Bootstrap Probability Research Articles

Accurate Inference of Tree Topologies from Multiple Sequence Alignments Using Deep Learning.

Reconstructing the phylogenetic relationships between species is one of the most formidable tasks in evolutionary biology. Multiple methods exist to reconstruct phylogenetic trees, each with their own strengths and weaknesses. Both simulation and empirical studies have identified several "zones" of parameter space where accuracy of some methods can plummet, even for four-taxon trees. Further, some methods can have undesirable statistical properties such as statistical inconsistency and/or the tendency to be positively misleading (i.e. assert strong support for the incorrect tree topology). Recently, deep learning techniques have made inroads on a number of both new and longstanding problems in biological research. In this study, we designed a deep convolutional neural network (CNN) to infer quartet topologies from multiple sequence alignments. This CNN can readily be trained to make inferences using both gapped and ungapped data. We show that our approach is highly accurate on simulated data, often outperforming traditional methods, and is remarkably robust to bias-inducing regions of parameter space such as the Felsenstein zone and the Farris zone. We also demonstrate that the confidence scores produced by our CNN can more accurately assess support for the chosen topology than bootstrap and posterior probability scores from traditional methods. Although numerous practical challenges remain, these findings suggest that the deep learning approaches such as ours have the potential to produce more accurate phylogenetic inferences.

Phylogeny of the supertribe Nebriitae (Coleoptera: Carabidae) based on analyses of molecular sequence data--some surprises.

To explore phylogenetic relationships among members of the carabid supertribe Nebriitae, we sampled DNA fragments extracted from adults of 244 nebriite species-group taxa, representing about 25% of known species-group taxa and 93% (38 of 41) of the currently accepted nebriite genus-group taxa. Sequence data were recorded for eight gene fragments (a total of 5654 bases) from nuclear ribosomal, mitochondrial, and nuclear protein coding genes. The resulting phylogeny is based on both individual gene and concatenated gene analyses using Maximum Likelihood and Bayesian methods, which produced nearly identical results. Most nodes are well supported by both high bootstrap and high posterior probability values, and many of them also by unique (autapomorphic) bases. Many of the previously recognized genus-group taxa are supported as monophyletic, but others are not. Alternative reclassifications that reflect monophyletic groups are still being considered, based on the molecular analyses along with considerations of ease of group identification and geographical distribution. Of the two alternative reclassifications being considered, one requires the designation of 16 new subgeneric synonymies and the other only nine new subgeneric synonymies plus the recognition of three new subgenera. However, under both schemes, Nippononebria Uéno (along with its subgenus, Vancouveria Kavanaugh) is re-established as a genus separate from Nebria and sister to Leistus Frölich; and Archastes is embedded within a monophyletic genus Nebria and closely related to subgenera Oreonebria Daniel and Orientonebria Shilenkov. Additional details will be reported in the presentation.

Selective Inference for Testing Trees and Edges in Phylogenetics

Selective inference is considered for testing trees and edges in phylogenetic tree selection from molecular sequences. This improves the previously proposed approximately unbiased test by adjusting the selection bias when testing many trees and edges at the same time. The newly proposed selective inference $p$-value is useful for testing selected edges to claim that they are significantly supported if $p>1-\alpha$, whereas the non-selective $p$-value is still useful for testing candidate trees to claim that they are rejected if $p<\alpha$. The selective $p$-value controls the type-I error conditioned on the selection event, whereas the non-selective $p$-value controls it unconditionally. The selective and non-selective approximately unbiased $p$-values are computed from two geometric quantities called signed distance and mean curvature of the region representing tree or edge of interest in the space of probability distributions. These two geometric quantities are estimated by fitting a model of scaling-law to the non-parametric multiscale bootstrap probabilities. Our general method is applicable to a wider class of problems; phylogenetic tree selection is an example of model selection, and it is interpreted as the variable selection of multiple regression, where each edge corresponds to each predictor. Our method is illustrated in a previously controversial phylogenetic analysis of human, rabbit and mouse.

Phylogeography of the red-bellied lizard, Darevskia parvula in Turkey

The groups of red-bellied lizards had a small distribution area in the Pontic zone. The several studies performed on these lizard groups are based on taxonomy and systematics. Although there were several taxonomic or systematic researches on some species of this group, the phylogeographical pattern and species disturbing boundaries of this group is still not clear. In the present study, we aimed to resolve the taxonomic and phylogenetic relationships of the red-bellied lizards in Turkey, based on two combined mitochondrial gene fragments and one protein-coding nuclear gene (rag1). Also, we evaluated ecological niches differentiations among red-bellied lizard groups. The mitochondrial DNA genes were found to be highly polymorphic in this group. One hundred and one variable nucleotide sites were detected on the combined gene sequences. According to phylogenetic trees based on the maximum likelihood (ML) and Bayesian inference (BI), the red-bellied lizards group have three species groups; Darevskia parvula, D. adjarica and unnamed Darevskia sp. (candidate species for Darevskia genus). This situation was supported by high bootstrap and posterior probability values in the trees of mitochondrial DNA gene fragments. However, no genetic variation was detected according to nuclear DNA (rag1) sequence. Because the species groups have no overlaps in terms of their ecological niches, ecological niche modelling (ENM) results revealed differences among the groups of D. parvula, D. adjarica, and unnamed Darevskia sp. Besides, we detected no geographical overlaps among three species groups, since there were geographical isolation zones among the species groups of red-bellied lizard.

Application of laser induced breakdown spectroscopy in food container glass discrimination

Although glass is primarily silicon dioxide, manufacturers produce different types of glass by blending various other oxides to impart desired functional properties. In this study, the application of laser induced breakdown spectroscopy (LIBS) as a rapid tool for glass discrimination was examined, focusing on glass used for food packaging applications (soda-lime-silicate glass). The LIBS instrument operational parameters and applicable calibration methods were evaluated using National Institute of Standards and Technology certified glass standards. LIBS-based spectra of emission lines for selected elements were recorded on a pool of mixed glass samples taken from assorted food containers and other non-food packaging glass including labware, electronic display glass and glass used for different types of lighting. Owing to a greater contrast in composition, an excellent discrimination between food container glass and the other types of glass was demonstrated by post-processing the emission line data with a correlation matrix, multivariate data visualization and clustering techniques. Some significant within-glass category difference were also detected when a set of glass jar samples taken from different food brands were evaluated using clustering analysis. The latter shows a sensitivity to distinguish smaller differences among the soda-lime-glass that could be attributed to a manufacturing variation. The significance and stability of the group association patterns, generated by the clustering analysis, were further confirmed with uncertainty analysis using the method of bootstrap re-sampling and bootstrap probability calculations. Results of the study suggest that LIBS measurement coupled with appropriate multivariate calibration, data visualization and clustering analysis can be utilized as a rapid tool for glass identification and to support food safety investigations by helping to trace the identity and source of glass fragment inclusions.

Analysis of mitochondrial genomes resolves the phylogenetic position of Chinese freshwater mussels (Bivalvia, Unionidae).

The Yangtze River basin is one of the most species-rich regions for freshwater mussels on Earth, but is gravely threatened by anthropogenic activities. However, conservation planning and management of mussel species has been hindered by a number of taxonomic uncertainties. In order to clarify the taxonomic status and phylogenetic position of these species, mitochondrial genomes of four species (Acuticostachinensis, Schistodesmuslampreyanus, Cuneopsisheudei and Cuneopsiscapitatus) were generated and analyzed along with data from 43 other mitogenomes. The complete F-type mitogenomes of A.chinensis, S.lampreyanus, C.heudei, and C.capitatus are 15652 bp, 15855 bp, 15892 bp, and 15844 bp, respectively, and all four F-type mitogenomes have the same pattern of gene arrangement. ML and BI trees based on the mitogenome dataset are completely congruent, and indicate that the included Unionidae belong to three subfamilies with high bootstrap and posterior probabilities, i.e., Unioninae (Aculamprotula, Cuneopsis, Nodularia, and Schistodesmus), Anodontinae (Cristaria, Arconaia, Acuticosta, Lanceolaria, Anemina, and Sinoanodonta), and Gonideinae (Ptychorhynchus, Solenaia, Lamprotula, and Sinohyriopsis). Results also indicate that A.chinensis has affinities with Arconaialanceolata and Lanceolariagrayii and is a member of the subfamily Anodontinae.

The critically endangered forest owlet Heteroglaux blewitti is nested within the currently recognized Athene clade: A century-old debate addressed.

Range-restricted species generally have specific niche requirements and may often have unique evolutionary histories. Unfortunately, many of these species severely lack basic research, resulting in poor conservation strategies. The phylogenetic relationship of the Critically Endangered Forest Owlet Heteroglaux blewitti has been the subject of a century-old debate. The current classifications based on non-phylogenetic comparisons of morphology place the small owls of Asia into three genera, namely, Athene, Glaucidium, and Heteroglaux. Based on morphological and anatomical data, H. blewitti has been alternatively hypothesized to belong within Athene, Glaucidium, or its own monotypic genus Heteroglaux. To test these competing hypotheses, we sequenced six loci (~4300 bp data) and performed phylogenetic analyses of owlets. Mitochondrial and nuclear trees were not congruent in their placement of H. blewitti. However, both mitochondrial and nuclear combined datasets showed strong statistical support with high maximum likelihood bootstrap (>/ = 90) and Bayesian posterior probability values (>/ = 0.98) for H. blewitti being nested in the currently recognized Athene group, but not sister to Indian A. brama. The divergence of H. blewitti from its sister taxa was between 4.3 and 5.7 Ma coinciding with a period of drastic climatic changes in the Indian subcontinent. This study presented the first genetic analysis of H. blewitti, a Critically Endangered species, and addressed the long debate on the relationships of the Athene-Heteroglaux-Glaucidium complex. We recommend further studies with more data and complete taxon sampling to understand the biogeography of Indian Athene species.

Pan-genome and phylogeny of Bacillus cereus sensu lato

BackgroundBacillus cereus sensu lato (s. l.) is an ecologically diverse bacterial group of medical and agricultural significance. In this study, I use publicly available genomes and novel bioinformatic workflows to characterize the B. cereus s. l. pan-genome and perform the largest phylogenetic and population genetic analyses of this group to date in terms of the number of genes and taxa included. With these fundamental data in hand, I identify genes associated with particular phenotypic traits (i.e., “pan-GWAS” analysis), and quantify the degree to which taxa sharing common attributes are phylogenetically clustered.MethodsA rapid k-mer based approach (Mash) was used to create reduced representations of selected Bacillus genomes, and a fast distance-based phylogenetic analysis of this data (FastME) was performed to determine which species should be included in B. cereus s. l. The complete genomes of eight B. cereus s. l. species were annotated de novo with Prokka, and these annotations were used by Roary to produce the B. cereus s. l. pan-genome. Scoary was used to associate gene presence and absence patterns with various phenotypes. The orthologous protein sequence clusters produced by Roary were filtered and used to build HaMStR databases of gene models that were used in turn to construct phylogenetic data matrices. Phylogenetic analyses used RAxML, DendroPy, ClonalFrameML, PAUP*, and SplitsTree. Bayesian model-based population genetic analysis assigned taxa to clusters using hierBAPS. The genealogical sorting index was used to quantify the phylogenetic clustering of taxa sharing common attributes.ResultsThe B. cereus s. l. pan-genome currently consists of ≈60,000 genes, ≈600 of which are “core” (common to at least 99% of taxa sampled). Pan-GWAS analysis revealed genes associated with phenotypes such as isolation source, oxygen requirement, and ability to cause diseases such as anthrax or food poisoning. Extensive phylogenetic analyses using an unprecedented amount of data produced phylogenies that were largely concordant with each other and with previous studies. Phylogenetic support as measured by bootstrap probabilities increased markedly when all suitable pan-genome data was included in phylogenetic analyses, as opposed to when only core genes were used. Bayesian population genetic analysis recommended subdividing the three major clades of B. cereus s. l. into nine clusters. Taxa sharing common traits and species designations exhibited varying degrees of phylogenetic clustering.ConclusionsAll phylogenetic analyses recapitulated two previously used classification systems, and taxa were consistently assigned to the same major clade and group. By including accessory genes from the pan-genome in the phylogenetic analyses, I produced an exceptionally well-supported phylogeny of 114 complete B. cereus s. l. genomes. The best-performing methods were used to produce a phylogeny of all 498 publicly available B. cereus s. l. genomes, which was in turn used to compare three different classification systems and to test the monophyly status of various B. cereus s. l. species. The majority of the methodology used in this study is generic and could be leveraged to produce pan-genome estimates and similarly robust phylogenetic hypotheses for other bacterial groups.

Inferring the shallow phylogeny of true salamanders (Salamandra) by multiple phylogenomic approaches

The rise of high-throughput sequencing techniques provides the unprecedented opportunity to analyse controversial phylogenetic relationships in great depth, but also introduces a risk of being misinterpreted by high node support values influenced by unevenly distributed missing data or unrealistic model assumptions. Here, we use three largely independent phylogenomic data sets to reconstruct the controversial phylogeny of true salamanders of the genus Salamandra, a group of amphibians providing an intriguing model to study the evolution of aposematism and viviparity. For all six species of the genus Salamandra, and two outgroup species from its sister genus Lyciasalamandra, we used RNA sequencing (RNAseq) and restriction site associated DNA sequencing (RADseq) to obtain data for: (1) 3070 nuclear protein-coding genes from RNAseq; (2) 7440 loci obtained by RADseq; and (3) full mitochondrial genomes. The RNAseq and RADseq data sets retrieved fully congruent topologies when each of them was analyzed in a concatenation approach, with high support for: (1) S. infraimmaculata being sister group to all other Salamandra species; (2) S. algira being sister to S. salamandra; (3) these two species being the sister group to a clade containing S. atra, S. corsica and S. lanzai; and (4) the alpine species S. atra and S. lanzai being sister taxa. The phylogeny inferred from the mitochondrial genome sequences differed from these results, most notably by strongly supporting a clade containing S. atra and S. corsica as sister taxa. A different placement of S. corsica was also retrieved when analysing the RNAseq and RADseq data under species tree approaches. Closer examination of gene trees derived from RNAseq revealed that only a low number of them supported each of the alternative placements of S. atra. Furthermore, gene jackknife support for the S. atra - S. lanzai node stabilized only with very large concatenated data sets. The phylogeny of true salamanders thus provides a compelling example of how classical node support metrics such as bootstrap and Bayesian posterior probability can provide high confidence values in a phylogenomic topology even if the phylogenetic signal for some nodes is spurious, highlighting the importance of complementary approaches such as gene jackknifing. Yet, the general congruence among the topologies recovered from the RNAseq and RADseq data sets increases our confidence in the results, and validates the use of phylotranscriptomic approaches for reconstructing shallow relationships among closely related taxa. We hypothesize that the evolution of Salamandra has been characterized by episodes of introgressive hybridization, which would explain the difficulties of fully reconstructing their evolutionary relationships.

Phylogeny and systematics of Anatolian mountain frogs

Anatolian mountain frogs (Rana macrocnemis, Rana camerani, Rana holtzi, and Rana tavasensis) are one of the most specious amphibian groups in Turkey containing two endemic taxa (R. holtzi and R. tavasensis). The taxonomy of this group remains controversial as there are several unresolved issues.In the present study, we aimed to resolve the taxonomic uncertainty of the Anatolian mountain frogs through two mitochondrial genes (CYTB, 481 bp and COI, 743 bp) and two protein-coding nuclear genes (POMC, 401 bp and RAG1, 717 bp). The mitochondrial DNA (mtDNA) markers were found to be highly polymorphic in this group. Haplotype network analysis revealed that R. tavasensis was different for at least 33 and 52 mutational steps according to CYTB and COI gene regions, respectively. High bootstrap and posterior probability values obtained from the mtDNA genes support the idea that Anatolian mountain frogs are represented by two distinct species in Anatolia: R. macrocnemis and R. tavasensis. However, no genetic variation was detected according to nuclear DNA (nDNA) markers.The analysis of molecular variance (AMOVA) revealed no differences among the groups of R. macrocnemis, R. camerani, and R. holtzi. Despite the low genetic distance among R. macrocnemis, R. camerani, and R. holtzi species, the pairwise distances estimated from R. tavasensis were higher compared with other Anatolian mountain frog lineages.

Genetic variability and differentiation among the populations of Dice snake, Natrix tessellata (Sepentes, Colubridae) in the Iranian Plateau

The Dice snake, Natrix tessellate, is one of the widely distributed but poorly studied reptiles of the Iranian plateau. Here, we sequenced a partial fragment of Cyt b for 14 samples of Natrix tessellata from Iran. The data were added to the previously published data from GenBank. The phylogenetic analyses indicated that the Iranian populations of N. tessellata are well divided into two major clades. The north, eastern and NE populations of Dice snake in Iran in an association with the Central Asian populations constitute the first clade which is in turn a sister clade to the European populations. The other clade that is placed in the base of the whole Dice snake clade, is formed by the southern and western Iranian populations. These are separated from the first group with high bootstrap and posterior probability values. Position of the western and southern Iranian populations in the phylogenetic tree and its close relation to the N. natrix samples, included in the analyses, imply that the group is most ancient among all populations of Dice snake in the Palearctic region with a long history in the area.

Phylogeny of Acianthera sect. Pleurobotryae (Orchidaceae: Pleurothallidinae), an endemic group of the Atlantic Forest

Among the members of subtribe Pleurothallidinae, Acianthera is one of the largest genera in Brazil with ca. 107 endemic species. A recent study of Acianthera, including a phylogeny based on nrITS and a systematic revision, proposed dividing the genus into nine sections. In this revision, the genus Pleurobotryum was reduced to Acianthera sect. Pleurobotryae. However, the true position of this section and its circumscription are debatable because the authors included only one species of the genus and did not include the type species (Pleurobotryum atropurpureum Barb. Rodr.) in the analysis. The objective of this work was to study the monophyly of the section and phylogenetic relationships among the species of A. sect. Pleurobotryae. The study included all of the species in this group and used the nuclear marker ITS (ITS1, 5.8S gene and ITS2) and an initial portion of the plastid marker matK for individual and combined maximum parsimony, Bayesian and maximum likelihood analyses. The results show that A. sect. Pleurobotryae is monophyletic with high bootstrap, decay index, and posterior probability support. Within the group, the results suggest, with moderate to strong support, the recognition of two sister clades: [(Acianthera hatschbachii (Schltr.) Chiron & van den Berg, Acianthera atropurpurea (Barb. Rodr.) Chiron & van den Berg), (Acianthera mantiquyrana (Barb. Rodr.) V. T. Rodrigues & F. Barros, Acianthera crepiniana (Cogn.) Chiron & van den Berg)]. The lack of resolution using only nrITS, the strong phylogenetic signal from the matK, and the combined analyses suggest that more studies are needed to verify the phylogenetic position and circumscription of some of the other sections in the genus. In addition, it is important that type species and morphological characters are incorporated into the definitions of the clades.

Genetic Diversity and Identification of Palearctic Black Flies in the Subgenus Wilhelmia (Diptera: Simuliidae).

Accurate species identifications are the essential first step in understanding the medical, economic, and ecological importance of black flies. The utility of DNA barcoding based on cytochrome c oxidase subunit 1 (COI) sequences was evaluated for identifying six common species of Palearctic black flies in the subgenus Wilhelmia, including several that are virulent pests. Chromosomally identified larvae from Turkey and Germany and COI sequences in GenBank were analyzed. Intraspecific genetic divergence was 0.7-3.5% (mean 1.6%), whereas interspecific genetic divergence was 2.7-16.9%. On the basis of COI barcodes, the six nominal species of Simulium (Wilhelmia) were clustered in three distinct clades with high levels of genetic divergence, using maximum likelihood and Bayesian analyses. All specimens of Simulium equinum (L.), Simulium pseudequinum Séguy, and Simulium paraequinum Puri were correctly identified. However, >75% of identifications were ambiguous for Simulium lineatum (Meigen) and Simulium turgaicum Rubtsov (Meigen) because of overlapping intra- and interspecific divergence of the two species and Simulium balcanicum (Enderlein), all three of which are chromosomally similar and nearly isomorphic. Phylogenetic evaluation showed that S. balcanicum, S. equinum, S. pseudequinum, and S. paraequinum were monophyletic, with high bootstrap and posterior probability values, but it also showed that S. lineatum and S. turgaicum were paraphyletic, each clustering in two distinct groups, suggesting the presence of cryptic taxa. Although DNA barcoding provided a partial means of identification and indications of additional biodiversity, other molecular markers are needed to clarify the limits of all pest species of the subgenus Wilhelmia.

The Reliability and Stability of an Inferred Phylogenetic Tree from Empirical Data.

The reliability of a phylogenetic tree obtained from empirical data is usually measured by the bootstrap probability (Pb) of interior branches of the tree. If the bootstrap probability is high for most branches, the tree is considered to be reliable. If some interior branches show relatively low bootstrap probabilities, we are not sure that the inferred tree is really reliable. Here, we propose another quantity measuring the reliability of the tree called the stability of a subtree. This quantity refers to the probability of obtaining a subtree (Ps) of an inferred tree obtained. We then show that if the tree is to be reliable, both Pb and Ps must be high. We also show that Ps is given by a bootstrap probability of the subtree with the closest outgroup sequence, and computer program RESTA for computing the Pb and Ps values will be presented.

A molecular phylogeny of the Laelia alliance (Orchidaceae) and a reassessment of Laelia and Schomburgkia

AbstractWe provide a molecular phylogenetic analysis of the Laelia alliance based on sampling of 20 of an estimated 24 species, and seven plastid regions plus nrITS 1 &amp; 2 analyzed using maximum parsimony, maximum likelihood, and Bayesian inference. Furthermore, we used coded indels to evaluate their effect on the phylogenetic relationships and/or support values (bootstrap and posterior probabilities). Our results confirm the existence of two clades, the long known Laelia (endemic to México at middle to high elevations) and Schomburgkia (a more widely distributed taxon). Both clades were strongly supported by PP values and moderate BS; thus, we preferred to recognize them as separate genera with a reappraised morphology and taxonomic delimitation. The inclusion of coded indels in the analyses was of great utility; we could identify the probable hybrid origin of L. gouldiana and L. halbingeriana. We propose new combinations to accommodate the taxonomic changes resulting from our study. We transfer L. anceps subsp. anceps, L. anceps subsp. dawsonii, L. aurea, L. mottae, and L. rubescens to Schomburgkia, with the second taxon elevated to species level; the molecular evidence together with previous published work on the morphology, phenology and geography of the group support these decisions. Lastly, the recently described genus Encabarcenia is considered as a synonym of Schomburgkia and, as a result, a new combination is proposed.

Phylogenetic position of Magnivitellinum Kloss, 1966 and Perezitrema Baruš & Moravec, 1967 (Trematoda: Plagiorchioidea: Macroderoididae) inferred from partial 28S rDNA sequences, with the establishment of Alloglossidiidae n. fam.

The systematic position of two genera of Macroderoididae McMullen, 1937, Perezitrema Baruš & Moravec, 1967 and Magnivitellinum Kloss, 1966 is reviewed based on a phylogenetic analysis of the interrelationships of 15 species of the family allocated into six genera, along with 44 species of plagiorchioid trematodes, using partial sequences of the 28S rRNA gene. Sequences were analysed through parsimony, maximum likelihood and Bayesian inference. The obtained topologies show Perezitrema as the sister taxon of three species of Macroderoides Pearse, 1924; the latter genus appears to be paraphyletic since another three species are not included in this group. Instead, Magnivitellinum was placed as the sister taxon of Alloglossidium Simer, 1929. These relationships are well supported by high bootstrap and posterior probability values. The resulting trees demonstrate that the family Macroderoididae, as currently conceived in taxonomic treatments, is not monophyletic. Magnivitellinum simplex Kloss, 1966 and Alloglossidium spp. were nested as sister taxa of members of the family Leptophallidae Dayal, 1938, whereas Perezitrema bychowskii Baruš & Moravec, 1967 and species of Macroderoides and Paramacroderoides Venard, 1941 were grouped with Auridistomum chelydrae (Stafford, 1900), a monotypic member of Auridistomidae Stunkard, 1924. Based on our results, a new family, Alloglossidiidae n. fam. was established to accommodate the genera Magnivitellinum and Alloglossidium.

Pinworm diversity in free-ranging howler monkeys (Alouatta spp.) in Mexico: Morphological and molecular evidence for two new Trypanoxyuris species (Nematoda: Oxyuridae)

Two new species of Trypanoxyuris are described from the intestine of free-ranging howler monkeys in Mexico, Trypanoxyuris multilabiatus n. sp. from the mantled howler Alouatta palliata, and Trypanoxyuris pigrae n. sp. from the black howler Alouatta pigra. An integrative taxonomic approach is followed, where conspicuous morphological traits and phylogenetic trees based on DNA sequences are used to test the validity of the two new species. The mitochondrial cytochrome oxidase subunit 1 gene, and the nuclear ribosomal 18S and 28S rRNA genes were used for evolutionary analyses, with the concatenated dataset of all three genes used for maximum likelihood and Bayesian phylogenetic analyses. The two new species of pinworms from howler monkeys were morphologically distinct and formed reciprocally monophyletic lineages in molecular phylogenetic trees. The three species from howler monkeys, T. multilabiatus n. sp., T. pigrae n. sp., and Trypanoxyuris minutus, formed a monophyletic group with high bootstrap and posterior probability support values. Phylogenetic patterns inferred from sequence data support the hypothesis of a close evolutionary association between these primate hosts and their pinworm parasites. The results suggest that the diversity of pinworm parasites from Neotropical primates might be underestimated.

Evaluating the Phylogenetic Status of the Extinct Japanese Otter on the Basis of Mitochondrial Genome Analysis.

The Japanese otter lived throughout four main Japanese islands, but it has not been observed in the wild since 1979 and was declared extinct in 2012. Although recent taxonomic and molecular phylogenetic studies suggest that it should be treated as an independent species, International Union for Conservation of Nature Red List considers it as subspecies of Lutra lutra. Therefore, the taxonomic status of this species needs to be resolved. Here we determined the complete mitochondrial genome of two Japanese otters caught in Kanagawa and Kochi prefectures and five Eurasian otters (L. lutra). We reconstructed a molecular phylogenetic tree to estimate the phylogenetic position of the Japanese otter in Lutrinae using the Japanese otters and the other 11 Lutrinae species on the basis of ND5 (692 bp) and cytochrome b (1,140 bp) sequences. We observed that the two Japanese otters had close relationships with Eurasian otters, forming a monophyletic group (100% bootstrap probability). To elucidate detailed phylogenetic relationships among the Japanese and Eurasian otters, we reconstructed a maximum likelihood tree according to mitochondrial genome sequences (14,740 bp). The Japanese otter (JO1) collected in Kanagawa was deeply nested in the Eurasian otter clade, whereas the Japanese otter (JO2) collected in Kochi formed a distinct independent lineage in the Lutra clade. The estimated molecular divergences time for the ancestral lineages of the Japanese otters was 0.10 Ma (95%: 0.06–0.16 Ma) and 1.27 Ma (95%: 0.98–1.59 Ma) for JO1 and JO2 lineages, respectively. Thus, JO1 was identified as a member of L. lutra; JO2 represented the old Japanese otter lineage, which may be a distinct new species or subspecies of Lutra. We suggest that the ancestral population of the JO2 lineage migrated to Japan via the land bridge that existed between western Japanese islands and Asian continent at 1.27 Ma.

Morphological and molecular characterization of the cyanobiont-bearing dinoflagellate Sinophysis canaliculata from the Canary Islands (eastern central Atlantic)

Morphological and molecular characterization of the cyanobiont-bearing dinoflagellate Sinophysis canaliculata from the Canary Islands (eastern central Atlantic)

Abstract 3290: Limited intra-tumor versus inter-tumor heterogeneity as assessed by proteomic profiling of high-risk neuroblastoma

Abstract Introduction: Tumor heterogeneity at a histological and molecular level has been previously described in neuroblastoma (NB) and therefore concern exists about how representative the molecular profile of a single tissue section can be of an entire tumor. We hypothesized that a single tissue section would provide a representative proteomic signature of the whole tumor. Methods: As part of our effort to assess differential protein expression patterns from two maximally divergent groups with high-risk NB (i.e.: patients with early death from disease versus long-term survivors), we sampled multiple non-adjacent sections from blocks of 5 tumors within our larger cohort of 58 single section tumors. De-identified samples were obtained from the Children's Oncology Group biorepository. Using 5-10μ thick formalin fixed paraffin embedded tumor sections on a glass slide, we extracted 1 microgram of protein, performed tryptic digestion and desalting, and loaded peptides onto a reverse-phase column for separation by nanoflow chromatography. We performed peptide tandem mass spectrometry and protein expression vectors were identified using MaxQuant. The 1,495 most abundant proteins were evaluated across samples. Data were normalized and imputed. Hierarchical clustering analysis was performed using the pvclust R package between subjects with multiple sections and between all samples. We used average clustering criteria, correlation methods for distance, and pairwise covariance measures. Confidence of clusters was measured using approximately unbiased and bootstrap probability (1000 permutations) methods. Results: Intra-tumor distance was significantly shorter than the inter-tumor distance. When testing only subjects with multiple tumor samples, 4 out of 5 samples from the same subject clustered with &amp;gt;95% confidence. The samples from the remaining subject have a confidence &amp;gt;80% but seem to have differing rates of missing data, suggesting experimental errors. When using all samples available, tumor samples from the same subject still clustered together and had a greater correlation than tumor samples from different subjects. A validation cohort of 7 distinct sections from separate blocks of tumors of 3 high-risk patients confirmed that tumors cluster by protein expression. Conclusion and Future Directions: Intra-tumor heterogeneity exists but is significantly less than inter-tumor heterogeneity as assessed by proteomic profiling. We provide rationale to use single tissue section proteomics in ongoing research to define biologic drivers of primary refractory NB, a clinically important subgroup of patients who have highly lethal disease. Citation Format: Raquel Castellanos, Katherine Heaton-Johnson, Jonathan Chung, Edward Nieves, Michael Fremed, Stephen R. Master, Daniel Weiser. Limited intra-tumor versus inter-tumor heterogeneity as assessed by proteomic profiling of high-risk neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3290. doi:10.1158/1538-7445.AM2015-3290