Molecular and morphological criteria for revision of the genus Microcoleus (Oscillatoriales,Cyanobacteria)

Ninety‐two strains of Microcoleus vaginatus (=nomenclatural‐type species of the genus Microcoleus Desmazières ex Gomont) and Phormidium autumnale Trevisan ex Gomont from a wide diversity of regions and biotopes were examined using a combination of morphological and molecular methods. Phylogenies based on the 16S rDNA and 16S‐23S ITS (partial) demonstrated that the 92 strains, together with a number of strains in GenBank, were members of a highly supported monophyletic clade of strains (Bayesian posterior probability = 1.0) distant from the species‐cluster containing the generitype of Phormidium. Similarity of the 16S rRNA gene exceeded 95.5% among all members of the Microcoleus clade, but was less than 95% between any Microcoleus strains and species outside of the clade (e.g., Phormidium sensu stricto). These findings, which are in agreement with earlier studies on these taxa, necessitate the revision of Microcoleus to include P. autumnale. Furthermore, the cluster of Phormidium species in the P. autumnale group (known as Group VII) must be moved into Microcoleus as well, and these nomenclatural transfers are included in this study. The main diacritical characters defining Microcoleus are related to the cytomorphology of trichomes, including: narrowed trichome ends, calyptra, cells shorter than wide up to more or less isodiametric, and facultative presence of sheaths. The majority of species are 4–10 μm in diameter. The possession of multiple trichomes in a common sheath is present facultatively in many but not all species.     

Phylogeny and species delineation in the marine diatom Pseudo‐nitzschia (Bacillariophyta) using cox1, LSU,and ITS2 rRNA genes: A perspective in character evolution

Analyses of the mitochondrial cox1, the nuclear‐encoded large subunit (LSU), and the internal transcribed spacer 2 (ITS2) RNA coding region of Pseudo‐nitzschia revealed that the P. pseudodelicatissima complex can be phylogenetically grouped into three distinct clades (Groups I–III), while the P. delicatissima complex forms another distinct clade (Group IV) in both the LSU and ITS2 phylogenetic trees. It was elucidated that comprehensive taxon sampling (sampling of sequences), selection of appropriate target genes and outgroup, and alignment strategies influenced the phylogenetic accuracy. Based on the genetic divergence, ITS2 resulted in the most resolved trees, followed by cox1 and LSU. The morphological characters available for Pseudo‐nitzschia, although limited in number, were overall in agreement with the phylogenies when mapped onto the ITS2 tree. Information on the presence/absence of a central nodule, number of rows of poroids in each stria, and of sectors dividing the poroids mapped onto the ITS2 tree revealed the evolution of the recently diverged species. The morphologically based species complexes showed evolutionary relevance in agreement with molecular phylogeny inferred from ITS2 sequence–structure data. The data set of the hypervariable region of ITS2 improved the phylogenetic inference compared to the cox1 and LSU data sets. The taxonomic status of P. cuspidata and P. pseudodelicatissima requires further elucidation.     

Molecular and morphological delimitation and generic classification of the family Oocystaceae (Trebouxiophyceae,Chlorophyta)

The family Oocystaceae (Chlorophyta) is a group of morphologically and ultrastructurally distinct green algae that constitute a well‐supported clade in the class Trebouxiophyceae. Despite the family's clear delimitation, which is based on specific cell wall features, only a few members of the Oocystaceae have been examined using data other than morphological. In previous studies of Trebouxiophyceae, after the establishment of molecular phylogeny, the taxonomic status of the family was called into question. The genus Oocystis proved to be paraphyletic and some species were excluded from Oocystaceae, while a few other species were newly redefined as members of this family. We investigated 54 strains assigned to the Oocystaceae using morphological, ultrastructural and molecular data (SSU rRNA and rbcL genes) to clarify the monophyly of and diversity within Oocystaceae. Oonephris obesa and Nephrocytium agardhianum clustered within the Chlorophyceae and thus are no longer members of the Oocystaceae. On the other hand, we transferred the coenobial Willea vilhelmii to the Oocystaceae. Our findings combined with those of previous studies resulted in the most robust definition of the family to date. The division of the family into three subfamilies and five morphological clades was suggested. Taxonomical adjustments in the genera Neglectella, Oocystidium, Oocystis, and Ooplanctella were established based on congruent molecular and morphological data. We expect further taxonomical changes in the genera Crucigeniella, Eremosphaera, Franceia, Lagerheimia, Oocystis, and Willea in the future.     

Systematics and phylogeny of Vasseuromys (Mammalia,Rodentia, Gliridae) with a description of a new species from the late Miocene of eastern Europe

Vasseuromys is a species‐rich genus of small‐ to medium‐sized glirids spanning the latest Oligocene to late Miocene of Europe and western Asia. Despite extensive discoveries over the past 50 years, little phylogenetic work has been done on Vasseuromys. This study presents the first phylogenetic analysis of the genus that includes all the described species and a new taxon Vasseuromys tectus sp. nov. from the late Miocene of eastern Europe, providing the first insights into the evolutionary relationships within the clade. Results suggest that the genus is clearly paraphyletic. Two strongly supported genus‐level clades are recognized within ‘Vasseuromys’: a restricted Vasseuromys clade (containing the three species, V. pannonicus, V. rugosus and V. tectus) and the Glirulus clade that includes ‘Vasseuromys’ duplex. The remaining ‘Vasseuromys’ species are found to constitute a set of paraphyletic taxa, with the polyphyletic ‘Ramys’ nested within it. The genus Gliruloides is synonymized with Glirulus. Vasseuromys tectus sp. nov. is the most derived member of the genus in having a greater number of cheek teeth ridges including constantly present anterotrope, centrotrope, second prototrope on M1–2, third metatrope on M2, two to three posterotropids on p4 and strong ectolophids on lower molars. The results of the study confirm a European origin for Vasseuromys while suggesting that the late Miocene species of the genus dispersed from the east in the early Turolian.     

Molecular phylogeny and morphological evolution of the Acantharia (Radiolaria)

Acantharia are ubiquitous and abundant rhizarian protists in the world ocean. The skeleton made of strontium sulphate and the fact that certain harbour microalgal endosymbionts make them key planktonic players for the ecology of marine ecosystems. Based on morphological criteria, the current taxonomy of Acantharia was established by W.T. Schewiakoff in 1926, since when no major revision has been undertaken. Here, we established the first comprehensive molecular phylogeny from single morphologically-identified acantharian cells, isolated from various oceans. Our phylogenetic analyses based on 78 18S rDNA and 107 partial 28S rDNA revealed the existence of 6 main clades, sub-divided into 13 sub-clades. The polyphyletic nature of acantharian families and genera demonstrates the need for revision of the current taxonomy. This molecular phylogeny, which highlights the taxonomic relevance of specific morphological criteria, such as the presence of a shell and the organisation of the central junction, provides a robust phylogenetic framework for future taxonomic emendation. Finally, mapping all the existing environmental sequences available to date from different marine ecosystems onto our reference phylogeny unveiled another 3 clades and improved the understanding of the biogeography and ecology of Acantharia.     

Molecular evolution and diversification of the moss family Daltoniaceae (Hookeriales,Bryophyta) with emphasis on the unravelling of the phylogeny of Distichophyllum and its allies

Phylogenetic relationships in Daltoniaceae (～200 species in 14 genera) are inferred from nucleotide sequences from five genes, representing all genomic compartments, using parsimony, likelihood and Bayesian methods. Alternative classifications for Daltoniaceae have favoured traits from either sporophytes or gametophytes; phylogenetic transitions in gametophytic leaf limbidia and sporophytic exostome ornamentation were evaluated using ancestral state reconstruction to assess the levels of conflict between these generations. Elimbate leaves and the cross‐striate exostome are reconstructed as plesiomorphic states. Limbate leaves and papillose exostomes evolved at least two and six times, respectively, without reversals. The evolution of leaf limbidia is relatively conserved, but exostome ornamentation is highly homoplasious, indicating that superficial similarity in peristomes gives unreliable approximations of phylogenetic relatedness. Our phylogenetic analyses show that Achrophyllum and Calyptrochaeta are reciprocally monophyletic. Within core Daltoniaceae, relationships among taxa with elimbate leaves are generally well understood. However, taxa with limbate leaves form a monophyletic group, but resolved subclades correspond to biogeographical entities, rather than to traditional concepts of genera. Daltonia (～21 species), Distichophyllum (～100 species) and Leskeodon (～20 species) are polyphyletic. Seven nomenclatural changes are proposed here. As the current taxonomy of Daltoniaceae lacks phylogenetic consistency, critical generic revisions are needed. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, ?? , ??–??.     

A polyphasic approach to the delimitation of diatom species: a case study for the genus Pinnularia (Bacillariophyta)

Diatoms are one of the most abundant and arguably the most species‐rich group of protists. Diatom species delimitation has often been based exclusively on the recognition of morphological discontinuities without investigation of other lines of evidence. Even though DNA sequences and reproductive experiments have revealed several examples of (pseudo)cryptic diversity, our understanding of diatom species boundaries and diversity remains limited. The cosmopolitan pennate raphid diatom genus Pinnularia represents one of the most taxon‐rich diatom genera. In this study, we focused on the delimitation of species in one of the major clades of the genus, the Pinnularia subgibba group, based on 105 strains from a worldwide origin. We compared genetic distances between the sequences of seven molecular markers and selected the most variable pair, the mitochondrial cox1 and nuclear encoded LSU rDNA, to formulate a primary hypothesis on the species limits using three single‐locus automated species delimitation methods. We compared the DNA‐based primary hypotheses with morphology and with other available lines of evidence. The results indicate that our data set comprised 15 species of the P. subgibba group. The vast majority of these taxa have an uncertain taxonomic identity, suggesting that several may be unknown to science and/or members of (pseudo)cryptic species complexes within the P. subgibba group.     

Molecular phylogeny of the superfamily Tephritoidea (Insecta: Diptera) reanalysed based on expanded taxon sampling and sequence data

Phylogenenetic relationships of the superfamily Tephritoidea (Diptera: Tephritidae) were reanalysed based upon four mitochondrial gene fragments (12S, 16S, cytochrome c oxidase I and cytochrome c oxidase II) from 53 tephritoid (10 families) and 30 outgroup (14 families) species. The data set of Han and Ro (Mol Phylogenet Evol, 39, 2005, 416) was expanded in terms of the number of taxa as well as molecular characters. We were able to sample the enigmatic families Ctenostylidae and Eurygnathomyiidae for the first time. Based on increased taxon sampling (from 49 to 83 species) and additional sequences (combined length of DNA fragments increased from 2451 to 4490 bp), the inferred phylogenetic trees suggest a number of interesting phylogenetic relationships, some of which were not recovered from the previous study. Some of the important findings are as follows: (1) monophyly of the superfamily Tephritoidea; (2) all the included tephritoid families except for Tephritidae were recovered as monophyletic groups; (3) Tephritoidea can be divided into two monophyletic groups – the Piophilidae Family Group (Pallopteridae, Circumphallidae?, Lonchaeidae, Piophilidae and Eurygnathomyiidae) and the Tephritidae Family Group (Richardiidae, Ulidiidae, Platystomatidae, Tephritidae, Ctenostylidae and Pyrgotidae); (4) Eurygnathomyiidae is recognized as an independent monophyletic family apart from Pallopteridae; (5) the enigmatic family Ctenostylidae is a member of the superfamily Tephritoidea; (6) parasitic Pyrgotidae + Ctenostylidae + Tachiniscinae and mostly phytophagous Tephritidae are recovered within a monophyletic group; and (7) according to an inferred chronogram, the first Tephritoidea might have evolved around the middle of Paleocene Epoch [~59 Million years ago (mya)] and the family Tephritidae around the late Eocene (~36 mya).     

A polyphasic approach to the study of the genus Nitzschia (Bacillariophyta): three new planktonic species from the Adriatic Sea

The paraphyletic diatom genus Nitzschia comprises over 1000 morphologically distinct pennate taxa, known from the benthos and plankton of freshwater, brackish, and marine environments. The principal diagnostic characters for delimitation of Nitzschia species include valve shape, the position and structure of the raphe, presence/absence and shape of the proximal raphe endings and terminal raphe fissures, areola structure, and specific morphometric features such as cell size, and stria and fibula density. In this study, we isolated 12 diatom strains into culture from samples collected at the surface or greater depths of the southeastern Adriatic Sea. Morphological analyses included LM, SEM, and TEM observations, which, along with specific morphometric features, allowed us to distinguish three new Nitzschia species. These findings were congruent with the results of phylogenetic analyses performed on nuclear‐encoded SSU (18S) rDNA and chloroplast‐encoded rbcL and psbC genes. One of the new species (Nitzschia dalmatica sp. nov.) formed a lineage within a clade of Bacillariaceae containing members of the Nitzschia sect. Dubiae, which was sister to Psammodictyon. A second lineage was part of a novel clade that is significantly distinct from other Nitzschia species sequenced so far and includes Nitzschia adhaerens sp. nov. and N. cf. adhaerens. A further new species was found, Nitzschia inordinata sp. nov., which appeared as the sister group to the N. adhaerens clade and the conopeoid Nitzschia species in our phylogenetic trees. Our findings contribute to the overall diversity of genus Nitzschia, especially in identifying some deep branches within the Bacillariaceae, and highlight under‐scoring of this genus in marine plankton.     

Cranial morphology and taxonomic resolution of some dolphin taxa (Delphinidae) in Australian waters,with a focus on the genus Tursiops

Phylogenetic relationships in the family Delphinidae have been widely debated. We examined 347 skulls of Tursiops, Stenella, Delphinus, Steno, Lagenodelphis, and Sousa in order to resolve the phylogenetic position of Australian species of Tursiops. Five Tursiops type specimens were included. Cranial morphology was described using 2‐dimensional (2‐D) and 3‐dimensional geometric morphometrics (3‐GM), counts and categorical data. Analyses showed a clear morphological separation of Tursiops, including type specimens, from other genera. The three Stenella species did not cluster together. Stenella attenuata clustered with Delphinus delphis, and Stenella coeruleoalba with Lagenodelphis hosei. Length and width of the skull and rostrum were important discriminators in both methods. For 3‐D data, round vs. angular posterior skull shape distinguished some genera. Taxa that overlapped in the multivariate analyses had different mean tooth counts. Our study challenges genetic studies that identified Tursiops as polyphyletic, with T. aduncus closer to S. attenuata.     

Molecular phylogeny of Zeacarpa (Ralfsiales,Phaeophyceae) proposing a new family Zeacarpaceae and its transfer to Nemodermatales

Zeacarpa leiomorpha is a crustose brown alga endemic to South Africa. The species has been tentatively placed in Ralfsiaceae, but its ordinal assignment has been uncertain. The molecular phylogeny of brown algae based on concatenated DNA sequences of seven chloroplast and mitochondrial gene sequences (atpB, psaA, psaB, psbA, psbC, rbcL, and cox1) of taxa covering most of the orders revealed the most related phylogenetic relationship of Z. leiomorpha to Nemoderma tingitanum (Nemodermatales) rather than Ralfsiaceae (Ralfsiales). Morphologically, Zeacarpa and Nemoderma share crustose thallus structure and multiple discoidal chloroplasts without pyrenoids in each cell, however, the formation of lateral unilocular zoidangia in tufts in loose upright filaments in Zeacarpa is distinctive in brown algae. Considering the relatively distant genetic divergence between the two taxa, comparable to that among families or orders in representative brown algae, in addition to the above‐mentioned unique morphological features, we propose the classification of Zeacarpa in a new family Zeacarpaceae in the order Nemodermatales.     

Fine structure and 18S rDNA phylogeny of a marine araphid pennate diatom Plagiostriata goreensis gen. et sp. nov. (Bacillariophyta)

A marine araphid pennate diatom Plagiostriata goreensis is described from the sand grains of Goree Island, Dakar, Republic of Senegal, based on observations of fine structure of its frustule. The most striking feature of the species is its striation, which is angled at approximately 60° across the robust sternum. The other defining features of the species are its one highly reduced rimoportula and apical pores located at both ends of the valve margin. In the 18S rDNA phylogeny, the species appears as a member of a ‘small‐celled clade’ of araphid pennate diatoms that consist of Nanofrustulum, Opephora and Staurosira. The results of the phylogenetic analyses suggest that the distinct characters of the diatom; namely, oblique striae and apical pores, may have been acquired independently. However, it remains unclear whether the rimoportula of P. goreensis is a reduced state or P. goreensis acquired its morphologically curious rimoportula independently after the loss of an ancient rimoportula at the root of the small‐celled clade.     

A molecular investigation of the genus Ecklonia (Phaeophyceae,Laminariales) with special focus on the Southern Hemisphere

Brown algae of the order Laminariales, commonly referred to as kelps, are the largest and most productive primary producers in the coastal inshore environment. The genus Ecklonia (Lessoniaceae, Phaeophyceae) consists of seven species with four species in the Northern Hemisphere and three in the Southern Hemisphere. It was recently transferred to the family Lessoniaceae based on phylogenetic analyses of nuclear and chloroplastic markers, though the type of the genus was not included and its relationship with allied genera Eckloniopsis and Eisenia remained unresolved. The present study is the first to produce a phylogeny focussed on the genus Ecklonia. It included sequences from nuclear, mitochondrial, and chloroplastic DNA, for most of the distribution range of the three current Southern Hemisphere species (Ecklonia radiata, Ecklonia maxima, and a sample of a putative Ecklonia brevipes specimen), sequences for East Asiatic species (Ecklonia cava, Ecklonia kurome, and Ecklonia stolonifera), as well as the closely related genera Eckloniopsis and Eisenia. Results confirmed E. radiata and E. maxima as two distinct species in South Africa, E. radiata as a single species throughout the Southern Hemisphere (in South Africa, Australia, and New Zealand) and East Asiatic species as a distinct lineage from the Southern Hemisphere clade. Results further pointed out a close sister relationship between Eckloniopsis radicosa and two Eisenia species (including the type species: Eisenia arborea) to the genus Ecklonia suggesting that the genera Eckloniopsis and Eisenia are superfluous.     

Molecular phylogeny and possible scenario of ponyfish (Perciformes:Leiognathidae) evolution

The family Leiognathidae, commonly known as ponyfish or slip mouth, comprises three genera, each being characterized mainly by mouth morphology. To date, however, neither the phylogenetic relationships within the family nor monophyly of the genera has been tested. The phylogenetic relationships among 14 species of Leiognathidae, inferred from two protein coding mitochondrial genes (ND4 and 5), indicated monophyly of the studied species form genera Gazza and Secutor, and paraphyly of the genus Leiognathus, with L. equulus occupying a basal branch of the family. The relationships allowed phylogenetic analyses of mouthpart structures and light organ systems. The results suggested that the morphology of the upwardly and forwardly protractile mouth types (latter with canine-like teeth) are phylogenetically informative, and the downwardly protractile mouth type being ancestral in the family. The results also suggested that internal sexual dimorphism of the light organ system was present in the common ancestor of a sister clade to L. equulus, whereas external sexual dimorphism seems to have evolved subsequently in two monophyletic subgroups.     

Molecular approach to the phylogeny and systematics of Cytisus (Leguminosae) and related genera based on nucleotide sequences of nrDNA (ITS region) and cpDNA (trnL-trnF intergenic spacer)

 Phylogenetic relationships of Cytisus and allied genera (Argyrocytisus, Calicotome, Chamaecytisus, Cytisophyllum, and Spartocytisus) were assessed by analysis of sequences of the nrDNA internal transcribed spacer (ITS) and the cpDNA trnL-trnF intergenic spacer. Genera of the Genista-group (Chamaespartium, Echinospartum, Genista, Pterospartum, Spartium, Teline and Ulex) were included to check the position of Cytisus species transferred to Teline. The tree obtained by combining both sets of data indicates that the Genista and Cytisus groups form two separate clades. Cytisus heterochrous and C. tribracteolatus are more closely related to the Cytisus-group, thus their transfer to Teline is not supported by molecular data. Cytisus fontanesii (syn. Chronanthos biflorus) groups with Cytisophyllum sessilifolium and Cytisus heterochrous within the Cytisus-group. Similarly, Argyrocytisus battandieri falls within the Cytisus-group as a well differentiated taxon. All these taxa seem to have early diverged from the Cytisus-group. Their taxonomic rank should be reconsidered to better reflect their phylogenetic separation from Cytisus. On the contrary, Chamaecytisus proliferus and Spartocytisus supranubius enter in the main core of Cytisus, and they should better be included in sections of Cytisus (sect. Tubocytisus and Oreosparton, respectively). Sect. Spartopsis is not monophyletic and the position of several species, currently included in this section, deserves reevaluation: C. arboreus aggregate is closely related to C. villosus (sect. Cytisus) and to Calicotome; C. striatus is closely related to Cytisus sect. Alburnoides; and the position of C. commutatus (incl. C. ingramii) remains unclear. The relationships and positioning of several minor taxa (C. transiens, C. megalanthus, and C. maurus) are also discussed. Received November 22, 2001; accepted March 16, 2002 Published online: October 14, 2002 Addresses of the authors: Paloma Cubas (e-mail: cubas@farm.ucm.es) and Cristina Pardo (e-mail: cpardo@farm.ucm.es), Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense, E-28040 Madrid, Spain. Hikmat Tahiri Faculté des Sciences, Université Mohammed V, BP 1014 Rabat, Morocco (e-mail: tahiri@ fsr.ac.ma).