A molecular phylogenetic investigation of Opisthonecta and related genera (Ciliophora, Peritrichia, Sessilida)

The gene encoding 18S small subunit ribosomal RNA (ssu rRNA) was sequenced in the sessiline peritrichs Opisthonecta minima and Opisthonecta matiensis, whose free-swimming, paedomorphic trophonts resemble telotrochs. Using these new sequences, phylogenetic trees were constructed with four different methods to test a previously published association between Opisthonecta henneguyi and members of the families Vorticellidae and Astylozoidae. All trees had similar topologies, with O. minima, O. henneguyi, Vorticella microstoma, and Astylozoon enriquesi forming a well-supported, certainly monophyletic clade. On the basis of genetic evidence, genera of the families Opisthonectidae and Astylozoidae are assigned to the family Vorticellidae, which already includes some species with free-swimming morphotypes. The ssu rRNA sequence of O. matiensis places it in the family Epistylididae; its taxonomic revision will be left to another group of authors. A close association of Ophrydium versatile with members of the family Vorticellidae was confirmed, casting doubt on the validity of the family Ophrydiidae. Epistylis galea, Campanella umbellaria, and Opercularia microdiscum are confirmed as comprising an extremely distinct, monophyletic, but morphologically heterogeneous clade that is basal to other clades of sessiline peritrichs.     

Insights into the phylogenetic and taxonomy of philasterid ciliates (Protozoa, Ciliophora, Scuticociliatia) based on analyses of multiple molecular markers

Scuticociliates are a rich assemblage of species with mostly unresolved phylogenetic relationships, especially in the order Philasterida. In the present work, 48 new sequences for three linked genes are characterized and phylogenetic trees are constructed to assess the inter- and intra-generic relationships of philasterids. Results reveal the following: (1) the combined three-gene tree provides more resolution in nodes than in the SSU-rDNA topologies; (2) the family Orchitophryidae is non-monophyletic as it is split into two parts and Paranophrys magna, Metanophrys sp. and Metanophrys sinensis are designated incertae sedis at the familial level; (3) Uronematidae is non-monophyletic and Homalogastra setosa is designated incertae sedis; (4) Parauronematidae becomes a junior synonym of Uronematidae and the clade containing A. haemophila, Miamiensis avidus, and Glauconema trihymene might stand for a new family; (5) Parauronema being a junior synonym of Uronema is supported and P. longum should be removed from the genus Parauronema; (6) Uronema is not monophyletic and molecular analyses reveal that Uronema sp. QD shares a more recent common ancestor with Uronemella species than with other Uronema species; (7) Metanophrys is polyphyletic; (8) multiple samples of two highly controversial species, viz., Mesanophrys pugettensis and M. chesapeakensis have identical ITS1-5.8S-ITS2 region sequence and we propose they should be synonymous with M. carcini, and (9) there may be cryptic species in M. carcini and M. avidus.     

Ultrastructural and molecular phylogenetic delineation of a new order,the Rhizophydiales (Chytridiomycota)

In the order Chytridiales, Rhizophydium is a morphologically defined genus based upon the production of a monocentric, inoperculate, epibiotic sporangium, an endobiotic rhizoidal axis which branches, and an epibiotic resting spore. Despite its simple morphology, over 220 species of Rhizophydium have been described. Recent phylogenetic analyses using nuLSU rRNA (28 S rRNA) gene sequences of a geographically diverse sampling of Rhizophydium cultures revealed that the classical genus Rhizophydium is genetically more variable than previously understood and actually represents multiple genera. In the present study, we use zoospore ultrastructural characters and 28 S rRNA and 5.8 S ribosomal gene sequences of 96 isolates in culture to circumscribe the monophyletic Rhizophydium clade as a new order, Rhizophydiales. Correspondingly, zoospores of members of the Rhizophydiales exhibit a unique suite of ultrastructural character states that further define the order and distinguish it from the order Chytridiales. Molecular analyses reveal several strongly supported clades within the Rhizophydiales. Three of those clades encompass a broad range of isolates and are defined as new families Rhizophydiaceae, Terramycetaceae, and Kappamycetaceae. To resolve close relationships within Terramycetaceae, combined 28 S rRNA and ITS1–5.8 S–ITS2 sequences were analysed and details of zoospore ultrastructural character states determined, with two new genera, Terramyces and Boothiomyces, described. Two species formerly classified in Rhizophydium are transferred to the new genera. This work provides a framework for additional taxonomic revisions within the new order Rhizophydiales and compares genetic variation useful in defining genera, species, and populations within this lineage of chytrids. A broader sampling of representatives is needed before taxonomic decisions can be made for remaining clades within the Rhizophydiales.     

The revision of species-rich genera: a phylogenetic framework for the strategic revision of Pilea (Urticaceae) based on cpDNA, nrDNA, and morphology

The revision of species-rich genera underpins research and supports the sustainable use and monitoring of biological diversity. One fifth to one quarter of the diversity of all seed plant species occurs in such genera, but difficulties with the revision of species-rich genera has resulted in many of them being ignored since the late 1800s. Pilea, with 600-715 species is in need of revision. The only realistic approach is in manageable subunits, which requires confirmation of monophyly and identification of monophyletic subdivisions. Parsimony analyses of trnL-F, ITS, and morphology data were used to test the monophyly of, and explore intrageneric relationships within, Pilea. Analysis of trnL-F data confirms and recovers two morphologically diagnosable monophyletic clades that include all of the taxa within Pilea. Overlaying geographic distribution on a most parsimonious tree indicates a strong association between geography and phylogenetic relatedness. It is suggested that a strategic revision within the framework of morphologically and geographically diagnosable units might enable the revision of the group using an iterative approach. Analysis of the outgroup taxa supports the inclusion of Poikilospermum within the Urticaceae and suggests that the Urticaceae tribes could be placed into two clades that are supported by floral morphology.     

Convergent evolution and paraphyly of the hawk-eagles of the genus Spizaetus (Aves, Accipitridae) – phylogenetic analyses based on mitochondrial markers

The phylogenetic relationships within the New and Old World hawk-eagle assemblage (genus Spizaetus ; Aves: Accipitridae) were studied using mitochondrial DNA sequences ( cytochrome b , control region). Eighty-four specimens representing all Spizaetus species and almost all currently distinguished subspecies as well as 11 other booted and non-booted 'eagle' genera from the Neotropics, Africa, Eurasia, South Asia and Australasia ( Oroaetus , Harpia , Morphnus , Lophaetus , Stephanoaetus , Hieraaetus , Aquila , Ictinaetus , Spilornis , Pithecophaga , Harpyopsis ) were investigated. Although the basal branching could not be resolved, our investigations clearly indicate that hawk-eagles represent a paraphyletic assemblage and thus their external similarities have to be ascribed to convergent evolution. The New World taxa of Spizaetus cluster together, but the South American species Oroaetus isidori appears embedded within this clade. The taxa from Southeast to East Asia form a clearly separated monophyletic group. It is further divided into two subgroups, which are also characterized by distinct juvenile plumage patterns. Spizaetus africanus , the only African representative of the genus, is found in a mixed cluster consisting of members of the genera Aquila and Hieraaetus . These findings are in accordance with previous studies of other authors based on various molecular markers and different sets of taxa, but disagree with current taxonomy. Therefore, we suggest assigning the species of the genus Spizaetus to three different genera: (1) Spizaetus (including Oroaetus isidori ) in Central and South America and (2) Nisaetus for the Southeast to East Asian group. (3) The African taxon ( Spizaetus africanus ) is discussed to be included into the genus Aquila. Furthermore, we propose to use the former genus name Lophotriorchis Sharpe, 1874, for the monotypic species Hieraaetus kienerii , which has an isolated phylogenetic position.     

Molecular phylogenetic analyses of mullets (Mugilidae, Mugiliformes) based on two mitochondrial genes

Molecular phylogenetic studies are very scarce for the Mugilidae family; the present analysis using DNA sequences of the mitochondrial 16S rRNA and cytochrome b genes is the first study involving Brazilian mugilids. The results corroborate the monophyly of Mugil and are elucidative for the taxonomy of Brazilian mugilids. Mugil curema is clearly divided into two genetically distinct taxa, M. curema type I being closer related to Mugil hospes and the true M. curema (type II) grouping significantly with M. incilis. The results also suggest that Mugil liza and Mugil platanus should be treated as a single species or even populations of Mugil cephalus.     

Tortula and some related genera (Pottiaceae, Musci): phylogenetic relationships based on chloroplast rps4 sequences

 According to chloroplast rps4 sequence data the genus Syntrichia forms a monophyletic clade clearly separated from Tortula, while Pottia is shown to be polyphyletic and the Rhynchostegiae species (with rostrate lid) are very similar to Tortula. Crossidium is probably polyphyletic. The close affinity of Desmatodon, Stegonia, Pterygoneurum and some of the species of Phascum with Tortula is confirmed by the molecular data. Received May 23, 2002; accepted August 23, 2002 Published online: November 22, 2002 Address of the authors: O. Werner, R. M. Ros, M. J. Cano, J. Guerra (E-mail: werner@um.es) Departamento de Biología Vegetal (Botánica), Facultad de Biología, Universidad de Murcia, Campus de Espinardo, E-30100-Murcia, Spain.     

Molecular identification and phylogenetic relationship of green algae,Spirogyra ellipsospora (Chlorophyta) using ISSR and rbcL markers

Spirogyra is found in a wide range of habitats, including small stagnant water bodies, rivers, and streams. Spirogyra ellipsospora is common in northern Thailand. Species identification of the Spirogyra species based only on morphological characteristics can be difficult. A reliable and accurate method is required to evaluate genetic variations. This study aims to apply molecular approaches for the identification of S. ellipsospora using microsatellites and rbcL markers. Based on DNA sequencing, the rbcL gene was sequenced and the data was analyzed using the BLAST (Basic Local Alignment Search Tool) program in the NCBI (National Center for Biotechnology Information) database. The sequence of S. ellipsospora from this study revealed definitive identity matches in the range of 99% for the consensus sequences of S. ellipsospora. The 10 primers of ISSR could be amplified by 92 amplification fragments. The DNA fragments and the rbcL sequence data grouped the Spirogyra specimens into two distinct clusters.     

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.     

Genetic identification of bucktooth parrotfish Sparisoma radians (Valenciennes, 1840) (Labridae,Scarinae) by chromosomal and molecular markers

Parrotfishes (Labridae, Scarinae) comprise a large marine fish group of difficult identification, particularly during juvenile phase when the typical morphology and coloration of adults are absent. Therefore, the goal of this study was to test cytogenetic markers and DNA barcoding in the identification of bucktooth parrtotfish Sparisoma radians from the northeastern coast of Brazil. Sequencing of cytochrome c oxidase subunit I (COI) confirmed all studied samples as S. radians, and all showed high similarity (99–100%) with Caribbean populations. The karyotype of this species was divergent from most marine Perciformes, being composed of 2n = 46 chromosomes. These consisted of a large number of metacentric and submetacentric pairs with small amounts of heterochromatin and GC-rich single nucleolar organizer regions (NORs) not syntenic to 5S rDNA clusters. These are the first data about DNA barcoding in parrotfish from the Brazilian province and the first refined chromosomal analysis in Scarinae, providing useful data to a reliable genetic identification of S. radians.     

A molecular phylogenetic investigation of Pseudoamphisiella and Parabirojimia (Protozoa, Ciliophora, Spirotrichea), two genera with ambiguous systematic positions

The gene coding for the small subunit ribosomal RNA molecule was sequenced in three marine spirotrichs: Pseudoamphisiella lacazei, P. alveolata, and Parabirojimia similis, the systematic positions of which remain unclear in spite of recent progress. Phylogenetic trees were constructed by means of neighbor-joining (NJ), least-squares (LS), maximum parsimony (MP), and Bayesian inference (BI) methods in order to test previously inferred phylogenetic assignments of Pseudoamphisiella and Parabirojimia based on morphological and morphogenetic data. The main results are that: (1) all trees constructed have similar topologies in which the genus Parabirojimia derives at the base of the class Stichotrichia, which suggests that it might represent a unique taxon at about suborder or even-order level near urostylids; (2) the present study supports the conclusion reported by previous researchers that urostylids could be a paraphyletic assemblage and that Uroleptus and Paruroleptus should be removed from the order Urostylida; (3) Pseudoamphisiellidae/Pseudoamphisiella always clusters with Prodiscocephalus borrori rather than with urostylids, which indicates that they might represent an ancestral form for both discocephaline and urostylid species; (4) the monophyly of the traditional suborder Discocephalina and typical euplotids is consistently rejected. Based on both morphological/morphogenetical and molecular data obtained, a new suborder Parabirojimina n. subord. is suggested, which currently includes one family, Parabirojimidae n. fam., and the single genus Parabirojimia.     

Molecular phylogenetic analyses of Ulva (Chlorophyta, Ulvophyceae) mats in the Xiangshan Bay of China using high-resolution DNA markers

Intertidal Ulva mats occur annually in winter and spring in the Xiangshan Bay (29°26′–29°34′ N, 121°27′–121°50′ E) of China. Thousands of tons of Ulva biomass have been harvested as edible seaweeds for human consumption for several decades in this region. This investigation was designed to quantify Ulva microscopic propagules associated with the mat, identify species composition, and to analyze intra-species relationships using three molecular markers. Phylogenetic analysis based on the nuclear encoded rDNA internal transcribed spacer region, the plastid encoded large subunit of the ribulose 1,5-bisphosphate carboxylase gene, and the 5S rDNA spacer region showed that the mat was principally composed of Ulva prolifera and Ulva flexuosa. Their propagules were detected in both the water column and sediment. Based on phylogenetic analyses of the 5S rDNA spacer region, mat samples of U. prolifera and U. flexuosa were genetically distinct from the green tide samples in the Yellow Sea and U. flexuosa samples from Jiangsu coasts, respectively, revealing that isolated geographical position of the Xiangshan Bay might result in the maintenance of a distinct Ulva population. The results demonstrate that high-resolution DNA markers have great potential in identification and discrimination at and below the species level.     

Polyphyly of the grass tribe Hainardieae (Poaceae: Pooideae): identification of its different lineages based on molecular phylogenetics, including morphological and cytogenetic characteristics

The small pooid grass tribe Hainardieae comprises six genera with approximately ten species; however, this tribe was not accepted by all previous taxonomic treatments. To study the relationships among these genera and to infer the phylogeny and evolutionary patterns, we used sequence variation of the internal transcribed spacers (ITS) of nuclear ribosomal and chloroplast (cp) matK DNA and morphology. Many genera of the Aveneae/Poeae tribe complex additionally were included. Both molecular datasets showed Hainardieae to be highly polyphyletic, and its genera to branch with different groups of the Aveneae/Poeae. Parapholis and Hainardia are corroborated as being closely related, and belonging to a firmly supported Eurasian clade together with Catapodium incl. Scleropoa, Cutandia, Desmazeria, Sphenopus, Vulpiella (subtribe Parapholiinae) and with Cynosurus as sister to this assemblage. The other genera of traditionally recognised Hainardieae are positioned phylogenetically distant: Mediterranean Narduroides is verified as more or less related to Festuca and relatives (subtribe Loliinae), whereas the west Eurasian Pholiurus is close to the lineage of Poa and relatives (subtribe Poinae). North American Scribneria is sister to Deschampsia and both genera should be unified under a common subtribe (Aristaveninae or Holcinae). The phylogenetic position of the Algerian genus Agropyropsis (close to Narduroides and within the Loliinae) is suggested on morphology only, because no molecular data was obtained for it. Considering classification, we support the abandonment of tribe Hainardieae and argue to abandon Poeae subtribe Scribneriinae. Poeae subtribe Parapholiinae is redefined with a novel genus content, due to the exclusion of Agropyropsis and Pholiurus and the inclusion of Vulpiella.     

A new karyological variant of Oecomys (Rodentia: Sigmodontinae) and its phylogenetic relationship based on molecular data.

A new karyological variant of the Sigmodontinae genus Oecomys was detected in specimens from Brazilian Pantanal. Karyologic analyses showed 2n = 72 and FN = 90, which differs from all known chromosomal complements of the genus. This new karyomorph showed distinctive morphological attributes and could not be attributed to any described Oecomys species. Molecular analysis using cytochrome-b sequence data suggested the monophyly of Oecomys. Maximum-likelihood analyses placed the Oecomys sp. haplotype clade as the sister branch to Oecomys bicolor, whereas maximum-parsimony analyses did not resolve its relationship with the other Oecomys species. Kimura's two parameters distance estimates between this and other Oecomys species are high, and active gene flow has been found not to coexist with these divergence estimates.     

A molecular method for identification of the morphologically plastic invasive algal genera Eucheuma and Kappaphycus (Rhodophyta, Gigartinales) in Hawaii

A paucity of diagnostic morphological characters for identification and high morphological plasticity within the genera Eucheuma and Kappaphycus has led to confusion about the distributions and spread of three introduced eucheumoid species in Hawaii. Entities previously identified as E. denticulatum, K. alvarezii, and K. striatum have had profound negative effects on Oahu’s coral reef ecosystems. The use of molecular tools to aid identification of algal species has been promising in other morphologically challenging taxa. We used three molecular markers (partial nuclear 28S rRNA, partial plastid 23S rRNA, and mitochondrial 5′ COI) and followed a DNA barcoding-like approach to identify Eucheuma and Kappaphycus samples from Hawaii. Neighbor-joining analyses were congruent in their separation of Eucheuma and Kappaphycus, and the resulting clusters were consistent with those revealed for global comparisons with the mitochondrial cox2-3 spacer and GenBank data. Based on these results, new insights were revealed into the distribution of these groups in Hawaii.     

Development of phylogenetic markers from single-copy nuclear genes for multi locus, species level analyses in the mint family (Lamiaceae)

The use of single copy nuclear markers is of increasing importance in plant phylogenetics. The generally higher level of variability compared to chloroplast DNA and the ability to use incongruence in a multilocus analysis to determine reticulation patterns makes these kinds of sequence based markers especially useful for species level investigations. However, the prevalence of gene duplication that results from the high frequency of polyploidization events during the evolution of higher plants can impede marker development especially for groups lacking model organisms. Here, we present the strategy and results of marker development for phylogenetic analysis in Micromeria, using publicly available DNA sequences and ESTs from related genera from Lamiaceae, subfamily Nepetoideae. By eliminating markers with signatures of duplication during four steps of marker development, we were able to select 19 primer pairs that resulted in orthologous products for all the species studied. This corresponds to 23% of the initial 84 primer pairs designed. Using an initial sampling of eight individuals, we tested the markers for support of phylogenetic hypotheses related to the evolution of Micromeria on the Canary Islands. While some hypotheses were supported by all markers, an east west split, with a closer relationship between the species of Tenerife and Madeira on one hand and the ones from Gran Canaria and the eastern islands on the other is supported by 12 markers but contradicted by the remaining seven. This indicates that reticulation and inter-island gene flow played a role in the evolution of Micromeria.     

Comparative and phylogenetic analyses of Swertia L. (Gentianaceae) medicinal plants (from Qinghai,China) based on complete chloroplast genomes

Swertia L. is a large genus in Swertiinae (Gentianaceae). In China, many Swertia species are used as traditional Tibetan medicines, known as “Zangyinchen” or “Dida”. However, the phylogenetic relationships among Swertia medicinal plants and their wild relatives have remained unclear. In this study, we sequenced and assembled 16 complete chloroplast (cp) genomes of 10 Swertia species, mainly distributed in Qinghai Province, China. The results showed that these species have typical structures and characteristics of plant cp genomes. The sizes of Swertia cp genomes are ranging from 149,488 bp to 154,097 bp. Most Swertia cp genomes presented 134 genes, including 85 protein coding genes, eight rRNA genes, 37 tRNA genes, and four pseudogenes. Furthermore, the GC contents and boundaries of cp genomes are similar among Swertia species. The phylogenetic analyses indicated that Swertia is a complex polyphyletic group. In addition, positive selection was found in psaI and petL genes, indicating the possible adaptation of Qinghai Swertia species to the light environment of the Qinghai-Tibet plateau. These new cp genome data could be further investigated to develop DNA barcodes for Swertia medicinal plants and for additional systematic studies of Swertia and Swertiinae species.