Nuclear rDNA Research Articles

Conflicting phylogenetic signals in genomic data of the coffee family (Rubiaceae)

AbstractReconstructions of phylogenetic relationships in the flowering plant family Rubiaceae have up until now relied heavily on single‐ or multi‐gene data, primarily from the plastid compartment. With the availability of cost‐ and time‐efficient techniques for generating complete genome sequences, the opportunity arises to resolve some of the relationships that, up until now, have proven problematic. Here, we contribute new data from complete 58 plastid genome sequences, representing 55 of the currently 65 recognized tribes of the Rubiaceae. Also contributed are new data from the nuclear rDNA cistrons for corresponding taxa. Phylogenetic analyses are conducted on two plastid data sets, one including data from the protein coding genes only, and a second where protein coding data are combined with non‐coding regions, and on a nuclear rDNA data set. Our results clearly show that simply adopting a “more characters” approach does not resolve the relationships in the Rubiaceae. More importantly, we identify conflicting phylogenetic signals in the data. Analyses of the same plastid data, treated as nucleotides or as codon‐degenerated data, resolve and support conflicting topologies in the subfamily Cinchonoideae. As these analyses use the same data, we interpret the conflict to result from erroneous assumptions in the models used to reconstruct our phylogenies. Conflicting signals are also identified in the analyses of the plastid versus the nuclear rDNA data sets. These analyses use data from different genomic compartments, with different inheritance patterns, and we interpret the conflicts as representing “real” conflicts, reflecting biological processes of the past.

Recognition of the Trachypetidae stat.n. as a new extant family of Ichneumonoidea (Hymenoptera), based on molecular and morphological evidence

AbstractThe Trachypetinae (type genus Trachypetus Guérin de Méneville) comprise seven species of large‐bodied wasps in three genera (Cercobarcon Tobias, Megalohelcon Turner and Trachypetus) endemic to continental Australia. Historically they have been variously treated, as members of the Helconinae in the case of Megalohelcon, or as separate subfamilies (Cercobarconinae and Trachypetinae). Some 25 years ago they were united in a single subfamily, the Trachypetinae, based on a number of characters. Although there has been conflicting evidence from morphological and molecular phylogenetic studies as to how best to treat the group, there has been a growing consensus that they fall outside the rest of the Braconidae, although taxon sampling has been a limiting factor for molecular studies. We generated a molecular dataset comprising five gene fragments (nuclear 28S ribosomal rDNA, nuclear 18S, elongation factor 1‐alpha, mitochondrial 16S rDNA, and mitochondrial cytochrome oxidase subunit 1) for a taxonomically broad range of Braconidae, Ichneumonidae, trachypetines and outgroup hymenopterans including the first molecular data for the trachypetines Cercobarcon and Trachypetus obtained using specially designed internal primers. Molecular and combined molecular and morphological analyses confirm the monophyly of the Trachypetinae and robustly place them as sister to the Braconidae. Detailed morphological analysis including newly recognized characters shows that trachypetines lack several synapomorphies that define the Braconidae, and that they possess a number of symplesiomorphies absent from this family but found in some ichneumonids. We conclude that family‐level status is warranted for the group based on both molecular and morphological criteria, and hence we propose the new family, Trachypetidae Schulz stat.n. (type genus Trachypetus Guérin de Méneville), for it. As a result, the remaining extant Braconidae become clearly defined based on synapomorphies not present in Trachypetidae stat.n.This published work has been registered on ZooBank, http://zoobank.org/urn:lsid:urn:lsid:zoobank.org:pub:5418F709‐D724‐4F14‐89D8‐1E054D1D27D0.

Molecular phylogenetics of deep-sea amphipods (Eurythenes) reveal a new undescribed species at the Porcupine Abyssal Plain, North East Atlantic Ocean

The genus Eurythenes S. I. Smith in Scudder, 1882, has been the focus of integrated molecular and morphological taxonomy studies in recent years, resulting in the number of species in the genus increasing from three to eight. Samples of Eurythenes spp. collected using free-fall baited traps from the Porcupine Abyssal Plain (PAP), North East Atlantic Ocean, at 4850 m were examined in this study to investigate the identity of the species found using molecular barcoding methods. Mitochondrial COI analysis confirms the presence of E. maldoror at PAP and data from the nuclear 28S rDNA gene provides independent confirmation of this observation. A new, undescribed species that is clearly divergent from all other known species and molecular lineages was identified, which can be distinguished morphologically from known species. A specimen within the E. magellanicus lineage was also found at the site. We discuss the presence of these three Eurythenes species at the Porcupine Abyssal Plain.

A new Dondice Marcus Er. 1958 (Gastropoda: Nudibranchia) from the Mediterranean Sea reveals interesting insights into the phylogenetic history of a group of Facelinidae taxa.

The diversity of Mediterranean nudibranchs has yet to be thoroughly studied: new species are constantly described, and molecular approaches have revealed some cryptic species. A new facelinid species has been discovered based on specimens collected from the Tyrrhenian Sea (Mediterranean Sea). Integrative results of molecular analyses and of anatomical investigations support the description of Dondice trainitoi sp. nov. The characteristic chromatic body pattern and the black epithelium covering the masticatory jaws allow an unambiguous identification of the new taxon. Preliminary phylogenetic analyses based on multi-locus molecular markers (nuclear H3 gene and mitochondrial markers 16S rDNA and COI) surprisingly revealed paraphyly of the genus Dondice and the need of further studies including more taxa assigned to the currently accepted family Facelinidae. Furthermore, following an integrative taxonomy approach, considerations on the ecological behaviour characterizing most of the species involved in this study provide useful insights for understanding the evolutionary history of this facelinid group.

Full Mitochondrial Genome and Nuclear 18S rDNA Sequences Refine the Taxonomic Placement of Choleoeimeria taggarti n. comb. from the Prostate of Antechinus flavipes (Yellow-Footed Antechinus)

An unusual coccidian parasite was described previously from the prostate of a male Antechinus flavipes (family: Dasyuridae; common name: yellow-footed antechinus). Morphometrics and a partial nuclear 18S small subunit rDNA (18S rDNA) sequence were used to assign this parasite to the genus Eimeria; it was named Eimeria taggarti. We generated full nuclear 18S rDNA and mitochondrial genome sequences from this parasite and used the newly completed 18S rDNA and mitochondrial cytochrome c oxidase subunit I (COI) sequences to perform a more in-depth phylogenetic analysis. The parasite clustered closely with Choleoeimeria spp. and Acroeimeria spp. infecting herptiles in a well-supported clade that was the sister lineage to the Eimeriidae sensu stricto. The mitochondrial genome of this parasite contained 2 inverted segments compared to mitochondrial genomes from parasites in the Eimeriidae sensu stricto (i.e., Stieda body-possessing coccidia with 4 dizoic sporocysts); this mitochondrial genome arrangement was shared with the only Choleoeimeria species for which sequence data were available publicly. Examination of histological preparations and TEM images uncovered bivalvate sporocysts and otherwise confirmed previously described morphological features of the parasite. Based on our phylogenetic analyses and histological observations, we propose the generic reclassification of E. taggarti to Choleoeimeria taggarti n. comb.

Identification of Rosellinia species as producers of cyclodepsipeptide PF1022 A and resurrection of the genus Dematophora as inferred from polythetic taxonomy.

Rosellinia (Xylariaceae) is a large, cosmopolitan genus comprising over 130 species that have been defined based mainly on the morphology of their sexual morphs. The genus comprises both lignicolous and saprotrophic species that are frequently isolated as endophytes from healthy host plants, and important plant pathogens. In order to evaluate the utility of molecular phylogeny and secondary metabolite profiling to achieve a better basis for their classification, a set of strains was selected for a multi-locus phylogeny inferred from a combination of the sequences of the internal transcribed spacer region (ITS), the large subunit (LSU) of the nuclear rDNA, beta-tubulin (TUB2) and the second largest subunit of the RNA polymerase II (RPB2). Concurrently, various strains were surveyed for production of secondary metabolites. Metabolite profiling relied on methods with high performance liquid chromatography with diode array and mass spectrometric detection (HPLC-DAD/MS) as well as preparative isolation of the major components after re-fermentation followed by structure elucidation using nuclear magnetic resonance (NMR) spectroscopy and high resolution mass spectrometry (HR-MS). Two new and nine known isopimarane diterpenoids were identified during our mycochemical studies of two selected Dematophora strains and the metabolites were tested for biological activity. In addition, the nematicidal cyclodepsipeptide PF1022 A was purified and identified from a culture of Rosellinia corticium, which is the first time that this endophyte-derived drug precursor has been identified unambiguously from an ascospore-derived isolate of a Rosellinia species. While the results of this first HPLC profiling were largely inconclusive regarding the utility of secondary metabolites as genus-specific chemotaxonomic markers, the phylogeny clearly showed that species featuring a dematophora-like asexual morph were included in a well-defined clade, for which the genus Dematophora is resurrected. Dematophora now comprises all previously known important plant pathogens in the genus such as D. arcuata, D. bunodes, D. necatrix and D. pepo, while Rosellinia s. str. comprises those species that are known to have a geniculosporium-like or nodulisporium-like asexual morph, or where the asexual morph remains unknown. The extensive morphological studies of L.E. Petrini served as a basis to transfer several further species from Rosellinia to Dematophora, based on the morphology of their asexual morphs. However, most species of Rosellinia and allies still need to be recollected in fresh state, cultured, and studied for their morphology and their phylogenetic affinities before the infrageneric relationships can be clarified.

Redescription of Gyrinicola japonica, a Tadpole-Endoparasitic Nematode from Japan, with Resurrection of the Family Gyrinicolidae (Nematoda: Oxyurina).

The taxonomic account of the tadpole-parasitic nematode Gyrinicola japonica Yamaguti, 1938, which is the type species of the genus, was reassessed based on syntypes and newly-collected specimens from the type locality. Our redescription of G. japonica addresses the erroneous original description of a spicule in this nematode, and emends the diagnosis of the species. Additionally, molecular phylogenetic trees based on nuclear 18S and 28S rDNA sequences revealed that G. japonica forms a distinctive lineage within the suborder Oxyurina, and this tadpole-specialist is phylogenetically close to the lizard-parasitic nematodes that belong to the family Pharyngodonidae. The results of morphological examination with the aid of molecular phylogenetic trees highlight the systematic uniqueness of this tadpole-parasitic group within Oxyurina, and Gyrinicolidae is accordingly resurrected as a distinctive oxyurinan family, with redefinition of the family and the genus Gyrinicola.

CARDUUS CINEREUS (ASTERACEAE) – NEW TO NORTH AMERICA

Carduus cinereus M.Bieb. populations have been discovered in the Hells Canyon Wilderness of northeastern Oregon and western Idaho. These plants were initially identified as C. pycnocephalus L. They are distinguished from that species by loosely clustered, usually pedunculate, heads and by scarious-margined phyllaries. These plants are genetically distant from other North American Carduus species (<94% similarity) based on DNA sequence comparisons of the internal transcribed spacers between the 18S and 26S nuclear rDNA regions. Carduus cinereus is new to North America. A revised key to the species of Carduus in North America is presented.

The biogeography of the caribou lungworm, Varestrongylus eleguneniensis (Nematoda: Protostrongylidae) across northern North America.

Varestrongylus eleguneniensis (Nematoda; Protostrongylidae) is a recently described species of lungworm that infects caribou (Rangifer tarandus), muskoxen (Ovibos moschatus) and moose (Alces americanus) across northern North America. Herein we explore the geographic distribution of V. eleguneniensis through geographically extensive sampling and discuss the biogeography of this multi-host parasite. We analyzed fecal samples of three caribou subspecies (n = 1485), two muskox subspecies (n = 159), and two moose subspecies (n = 264) from across northern North America. Protostrongylid dorsal-spined larvae (DSL) were found in 23.8%, 73.6%, and 4.2% of these ungulates, respectively. A portion of recovered DSL were identified by genetic analyses of the ITS-2 region of the nuclear rDNA or the cytochrome oxidase c subunit I (COI) region of the mtDNA. We found V. eleguneniensis widely distributed among caribou and muskox populations across most of their geographic prange in North America but it was rare in moose. Parelaphostrongylus andersoni was present in caribou and moose and we provide new geographic records for this species. This study provides a substantial expansion of the knowledge defining the current distribution and biogeography of protostrongylid nematodes in northern ungulates. Insights about the host and geographic range of V. eleguneniensis can serve as a geographically extensive baseline for monitoring current distribution and in anticipating future biogeographic scenarios under a regime of accelerating climate and anthropogenic perturbation.

Two new species and a new record of Crepidotus (Agaricomycetes) from India

During ongoing studies of the mycoflora of Kerala State, India, three species of Crepidotus, namely, C. exilis A.M.Kumar &amp;amp; C.K.Pradeep sp. nov., C. globisporus A.M.Kumar &amp;amp; C.K.Pradeep sp. nov. and C. croceotinctus Peck, were discovered. These species are described and illustrated. A phylogenetic reconstruction based on maximum-likelihood analyses of nuclear ribosomal large subunit rDNA sequences are provided.

The more the merrier: unparalleled sympatric species richness in a sea spider genus (Pycnogonida : Callipallenidae : Pallenella) from Tasmanian waters

Southern Australian waters feature remarkably diverse assemblages of the sea spider family Callipallenidae Hilton, 1942. The most speciose of the three Australian-endemic genera currently recognised has been known as Meridionale Staples, 2014, but is here reinstated under the name Pallenella Schimkewitsch, 1909 based on its type species Pallenella laevis (Hoek, 1881). This genus includes several brightly coloured forms that occur in high abundance on arborescent bryozoans. However, considerable similarity of congeners and scarcity of diagnostic characters continue to render species delineation in this genus challenging. Using an integrative taxonomic approach, we combine detailed morphological investigation with analysis of two genetic markers (mitochondrial cytochrome c oxidase subunit I, and nuclear rDNA including internal transcribed spacers 1 and 2) to explore the extraordinary species richness of the genus Pallenella in south-east Tasmania. In agreement with our morphology-based segregation of different species and morphotypes, we recovered well-supported corresponding clades in the genetic analyses. Strong mito-nuclear concordance in the two markers supports the inference of sustained reproductive isolation between the sympatrically occurring forms. Based on these findings, we distinguish a total of 13 Tasmanian congeners, representing the most diverse assemblage of sympatric species in the same microhabitat reported for a single pycnogonid genus. Within this assemblage, we (1) record the type species P. laevis for the first time after almost 150 years, (2) delineate the two Tasmanian morphotypes of the provisional ‘variabilis’ complex, and (3) describe two species new to science (P. karenae, sp. nov., P. baroni, sp. nov.). Despite considerable genetic divergences between most congeners, only few and often subtle characters are found to be suitable for morphology-based delineation. Notably, colouration of living specimens is suggested to be informative in some cases. For morphology-based species identification of preserved specimens, a key relying on combinations of characters rather than single diagnostic features is proposed.

Diversity of a phosphate transporter gene among species and isolates of arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF) are a key group of beneficial obligate biotrophs, establishing a mutualistic symbiosis with the roots of most land plants. The molecular markers generally used for their characterization are mainly based on informative regions of nuclear rDNA (SSU-ITS-LSU), although protein-encoding genes have also been proposed. Within functional genes, those encoding for phosphate transporters (PT) are particularly important in AMF, given their primary ability to take up Pi from soil, and to differentially affect plant phosphate nutrition. In this work, we investigated the genetic diversity of PT1 gene sequences and sequences of the taxonomically relevant SSU-ITS-LSU region in two isolates of the species Funneliformis coronatus, three isolates of the species Funneliformis mosseae and two species of the genus Rhizoglomus, originated from geographically distant areas and cultured in vivo. Our results showed that partial PT1 sequences not only successfully differentiated AMF genera and species like ribosomal gene sequences but also highlighted intraspecific diversity among F. mosseae and F. coronatus isolates. The study of functional genes related to the uptake of key mineral nutrients for the assessment of AMF diversity represents a key step in the selection of efficient isolates to be used as inocula in sustainable agriculture.

Widening the area of a Baikal endemic Leymus secalinus (Georgi) Tzvelev. (Poaceae) northward to the Aldan River basin (Yakutia) based on the molecular genetic study results

The paper presents the results of studying the taxonomic relationships of Leymus secalinus (Georgi) Tzvelev. and L. littoralis (Griseb.) Peschkova in different geographical sites based on a comparison of ITS1-5.8S-ITS2 sequences of nuclear rDNA and cpDNA matK. Molecular genetic research of these species allowed us to expand the area of L. secalinus to the Aldan River basin (Yakutia).

Molecular and Morphological Delimitation of Species in the Group of Lepocinclis Ovum‐like taxa (Euglenida)

Although Lepocinclis ovum is recognized as a cosmopolitan and common species, and Lepocinclis globulus is the type species of the genus Lepocinclis, their correct identification is nearly impossible. The reason is that over 30 morphologically similar taxa appear in the literature, but no good diagnostic features exist to distinguish amongst them. Using environmental sampling and nuclear SSU rDNA sequencing, we delimited species within the group of Lepocinclis ovum-like taxa. Morphological and molecular features were analyzed for taxa isolated from Poland and six cultured strains from algal collections. In the case of environmental sampling, DNA was obtained from a small number of cells (20-400) isolated with a micropipette without setting up laboratory cultures (52 isolates), and phylogenetic analyses were based on the variation in nSSU rDNA. Apart from L. ovum and L. globulus, 13 other species were distinguished and four taxa (Lepocinclis conica comb. nov., L. fominii comb. nov., L. gracilicauda comb. nov., and L. pseudofominii nom. nov.) had their taxonomic ranks changed. For all verified species, diagnostic descriptions were emended and epitypes designated. The only exception was L. ovum, for which the epitype was questioned and thus, a new candidate for the epitype was suggested for future adoption.

Genotyping-by-Sequencing for Analysis of the Genetic Variation of Podosphaera xanthii, Incitant of Cucurbit Powdery Mildew.

This research was conducted to identify species causing powdery mildew on cucurbits and to determine genetic variations among isolates of the pathogen. We collected 109 isolates from six cucurbit species hosts (Cucumis melo, Cucumis sativus, Cucurbita maxima, Cucurbita moschata, Cucurbita pepo, and Lagenaria siceraria) in California, Illinois, Indiana, Michigan, New York, Texas, Washington, and Wisconsin in the United States and in Italy. By sequencing the internal transcribed spacer region of the nuclear rDNA of these 109 isolates, Podosphaera xanthii was found as the only species causing powdery mildew on cucurbits in the United States. Genotyping-by-sequencing was applied to these 109 isolates to investigate their genetic diversity, which showed a trend of isolates clustering from New York and Italy. In addition, the virulence of 36 isolates was compared and a significant difference (P < 0.0001) was found among them. Furthermore, results of the virulence tests of 28 isolates from Illinois showed significant effects of collection years, hosts, and locations on the virulence of the isolates.

Phylogenomic insights into the Fascicularia-Ochagavia group (Bromelioideae, Bromeliaceae)

AbstractOchagavia (four species) and Fascicularia (one species) form a well-supported clade of the early-diverging Bromelioideae. The two genera are morphologically similar, but they can be easily discerned on the basis of generative characters. Besides the species distributed on the Chilean mainland, the group includes O. elegans, endemic to the Robinson Crusoe Island of the Juan Fernández Islands. In previous molecular phylogenetic studies, O. elegans formed a sister clade to the remainder of Fascicularia and Ochagavia. A phylogenomic approach, including nearly complete and, in five cases, full plastomes (c. 160 kbp) and the nuclear rDNA cistron (c. 6 kbp), and scanning electron microscope (SEM) images of pollen were used to analyse relationships in the Fascicularia-Ochagavia group. Plastome and nuclear trees were largely congruent and supported previous phylogenetic analyses of O. elegans being sister to the remainder of the group. A divergent phylogenetic position was suggested for O. carnea using different organellar trees. SEM analysis of pollen supported the division of Fascicularia and Ochagavia. Evolutionary and taxonomic implications of our results are discussed.

Skim-Sequencing Reveals the Likely Origin of the Enigmatic Endangered Sunflower Helianthus schweinitzii.

Resolving the origin of endangered taxa is an essential component of conservation. This information can be used to guide efforts of bolstering genetic diversity, and also enables species recovery and future evolutionary studies. Here, we used low-coverage whole genome sequencing to clarify the origin of Helianthus schweinitzii, an endangered tetraploid sunflower that is endemic to the Piedmont Plateau in the eastern United States. We surveyed four accessions representing four populations of H. schweinitzii and 38 accessions of six purported parental species. Using de novo approaches, we assembled 87,004 bp of the chloroplast genome and 6770 bp of the nuclear 35S rDNA. Phylogenetic reconstructions based on the chloroplast genome revealed no reciprocal monophyly of taxa. In contrast, nuclear rDNA data strongly supported the currently accepted sections of the genus Helianthus. Information from combined cpDNA and rDNA provided evidence that H. schweinitzii is likely an allo-tetraploid that formed as a result of hybridization between the diploids Helianthus giganteus and Helianthus microcephalus.

&lt;p&gt;&lt;strong&gt;Two new species of &lt;em&gt;Calocybe&lt;/em&gt; (Lyophyllaceae) from northeast China&lt;/strong&gt;&lt;/p&gt;

Two new species of Calocybe (Calocybe erminea sp. nov. and C. badiofloccosa sp. nov.) are described from Liaoning province, China. Calocybe erminea is characterized mainly by its light khaki to dirty white pileus, slightly hygrophanous stipe, small basidiospores and cellular epicutis. The key characters of C. badiofloccosa are its light ocher yellow and rough pileus, villose stipe and large basidiospores. The results of phylogenetic analyses based on the internal transcribed spacer (ITS) and the nuclear large subunit rDNA (nrLSU) region indicated that they belong to Calocybe, but they are distinct from other species in the genus. They are compared morphologically with other species which are phenotypically similar and the phylogenetic relationships between them and allied taxa are discussed. These results confirm the species is new to science.

Redescription of Synactinernus flavus for the First Time after a Century and Description of Synactinernus churaumi sp. nov. (Cnidaria: Anthozoa: Actiniaria).

Two species of Synactinernus sea anemones were found in Japanese waters. Synactinernus flavus Carlgren, 1918, the only described species of this genus, is rediscovered from off the Goto Islands a century after the original description. Synactinernus flavus was once synonymized with Isactinernus quadrilobatus Carlgren, 1918; however, we show that, based on morphological (including examination of type specimens) and molecular (using nuclear 18S rDNA) evidence, these species are completely different. The other species, Synactinernus churaumi sp. nov., was found off Ishigaki Island and Okinawa Island by a remotely operated vehicle (ROV), and had been kept for 15 years in a tank at the Okinawa Churaumi Aquarium. There are clear differences between these two species; therefore, we describe the second species and revise the diagnosis of Synactinernus.

Optimal markers for the identification of Colletotrichum species

Colletotrichum is among the most important genera of fungal plant pathogens. Molecular phylogenetic studies over the last decade have resulted in a much better understanding of the evolutionary relationships and species boundaries within the genus. There are now approximately 200 species accepted, most of which are distributed among 13 species complexes. Given their prominence on agricultural crops around the world, rapid identification of a large collection of Colletotrichum isolates is routinely needed by plant pathologists, regulatory officials, and fungal biologists. However, there is no agreement on the best molecular markers to discriminate species in each species complex. Here we calculate the barcode gap distance and intra/inter-specific distance overlap to evaluate each of the most commonly applied molecular markers for their utility as a barcode for species identification. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone-3 (HIS3), DNA lyase (APN2), intergenic spacer between DNA lyase and the mating-type locus MAT1-2-1 (APN2/MAT-IGS), and intergenic spacer between GAPDH and a hypothetical protein (GAP2-IGS) have the properties of good barcodes, whereas sequences of actin (ACT), chitin synthase (CHS-1) and nuclear rDNA internal transcribed spacers (nrITS) are not able to distinguish most species. Finally, we assessed the utility of these markers for phylogenetic studies using phylogenetic informativeness profiling, the genealogical sorting index (GSI), and Bayesian concordance analyses (BCA). Although GAPDH, HIS3 and β-tubulin (TUB2) were frequently among the best markers, there was not a single set of markers that were best for all species complexes. Eliminating markers with low phylogenetic signal tends to decrease uncertainty in the topology, regardless of species complex, and leads to a larger proportion of markers that support each lineage in the Bayesian concordance analyses. Finally, we reconstruct the phylogeny of each species complex using a minimal set of phylogenetic markers with the strongest phylogenetic signal and find the majority of species are strongly supported as monophyletic.