Horizontal Transfer and Gene Loss Shaped the Evolution of Alpha-Amylases in Bilaterians.

The subfamily GH13_1 of alpha-amylases is typical of Fungi, but it is also found in some unicellular eukaryotes (e.g., Amoebozoa, choanoflagellates) and non-bilaterian Metazoa. Since a previous study in 2007, GH13_1 amylases were considered ancestral to the Unikonts, including animals, except Bilateria, such that it was thought to have been lost in the ancestor of this clade. The only alpha-amylases known to be present in Bilateria so far belong to the GH13_15 and 24 subfamilies (commonly called bilaterian alpha-amylases) and were likely acquired by horizontal transfer from a proteobacterium. The taxonomic scope of Eukaryota genomes in databases has been greatly increased ever since 2007. We have surveyed GH13_1 sequences in recent data from ca. 1600 bilaterian species, 60 non-bilaterian animals and also in unicellular eukaryotes. As expected, we found a number of those sequences in non-bilaterians: Anthozoa (Cnidaria) and in sponges, confirming the previous observations, but none in jellyfishes and in Ctenophora. Our main and unexpected finding is that such fungal (also called Dictyo-type) amylases were also consistently retrieved in several bilaterian phyla: hemichordates (deuterostomes), brachiopods and related phyla, some molluscs and some annelids (protostomes). We discuss evolutionary hypotheses possibly explaining the scattered distribution of GH13_1 across bilaterians, namely, the retention of the ancestral gene in those phyla only and/or horizontal transfers from non-bilaterian donors.

Homoplasy-Based Partitioning Outperforms Alternatives in Bayesian Analysis of Discrete Morphological Data.

Bayesian analysis of morphological data is becoming increasingly popular mainly (but not only) because it allows for time-calibrated phylogenetic inference using relaxed morphological clocks and tip dating whenever fossils are available. As with molecular data, recent studies have shown that modeling among-character rate variation (ACRV) in morphological matrices greatly improves phylogenetic inference. In a likelihood framework this may be accomplished, for instance, by employing a hidden Markov model to assign characters to rate categories drawn from a (discretized) $\Gamma$ distribution and/or by partitioning data sets according to rate heterogeneity and estimating per-partition branch lengths, conditioned on a single topology. While the first approach is available in many phylogenetic analysis software, there is still no clear consensus on how to partition data, except perhaps in the simplest cases (e.g., "by codon" partitioning of coding sequences). Additionally, there is a trade-off between improvement in likelihood scores and the number of free parameters in the analysis, which rises quickly with the number of partitions. This trade-off may be dealt with by employing statistics that penalize overfitting of complex models, such as Akaike or Bayesian information criteria, or the more recently introduced stepping-stone method for marginal likelihood approximation. We applied the latter to three distinct matrices of discrete morphological data and demonstrated that sorting characters by homoplasy scores (obtained from implied weighting parsimony analysis) outperformed other partitioning strategies (anatomically-based and PartitionFinder2). The method was in fact so efficient in segregating characters by rates of evolution that no within-partition ACRV modeling was necessary, while among-partition rate variation was adequately accommodated by rate multipliers. We conclude that partitioning by homoplasy is a powerful and easy-to-implement strategy to address ACRV in complex data sets. We provide some guidelines focusing on morphological matrices, although this approach may be also applicable to molecular data sets.

SFREEMAP - A simulation-free tool for stochastic mapping.

BACKGROUND: Stochastic mapping is frequently used in comparative biology to simulate character evolution, enabling the probabilistic computation of statistics such as number of state transitions along a tree and distribution of states in its internal nodes. Common implementations rely on Continuous-time Markov Chain simulations whose parameters are difficult to adjust and subjected to inherent inaccuracy. Thus, researchers must run a large number of simulations in order to obtain adequate estimates. Although execution time tends to be relatively small when simulations are performed on a single tree assumed to be the "true" topology, it may become an issue if analyses are conducted on several trees, such as the ones that make up posterior distributions obtained via Bayesian phylogenetic inference. Working with such distributions is preferable to working with a single tree, for they allow the integration of phylogenetic uncertainty into parameter estimation. In such cases, detailed character mapping becomes less important than parameter integration across topologies. Here, we present an R-based implementation (SFREEMAP) of an analytical approach to obtain accurate, per-branch expectations of numbers of state transitions and dwelling times. We also introduce an intuitive way of visualizing the results by integrating over the posterior distribution and summarizing the parameters onto a target reference topology (such as a consensus or MAP tree) provided by the user. RESULTS: We benchmarked SFREEMAP's performance against make.simmap, a popular R-based implementation of stochastic mapping. SFREEMAP confirmed theoretical expectations outperforming make.simmap in every experiment and reducing computation time of relatively modest datasets from hours to minutes. We have also demonstrated that SFREEMAP returns estimates which were not only similar to the ones obtained by averaging across make.simmap mappings, but also more accurate, according to simulated data. We illustrate our visualization strategy using previously published data on the evolution of coloniality in scleractinian corals. CONCLUSION: SFREEMAP is an accurate and fast alternative to ancestral state reconstruction via simulation-based stochastic mapping.

Towards a phylogenetic classification of Leptothecata (Cnidaria, Hydrozoa).

Leptothecata are hydrozoans whose hydranths are covered by perisarc and gonophores and whose medusae bear gonads on their radial canals. They develop complex polypoid colonies and exhibit considerable morphological variation among species with respect to growth, defensive structures and mode of development. For instance, several lineages within this order have lost the medusa stage. Depending on the author, traditional taxonomy in hydrozoans may be either polyp- or medusa-oriented. Therefore, the absence of the latter stage in some lineages may lead to very different classification schemes. Molecular data have proved useful in elucidating this taxonomic challenge. We analyzed a super matrix of new and published rRNA gene sequences (16S, 18S and 28S), employing newly proposed methods to measure branch support and improve phylogenetic signal. Our analysis recovered new clades not recognized by traditional taxonomy and corroborated some recently proposed taxa. We offer a thorough taxonomic revision of the Leptothecata, erecting new orders, suborders, infraorders and families. We also discuss the origination and diversification dynamics of the group from a macroevolutionary perspective.

Hidden among sea anemones: the first comprehensive phylogenetic reconstruction of the order Actiniaria (Cnidaria, Anthozoa, Hexacorallia) reveals a novel group of hexacorals.

Sea anemones (order Actiniaria) are among the most diverse and successful members of the anthozoan subclass Hexacorallia, occupying benthic marine habitats across all depths and latitudes. Actiniaria comprises approximately 1,200 species of solitary and skeleton-less polyps and lacks any anatomical synapomorphy. Although monophyly is anticipated based on higher-level molecular phylogenies of Cnidaria, to date, monophyly has not been explicitly tested and at least some hypotheses on the diversification of Hexacorallia have suggested that actiniarians are para- or poly-phyletic. Published phylogenies have demonstrated the inadequacy of existing morphological-based classifications within Actiniaria. Superfamilial groups and most families and genera that have been rigorously studied are not monophyletic, indicating conflict with the current hierarchical classification. We test the monophyly of Actiniaria using two nuclear and three mitochondrial genes with multiple analytical methods. These analyses are the first to include representatives of all three currently-recognized suborders within Actiniaria. We do not recover Actiniaria as a monophyletic clade: the deep-sea anemone Boloceroides daphneae, previously included within the infraorder Boloceroidaria, is resolved outside of Actiniaria in several of the analyses. We erect a new genus and family for B. daphneae, and rank this taxon incerti ordinis. Based on our comprehensive phylogeny, we propose a new formal higher-level classification for Actiniaria composed of only two suborders, Anenthemonae and Enthemonae. Suborder Anenthemonae includes actiniarians with a unique arrangement of mesenteries (members of Edwardsiidae and former suborder Endocoelantheae). Suborder Enthemonae includes actiniarians with the typical arrangement of mesenteries for actiniarians (members of former suborders Protantheae, Ptychodacteae, and Nynantheae and subgroups therein). We also erect subgroups within these two newly-erected suborders. Although some relationships among these newly-defined groups are still ambiguous, morphological and molecular results are consistent enough to proceed with a new higher-level classification and to discuss the putative functional and evolutionary significance of several morphological attributes within Actiniaria.

Toward a natural classification: phylogeny of acontiate sea anemones (Cnidaria, Anthozoa, Actiniaria).

Acontia-nematocyst-dense, thread-like extensions of the mesenterial filaments-are the characteristic feature of the actiniarian group Acontiaria. Phylogenetic analyses have shown that acontiate taxa form a clade that also includes some taxa without acontia. We analyse five molecular markers from 85 actiniarians to explore the phylogenetic relationships among families in Acontiaria, including acontiate species assigned to other higher taxa and species without acontia that have been allied to Acontiaria. Based on our results, we redefine the group to accommodate those lineages that have lost acontia, and formalize it as superfamily Metridioidea. Based on stable and well supported clades, we resurrect Phelliidae and Amphianthidae, redefine Kadosactinidae and Actinoscyphiidae, and move two species to new genera: that previously termed Sagartiogeton erythraios belongs in Jasonactis gen. nov.; and that previously termed Anthosactis pearseae belongs in Ostiactis gen. nov., type genus of Ostiactinidae fam. nov. We also synonymized Halcampoididae and Halcampidae (as Halcampidae) and Andvakiidae and Isophelliidae (as Andvakiidae). The results of our phylogenetic analyses indicate that the diagnostic morphological characters used in the family-level taxonomy of acontiate actiniarians such as the nematocysts of the acontia, the marginal sphincter muscle, and mesenteries divisible into macro- and micro-cnemes, have to be revisited, as these features are highly homoplasious.

Quantifying complex shapes: elliptical fourier analysis of octocoral sclerites.

Species descriptions of most alcyonacean octocorals rely heavily on the morphology of sclerites, the calcium carbonate spicules embedded in the soft tissue. Sclerites provide taxonomic characters for species delineation but require qualitative descriptions, which introduce ambiguities in recognizing morphological features. Elliptical Fourier analysis of the outline of sclerites was used to quantify the morphology of eight species of gorgoniid octocoral in the genus Pseudopterogorgia. Sclerites from one to seven colonies of each species were compared. Scaphoids and spindles were examined separately; rods and octoradiates were excluded from the analyses because of their morphologic similarity across all species. Discriminant analysis of elliptical Fourier descriptors (EFDs) was used to determine whether the elliptical Fourier analysis could be used to identify the specimens. Sclerites were highly variable even within a single colony. Correct species assignments of individual sclerites were greater than 50% for both scaphoids and spindles. Species assignments based on averages of the EFDs for each colony approached 90%. Elliptical Fourier analysis quantifies morphological differences between species and measures colony variance in sclerite size and shape among colonies and species. Phylogenetic analysis based on EFDs did not capture monophyletic groups. The quantification of complex shapes such as sclerites provides an important tool in alpha taxonomy but may be less useful in phylogenetic analyses.

The phylogenetic position of Myxozoa: exploring conflicting signals in phylogenomic and ribosomal data sets.

Myxozoans are a diverse group of microscopic endoparasites that have been the focus of much controversy regarding their phylogenetic position. Two dramatically different hypotheses have been put forward regarding the placement of Myxozoa within Metazoa. One hypothesis, supported by ribosomal DNA (rDNA) data, place Myxozoa as a sister taxon to Bilateria. The alternative hypothesis, supported by phylogenomic data and morphology, place Myxozoa within Cnidaria. Here, we investigate these conflicting hypotheses and explore the effects of missing data, model choice, and inference methods, all of which can have an effect in placing highly divergent taxa. In addition, we identify subsets of the data that most influence the placement of Myxozoa and explore their effects by removing them from the data sets. Assembling the largest taxonomic sampling of myxozoans and cnidarians to date, with a comprehensive sampling of other metazoans for 18S and 28S nuclear rDNA sequences, we recover a well-supported placement of Myxozoa as an early diverging clade of Bilateria. By conducting parametric bootstrapping, we find that the bilaterian placement of Buddenbrockia could not alone be explained by long-branch attraction. After trimming a published phylogenomic data set, to circumvent problems of missing data, we recover the myxozoan Buddenbrockia plumatellae as a medusozoan cnidarian. In further explorations of these data sets, we find that removal of just a few identified sites under a maximum likelihood criterion employing the Whelan and Goldman amino acid substitution model changes the placement of Buddenbrockia from within Cnidaria to the alternative hypothesis at the base of Bilateria. Under a Bayesian criterion employing the CAT model, the cnidarian placement is more resilient to data removal, but under one test, a well-supported early diverging bilaterian position for Buddenbrockia is recovered. Our results confirm the existence of two relatively stable placements for myxozoans and demonstrate that conflicting signal exists not only between the two types of data but also within the phylogenomic data set. These analyses underscore the importance of careful model selection, taxon and data sampling, and in-depth data exploration when investigating the phylogenetic placement of highly divergent taxa.

Repeated loss of coloniality and symbiosis in scleractinian corals.

The combination of coloniality and symbiosis in Scleractinia is thought to confer competitive advantage over other benthic invertebrates, and it is likely the key factor for the dominance of corals in tropical reefs. However, the extant Scleractinia are evenly split between zooxanthellate and azooxanthellate species. Most azooxanthellate species are solitary and nearly absent from reefs, but have much wider geographic and bathymetric distributions than reef corals. Molecular phylogenetic analyses have repeatedly recovered clades formed by colonial/zooxanthellate and solitary/azooxanthellate taxa, suggesting that coloniality and symbiosis were repeatedly acquired and/or lost throughout the history of the Scleractinia. Using Bayesian ancestral state reconstruction, we found that symbiosis was lost at least three times and coloniality lost at least six times, and at least two instances in which both characters were lost. All of the azooxanthellate lineages originated from ancestors that were reconstructed as symbiotic, corroborating the onshore-offshore diversification trend recorded in marine taxa. Symbiotic sister taxa of two of these descendant lineages are extant in Caribbean reefs but disappeared from the Mediterranean before the end of the Miocene, whereas extant azooxanthellate lineages have trans-Atlantic distributions. Thus, the phyletic link between reef and nonreef communities may have played an important role in the dynamics of extinction and recovery that marks the evolutionary history of scleractinians, and some reef lineages may have escaped local extinction by diversifying into offshore environments. However, this macroevolutionary mechanism offers no hope of mitigating the effects of climate change on coral reefs in the next century.

Rethinking the phylogeny of scleractinian corals: a review of morphological and molecular data.

Scleractinian corals, which include the architects of coral reefs, are found throughout the world's oceans and have left a rich fossil record over their 240 million year history. Their classification has been marked by confusion but recently developed molecular and morphological tools are now leading to a better understanding of the evolutionary history of this important group. Although morphological characters have been the basis of traditional classification in the group, they are relatively few in number. In addition, our current understanding of skeletal growth and homology is limited, and homoplasy is rampant, limiting the usefulness of morphological phylogenetics. Molecular phylogenetic hypotheses for the order, which have been primarily focused on reef-building corals, differ significantly from traditional classification. They suggest that the group is represented by two major lineages and do not support the monophyly of traditional suborders and most traditional families. It appears that once a substantial number of azooxanthellate taxa are included in molecular phylogenetic analyses, basal relationships within the group will be clearly defined. Understanding of relationships at lower taxonomic levels will be best clarified by combined analyses of morphological and molecular characters. Molecular phylogenies are being used to inform our understanding of the evolution of morphological characters in the Scleractinia. Better understanding of the evolution of these characters will help to integrate the systematics of fossil and extant taxa. We demonstrate how the combined use of morphological and molecular tools holds great promise for ending confusion in scleractinian systematics.

Evolutionary relationships among scyphozoan jellyfish families based on complete taxon sampling and phylogenetic analyses of 18S and 28S ribosomal DNA.

A stable phylogenetic hypothesis for families within jellyfish class Scyphozoa has been elusive. Reasons for the lack of resolution of scyphozoan familial relationships include a dearth of morphological characters that reliably distinguish taxa and incomplete taxonomic sampling in molecular studies. Here, we address the latter issue by using maximum likelihood and Bayesian methods to reconstruct the phylogenetic relationships among all 19 currently valid scyphozoan families, using sequence data from two nuclear genes: 18S and 28S rDNA. Consistent with prior morphological hypotheses, we find strong evidence for monophyly of subclass Discomedusae, order Coronatae, rhizostome suborder Kolpophorae and superfamilies Actinomyariae, Kampylomyariae, Krikomyariae, and Scapulatae. Eleven of the 19 currently recognized scyphozoan families are robustly monophyletic, and we suggest recognition of two new families pending further analyses. In contrast to long-standing morphological hypotheses, the phylogeny shows coronate family Nausithoidae, semaeostome family Cyaneidae, and rhizostome suborder Daktyliophorae to be nonmonophyletic. Our analyses neither strongly support nor strongly refute monophyly of order Rhizostomeae, superfamily Inscapulatae, and families Ulmaridae, Catostylidae, Lychnorhizidae, and Rhizostomatidae. These taxa, as well as familial relationships within Coronatae and within rhizostome superfamily Inscapulatae, remain unclear and may be resolved by additional genomic and taxonomic sampling. In addition to clarifying some historically difficult taxonomic questions and highlighting nodes in particular need of further attention, the molecular phylogeny presented here will facilitate more robust study of phenotypic evolution in the Scyphozoa, including the evolution characters associated with mass occurrences of jellyfish.

Phylogeography and morphological variation of the branching octocoral Pseudopterogorgia elisabethae.

Coral reef anthozoans exhibit extensive morphological variation across and within environmental clines making it difficult to define species boundaries. The relative contributions of genetic variation and ecophenotypic plasticity to the observed phenotypic variation are unknown in most cases. The branching octocoral Pseudopterogorgia elisabethae is widely distributed throughout the Caribbean and colonies vary in appearance within and among populations. We performed genetic and morphological analyses of P.elisabethae from multiple locations within the Bahamas, as well as a Florida Keys and a distant western Caribbean location to determine the levels of genetic and morphological variation (colony form and sclerites characteristics) across populations from different sites, and assessed whether there was congruence between the genetic and morphological variation. Based on sequences of the internal transcribed spacer region of the ribosomal DNA, four groups were found that generally correspond to the geography of the Bahamas. Morphometric analysis of branch and branchlet characteristics indicated that colonies from two of the sites differed from the rest, but there was no clear correspondence between genetic and morphological variation. In general, there were no qualitative differences in the sclerites from the different populations. However, there were some differences in the dimensions of scaphoids and rods of colonies from different sites. This study has shown that P. elisabethae displays genetic and morphologic variation among some populations of the Bahamas, Florida and San Andres, Colombia. P. elisabethae is harvested in the Bahamas and these findings should be considered in management plans and conservation efforts for the species.

Identifying functional neighborhoods within the cell nucleus: proximity analysis of early S-phase replicating chromatin domains to sites of transcription, RNA polymerase II, HP1gamma, matrin 3 and SAF-A.

Higher order chromatin organization in concert with epigenetic regulation is a key process that determines gene expression at the global level. The organization of dynamic chromatin domains and their associated protein factors is intertwined with nuclear function to create higher levels of functional zones within the cell nucleus. As a step towards elucidating the organization and dynamics of these functional zones, we have investigated the spatial proximities among a constellation of functionally related sites that are found within euchromatic regions of the cell nucleus including: HP1gamma, nascent transcript sites (TS), active DNA replicating sites in early S-phase (PCNA) and RNA polymerase II sites. We report close associations among these different sites with proximity values specific for each combination. Analysis of matrin 3 and SAF-A sites demonstrates that these nuclear matrix proteins are highly proximal with the functionally related sites as well as to each other and display closely aligned and overlapping regions following application of the minimal spanning tree (MST) algorithm to visualize higher order network-like patterns. Our findings suggest that multiple factors within the nuclear microenvironment collectively form higher order combinatorial arrays of function. We propose a model for the organization of these functional neighborhoods which takes into account the proximity values of the individual sites and their spatial organization within the nuclear architecture.

Ants and their effects on an insect herbivore community associated with the inflorescences of Byrsonima crassifolia ( Linnaeus ) H B K ( Malpighiaceae )

Os efeitos de formigas na comunidade de insetos em inflorescências de Byrsonima crassifolia (Malpighiaceae) foram testados em um experimento de exclusão em uma vegetacão de cerrado no Sudeste do Brasil. Quarenta e quatro espécies de insetos (23 famílias) e nove espécies de formigas (seis gêneros e três subfamílias) foram encontradas nas inflorescências de B. crassifolia. A exclusão das formigas, principalmente de Camponotus sericeiventris e de Camponotus spp. reduziu a populacão de membracídeos para 20por cento da abundância original. Exclusão das formigas e o tempo influenciaram a abundância de insetos mastigadores (exclusão, P<0,001; tempo, P<0,002) e sugadores (exclusão, P<0,02; tempo, P<0,01). Insetos mastigadores e sugadores foram encontrados duas vezes mais em inflorescências com formigas excluídas quando comparados com inflorescências controle (P<0,001). Insetos sugadores foram encontrados 1,5 vezes mais em inflorescências com formigas excluídas do que no controle. Apenas o tempo influenciou significativamente a riqueza de insetos mastigadores e sugadores associados com as inflorescências de B. crassifolia. Inflorescências em ramos controle foram significativamente menos atacadas por herbívoros do que inflorescências em ramos com formigas excluídas (P<0,001). Portanto, estes resultados sugerem que a presenca das formigas influencia a estrutura da comunidade de insetos herbívoros associados com B. crassifolia.