Sperm morphology and phylogeny of lagriids (Coleoptera,Tenebrionidae)

The systematics of tenebrionids remain unclear, principally at the subfamily level, as is the case of the Lagriinae. Considering that sperm morphology has contributed to the various insect group phylogenies, in this work we describe the structure and ultrastructure of these cells in Lagria villosa. Sperm in this species exhibit a strong morphological similarity to those of Tenebrio molitor and Tribolium castaneum, the only two species of Tenebrionidae with previously described sperm. In tenebrionids, the flagellar components offer good diagnostic characters, e.g. the symmetry of mitochondrial derivatives in L. villosa differentiates sperm of this species from those of Te. molitor and Tr. castaneum. However, the lateral association of the nucleus with flagellar components, the form of accessory bodies, and the presence of material connecting the axoneme to the accessory bodies and mitochondrial derivatives indicate that the three species form a sister group. Therefore, the sperm morphology of L. villosa support lagriid beetles as a subfamily (Lagriinae) of Tenebrionidae.     

DNA phylogeny, morphology and pathogenicity of Botryosphaeria species on grapevines

Several species of Botr yosphaeria are known to occur on grapevines, causing a wide range of disorders including bud mortality, dieback, brown wood streaking and bunch rot. In this study the 11 Botryosphaeria spp. associated with grapevines growing in various parts of the world, but primarily in South Africa, are distinguished based on morphology, DNA sequences (ITS-1, 5.8S, ITS-2 and EF1-α) and pathological data. Botryosphaeria australis, B. lutea, B. obtusa, B. parva, B. rhodina and a Diplodia sp. are confirmed from grapevines in South Africa, while Diplodia porosum, Fusicoccum viticlavatum and F. vitifusiforme are described as new. Although isolates of B. dothidea and B. stevensii are confirmed from grapevines in Portugal, neither of these species occurred in South Africa, nor were any isolates of B. ribis confirmed from grapevines. All grapevine isolates from Portugal, formerly presumed to be B. ribis, are identified as B. parva based on their EF1-α equence data. From artificial inoculations on grapevine shoots, we conclude that B. australis, B. parva, B. ribis and B. stevensii are more virulent than the other species studied. The Diplodia sp. collected from grapevine canes is morphologically similar but phylogenetically distinct from D. sarmentorum. Diplodia sarmentorum is confirmed as anamorph of Otthia spiraeae, the type species of the genus Otthia (Botryosphaeriaceae). A culture identified as O. spiraeae clustered within Botryosphaeria and thus is regarded as probable synonym. These findings confirm earlier suggestions that the generic concept of Botryosphaeria should be expanded to include genera with septate ascospores and Diplodia anamorphs.     

Ribes (Grossulariaceae) phylogeny as indicated by restriction-site polymorphisms of PCR-amplified chloroplast DNA

We surveyed exemplars from all 12 infrageneric taxa ofRibes (Grossulariaceae) for restriction site variation in two cpDNA regions, fromrbcL toaccD and fromrpoC1 torpoC2, in order to develop an explicit phylogenetic hypothesis and to assess the validity of infrageneric classifications. Maximum parsimony analysis resolves sect.Ribes (red currants), sect.Berisia (European alpine currants), sect.Symphocalyx (golden currants), sect.Grossularia plus sect.Grossularioides (true gooseberries and spiny currants), andHesperia, Lobbia, and probably sect.Robsonia (west North American gooseberries) as well-supported monophyletic groups. The clade of sectionsGrossularioides andGrossularia is unexpected, and suggests that subgenusGrossularia is not monophyletic. Alternatively, sect.Grossularioides may have acquired its cpDNA via hybridization and introgression. SectionsCoreosma (black currants) andHeritiera (dwarf currants) are apparently non monophyletic. Relationships among the well-supported lineages and the other sampled taxa remain unresolved. Maximum likelihood analysis is consistent with the parsimony results.     

Pollen morphology and phylogeny of the Alyxieae (Apocynaceae)

The pollen of the tribe Alyxieae, including Alyxia , Chilocarpus , Condylocarpon , Lepinia , Lepiniopsis , Plectaneia and Pteralyxia , has been studied with light, scanning electron and transmission electron microscopy. It is characterised by relatively large, irregularly disposed and - unequal-sized pores. 2-porate grains are most common in the Alyxieae, and occur in all genera except for Condylocarpon . 1-, 3-, 4- and 5-porate pollen grains are less common. The orientation of the polar axis and the equatorial plane could not be determined in any of the genera. Within the Alyxieae, Chilocarpus and Plectaneia are the nearest to other Apocynaceae, showing an essentially similar exine ultrastructure. Alyxia , Lepinia , Lepiniopsis and Pteralyxia make up a derived group, characterised by large pollen grain size and a remarkable nexine structure. Pollen of Lepinia and Lepiniopsis might be derived on the basis of their fossulate-verrucate ornamentation. Condylocarpon pollen shows a deviating set of pollen features (tetrads, inaperturate, reduced exine) that suggest it to be neotenic. Pollen morphology supports preliminary results of cladistic analyses of chloroplast data, in which the tribe is a monophyletic group nested in a 3-colporate environment, Chilocarpus and Condylocarpon are sisters at the base of the tree, Plectaneia is sister to a clade made up by Alyxia , Pteralyxia , Lepinia and Lepiniopsis , of which Lepinia and Lepiniopsis are the most derived.     

Floral morphology and phylogeny of the Disinae (Orchidaceae)

Previous classification of the Disinae has been based almost entirely on floral morphological data. These data are critically assessed by various methods to determine to what extent they support a classification resolved to sectional level. The variation in the characters relative to the sections and genera is mapped, homologies are established by careful morphological observation and ontogenetic studies, and finally hypotheses of homology are tested by congruence in several cladistic analyseS. It is found that although floral morphology allows the recognition of some groups, for others the results are ambiguouS. Floral morphological data are clearly inadequate to establish the taxonomy of the Disinae on a sound footing.     

Comparative morphology and molecular phylogeny of aplanochytrids (Labyrinthulomycota)

Aplanochytrids comprise one of three major subgroups within the Labyrinthulomycota. We have surveyed the diversity of aplanochytrids and have discovered that most isolates are difficult to identify to species because of character plasticity and ambiguity. Ten isolates were studied using molecular phylogenies based on small subunit ribosomal gene sequences (SSU rDNA) and morphological characters derived from light microscopy, SEM and TEM (e.g., colony size, colony shape, colony pattern, agar penetration, cell shape, cell surface patterns, cell inclusion characteristics and ectoplasmic net morphology). Of these isolates, we could positively identify two of them to species, namely Aplanochytrium yorkensis (Perkins, 1973) Leander and Porter, 2000 and A. minuta (Watson and Raper, 1957) Leander and Porter, 2000. We used standardized conditions for growing aplanochytrid isolates in order to minimize environmentally induced phenotypic plasticity in our comparative studies of morphology. By mapping the morphological characters listed above onto a conservative phylogenetic topology derived from SSU rDNA sequences, we were able to identify several synapomorphies (e.g., gross colony characteristics and cell surface patterns) that serve as valuable taxonomic characters for the identification of species and specific clades of aplanochytrids.     

Reproductive success of polygynous male corn buntings (Miliaria calandra) as confirmed by DNA fingerprinting

Despite a sex ratio approximating to unity, female corn buntingswere not equally distributed among males. In 1989 and 1990,41.2% of 50 males were monogamously paired, 29.4% were polygynous,and 23.5% were unpaired. Polygynous males usually paired withtwo females, although in 1990 three males were trigamous. Polygynousmales fledged more offspring from their territories than didmonogamous males, mainly because they had more mates. The fledgingsuccess per nesting female was slightly higher in territoriesof polygynous males, but not significantly so. DNA fingerprintingwas used to confirm the true paternity of 44 offspring from15 broods and the true maternity of 50 offspring from 16 broods.A further 12 offspring from three broods for which neither putativeparent was available were also fingerprinted. Actual reproductivesuccess of parents was close to that inferred from observationsof number of young raised. There was only one brood, containingtwo chicks (4.5% of offspring, or in 6.7% of broods), wherethe chicks were not fathered by the male defending the territory.However, this nest was close to the territory boundary, andthe defending male may have been assigned incorrectly. Therewere no cases of intraspecific brood parasitism (n = 16 broods).The copulation rate was low, and extrapair copulation attemptswere rare, probably because of the poor chances of sneakingonto a neighbor's territory undetected and the costs of leavinga territory unguarded.     

A phylogeny of sparoid fishes (Perciformes, Percoidei) based on morphology

 The putative percoid superfamily Sparoidea includes the Nemipteridae, Lethrinidae, Sparidae, and Centracanthidae. Although a rigorous cladistic analysis has never been attempted, two hypotheses regarding relationships among these families have been proposed. One early noncladistic hypothesis considered the Sparidae to be intermediate between the more primitive Nemipteridae and the more derived Lethrinidae. A later nonformal phylogenetic treatment provided evidence for a close relationship between Sparidae and Centranthidae and suggested a closer affinity between the Nemipteridae and Lethrinidae. We examine 54 osteological, ligament, and squamation characters in representatives of all 45 genera of these families and 4 outgroup taxa. The results of our cladistic analysis are congruent with a cladistic interpretation of the earlier hypothesis, with strong support for the phyletic sequence Nemipteridae, Lethrinidae, Sparidae plus Centracanthidae, with placement of centracanthids unresolved with respect to sparid genera. Received: May 21, 2001 / Revised: October 26, 2001 / Accepted: November 19, 2001     

Variation of pollen morphology, and its implications in the phylogeny of Clematis (Ranunculaceae)

Clematis s.l. (including Archiclematis and Naravelia) is a genus of approximately 300 species with cosmopolitan distribution. The diversity of its pollen was surveyed in 162 taxa belonging to all infrageneric groups of Clematis s.l. Pollen morphology was investigated by use of scanning electron microscopy to identify useful characters, test taxonomic and systematic hypotheses, and elucidate pollen character evolution on the basis of the molecular phylogeny. Clematis pollen is small to medium (14.8–32.1?μm?×?14.2–28.7?μm), oblate to prolate (P/E?=?0.9–1.4) in shape. The apertures may be tricolpate and pantoporate sometimes with 4-zonocolpate and pantocolpate pollen grains as transitional forms. The tricolpate pollen grains are predominant and occur in all the sections of the genus, whereas pantoporate pollen grains can be found in sect. Tubulosae, sect. Viorna, sect. Viticella, and Naravelia only. Phylogenetic mapping of aperture types reveals that the pantoporate pollen type may be the apomorphy in the genus and evolved several times. The surface ornamentation in all taxa studied is similar and characterized by microechinae evenly distributed on the microperforate tectum. The size and density of spinules on the tectum vary greatly but successive in the whole genus. According to the character syndromes of the ornamentation, separating sect. Brachiata from sect. Meclatis is supported. Though pollen morphology may contribute to investigation of problematic taxa, the taxonomic value of pollen morphology is limited at the species level.     

Chloroplast DNA phylogeny and biogeography of Lepidium (Brassicaceae)

Two intergenic spacers, trnT-trnL and trnL-trnF, and the trnL intron of cpDNA were sequenced to study phylogenetic relationships and biogeography of 73 Lepidium taxa. Insertions/deletions of ≥3 bp (base pairs) provided reliable phylogenetic information whereas indels ≤2 bp, probably originating from slipped-strand mispairing, are prone to parallelism in the context of our phylogenetic framework. For the first time, an hypothesis of the genus Lepidium is proposed based on molecular phylogeny, in contrast to previous classification schemes into sections and greges (the latter category represents groups of related species within a given geographic region), which are based mainly on fruit characters. Only a few of the taxa as delimited in the traditional systems represent monophyletic lineages. The proposed phylogeny would suggest three main lineages, corresponding to (1) sections Lepia and Cardaria, (2) grex Monoplocoidea from Australia, and (3) remaining taxa, representing the bulk of Lepidium species with more or less resolved sublineages that sometimes represent geographical correspondence. The fossil data, easily dispersible mucilaginous seeds, widespread autogamous breeding systems, and low levels of sequence divergence between species from different continents or islands suggest a rapid radiation of Lepidium by long-distance dispersal in the Pliocene/Pleistocene. As a consequence of climatic changes in this geological epoch, arid/semiarid areas were established, providing favorable conditions for the radiation of Lepidium by which the genus attained its worldwide distribution.     

A new lichenicolous teleomorph is related to plant pathogens in Laetisaria and Limonomyces (Basidiomycota, Corticiales)

Molecular and morphological data were used to assess the taxonomic placement of an undescribed lichenicolous basidiomycete teleomorph collected in Luxembourg, Belgium and Germany. The new species is ecologically and morphologically similar to Marchandiobasidium aurantiacum, teleomorph of the common bulbilliferous lichen pathogen Marchandiomyces aurantiacus. However phylogenetic analysis of nuclear and mitochondrial rDNA sequences indicated a close relationship of the new species-not to M. aurantiacum but to turf grass pathogens in genera Laetisaria and Limonomyces, including the generic type of Laetisaria. We are including the new species in Laetisaria based on strong statistical support for the clade containing these teleomorphs and several Marchandiomyces anamorphs. The morphological and ecological diversity of this clade indicates a potentially significant evolutionary role played by lichen-associated species in the Corticiales.     

Sperm morphology of salamandrids (Amphibia, Urodela): implications for phylogeny and fertilization biology

Mature spermatozoa belonging to four salamander species, Salamandrina terdigitata, Triturus alpestris, Triturus carnifex and Triturus vulgaris, have been investigated by electron microscopy. The sperm ultrastructure of these species was compared with that of previously examined urodeles (36 species and 20 genera) and with that of anurans and caecilians. Many phylogenetic considerations may be inferred as a consequence of comparative spermatology. Urodela appears to be a monophyletic order characterized by three sperm synapomorphies: the acrosomal barb, nuclear ridge and marginal filament. Cryptobranchoidea are confirmed to form a monophyletic suborder having two synapomorphic characters: absence of mitochondria in the tail, and cylindrical shape of the tail axial rod. Within the family Salamandridae, sperm morphology confirms the phylogenetic distance between Salamandrina and Triturus, as already pointed out on the basis of molecular and morphological characters. The very complex ultrastructure of spermatozoa confirms a previous opinion that internal fertilization is the ancestral condition of the Amphibia.     

First reported case of lysinuric protein intolerance (LPI) in Lithuania,confirmed biochemically and by DNA analysis

We report on an 18-year-old Lithuanian girl with hepatosplenomegaly noticed at birth, which progressed thereafter. The patient had to wait about 17 years for an accurate diagnosis and appropriate therapy. Lactase deficiency, congenital cataract of the right eye, and osteoporosis were observed. Episodes of drowsiness were caused by intake of high-protein food. Laboratory findings included slight hyperammonaemia, high plasma Citr, Ala, Gly, Glu, Ser levels, as well as citrullinuria, lysinuria, glutaminuria, alaninuria, argininuria, prolinuria, hydroxyprolinuria, ornithinuria, and orotic aciduria. Aversion to high-protein diet strongly suggested a disorder resulting in hyperammonaemia. Citrullinaemia was suspected. Subsequently the diagnosis of LPI was made on the basis of biochemical and clinical features. Molecular genetic testing revealed a mutation in the SLC7A7 gene, confirming the diagnosis.     

Molecular phylogeny of Old World monkeys (Cercopithecidae) as inferred from gamma-globin DNA sequences

DNA sequence data of the nuclear-encoded gamma1-gamma2-globin duplication region were used to examine the phylogenetic relationships of 16 cercopithecid (Old World monkey) species representing 12 extant genera. Morphology- and molecular-based hypotheses of Old World monkey branching patterns are generally congruent, except for generic relationships within the subtribe Papionina. The cercopithecids divide into colobines (leaf-eating monkeys) and cercopithecines (cheek-pouched monkeys). The colobines examined by the DNA data divide into an Asian clade (Nasalis, proboscis monkeys; Trachypithecus, langurs) and an African clade (Colobus, colobus monkeys). The cercopithecines divide into tribes Cercopithecini (Erythrocebus, patas monkey; Chlorocebus, green monkeys; Cercopithecus, guenons) and Papionini. Papionins divide into subtribes Macacina (Macaca, macaques) and Papionina (Papio, hamadryas baboons; Mandrillus, drills and mandrills; Theropithecus, gelada baboons; Lophocebus, arboreal mangabeys; Cercocebus, terrestrial mangabeys). In a morphologically based classification, Mandrillus is a subgenus of Papio, whereas Lophocebus is a subgenus of Cercocebus. In contrast, the molecular evidence treats Mandrillus as a subgenus of Cercocebus, and treats both Theropithecus and Lophocebus as subgenera of Papio. Local molecular clock divergence time estimates were used as a yardstick in a "rank equals age" system to propose a reduction in taxonomic rank for most clades within Cercopithecidae.     

Mitochondrial DNA phylogeny of West African aplocheiloid killifishes (Cyprinodontiformes, Aplocheilidae)

African killifishes (Cyprinodontiformes, Aplocheilidae) historically associated with the genus Aphyosemion occur in two geographically distinct regions. One assemblage from far West Africa has been previously shown to be monophyletic and not closely related to the remaining eastern species of Aphyosemion (W. J. Murphy and G. E. Collier, 1997, Mol. Biol. Evol. 14, 790-799). This is supported by further analysis of mitochondrial DNA sequences from 19 species from 21 different localities, representing 19 of the putative 22 species of this western group. Phylogenetic analyses of these data corroborate the monophyly and sister-group relationship of two distinct groups of taxa: Callopanchax and Scriptaphyosemion. Many of the relationships within Scriptaphyosemion suggest that these taxa may have radiated within a short period of time relative to the rate of substitutions within these sequences. A third, and possibly paraphyletic group of species, Archiaphyosemion, is suggested to be the sister taxon to the first two groups. These three groups are elevated to generic rank and together represent the sister group to the genus Epiplatys. Biogeographic inference suggests that the ancestors of this group diversified westward through upland habitat and have only relatively recently entered the lowland habitats in which Scriptaphyosemion and Callopanchax have diversified, with the latter genus reacquiring a suite of traits collectively referred to as annualism.     

Molecular phylogeny, recent radiation and evolution of gross morphology of the rhubarb genus Rheum (Polygonaceae) inferred from chloroplast DNA trnL-F sequences

BACKGROUND AND AIMS: Rheum, a highly diversified genus with about 60 species, is mainly confined to the mountainous and desert regions of the Qinghai-Tibetan plateau and adjacent areas. This genus represents a good example of the extensive diversification of the temperate genera in the Qinghai-Tibetan plateau, in which the forces to drive diversification remain unknown. To date, the infrageneric classification of Rheum has been mainly based on morphological characters. However, it may have been subject to convergent evolution under habitat pressure, and the systematic position of some sections are unclear, especially Sect. Globulosa, which has globular inflorescences, and Sect. Nobilia, which has semi-translucent bracts. Recent palynological research has found substantial contradictions between exine patterns and the current classification of Rheum. Two specific objectives of this research were (1) to evaluate possible relationships of some ambiguous sections with a unique morphology, and (2) to examine possible occurrence of the radiative speciation with low genetic divergence across the total genus and the correlation between the extensive diversification time of Rheum and past geographical events, especially the recent large-scale uplifts of the Qinghai-Tibetan Plateau. METHODS: The chloroplast DNA trnL-F region of 29 individuals representing 26 species of Rheum, belonging to seven out of eight sections, was sequenced and compared. The phylogenetic relationships were further constructed based on the sequences obtained. KEY RESULTS: Despite the highly diversified morphology, the genetic variation in this DNA fragment is relatively low. The molecular phylogeny is highly inconsistent with gross morphology, pollen exine patterns and traditional classifications, except for identifying all samples of Sect. Palmata, three species of Sect. Spiciformia and a few species of Sect. Rheum as corresponding monophyletic groups. The monotypic Sect. Globulosa showed a tentative position within the clade comprising five species of Sect. Rheum. All of the analyses revealed the paraphyly of R. nobile and R. alexandrae, the only two species of Sect. Nobilia circumscribed by the possession of large bracts. The crude calibration of lineages based on trnL-F sequence differentiation implied an extensive diversification of Rheum within approx. 7 million years. CONCLUSIONS: Based on these results, it is suggested that the rich geological and ecological diversity caused by the recent large-scale uplifts of the Qinghai-Tibetan Plateau since the late Tertiary, coupled with the oscillating climate of the Quaternary stage, might have promoted rapid speciation in small and isolated populations, as well as allowing the fixation of unique or rare morphological characters in Rheum. Such a rapid radiation, combined with introgressive hybridization and reticulate evolution, may have caused the transfer of cpDNA haplotypes between morphologically dissimilar species, and might account for the inconsistency between morphological classification and molecular phylogeny reported here.     

Chloroplast DNA phylogeny of Asian Bamboos (Bambusoideae,Poaceae) and its systematic implication

The bamboo is usually classified as a subfamily Bambusoideae of Poaceae, and includes approximately 20 genera and 300 species. To estimate phylogenetic relationships among these genera, we examined restriction site mutations of cpDNA for 16 Asian genera. In the cladogram obtained, the Bambosoideae was divided into two major lineages, one includingPleioblastus, Pseudosasa, Semiarundinaria, Shibataea, Phyllostachys, Sasa, Sinobambusa, Chimonobambusa, Arthrostylidium, andYushania, and the other consisting ofBambusa, Gigantochloa, Dendrocalamus, Thyrostachys, Melocanna, andSchizostachyum. Monophylly of each clade was supported by 83% and 98% bootstrap probability, respectively. The present result supports monophylly of Arundinarieae of Potztal's (1964) classical system, but does not support his treatment to recognize Dendrocalameae.     

Cloning and characterization of the mating type (MAT) locus from Ascochyta rabiei (teleomorph: Didymella rabiei) and a MAT phylogeny of legume-associated Ascochyta spp

Degenerate primers designed to correspond to conserved regions of the high mobility group (HMG) protein encoded by the MAT1-2 gene of Cochliobolus heterostrophus, Cochliobolus sativus, and Alternaria alternata were used to amplify the portion of the sequence corresponding to the HMG box motif from Ascochyta rabiei (teleomorph: Didymella rabiei). A combination of TAIL and inverse PCR extended the MAT1-2 sequence in both directions, then primers designed to MAT1-2 flanking DNA were used to amplify the entire MAT1-1 idiomorph. MAT1-1 and MAT1-2 idiomorphs were 2294 and 2693 bp in length, respectively, and each contained a single putative open reading frame (ORF) and intron similar to MAT loci of other loculoascomycete fungi. MAT genes were expressed at high levels in rich medium. MAT-specific PCR primers were designed for use in a multiplex PCR assay and MAT-specific PCR amplicons correlated perfectly to mating phenotype of 35 ascospore progeny from a cross of MAT1-1 by MAT1-2 isolates and to the mating phenotype of field-collected isolates from diverse geographic locations. MAT-specific PCR was used to rapidly determine the mating type of isolates of A. rabiei sampled from chickpea fields in the US Pacific Northwest. Mating type ratios were not significantly different from 1:1 among isolates sampled from two commercial chickpea fields consistent with the hypothesis that these A. rabiei populations were randomly mating. The mating type ratio among isolates sampled from an experimental chickpea field where asexual reproduction was enforced differed significantly from 1:1. A phylogeny estimated among legume-associated Ascochyta spp. and related loculoascocmycete fungi using sequence data from the nuclear ribosomal internal transcribed spacer (ITS) demonstrated the monophyly of Ascochyta/Didymella spp. associated with legumes but was insufficiently variable to differentiate isolates associated with different legume hosts. In contrast, sequences of the HMG region of MAT1-2 were substantially more variable, revealing seven well-supported clades that correlated to host of isolation. A. rabiei on chickpea is phylogenetically distant from other legume-associated Ascochyta spp. and the specific status of A. rabiei, A. lentis, A. pisi, and A. fabae was confirmed by the HMG phylogeny