Low Statistical Support Research Articles

Ultraconserved elements-based phylogenomic systematics of the snake superfamily Elapoidea, with the description of a new Afro-Asian family

The highly diverse snake superfamily Elapoidea is considered to be a classic example of ancient, rapid radiation. Such radiations are challenging to fully resolve phylogenetically, with the highly diverse Elapoidea a case in point. Previous attempts at inferring a phylogeny of elapoids produced highly incongruent estimates of their evolutionary relationships, often with very low statistical support. We sought to resolve this situation by sequencing over 4,500 ultraconserved element loci from multiple representatives of every elapoid family/subfamily level taxon and inferring their phylogenetic relationships with multiple methods. Concatenation and multispecies coalescent based species trees yielded largely congruent and well-supported topologies. Hypotheses of a hard polytomy were not retained for any deep branches. Our phylogenies recovered Cyclocoridae and Elapidae as diverging early within Elapoidea. The Afro-Malagasy radiation of elapoid snakes, classified as multiple subfamilies of an inclusive Lamprophiidae by some earlier authors, was found to be monophyletic in all analyses. The genus Micrelaps was consistently recovered as sister to Lamprophiidae. We establish a new family, Micrelapidae fam. nov., for Micrelaps and assign Brachyophis to this family based on cranial osteological synapomorphy. We estimate that Elapoidea originated in the early Eocene and rapidly diversified into all the major lineages during this epoch. Ecological opportunities presented by the post-Cretaceous-Paleogene mass extinction event may have promoted the explosive radiation of elapoid snakes.

Comparative plastomes and phylogenetic analysis of seven Korean endemic Saussurea (Asteraceae)

BackgroundSaussurea is one of the most species-rich genera in the Cardueae, Asteraceae. There are approximately 40 Saussurea species distributed in Korea, with nearly 40% of them endemics. Infrageneric relationships remain uncertain due to insufficient resolutions and low statistical support. In this study, we sequenced the plastid genomes of five Korean endemic Saussurea (S. albifolia, S. calcicola, S. diamantica, S. grandicapitula, and S. seoulensis), and comparative analyses including two other endemics (S. chabyoungsanica and S. polylepis) were conducted.ResultsThe plastomes of Korean endemics were highly conserved in gene content, order, and numbers. Exceptionally, S. diamantica had mitochondrial DNA sequences including two tRNAs in SSC region. There were no significant differences of the type and numbers of SSRs among the seven Korean endemics except in S. seoulensis. Nine mutation hotspots with high nucleotide diversity value (Pi > 0.0033) were identified, and phylogenetic analysis suggested that those Korean endemic species most likely evolved several times from diverse lineages within the genus. Moreover, molecular dating estimated that the Korean endemic species diverged since the late Miocene.ConclusionsThis study provides insight into understanding the plastome evolution and evolutionary relationships of highly complex species of Saussurea in Korean peninsula.

Hidden diversity in Selenastraceae (Sphaeropleales, Chlorophyceae): Molecular phylogeny revealed a nannoplanktonic species, with the description of Viridiparva madeirensis gen. et sp. nov.

The simple morphology of the members of the family Selenastraceae, common in freshwater phytoplankton, has led to misidentifications. But the use of molecular tools is untangling their phylogeny and unveiling new genera and species hidden under the same morphotype or overlooked by microscopic analysis. Here we propose a description of Viridiparva madeirensis gen. et sp. nov., a small Selenastraceae member, based on the study of three strains by light, scanning and transmission microscopy and 18S rDNA and rbcL phylogenies. The phylogenetic analyses by 18S rDNA and rbcL show, respectively, that the closest relatives to Viridiparva madeirensis are Monoraphidium dybowskii and Raphidocelis microscopica, but with no or low statistical support. V. madeirensis is smaller than M. dybowski and has no cell wall incrustations typical for Raphidocelis. Thus, phylogeny and morphological analyses indicate a novel lineage within Selenastraceae. Furthermore, also reinforce the necessary revision of the genera within Selenastraceae, as Monoraphidium and Raphidocelis. Therefore study contributes to clarify the phylogeny and taxonomy of the family and add genetic sequences of neglected nanophytoplankton to GenBank, including one strain with known potential for biodiesel production.

First molecularly substantiated records of the pond snail Ampullaceana fontinalis in the Siverskyi Donets River Basin, Ukraine (Gastropoda: Lymnaeidae)

Ampullaceana fontinalis (Studer, 1820) has not been considered as a separate species for a long time. Here we present new records of A. fontinalis for tributaries of the Siverskyi Donets River in Donetsk and Luhansk regions of Ukraine based on sequences of the cytochrome c oxidase gene and morphological characters. The newly generated sequences joined the clade of A. fontinalis and entered different subclades within it. The genetic diversity observed in the study area suggests separate evolutionary histories of geographically proximal populations of A. fontinalis. Our phylogenetic analysis confirmed species-level monophyletic groups of Ampullaceana ampla (Hartmann, 1821), Ampullaceana dipkunensis (Gundrizer & Starobogatov 1979), Ampullaceana balthica (Linnaeus, 1758), Ampullaceana lagotis (Schrank, 1803) and Ampullaceana fontinalis (Studer, 1820). The low statistical support of the latter may be attributed to the single-gene phylogeny and may be enhanced by analyzing more loci and samples.

Phylogenomic data resolve the historical biogeography and ecomorphs of Neotropical forest lizards (Squamata, Diploglossidae)

Few studies have been conducted on the biogeography and phylogenetic relationships of Neotropical forest lizards (Diploglossidae) because of incomplete taxon sampling, conflicting datasets, and low statistical support at phylogenetic nodes. Here, we enhance a recent nine-gene dataset with a genomic dataset of 3,232 loci and 642,775 aligned base pairs. The resulting phylogeny includes 30 diploglossid species, 10 of the 11 genera, and the three subfamilies. It shows significant support for all supra-specific taxa in either maximum likelihood or Bayesian analyses or both. With this well-supported phylogeny, we further investigate the historical biogeography of the group and how diploglossids reached the Caribbean islands. Our analyses indicate that Antillean diploglossid lizards originated from at least two overwater dispersals from South America. Our tests for the strength of convergent evolution between morphologically similar taxa support the recognition of a soil and a tree ecomorph. In addition, we propose grass, ground, rock, and swamp ecomorphs for species in this family based on ecological and morphological data and analyses.

New phylogenetic insights into the African catfish families Mochokidae and Austroglanididae.

Several hundred catfish species (order: Siluriformes) belonging to 11 families inhabit Africa, of which at least six families are endemic to the continent. Although four of those families are well‐known to belong to the ‘Big‐Africa clade’, no previous study has addressed the phylogenetic placement of the endemic African catfish family Austroglanididae in a comprehensive framework with molecular data. Furthermore, interrelationships within the ‘Big‐Africa clade’, including the most diverse family Mochokidae, remain unclear. This study was therefore designed to help reconstruct inter‐ and intrarelationships of all currently valid mochokid genera, to infer their position within the ‘Big Africa clade’ and to establish a first molecular phylogenetic hypothesis of the relationships of the enigmatic Austroglanididae within the Siluriformes. We assembled a comprehensive mitogenomic dataset comprising all protein coding genes and representing almost all recognized catfish families (N = 33 of 39) with carefully selected species (N = 239). We recovered the monophyly of the previously identified multifamily clades ‘Big Asia’ and ‘Big Africa’ and determined Austroglanididae to be closely related to Pangasiidae, Ictaluroidea and Ariidae. Mochokidae was recovered as the sister group to a clade encompassing Auchenoglanididae, Claroteidae, Malapteruridae and the African Schilbeidae, albeit with low statistical support. The two mochokid subfamilies Mochokinae and Chiloglanidinae as well as the chiloglanid tribe Atopochilini were recovered as reciprocally monophyletic. The genus Acanthocleithron forms the sister group of all remaining Mochokinae, although with low support. The genus Atopodontus is the sister group of all remaining Atopochilini. In contrast to morphological reconstructions, the monophyly of the genus Chiloglanis was strongly supported in our analysis, with Chiloglanis macropterus nested within a Chiloglanis sublineage encompassing only other taxa from the Congo drainage. This is an important result because the phylogenetic relationships of C. macropterus have been controversial in the past, and because we and other researchers assumed that this species would be resolved as sister to most or all other members of Chiloglanis. The apparent paraphyly of Synodontis with respect to Microsynodontis provided an additional surprise, with Synodontis punu turning out to be the sister group of the latter genus.

Discordance in a South African Memecylon Clade (Melastomataceae): Evidence for Reticulate Evolution

Premise of research. Evergreen forests in eastern South Africa have high biodiversity but are limited in extent and have a highly fragmented distribution. Populations of forest plants are thus geographically isolated, and fine-scale evolutionary studies of these lineages might yield important insights into the history and assembly of the forests themselves. A prior study showed that, despite their morphological diversity, three South African Memecylon taxa in Melastomataceae (Memecylon natalense, M. bachmannii, and M. australissimum) had almost identical nuclear ribosomal spacer sequences. Our study investigates phylogenetic relationships within this clade using multiple samples collected across populations and a next-generation phylogenomic approach.Methodology. We used 87 low-copy nuclear (LCN) loci to examine relationships among these taxa using both concatenated and coalescent methods. We further used LCN loci to estimate phylogenetic networks and single-nucleotide polymorphisms (SNPs) derived from LCN genes for STRUCTURE analysis of South African Memecylon populations. Finally, we employed two approaches (flow cytometry and SNPs) to infer the ploidy levels of these three taxa.Pivotal results. Our investigations showed discordance among gene trees and the species tree and low statistical support for relationships, indicating that species monophyly cannot be recovered from this phylogenomic analysis. Phylogenetic networks and population structures showed that the South African Memecylon clade may be affected by gene flow and reticulate evolution. Flow cytometry and SNP-based estimations provided evidence for polyploidy within this group.Conclusions. We found no evidence of monophyly for species within the South African Memecylon clade, which we infer to be the consequence of reticulation and recent and rapid evolution. More cytological studies and genomic data are needed to elucidate the evolutionary history of this group. Additionally, our study identifies priority populations for conservation within the South African Memecylon clade.

Efficient algorithms for robustness analysis of maximum a posteriori inference in selective sum-product networks

Sum-Product Networks (SPN) are deep probabilistic models with demonstrated excellent performance in several machine learning tasks. As with many other probabilistic models, performing Maximum-A-Posteriori inference in SPNs is NP-hard. Selective SPNs are a subclass of SPNs that allow for efficient Maximum-A-Posteriori inference and closed-form parameter learning. Due to the high number of parameters, SPNs learned from data can produce unreliable and overconfident inferences, especially for instances with low statistical support. This issue can be partially mitigated by performing a robustness analysis of inferences with respect to small changes in the parameters. In this work, we address the problem of assessing the robustness of Maximum-A-Posteriori inferences produced with Selective SPNs to global perturbations of the parameters. We consider such an inference robust if it remains the single maximizer under small perturbations of the model parameters. We present efficient algorithms and an empirical analysis with realistic problems involving missing data completion and multilabel classification. The experiments show that our criteria are informative with respect to the inference accuracy, suggesting that it indeed discriminate robust and non-robust instances.

A ddRAD-based population genetics and phylogenetics of an endangered freshwater fish from Japan

Rhodeus ocellatus kurumeus is a small cyprinid fish endemic to the Japanese archipelago. This fish frequently hybridizes with an exotic subspecies Rhodeus ocellatus ocellatus, and non-introgressed populations of R. o. kurumeus have greatly decreased. Previous studies based on mitochondrial and nuclear DNA identified introgressed populations and inferred phylogenetic relationships, but these approaches may lead to underestimates of introgression or ambiguous results owing to the small number of genetic markers used. The present study applied double digest restriction site-associated DNA (ddRAD) sequencing to assignment tests and phylogenetic inferences. Data matrices can vary in size and content depending on the strategies used to process ddRAD sequences; therefore, we prepared 25 data matrices with various processing strategies. The assignment tests based on the 25 data matrices resulted in similar assignment patterns and provided evidence for introgression in some populations that had been identified as non-introgressed populations in previous studies. The maximum-likelihood (ML) phylogenetic trees differed in robustness and topology among the 25 matrices; interestingly, ML trees with low statistical support reflected geographical distributions of fish better than ML trees with high statistical support. These results indicate that ddRAD sequencing can detect introgression with greater sensitivity than conventional DNA markers, that ddRAD sequencing is useful for phylogenetic inference among closely related populations within a subspecies, and that statistically robust ML trees do not necessarily reflect the true phylogeny. Application of ddRAD data to conservation genetics and evolutionary history of this subspecies are also discussed.

Capanemia (Oncidiinae): an orchid genus revised and simplified

A taxonomic revision of Capanemia (Orchidaceae) was completed after morphological, nomenclatural and phylogenetic studies. Our analyses do not support the infrageneric classification (Capanemia sect. Capanemia and Capanemia sect. Planifolia) proposed by Pabst in the seventies and recovered Sanderella as the sister group of Capanemia, albeit with low statistical support. We herein recognized six species of Capanemia, proposed five new synonyms, designated one lectotype and excluded one name.

Internet use and labor productivity growth: recent evidence from the U.S. and other OECD countries

This study provides evidence on the nature and extent of the effect of Internet usage or penetration on labor productivity growth, while focusing on the recent experience in OECD countries. The basic model modifies the traditional labor-augmented production function approach by integrating Internet usage as a factor that improves labor quality. Variables on output, capital, and labor are extracted from official OECD publications, and data on the penetration ratio, a proxy for Internet usage, is obtained from an online source. The result is a panel that covers 28 OECD countries, including the U.S., over the time period from 2001 through 2016. Parameter estimates for Internet usage across the models are found to be positive, albeit with low statistical support. Based on a unique historical survey data set for the U.S. that separates Internet use at home from the use at work, a descriptive analysis for the U.S. suggests that growth of Internet use at work lowers productivity growth.

Assessing Efficiency of the Mitochondrial 12S Marker Fragment for the Use in Reconstruction of the Phylogeny of Acanthobdellids

For many years, methods based on the molecular analysis of conservative genome segments have been used to solve controversial issues of phylogeny by calculating the divergence of organisms. In the present study, a marker fragment of the 12S rRNA gene was used to clarify the phylogenetic position of a poorly studied faunistic group, ancient leech-like parasites of the genus Acanthobdella. The mosaic combination of oligochaete (Oligochaeta) and modern leech (Hirudinea) traits points to the intermediate evolutionary position of acanthobdells. Our results suggest that the 12S marker fragment is efficient for phylogenetic analysis at the genus and species levels. The phylogenetic history of acanthobdellids reconstructed in the present study is not consistent with the results of earlier studies. Low statistical support suggests that the 12S marker is not efficient for phylogenetic analysis at the supergenus level.

Evolution of green lacewings (Neuroptera: Chrysopidae): a molecular supermatrix approach

AbstractWe present a time‐calibrated phylogeny of the charismatic green lacewings (Neuroptera: Chrysopidae). Previous phylogenetic studies on the family using DNA sequences have suffered from sparse taxon sampling and/or limited amounts of data. Here we combine all available previously published DNA sequence data and add to it new DNA sequences generated for this study. We analysed these data in a supermatrix using Bayesian and maximum likelihood methods and provide a phylogenetic hypothesis for the family that recovers strong support for the monophyly of all subfamilies and resolves relationships among a large proportion of chrysopine genera. Chrysopinae tribes Leucochrysini and Belonopterygini were recovered as monophyletic sister clades, while the species‐rich tribe Chrysopini was rendered paraphyletic by Ankylopterygini. Relationships among the subfamilies were resolved, although with relatively low statistical support, and the topology varied based on the method of analysis. Greatest support was found for Apochrysinae as sister to Nothochrysinae and Chrysopinae, which is in contrast to traditional concepts that place Nothochrysinae as sister to the rest of the family. Divergence estimates suggest that the stem groups to the various subfamilies diverged during the Triassic‐Jurassic, and that stem groups of the chrysopine tribes diverged during the Cretaceous.

Morphological and molecular characterization of Bysmatrum subsalsum (Dinophyceae) from the western Mediterranean Sea reveals the existence of cryptic species.

Bysmatrum subsalsum is a cosmopolitan dinoflagellate species that inhabits marine and transitional habitats. Despite its wide distribution, information on the morphological variability, phylogeny and ecology of B.subsalsum is scarce. In this study, we provide morphological and molecular data on B.subsalsum strains and wild cells from different locations in the Mediterranean Basin. The dynamics of cell abundances and the associated environmental conditions during a field bloom are also described. Genetic sequences of B.subsalsum obtained in this study showed large intraspecific differences, clustering in two well-differentiated clades. Despite a certain degree of variation with respect to cell size, apical pore complex (APC) morphology and size, and cingulum displacement, cells from the two clades showed similar morphological traits. These findings indicated the occurrence of cryptic species. Comparisons of themorphology of our B.subsalsum specimens with the few descriptions available in the literature revealed larger than previously known intraspecific morphological variability. Phylogenetic trees inferred from the concatenated SSU, 5.8S-ITS, and LSU rRNA and the individual 5.8S-ITS regions suggested the inclusion of Bysmatrum in the Peridiniales and a close phylogenetic relationship with Peridinium sensu stricto. However, the low statistical support prevented the assignment of Bysmatrum to a particular family of Peridiniales. Ecological data obtained from a bloom in La Pletera salt marshes (Catalan Coast, Spain) suggested the species reaches high cell abundances at water temperatures >20°C and salinity levels >30. Our results add new information regarding the morphology, phylogeny, and ecology of B.subsalsum.

Phylogenetic and Taxonomic Status Analyses of the Abaso Section from Multiple Nuclear Genes and Plastid Fragments Reveal New Insights into the North America Origin of Populus (Salicaceae)

Although, the Abaso section is widely accepted as an independent section, the taxonomic status of Populus mexicana (section Abaso) has not yet been resolved due to the limited availability markers and/or the lack of P. mexicana specimens in previous studies. Thirty-one poplar species that represent six sections of the Populus genus were sampled, and 23 single-copy nuclear DNA and 34 chloroplast fragments were sequenced. The present study obtained two updated phylogenies of Populus. We found that monophyly of the genus Populus is strongly supported by nuclear and plastid gene, which is consistent with previous studies. P. mexicana, diverged first in the nuclear DNA tree, which occupied the basal position, implying that the section Abaso may be the most ancestral lineage in extant populous species. Given that the short branches and low statistical support for the divergence of sections Abaso and Turanga, this observation probably indicated that a rapid radiation evolution following the early split of the genus Populus. In the plastid tree, P. mexicana clustered with modern-day species of section Tacamahaca in the plastid tree. Based on cytoplasmic and single-copy nuclear marker sequences, we hypothesized that chloroplast capture resulted in the inconsistent position of P. mexicana between the phylogenetic trees. Given the first unequivocal records of poplar fossils from the Eocene with similar leaf morphology to the extant P. mexicana and the phylogenetic positions of P. mexicana in our study, we support the hypothesis that the Populus genus originated in North America, which will provide new insights to the development of the origin of Populus species.

Morphological Phylogenetic Analysis of Two Early-Diverging Genera of Bromelioideae (Bromeliaceae)

Abstract Bromelia includes 66 species, grouped in three subgenera: Bromelia, Distiacanthus and Karatas. The genus is one of the earlier divergent groups within Bromelioideae, a subfamily with innumerable problems of generic delimitation. Considering that few phylogenetic studies have included more than one species of Bromelia, the objectives of this study were to carry out a phylogenetic analysis for the genus, seeking to evaluate its monophyly as well as its subgenera, and to better understand the interspecific evolutionary relationships within the genus and the intergeneric relationships within Bromelioideae. Also included in the analysis were the two species of Fernseea, a genus originally described from a species placed in Bromelia. The phylogenetic analysis was conducted with combined micro- and macromorphological data, including 39 taxa with 116 characters. As in other studies on Bromeliaceae, the clades have low statistical support and many questions regarding the relationships of the groups remain. The circumscription of Bromelia is still uncertain, due to the position of B. irwinii; Fernseea seems to emerge within this genus, as the marority-rule consensus suggests, and the infrageneric categories are debatable because they do not constitute monophyletic groups. However, Bromelia seems to be paraphyletic and, without B. irwinii, the genus can be considered monophyletic. Beginning with the premise that the most recent common ancestor of Bromelioideae appeared first in the southern Andes, Bromelia and Fernseea seems to have dispersed along three biogeographical routes. The present work is an important contribution to understand the evolution and biogeography of the Bromelioideae.

Congruence between comparative morphology and molecular phylogenies: Evolution of the male genital skeletal/muscular system in the subtribe Polyommatina (Lepidoptera, Lycaenidae)

The skeleton and musculature of male genitalia were studied in species of a model butterfly group (subtribe Polyommatina, Lycaenidae). In total, we analyzed 45 species of the tribe Polyommatini most of which were previously used in the molecular phylogenetic study (Talavera et al., 2013). The studied morphological characters were mapped on the molecular trees, which allowed us to reveal trends of morphological changes and to estimate the age of their origin. As a result, chronology of evolution of skeleton and musculature traits was established. It was shown that periods of slow morphological evolution alternated in the subtribe Polyommatina with those of a high rate of origin of new traits. For example, topography of the intravalvar muscles has not changed for 26 MY preserving their initial fan-shaped attachment. The evolution of intravalvar muscles started 10 MYA, proceeded slowly during the first 5 MY, and then accelerated during the last 5 MY resulting in the extensive splitting of the musculature in most monophyletic lineages. Mapping the morphological characters on the phylogeny demonstrated that the rates of skeleton and muscle evolution within the skeleton/musculature apparatus were different. In most cases the intravalvar musculature evolved much faster than the skeleton. The cladistic interpretation of states of morphological traits was found to be consistent with phylogenetic reconstructions based on analysis of multiple molecular markers. Moreover, morphological synapomorphies were found for the lineages Alpherakya + Glabroculus and Aricia + (Alpherakya + Glabroculus), which had low statistical support in molecular phylogenetic analysis. Additionally, in some cases molecular studies helped to reveal trends in the evolution of morphological traits. For example, the unpaired uncus and the compact juxta are not plesiomorphic for Cupidina as previously thought; instead, they were shown to have evolved secondarily within this subtribe.

New host records for European Acroceridae (Diptera), with discussion of species limits of Acrocera orbiculus (Fabricius) based on DNA-barcoding

New European host records for the Acroceridae species Acrocera orbiculus (Fabricius) and Ogcodes reginae Trojan are reported. Acrocera orbiculus was reared from Amaurobius erberi (Keyserling), and O. reginae from Clubiona leucaspis (Simon) and Evarcha jucunda (Lucas). Where possible, DNA-barcodes are presented for reared endoparasitoids and their host specimens. Based on mitochondrial COI, the intraspecific genetic variability of 15 western Palaearctic A. orbiculus is discussed. Maximum likelihood analysis reveals two clades, though they have low statistical support and no distinct barcoding gap. Therefore, we consider all barcoded specimens of A. orbiculus to be a single biological species with a high degree of phenotypic plasticity regarding body size and coloration. Based on molecular and morphological evidence, Paracrocera kaszabi Majer, Paracrocera manevali Séguy and Paracrocera minuscula Séguy are placed in synonymy with A. orbiculus. The male of the Canary Islands endemic Acrocera cabrerae Frey is described for the first time.

Does comorbidity increase the risk of mortality among children under 3 years of age?

ObjectivesDiarrhoea and pneumonia remain leading causes of morbidity and mortality in children under 5 years of age. Little data is available to quantify the burden of comorbidity and the relationship...

Reconstruction of family-level phylogenetic relationships within Demospongiae (Porifera) using nuclear encoded housekeeping genes.

BackgroundDemosponges are challenging for phylogenetic systematics because of their plastic and relatively simple morphologies and many deep divergences between major clades. To improve understanding of the phylogenetic relationships within Demospongiae, we sequenced and analyzed seven nuclear housekeeping genes involved in a variety of cellular functions from a diverse group of sponges.Methodology/Principal FindingsWe generated data from each of the four sponge classes (i.e., Calcarea, Demospongiae, Hexactinellida, and Homoscleromorpha), but focused on family-level relationships within demosponges. With data for 21 newly sampled families, our Maximum Likelihood and Bayesian-based approaches recovered previously phylogenetically defined taxa: Keratosap, Myxospongiaep, Spongillidap, Haploscleromorphap (the marine haplosclerids) and Democlaviap. We found conflicting results concerning the relationships of Keratosap and Myxospongiaep to the remaining demosponges, but our results strongly supported a clade of Haploscleromorphap+Spongillidap+Democlaviap. In contrast to hypotheses based on mitochondrial genome and ribosomal data, nuclear housekeeping gene data suggested that freshwater sponges (Spongillidap) are sister to Haploscleromorphap rather than part of Democlaviap. Within Keratosap, we found equivocal results as to the monophyly of Dictyoceratida. Within Myxospongiaep, Chondrosida and Verongida were monophyletic. A well-supported clade within Democlaviap, Tetractinellidap, composed of all sampled members of Astrophorina and Spirophorina (including the only lithistid in our analysis), was consistently revealed as the sister group to all other members of Democlaviap. Within Tetractinellidap, we did not recover monophyletic Astrophorina or Spirophorina. Our results also reaffirmed the monophyly of order Poecilosclerida (excluding Desmacellidae and Raspailiidae), and polyphyly of Hadromerida and Halichondrida.Conclusions/SignificanceThese results, using an independent nuclear gene set, confirmed many hypotheses based on ribosomal and/or mitochondrial genes, and they also identified clades with low statistical support or clades that conflicted with traditional morphological classification. Our results will serve as a basis for future exploration of these outstanding questions using more taxon- and gene-rich datasets.