Species-level Lineages Research Articles

DNA sequencing of topotypes helps delineate species distributions in the Ischnocnema verrucosa complex (Anura, Brachycephalidae)

As many new evolutionary lineages are being discovered and formally named, sequencing topotypes when holotypes are not available becomes essential for taxonomy. This study uses a DNA-taxonomy approach to sequence new populations of the Ischnocnema verrucosa species complex (Brazilian Wart Frogs) from different locations, including, for the first time, individuals from the type localities. Phylogenetic analysis of the mitochondrial 16S gene recovered a monophyletic Ischnocnema verrucosa species series composed of three main clades. The most recent common ancestor was estimated to be 33.76 million years ago, and diversification within the three main clades occurred primarily during the Miocene. We delimited eight species-level lineages with high levels of sequence divergence (7% to 16%). Our study highlights the importance of DNA taxonomy and the necessity of protecting and sequencing topotypes in taxonomic studies. Our study also contributes to the conservation and understanding of the genus Ischnocnema and the biodiversity of the Brazilian Atlantic Forest region.

Disentangling disjunctus: an integrated analysis of the patent leather beetle, Odontotaenius disjunctus complex (Passalidae: Coleoptera) with the description of two new species

The categorical rank of species is the fundamental taxonomic unit in characterizing biodiversity, and the accurate identification of species-level lineages is vital to all fields of biological research. This is particularly true for conservation biology, where state and federal agencies make conservation assessments based on recognized species’ distribution, ecology, and demography. Currently, there are two species of bess beetle in the conterminous United States, Odontotaenius flordianus (Schuster) and O. disjunctus (Illeger). Phylogeographic studies have proposed that O. disjunctus may represent a species complex harboring cryptic diversity. This study combines multi-locus species delimitation methods and multivariate analyses of morphological characters used to distinguish O. floridanus, to assess species diversity within the O. disjunctus complex. Delimitation methods inferred four well-supported species that diverged during the Pleistocene and whose formation was driven by historical isolation associated with shifting climatic conditions. We found a correlation between increased morphological differentiation and time since divergence. We described two new species, Odontotaenius jackmanicus sp. nov., based on morphological and molecular characters, and the cryptic species, Odontotaenius cryptodisjunctus sp. nov., diagnosed exclusively on molecular characters. We propose that the morphological similarities between O. disjunctus and O. cryptodisjunctus are due to a recent speciation combined with morphological conservatism and evolutionary stasis.

Development of novel broad-range pan-genus PCR assays for the detection of Tropheryma species.

Introduction. Tropheryma whipplei is responsible for the classical Whipple's disease. Recently, a new Tropheryma species was described in a Belgian immunocompromised patient with pleuritis.Gap Statement. There is currently no specific molecular diagnostic test detecting other Tropheryma species than Tropheryma whipplei.Aim. To develop and validate two broad-range pan-Tropheryma genus PCRs detecting both T. whipplei and new Tropheryma species.Methodology. From shotgun sequencing data of the lung tissue biopsy of the Belgian subject, we designed two PCRs targeting the 23S rRNA and rnpB genes. Prospectively, requests for T. whipplei PCR were tested with T. whipplei-specific PCRs and the two Tropheryma broad-range PCRs from January 2019 to November 2022.Results. In total, 2605 samples were tested using both the pan-Tropheryma 23S rRNA PCR and the T. whipplei-specific PCR. In addition, 833 of the 2605 samples were also tested using the pan-Tropheryma rnpB PCRs. Sensitivity was 78.8% and 79.7% for 23S rRNA and rnpB PCRs, as compared with the species-specific T. whipplei PCR. Specificity was 99.9% and 99.7% for the 23S rRNA and the rnpB PCRs, respectively. We identified a patient whose bronchoalveolar lavage tested positive with the two broad-range PCRs with >105 copies ml-1. Specific T. whipplei PCRs were negative. Known for panuveitis, this 49-year-old male presented with an eye inflammation recurrence, and a CT scan showed multiple mediastino-hilar necrotic adenopathies. Doxycyclin (1 year), hydroxychloroquin (1 year) and co-trimoxazol (1 month) treatments led to a favourable outcome.Conclusion. Specific T. whipplei PCR exhibited better sensitivity than the pan-Tropheryma PCRs. However, both broad-range pan-Tropheryma PCRs demonstrated excellent specificity and were pivotal to identifying a new probable case of Tropheryma infection due to another species-level lineage.

Endless forms most frustrating: disentangling species boundaries in the Ramalina decipiens group (Lecanoromycetes, Ascomycota), with the description of six new species and a key to the group.

Oceanic islands have been recognized as natural laboratories in which to study a great variety of evolutionary processes. One such process is evolutionary radiations, the diversification of a single ancestor into a number of species that inhabit different environments and differ in the traits that allow them to exploit those environments. The factors that drive evolutionary radiations have been studied for decades in charismatic organisms such as birds or lizards, but are lacking in lichen-forming fungi, despite recent reports of some lineages showing diversification patterns congruent with radiation. Here we propose the Ramalina decipiens group as a model system in which to carry out such studies. This group is currently thought to be comprised of five saxicolous species, all of them endemic to the Macaronesian region (the Azores, Madeira, Selvagens, Canary and Cape Verde islands). Three species are single-island endemics (a rare geographic distribution pattern in lichens), whereas two are widespread and show extreme morphological variation. The latter are suspected to harbor unrecognized species-level lineages. In order to use the Ramalina decipiens group as a model system it is necessary to resolve the group's phylogeny and to clarify its species boundaries. In this study we attempt to do so following an integrative taxonomy approach. We constructed a phylogenetic tree based on six molecular markers, four of which are newly developed and generated competing species hypotheses based on molecular (species discovery strategies based on both single locus and multilocus datasets) and phenotypic data (unsupervised clustering algorithms based on morphology, secondary chemistry and geographic origin). We found that taxonomic diversity in the Ramalina decipiens group has been highly underestimated in previous studies. In consequence, we describe six new species, most of them single-island endemics and provide a key to the group. Phylogenetic relationships among species have been reconstructed with almost full support which, coupled with the endemic character of the group, makes it an excellent system for the study of island radiations in lichen-forming fungi. Citation: Blázquez M, Pérez-Vargas I, Garrido-Benavent I, et al. 2024. Endless forms most frustrating: disentangling species boundaries in the Ramalina decipiens group (Lecanoromycetes, Ascomycota), with the description of six new species and a key to the group. Persoonia 52: 44-93. https://doi.org/10.3767/persoonia.2024.52.03 .

Taxonomizing a truly morphologically cryptic complex of dwarf geckos from Madagascar: molecular evidence for new species-level lineages within the Lygodactylus tolampyae complex

Taxonomizing a truly morphologically cryptic complex of dwarf geckos from Madagascar: molecular evidence for new species-level lineages within the Lygodactylus tolampyae complex

What’s in a name? Using species delimitation to inform conservation practice for Chinese giant salamanders (Andrias spp.)

Abstract Genetically-defined biodiversity units must align with practical conservation frameworks, and most conservation is conducted at the species level. Chinese giant salamanders have traditionally been interpreted as the single widespread species Andrias davidianus, but molecular studies have reinterpreted this taxon as representing multiple allopatric clades, and competing taxonomic hypotheses support different numbers of candidate species. We conducted species delimitation analyses using tree-based models (General Mixed Yule Coalescent, Poisson Tree Processes) and alignment-based models (Bayesian Phylogenetics and Phylogeography) to interpret diversification across Andrias within a comparative systematic framework, using 30 mitogenomes representing all recognised Chinese clades. Nearly all tested models provide support for at least seven statistically-resolved Chinese species-level lineages, and most provide support for nine species. Only four species have available names. Chinese Andrias populations are Critically Endangered, but unnamed species cannot be incorporated into national or international conservation frameworks and risk being excluded from recovery efforts. We urge taxonomists and conservation practitioners to focus more attention on the world’s largest amphibians, and non-standard taxonomic approaches may be required to name these species before they disappear.

Similar looking sisters: A new sibling species in the Pristimantis danae group from the southwestern Amazon basin (Anura, Strabomantidae)

We describe a new frog species that is the sibling of Pristimantis reichlei. These two sister species inhabit the Amazonian lowlands and adjacent foothills of the Andes, from central Bolivia to central Peru. Pristimantis reichlei occurs from central Bolivia to southern Peru (Alto Purús National Park), while the new species occurs from northern Bolivia (Departamento Pando) to Panguana in central Peru (Departamento Huánuco), at elevations between 220 and 470 m a.s.l. In spite of their morphological crypsis, these siblings occur in syntopy without evidence of interbreeding (in the Alto Purús area) and are recovered as reciprocally monophyletic. Their uncorrected pairwise genetic distances in the 16S rRNA gene range from 9.5–13.5%, and their advertisement calls differ in both qualitative and quantitative traits. Moreover, our study found uncorrected pairwise distances within the new species of up to 5.0% and up to 9.3% within P. reichlei. We therefore cannot rule out the possible existence of hybrids or additional species-level lineages hidden in this complex. Furthermore, we found another potential pair of sibling species composed of P. danae and an unnamed lineage, with divergences of 9.4% in the 16S gene, whose in-depth analysis and taxonomic treatment are pending future revision. With the new nominal species, the Pristimantis danae species group now includes 20 species, distributed across the upper Amazon basin and in the eastern Andes, from western Brazil to Bolivia and Peru. Our study, together with an increasing number of other studies, indicates that sibling species are far from being rare among Amazonian amphibians and that species resolution remains low even for groups that have received considerable attention in recent years.

Molecular Diversity of the Genus Plagiorchis Lühe, 1899 in Snail Hosts of Central Europe with Evidence of New Lineages

Cercariae of Plagiorchis spp. are frequently reported in European freshwater snails, but their true diversity is difficult to estimate due to subtle differences in morphology. We molecularly characterized 67 isolates of Plagiorchis cercariae collected from four lymnaeid snail hosts, Ampullaceana balthica, Ampullaceana lagotis, Radix auricularia and Lymnaea stagnalis in freshwater ecosystems in the Czech Republic and Poland. Based on mitochondrial cox1 and nuclear 28S sequences, ten species or species-level lineages were identified, including the first molecular evidence of P. vespertilionis from snail hosts and two species-level lineages reported for the first time. Previously undescribed species and species-level lineages are characterized morphometrically. We confirm the overlapping spatial distribution of Plagiorchis spp. in their snail hosts from Central Europe with those from Western and sub-Arctic Europe. Our results increase the known diversity of Plagiorchis spp. in Europe to 25 species/lineages in snails, but further research is needed to establish links between life cycle stages and to assess the host specificity of these parasites.

Phylogenetic insight into the Lecidea atrobrunnea complex – evidence of narrow geographic endemics and the pressing need for integrative taxonomic revisions

AbstractSpecies of lichen-forming fungi (LFF) display an array of geographical distribution patterns. Among the broadly distributed lichen-forming fungal species, the degree of reproductive isolation and genetic substructure among populations varies widely, in some cases masking unrecognized diversity or meaningful biogeographical patterns. Lecidea atrobrunnea (Raymond ex Lam. & DC.) Schaer. s. lat. (Lecideaceae) is a widespread species complex that has been studied for over two centuries since its initial description. The diversity of the L. atrobrunnea group is highest in western North America, where a dizzying array of morphologies and chemistry can occur at local scales. Here we investigate whether the assumed cosmopolitan distribution of L. atrobrunnea s. lat. is an artifact of taxonomic limitations and masks biogeographical patterns in this species complex. To address these questions, we compiled sequence data from the standard fungal barcoding marker (ITS) for over 100 specimens within this complex, in addition to genome-scale data from a subset of these representing over 1600 single-copy nuclear genes spanning over 3 Mb of the genome. Our study corroborates the perspective that the morphologically and chemically variable Lecidea atrobrunnea group reflects a complex of distinct species-level lineages, with 42–83 candidate species inferred from the ITS region and high levels of diversity inferred from a subset of specimens using genome-scale data. However, both phenotype- and molecular-based species boundaries remained unsettled, with the most common nominal taxa recovered as highly polyphyletic and with conflict among different molecular species delimitation approaches. Our study also highlights the potential for geographically restricted species, with fascinating biogeographical patterns, challenging, in part, the assumed cosmopolitan distribution of L. atrobrunnea s. lat. This study provides valuable direction for future research that will be crucial in understanding diversification and establishing a robust taxonomy for this well-known species complex.

“Candidatus Intestinibacterium parameciiphilum”—member of the “Candidatus Paracaedibacteraceae” family (Alphaproteobacteria, Holosporales) inhabiting the ciliated protist Paramecium

Protists frequently host diverse bacterial symbionts, in particular those affiliated with the order Holosporales (Alphaproteobacteria). All characterised members of this bacterial lineage have been retrieved in obligate association with a wide range of eukaryotes, especially multiple protist lineages (e.g. amoebozoans, ciliates, cercozoans, euglenids, and nucleariids), as well as some metazoans (especially arthropods and related ecdysozoans). While the genus Paramecium and other ciliates have been deeply investigated for the presence of symbionts, known members of the family “Candidatus Paracaedibacteraceae” (Holosporales) are currently underrepresented in such hosts. Herein, we report the description of “Candidatus Intestinibacterium parameciiphilum” within the family “Candidatus Paracaedibacteraceae”, inhabiting the cytoplasm of Paramecium biaurelia. This novel bacterium is almost twice as big as its relative “Candidatus Intestinibacterium nucleariae” from the opisthokont Nuclearia and does not present a surrounding halo. Based on phylogenetic analyses of 16S rRNA gene sequences, we identified six further potential species-level lineages within the genus. Based on the provenance of the respective samples, we investigated the environmental distribution of the representatives of “Candidatus Intestinibacterium” species. Obtained results are consistent with an obligate endosymbiotic lifestyle, with protists, in particular freshwater ones, as hosts. Thus, available data suggest that association with freshwater protists could be the ancestral condition for the members of the “Candidatus Intestinibacterium” genus.

Unveiling underestimated species diversity within the Central American Coralsnake, a medically important complex of venomous taxa

Coralsnakes of the genus Micrurus are a diverse group of venomous snakes ranging from the southern United States to southern South America. Much uncertainty remains over the genus diversity, and understanding Micrurus systematics is of medical importance. In particular, the widespread Micrurus nigrocinctus spans from Mexico throughout Central America and into Colombia, with a number of described subspecies. This study provides new insights into the phylogenetic relationships within M. nigrocinctus by examining sequence data from a broad sampling of specimens from Mexico, Guatemala, Honduras, Nicaragua, Costa Rica, and Panama. The recovered phylogenetic relationships suggest that M. nigrocinctus is a species complex originating in the Pliocene and composed of at least three distinct species-level lineages. In addition, recovery of highly divergent clades supports the elevation of some currently recognized subspecies to the full species rank while others may require synonymization.

A new species of green-eyed Cordylus Laurenti, 1768 from the west-central highlands of Angola, and the rediscovery of Cordylus angolensis (Bocage, 1895) (Squamata: Cordylidae)

Cordylus angolensis (Bocage, 1895) was described 128 years ago on the basis of a single specimen collected at Caconda in the west-central highlands of Angola. Additional specimens referred to this species were collected at ‘Mombolo’ (also in the central highlands) during the Vernay Angola Expedition in 1925. As the holotype was apparently destroyed in the fire of 1978 at the Museu Bocage in Lisbon and no additional specimens have been collected, its taxonomic status and phylogenetic relationships has remained uncertain. The species has eluded all efforts aimed at its re-discovery in the vicinity of the type locality, with a single specimen from near Condé, north of Mombolo—collected in 1970 by Wulf Haacke—the only other specimen of Cordylus known from west-central Angola. Recent field work in the Angolan highlands resulted in the collection of a series of specimens from Taqueta Mountain (west of Caconda), Monte Verde (Sandula, ‘Mombolo’) and Uassamba (Vondo). A phylogenetic analysis, using three mitochondrial and six nuclear genes, indicated the existence of two distinct species-level lineages in the Angolan highlands. These two species are allopatric and morphologically distinct, differing especially in terms of their colour patterns, eye colour and certain scalation characteristics. We therefore confirm that C. angolensis is a valid species and designate a neotype, and describe a new species, Cordylus momboloensissp. nov.

Systematic revision of the Spotted and Northern Dusky Salamanders (Plethodontidae: Desmognathus conanti and D. fuscus), with six new species from the eastern United States.

Spotted and Northern Dusky Salamanders (Desmognathus conanti and D. fuscus) have a long and complex taxonomic history. At least 10 other currently recognized species in the genus were either described from populations previously considered D. fuscus, described as or later considered subspecies thereof, or later considered synonyms thereof, before ultimately being recognized as distinct. Recent molecular analyses have also revealed extensive cryptic diversity within both species, which are polyphyletic assemblages of 13 distinct mitochondrial lineages with 5.7-10.3% uncorrected 'p' distances in the COI barcode locus. Based on phylogenomic data and population-clustering analyses considering admixture between lineages, 11 candidate species were circumscribed by recent authors. Those within D. conanti are also ecomorphologically variable, comprising both large, robust, keel-tailed populations, and small, gracile, round-tailed forms. Evaluating their distinctiveness based on genetic, geographic, and morphological evidence, we conclude that six of the candidates represent new species: Desmognathus anicetus sp. nov., D. bairdi sp. nov., D. campi sp. nov., D. catahoula sp. nov., D. lycos sp. nov., and D. tilleyi sp. nov. Consequently, we recognize eight total species from populations formerly associated with the nominal species D. conanti and D. fuscus, the re-delimited concepts of which also contain additional phylogeographic lineage diversity that may represent further distinct species. In addition to existing mitochondrial and nuclear phylogenetic, network, and clustering results, we present preliminary analyses of linear morphometrics to bolster diagnostic specificity based on phenotypic characteristics. These changes stabilize the previously paraphyletic taxonomy of species-level lineages within Desmognathus, though additional cryptic diversity may exist both within the species considered here, and elsewhere in the genus.

Ocean crossers: A tale of disjunctions and speciation in the dwarf-fruticose Lichina (lichenized Ascomycota)

Lichens thrive in rocky coastal areas in temperate and cold regions of both hemispheres. Species of the genus Lichina, which form characteristic black fruiting thalli associated with cyanobacteria, often create distinguishable bands in the intertidal and supralittoral zones. The present study uses a comprehensive specimen dataset and four gene loci to (1) delineate and discuss species boundaries in this genus, (2) assess evolutionary relationships among species, and (3) infer the most likely causes of their current geographic distribution in the Northern and Southern hemispheres. A dated phylogeny describes the time frame in which extant disjunctions of species and populations were established. The results showed that the genus is integrated by four species, with Lichina pygmaea, L. confinis and the newly described L. canariensis from rocky seashores in the Canary Islands, occurring in the Northern Hemisphere, whereas L. intermedia is restricted to the Southern Hemisphere. Lichina intermedia hosted a much higher intraspecific genetic diversity than the other species, with subclades interpreted as species-level lineages by the different species delimitation approaches. However, a conservative taxonomic approach was adopted. This species showed a striking disjunct distribution between Australasia and southern South America. The timing for the observed interspecific and intraspecific divergences and population disjunctions postdated continental plate movements, suggesting that long-distance dispersal across body waters in the two hemispheres played a major role in shaping the current species distributions. Such ocean crossings were, as in L. canariensis, followed by speciation. New substitution rates for the nrITS of the genus Lichina were inferred using a tree spanning the major Ascomycota lineages calibrated using fossils. In conclusion, this work lays the foundation for a better understanding of the evolution through time and space of maritime lichens.

Molecular and geographic evaluations of the Heteromys pictus–spectabilis species complex indicate multiple cryptic species

Abstract The Heteromys pictus–spectabilis species complex currently consists of two species: H. pictus, which is distributed across western, central, and southern Mexico; and H. spectabilis, an endangered species which is restricted to a small region of southeastern Jalisco. Previous phylogenetic studies have indicated that H. spectabilis renders H. pictus as paraphyletic, and therefore revisions are needed to resolve this paraphyly. Phylogenetic trees based on mitochondrial DNA and nuclear DNA genes were constructed using maximum likelihood and Bayesian methods, grouping individuals into previously established Cytochrome b (Cytb) haplogroups across the geography. Phylogenetic trees were run through bPTP and GMYC analyses to estimate the number of predicted species that may be present, and when these lineages likely diverged based on the genes tested. To assist with species delimitation predictions (based on the Genetic Species Concept), intra- and interspecific Kimura two-parameter (K2P) values were calculated to predict species-level lineages within the species complex. We found evidence to support the hypothesis that there are multiple cryptic species present within H. pictus, with some K2P values between the Cytb haplogroups being on par with what is expected between different species and genera within Rodentia. This is further supported by the phylogenetic trees (individual and concatenated) constructed from Cytb and three nuclear genes (Bfib, IRBP, and PRKCI), which consistently group certain Cytb haplogroups together in ways that correspond to geographic filter barriers in Mexico.

The description of Haematococcus privus sp. nov. (Chlorophyceae, Chlamydomonadales) from North America

An enormous body of research is focused on finding ways to commercialize carotenoids produced by the unicellular green alga, Haematococcus, often without the benefit of a sound phylogenetic assessment. Evidence of cryptic diversity in the genus means that comparing results of pigment studies may be confounded by the absence of a phylogenetic framework. Moreover, previous work has identified unnamed strains that are likely candidates for species status. We reconstructed the phylogeny of an expanded sampling of Haematococcus isolates utilizing data from nuclear ribosomal markers (18S rRNA gene, 26S rRNA gene, internal transcribed spacer [ITS]-1, 5.8S rRNA gene, and ITS-2) and the rbcL gene. In addition, we gathered morphological, ultrastructural and pigment data from key isolates of Haematococcus. Our expanded data and taxon sampling support the concept of a new species, H. privus, found exclusively in North America. Despite overlap in numerous morphological traits, results indicate that ratios of protoplast length to width and akinete diameter may be useful for discriminating Haematococcus lineages. High growth rate and robust astaxanthin yield indicate that H. rubicundus (SAG 34-1c) is worthy of additional scrutiny as a pigment source. With the description of H. privus, the evidence supports the existence of at least five, species-level lineages in the genus. Our phylogenetic assessment provides the tools to frame future pigment investigations of Haematococcus in an updated evolutionary context. In addition, our investigation highlighted open questions regarding polyploidy and sexuality in Haematococcus which demonstrate that much remains to be discovered about this green flagellate.

Multiple transitions between realms shape relict lineages of Proteus cave salamanders.

In comparison to biodiversity on Earth's surface, subterranean biodiversity has largely remained concealed. The olm (Proteus anguinus) is one of the most enigmatic extant cave inhabitants, and until now little was known regarding its genetic structure and evolutionary history. Olms inhabit subterranean waters throughout the Dinaric Karst of the western Balkans, with a seemingly uniform phenotypic appearance of cave-specialized traits: an elongate body, snout and limbs, degenerated eyes and loss of pigmentation ("white olm"). Only a single small region in southeastern Slovenia harbours olms with a phenotype typical of surface animals: pigmented skin, eyes, a blunt snout and short limbs ("black olm"). We used a combination of mitochondrial DNA and genome-wide single nucleotide polymorphism data to investigate the molecular diversity, evolutionary history and biogeography of olms along the Dinaric Karst. We found nine deeply divergent species-level lineages that separated between 17 and 4 million years ago, while molecular diversity within lineages was low. We detected no signal of recent admixture between lineages and only limited historical gene flow. Biogeographically, the contemporaneous distribution of lineages mostly mirrors hydrologically separated subterranean environments, while the historical separation of olm lineages follows microtectonic and climatic changes in the area. The reconstructed phylogeny suggests at least four independent transitions to the cave phenotype. Two of the species-level lineages have miniscule ranges and may represent Europe's rarest amphibians. Their rarity and the decline in other lineages call for protection of their subterranean habitats.

DOP48 Key Anti-Inflammatory Faecalibacterium prausnitzii is Heterogeneous and Diverse in the East and West and in Health and Crohn’s disease. The ENIGMA study.

Abstract Background There is insufficient knowledge about the key characteristics of the pathogenic and protective bacteria in inflammatory bowel disease. The ENIGMA study aimed to identify the phylogenetic and functional characteristics of Faecalibacterium prausnitzii, considered a protective “anti-inflammatory” bacterium, in both health and Crohn’s disease in populations in the West (high Crohn’s incidence) and Hong Kong (rapidly increasing Crohn’s incidence). Methods Total DNA was extracted from tissue (right colon and terminal ileum) and stool collected from case-control cohorts in Hong Kong (n=51; 30 Crohn’s, 21 controls) and Australia (n=49; 29 Crohn’s, 20 controls). In parallel, the colonic mucosa-associated microbiota (MAM) for each subject was cultured from anaerobically preserved tissue. Shotgun metagenomic sequencing was used to characterise the stool and colonic MAM, augmented with 16S rRNA gene profiling and F. prausnitzii qPCR. The HUMANn3 and/or QIIME2 packages were used for the taxonomic and/or functional characterisation of the datasets. Metagenome-assembled genomes (MAG) were also produced from colonic MAM datasets using the MetaWRAP pipeline, and taxonomy deduced using the Genome Taxonomy Database-Tool kit (GTDB-tk). Results F. prausnitzii was detected in all case-control cohorts from Hong Kong and Australia. However, the prevalence was greater for the colonic MAM cultures of the Australian subjects, which provided 24/32 high-quality Faecalibacterium-affiliated MAG. No less than 6 different species-level lineages were predicted by GTDB-tk, and a similar phylogeny predicted using the gene encoding immunomodulatory peptides (mam). Functional features for the MAG from Crohn’s disease and control subjects were compared, and genes involved with histidine, riboflavin, purine and pyrimidine metabolism, pentose phosphate pathway, and oxidoreductases were enriched in the control subject MAG. In contrast, MAG derived from Crohn’s disease subjects possessed more functions putatively assigned to antibiotic resistance mechanisms and other protective functions. Differences were also observed in the carbohydrate-active enzyme profiles of the MAG recovered from Crohn’s disease and control subjects. Conclusion The populations of F. prausnitzii recovered from the colonic mucosa associated microbiota are both diverse and vary between Crohn’s disease and control subjects in both countries. The differences include metabolic and physiological characteristics. Therapeutic modification of F. prausnitzii needs to consider phylogenetic and functional characteristics. The ENIGMA study is supported by a grant from the Leona M. and Harry B. Helmsley Charitable Trust

Phylogenetic relationships and further unknown diversity of diplostomids (Diplostomida: Diplostomidae) parasitic in kingfishers.

Kingfishers (Alcedinidae Rafinesque) are common inhabitants of wetlands and are known to be definitive hosts to a wide range of digeneans that parasitize fish as second intermediate hosts. Among these digeneans, members of the Diplostomidae Poirier, 1886 (diplostomids) are particularly common. Recent studies of diplostomids collected from kingfishers have revealed that they are probably more diverse than currently known. This particularly concerns the genera Crassiphiala Van Haitsma, 1925 and Uvulifer Yamaguti, 1934. In the present work, we studied seven diplostomid taxa from kingfishers in Brazil, the USA and the Philippines. Partial DNA sequences of the nuclear large ribosomal subunit (28S) and mitochondrial cytochrome c oxidase I (cox1) genes were obtained, and 28S sequences were used to study the phylogenetic interrelationships of these diplostomids. We provide the first DNA sequences from Uvulifer semicircumcisus Dubois et Rausch, 1950 and a member of Subuvulifer Dubois, 1952. Pseudocrassiphiala n. gen. is erected for a previously recognized species-level lineage of Crassiphiala and a new generic diagnosis of Crassiphiala is provided. Crassiphiala jeffreybelli n. sp., Crassiphiala wecksteini n. sp. and Pseudocrassiphiala tulipifera n. sp. are described, and a description of newly collected, high-quality specimens of Crassiphiala bulboglossa Van Haitsma, 1925 (the type-species of the genus) is provided.

Phylogenomic and comparative genomic studies robustly demarcate two distinct clades of Pseudomonas aeruginosa strains: proposal to transfer the strains from an outlier clade to a novel species Pseudomonas paraeruginosa sp. nov.

The strains of Pseudomonas aeruginosa exhibit considerable differences in their genotypic and pathogenic properties. To clarify their evolutionary/taxonomic relationships, comprehensive phylogenomic and comparative genomic studies were conducted on the genome sequences of 212 P . aeruginosa strains covering their genetic diversity. In a phylogenomic tree based on 118 conserved proteins, the analysed strains formed two distinct clades. One of these clades, Clade-1, encompassing >70 % of the strains including the type strain DSM 50071T, represents the species P. aeruginosa sensu stricto. Clade-2, referred to in earlier work as the outlier group, with NCTC 13628T as its type strain, constitutes a novel species level lineage. The average nucleotide identity, average amino acid identity and digital DNA–DNA hybridization values between the strains from Clade-1 and Clade-2 are in the range of 93.4–93.7, 95.1–95.3 and 52–53 %, respectively. The 16S rRNA gene of P. aeruginosa DSM 50071T also shows 98.3 % similarity to that of NCTC 13628T. These values are lower than the suggested cut-off values for species distinction, indicating that the Clade-2 strains (NCTC 13628T) constitute a new species. We also report the identification of 12 conserved signature indels in different proteins and 24 conserved signature proteins that are exclusively found in either Clade-1 or Clade-2, providing a reliable means for distinguishing these clades. Additionally, in contrast to swimming motility, twitching motility is only present in Clade-1 strains. Based on earlier work, the strains from these two clades also differ in their pathogenic mechanisms (presence/absence of Type III secretion system), production of biosurfactants, phenazines and siderophores, and several other genomic characteristics. Based on the evidence from different studies, we propose that the Clade-2 strains constitute a novel species for which the name Pseudomonas paraeruginosa is proposed. The type strain is NCTC 13628T (=PA7T=ATCC 9027T). The description of Pseudomonas aeruginosa is also emended to include information for different molecular markers specific for this species.