orthoptera: Caelifera: Acrididae)

AbstractGrasshoppers (Orthoptera: Acrididae) are known for their significantly enlarged genome compared to other insects. However, our understanding of the evolutionary dynamics of genome size (GS) with this family is still limited. This study measured the GS of 62 grasshopper species using flow cytometry and assembled 10 new mitochondrial genomes for comparative phylogenetic analyses. An expanded species sampling discovered several grasshopper species with giant GS surpassing the previous insect record. We then applied recently developed methods to test more complicated, heterogeneous evolutionary models. We found that grasshopper GS has a strong phylogenetic signal and does not correlate with species' body size or flight ability. These results support the neutral or near‐neutral hypotheses of GS evolution. However, GS had accelerated rates of evolution on some grasshopper lineages, suggesting heterogeneity in its evolutionary dynamics. Ancestral state reconstruction indicates that the large genome evolved before the origin of the Acrididae family. Future studies with more species measurements will help assess the frequency of macroevolutionary shifts and identify possible mechanisms for these shifts in grasshopper GS evolution.

Does learning matter? Birdsong-learning program determines coping strategies for living in urban noisy environments

Urban noise limits perception by masking acoustic signals, with negative consequences for communication. Although animals relying on acoustic communication are affected, they have developed different strategies to reduce the masking effect of urban noise. Theoretically, birdsong vocal learning confers behavioral plasticity, which may be important for adapting to life in urban environments. To understand the role of vocal learning for adjusting to noisy places, we performed a field study combined with a phylogenetic comparative analysis, comparing passerine species that typically exhibit song learning (oscines) and those that do not (suboscines). Under the premise that vocal learning confers behavioral plasticity, we hypothesized that (1) while oscine species would vary song traits (acoustic parameters), under noisy conditions, suboscines would remain consistent; (2) suboscines may vary birdsong activity in relation to noise; and (3) song learning functions as an exaptation for inhabiting noisy urban environments. We found that oscines only shifted the minimum frequency of their song and did not vary song activity in noisy areas. In contrast, suboscines shifted their complete song upwards and decreased song activity in cities. Our phylogenetic analysis indicated that foraging stratum and song frequency, not learning, best explain adaptation to cities in an evolutionary context. If city noise functions as an ecological filter, frequency traits may serve as an exaptation for colonizing noisy environments. We provided clear evidence that passerine species, depending on their song-learning ability, use different strategies to cope with noise, suggesting that vocal learning determines how birds cope with the masking effect of urban noise.Significance statementSince birdsong learning may confer behavioral flexibility, we studied its role for adapting to urban noisy environments. We studied passerines that vary in vocal learning ability combining field data with a phylogenetic comparative analysis. Our methodology may provide information on both the response and the evolutionary advantages of vocal learning for living in noisy urban environments. Although both learner and non-learner birds varied their responses, they displayed different strategies for coping with urban noise. Moreover, differences in vocal learning might not limit colonization of noisy environments, and ecological and acoustic traits may explain adaptation to urbanization. Frequency parameters are conserved evolutionary traits among birds living in cities and may function as a preadaptation that facilitates the colonization of urban environments. Our study suggests that the birdsong-learning program does not help birds colonize cities but determines how they cope with the masking effect of urban noise.

Ascospore morphology of bryophilous Pezizales is closely associated with the place of infection and host ecology

Fungal spores are morphologically highly diverse and are therefore frequently used as diagnostic characters in taxonomy. However, the connection between spore morphology and fungal ecology remains poorly understood. Using phylogenetic comparative analyses, we investigated the putative relationships between four ascospore traits and the dominant place of infection, host ecology, and host taxonomic placement in 123 species of biotrophic parasites of bryophytes. Ascospore shape, ornamentation height and relative lipid content are significantly correlated in bryophilous Pezizales. Species attached by their hyphae to bryophyte rhizoids have more globose ascospores with higher ornamentation and relative lipid content than species attached to aboveground organs. Furthermore, some ascospore traits are significantly associated with host lifespan, habitat preferences, and taxonomic placement of their host bryophytes. Our results suggest that the ascospore morphology in this fungal group is closely linked to its ecology and several of the detected relationships point to the existence of distinct dispersal strategies. • Multiple ascospore traits are significantly correlated in bryophilous Pezizales • Ascospore morphology is closely associated with characteristics of host bryophytes • Diverse ascospores in this group likely reflect differences in dispersal strategies • Requirements for longer survival could play a crucial role in ascospore evolution

Correlated evolution of social organization and lifespan in mammals

Discerning the relationship between sociality and longevity would permit a deeper understanding of how animal life history evolved. Here, we perform a phylogenetic comparative analysis of ~1000 mammalian species on three states of social organization (solitary, pair-living, and group-living) and longevity. We show that group-living species generally live longer than solitary species, and that the transition rate from a short-lived state to a long-lived state is higher in group-living than non-group-living species, altogether supporting the correlated evolution of social organization and longevity. The comparative brain transcriptomes of 94 mammalian species identify 31 genes, hormones and immunity-related pathways broadly involved in the association between social organization and longevity. Further selection features reveal twenty overlapping pathways under selection for both social organization and longevity. These results underscore a molecular basis for the influence of the social organization on longevity.

Genome-wide annotation and expression analysis of WRKY and bHLH transcriptional factor families reveal their involvement under cadmium stress in tomato (Solanum lycopersicum L.).

The WRKY and bHLH transcription factors have been implicated in the regulation of gene expression during various physiological processes in plants, especially in plant stress responses. However, little information about the heavy metal-responsive SlWRKY and SlbHLH in tomato (Solanum lycopersicum) is available. We performed a genome-wide investigation for these two TF families in S. lycopersicum and determined their role in cadmium (Cd) stress tolerance. Furthermore, ortholog analysis with the Arabidopsis genome led to classifying WRKY and bHLH ortholog genes into nine and 11 clusters, respectively. The comparative phylogenetic analysis revealed duplication events and gene loss in Arabidopsis and S. lycopersicum, which occurred during evolution both before and after the last common ancestor of the two species. Orthologous relationships are also supported by additional evidence, such as gene structure, conserved motif compositions, and protein-protein interaction networks for the majority of genes, suggesting their similar functions. A comprehensive transcriptomics analysis revealed that both WRKY and bHLH genes were differentially expressed in response to cadmium stress as compared with control plants. A gene ontology analysis revealed that most WRKYs and bHLHs are DNA-binding essential proteins that regulate gene expression positively and negatively. Analyses of interaction networks revealed that both WRKYs and bHLHs mediate networks implicated in several stress-signaling pathways. The findings of this work may help us to comprehend the intricate transcriptional control of WRKY and bHLH genes and identify potential stress-responsive genes relevant to tomato genetic improvement. Moreover, identifying heavy metal stress-responsive WRKY and bHLH genes in S. lycopersicum will provide fundamental insights for developing new heavy metal stress-tolerant varieties of tomato crops.

Updated Virophage Taxonomy and Distinction from Polinton-like Viruses.

Virophages are small dsDNA viruses that hijack the machinery of giant viruses during the co-infection of a protist (i.e., microeukaryotic) host and represent an exceptional case of "hyperparasitism" in the viral world. While only a handful of virophages have been isolated, a vast diversity of virophage-like sequences have been uncovered from diverse metagenomes. Their wide ecological distribution, idiosyncratic infection and replication strategy, ability to integrate into protist and giant virus genomes and potential role in antiviral defense have made virophages a topic of broad interest. However, one limitation for further studies is the lack of clarity regarding the nomenclature and taxonomy of this group of viruses. Specifically, virophages have been linked in the literature to other "virophage-like" mobile genetic elements and viruses, including polinton-like viruses (PLVs), but there are no formal demarcation criteria and proper nomenclature for either group, i.e., virophage or PLVs. Here, as part of the ICTV Virophage Study Group, we leverage a large set of genomes gathered from published datasets as well as newly generated protist genomes to propose delineation criteria and classification methods at multiple taxonomic ranks for virophages 'sensu stricto', i.e., genomes related to the prototype isolates Sputnik and mavirus. Based on a combination of comparative genomics and phylogenetic analyses, we show that this group of virophages forms a cohesive taxon that we propose to establish at the class level and suggest a subdivision into four orders and seven families with distinctive ecogenomic features. Finally, to illustrate how the proposed delineation criteria and classification method would be used, we apply these to two recently published datasets, which we show include both virophages and other virophage-related elements. Overall, we see this proposed classification as a necessary first step to provide a robust taxonomic framework in this area of the virosphere, which will need to be expanded in the future to cover other virophage-related viruses such as PLVs.

Complete chloroplast genomes provide insights into evolution and phylogeny of Zingiber (Zingiberaceae)

BackgroundThe genus Zingiber of the Zingiberaceae is distributed in tropical, subtropical, and in Far East Asia. This genus contains about 100–150 species, with many species valued as important agricultural, medicinal and horticultural resources. However, genomic resources and suitable molecular markers for species identification are currently sparse.ResultsWe conducted comparative genomics and phylogenetic analyses on Zingiber species. The Zingiber chloroplast genome (size range 162,507–163,711 bp) possess typical quadripartite structures that consist of a large single copy (LSC, 86,986–88,200 bp), a small single copy (SSC, 15,498–15,891 bp) and a pair of inverted repeats (IRs, 29,765–29,934 bp). The genomes contain 113 unique genes, including 79 protein coding genes, 30 tRNA and 4 rRNA genes. The genome structures, gene contents, amino acid frequencies, codon usage patterns, RNA editing sites, simple sequence repeats and long repeats are conservative in the genomes of Zingiber. The analysis of sequence divergence indicates that the following genes undergo positive selection (ccsA, ndhA, ndhB, petD, psbA, psbB, psbC, rbcL, rpl12, rpl20, rpl23, rpl33, rpoC2, rps7, rps12 and ycf3). Eight highly variable regions are identified including seven intergenic regions (petA-pabJ, rbcL-accD, rpl32-trnL-UAG, rps16-trnQ-UUG, trnC-GCA-psbM, psbC-trnS-UGA and ndhF-rpl32) and one genic regions (ycf1). The phylogenetic analysis revealed that the sect. Zingiber was sister to sect. Cryptanthium rather than sect. Pleuranthesis.ConclusionsThis study reports 14 complete chloroplast genomes of Zingiber species. Overall, this study provided a solid backbone phylogeny of Zingiber. The polymorphisms we have uncovered in the sequencing of the genome offer a rare possibility (for Zingiber) of the generation of DNA markers. These results provide a foundation for future studies that seek to understand the molecular evolutionary dynamics or individual population variation in the genus Zingiber.

Human activities favour prolific life histories in both traded and introduced vertebrates

Species’ life histories determine population demographics and thus the probability that introduced populations establish and spread. Life histories also influence which species are most likely to be introduced, but how such ‘introduction biases’ arise remains unclear. Here, we investigate how life histories affect the probability of trade and introduction in phylogenetic comparative analyses across three vertebrate classes: mammals, reptiles and amphibians. We find that traded species have relatively high reproductive rates and long reproductive lifespans. Within traded species, introduced species have a more extreme version of this same life history profile. Species in the pet trade also have long reproductive lifespans but lack ‘fast’ traits, likely reflecting demand for rare species which tend to have slow life histories. We identify multiple species not yet traded or introduced but with life histories indicative of high risk of future trade, introduction and potentially invasion. Our findings suggest that species with high invasion potential are favoured in the wildlife trade and therefore that trade regulation is crucial for preventing future invasions.

Genomic analysis as a tool to infer disparate phylogenetic origins of dysembryoplastic neuroepithelial tumors and their satellite lesions

Dysembryoplastic neuroepithelial tumor (DNET) is a low-grade brain tumor commonly associated with drug-resistant epilepsy. About half of DNETs are accompanied by tiny nodular lesions separated from the main mass. The existence of these satellite lesions (SLs) has shown a strong association with tumor recurrence, suggesting that they are true tumors. However, it is not known whether SLs represent multiple foci of progenitor tumor cell extension and migration or a multifocal development of the main DNET. This study was designed to elucidate the histopathology and pathogenesis of SLs in DNETs. Separate biopsies from the main masses and SLs with DNET were analyzed. We performed comparative lesion sequencing and phylogenetic analysis. FGFR1 K656E and K655I mutations or duplication of the tyrosine kinase domain was found in all 3 DNET patients and the main masses and their SLs shared the same FGFR1 alterations. The phylogenic analysis revealed that the SLs developed independently from their main masses. It is possible that the main mass and its SLs were separated at an early stage in oncogenesis with shared FGFR1 alterations, and then they further expanded in different places. SLs of DNET are true tumors sharing pathogenic mutations with the main masses. It is plausible that multifocal tumor development takes place in the dysplastic cortex containing cells with a pathogenic genetic alteration.

Disentangling thermal from alternative drivers of reflectance in jewel beetles: A macroecological study

AbstractAimTo predict future colour–climate relationships, it is important to distinguish thermal drivers of reflectance from other evolutionary drivers. We aimed to achieve this by comparing relationships between climate and coloration in ultraviolet–visible (UV–Vis) and near‐infrared (NIR) light, separately.LocationSamples were distributed primarily across Australia and North America, with some from Africa and Asia.Major taxa studiedColeoptera: Buprestidae.MethodsWe used jewel beetles as models to identify climatic drivers of reflectance, because jewel beetles have highly diverse coloration and a wide distribution and are often active in hot conditions. Specifically, we tested the association between climate, body size and reflectance using a phylogenetic comparative analysis for three wavebands (UV–Vis, NIR and total).ResultsReflectance of jewel beetles was more strongly predicted by body size than by climate. NIR reflectance and total reflectance were not associated with climate, but larger beetles had higher NIR reflectance. For UV–Vis reflectance, small beetles were darker in warmer and more humid environments, whereas there was no association with climate for large beetles.Main conclusionsOur study suggests that variation in reflectance of jewel beetles is not driven by thermal requirements and highlights the importance of considering NIR reflectance when evaluating explanations of the effects of colour on thermoregulation.

Metamorphosis Imposes Variable Constraints on Genome Expansion through Effects on Development.

Genome size varies ∼100,000-fold across eukaryotes and has long been hypothesized to be influenced by metamorphosis in animals. Transposable element accumulation has been identified as a major driver of increase, but the nature of constraints limiting the size of genomes has remained unclear, even as traits such as cell size and rate of development co-vary strongly with genome size. Salamanders, which possess diverse metamorphic and non-metamorphic life histories, join the lungfish in having the largest vertebrate genomes-3 to 40 times that of humans-as well as the largest range of variation in genome size. We tested 13 biologically-inspired hypotheses exploring how the form of metamorphosis imposes varying constraints on genome expansion in a broadly representative phylogeny containing 118 species of salamanders. We show that metamorphosis during which animals undergo the most extensive and synchronous remodeling imposes the most severe constraint against genome expansion, with the severity of constraint decreasing with reduced extent and synchronicity of remodeling. More generally, our work demonstrates the potential for broader interpretation of phylogenetic comparative analysis in exploring the balance of multiple evolutionary pressures shaping phenotypic evolution.

Complete sequence and comparative analysis of the mitochondrial genome of the rare and endangered Clematis acerifolia, the first clematis mitogenome to provide new insights into the phylogenetic evolutionary status of the genus.

Clematis is one of the large worldwide genera of the Ranunculaceae Juss. Family, with high ornamental and medicinal value. China is the modern distribution centre of Clematis with abundant natural populations. Due to the complexity and high morphological diversity of Clematis, the genus is difficult to classify systematically, and in particular, the phylogenetic position of the endangered Clematis acerifolia is highly controversial. The use of the mitochondrial complete genome is a powerful molecular method that is frequently used for inferring plants phylogenies. However, studies on Clematis mitogenome are rare, thus limiting our full understanding of its phylogeny and genome evolution. Here, we sequenced and annotated the C. acerifolia mt genome using Illumina short- and Nanopore long-reads, characterized the species first complete mitogenome, and performed a comparative phylogenetic analysis with its close relatives. The total length of the C. acerifolia mitogenome is 698,247 bp and the main structure is multi-branched (linear molecule 1 and circular molecule 2). We annotated 55 genes, including 35 protein-coding, 17 tRNA, and 3 rRNA genes. The C. acerifolia mitogenome has extremely unconserved structurally, with extensive sequence transfer between the chloroplast and mitochondrial organelles, sequence repeats, and RNA editing. The phylogenetic position of C. acerifolia was determined by constructing the species mitogenome with 24 angiosperms. Further, our C. acerifolia mitogenome characteristics investigation included GC contents, codon usage, repeats and synteny analysis. Overall, our results are expected to provide fundamental information for C. acerifolia mitogenome evolution and confirm the validity of mitochondrial analysis in determining the phylogenetic positioning of Clematis plants.

Ancient deep ocean as a harbor of biotic innovation revealed by Carboniferous ophiuroid microfossils

Fossil-informed molecular phylogenies have emerged as the most powerful tool for correlating biotic evolution and Earth history. The accuracy of these trees, however, depends on the completeness of fossil sampling. For most organismal clades, the available fossil record is insufficiently sampled. This is especially true for groups with a multi-element skeleton, such as echinoderms and vertebrates, where sampling efforts focus largely on rare finds of intact skeletons. For these groups, inconspicuous but informative skeletal fragments are commonly neglected. This sampling bias excludes the numerous paleoenvironments in which preservation of intact skeletons is extremely unlikely, in particular deep-water settings. We describe new finds of brittle-star, or ophiuroid, fossils retrieved from sieving residues of sediments deposited during the Atokan (Upper Carboniferous) on the deep shelf to upper slope of the Ardmore Basin in present-day southern Oklahoma, USA. Although preserved as disarticulated, microscopic ossicles, the pristine preservation of the skeletal microstructure allows for precise identification of the remains. Comparative anatomical and phylogenetic analyses confirm the presence of basal representatives of the extant ophiuroid orders Ophioscolecida and Amphilepidida. Our finds provide the first unambiguous fossil evidence that the early crown-group diversification of the Ophiuroidea was well under way long before the end-Permian mass extinction, and that a significant part of this diversification took place in deep-water settings, as previously predicted by molecular evidence.

Ancient deep ocean as a harbor of biotic innovation revealed by Carboniferous ophiuroid microfossils

Abstract Fossil-informed molecular phylogenies have emerged as the most powerful tool for correlating biotic evolution and Earth history. The accuracy of these trees, however, depends on the completeness of fossil sampling. For most organismal clades, the available fossil record is insufficiently sampled. This is especially true for groups with a multi-element skeleton, such as echinoderms and vertebrates, where sampling efforts focus largely on rare finds of intact skeletons. For these groups, inconspicuous but informative skeletal fragments are commonly neglected. This sampling bias excludes the numerous paleoenvironments in which preservation of intact skeletons is extremely unlikely, in particular deep-water settings. We describe new finds of brittle-star, or ophiuroid, fossils retrieved from sieving residues of sediments deposited during the Atokan (Upper Carboniferous) on the deep shelf to upper slope of the Ardmore Basin in present-day southern Oklahoma, USA. Although preserved as disarticulated, microscopic ossicles, the pristine preservation of the skeletal microstructure allows for precise identification of the remains. Comparative anatomical and phylogenetic analyses confirm the presence of basal representatives of the extant ophiuroid orders Ophioscolecida and Amphilepidida. Our finds provide the first unambiguous fossil evidence that the early crown-group diversification of the Ophiuroidea was well under way long before the end-Permian mass extinction, and that a significant part of this diversification took place in deep-water settings, as previously predicted by molecular evidence.

Unraveling the macroevolution of horseshoe bats (Chiroptera: Rhinolophidae: Rhinolophus).

Unraveling the diversification mechanisms of organisms is a fundamental and important macroevolutionary question regarding the diversity, ecological niche, and morphological divergence of life. However, many studies have only explored diversification mechanisms via isolated factors. Here, based on comparative phylogenetic analysis, we performed a macroevolutionary examination of horseshoe bats (Chiroptera: Rhinolophidae: Rhinolophus), to reveal the inter-relationships among diversification, intrinsic/extrinsic factors, and climatic ecological niche characteristics. Results showed a general slowing trajectory during diversification, with two dispersal events from Asia into Southeast Asia and Africa playing key roles in shaping regional heterogeneous diversity. Morphospace expansions of the investigated traits (e.g., body size, echolocation, and climate niche) revealed a decoupled pattern between diversification trajectory and trait divergence, suggesting that other factors (e.g., biotic interactions) potentially played a key role in recent diversification. Based on ancestral traits and pathway analyses, most Rhinolophus lineages belonging to the same region overlapped with each other geographically and were positively associated with the diversification rate, implying a competitive prelude to speciation. Overall, our study showed that multiple approaches need to be integrated to address diversification history. Rather than a single factor, the joint effects of multiple factors (biogeography, environmental drivers, and competition) are responsible for the current diversity patterns in horseshoe bats, and a corresponding multifaceted strategy is recommended to study these patterns in the future.

Genomic evidence of SARS-CoV-2 reinfection cases in southern Brazil.

Cases of reinfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported worldwide. We investigated reinfection cases in a set of more than 30,000 samples, and the SARS-CoV-2 genomes from selected samples from four patients with at least two positive diagnoses with an interval ≥ 45 days between tests were sequenced and analyzed. Comparative genomic and phylogenetic analysis confirmed three reinfection cases and suggested that the fourth one was caused by a virus of the same lineage. Viral sequencing is crucial for understanding the natural course of reinfections and for planning public health strategies for management of COVID-19.

Systematic revision of the microcystid land snails endemic to Norfolk Island (Gastropoda: Stylommatophora) based on comparative morpho-anatomy and mitochondrial phylogenetics

Norfolk Island harbours a rich land snail diversity dominated by the Microcystidae and Helicarionidae that are currently represented by 10 endemic genera and 27 accepted species and subspecies. We comprehensively revise the taxonomy of these taxa using comparative morphology and phylogenetic analyses of the mitochondrial genes COI and 16S. We demonstrate that most ‘helicarionid’ species belong to Microcystidae with only a single species of Helicarionidae present (Dendrolamellaria mathewsi). The Norfolk Island microcystids comprise five major clades. These clades may have independently colonised the Norfolk Island group; however, clarity may only be achieved in a broader phylogenetic context that incorporates the study of extralimital groups. Three clades have radiated in situ into multiple endemic species. Based on our findings, we recognise the previously accepted genera Iredaleoconcha, Nancibella and Roybellia as junior synonyms of Allenoconcha, and Mathewsoconcha and Quintalia as junior synonyms of Advena, based on the close phylogenetic relationships. Furthermore, we confirm the previous treatment of Lutilodix, Parcolena and Dolapex as junior synonyms of Fanulena and Belloconcha as a synonym of Advena, bringing the total number of Norfolk Island microcystid genera to five. Secondly, we provide revised descriptions for each of these genera. Thirdly, we remove Allenoconcha belli, A. mathewsi, A. monspittensis, A. perdepressa and A. royana from the synonymy with Allenoconcha basispiralis. We also remove Helix patescens from synonymy with Quintalia flosculus, treating this as a member of Allenoconcha, and resurrect Advena campbellii nepeanensis and Quintalia stoddartii intermedia as accepted subspecies. Based on comparative morpho-anatomy, we treat Allenoconcha mathewsi and A. monspittensis as junior synonyms of Allenoconcha royana, A. inopina as a junior synonym of Allenoconcha caloraphe, Fanulena fraternus as a junior synonym of F. amiculus, Advena campbellii charon as a synonym of Advena campbellii campbellii, Mathewsoconcha belli, M. compacta and M. norfolkensis as junior synonyms of Advena suteri, M. elevata as a junior synonym of Advena grayi, and M. microstriatum as a junior synonym of Advena phillipii. Lastly, we describe three new species, Allenoconcha evansorum sp. nov., A. margaretae sp. nov. and A. varmani sp. nov. In summary, we accept 27 microcystid species and subspecies all of which are endemic to the Norfolk Island group. We provide recent and historical distribution data for each species and demonstrate that seven species or subspecies are probably extinct. All species are well differentiated in terms of basal branch lengths in the phylogenetic tree, but this distinction is not consistently reflected in the external morphology. Some closely related sister-taxa are very similar in shell morphology whereas others exhibit highly distinctive shells. We hypothesise that these stark differences in shell morphology may result from adaptation to different ecological niches, yet we currently lack a detailed understanding of the underlying evolutionary mechanisms. ZooBank: urn:lsid:zoobank.org:pub:BEFC0F76-4405-4EE7-9060-B7D9FB84BCB1

Molecular detection of Dirofilaria immitis and its Wolbachia endosymbionts in dogs from Myanmar

Heartworm disease in dogs and cats caused by Dirofilaria immitis continues to be a major clinical issue globally. This study focused on dogs suspicious of having tick-borne diseases (TBD) brought to a clinic and a veterinary teaching hospital in Myanmar. Blood samples were collected and initially screened using SNAP® 4Dx® Plus test kit. All dog blood samples were subjected to conventional PCR to detect both Dirofilaria spp. (cox1 gene) and Wolbachia spp. (16S rDNA) infections. Infection with D. immitis was detected in 14 (28.0%) of 50 examined samples, while the detection rate of TBD causative agents, including Anaplasma phagocytophilum and Ehrlichia canis, was 26.0% (13/50) and 26.0% (13/50), respectively, as determined by ELISA rapid test. In this study, D. immitis infection was moderately but significantly correlated with TBD infections (Pearson’s r = 0.397, P = 0.008). Comparative sequence and phylogenetic analyses provided molecular identification of D. immitis in Myanmar and confirmed the identity of its Wolbachia endosymbiont with Wolbachia endosymbionts isolated from D. immitis, Rhipicephalus sanguineus and Aedes aegypti. The present study contributes to our understanding of the coexistence of D. immitis and Wolbachia endosymbiosis in dogs, and the findings may benefit the future prevention and control of dirofilariasis in dogs.