The New Zealand genus Tetrachaetus (Diptera: Dolichopodidae): synonymisation of Tetrachaetus bipunctatus and T. simplex

Early detection and treatment of ovarian cancer: shifting from early stage to minimal volume of disease based on a new model of carcinogenesis

Hypogeococcus pungens, a mealybug native of southern South America, is devastating native cacti in Puerto Rico and threatening cactus diversity in the Caribbean, and potentially in Central and North America. The taxonomic status of H. pungens is controversial since it has been reported feeding not only on Cactaceae but also on other plant families throughout its distribution range. However, in Australia, where the species had been exported from Argentina to control weedy American cacti, it was never found on host plants other than Cactaceae. These conflicting pieces of evidence not only cast doubt on the species identity that invaded Puerto Rico, but also have a negative impact on the search for natural enemies to be used in biological control programs against this pest. Here we present reproductive incompatibility and phylogenetic evidences that give support to the hypothesis that H. pungens is a species complex in which divergence appears to be driven by the host plants. The nuclear EF1α and 18S and the mitochondrial COI genes were used as markers to evaluate the phylogenetic relationships among H. pungens populations collected in Argentina, Australia and Puerto Rico feeding on Cactaceae and/or Amaranthaceae. Additionally, we conducted reciprocal crosses between mealybugs from both hosts. Species delimitation analysis revealed two well-supported putative species within H. pungens, one including mealybugs feeding on Amaranthaceae (H. pungens sensu stricto), and a new undescribed species using Cactaceae as hosts. Additionally, we found asymmetric reproductive incompatibility between these putative species suggesting recent reproductive isolation. The Bayesian species delimitation also suggested that the Australian mealybug population may derive from another undescribed species. Overall, the patterns of genetic differentiation may be interpreted as the result of recent speciation events prompted by host plant shifts. Finally, the finding of a single haplotype in the Puerto Rico population suggests only one invasive event. We still need to identify the geographical origin of the pest in order to enable the use of biological control to reduce the threat to cacti diversity in the Caribbean.

The uncertainty about whether, in China, the genus Melia (Meliaceae) consists of one species (M. azedarach Linnaeus) or two species (M. azedarach and M. toosendan Siebold & Zuccarini) remains to be clarified. Although the two putative species are morphologically distinguishable, genetic evidence supporting their taxonomic separation is lacking. Here, we investigated the genetic diversity and population structure of 31 Melia populations across the natural distribution range of the genus in China. We used sequence-related amplified polymorphism (SRAP) markers and obtained 257 clearly defined bands amplified by 20 primers from 461 individuals. The polymorphic loci (P) varied from 35.17% to 76.55%, with an overall mean of 58.24%. Nei’s gene diversity (H) ranged from 0.13 to 0.31, with an overall mean of 0.20. Shannon’s information index (I) ranged from 0.18 to 0.45, with an average of 0.30. The genetic diversity of the total population (Ht) and within populations (Hs) was 0.37 ± 0.01 and 0.20 ± 0.01, respectively. Population differentiation was substantial (Gst = 0.45), and gene flow was low. Of the total variation, 31.41% was explained by differences among putative species, 19.17% among populations within putative species, and 49.42% within populations. Our results support the division of genus Melia into two species, which is consistent with the classification based on the morphological differentiation.

Summary The Canary Islands and Madeira are reportedly home to seven recognised species of baetid mayflies (Ephemeroptera, Baetidae), two of which also occur on the European mainland. Their species status remains unsure, and loss of habitat suggests they are of conservation concern. We applied morphological characters and a general mixed Yule‐coalescent (gmyc) model analysis of the cytochrome c oxidase subunit 1 (cox1) gene to delineate putative species within morphologically cryptic species groups Baetis (Rhodobaetis) and Cloeon dipterum s.l. We used a three‐gene mitochondrial data set (1450 base pairs) to infer phylogenetic relationships and a molecular clock calibrated using island geological ages to infer colonisation history. Genetic and morphological evidence indicated the presence of 12 putative species, 11 of which were endemic to the islands. Only Baetis atlanticus, on Madeira, also occurs on the European mainland. Two lineages (B. pseudorhodani s.l. and B. canariensis s.l.) appear to have arisen in the past 15 million years (mya) and diversified in parallel throughout the Canary Islands. Within the canariensis lineage, sister species occur on the island of Gran Canaria and in North Africa. Pronounced island endemism contradicts previous taxonomic work, which reported a depauperate fauna that included several mainland species. Recent diversification among islands and a close link to North Africa suggest a complex evolutionary history. Owing to their small population size and ongoing habitat alteration, several of these island endemics are among the most endangered aquatic insects in Europe.

A recent taxonomic revision of the Neotropical catfish genus Rhamdia (Pisces: Siluriformes: Heptapteridae) reduced a number of described species to synonymy, especially under a broadly circumscribed R. quelen. Evidence is presented here from DNA sequence data, external morphology, and morphometrics that argues for the recognition of R. guatemalensis in Central and northern South America and R. saijaensis and R. cinerascens in the Pacific drainages of Colombia and Ecuador, respectively. The DNA data indicate that all trans-Andean samples form a monophyletic group, within which there are separate clades corresponding to R. laticauda and the synonymized R. guatemalensis, R. saijaensis, and R. cinerascens. The morphometric data substantiate the phylogenetic groupings, and in external morphology, each putative species has diagnostic characters. Rhamdia guatemalensis is characterized by insertion of the adipose fin closer to the dorsal fin than to the caudal fin and presence of a conspicuous lateral longitudinal dark band; R. saijaensis is characterized by a small head with head length 20.8–23.4% of standard length and by lacking a lateral longitudinal band; and R. cinerascens is characterized by a large head with head length 25.8–30.1% of standard length, base of the adipose fin 30.3–33.3% of standard length, outer mental barbels extending to the base of the pectoral rays, and presence of a faint lateral longitudinal band. The external morphological differences and phylogenetic relationships indicate that these groups are both recognizable and represent independent lineages, which argue for their recognition as species.

Molecular, morphological, and biogeographic resolution of cryptic taxa in the Greenside Darter Etheostoma blennioides complex

Eucolaspis Sharp 1886 is a New Zealand native leaf beetle genus (Coleoptera: Chrysomelidae: Eumolpinae) with poorly described species and a complex taxonomy. Many economically important fruit crops are severely damaged by these beetles. Uncertain species taxonomy of Eucolaspis is leaving any biological research, as well as pest management, tenuous. We used morphometrics, mitochondrial DNA and male genitalia to study phylogenetic and geographic diversity of Eucolaspis in New Zealand. Freshly collected beetles from several locations across their distribution range, as well as identified voucher specimens from major museum collections were examined to test the current classification. We also considered phylogenetic relationships among New Zealand and global Eumolpinae (Coleoptera: Chyrosomelidae). We demonstrate that most of the morphological information used previously to define New Zealand Eucolaspis species is insufficient. At the same time, we show that a combination of morphological and genetic evidence supports the existence of just 3 mainland Eucolaspis lineages (putative species), and not 5 or 15, as previously reported. In addition, there may be another closely related lineage (putative species) on an offshore location (Three Kings Islands, NZ). The cladistic structure among the lineages, conferred through mitochondrial DNA data, was well supported by differences in male genitalia. We found that only a single species (lineage) infests fruit orchards in Hawke’s Bay region of New Zealand. Species-host plant associations vary among different regions.

Analysis of human genetic variation in mountain communities can shed light on the peopling of mountainous regions, perhaps revealing whether the remote geographic location spared them from outside invasion and preserved their gene pool from admixture. In this study, we created a model to assess genetic traces of historical events by reconstructing the paternal and maternal genetic history of seven small mountain villages in inland valleys of Central Italy. The communities were selected for their geographic isolation, attested biodemographic stability, and documented history prior to the Roman conquest. We studied the genetic structure by analyzing two hypervariable segments (HVS-I and HVS-II) of the mtDNA D-loop and several informative single nucleotide polymorphisms (SNPs) of the mtDNA coding region in 346 individuals, in addition to 17 short tandem repeats (STRs) and Y-chromosome SNPs in 237 male individuals. For both uniparental markers, most of the haplogroups originated in Western Europe while some Near Eastern haplogroups were identified at low frequencies. However, there was an evident genetic similarity between the Central Italian samples and Near Eastern populations mainly in the male genetic pool. The samples highlight an overall European genetic pattern both for mtDNA and Y chromosome. Notwithstanding this scenario, Y chromosome haplogroup Q, a common paternal lineage in Central/Western Asia but almost Europe-wide absent, was found, suggesting that Central Italy could have hosted a settlement from Anatolia that might be supported by cultural, topographic and genetic evidence.

Natural hybridization may result in the exchange of genetic material between divergent lineages and even the formation of new taxa. Many of the Neo-Darwinian architects argued that, particularly for animal clades, natural hybridization was maladaptive. Recent evidence, however, has falsified this hypothesis, instead indicating that this process may lead to increased biodiversity through the formation of new species. Although such cases of hybrid speciation have been described in plants, fish and insects, they are considered exceptionally rare in mammals. Here we present evidence for a marine mammal, Stenella clymene, arising through natural hybridization. We found phylogenetic discordance between mitochondrial and nuclear markers, which, coupled with a pattern of transgressive segregation seen in the morphometric variation of some characters, support a case of hybrid speciation. S. clymene is currently genetically differentiated from its putative parental species, Stenella coerueloalba and Stenella longisrostris, although low levels of introgressive hybridization may be occurring. Although non-reticulate forms of evolution, such as incomplete lineage sorting, could explain our genetic results, we consider that the genetic and morphological evidence taken together argue more convincingly towards a case of hybrid speciation. We anticipate that our study will bring attention to this important aspect of reticulate evolution in non-model mammal species. The study of speciation through hybridization is an excellent opportunity to understand the mechanisms leading to speciation in the context of gene flow.

Hybridisation is common among plants and is considered to be an important process in evolution. However, there is much debate as to the role of hybridisation in conservation, particularly whether species of hybrid origin should be protected. In general, conservation policy allows for the protection of hybrids and hybrid progeny if they are shown to be taxonomically distinct, stable and capable of self-perpetuation, and naturally produced. The rare species Adenanthos cunninghamii was suspected to be a hybrid between putative parents, Adenanthos sericeus and Adenanthos cuneatus, as it only occurs where these species co-occur and it displays intermediate and variable morphology. Genetic analysis of A. cunninghamii and the two putative parent species was consistent with this species being a hybrid between A. sericeus and A. cuneatus. Direct analysis of diagnostic loci and phenetic analysis indicated that A. cunninghamii was not genetically uniform and was genetically intermediate between the putative parents. A. cunninghamii is not a distinguishable taxon, morphologically or genetically, and does not produce offspring with morphology within the taxonomic description of the species, thus the species does not satisfy the criteria for protection of hybrids and listing as a rare species in Western Australia.

Homoploid hybrid speciation is thought to require unusual circumstances to yield reproductive isolation from the parental species, and few examples are known from nature. Here, we present genetic evidence for this mode of speciation in birds. Using Bayesian assignment analyses of 751 individuals genotyped for 14 unlinked, nuclear microsatellite loci, we show that the phenotypically intermediate Italian sparrow (Passer italiae) does not form a cluster of its own, but instead exhibits clear admixture (over its entire breeding range) between its putative parental species, the house sparrow (P.domesticus) and the Spanish sparrow (P.hispaniolensis). Further, the Italian sparrow possesses mitochondrial (mt) DNA haplotypes identical to both putative parental species (although mostly of house sparrow type), indicating a recent hybrid origin. Today, the Italian sparrow has a largely allopatric distribution on the Italian peninsula and some Mediterranean islands separated from its suggested parental species by the Alps and the Mediterranean Sea, but co-occurs with the Spanish sparrow on the Gargano peninsula in southeast Italy. No evidence of interbreeding was found in this sympatric population. However, the Italian sparrow hybridizes with the house sparrow in a sparsely populated contact zone in the Alps. Yet, the contact zone is characterized by steep clines in species-specific male plumage traits, suggesting that partial reproductive isolation may also have developed between these two taxa. Thus, geographic and reproductive barriers restrict gene flow into the nascent hybrid species. We propose that an origin of hybrid species where the hybrid lineage gets geographically isolated from its parental species, as seems to have happened in this system, might be more common in nature than previously assumed.

Riparian formations encompass a diverse suite of transitional zones between terrestrial and aquatic ecosystems. During the last decades, these formations have been impacted by several emerging diseases. The first outbreaks were detected on alder formations, but have progressively also been observed on other plant species such as Betula pubescens, Nerium oleander, Populus alba, Salix alpina, Salix purpurea and Tamarix gallica. Declining plants showed a plethora of symptoms (leaf spot, shoot blight, bleeding cankers and root rot) indicative of Phytophthora infections. Since there is little information about the aetiology of these pathosystems, from November 2019 to March 2023, an in-depth study was conducted in 46 riparian ecosystems spanning from the Mediterranean to Alpine regions. Overall, 744 symptomatic samples (stem bleeding cankers and root with rhizosphere) from 27 host species were collected for Phytophthora isolation. Based on morphology and DNA sequence data, 20 known Phytophthora species belonging to seven phylogenetic clades have been identified: P. plurivora (202 isolates), P. gonapodyides (156), P. pseudosyringae (84), P. lacustris (57), P. acerina (31), P. idaei (30), P. alpina (20), P. pseudocryptogea (19), P. cambivora (13), P. pseudotsugae (13), P. cactorum (9), P. honggalleglyana (6), P. pseudogregata (6), P. debattistii (4), P. multivora (4), P. cinnamomi (3), P. bilorbang (2) P. crassamura (2), P. ilicis (2) and P. inundata (2). In addition, 26 isolates of a new putative species obtained from Alnus incana and Pinus sylvestris are described here as Phytophthora heteromorpha sp. nov. The new species proved to be pathogenic on grey alder causing symptoms congruent with field observations. This study represents the most comprehensive investigation on the Phytophthora species associated with declining riparian vegetation in Italy and highlights that the polyphagous pathogen P. plurivora represents a growing threat to Mediterranean, temperate and alpine ecosystems.

Microgastrinae parasitoid wasps (Hymenoptera: Braconidae) were studied in the St. Lawrence Lowlands ecoregion (~14,100 km2) in Ontario, Canada. This subfamily is one of (if not the) most species-rich clades of Lepidoptera parasitoids and has important applications in the biological control of agricultural pests. The St. Lawrence Lowlands ecoregion is one of the nine southern Canadian ecoregions to be identified as a “crisis ecoregion,” having high biodiversity, high risk of biodiversity loss, and low proportion of land included in protected areas. A total of 3,481 specimens collected from 1905 to 2021 within the region were studied. Two species are recorded for the first time in the Nearctic: Apanteles minornavarroi Fernandez-Triana, 2014 and Protapanteles anchisiades (Nixon, 1973); two species are recorded for the first time in Canada: Promicrogaster virginiana Fernandez-Triana, 2019 and Protapanteles immunis (Haliday, 1834); and two are recorded for the first time in Ontario: Cotesia plathypenae (Muesebeck, 1921) and Alphomelon winniewertzae Deans, 2003. DNA-barcode sequences for 2,173 specimens and 66% of the formally described species were successfully recovered. Using a combination of DNA barcodes and morphological assessment, we document herein a minimum putative species count of 228 and a maximum count of 304. We assess the accuracy of species identification in the ecoregion through DNA barcodes and discuss the use of Barcode Index Numbers (BINs) for species discovery in this taxon. Using BINs, 83% of the formally described species with molecular data can be successfully discriminated. The incredible diversity revealed by DNA-barcoding and the high risk of biodiversity loss in the ecoregion highlight the need for increased taxonomic efforts in this taxon to catalog species before they are potentially lost. Several species are present solely in unique habitats within the study area, such as Sphagnum bogs and wetlands. Other (semi) natural features important for these beneficial insects include hedgerows, riparian zones, ditch banks, and wooded areas. Enrichment of these habitats in proximity to field crops could help maintain microgastrine populations and control Lepidoptera crop pests.

Although oysters are commercially very important in Brazil, there is still much dispute about the number of Crassostrea species occurring on the Brazilian coast. The dispute is centered around C. brasiliana, considered by some authors to be a junior synonym of C. rhizophorae. In this paper we compared, by allozyme electrophoresis, sympatric and allopatric populations of the two putative species. Of the 17 loci analysed, five were diagnostic for the two species in sympatry (gene identity = 0.46 to 0.47), clearly demonstrating that they are distinct biological species. Heterozygosity (h) levels were high for both species (h = 0.24 to 0.28), and no heterozygote deficiencies were observed in any population (local inbreeding, F IS = 0.141; P > 0.70). Levels of population structure in C. rhizophorae along 1300 km of coast were very low (population inbreeding, F ST = 0.026; P > 0.15), indicating that the planktonic, planktotrophic larvae of these species are capable of long-range dispersal.

Article Figures and data Abstract eLife digest Introduction Results and discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The phylogenetic placement of the morphologically simple placozoans is crucial to understanding the evolution of complex animal traits. Here, we examine the influence of adding new genomes from placozoans to a large dataset designed to study the deepest splits in the animal phylogeny. Using site-heterogeneous substitution models, we show that it is possible to obtain strong support, in both amino acid and reduced-alphabet matrices, for either a sister-group relationship between Cnidaria and Placozoa, or for Cnidaria and Bilateria as seen in most published work to date, depending on the orthologues selected to construct the matrix. We demonstrate that a majority of genes show evidence of compositional heterogeneity, and that support for the Cnidaria + Bilateria clade can be assigned to this source of systematic error. In interpreting these results, we caution against a peremptory reading of placozoans as secondarily reduced forms of little relevance to broader discussions of early animal evolution. https://doi.org/10.7554/eLife.36278.001 eLife digest Filter-feeding sponges and tiny gliding, pancake-like animals called placozoans are the only two major groups of animals that lack muscles, nerves and an internal gut. Sponges have historically been seen as the first to have branched off in animal phylogeny – the family tree of living organisms that shows how species are related. This is because it is assumed that they split from the other animals before features including muscles, nerves and internal guts evolved. Sequences of their genetic material (the genome) support this view, although some argue that jellyfish-like animals called ctenophores branched first. One explanation for this disagreement is that ctenophores use different proportions of amino acids in their proteins, known as compositional heterogeneity. Computer algorithms that assume amino acid usage is the same universally throughout evolution may therefore place ctenophores incorrectly. In contrast, so far the only genome from a placozoan shows that they are equally closely related to jellyfish and corals (cnidarians) and bilaterians, which includes worms, insects and vertebrates. To test whether this view of the first branches of the animal tree of life is correct, Laumer et al. included the genomes from several undescribed species of placozoans in a phylogenetic analysis. These analyses showed a relationship that had not previously been seen. The placozoans were the closest living relative to cnidarians. However, when looking at the level of genes rather than whole genomes, the more usual relationship of placozoans being equally related to cnidarians and bilaterians re-emerged. To resolve this conflict, Laumer et al. focused on the genes that had the least compositional heterogeneity. When doing this, the relationship appeared to be the newly identified one of placozoans being most closely related to cnidarians. Researchers studying cnidarians often hope to find some clues as to how the complex features they seem to share with bilaterians originated. The findings of Laumer et al. may suggest that the ancestors of the placozoans did in fact have muscles, nerves and guts, but they lost these traits in favor of a simpler lifestyle. An alternative, but controversial possibility is that the ancestor of cnidarians and bilaterians was a simple organism like a placozoan, and the two evolved their complex traits independently. The findings show a complex picture of early animal evolution. Further study of placozoans may well clarify this picture. https://doi.org/10.7554/eLife.36278.002 Introduction The discovery (Schulze, 1883) and mid-20th century rediscovery (Grell and Benwitz, 1971) of the enigmatic, amoeba-like placozoan Trichoplax adhaerens did much to ignite the imagination of zoologists interested in early animal evolution (Bütschli, 1884). As microscopic animals adapted to extracellular grazing on the biofilms over which they creep (Wenderoth, 1986), placozoans have a simple anatomy suited to exploit passive diffusion for many physiological needs, with only six morphological cell types discernible even to intensive microscopical scrutiny (Grell and Ruthmann, 1991; Smith et al., 2014), albeit a greater diversity of cell types is apparent through single-cell RNA-seq (Sebé-Pedrós, 2018a). They have no conventional muscular, digestive, or nervous systems, yet show tightly-coordinated behaviour regulated by peptidergic signaling (Smith et al., 2015; Senatore et al., 2017; Varoqueaux, 2018; Armon et al., 2018). In laboratory conditions, they proliferate through fission and somatic growth. Evidence for sexual reproduction remains elusive, despite genetic evidence of recombination (Srivastava et al., 2008) and descriptions of early abortive embryogenesis (Eitel et al., 2011; Grell, 1972), with the possibility that sexual phases of the life cycle may occur only under poorly understood field conditions (Pearse and Voigt, 2007; McFall-Ngai et al., 2013) Given their simple, puzzling morphology and dearth of embryological clues, molecular data are crucial in placing placozoans phylogenetically. The position of Placozoa in the animal tree proved recalcitrant to early standard-marker analyses (Kim et al., 1999; Silva et al., 2007; Wallberg et al., 2004), although this paradigm did reveal a large degree of molecular diversity in placozoan isolates from around the globe, clearly indicating the existence of many cryptic species (Pearse and Voigt, 2007; Eitel et al., 2013; Signorovitch et al., 2007) with up to 27% genetic distance in 16S rRNA alignments (Eitel and Schierwater, 2010). An apparent answer to the question of placozoan affinities was provided by analysis of a nuclear genome assembly (Srivastava et al., 2008), which strongly supported a position as the sister group of a clade of Cnidaria + Bilateria (sometimes called Planulozoa). However, this effort also revealed a surprisingly bilaterian-like (Dunn et al., 2015) developmental gene toolkit in placozoans, a paradox for such a simple animal. As metazoan phylogenetics has pressed onward into the genomic era, perhaps the largest controversy has been the debate over the identity of the sister group to the remaining metazoans, traditionally thought to be Porifera, but considered to be Ctenophora by Dunn et al (Dunn et al., 2008). and subsequently by additional studies (Hejnol et al., 2009; Moroz et al., 2014; NISC Comparative Sequencing Program et al., 2013; Whelan et al., 2015; Whelan et al., 2017). Others have suggested that this result arises from artifacts with potentially additive effects, such as inadequate taxon sampling, flawed matrix husbandry (undetected paralogy or contamination), and use of poorly fitting substitution models (Philippe et al., 2009; Pick et al., 2010; Pisani et al., 2015; Simion et al., 2017; Feuda et al., 2017). A third view has emphasized that using different sets of genes can lead to different conclusions, with only a small number sometimes sufficient to drive one result or another (Nosenko et al., 2013; Shen et al., 2017). This controversy, regardless of its eventual resolution, has spurred serious contemplation of possibly independent origins of several hallmark traits such as striated muscles, digestive systems, and nervous systems (Moroz et al., 2014; Dayraud et al., 2012; Hejnol and Martín-Durán, 2015; Liebeskind et al., 2017; Moroz and Kohn, 2016; Presnell et al., 2016; Steinmetz et al., 2012). Driven by this controversy, new genomic and transcriptomic data from sponges, ctenophores, and metazoan outgroups have accrued, while new sequences and analyses focusing on the position of Placozoa have been slow to emerge. Here, we provide a novel test of the phylogenetic position of placozoans, adding draft genomes from three putative species that span the root of this clade’s known diversity (Eitel et al., 2013) (Table 1), and critically assessing the role of systematic error in placing of these enigmatic organisms (Laumer, 2018). Table 1 Summary statistics describing the contiguity and completeness of the draft host metagenome bins from the three clade A placozoan isolates utilized in this paper, presented in comparison to the reference H1 strain. https://doi.org/10.7554/eLife.36278.003 H11H4H6H1assembly span (Mbp)56.6383.3976.798.06scaffold number5813533783101415scaffold N50 (kbp)12.73825.9712.845790GC%30.7630.8429.929.37BUSCO2 Eukaryota complete (of 303)220276239294BUSCO2 Eukaryota complete + partial (of 303)246282265298Average # of hits per BUSCO1.001.041.001.00% of BUSCOs with more than one match0.453.990.420.34 Results and discussion Orthology assignment on sets of predicted proteomes derived from 59 genome and transcriptome assemblies yielded 4294 gene trees with at least 20 sequences each, sampling all five major metazoan clades and outgroups, from which we obtained 1388 well-aligned orthologues. Within this set, individual maximum-likelihood (ML) gene trees were constructed, and a set of 430 most-informative orthologues were selected on the basis of tree-likeness scores (Misof et al., 2013). This yielded an amino-acid matrix of 73,547 residues with 37.55% gaps or missing data, with an average of 371.92 and 332.75 orthologues represented for Cnidaria and Placozoa, respectively (with a maximum of 383 orthologues present for the newly sequenced placozoan H4 clade representative; Figure 1). Figure 1 with 2 supplements see all Download asset Open asset Consensus phylogram showing deep metazoan interrelationships under Bayesian phylogenetic inference of the 430-orthologue amino acid matrix, using the CAT + GTR + Г4 mixture model. All nodes received full posterior probability. Numerical annotations of given nodes represent Extended Quadripartition Internode Certainty (EQP-IC) scores, describing among-gene-tree agreement for both the monophyly of the five major metazoan clades and the given relationships between them in this reference tree. A bar chart on the right depicts the proportion of the total orthologue set each terminal taxon is represented by in the concatenated matrix. ‘Placozoa H1’ in this and all other figures refers to the GRELL isolate sequenced in Srivastava et al., 2008, which has there and elsewhere been referred to as Trichoplax adhaerens, despite the absence of type material linking this name to any modern isolate. Line drawings of clade representatives are taken from the BIODIDAC database (http://biodidac.bio.uottawa.ca/). https://doi.org/10.7554/eLife.36278.004 Our Bayesian analyses of this matrix place Cnidaria and Placozoa as sister groups with full posterior probability under the general site-heterogeneous CAT + GTR + Г4 model (Figure 1). Under ML inference with the C60 +LG + FO + R4 profile mixture model (Wang et al., 2018) (Figure 1—figure supplement 1), we again recover Cnidaria + Placozoa, albeit with more marginal resampling support. Both Bayesian and ML analyses show little internal branch diversity within Placozoa. Accordingly, deleting all newly-added placozoan genomes from our analysis has no effect on topology and only a marginal effect on support in ML analysis (Figure 1—figure supplement 2). Quartet-based concordance analyses (Zhou, 2017) show no evidence of strong phylogenetic conflicts among ML gene trees in this 430-gene set (Figure 1), although internode certainty metrics are close to 0 for many key clades including Cnidaria + Placozoa, indicating that support for some ancient relationships may be masked by gene-tree estimation errors, emerging only in combined analysis (Gatesy and Baker, 2005). Compositional heterogeneity of amino-acid frequencies along the tree is a source of phylogenetic error not modelled by even complex site-heterogeneous substitution models such as CAT+GTR (Blanquart and Lartillot, 2008; Foster, 2004; Lartillot and Philippe, 2004; Lartillot et al., 2013). Furthermore, previous analyses (Nosenko et al., 2013) have shown that placozoans and choanoflagellates in particular, both of which taxa our matrix samples intensively, deviate strongly from the mean amino-acid composition of Metazoa, perhaps as a result of genomic GC content discrepancies. As a measure to at least partially ameliorate such nonstationary substitution, we recoded the amino-acid matrix into the 6 ‘Dayhoff’ categories, a common strategy previously shown to reduce the effect of compositional variation among taxa, albeit the Dayhoff-6 groups represent only one of many plausible recoding strategies, all of which sacrifice information (Feuda et al., 2017; Nesnidal et al., 2010; Rota-Stabelli et al., 2013; Susko and Roger, 2007). Analysis of this recoded matrix under the CAT + GTR model again recovered full support (pp = 1) for Cnidaria + Placozoa (Figure 2). Indeed, under Dayhoff-6 recoding, the only major change is in the relative positions of Ctenophora and Porifera, with the latter here constituting the sister group to all other animals with full support. Similar recoding-driven effects on relative positions of Porifera and Ctenophora have also been seen in other recent work (Feuda et al., 2017), and have been interpreted to indicate a role for compositional bias in misplacing Ctenophora as sister group to all other animals Figure 2 Download asset Open asset Consensus phylogram under Bayesian phylogenetic inference under the CAT + GTR + Г4 mixture model, on the 430-orthologue concatenated amino acid matrix, recoded into 6 Dayhoff groups. Nodes annotated with posterior probability; unannotated nodes received full support. https://doi.org/10.7554/eLife.36278.007 Many research groups, using good taxon sampling and genome-scale datasets, and even recently including data from a new divergent placozoan species (Whelan et al., 2017; Feuda et al., 2017; Eitel, 2017), have consistently reported strong support for Planulozoa under the CAT + GTR model. Indeed, when we construct a supermatrix from our predicted peptide catalogues using a different strategy, relying on complete sequences of 303 pan-eukaryote ‘Benchmarking Universal Single-Copy Orthologs’ (BUSCOs) (Simão et al., 2015), we also see full support in a CAT + GTR + Г analysis for Planulozoa, in both amino-acid (Figure 3a) and Dayhoff-6 recoded alphabets (Figure 3b). Which phylogeny is correct, and what process drives support for the incorrect topology? Posterior predictive tests, which compare the observed among-taxon usage of amino-acid frequencies to expected distributions simulated using the sampled posterior distribution and a single composition vector, may provide insight (Feuda et al., 2017; Lartillot and Philippe, 2004). Both the initial 430-gene matrix and the 303-gene BUSCO matrix fail these tests, but the BUSCO matrix fails it more profoundly, with z-scores (measuring mean-squared across-taxon heterogeneity) scoring in the range of 330–340, in contrast to the range of 176–187 seen in the 430-gene matrix (Table 2). Furthermore, inspecting z-scores for individual taxa in representative chains from both matrices shows that a large amount of this global difference in z-scores can be attributed to placozoans, with additional contributions from choanoflagellates and select isolated representatives of other clades (Figure 3C). Figure 3 Download asset Open asset Posterior consensus trees from CAT + GTR + Г4 mixture model analysis of a 94,444 amino acid supermatrix derived from the 303 single-copy conserved eukaryotic BUSCO orthologs, analysed in A. amino acid space or (B) the Dayhoff-6 reduced alphabet space. Nodal support values comprise posterior probabilities; nodes with full support not annotated. Taxon colourings as in previous Figures. (C) Plot of z-scores (summed absolute distance between taxon-specific and global empirical frequencies) from representative posterior predictive tests of amino acid compositional bias, from both the BUSCO 303-orthologue matrix (red) and the initial 430-orthologue matrix (blue). Placozoan taxon abbreviations are shown in blue font. https://doi.org/10.7554/eLife.36278.008 Table 2 Mean (and standard deviation of) z-scores from posterior predictive tests of per-site amino acid diversity and among-lineage compositional homogeneity, called for amino-acid alignments using the PhyloBayes-MPI v1.8 readpb_mpi –div and –comp options, respectively, with burn-ins selected as per the posterior consensus summaries shown elsewhere. Except for the diversity statistic in the test-passing matrix, all tests reject (at p=0.05) the adequacy of the inferred CAT + GTR + Г4 model to describe the data. https://doi.org/10.7554/eLife.36278.009 DiversityComposition (mean)Composition (maximum)430 matrix1.94 (0.09)181.35 (7.50)105.04 (3.13)BUSCO 303-gene matrix11.27 (0.73)334.98 (4.56)107.56 (6.17)comp-failed matrix2.51 (0.19)270.16 (12.03)173.87 (9.15)comp-passed matrix0.81 (0.18)107.67 (10.10)63.19 (6.95) As a final measure to describe the influence of compositional heterogeneity in this dataset, we applied a null-simulation test for compositional bias to each alignment in our set of 1388 orthologues. This test, which compares the real data to a null distribution of amino-acid frequencies simulated along assumed gene trees with a substitution model using a single composition vector, is less prone to Type II errors than the more conventional X (Grell and Benwitz, 1971) test (Foster, 2004). Remarkably, at a conservative significance threshold of α = 0.10, the majority (764 genes or ~55%) of this gene set is identified as compositionally biased by this test, highlighting the importance of using appropriate statistical tests to control this source of systematic error, rather than applying arbitrary heuristic cutoffs (Kück and Struck, 2014). Building informative matrices from gene sets on either side of this significance threshold, and again applying both CAT + GTR mixture models and ML profile mixtures, we see strong support for Cnidaria + Placozoa in the test-passing supermatrix, and conversely, strong support for Cnidaria + Bilateria in the test-failing supermatrix (Figure 4, Figure 4—figure supplement 1, Figure 4—figure supplement 2). Interestingly, in trees built through CAT + GTR + Г4 analysis of the test-failing supermatrix (Figure 4A,C), in both amino-acid and Dayhoff-6 alphabets, we also observe full support for Porifera as sister to all other animals. In contrast, analysis of this amino acid matrix under a profile mixture model recovers support for Ctenophora in this position (Figure 4—figure supplement 1), indicating that, at least for this alignment, compositional heterogeneity need not be invoked to explain why outcomes differ among analyses, as some have argued (Feuda et al., 2017): both CAT + GTR and the C60 +LG + FO + R4 profile mixture model assume a single composition vector over time, but the CAT + GTR model is better able to accommodate site-heterogeneous substitution patterns (Lartillot et al., 2013; Quang et al., 2008). In the context of this experiment, Dayhoff-6 recoding appears impactful only for the test-passing supermatrix (Figure 4B,D), where it obviates support for Ctenophora-sister (Figure 4B, Figure 4—figure supplement 2) in favour of (albeit, with marginal support) Porifera-sister (Figure 4D), and also diminishes support for Placozoa + Cnidaria (in contrast to the 430-gene matrix; Figure 2), perhaps reflecting the inherent information loss of using a reduced amino-acid alphabet for this relatively shorter matrix. Figure 4 with 2 supplements see all Download asset Open asset Schematic depiction of deep metazoan interrelationships in posterior consensus trees from CAT + GTR + Г4 mixture model analyses of matrices made from subsets of genes passing or failing a sensitive null-simulation test of compositional heterogeneity. Panels correspond to (A) the amino acid matrix made within the failing set; (B) the amino acid matrix derived from the passing set; (C) the Dayhoff-6 recoded matrix from the failing set; (D) the Dayhoff-6 recoded matrix from the passing set. nodes with posterior probability less than are annotated A possible related to the phylogenetic we the significance of which remains is mean alignment both the test-passing and the 430-gene matrix are of shorter alignments than the test-failing and the 303-gene BUSCO matrix Materials and Indeed, alignment has been previously shown to be predictive of a number of other metrics of phylogenetic relevance et al., the and of such relationships in empirical at of is clearly of The previously cryptic phylogenetic between cnidarians and placozoans seen in gene sets less by compositional bias with other analyses and data such as genomic which be more as assemblies to be reported from animals (Eitel et al., 2018; et al., 2018; 2018; 2018). However, this relationship to on the of traits Many the known (Eitel et al., 2011; and Voigt, 2007) and relatively bilaterian-like gene content of placozoans (Srivastava et al., 2008; Eitel, 2017), that these organisms have a more and life cycle et al., or are that have significance to any their Indeed, it is to this new phylogenetic position as such as much work on models in the paradigm is on the that cnidarians and bilaterians more or many morphological and 2017; et al., nervous systems et al., 2017; Moroz and Kohn, 2016; et al., 2015; 2016; et al., et al., 2017; and et al., et al., 2017), and internal et al., 2016; et al., 2007; Hejnol and 2008; and we not as some have and Schierwater, that placozoans metazoan we to them a as to understanding early evolution in although simpler and less placozoans have to cnidarians as an we see in assumed of by by between bilaterians and cnidarians to placozoans, an which the large in and molecular genetic between these taxa, which recent has been made using both methods such as in et al., 2018) and analysis as well as new such as single-cell RNA-seq (Sebé-Pedrós, et al., we another of this that can be as at any level between Bilateria and Cnidaria have in animal evolution than previously and either occur in modern placozoans or have been lost at some in their In this of early animal evolution on of forms 2018; 2017; and 2018; and 2010; et al., 2017) and molecular et al., 2017; et al., 2015; and 2017; et al., may Materials and methods and reference genomes from previously placozoans a H4 and placozoans were from at the of in placozoans were from the show in the in All placozoans were sampled by placing or in into the for (Pearse and Voigt, 2007). were identified under a and single were to of as per was from 3 of and of using the and and from three H4 were using the with both to and was by the as of an in In was with the and the genomic was to an average of the the was and an was using the The the the H4 the RNA-seq was to and were with the for All were selected by and the recovered and by each – or were sequenced on or for the H4 – single were and were with with a of two and a of and single were from the analysis. was error using et al., 2013). A combined assembly of all for each was using et al., 2012). and data were from the full set with standard and The data was to all with an average using and with and from each were to the using with the = were on the data using et al., 2015) with and the The Trichoplax host bins were using et al., on and to the Trichoplax H1 reference assembly The metrics were with (Simão et al., 2015) (Table 1) and et al., 2013). Both the also in other organisms et al., and the proportion of orthologue hits in the (Table 1) to the lack of evidence for within the for gene proteomes from transcriptome and genome assemblies a proteomes from species with published draft genome assemblies were from the or in A placozoans, host bins were for gene the and was with et al., for the H4 total RNA-seq obtained from three isolates were to genomic with et al., 2013) under were to genomic and predicted with et al., under et al., 2017) were provided as data from from a