Intermediate Lineage Research Articles

Afrosalsola (Amaranthaceae-Chenopodiaceae), an intermediate C3-C4 lineage from NW Africa and the Canary Islands, and some new combinations in African species of Caroxylon and Soda

The segregation of the polyphyletic genus Salsola (Amaranthaceae-Chenopodiaceae) caused enormous nomenclatural changes during the last two decades. A lineage, that is still awaiting to be validated by a new valid name comprises four interesting taxa endemic to the Canary Islands and NW Africa. These are shrubby plants with opposite and alternate branches and leaves, specific leaf-anatomy with a hypodermis and Kranz-like cells performing a C3-C4 intermediate photosynthetic pathway (C2 type). The new genus Afrosalsola is described with the inclusion of 2 species [Afrosalsola divaricata (Mass. ex Link) Akhani comb. nov., and A. deschaseauxiana (Litard. & Maire) Akhani comb. nov.] and three subspecies of the former [Afrosalsola divaricata subsp. divaricata, A. divaricata subsp. verticillata (Schousboe) Akhani and A. divaricata subsp. gymnomaschala (Maire) Akhani]. The synonymy of Salsola deschaseauxiana Litard. & Maire with S. verticillata Mass. ex Link is rejected based on typical alternate leaves of the former. Salsola divaricata is typified based on its original collection in BM. Additional new combinations of African species belonging to Caroxylon and Soda are provided.

Backcrossing to different parents produced two distinct hybrid species.

Repeated homoploid hybrid speciation (HHS) events with the same parental species have rarely been reported. In this study, we used population transcriptome data to test paraphyly and HHS events in the conifer Picea brachytyla. Our analyses revealed non-sister relationships for two lineages of P. brachytyla, with the southern lineage being placed within the re-circumscribed P. likiangensis species complex (PLSC) and P. brachytyla sensu stricto (s.s.) consisted solely of the northern lineage, forming a distinct clade that is paratactic to both the PLSC and P. wilsonii. Our phylogenetic and coalescent analyses suggested that P. brachytyla s.s. arose from HHS between the ancestor of the PLSC before its diversification and P. wilsonii through an intermediate hybrid lineage at an early stage and backcrossing to the ancestral PLSC. Additionally, P. purpurea shares the same parents and an extinct lineage with P. brachytyla s.s. but backcrossing to the other parent, P. wilsonii at a later stage. We reveal the first case that backcrossing to different parents of the same extinct hybrid lineage produced two different hybrid species. Our results highlight the existence of more reticulate evolution during species diversification in the spruce genus and more complex homoploid hybrid events than previously identified.

Cenozoic colonisation of the Indian Ocean region by the Australian freshwater-originating glassperch family Ambassidae (Teleostei)

We examined the phylogeny and biogeography of the glassperch family Ambassidae (Teleostei), which is widely distributed in the freshwater, brackish and marine coastal habitats across the Indo-West Pacific region. We first built a comprehensive time-calibrated phylogeny of Ambassidae using five genes. We then used this tree to reconstruct the evolution of the salinity preference and ancestral areas. Our results indicate that the two largest genera of Ambassidae, Ambassis and Parambassis, are each not monophyletic. The most recent common ancestor of Ambassidae was freshwater adapted and lived in Australia about 56 million years ago. Three independent freshwater-to-marine transitions are inferred, but no marine-to-freshwater ones. To explain the distribution of ambassids, we hypothesise two long-distance marine dispersal events from Australia. A first event was towards Southeast Asia during the early Cenozoic, followed by a second one towards Africa during mid-Cenozoic. The phylogenetic signal associated with the salinity adaptation of these events was not detected, possibly because of the selective extinction of intermediate marine lineages. The Ambassidae shares two characteristics with other freshwater fish groups distributed in continental regions surrounding the Indian Ocean: They are too young to support the hypothesis that their distribution is the result of the fragmentation of Gondwana, but they did not retain the phylogenetic signal of their marine dispersal.

Unwarranted Exclusion of Intermediate Lineage A-B SARS-CoV-2 Genomes Is Inconsistent with the Two-Spillover Hypothesis of the Origin of COVID-19

Pekar et al. (2022) propose that SARS-CoV-2 was a zoonotic spillover that first infected humans in the Huanan Seafood Market in Wuhan, China. They propose that there were two separate spillovers of the closely related lineages A and lineage B in a short period of time. The two lineages are differentiated by two SNVs; hence, a single-SNV A-B intermediate must have occurred in an unsampled animal host if the two-spillover hypothesis is correct. Consequently, confirmation of the existence of an intermediate A-B genome from humans would falsify their hypothesis of two spillovers. Pekar et al. identified and excluded 20 A-B intermediate genomes from their analysis. A variety of exclusion criteria were applied, including low read depth and the assertion of repeated erroneous base calls at lineage-defining positions 8782 and 28144. However, data from GISAID show that most of the genomes were sequenced to high average sequencing depth, appearing inconsistent with these criteria. The decision to exclude the majority of genomes was based on personal communications, with raw data unavailable for inspection. Multiple errors, biases, and inconsistencies were observed in the exclusion process. For example, 12 intermediate genomes from one study were excluded; however, 54 other genomes from the same study were included, indicating selection bias. Puzzlingly, two intermediate genomes from Beijing were discarded despite an average sequencing depth of 2175X; however, four genomes from the same sequencing study were included in the analysis. Lastly, we discuss 14 additional possible intermediate genomes not discussed by Pekar et al. and note that genome sequence filtration is inappropriate when considering the presence or absence of a specific SNV pair in an outbreak. Consequently, we find that the exclusion of many of the intermediate genomes is unfounded, leaving the conclusion of two natural zoonoses unsupported.

Olig1/2-Expressing Intermediate Lineage Progenitors Are Predisposed to PTEN/p53-Loss-Induced Gliomagenesis and Harbor Specific Therapeutic Vulnerabilities.

Multiple progenitor-state mutagenesis reveal that Olig1/2-expressing intermediate precursors are highly susceptible to PTEN/p53-loss-mediated transformation and impart differential drug sensitivity, indicating tumor-initiating cell states and genetic drivers dictate glioma phenotypes and drug responses. See related commentary by Zamler and Hu, p. 807.

Complete genome sequencing and comparative genomic analyses of a new spotted-fever Rickettsia heilongjiangensis strain B8

ABSTRACT Rickettsia heilongjiangensis, a tick-borne obligate intracellular bacterium and causative agent of spotted fever in China, has attracted increasing concern regarding its capability in causing human rickettsiosis. Here, we conducted a genomic analysis of a new R. heilongjiangensis strain B8 (B8) isolated from the serum of a patient who had been bitten by a Haemaphysalis longicornis tick in Anhui Province, China. The present study sought to identify exclusive genes that might be associated with the pathogenicity of B8 using comparative genomics. Specifically, the sequences of B8 were assembled into one circular chromosome of 1,275,081 bp and predicted to contain 1447 genes. Comparative genome analyses were performed based on the genome of B8 and 28 spotted fever group (SFG) rickettsial genomes deposited in NCBI. Phylogenomic analyses indicated the B8 strain was clustered within the R. heilongjiangensis species; however, a sum of 112 and 119 B8-unique genes was identified when compared with R. heilongjiangensis and R. japonica strains, respectively. Functional annotation analyses revealed that these B8-unique genes were mainly annotated to defence mechanisms, lipid transport and metabolism, cell wall/membrane/envelope biogenesis. These data indicate B8 rather represents a previously undescribed human-pathogenic SFG rickettsia lineage, which may be an intermediate lineage of R. heilongjiangensis and R. japonica. Overall, this study isolated a new strain of R. heilongjiangensis in East-Central China for the first time, and provided potential B8-unique genetic loci that could be used for the discrimination of B8 from other R. heilongjiangensis and closely related SFG Rickettsial strains.

P192 Investigating the link between pleomorphism and virulence in Cryptococcus

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjectivesFungal pathogens Cryptococcus neoformans and C. gattii are responsible for hundreds of thousands of annual deaths in immunocompromised individuals. Considerable phenotypic variation is exhibited by strains in response to stresses encountered during host infection, including increased capsule and cell size, the release of shed capsule, and the production of giant (>15 μm), micro (<1 μm), and irregular cells. We aimed to investigate whether the production of these morphological variants is associated with virulence using two sets of strains. The first is a collection of diverse clinical isolates obtained from HIV/AIDS patients in Botswana with accompanying clinical data. The second is a collection of lineages derived from the C. neoformans type strain H99 with high genetic similarity but differing levels of virulence. Some lineages in this set possess a mutation in SGF29, which encodes a component of the SAGA histone acetylation complex that has previously been implicated in their hypervirulence.MethodsIsolates were cultured under conditions that simulate stresses encountered in vivo (DMEM, 5% CO2, 37°C) as these are known to enhance capsule production and induce cell size changes. Cells were counterstained with India Ink, visualized by light microscopy, and phenotypes were scored. For clinical isolates, MLST analysis was performed to determine their relatedness. For H99 strains, Galleria mellonella larval infection assays, growth curves, and antifungal susceptibility testing was performed to confirm their relative virulence and growth profiles. Serial block face and regular scanning electron microscopy were used to investigate the internal morphology of the giant, micro, and irregular cells to confirm that they possess attributes of functional cells.ResultsSubstantial pleomorphism was seen across both collections. In the clinical strain set, phenotypic variables fell into two groups associated with differing symptoms. The production of ‘large’ phenotypes was associated with a higher CD4 count and was negatively correlated with intracranial pressure indicators, suggesting that these are induced in early-stage infection. ‘Small’ phenotypes were associated with lower CD4 counts, negatively correlated with meningeal inflammation indicators, and positively correlated with intracranial pressure indicators, suggesting that they are produced later during infection and may promote proliferation and dissemination. Isolates possessing giant cells, microcells, and shed capsule were rare, but strikingly, they were associated with patient death.In the H99 set, strains from hypervirulent lineages had larger average capsule size, greater variation in cell size, and increased production of microcells and shed capsule. Deletion of SGF29 in an intermediate virulence lineage substantially increased its production of microcells and released capsule, consistent with a switch to hypervirulence. SGF29 loss-of-function mutations were subsequently identified in clinical isolates and were found to be significantly correlated with patient death. Expansion of a TA repeat in the second intron of SGF29 in clinical isolates was positively correlated with cell and capsule size, suggesting it also affects SGF29 function.ConclusionOur results extend the evidence for a link between pleomorphism and virulence, with a likely role for epigenetic mechanisms mediated by SAGA-induced histone acetylation.

S5.3c Investigating the link between pleomorphism and virulence in Cryptococcus

S5.3 Cellular pleomorphism and fungal virulence, September 22, 2022, 3:00 PM - 4:30 PMObjectivesFungal pathogens Cryptococcus neoformans and Cryptococcus gattii are responsible for hundreds of thousands of annual deaths in immunocompromised individuals. Considerable phenotypic variation is exhibited by strains in response to stresses encountered during host infection, including increased capsule and cell size, the release of shed capsule, and the production of giant (> 15 μm), micro (< 1 μm), and irregular cells. We aimed to investigate whether the production of these morphological variants is associated with virulence using two sets of strains. The first is a collection of diverse clinical isolates obtained from HIV/AIDS patients in Botswana with accompanying clinical data. The second is a collection of lineages derived from the C. neoformans type strain H99 with high genetic similarity but differing levels of virulence. Some lineages in this set possess a mutation in SGF29, which encodes a component of the SAGA histone acetylation complex that has previously been implicated in their hypervirulence.MethodsIsolates were cultured under conditions that simulate stresses encountered in vivo (DMEM, 5% CO2, 37°C) as these are known to enhance capsule production and induce cell size changes. Cells were counterstained with india ink, visualized by light microscopy, and phenotypes were scored. For clinical isolates, MLST analysis was performed to determine their relatedness. For H99 strains, Galleria mellonella larval infection assays, growth curves, and antifungal susceptibility testing was performed to confirm their relative virulence and growth profiles. Serial block face and regular scanning electron microscopy were used to investigate the internal morphology of the giant, micro, and irregular cells to confirm that they possess attributes of functional cells.ResultsSubstantial pleomorphism was seen across both collections. In the clinical strain set, phenotypic variables fell into two groups associated with differing symptoms. The production of ‘large’ phenotypes was associated with a higher CD4 count and was negatively correlated with intracranial pressure indicators, suggesting that these are induced in early-stage infection. ‘Small’ phenotypes were associated with lower CD4 counts, negatively correlated with meningeal inflammation indicators, and positively correlated with intracranial pressure indicators, suggesting that they are produced later during infection and may promote proliferation and dissemination. Isolates possessing giant cells, microcells, and shed capsules were rare, but strikingly, they were associated with patient death.In the H99 set, strains from hypervirulent lineages had larger average capsule size, greater variation in cell size, and increased production of microcells and shed capsules. Deletion of SGF29 in an intermediate virulence lineage substantially increased its production of microcells and released capsule, consistent with a switch to hypervirulence. SGF29 loss-of-function mutations were subsequently identified in clinical isolates and were found to be significantly correlated with patient death. Expansion of a TA repeat in the second intron of SGF29 in clinical isolates was positively correlated with cell and capsule size, suggesting it also affects Sgf29 function.ConclusionOur results extend the evidence for a link between pleomorphism and virulence, with a likely role for epigenetic mechanisms mediated by SAGA-induced histone acetylation.

The genome of Orychophragmus violaceus provides genomic insights into the evolution of Brassicaceae polyploidization and its distinct traits

Orychophragmus violaceus, referred to as “eryuelan” (February orchid) in China, is an early-flowering ornamental plant. The high oil content and abundance of unsaturated fatty acids in O. violaceus seeds make it a potential high-quality oilseed crop. Here, we generated a whole-genome assembly for O. violaceus using Nanopore and Hi-C sequencing technologies. The assembled genome of O. violaceus was ∼1.3 Gb in size, with 12 pairs of chromosomes. Through investigation of ancestral genome evolution, we determined that the genome of O. violaceus experienced a tetraploidization event from a diploid progenitor with the translocated proto-Calepineae karyotype. Comparisons between the reconstructed subgenomes of O. violaceus identified indicators of subgenome dominance, indicating that subgenomes likely originated via allotetraploidy. O. violaceus was phylogenetically close to the Brassica genus, and tetraploidy in O. violaceus occurred approximately 8.57 million years ago, close in time to the whole-genome triplication of Brassica that likely arose via an intermediate tetraploid lineage. However, the tetraploidization in Orychophragmus was independent of the hexaploidization in Brassica, as evidenced by the results from detailed phylogenetic analyses and comparisons of the break and fusion points of ancestral genomic blocks. Moreover, identification of multi-copy genes regulating the production of high-quality oil highlighted the contributions of both tetraploidization and tandem duplication to functional innovation in O. violaceus. These findings provide novel insights into the polyploidization evolution of plant species and will promote both functional genomic studies and domestication/breeding efforts in O. violaceus.

Unwarranted exclusion of intermediate lineage A/B SARS-CoV-2 genomes is inconsistent with the two spillover hypothesis of the origin of COVID-19

Unwarranted exclusion of intermediate lineage A/B SARS-CoV-2 genomes is inconsistent with the two spillover hypothesis of the origin of COVID-19

Unwarranted exclusion of intermediate lineage A-B SARS-CoV-2 genomes is inconsistent with the two spillover hypothesis of the origin of COVID-19

Unwarranted exclusion of intermediate lineage A-B SARS-CoV-2 genomes is inconsistent with the two spillover hypothesis of the origin of COVID-19

Unwarranted exclusion of intermediate lineage A/B SARS-CoV-2 genomes is inconsistent with the two spillover hypothesis of the origin of COVID-19

Unwarranted exclusion of intermediate lineage A/B SARS-CoV-2 genomes is inconsistent with the two spillover hypothesis of the origin of COVID-19

Emergence and adaptive evolution of influenza D virus

As a novel member of the Orthomyxoviridae, influenza D virus (IDV) was firstly isolated from swine. However, cattle were found to serve as its primary reservoir. The study of IDV emergence can shed light into the dynamics of zoonotic infections and interspecies transmission. Although there is an increasing number of strains and sequenced IDV strains, their origin, epidemiology and evolutionary dynamics remain unclear. In this study, we reconstruct the diversity and evolutionary dynamics of IDVs. Molecular detection of swine tissue samples shows that six IDV positive samples were identified in the Eastern China. Phylogenetic analyses suggest three major IDV lineages designated as D/Japan, D/OK and D/660 as well as intermediate lineages. IDVs show strong association with geographical location indicating a high level of local transmission, which suggests IDVs tend to establish a local lineage of in situ evolution. In addition, the D/OK lineage widely circulates in swine in Eastern China, and all of the Chinese virus isolates form a distinct sub-clade (D/China sub-lineage). Furthermore, we identified important amino acids in the HEF gene under positive selection that might affect its receptor binding cavity relevant for its broader cell tropism. The combined results highlight that more attention should be paid to the potential threat of IDV to livestock and farming in China.

Systematic Position of Heterotermitinae and Coptotermitinae (Blattodea: Isoptera: Rhinotermitidae)

AbstractRhinotermitidae (Isoptera) is an economically important termite family globally. The family is not monophyletic, and the relationships among some of its taxa remain confusing. The taxonomic status of some subfamilies is likely questionable because of various morphological characters of the soldiers and imagoes. Twenty species in 10 genera, representing all six subfamilies of Rhinotermitidae were studied herein. Morphological phylogenetic trees based on 65 characters and molecular phylogenetic trees based on mitochondrial COII and 16SrRNA genes were reconstructed. The sequence composition and genetic distance were analyzed. Based on these results, Rhinotermitidae is polyphyletic. In the morphological trees, the family was divided into five clades. Heterotermitinae and Coptotermitinae were grouped into the most apical one of them and constituted the sister group to Termitidae in the Bayesian inference (BI) tree. In the combined molecular tree, Rhinotermitidae was divided into three clades. The position of the clade composing Heterotermitinae and Coptotermitinae was the same as in the morphological BI tree. The nucleotide sequence analyses also showed that the genetic distances between Heterotermitinae or Coptotermitinae with Termitidae might be closer than those between Heterotermitinae or Coptotermitinae with other subfamilies in Rhinotermitidae. It is postulated that the relationship of Heterotermitinae and Coptotermitinae with other subfamilies of Rhinotermitidae is not as close as that indicated in the current taxonomic system. They might be the real representative of the intermediate lineage from lower to higher termites. These findings further suggest that Heterotermitinae and Coptotermitinae should be separated from Rhinotermitidae and be upgraded as a whole to family rank.

Five Mitochondrial Genomes of the Genus Eysarcoris Hahn, 1834 with Phylogenetic Implications for the Pentatominae (Hemiptera: Pentatomidae).

Simple SummaryPentatominae is the largest subfamily in the Pentatomidae, and most of its species are considered important agricultural pests. The phylogenetic relationships of tribes within Pentatominae remain controversial despite the fact that many studies have been performed using various molecular markers. In this study, five mitogenomes of the genus Eysarcoris were sequenced and analyzed, and the phylogenetic relationships of tribes within Pentatominae were reconstructed. The gene arrangement of the five mitochondrial genomes were found to be conserved and identical to other heteropteran mitogenomes. Differences in start codon usage and tandem repeats within control regions were found between E. gibbosus and the other four Eysarcoris species. In addition, the phylogenetic analyses showed that E. gibbosus is the first diverging clade within Eysarcorini. The results support the proposal to transfer E. gibbosus to the Stagonomus, another genus of Eysarcorini. Our results clarified the phylogenetic relationships among tribes of Pentatominae and laid a foundation for the further studies of Pentatominae.Four complete mitogenomes of Eysarcoris rosaceus, E. montivagus, E. gibbosus, E. annamita and one near-complete mitochondrial genome of E. ventralis were sequenced and used to explore the phylogenetic relationships of tribes within the subfamily Pentatominae. The mitogenomes range from 15,422 to 16,043 base pairs (bp) in length and encode 37 genes, including 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes (21 in E. ventralis), and a control region. Similar to other heteropteran species, the AT contents of the sequenced species were higher than their GC contents. The most frequently used start/stop codon was ATN/TAA. GTG was only found in atp6 and atp8 of E. gibbosus. All transfer RNA genes (tRNAs) exhibit the typical cloverleaf secondary structure, except for the trnS1 and trnV, which lacks the stem of the DHU arm. The length and copy number of repeat units were conserved within Eysarcoris, with the exception of E. gibbosus. Phylogenetic analyses based on mitogenomes using both maximum likelihood (ML) and Bayesian inference (BI) methods strongly supported the relationship among tribes within Pentatominae and confirmed that Graphosoma should be an intermediate lineage of Pentatominae. The relationship between Eysarcoris and Carbula was strongly supported and combined with our previous geometric morphometrics and chromosomal studies, suggest the Eysarcoris should belong to the tribe Eyasrcorini. This work will help to enhance our understanding of mitochondrial genomic evolution and phylogenetic relationships in Pentatominae.

Whole plastomes are not enough: phylogenomic and morphometric exploration at multiple demographic levels of the bee orchid clade Ophrys sect. Sphegodes.

Plastid sequences have long dominated phylogeny reconstruction at all time depths, predicated on a usually untested assumption that they accurately represent the evolutionary histories of phenotypically circumscribed species. We combined detailed in situ morphometrics (124 plants) and whole-plastome sequencing through genome skimming (71 plants) in order to better understand species-level diversity and speciation in arguably the most challenging monophyletic group within the taxonomically controversial, pseudo-copulatory bee orchid genus Ophrys. Using trees and ordinations, we interpreted the data at four nested demographic levels-macrospecies, mesospecies, microspecies, and local population-seeking the optimal level for bona fide species. Neither morphological nor molecular discontinuities are evident at any level below macrospecies, the observed overlap among taxa suggesting that both mesospecies and microspecies reflect arbitrary division of a continuum of variation. Plastomes represent geographic location more strongly than taxonomic assignment and correlate poorly with morphology, suggesting widespread plastid capture and possibly post-glacial expansion from multiple southern refugia. As they are rarely directly involved in the speciation process, plastomes depend on extinction of intermediate lineages to provide phylogenetic signal and so cannot adequately document evolutionary radiations. The popular 'ethological' evolutionary model recognizes as numerous 'ecological species' (microspecies) lineages perceived as actively diverging as a result of density-dependent selection on very few features that immediately dictate extreme pollinator specificity. However, it is assumed rather than demonstrated that the many microspecies are genuinely diverging. We conversely envisage a complex four-dimensional reticulate network of lineages, generated locally and transiently through a wide spectrum of mechanisms, but each unlikely to maintain an independent evolutionary trajectory long enough to genuinely speciate by escaping ongoing gene flow. The frequent but localized microevolution that characterizes the Ophrys sphegodes complex is often convergent and rarely leads to macroevolution. Choosing between the contrasting 'discontinuity' and 'ethology' models will require next-generation sequencing of nuclear genomes plus ordination of corresponding morphometric matrices, seeking the crucial distinction between retained ancestral polymorphism-consistent with lineage divergence-and polymorphisms reflecting gene flow through 'hybridization'-more consistent with lineage convergence.

Hematopoietic stem cells produce intermediate lineage adipocyte progenitors that simultaneously express both myeloid and mesenchymal lineage markers in adipose tissue

ABSTRACT Some adipocytes are produced from bone marrow hematopoietic stem cells. In vitro studies previously indicated that these bone marrow-derived adipocytes (BMDAs) were generated from adipose tissue macrophage (ATM) that lose their hematopoietic markers and acquire mesenchymal markers prior to terminal adipogenic differentiation. Here we interrogated whether this hematopoietic-to-mesenchymal transition drives BMDA production In vitro. We generated transgenic mice in which the lysozyme gene promoter (LysM) indelibly labeled ATM with green fluorescent protein (GFP). We discovered that adipose stroma contained a population of LysM-positive myeloid cells that simultaneously expressed hematopoietic/myeloid markers (CD45 and CD11b), and mesenchymal markers (CD29, PDGFRa and Sca-1) typically found on conventional adipocyte progenitors. These cells were capable of adipogenic differentiation In vitro and In vitro, while other stromal populations deficient in PDGFRa and Sca-1 were non-adipogenic. BMDAs and conventional adipocytes expressed common fat cell markers but exhibited little or no expression of hematopoietic and mesenchymal progenitor cell markers. The data indicate that BMDAs are produced from ATM simultaneously expressing hematopoietic and mesenchymal markers rather than via a stepwise hematopoietic-to-mesenchymal transition. Because BMDA production is stimulated by high fat feeding, their production from hematopoietic progenitors may maintain adipocyte production when conventional adipocyte precursors are diminished.

The type species of Amphorellopsis and Tintinnopsis (Protozoa:Ciliophora): A new ciliary pattern and some comments in Tintinnina

BackgroundAs dominant components of planktonic microeukaryotes, the tintinnine ciliates act as a trophic link between microbial food web and traditional planktonic food chain in aquatic ecosystems. However, traditionally the taxonomy and systematics of tintinnines have been based on the lorica. It is now accepted that lorica features alone are insufficient and that cytological and molecular information are also needed. The systematics of the genera Tintinnopsis and Amphorellopsis is ambiguous, mainly due to the lack of ciliary and molecular information on their type species, T. beroidea and A. acuta. ResultIn the present study, specimens of both species were collected from coastal waters of Qingdao, China. The morphology of each was investigated based on observations of live and protargol-stained specimens, and their SSU rDNA- and LSU rDNA-based phylogenies were analyzed. Tintinnopsis beroidea presents the genus-typical ciliature. The somatic kineties in Amphorellopsis acuta were found to be evenly distributed without differentiation into distinct ciliary fields. The phylogenetic analyses revealed that Tintinnopsis is non-monophyletic, and that T. beroidea has a close relationship with T. nana and T. baltica. The monophyly of Amphorellopsis is also not supported by SSU rDNA analysis. ConclusionThis study first reveals the ciliary pattern and two nuclear rRNA gene sequences of two tintinnine type species, and thus expands knowledge and database of tintinnines. Amphorellopsis represents an intermediate lineage between tintinnines (loricate form) and aloricate choreotrichids.

An updated genetic marker for detection of Lake Sinai Virus and metagenetic applications.

BackgroundLake Sinai Viruses (LSV) are common RNA viruses of honey bees (Apis mellifera) that frequently reach high abundance but are not linked to overt disease. LSVs are genetically heterogeneous and collectively widespread, but despite frequent detection in surveys, the ecological and geographic factors structuring their distribution in A. mellifera are not understood. Even less is known about their distribution in other species. Better understanding of LSV prevalence and ecology have been hampered by high sequence diversity within the LSV clade.MethodsHere we report a new polymerase chain reaction (PCR) assay that is compatible with currently known lineages with minimal primer degeneracy, producing an expected 365 bp amplicon suitable for end-point PCR and metagenetic sequencing. Using the Illumina MiSeq platform, we performed pilot metagenetic assessments of three sample sets, each representing a distinct variable that might structure LSV diversity (geography, tissue, and species).ResultsThe first sample set in our pilot assessment compared cDNA pools from managed A. mellifera hives in California (n = 8) and Maryland (n = 6) that had previously been evaluated for LSV2, confirming that the primers co-amplify divergent lineages in real-world samples. The second sample set included cDNA pools derived from different tissues (thorax vs. abdomen, n = 24 paired samples), collected from managed A. mellifera hives in North Dakota. End-point detection of LSV frequently differed between the two tissue types; LSV metagenetic composition was similar in one pair of sequenced samples but divergent in a second pair. Overall, LSV1 and intermediate lineages were common in these samples whereas variants clustering with LSV2 were rare. The third sample set included cDNA from individual pollinator specimens collected from diverse landscapes in the vicinity of Lincoln, Nebraska. We detected LSV in the bee Halictus ligatus (four of 63 specimens tested, 6.3%) at a similar rate as A. mellifera (nine of 115 specimens, 7.8%), but only one H. ligatus sequencing library yielded sufficient data for compositional analysis. Sequenced samples often contained multiple divergent LSV lineages, including individual specimens. While these studies were exploratory rather than statistically powerful tests of hypotheses, they illustrate the utility of high-throughput sequencing for understanding LSV transmission within and among species.

Complete mitochondrial genomes of two deep-sea pandalid shrimps, Heterocarpus ensifer and Bitias brevis: insights into the phylogenetic position of Pandalidae (Decapoda: Caridea)

The mitochondrial genome (mitogenome) analysis is a significant tool for investigating the evolutionary history of metazoan animals. The family Pandalidae is a diverse caridean group containing mainly deep-sea species. Until May 30 2019, only two complete mitogenomes are available in GenBank. Here we present the complete mitogenome sequences of two deep-sea pandalid shrimps, Heterocarpus ensifer and Bitias brevis through Illumina sequencing. The mitochondrial genomes were determined to be 15 939 bp and 15 891 bp long, and both consist of 13 protein-coding genes (PCGs), 23 transfer-RNA genes (tRNAs), two ribosomal-RNA genes (rRNAs), and one control region. The nucleotide composition is biased toward adenine and thymine. Overall, the gene contents and arrangements are consistent with the pancrustacean ground pattern. The alignment of the control regions of four pandalids reveals a conserved sequence block (CSB) (104 bp in length, average GC%=29.47% and 69.23% similarity). A phylogenetic analysis based on 51 Caridea complete mitogenomes revealed that the deep-sea pandalid shrimps are situated an intermediate lineage, with a tendency to originated from those living in shallow sea area.