Phylogenetic analyses of marine sponges within the order Verongida: a comparison of morphological and molecular data

Abstract. Because the taxonomy of marine sponges is based primarily on morphological characters that can display a high degree of phenotypic plasticity, current classifications may not always reflect evolutionary relationships. To assess phylogenetic relationships among sponges in the order Verongida, we examined 11 verongid species, representing six genera and four families. We compared the utility of morphological and molecular data in verongid sponge systematics by comparing a phylogeny constructed from a morphological character matrix with a phylogeny based on nuclear ribosomal DNA sequences. The morphological phylogeny was not well resolved below the ordinal level, likely hindered by the paucity of characters available for analysis, and the potential plasticity of these characters. The molecular phylogeny was well resolved and robust from the ordinal to the species level. We also examined the morphology of spongin fibers to assess their reliability in verongid sponge taxonomy. Fiber diameter and pith content were highly variable within and among species. Despite this variability, spongin fiber comparisons were useful at lower taxonomic levels (i.e., among congeneric species); however, these characters are potentially homoplasic at higher taxonomic levels (i.e., between families). Our molecular data provide good support for the current classification of verongid sponges, but suggest a re-examination and potential reclassification of the genera Aiolochroia and Pseudoceratina . The placements of these genera highlight two current issues in morphology-based sponge taxonomy: intermediate character states and undetermined character polarity.     

Phylogeography and morphological variability in land snails: the Sicilian Marmorana (Pulmonata, Helicidae)

Land snails have long been recognized as suitable organisms for studying phenotypic differentiation and phylogeny in relation to geographical distribution. Morphological data (shell and anatomy biometry on different geographical scales) and partial sequences from mitochondrial genes (cytochrome oxidase subunit I , 16S rDNA) were used to test whether morphological patterns match phylogeny in a diversified group of Sicilian rock-dwelling land snails belonging to the genus Marmorana . The taxonomic implications of the three character sets (shell and anatomical biometry and molecular data) were also considered. The inferred phylogenetic relationships do not match morphological (shell and genitalia) patterns. This result may significantly modify the current taxonomy. Mitochondrial based reconstructions define several supported clades well correlated with geographic distribution and populations were found to be distributed parapatrically. The progressive decline in mitochondrial DNA sequence similarity over a distance of 250 km is consistent with a model of isolation by distance, a pattern previously recognized for other groups of land snails. For one clade of Marmorana , colonization along Mediterranean trade routes appears to be a possibility.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 809–823.     

Molecular phylogeny of solitary shell-bearing Polycystinea (Radiolaria)

The phylogeny of the Family Spongodiscidae (polycystine Radiolaria), which includes Dictyocoryne profunda Ehrenberg, Dictyocoryne truncatum (Ehrenberg) and Spongaster tetras Ehrenberg, was examined using 18S ribosomal DNA (small-subunit ribosomal DNA) sequence analysis. Three types of tree construction methods, the neighbor joining (NJ), maximum parsimony (MP), and maximum likelihood (ML) methods, were used to infer the phylogenetic relationships of the polycystine and acantharian Radiolaria among eukaryotes. The obtained 18S rDNA molecular phylogenetic tree argues for the monophyly of the two groups. Furthermore, the Polycystinea is divided into at least two distinct lineages consisting of: (1) colonial and skeletonless Polycystinea, including Thalassicollidae, Collospaeridae, and Sphaerozoidae; and (2) shell-bearing solitary Polycystinea, including Spongodiscidae. The Polycystinea thus show a paraphyly among Radiolaria. Moreover, the monophyly of the clade including the acantharians and the spongodiscid polycystines was supported by bootstrap values, which were 94%, 53%, and 59% in the NJ, MP, and ML analyses, respectively. This lineage is characterized by having latticed or spongy skeletons of different chemical composition, namely SiO₂ (Class Polycystinea) or SrSO₄ (Class Acantharea). According to the present taxonomic scheme, the Acantharea and the Polycystinea have not been placed in different classes, but the results of our molecular study show the opposite. We therefore suggest, based on the monophyly of the two clades, that a new taxonomic group of Radiolaria can be established. Our molecular data also suggest that the currently used radiolarian taxonomic system may need serious revisions.     

Outer membrane efflux protein (OMEP) is a suitable molecular marker for resolving the phylogeny and taxonomic status of closely related cyanobacteria

Taxonomy of Cyanobacteria, the oldest phototrophic prokaryotes, is problematic for many years due to their simple morphology, high variability and adaptability to diverse ecological niches. After introduction of the polyphasic approach, which is based on the combination of several criteria (molecular sequencing, morphological and ecological), the whole classification system of these organisms is subject to reorganization. The aim of this study was to evaluate whether the outer membrane efflux protein (OMEP) sequences can be used as a molecular marker for resolving the phylogeny and taxonomic status of closely related cyanobacteria. We have performed phylogenetic analyses based on the amino acid sequences of the OMEP and the DNA sequences of the 16S rRNA gene from 86 cyanobacterial species/strains with completely sequenced genomes. Phylogenetic trees based on the OMEP showed that most of the cyanobacterial species/strains belonging to different genera are clustered in separate clades supported by high bootstrap values. Comparing the OMEP trees with the 16S rDNA tree clearly showed that the OMEP is more suitable marker in resolving phylogenetic relationships within Cyanobacteria at generic and species level.     

Phylum Tardigrada: an “individual” approach

Phylum Tardigrada consists of ～1000 tiny, hardy metazoan species distributed throughout terrestrial, limno‐terrestrial and oceanic habitats. Their phylogenetic status has been debated, with current evidence placing them in the Ecdysozoa. Although there have been efforts to explore tardigrade phylogeny using both morphological and molecular data, limitations such as their few morphological characters and low genomic DNA concentrations have resulted in restricted taxonomic coverage. Using a protocol that allows us to identify and extract DNA from individuals, we have sequenced 18S rDNA from 343 tardigrades from across the globe. Using maximum parsimony and Bayesian analyses we have found support for dividing Order Parachela into three super‐families and further evidence that indicates the traditional taxonomic perspective of families in the class Eutardigrada are nonmonophyletic and require re‐working. It appears that conserved morphology within Tardigrada has resulted in conservative taxonomy as we have found cases of several discrete lineages grouped into single genera. Although this work substantially adds to the understanding of the evolution and taxonomy of the phylum, we highlight that inferences gained from this work are likely to be refined with the inclusion of further taxa—specifically representatives of the nine families yet to be sampled. © The Willi Hennig Society 2008.     

Molecular systematics and species limits in the Philippine fantails (Aves: Rhipidura)

Islands have long-attracted scientists because of their relatively simple biotas and stark geographic boundaries. However, for many islands and archipelagos, this simplicity may be overstated because of methodological and conceptual limitations when these biotas were described. One archipelago that has received relatively little recent attention is the Philippine islands. Although much of its biota was documented long ago, taxonomic revision and evolutionary study has been surprisingly scarce, and only a few molecular phylogenetic studies are beginning to appear. We present a molecular phylogeny and taxonomic revision for the Philippine fantails (Aves: Rhipidura) using nuclear and mitochondrial DNA sequences. Our results suggest that current taxonomy underestimates diversity in the group. Some morphologically distinct subspecies warrant species status, whereas one was indistinguishable genetically and morphologically and should not be retained. A few taxa require additional sampling for thorough taxonomic assessment. Patterns of diversity within Philippine Rhipidura mostly corroborate predictions of the Pleistocene aggregate island complex (PAIC) hypothesis, in which diversity is expected to be partitioned by deep water channels separating Pleistocene aggregate islands rather than by current islands. Substantial structure within PAIC clades indicates that additional drivers of diversification should be considered.     

New insights into and limitations of the molecular phylogeny in the taxon-rich land snail genus Montenegrina (Mollusca: Gastropoda: Clausiliidae)

Rock-dwelling gastropods are usually patchily distributed in limestone habitats, presumably have low active and passive dispersal ability and often represent narrow-ranged endemic taxa. Their current taxonomy is predominantly shell morphology based, and it remains unknown whether the morphologically differentiated and geographically separated populations represent phylogenetic clades. In this study, we analysed the hyperdiverse, terrestrial door snail genus Montenegrina. Based on the current taxonomy defined by shell morphology, it contains 29 species and 106 subspecies distributed in the Balkan region. The constructed phylogenetic tree using three mitochondrial markers was used to test whether it agrees with the current taxonomy. In this comprehensive tree, about half of the species and subspecies are monophyletic. Some of the paraphylies could be reasonably resolved by taxonomic changes; that is, some subspecies should be reassigned or raised to species level. Other incongruencies probably arose due to introgression even between distant clades. The histone genes turned out to be unsuitable for elucidating the phylogeny of Montenegrina. In the species-delimitation tests, considerably more molecular operational taxonomic units were delimited than the number of presently described species. The present data indicate that (a) shell morphology-based taxonomy and taxon recognition can be problematic in such a large and morphologically highly variable genus; (b) the potential error due to incomplete sampling presents a problem in a genus as variable as Montenegrina; (c) multi-locus analyses should be conducted to arrive at a better basis for species delimitation; and (d) integrative approaches including genetic as well as morphological/anatomical data from a comprehensive geographic sample are necessary.     

Molecular systematics of Volvocales (Chlorophyceae, Chlorophyta) based on exhaustive 18S rRNA phylogenetic analyses

The taxonomy of Volvocales (Chlorophyceae, Chlorophyta) was traditionally based solely on morphological characteristics. However, because recent molecular phylogeny largely contradicts the traditional subordinal and familial classifications, no classification system has yet been established that describes the subdivision of Volvocales in a manner consistent with the phylogenetic relationships. Towards development of a natural classification system at and above the generic level, identification and sorting of hundreds of sequences based on subjective phylogenetic definitions is a significant step. We constructed an 18S rRNA gene phylogeny based on 449 volvocalean sequences collected using exhaustive BLAST searches of the GenBank database. Many chimeric sequences, which can cause fallacious phylogenetic trees, were detected and excluded during data collection. The results revealed 21 strongly supported primary clades within phylogenetically redefined Volvocales. Phylogenetic classification following PhyloCode was proposed based on the presented 18S rRNA gene phylogeny along with the results of previous combined 18S and 26S rRNA and chloroplast multigene analyses.     

18S rDNA phylogeny of lamproderma and allied genera (Stemonitales, Myxomycetes, Amoebozoa)

The phylogenetic position of the slime-mould genus Lamproderma (Myxomycetes, Amoebozoa) challenges traditional taxonomy: although it displays the typical characters of the order Stemonitales, it appears to be sister to Physarales. This study provides a small subunit (18S or SSU) ribosomal RNA gene-based phylogeny of Lamproderma and its allies, with new sequences from 49 specimens in 12 genera. We found that the order Stemonitales and Lamproderma were both ancestral to Physarales and that Lamproderma constitutes several clades intermingled with species of Diacheopsis, Colloderma and Elaeomyxa. We suggest that these genera may have evolved from Lamproderma by multiple losses of fruiting body stalks and that many taxonomic revisions are needed. We found such high genetic diversity within three Lamproderma species that they probably consist of clusters of sibling species. We discuss the contrasts between genetic and morphological divergence and implications for the morphospecies concept, highlighting the phylogenetically most reliable morphological characters and pointing to others that have been overestimated. In addition, we showed that the first part (~600 bases) of the SSU rDNA gene is a valuable tool for phylogeny in Myxomycetes, since it displayed sufficient variability to distinguish closely related taxa and never failed to cluster together specimens considered of the same species.     

PhytoREF: a reference database of the plastidial 16S rRNA gene of photosynthetic eukaryotes with curated taxonomy

Photosynthetic eukaryotes have a critical role as the main producers in most ecosystems of the biosphere. The ongoing environmental metabarcoding revolution opens the perspective for holistic ecosystems biological studies of these organisms, in particular the unicellular microalgae that often lack distinctive morphological characters and have complex life cycles. To interpret environmental sequences, metabarcoding necessarily relies on taxonomically curated databases containing reference sequences of the targeted gene (or barcode) from identified organisms. To date, no such reference framework exists for photosynthetic eukaryotes. In this study, we built the PhytoREF database that contains 6490 plastidial 16S rDNA reference sequences that originate from a large diversity of eukaryotes representing all known major photosynthetic lineages. We compiled 3333 amplicon sequences available from public databases and 879 sequences extracted from plastidial genomes, and generated 411 novel sequences from cultured marine microalgal strains belonging to different eukaryotic lineages. A total of 1867 environmental Sanger 16S rDNA sequences were also included in the database. Stringent quality filtering and a phylogeny‐based taxonomic classification were applied for each 16S rDNA sequence. The database mainly focuses on marine microalgae, but sequences from land plants (representing half of the PhytoREF sequences) and freshwater taxa were also included to broaden the applicability of PhytoREF to different aquatic and terrestrial habitats. PhytoREF, accessible via a web interface ( http://phytoref.fr ), is a new resource in molecular ecology to foster the discovery, assessment and monitoring of the diversity of photosynthetic eukaryotes using high‐throughput sequencing.     

Phylogeny of coral-inhabiting barnacles (Cirripedia; Thoracica; Pyrgomatidae) based on 12S, 16S and 18S rDNA analysis

The traditional phylogeny of the coral-inhabiting barnacles, the Pyrgomatidae, is based on morphological characteristics, mainly of the hard parts. It has been difficult to establish the phylogenetic relationships among Pyrgomatidae because of the apparent convergence of morphological characteristics, and due to the use of non-cladistic systematics, which emphasize ancestor-descendant relationships rather than sister-clade relationships. We used partial sequences of two mithochondrial genes, 12S rDNA and 16S rDNA, and a nuclear gene, 18S rDNA, to infer the molecular phylogeny of the pyrgomatids. Our phylogenetic results allowed us to reject previous classifications of Pyrgomatidae based on morphological characteristics. Our results also suggested the possibility of paraphyly of the Pyrgomatidae. The hydrocoral barnacle Wanella is not found on the same clade as the other pyrgomatids, but rather, with the free-living balanids. The basal position of Megatrema and Ceratoconcha is supported. The archeaobalanid Armatobalanus is grouped with Cantellius at the base of the Indo-Pacific pyrgomatines. Fusion of the shell plate and modification of the opercular valves are homoplasious features that occurred more than three times on different clades. The monophyly of the "Savignium" group, comprising four nominal genera, is also not supported, and the different taxa are placed on different clades.     

Evolution of helotialean fungi (Leotiomycetes, Pezizomycotina): a nuclear rDNA phylogeny

The highly divergent characters of morphology, ecology, and biology in the Helotiales make it one of the most problematic groups in traditional classification and molecular phylogeny. Sequences of three rDNA regions, SSU, LSU, and 5.8S rDNA, were generated for 50 helotialean fungi, representing 11 out of 13 families in the current classification. Data sets with different compositions were assembled, and parsimony and Bayesian analyses were performed. The phylogenetic distribution of lifestyle and ecological factors was assessed. Plant endophytism is distributed across multiple clades in the Leotiomycetes. Our results suggest that (1) the inclusion of LSU rDNA and a wider taxon sampling greatly improves resolution of the Helotiales phylogeny, however, the usefulness of rDNA in resolving the deep relationships within the Leotiomycetes is limited; (2) a new class Geoglossomycetes, including Geoglossum, Trichoglossum, and Sarcoleotia, is the basal lineage of the Leotiomyceta; (3) the Leotiomycetes, including the Helotiales, Erysiphales, Cyttariales, Rhytismatales, and Myxotrichaceae, is monophyletic; and (4) nine clades can be recognized within the Helotiales.     

Diversity and phylogeny of percolomonads based on newly discovered species from hypersaline and marine waters

Percolomonads are common freshwater, marine and hypersaline tetraflagellated organisms. Current phylogenetic analyses of eukaryotes comprise only two species of this underinvestigated family. Here, we studied the morphology, salinity tolerance and 18S rDNA gene-based phylogeny of seven percolomonad cultures. We describe three new genera and five novel species of Percolomonadida based on phylogenetic distances and morphological characteristics: Barbelia atlantica, B. abyssalis, Lula jakobsenorum, Nakurumonas serrata and Percolomonas doradorae.The new species show features typical for percolomonads, one long flagellum for skidding, three shorter flagella of equal length and a ventral feeding groove. The new species comprise organisms living in marine and athalassic hypersaline waters with salinity ranging from 10 to 150 PSU. Based on these novel taxa, the taxonomy and phylogeny of Percolatea was extended and further resolved.     

Phylogenetic utility of protein (RPB2, β-tubulin) and ribosomal (LSU,SSU) gene sequences in the systematics of Sordariomycetes (Ascomycota,Fungi)

The Sordariomycetes is an important group of fungi whose taxonomic relationships and classification is obscure. There is presently no multi-gene molecular phylogeny that addresses evolutionary relationships among different classes and orders. In this study, phylogenetic analyses with a broad taxon sampling of the Sordariomycetes were conducted to evaluate the utility of four gene regions (LSU rDNA, SSU rDNA, beta-tubulin and RPB2) for inferring evolutionary relationships at different taxonomic ranks. Single and multi-gene genealogies inferred from Bayesian and Maximum Parsimony analyses were compared in individual and combined datasets. At the subclass level, SSU rDNA phylogenies demonstrate their utility as a marker to infer phylogenetic relationships at higher levels. All analyses with SSU rDNA alone, combined LSU rDNA and SSU rDNA, and the combined 28 S rDNA, SSU rDNA and RPB2 datasets resulted in three subclasses: Hypocreomycetidae, Sordariomycetidae and Xylariomycetidae, which correspond well to established morphological classification schemes. At the ordinal level, the best resolved phylogeny was obtained from the combined LSU rDNA and SSU rDNA datasets. Individually, the RPB2 gene dataset resulted in significantly higher number of parsimony informative characters. Our results supported the recent separation of Boliniaceae, Chaetosphaeriaceae and Coniochaetaceae from Sordariales and placement of Coronophorales in Hypocreomycetidae. Microascales was found to be paraphyletic and Ceratocystis is phylogenetically associated to Faurelina, while Microascus and Petriella formed another clade and basal to other members of Halosphaeriales. In addition, the order Lulworthiales does not appear to fit in any of the three subclasses. Congruence between morphological and molecular classification schemes is discussed.     

Radiolaria divided into Polycystina and Spasmaria in combined 18S and 28S rDNA phylogeny

Radiolarians are marine planktonic protists that belong to the eukaryote supergroup Rhizaria together with Foraminifera and Cercozoa. Radiolaria has traditionally been divided into four main groups based on morphological characters; i.e. Polycystina, Acantharia, Nassellaria and Phaeodaria. But recent 18S rDNA phylogenies have shown that Phaeodaria belongs within Cerocozoa, and that the previously heliozoan group Taxopodida should be included in Radiolaria. 18S rDNA phylogenies have not yet resolved the sister relationship between the main Radiolaria groups, but nevertheless suggests that Spumellaria, and thereby also Polycystina, are polyphyletic. Very few sequences other than 18S rDNA have so far been generated from radiolarian cells, mostly due to the fact that Radiolaria has been impossible to cultivate and single cell PCR has been hampered by low success rate. Here we have therefore investigated the mutual evolutionary relationship of the main radiolarian groups by using the novel approach of combining single cell whole genome amplification with targeted PCR amplification of the 18S and 28S rDNA genes. Combined 18S and 28S phylogeny of sequences obtained from single cells shows that Radiolaria is divided into two main lineages: Polycystina (Spumellaria+Nassellaria) and Spasmaria (Acantharia+Taxopodida). Further we show with high support that Foraminifera groups within Radiolaria supporting the Retaria hypothesis.     

Molecular Phylogenetic Positions and Ultrastructure of Marine Gregarines (Apicomplexa) Cuspisella ishikariensis n. gen., n. sp. and Loxomorpha cf. harmothoe from Western Pacific scaleworms (Polynoidae)

Marine gregarines are unicellular parasites of invertebrates commonly found infecting the intestine and coelomic spaces of their hosts. Situated at the base of the apicomplexan tree, marine gregarines offer an opportunity to explore the earliest stages of apicomplexan evolution. Classification of marine gregarines is often based on the morphological traits of the conspicuous feeding stages (trophozoites) in combination with host affiliation and molecular phylogenetic data. Morphological characters of other life stages such as the spore are also used to inform taxonomy when such stages can be found. The reconstruction of gregarine evolutionary history is challenging, due to high levels of intraspecific variation of morphological characters combined with relatively few traits that are taxonomically unambiguous. The current study combined morphological data with a phylogenetic analysis of small subunit rDNA sequences to describe and establish a new genus and species (Cuspisella ishikariensis n. gen., n. sp.) of marine gregarine isolated from the intestine of a polynoid host (Lepidonotus helotypus) collected from Hokkaido, Japan. This new species possesses a set of unusual morphological traits including a spiked attachment apparatus and sits on a long branch on the molecular phylogeny. Furthermore, this study establishes a molecular phylogenetic position for Loxomorpha cf. harmothoe, a previously described marine gregarine, and reveals a new group of gregarines that infect polynoid hosts.     

Systematics of the grey mullets (Teleostei: Mugiliformes: Mugilidae): molecular phylogenetic evidence challenges two centuries of morphology-based taxonomy

The family Mugilidae comprises mainly coastal marine species that are widely distributed in all tropical, subtropical and temperate seas. Mugilid species are generally considered to be ecologically important and they are a major food resource for human populations in certain parts of the world. The taxonomy and systematics of the Mugilidae are still much debated and based primarily on morphological characters. In this study, we provide the first comprehensive molecular systematic account of the Mugilidae using phylogenetic analyses of nucleotide sequence variation at three mitochondrial loci (16S rRNA, cytochrome oxidase I, and cytochrome b) for 257 individuals from 55 currently recognized species. The study covers all 20 mugilid genera currently recognized as being valid. The family comprises seven major lineages that radiated early on from the ancestor to all current forms. All genera that were represented by two species or more, except Cestraeus, turned out to be paraphyletic or polyphyletic. Thus, the present phylogenetic results generally disagree with the current taxonomy at the genus level and imply that the anatomical characters used for the systematics of the Mugilidae may be poorly informative phylogenetically. The present results should provide a sound basis for a taxonomic revision of the mugilid genera. A proportion of the species with large distribution ranges (including Moolgarda seheli, Mugil cephalus and M. curema) appear to consist of cryptic species, thus warranting further taxonomic and genetic work at the infra-generic level.     

杨属植物分类的研究进展和展望

自林奈建立杨属（Populus）以来,众多的学者对杨属的分类进行了研究,并建立了较多的分类系统。目前的国际杨属分类,总体上还停留在传统形态分类阶段,存在明显的地域和国别特征。尽管各国在主流上都采用“属—组—种”的分类阶元,但在种、亚种、变种的划分上,却存在很大的不同。中国和美国分别是细分派和聚合派的代表,两国的分类系统存在很大分歧,但各有优点和缺点。即使在国内,杨属分类也还有很多需要解决的问题。新兴的分子系统学研究,为杨属的分类修订提供了一些有益参考,但总体贡献不大。国际杨属分类的趋势和终极目标是建立基于系统发生的统一分类系统。这需要基于种群和综合物种概念,融合形态学、生态学、生物地理学、基因组生物信息学和进化生物学等的综合系统分类来实现。