Predicting the Impact of Describing New Species on Phylogenetic Patterns.

Although our inventory of Earth's biodiversity remains incomplete, we still require analyses using the Tree of Life to understand evolutionary and ecological patterns. Because incomplete sampling may bias our inferences, we must evaluate how future additions of newly discovered species might impact analyses performed today. We describe an approach that uses taxonomic history and phylogenetic trees to characterize the impact of past species discoveries on phylogenetic knowledge using patterns of branch-length variation, tree shape, and phylogenetic diversity. This provides a framework for assessing the relative completeness of taxonomic knowledge of lineages within a phylogeny. To demonstrate this approach, we use recent large phylogenies for amphibians, reptiles, flowering plants, and invertebrates. Well-known clades exhibit a decline in the mean and range of branch lengths that are added each year as new species are described. With increased taxonomic knowledge over time, deep lineages of well-known clades become known such that most recently described new species are added close to the tips of the tree, reflecting changing tree shape over the course of taxonomic history. The same analyses reveal other clades to be candidates for future discoveries that could dramatically impact our phylogenetic knowledge. Our work reveals that species are often added non-randomly to the phylogeny over multiyear time-scales in a predictable pattern of taxonomic maturation. Our results suggest that we can make informed predictions about how new species will be added across the phylogeny of a given clade, thus providing a framework for accommodating unsampled undescribed species in evolutionary analyses.

A combined approach to the phylogeny of Cephalopoda (Mollusca).

Cephalopoda represents a highly diverse group of molluscs, ranging in habitat from coastal regions to deep benthic waters. While cephalopods remain at the forefront of modern biology, in providing insight into fields such as neurobiology and population genetics, little is known about the relationships within the group. This study provides a comprehensive phylogenetic analysis of Cephalopoda (Mollusca) using a combination of molecular and morphological data. Four loci (three nuclear 18S rRNA, fragments of 28S rRNA and histone H3 and one mitochondrial cytochrome c oxidase subunit I) were combined with 101 morphological characters to test the relationships of 60 species of cephalopods, with emphasis within Decabrachia (squids and cuttlefishes). Individual and combined data sets were analyzed using the direct optimization method, with parsimony as the optimality criterion. Analyses were repeated for 12 different parameter sets accounting for a range of indel/change and transversion/transition cost ratios. Most analyses support the monophyly of Cephalopoda, Nautiloidea, Coleoidea and Decabrachia, however, the monophyly of Octobrachia was refuted due to the lack of support for a Cirroctopoda + Octopoda group. When analyzing all molecular evidence in combination and for total evidence analyses, Vampyromorpha formed the sister group to Decabrachia under the majority of parameters, while morphological data and some individual data sets supported a sister relationship between Vampyromorpha and Octobrachia. Within Decabrachia, a relationship between the sepioids Idiosepiida, Sepiida, Sepiolida and the teuthid Loliginidae was supported. Spirulida fell within the teuthid group in most analyses, further rendering Teuthida paraphyletic. Relationships within Decabrachia and specifically Oegopsida were found to be highly parameter-dependent.

[Diagnosis by directed mutagenesis of a mutation at the hMSH2 gene associated with hereditary nonpolyposis colorectal cancer]. / Diagnóstico por mutagénesis dirigida de una mutación en el gen hMSH2 vinculada a cáncer colorrectal hereditario no poliposo.

Hereditary nonpolyposis colorectal cancer (NHPCC) is the most common form of inherited colon cancer and one of the most frequent autosomal dominant disorders. HNPCC presents an early onset of colorectal cancer (< 50 years), proximal localization of the colonic tumors, and high risk of developing multiple primary colorectal tumors as well as extracolonic tumors. This disease is caused by mutations in at least four DNA mismatch repair genes, (hMSH2, hMLH1, hPMS1 and hPMS2) and estimations indicate that it affects 1:200-1:2,000 people in the Western populations. The identification of the genes responsible for HNPCC has prompted the search for mutations in affected individuals. DNA from an affected member of a family was sent to a Dutch HNPCC Diagnosis Centre. This Centre reported a germinal mutation, which introduces a premature stopcodon and causes the production of a truncated protein. This particular mutation has not been previously registered in the database of mutations related to this disease. After the identification of the mutation in the index patient, we have developed a quick and efficient procedure for detecting mutations in the rest of the family. The methodology is based on the amplification of the exon 13 in the hMSH2 gene using a forward primer that abuts the mutation site and introduces the cutting sequence of the enzyme Haelll++ only in the wild type allele. At present, seventeen members of the family have been diagnosed and nine have been found to be affected. The methodology is simple, specific, sensitive, inexpensive and applicable in low complexity laboratories.

A NGS approach to the encrusting Mediterranean sponge Crella elegans (Porifera, Demospongiae, Poecilosclerida): transcriptome sequencing, characterization and overview of the gene expression along three life cycle stages.

Sponges can be dominant organisms in many marine and freshwater habitats where they play essential ecological roles. They also represent a key group to address important questions in early metazoan evolution. Recent approaches for improving knowledge on sponge biological and ecological functions as well as on animal evolution have focused on the genetic toolkits involved in ecological responses to environmental changes (biotic and abiotic), development and reproduction. These approaches are possible thanks to newly available, massive sequencing technologies-such as the Illumina platform, which facilitate genome and transcriptome sequencing in a cost-effective manner. Here we present the first NGS (next-generation sequencing) approach to understanding the life cycle of an encrusting marine sponge. For this we sequenced libraries of three different life cycle stages of the Mediterranean sponge Crella elegans and generated de novo transcriptome assemblies. Three assemblies were based on sponge tissue of a particular life cycle stage, including non-reproductive tissue, tissue with sperm cysts and tissue with larvae. The fourth assembly pooled the data from all three stages. By aggregating data from all the different life cycle stages we obtained a higher total number of contigs, contigs with blast hit and annotated contigs than from one stage-based assemblies. In that multi-stage assembly we obtained a larger number of the developmental regulatory genes known for metazoans than in any other assembly. We also advance the differential expression of selected genes in the three life cycle stages to explore the potential of RNA-seq for improving knowledge on functional processes along the sponge life cycle.

Exploring the behavior of POY, a program for direct optimization of molecular data.

The performance of the computer program for phylogenetic analysis, POY, and its two implemented methods, "optimization alignment" and "fixed-states optimization," are explored for four data sets. Four gap costs are analyzed for every partition; some of the partitions (the 18S rRNA) are treated as a single fragment or in increasing fragments of 3, 10, and 30. Comparisons within and among methods are undertaken according to gap cost, number of fragments in which the sequences are divided, tree length, character congruence, topological congruence, primary homology statements, and computation time.

The position of arthropods in the animal kingdom: a search for a reliable outgroup for internal arthropod phylogeny.

Morphological evidence for the phylogeny of the animal kingdom has been discussed by numerous authors. DNA sequencing and phylogenetic methods for analyzing these data are alternative approaches to animal phylogeny, but the phenomenon of long branch attraction and poor taxonomic sampling have caused misinterpretations of metazoan relationships. Here we report a cladistic approach to metazoan evolution including 133 18S rDNA sequences of 31 animal phyla. Despite the difficulties associated with analyzing large data sets, our data suggest that the Bilateria and Protostomia are monophyletic. The internal phylogeny of the protostomes is divided into two main clades. One clade includes the classical protostome worms (annelids, sipunculans, echiurans, pogonophorans, and vestimentiferans), mollusks, nemerteans, "lophophorates," platyhelminths, rotiferans, and acanthocephalans, although the internal resolution of the clade is very low. The second clade includes arthropods and other molting animals: tardigrades, onychophorans, nematodes, nematomorphans, kinorhynchs, and priapulans. The arthropods and related phyla lack a ciliated larvae, lack a multiciliate (locomotory) epithelium, and share many features, notably, a reduced coelomic cavity and the presence of a cuticle which molts. The use of these outgroups within the molting clade to root arthropod phylogenies is recommended instead of using annelids or other spiralians. The data are quite conclusive in those phyla with a good taxonomic sampling (i.e., platyhelminths and arthropods).

Phylogeographical history of the sponge Crambe crambe (Porifera, Poecilosclerida): range expansion and recent invasion of the Macaronesian islands from the Mediterranean Sea.

We studied sequence variation in the nuclear ribosomal internal transcribed spacers (ITS-1 and ITS-2) in 111 individuals from 11 populations/localities of the sponge Crambe crambe across the core species range in the western Mediterranean Sea and Atlantic Ocean. We report the first confirmed instance of intragenomic variation in sponges. Phylogeographical, nested clade and population genetic analyses were used to elucidate the species' evolutionary history. The study revealed highly structured populations affected by restricted gene flow and isolation-by-distance. A contiguous range expansion in the whole distribution area of the sponge was inferred. Phylogenetic analyses indicate a recent origin of most sequence types that could be explained by a recent origin of the species or a by recent bottleneck event in the studied area. A recent expansion of the distribution range to the Macaronesian region from the Mediterranean Sea was also detected, suggesting that C. crambe was recently introduced from the Mediterranean Sea to the Atlantic Ocean via human-mediated transport, and that the pattern observed is not the result of a natural biogeographical relationship between these zones.

On gaps.

Gaps result from the alignment of sequences of unequal length during primary homology assessment. Viewed as character states originating from particular biological events (mutation), gaps contain historical information suitable for phylogenetic analysis. The effect of gaps as a source of phylogenetic data is explored via sensitivity analysis and character congruence among different data partitions. Example data sets are provided to show that gaps contain important phylogenetic information not recovered by those methods that omit gaps in their calculations. However, gap cost schemes are arbitrary (although they must be explicit) and thus data exploration is a necessity of molecular analyses, while character congruence is necessary as an external criterion for hypothesis decision.

Evidence that two types of 18S rDNA coexist in the genome of Dugesia (Schmidtea) mediterranea (Platyhelminthes, Turbellaria, Tricladida).

Sequences of 18S ribosomal DNA (rDNA) are increasingly being used to infer phylogenetic relationships among living taxa. Although the 18S rDNA belongs to a multigene family, all its copies are kept homogeneous by concerted evolution (Dover 1982; Hillis and Dixon 1991). To date, there is only one well-characterized exception to this rule, the protozoan Plasmodium (Gunderson et al. 1987; Waters, Syin, and McCutchan 1989; Qari et al. 1994). Here we report the 1st case of 18S rDNA polymorphism within a metazoan species. Two types (I and II) of 18S rDNA have been found and sequenced in the platyhelminth Dugesia (Schmidtea) mediterranea (Turbellaria, Seriata, Tricladida). Southern blot analysis suggested that both types of rDNA are present in the genome of this flatworm. This was confirmed through sequence comparisons and phylogenetic analysis using the neighbor-joining method and bootstrap test. Although secondary structure analysis suggests that both types are functional, only type I seems to be transcribed to RNA, as demonstrated by Northern blot analysis. The finding of different types of 18S rDNAs in a single genome stresses the need for analyzing a large number of clones whenever 18S sequences obtained by PCR amplification and cloning are being used in phylogenetic reconstruction.

Arthropod phylogeny based on eight molecular loci and morphology.

The interrelationships of major clades within the Arthropoda remain one of the most contentious issues in systematics, which has traditionally been the domain of morphologists. A growing body of DNA sequences and other types of molecular data has revitalized study of arthropod phylogeny and has inspired new considerations of character evolution. Novel hypotheses such as a crustacean-hexapod affinity were based on analyses of single or few genes and limited taxon sampling, but have received recent support from mitochondrial gene order, and eye and brain ultrastructure and neurogenesis. Here we assess relationships within Arthropoda based on a synthesis of all well sampled molecular loci together with a comprehensive data set of morphological, developmental, ultrastructural and gene-order characters. The molecular data include sequences of three nuclear ribosomal genes, three nuclear protein-coding genes, and two mitochondrial genes (one protein coding, one ribosomal). We devised new optimization procedures and constructed a parallel computer cluster with 256 central processing units to analyse molecular data on a scale not previously possible. The optimal 'total evidence' cladogram supports the crustacean-hexapod clade, recognizes pycnogonids as sister to other euarthropods, and indicates monophyly of Myriapoda and Mandibulata.

First molecular evidence for the existence of a Tardigrada + Arthropoda clade.

The complete 18S rDNA gene sequence of Macrobiotus group hufelandi (Tardigrada) was obtained and aligned with 18S rDNA and rRNA gene sequences of 24 metazoans (mainly protostomes). Discrete character (maximum-parsimony) and distance (neighbor-joining) methods were used to infer their phylogeny. The evolution of bootstrap proportions with sequence length (pattern of resolved nodes, PRN) was studied to test the resolution of the nodes in neighbor-joining trees. The results show that arthropods are monophyletic. Tardigrades represent the sister group of arthropods (in parsimony analyses) or they are related with crustaceans (distance analysis and PRN). Arthropoda are divided into two main evolutionary lines, the Hexapoda + Crustacea line (weakly supported), and the Myriapoda + Chelicerata line. The Hexapoda + Crustacea line includes Pentastomida, but the internal resolution is far from clear. The Insecta (Ectognatha) are monophyletic, but no evidence for the monophyly of Hexapoda is found. The Chelicerata are a monophyletic group and the Myriapoda cluster close to Arachnida. Overall, the results obtained represent the first molecular evidence for a Tardigrada + Arthropoda clade. In addition, the congruence between molecular phylogenies of the Arthropoda from other authors and this obtained here indicates the need to review those obtained solely on morphological characters.

Triploblastic relationships with emphasis on the acoelomates and the position of Gnathostomulida, Cycliophora, Plathelminthes, and Chaetognatha: a combined approach of 18S rDNA sequences and morphology.

Triploblastic relationships were examined in the light of molecular and morphological evidence. Representatives for all triploblastic "phyla" (except Loricifera) were represented by both sources of phylogenetic data. The 18S ribosomal (rDNA) sequence data for 145 terminal taxa and 276 morphological characters coded for 36 supraspecific taxa were combined in a total evidence regime to determine the most consistent picture of triploblastic relationships for these data. Only triploblastic taxa are used to avoid rooting with distant outgroups, which seems to happen because of the extreme distance that separates diploblastic from triploblastic taxa according to the 18S rDNA data. Multiple phylogenetic analyses performed with variable analysis parameters yield largely inconsistent results for certain groups such as Chaetognatha, Acoela, and Nemertodermatida. A normalized incongruence length metric is used to assay the relative merit of the multiple analyses. The combined analysis having the least character incongruence yields the following scheme of relationships of four main clades: (1) Deuterostomia [((Echinodermata + Enteropneusta) (Cephalochordata (Urochordata + Vertebrata)))]; (2) Ecdysozoa [(((Priapulida + Kinorhyncha) (Nematoda + Nematomorpha)) ((Onychophora + Tardigrada) Arthropoda))]; (3) Trochozoa [((Phoronida + Brachiopoda) (Entoprocta (Nemertea (Sipuncula (Mollusca (Pogonophora (Echiura + Annelida)))))))]; and (4) Platyzoa [((Gnathostomulida (Cycliophora + Syndermata)) (Gastrotricha + Plathelminthes))]. Chaetognatha, Nemertodermatida, and Bryozoa cannot be assigned to any one of these four groups. For the first time, a data analysis recognizes a clade of acoelomates, the Platyzoa (sensu Cavalier-Smith, Biol. Rev. 73:203-266, 1998). Other relationships that corroborate some morphological analyses are the existence of a clade that groups Gnathostomulida + Syndermata (= Gnathifera), which is expanded to include the enigmatic phylum Cycliophora, as sister group to Syndermata.

Phylogeny of the arachnid order Opiliones (Arthropoda) inferred from a combined approach of complete 18S and partial 28S ribosomal DNA sequences and morphology.

The phylogenetic relationships among the main evolutionary lines of the arachnid order Opiliones were investigated by means of molecular (complete 18S rDNA and the D3 region of the 28S rDNA genes) and morphological data sets. Equally and differentially weighted parsimony analyses of independent and combined data sets provide evidence for the monophyly of the Opiliones. In all the analyses, the internal relationships of the group coincide in the monophyly of the following main groups: Cyphophthalmi, Eupnoi Palpatores, Dyspnoi Palpatores, and Laniatores. The Cyphophthalmi are monophyletic and sister to a clade that includes all the remaining opilionid taxa (=Phalangida). Within the Phalangida the most supported hypothesis suggests that Palpatores are paraphyletic, as follows: (Eupnoi (Dyspnoi + Laniatores)), but the alternative hypothesis (Laniatores (Eupnoi + Dyspnoi)) is more parsimonious in some molecular data analyses. Relationships within the four main clades are also addressed. Evolution of some morphological characters is discussed, and plesiomorphic states of these characters are evaluated using molecular data outgroup polarization. Finally, Martens' hypothesis of opilionid evolution is assessed in relation to our results.

Internal phylogeny of the Chilopoda (Myriapoda, Arthropoda) using complete 18S rDNA and partial 28S rDNA sequences.

The internal phylogeny of the 'myriapod' class Chilopoda is evaluated for 12 species belonging to the five extant centipede orders, using 18S rDNA complete gene sequence and 28S rDNA partial gene sequence data. Equally and differentially weighted parsimony, neighbour-joining and maximum-likelihood were used for phylogenetic reconstruction, and bootstrapping and branch support analyses were performed to evaluate tree topology stability. The results show that the Chilopoda constitute a monophyletic group that is divided into two lines, Notostigmophora (= Scutigeromorpha) and Pleurostigmophora, as found in previous morphological analyses. The Notostigmophora are markedly modified for their epigenic mode of life. The first offshoot of the Pleurostigmophora are the Lithobiomorpha, followed by the Craterostigmomorpha and by the Epimorpha s. str. (= Scolopendromorpha + Geophilomorpha), although strong support for the monophyly of the Epimorpha s. lat. (= Craterostigmomorpha + Epimorpha s. str.) is only found in the differentially weighted parsimony analysis.

Interferencia de valores bajos de autoanticuerpos antitiroglobulina en la determinación de tiroglobulina / Interferences of low levels of thyroglobulin antibodies on thyroglobulin determination

Antecedentes. La tiroglobulina es sintetizada exclusivamente en las células foliculares tiroideas, y su medición sérica se utiliza en el seguimiento del cáncer diferenciado de tiroides tratado. La presencia de autoanticuerpos antitiroglobulina (AcTG) en el paciente dificulta la detección exacta de tiroglobulina por métodos inmunoanalíticos. Métodos. En este trabajo se evaluó el efecto de los AcTG, determinados por aglutinación de partículas (AGP) y por un método inmunométrico quimioluminiscente (IMQ), en la valoración de la tiroglobulina medida por dos técnicas de alta sensibilidad, pero basadas en distintos principios: inmunocompetencia (RIA) e inmunometría (quimioluminiscencia [QMO]). Se evaluó en el suero de 60 pacientes la tiroglobulina antes y después del agregado de una cantidad conocida de tiroglobulina, y se calculó el porcentaje de recuperación de la tiroglobulina agregada a cada suero. Resultados. Los resultados mostraron que la presencia de AcTG, aun en bajas concentraciones detectadas por IMQ pero no por AGP, interfiere en la determinación de tiroglobulina, tanto por RIA como por QMO. Conclusión. La determinación de tiroglobulina como marcador para el seguimiento del cáncer diferenciado de tiroides debe ir acompañada por la medición de AcTG por un método de alta sensibilidad. En caso de detectarse AcTG, aun en bajas concentraciones, la determinación de tiroglobulina debe ser considerada críticamente por la posibilidad de interferencia con los métodos de determinación (AU)

Diagnóstico por mutagénesis dirigida de una mutación en el gen hMSH2 vinculada a cáncer colorrectal hereditario no poliposo. / [Diagnosis by directed mutagenesis of a mutation at the hMSH2 gene associated with hereditary nonpolyposis colorectal cancer]

Hereditary nonpolyposis colorectal cancer (NHPCC) is the most common form of inherited colon cancer and one of the most frequent autosomal dominant disorders. HNPCC presents an early onset of colorectal cancer (< 50 years), proximal localization of the colonic tumors, and high risk of developing multiple primary colorectal tumors as well as extracolonic tumors. This disease is caused by mutations in at least four DNA mismatch repair genes, (hMSH2, hMLH1, hPMS1 and hPMS2) and estimations indicate that it affects 1:200-1:2,000 people in the Western populations. The identification of the genes responsible for HNPCC has prompted the search for mutations in affected individuals. DNA from an affected member of a family was sent to a Dutch HNPCC Diagnosis Centre. This Centre reported a germinal mutation, which introduces a premature stopcodon and causes the production of a truncated protein. This particular mutation has not been previously registered in the database of mutations related to this disease. After the identification of the mutation in the index patient, we have developed a quick and efficient procedure for detecting mutations in the rest of the family. The methodology is based on the amplification of the exon 13 in the hMSH2 gene using a forward primer that abuts the mutation site and introduces the cutting sequence of the enzyme Haelll++ only in the wild type allele. At present, seventeen members of the family have been diagnosed and nine have been found to be affected. The methodology is simple, specific, sensitive, inexpensive and applicable in low complexity laboratories.