Journal of Eukaryotic Microbiology

Holomycota, together with Holozoa, represents one of the two main branches of the supergroup Opisthokonta. Fungi, as well as three basal protist lineages Nuclearida, Rosellida, and Aphelida belong to Holomycota. Surprisingly, among all these holomycotan groups, only the most basal lineage, Nuclearida, lacks any traces of flagella in its life cycle and is represented by free-living amoebae, in contrast to the obligately parasitic and flagellated Rosellida and Aphelida, which evolved later. As the earliest-branching lineage, nucleariids play a key role in understanding the origin and diversification of Opisthokonta, which is presumed to have evolved from a uniflagellated ancestor. In this study, we describe the first known nucleariid that is parasitic on algae and possesses a flagellated stage in its life cycle. We studied the life cycle, and showed the ultrastructure of main life cycle stages of this parasite. The multigene phylogenetic tree based on the newly sequenced transcriptome demonstrated its basal position among nucleariids, forming a clade of environmental OTUs. This finding together with unique ultrastructural features, parasitic style of life, and presence of flagellum allowed us to describe a new species, genus, and family in the order Nuclearidida. We emended the diagnoses of the order, class, and phylum Nuclearida. Based on the ribosomal phylogenetic tree where the Insolitus nuclearius has a basal position among the nucleariids we discussed the evolution of this group from the uniflagellated ancestor and provided new insights into the early evolution of Holomycota in a whole, revising hypotheses regarding the opisthokont ancestor.

Arcellidae is a family of testate amoebae within Arcellinida (Amoebozoa), comprising three recognized genera: Arcella, Galeripora, and Antarcella. Although species in the family have been studied for nearly two centuries, many historically described taxa and major morphological groups remain unsampled at the molecular level. Here, we provide a comprehensive review of Arcellidae and generate new cytochrome c oxidase subunit I (COI) sequences for arcellid species from Canadian peatlands, focusing on tall-shelled Arcella historically classified in section Altae sensu Deflandre. COI phylogenetic analyses recover a strongly supported monophyletic clade corresponding to North American representatives of Altae, providing the first molecular corroboration of this morphologically defined group. Within this clade, we redescribe Arcella leidyana based on modern material from Eeyou Istchee (Quebec). We further describe Galeripora purdoni sp. nov. from a calcareous fen in eastern Ontario, representing a novel terrestrial lineage within the genus, and redescribe Galeripora artocrea, which we transfer to Arcella based on congruent molecular and morphological evidence. Phylogenomic analyses of Arcellidae isolates from the Protist 10,000 Genomes Project reveal an additional deep lineage basal to Arcella and Galeripora. Together, these results highlight hidden diversity and demonstrate the importance of integrative approaches for resolving arcellid systematics and refining its classification.

The obscure life histories of many kickxellalean genera remain a bottleneck for comprehensive taxon sampling and phylogenetic reconstruction of the order. While Kickxellales has long been regarded as saprobes inhabiting soil or dung, the discovery of "amphibious fungi" such as Unguispora, which exhibits dimorphic growth between the animal gut and feces, suggests a cryptic gut-dwelling stage within these genera. Given its phylogenetic proximity to Unguispora, ecophysiological traits of Linderina were investigated to evaluate its potential association with the animal gut. Two isolates of L. macrospora were obtained from Japanese soil, representing the first record of this species in Japan. Physiological assays revealed that the optimal temperature for both vegetative growth and sporulation was 25-30 {degrees}C. Furthermore, comparative growth assays on different media demonstrated that sporocladium abundance per sporangiophore is sensitive to nutrient availability, and nutrient-poor media were determined to be the most suitable for evaluating morphological characterization. Under anaerobic, nutrient-rich conditions which are known to induce yeast-like growth in Unguispora, sporangiospores of L. macrospora produce arthrospores. Although marked morphological plasticity was observed during the arthrospore formation, the occurrence of yeast-like unicellular proliferation suggests a potential relationship with the animal gut. Additionally, vegetative growth and sporulation were markedly inhibited by white light exposure; notably, a lethal effect on growth was observed during incubation at 20 {degrees}C, indicating that the natural niche of the species is restricted to light-shielded environments. Our findings will help to elucidate the cryptic life cycles and evolutionary trajectories within Kickxellales.

High-quality reference genomes provide an essential foundation for elucidating the molecular basis of organismal ecophysiology. Here, we sequenced and assembled chromosome-scale genomes of two Heterosigma akashiwo strains isolated from coastal waters of Japan and France. The assembly sizes were 1.18 Gb and 1.43 Gb for the Japanese and French strains, respectively. The scaffold N50 of the Japanese strain assembly was 66 Mb, whereas the one of the unscaffolded French strain assembly was 33 Mb. To our knowledge, these assemblies represent among the largest and most contiguous genome resources currently available for members of the Stramenopiles (Ochrophyta). Evidence-based gene prediction in the Japanese strain recovered approximately 90% of conserved stramenopile core genes, indicating a highly complete gene repertoire, and was complemented by extensive functional annotation. In the French strain, homology-based gene prediction recovered approximately 80% of conserved core genes. Comparative genome analysis revealed extensive synteny conservation between the two strains, although several putative duplication and translocation events were detected. These genomic resources provide a robust framework for investigating the molecular, cellular, and ecological mechanisms underlying the physiology, adaptation, and bloom-forming capacity of H. akashiwo.

During mycological surveys in Yunnan Province, China, specimens of a fungus producing massive, upright stromata up to 50 cm high and individually 2.2 Kg in weight were sampled. Through an integrative taxonomic approach combining detailed morphology, multilocus phylogeny (ITS, LSU, RPB2, TUB2), and phylogenomic analyses, this fungus is proposed as the new species Dianjunus rex gen. et sp. nov., the type of the new family Dianjunaceae (Xylariales). Phylogenetic analyses robustly place Dianjunaceae as a distinct sister clade to Graphostromataceae. Divergence time estimation dates the origin of this family to the early Paleocene (~65 Mya), coinciding with the post-K-Pg extinction period, when an estimated 75% of all plant and animal species went extinct, and a significant ecological reorganization of life on earth happened. The stromata of D. rex represent the largest fructifications documented within the Ascomycota, significantly expanding the known morphological range of the Xylariales. The study provides a comprehensive description, including a nodulisporium-like anamorph with periconiella-like branching patterns, and discusses the taxon's phylogenetic placement, and distinctive morphology. This discovery highlights the unexplored fungal diversity in East Asian forests.

The marine sponge Hymeniacidon perlevis is a globally-distributed species, likely because it has been extensively spread through human means. The status of this species in the Western Atlantic has been difficult to discern due to the presence of a morphologically similar species Hymeniacidon heliophila. Here, we have collected Hymeniacidon specimens from the H. heliophila type location and compared their morphology and genotypes to material from Europe, North and South America, and publicly available datasets. Specimens from the type location closely matched the original description of H. heliophila, and were morphologically indistinguishable from H. perlevis. Multilocus sequencing data confirm that these samples are indistinguishable from H. perlevis, and that the name H. heliophila should therefore be considered a junior synonym of H. perlevis. Analysis of a global cox1 dataset further revealed significantly higher genetic diversity in Europe than in the remainder of the world combined, supporting a European origin for the species and suggesting that all other genotyped populations are introductions. In the Western Atlantic, we confirm the presence of H. perlevis in the Gulf of Mexico, but contrary to some past reports, are unable to confirm its presence in the Caribbean. Our results clarify the taxonomy, biogeography, and recent history of one of the worlds most widespread marine sponges.

Scanning electron microscopy (SEM) is widely used to investigate bacterial surface architecture; however, sample preparation protocols may introduce structural artefacts that compromise the interpretation of morphometric and ultrastructural features. This issue becomes especially relevant for extremophilic microorganisms, whose specialized cell envelopes may respond differently to chemical fixation. In this study, we evaluated the effects of two aldehyde-based fixation protocols, 2.5% glutaraldehyde and Karnovskys solution, combined with different fixation times (1, 3, and 24 h) and the presence or absence of osmium tetroxide (OsO4) post-fixation, on three Gram-positive bacterial strains: the polyextremophiles Exiguobacterium sp. S17 and Nesterenkonia sp. Act20, and the mesophile Kocuria rosea CH-021. Morphological preservation was assessed using morphometric parameters, including cellular area and surface-to-volume ratio, together with texture analysis based on Haralick descriptors derived from grey-level co-occurrence matrices (GLCM). Results showed that fixation conditions significantly affected morphometric and textural features in a strain-dependent manner. Although overall morphology appeared preserved, quantitative analyses revealed marked differences in surface texture and structural integrity among treatments. Osmium tetroxide modified morphometric and textural parameters, although its effects varied among strains and fixation conditions. These findings highlight the importance of integrated quantitative approaches combining cellular geometry and surface texture analysis for the evaluation of SEM preparation protocols in structurally specialized microorganisms.

This study presents a systematic revision of the suborder Astrophorina for the temperate Pacific coast of the United States and Canada. Major findings include a reduction in the number of species previously thought to range into the region from Japan; validation of most Geodia species erected by Lendenfeld (1910), which were later synonymized by de Laubenfels (1932); the formal description of 10 new species (Poecillastra alaskensis sp. nov., Vulcanella explorata sp. nov., Vulcanella rupta sp. nov., Stelletta cardenasi sp. nov., Stelletta nicolenya sp. nov., Stelletta limuwensis sp. nov., Dercitus (Stoeba) giveni sp. nov., Penares anyapax sp. nov., Penares foxi sp. nov., and Thenea diastra sp. nov.); and one new combination, Penares orientalis comb. nov. Extensive SCUBA-based collection efforts yielded new samples for 11 of the 26 species identified in the region, which enabled an integrative taxonomic approach that combined field photography, fresh material for DNA sequencing, and improved characterization of species ranges and morphological variability in previously described taxa. Illumina sequencing generated complete nuclear ribosomal haplotypes for five species, while Sanger sequencing of the 28S and cox1 loci placed 20 of the 26 species within molecular phylogenies. The use of very short "mini-barcode" amplicons also enabled sequence recovery from historic type specimens up to 137 years old. This study additionally reports the discovery of sponge grounds of abundant, large Geodia at diving depths in Southern California. Together, these results substantially advance our understanding of global astrophorid diversity and systematics, and the biogeography of sponge diversity in the Northeast Pacific. Note about species names: this pre-print is not intended to be a publication of the associated species names for the purposes of zoological nomenclature.

The marine bacterium Vibrio alginolyticus and the food poisoning bacterium V. parahaemolyticus possess two types of flagella in one cell: proton-driven lateral flagella (Laf) extending from the periphery of the cell body, and sodium ion-driven polar flagella (Pof) extending from a cell pole. For swimming in seawater they use polar flagella, suppressing the expression of lateral flagella. When they attach to the body surface of fish or intestinal tract, lateral flagella are induced, allowing it to crawl along the surface or through mucus. The dynamometer hypothesis, which proposes that polar flagella sense rotation and control the expression of lateral flagellar genes, has been widely accepted. However, how rotation is sensed and how expression is controlled remains unclear. Although swarming has recently been analyzed by physical, biological, or biochemical perspectives, it remains unclear how this motility is controlled, or which substances and conditions are necessary for swarming ability. In this study, we discovered that adding gelatin to agar medium promotes swarming on the agar surface by the lateral flagella of Vibrio. Our data suggested that surfactants or viscous polysaccharides secreted extracellularly are important for promoting swarming on the agar surface and we identified that swarming is likely to be driven by S (social)-motility, in which bacteria move by interacting with each other, and A (adventure)-motility, in which bacteria move by interacting with the agar surface. Our study provides clues that help clarify the mechanism of bacterial swarming IMPORTANCEWe discovered that adding gelatin to hard agar medium promoted swarming on agar surfaces by the lateral flagella of Vibrio cells. The surfactants or viscous polysaccharides secreted extracellularly seem to be important for swarming ability on agar surfaces. We proposed that the swarming is thought to occur through S(social)-motility, where cells move by interacting with cell bodies each other, and A(adventure)-motility, where cells move by interacting with the agar surface and cell body. The present study should provide the clues to clarify the mechanism of bacterial swarming and how to move in a viscous environment.

AO_SCPLOWBSTRACTC_SCPLOWUnderstudied bacterial genera present a dynamic phylogenetic landscape and opportunities for discovering new taxa as more strains are isolated and genomic data is added. Here, through phylogenomic analysis, we describe three novel species of the globally distributed cosmopolitan marine bacterial genus Microbulbifer. This genus is ubiquitous in saltwater microbiomes and is a validated source of biodegradation enzymes as well as high value small molecule natural products. Average nucleotide identity (ANI) to the closest known species, Microbulbifer variabilis ATCC 700307T, was less than 88.4% for all three novel species. Isolates of the three novel species, designated as PAAF003T (T = type strain), ZKSA006T, and SSSA003T were imaged to reveal their phormological characteristics. Based on phylogenetic data, strains PAAF003T, ZKSA006T, and SSSA003T represent three new species of the genus Microbulbifer, for which the names Microbulbifer maximicatervae sp. nov., Microbulbifer regidiadema sp. nov., and Microbulbifer mixtoriginis sp. nov. are proposed, respectively, under the SeqCode. We also reconstructed a robust phylogeny of available Microbulbifer genomes, which should faciliatate future isolation and strain description studies.

The marine biodiversity inhabiting rocky shores in the Colombian Pacific remains largely undocumented, primarily due to geographic isolation, logistical challenges, and socio-political constraints. To address the existing knowledge gap, we conducted an expedition to enhance baseline biodiversity knowledge in rocky shores by integrating multiple complementary approaches, including visual censuses, specimen collection with morphological identification, environmental DNA (eDNA) metabarcoding and DNA barcodes. eDNA samples were collected at four coastal sites adjacent to rocky substrates, along with biological specimens obtained from fourteen locations through SCUBA diving at depths ranging from 1 to 25 meters. Tissue samples were subjected to genomic DNA isolation, followed by the generation and validation of cytochrome c oxidase subunit I (COI) barcode sequences, which were subsequently corroborated through taxonomic assessment to ensure accurate species identification. eDNA metabarcoding analyses yielded over 7 million high-quality sequence reads. Although taxonomic resolution at the species level was constrained by the limited completeness of reference sequence databases, a total of 106 species and 83 families were successfully identified, predominantly within the classes Actinopteri, Chondrichthyes, and marine mammals. From the 769 specimens obtained we generated 871 sequences, including 414 validated COI barcodes representing 76 species across 64 families. The integration of DNA barcoding and eDNA approaches resulted in over 1,400 taxonomic detections spanning five phyla, with only six species shared between methodologies. Richness and diversity varied among sites, and revealed significant differences along the coastline between Jurado and Cupica Gulf. All sequences were deposited in BOLDsystems database under the CCBIO project and were visualized through OBIS and GBIF databases. These findings provide the first molecular-based baseline for rocky shore biodiversity in the Colombian Pacific, highlighting the value of integrative approaches for monitoring and conservation.

Corals associate with many bacteria whose evolutionary histories and holobiont roles are unknown due to a lack of genomic resources. An example is the BD1-7 clade, which is found in some microbial metabarcoding libraries of corals and has been speculated to be phototrophic. To evaluate its phylogenetic position and assess its metabolic capabilities, we assembled and annotated the genome of an octocoral associate classified as BD1-7. Its full genome revealed that it instead represents a distinct and divergent clade of widespread coral associates. We propose the name Ca. Steroidedax gorgoniicola for this associate of Swiftia exserta. Unlike the true BD1-7 clade, its genome encoded no pathways to generate ATP from light and instead reveals that it is likely a heterotroph that can degrade steroids, chitin, and collagen as well as produce toxins or antimicrobial compounds and detoxify several reactive oxygen and nitrogen species. In addition, we identified several genes that were likely horizontally transmitted from Endozoicomonadaceae, including transposases and genes involved in virulence and cell adhesion. This work sheds light on the potential role of horizontal genetransfer in the evolution of symbiosis and highlights the importance of obtaining genomes to resolve coral-associated lineages and their metabolic capabilities.

The cyanobacterium Trichodesmium is important in ocean nitrogen (N) and carbon (C) biogeochemistry through fixation of N2 gas and CO2 into NH3, amino acids, and carbohydrates. These essential compounds then fuel primary productivity, bacterial and grazing communities, and upper trophic levels. In the oligotrophic surface oceans, they are one of few taxa capable of utilizing both atmospheric C and N. Trichodesmium fall into 2 diazotrophic phylogenomic clades named "Thieb" and "Tery" after the most well characterized botanical species, Trichodesmium thiebautii and Trichodesmium erythraeum. Thieb is commonly the most abundant clade in all major ocean regions and paradoxically, is also the least characterized in terms of morphology, genomics, and ecophysiology due to underrepresentation in culture collections. In this study, 25 novel strains of Thieb were enriched in culture from the Sargasso Sea, phenotypically characterized, and whole genome sequenced. Cultures were found to belong to four well-supported subclades (i.e., ThiebA, ThiebB, ThiebC, and ThiebD). Trichodesmium subclades were found to have environmentally relevant differences in colony shape, gene content, cell dimensions, and inferred temperature range. Specifically, ThiebD and ThiebC had the largest biovolumes whereas ThiebA and ThiebB had the smallest. Importantly ThiebB also became relatively more abundant only in bulk seawater metagenomes collected at high temperature (>28{degrees}C). This finding indicates that as ocean warming continues, there may be shifts in Trichodesmium community structure towards smaller cells, which would affect overall C+N standing stocks.

Trichodesmium is an important oceanic N2 fixing cyanobacterial genus that has been shown to provide up to 50% of new N to oligotrophic regimes. Despite its importance, we know relatively little about the genomic potential and functional diversity of the two major clades of Trichodesmium (Clade I and Clade III, hereafter Thieb and Tery, respectively). With the expanded collection of Trichodesmium isolates in the USCTCC (University of Southern California Trichodesmium Culture Collection), we sequenced genomes from seven cultivated strains to further characterize the genomic diversity within the genus. For example, sequencing the genome of the "gigantic", red Trichodesmium contortum surprisingly shows that they are closely related to the smallest Trichodesmium clade, TeryA. The high genomic identity between TeryA and T. contortum (>98.5% ANI) and lack of implicated auxiliary genes suggests their large biovolume differences might be transcriptional or epigenetic in origin. Furthermore, these data show that using Tery-subclades are a more accurate designation than the classical Trichodesmium species delineation. Finally, we placed the analysis of these genomes in an ecological context via read mapping with globally distributed RNA and DNA datasets. Our data show that Tery clades (A&B) are both lower in relative abundance compared to Thieb in global oceans, generally co-occur when detected in the field, and are highly linked to decreased salinity and increased temperatures, especially for sampling locations in the Bay of Bengal. Lastly, even though TeryA members are undersaturated with respect to current CO2 concentrations, our phenotypic and biogeography data suggests that salinity/ocean color could limit their predicted global impact as climate changes.

Members of the amoebozoan genus Acanthamoeba are unicellular heterotrophic protists that inhabit a wide range of terrestrial and aquatic environments. They are opportunistic pathogens known to host disease-causing bacteria as well large DNA viruses. While Acanthamoeba is commonly used to study cellular locomotion, phagocytosis, and disease, genetic tools for precise editing of the Acanthamoeba genome are still in their infancy. Here we have developed CRISPR/Cas9 tools for genetically engineering the model acanthamoebid Acanthamoeba castellanii strain Neff. In proof-of-principle experiments, we targeted the myosin-II heavy chain gene. Evidence for editing was found using reverse transcriptase PCR, microscopy, and DNA sequencing, which also identified unique alleles for the target locus. Collectively, we demonstrate that CRISPR can be employed to edit Acanthamoeba genes using a knock-in approach. This work serves as a foundation to further develop A. castellanii as a model system with which to study diverse questions in cell and molecular biology, biochemistry and evolution.

Most cockroaches and the termite Mastotermes darwiniensis are associated with Blattabacterium, an ancient obligate endosymbiont that participates in the nitrogen metabolism of its host. Blattabacterium has been vertically transmitted since it was acquired by the common ancestor of cockroaches and termites and was reportedly lost twice, once in the cockroach genus Nocticola and once in all termites except Mastotermes darwiniensis. Here, we acquired cockroach specimens spanning most of the cockroach phylogenetic tree to study Blattabacterium using shotgun sequencing. We found no traces of Blattabacterium in 64 specimens from ten independent lineages of cockroaches across three families: Blattellidae, Pseudophyllodromiidae, and Anaplectidae. The absence of Blattabacterium was confirmed with three PCR amplifications targeting the 16S and 23S ribosomal genes with primers specific to Blattabacterium. Notably, cockroaches lacking Blattabacterium were often infected by Rickettsia and Wolbachia, many of which were related to the mutualistic Wolbachia strain of Cimex lectularius, the common bed bug. These results indicate that cockroaches from Blattellidae, Pseudophyllodromiidae and Anaplectidae have lost their ancestral Blattabacterium endosymbiont at least ten times independently, with many of these losses possibly facilitated and compensated by new associations with mutualistic Wolbachia strains that may help provision the host with B vitamins.

A strictly anaerobic bacterial strain, F2T, was isolated from an anaerobic bioreactor fermenting source-separated organic waste. Cells of strain F2T are non-spore-forming, rod-shaped (1.5-2.5 x 0.27-0.33 m), and Gram-negative, although they possess a monoderm cell wall architecture. The strain grew at 37 degrees C within a pH range of 5 to 8 and produced short-, branched-, and medium-chain carboxylates as well as ammonium, H2 and CO2, with acetate and propanoate produced or consumed depending on fermentation conditions. The genome consists of a single 2.4 Mbp chromosome with a G+C content of 50.2% and 2,131 predicted genes. Phylogenetic analysis of the 16S rRNA gene against other isolates revealed that strain F2T is most similar to Eubacterium pyruvativorans I-6T (92.06% 16S rRNA identity). Based on further phenotypic, genomic, and phylogenetic analysis, strain F2T represents a novel genus and species within the family Anaerovoracaceae with the proposed name Peptonella octanoica gen. nov. sp. nov. The type strain is F2T (strain accession pending). As a member of this same genus-level clade, we propose reclassifying Eubacterium pyruvativorans as Peptonella pyruvativorans comb. nov. These findings disambiguate Peptonella spp. from the phylogenetically distant and phenotypically distinct Eubacterium limosum ATCC 8486T.

BackgroundThe tribal classification of scarab dung beetles (Coleoptera: Scarabaeinae) is currently largely incomplete due to the lack of robust phylogenetic evidence supporting the assignment of many Afro-Eurasian and American genera to tribes. MethodsWe used ultraconserved elements (UCEs) to infer phylogenetic relationships across most Afro-Eurasian dung beetle lineages, including 25 of the 27 extant genera currently incertae sedis. ResultsWe recovered full support for the monophyly of previously recognised tribes and for several phylogenetically and morphologically clearly delimited new tribal-level clades, allowing us to propose a complete tribal classification for all Afro-Eurasian dung beetle genera. Thirteen new tribes are described and diagnosed: Aphengoecini trib. nov., Bohepilissini trib. nov., Catharsiini trib. nov., Chalconotini trib. nov., Circelliini trib. nov., Dwesasilvasedini trib. nov., Haroldiini trib. nov., Heliocoprini trib. nov., Janssensantini trib. nov., Macroderini trib. nov., Nesovinsoniini trib. nov., Pycnopanelini trib. nov. and Tanzanolini trib. nov. The tribe Coprini sensu novo is redefined as comprising three subtribes: Coprina, Onychothecina subtrib. nov. and Pedariina subtrib. nov. The tribe Odontolomini is downranked to a subtribe of Endroedyolini sensu novo, becoming Endroedyolini Odontolomina stat. nov. The tribe Onthophagini sensu novo is redefined and divided into three subtribes: Helictopleurina stat. nov., Oniticellina stat. nov. and Onthophagina; the remaining former subtribes of Oniticellini (Attavicinina, Drepanocerina and Liatongina) are synonymised with Oniticellina; the subtribe Alloscelina of Onthophagini is synonymised with Onthophagina (syn. nov.). The tribe Panelini stat. rev. and sensu novo, comprising the single genus Panelus, is revalidated and redefined. Updated morphological diagnoses of Elassocanthonini, Gymnopleurini, Onitini and Scarabaeini are provided. The genus Phaedotrogus is synonymised with Haroldius (Haroldiini) (syn. nov.). An identification key to all Afro-Eurasian dung beetle tribes is provided. DiscussionOur results establish a robust phylogenetic framework and revised tribal classification for Afro-Eurasian Scarabaeinae dung beetles, providing a foundation for future taxonomic, comparative and macroevolutionary research.

Species-level delimitation within the genus Trichomycterus remains one of the main systematic challenges within the Trichomycterinae, particularly in lineages characterised by conservative external morphology, high apparent intraspecific variation, and historical diagnostic criteria based primarily on body proportions, colouration and a few meristic characters. In central Chile, Trichomycterus areolatus has traditionally been interpreted as a widely distributed and morphologically variable species, whilst T. maculatus, originally described from "Santiago du Chili", has remained subordinate to this broad conception without a modern phylogenetic reassessment. Here we reassess the specific boundaries of T. areolatus sensu lato using an integrative approach that combines complete mitogenomes, estimates of genetic divergence and comparative morphology of the cephalic laterosensory system associated with the neurocranium. Phylogenetic analyses reveal T. areolatus sensu lato to be non-monophyletic and identify a deeply divergent lineage, geographically coherent and attributable to T. maculatus. This lineage differs from restricted T. areolatus by extensive mitochondrial divergence, comparable to that observed between recognised species of Trichomycterinae, and by discrete characters of the cephalic lateral line system, primarily related to the continuity of the supraorbital canal and the arrangement of the associated pores. The congruence between mitogenomic, nuclear and morphological evidence supports the revalidation of Trichomycterus maculatus Valenciennes, 1846, and calls for a more restricted geographic circumscription of T. areolatus. These results demonstrate that the diversity of Trichomycterinae in central Chile has been underestimated, modify previous interpretation of the distribution of the species involved, and highlight the value of integrating mitogenomics and neurocranial/laterosensory characters into the taxonomy of morphologically conservative siluriform lineages.

A facultatively anaerobic, hydrogen-oxidizing, thermophilic bacterium (strain KUI-RBT) was isolated from a geothermal spring biofilm in Rotorua, New Zealand. Strain KUI-RBT is a motile, straight rod, measuring approximately 0.7 {micro}m by 1.0 to 1.5 {micro}m with a diderm cell wall. Growth of KUI-RBT occurred from 39 to 74 {degrees}C (Topt 64.5 {degrees}C), pH 5.0 to 7.5 (pHopt 6.5), and 0 to 1% (w/v) NaCl (NaClopt 0.4-0.7%, w/v). KUI-RBT utilizes carbon dioxide and various organic carbon substrates as carbon sources and hydrogen as an electron donor. KUI-RBT can use oxygen (0-21%, v/v), elemental sulfur, thiosulfate, sulfite, nitrate, arsenate, and selenate as terminal electron acceptors. Major fatty acids of strain KUI-RBT include C20:1, C18:1, and C18:0 and the primary quinone is MTK-7. The whole genome G+C content is 34.23 mol%. Phylogenetic analyses indicate KUI-RBT to be a member of the family Hydrogenothermaceae, with Sulfurihydrogenibium azorense Az-Fu1T its closest characterised relative (94.51% 16S rRNA gene sequence similarity, 78.01% whole genome ANI, 61.34% whole genome AAI). Based on phylogenetic and phenotypic analyses, we propose KUI-RBT represents a novel genus and species within the family Hydrogenothermaceae, for which we propose the name Reysenbachia aerophila gen. nov., sp. nov. The type strain is KUI-RBT (=KCTC accession =JCM accession). The GenBank accession number for the 16S rRNA gene sequence of strain KUI-RBT is PZ052650. The GenBank accession number for the whole genome of strain KUI-RBT is JBVODP000000000.