Phylogenetic perspectives on diversification and character evolution in the species‐rich genus Erysimum (Erysimeae; Brassicaceae) based on a densely sampled ITS approach

Erysimum includes 150–350 species distributed in the Northern Hemisphere, with Eurasia being the centre of greatest diversity. It is well known for its taxonomic complexity as a result of overlapping morphological characters. We present the first densely sampled phylogenetic analysis of Erysimum using internal transcribed spacer (ITS) DNA sequences from c. 85% of the species (117 for the first time), representing the full range of morphological variation and geographical distribution. We used several approaches to reconstruct phylogenetic relationships, dating of diversification and patterns of evolution of morphological characters in the genus. Ancestral‐state reconstructions of four morphological diagnostic characters were performed using maximum parsimony, maximum likelihood and Bayesian methods. Our phylogenetic framework strongly supports the monophyly of Erysimum and recovers some well‐supported clades that are geographically, rather than morphologically, correlated. Our study confirms the placement of Erysimum in lineage I and reveals two Malcolmia spp. (M. maritima and M. orsiniana) as its sister taxa. The results suggest that the biennial duration and caespitose habit (vs. annual or perennial duration and herbaceous or woody habit) and large, yellow, glabrous (vs. small, non‐yellow, pubescent) petals are ancestral in Erysimum. The ancestral‐state reconstruction results show that annual vs. perennial and woody vs. herbaceous features have been independently derived several times. The dating analyses suggest an early radiation of Erysimum during the late Pliocene or early Pleistocene. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 175 , 497–522.     

Phylogenetic and systematic study of Korean <Emphasis Type="Italic">Orius</Emphasis> species (Heteroptera: Anthocoridae) on the basis of molecular and morphological data

A phylogenetic and systematic study of Orius species (Heteroptera: Anthocoridae) from Korea has been conducted using both morphological and molecular characters. Thirty morphological character states were coded for 10 strains of 9 species. Five molecular markers, partial cytochrome c oxidase I (COI), cytochrome b (CytB), 16S rRNA (16S), 18S rRNA (18S), and 28S rRNA (28S), from mitochondrial and nuclear genes, were tested. Phylogenetic analyses based on molecular data were conducted by minimum evolution, maximum parsimony, maximum likelihood, and Bayesian phylogenetic (BP) analyses. Analysis of morphological data was performed using the parsimony programs NONA, and the combined dataset of morphological and molecular data was analyzed using BP analyses. The results of this study indicate that use of COI and CytB enabled relatively effective identification of species, whereas the sequences of 16S, 18S and 28S did not enable identification of closely related species such as Orius minutus and O. strigicollis. We discuss the usefulness of the five molecular markers for determining phylogenetic relationships and identifying the species.     

Pyramidodinium spinulosum sp. nov. (Dinophyceae), a sand‐dwelling non‐motile dinoflagellate from the seafloor (36 m deep) off Mageshima Island,Kagoshima, Japan

A new species of benthic marine dinoflagellate, Pyramidodinium spinulosum Horiguchi, Moriya, Pinto & Terada is described from the deep (36 m) seafloor off Mageshima Island, Kagoshima Prefecture, Japan in the subtropical region of the northwest Pacific. The life cycle of the dinoflagellate consists of a dominant, attached, dome‐shaped, vegetative form and short‐lasting, motile cell. Asexual reproduction takes place by the formation of two motile cells within each non‐motile cell. The released motile cells swim only for a short period and transform directly into the dome‐shaped vegetative form. The duration of the cell cycle varies and can be extremely long, ranging 5–38 days under culture conditions. The non‐motile cell is enclosed by a cell wall and its surface is covered with many (80 – 130) spines of various length. The dinoflagellate is photosynthetic and contains many (more than 50) discoidal chloroplasts. Phylogenetic analysis reveals that the dinoflagellate is closely related to the type species of the genus Pyramidodinium, P. atrofuscum which also possesses a dominant, attached, non‐motile form. However, P. spinulosum can be clearly distinguished from P. atrofuscum by the cell shape (dome‐shaped vs. pyramid‐shaped) and surface ornamentation (spines vs. wart‐like processes) of the non‐motile form. Based on these morphological differences together with molecular evidence, it was concluded that this organism from a deep water sand sample should be described as a second species of the genus Pyramidodinium, P. spinulosum.     

Morphological and molecular diagnostics of <Emphasis Type="Italic">Phytoseiulus persimilis</Emphasis> and <Emphasis Type="Italic">Phytoseiulus macropilis</Emphasis> (Acari: Phytoseiidae)

This study focuses on the diagnostics of two natural enemy species, belonging to the genus Phytoseiulus in the family Phytoseiidae (sub-family Amblyseiinae): P. macropilis and P. persimilis. These two species are of primary importance in biological control all over the world. However, they are morphologically very similar and specific diagnostics is difficult. This study utilizes mitochondrial molecular markers (12S rRNA and Cytb mtDNA) to differentiate these two species. Morphological analyses showed significant differences between P. persimilis and P. macropilis for 17 morphological characters of the 32 considered. However, despite these significant differences, the ranges of all characters overlap. Only the serration of the macroseta on the basitarsus (StIV) allows the differentiation between P. persimilis and P. macropilis. Despite these small morphological differences, molecular results, for both mitochondrial DNA fragments considered (rRNA and Cytb mtDNA), showed a clear delineation between the specimens of P. macropilis and P. persimilis. This study emphasizes (i) that only one morphological character (serration of the seta StIV) clearly separates these two species, and (ii) the usefulness of an automatical molecular and simple diagnostic tool for accurate differentiation of the two species and ensure the morphological diagnostics. Further studies are proposed, including more DNA sequences especially for P. macropilis.     

Genetic relatedness among some wild cherry (<Emphasis Type="Italic">Prunus</Emphasis> subgenus <Emphasis Type="Italic">Cerasus</Emphasis>) genotypes native to Iran assayed by morphological traits and random amplified polymorphic DNA analysis

Subgenus Cerasus species are useful genetic resources for cherry breeding programs. A total of 17 morphological traits together with 19 random amplified polymorphic DNA (RAPD) primers were used to study 39 accessions including 34 wild Cerasus subgenus genotypes belonging to Prunus avium L., P. cerasus L., P. mahaleb L., P. microcarpa Boiss., P. incana Pall., and P. brachypetala Boiss. species, along with an unknown wild Cerasus sample, two advanced cherry cultivars (‘Lambert’ and ‘Bulgar’), and two rootstocks (‘Colt’ and ‘Gisela 6’). Genotypes were separated into different groups according to their species and collection sites using cluster analysis performed by Ward’s clustering method based on morphological data. Nineteen RAPD primers from 60 screened produced 304 polymorphic reproducible bands (98.15% polymorphism). According to the similarity matrix, the lowest similarity was obtained between P. avium and P. microcarpa samples. A dendrogram was prepared by the unweighted pair-group method with arithmetic average (UPGMA), and the accessions were separated according to their species and geographic origin. In both morphological and molecular results, the advanced cultivars and rootstocks were separated from wild genotypes, and the unknown genotype was grouped with P. mahaleb accessions. Grouping by morphological characteristics was compared with the results of RAPD analysis, with no significant correlations between morphological and molecular data being found. This is the first report of molecular (RAPD) genetic diversity study in wild Cerasus subgenus genotypes from Iran, and the results demonstrate the high potential of RAPD analysis for discrimination of Cerasus subgenus genotypes.     

Legs of deception: disagreement between molecular markers and morphology of long‐legged flies (Diptera,Dolichopodidae)

Conflicting hypotheses in phylogenetics and systematics, generated by different data sets (e.g. morphological versus molecular), are common in biology. The clarification of such instances may allow understanding general mechanisms involved in the speciation process in an evolutionary light. Here, we present and discuss the case of the Dolichopus plumipes species group in the long‐legged flies, Dolichopodidae. A phylogenetic survey was performed based on both morphological and molecular data. The full data set comprises 31 morphological characters and 2252 molecular characters (mitochondrial – COI: 810; 12S: 343; 16S: 514; nuclear – ITS2: 585) of 49 different species, represented by 82 specimens. The molecular phylogenetic analysis revealed a clade (composed by the species D. plumipes, Dolichopus wahlbergi, Dolichopus polleti, Dolichopus simplex, and Dolichopus nigricornis) that disagrees with the traditional morphological view based on external characters. In particular, specimens of the species D. plumipes and D. simplex were indistinguishable with the molecular markers used here. Yet, we still consider D. plumipes and D. simplex as two distinct taxa and provide explanatory hypotheses on the evolutionary background. The conspicuous male secondary sexual characters (present in plumipes but not in simplex) are key factors in sexual selection and their presumably rapid reduction in D. simplex is thought to be of main importance for the explanation of the speciation process. The plumipes–simplex case may therefore be viewed as a paradigmatic illustration showing that a better integration of the molecular and morphological approaches is needed to understand and clarify the, in some cases, complex systematics and phylogeny of organisms.     

Construction of genetic linkage map of the medicinal and ornamental plant <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

An integrated genetic linkage map of the medicinal and ornamental plant Catharanthus roseus, based on different types of molecular and morphological markers was constructed, using a F₂ population of 144 plants. The map defines 14 linkage groups (LGs) and consists of 131 marker loci, including 125 molecular DNA markers (76 RAPD, 3 RAPD combinations; 7 ISSR; 2 EST-SSR from Medicago truncatula and 37 other PCR based DNA markers), selected from a total of 472 primers or primer pairs, and six morphological markers (stem pigmentation, leaf lamina pigmentation and shape, leaf petiole and pod size, and petal colour). The total map length is 1131.9 cM (centiMorgans), giving an average map length and distance between two markers equal to 80.9 cM and 8.6 cM, respectively. The morphological markers/genes were found linked with nearest molecular or morphological markers at distances varying from 0.7 to 11.4 cM. Linkage was observed between the morphological markers concerned with lamina shape and petiole size of leaf on LG1 and leaf, stem and petiole pigmentation and pod size on LG8. This is the first genetic linkage map of C. roseus.     

Combining molecular and morphological analyses of water strider phylogeny (Hemiptera–Heteroptera, Gerromorpha): effects of alignment and taxon sampling

Abstract. The semiaquatic bugs (Hemiptera–Heteroptera, infraorder Gerromorpha), comprising water striders and their allies (c. 1900 described species), are familiar inhabitants of water surfaces in all continents. Recent fossil evidence indicates that the evolutionary history of semiaquatic bugs spans more than 120 million years of geological time. At present, our insight into the phylogeny of higher taxa is based upon Andersen's manual cladistic analysis of a suite of morphological characters. The present work expands the phylogenetic insight with numerical cladistic analyses of morphological and molecular datasets (partial sequences of 16S and 28S rDNA) for forty species of Gerromorpha covering most higher taxa (families, subfamilies), estimates of branch support, character incongruence, and topological congruence (nodal stability). For the molecular data we apply different alignment options (manual vs numerical alignment; multiple alignment vs direct optimization), treat insertion–deletion events (indels) as either missing data or as a fifth character state, subject the data to a sensitivity analysis, and estimate topological congruence between different analysis trees. Relationships change considerably under different analysis conditions, which means that there is little node stability, and for selecting preferred analysis conditions there is conflicting evidence from rescaled incongruence length difference and the key node criterion. Based on the analysis of the combined morphological and molecular datasets, this study supports the close relationship between the families Gerridae, Hermatobatidae and Veliidae (superfamily Gerroidea), but not the monophyly of the family Veliidae. The results suggest that the genus Ocellovelia (Ocelloveliinae) should be excluded from this family and placed as a sister group to Gerridae + the remaining species of Veliidae. Our study also supports a close relationship between the subfamilies Halobatinae and Ptilomerinae (Gerridae), and that the subfamily Veliinae is probably nonmonophyletic.     

A new species of <Emphasis Type="Italic">Volvocisporium</Emphasis> from Namibia, <Emphasis Type="Italic">V. grewiae</Emphasis> sp. nov. (Microstromatales,Ustilaginomycota)

A new species of Volvocisporium (Ustilaginomycota, Microstromatales) has been found on Grewia cf. flavescens (Tiliaceae) in Namibia. V. grewiae is the second representative of this genus worldwide and the first one from the African continent based on morphological and molecular data. Taxonomical novelties Volvocisporium grewiae Ritschel & Oberw.     

CULTIVATION‐INDEPENDENT SPECIES IDENTIFICATION OF DINOBRYON SPECIES (CHRYSOPHYCEAE) BY MEANS OF MULTIPLEX SINGLE‐CELL PCR1

With the discovery of a high molecular diversity of protists, a discrepancy between morphological and molecular species richness estimates became apparent. Solving the current concerns requires a comparative analysis of different sequences combined with morphological analyses of single cells originating from preserved field samples. We refined a single‐cell PCR (SC‐PCR) protocol for analyzing cells from field samples preserved with Lugol’s iodine solution. We linked microscopic screening with multiplex PCR targeting the SSU rDNA, internal transcribed spacer 1 (ITS1), 5.8S rDNA, internal transcribed spacer 2 (ITS2), and the mitochondrial cytochrome oxidase 1 (CO1) in a single PCR reaction. Using this method, we investigated the intraspecific molecular variation in Dinobryon populations originating from two lakes in the Salzkammergut area of Austria. All investigated genetic markers showed two separated clusters within the investigated populations of Dinobryon divergens O. E. Imhof, indicating a reproductive isolation of the two coexisting populations. Based on these findings, we describe a lineage, which is morphologically similar to D. divergens but, based on the molecular data, is reproductively isolated.     

<Emphasis Type="Italic">Steinernema boemarei</Emphasis> n. sp. (Nematoda: Steinernematidae), a new entomopathogenic nematode from southern France

A new entomopathogenic nematode, Steinernema boemarei n. sp., is described from southern France. Morphological, molecular (28S and ITS rDNA sequence data) and cross-hybridisation studies were used for diagnostics and identification purposes. Both molecular and morphological data indicate that the new species belongs to the ‘glaseri-group’ of Steinernema spp. Key morphological diagnostic traits for S. boemarei n. sp. include the presence of prominent deirids (cervical papillae) on adult males, the morphology of the spicules and gubernaculum, and the arrangement of the 23 genital papillae of the first generation males. Additionally, morphometric traits of the third-stage infective juvenile, including total body length (mean 1,103 μm), tail length (mean 86 μm), location of the excretory pore (mean 91 μm), and D% (mean 63), E% (mean 106) and H% (mean 41) values are definitive. In addition to these morphological characters, analysis of both 28S and ITS rDNA sequences depict this Steinernema species as a distinct and unique entity.     

Densely sampled phylogenetic analyses of the Lesser Short-toed Lark (Alaudala rufescens) — Sand Lark (A. raytal) species complex (Aves,Passeriformes) reveal cryptic diversity

The taxonomy of the Lesser/Asian Short-toed Lark Alaudala rufescens–cheleensis complex has been debated for decades, mainly because of minor morphological differentiation among the taxa within the complex, and different interpretations of the geographical pattern of morphological characters among different authors. In addition, there have been few studies based on non-morphological traits. It has recently been suggested based on a molecular study of the lark family Alaudidae that the Sand Lark A. raytal is nested within this complex. We here analysed mitochondrial cytochrome b (cyt b) from 130 individuals across the range of this complex (hereafter called Alaudala rufescens–raytal complex), representing all except two of the 18 currently recognized subspecies. We also analysed 11 nuclear markers from a subsample of these individuals, representing all of the clades found in the cyt b tree. Five primary clades were recovered, which confirmed that A. raytal is nested within this complex. Divergence time estimates among these five clades ranged from 1.76 to 3.16 million years (my; 95% highest posterior density [HPD] 1.0–4.51 my) or 1.99–2.53 my (95% HPD 0.96–4.3 my) in different analyses. Only four of the currently recognized subspecies were recovered as monophyletic in the cyt b tree. Our results call for a taxonomic revision, and we tentatively suggest that at least four species should be recognized, although we stress the need for an approach integrating molecular, morphological and other data that are not yet available.     

Morphological and phylogenetic analyses of <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> and <Emphasis Type="Italic">U. vignae</Emphasis> on legumes in Japan

Uromyces appendiculatus, inclusive of three varieties, is distinguished from U. vignae primarily by the position of urediniospore germ pores and putative host specificity. However, opinions concerning these morphological and physiological features as taxonomic characters have varied greatly, and distinction of these species has often been confused. To clarify the taxonomy of these two species, morphological features of urediniospores and teliospores of 225 rust fungus specimens on species of Phaseolus, Vigna, Apios, Lablab, and Dunbaria were examined by light microscopy and scanning electron microscopy. Forty-five specimens were subjected to molecular phylogenetic analyses. As a result, the position of germ pores in urediniospores and the teliospore-wall thickness were considered as good characters to separate three morphological groups. In molecular analyses, the specimens fell into two and three clades based on the nucleotide sequence at D1/D2 domain of LSU rDNA and ITS regions, respectively. One of the D1/D2 clades corresponded to one morphological group whereas another D1/D2 clade included two other morphological groups. In contrast, each of the three ITS clades corresponded to a separate morphological group. Neither morphological groups nor molecular clades were host limited. It is suggested that the three morphological groups that corresponded to three distinct ITS clades constitute distinct species.Contribution no. 186 from the Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Revision of the Indo-West Pacific polynemid fish genus <Emphasis Type="Italic">Eleutheronema</Emphasis> (Teleostei: Perciformes)

A taxonomic revision of the polynemid fish genus Eleutheronema, which is redefined, resulted in three species of the genus being regarded as valid: Eleutheronema rhadinum (Jordan and Evermann, 1902), having to date been treated as a junior synonym of E. tetradactylum (Shaw, 1804) and currently known only from East Asia (China and Japan) where it is endemic; E. tetradactylum, a senior synonym of both Polynemus teria Hamilton, 1822 and Polynemus coecus Macleay, 1878, being a widely distributed Indo-West Pacific species, which ranges from the Persian Gulf to Australia; and E. tridactylum (Bleeker, 1845), distributed in Southeast Asia (Thailand, Malaysia, and Indonesia). Eleutheronema tridactylum is easily distinguished from both E. rhadinum and E. tetradactylum owing to the vomer lacking tooth plates in the former [vs. vomer with 2 deciduous tooth plates (in specimens at least over ca. 70 mm SL) in the latter] and lower counts of pectoral filaments (free lower rays, 3 vs. 4) and gill rakers [mode 8 (range 4–10) vs. 12 (10–17) and 13 (6–18) in E. rhadinum and E. tetradactylum, respectively]. Eleutheronema rhadinum clearly differs from E. tetradactylum in having higher counts of pored lateral line scales [mode 95 (range 82–95) vs. 73 (71–80) in the latter] and higher scale counts above and below the lateral line [12 (11–14) and 16 (15–17), respectively, vs. 10 (9–12) and 14 (13–15), respectively]. Furthermore, E. rhadinum is distinguished from E. tetradactylum by having a dense black pectoral fin [vs. vivid yellow in life (except in specimens over ca. 350 mm SL, pectoral fin dusky-yellow) in the latter]. Intraspecific variations and morphological changes with growth of the three species are also discussed. Received: June 13, 2001 / Revised: October 11, 2001 / Accepted: October 17, 2001     

Teleomorph-anamorph relationships and reclassification of <Emphasis Type="Italic">Cordyceps cuboidea</Emphasis> and its allied species

Based on morphological characteristics and molecular phylogeny, we reclassified Cordyceps cuboidea and allied species C. alboperitheciata, C. prolifica, and Ophiocordyceps ryogamiensis. We investigated their teleomorph-anamorph relationships and revealed that these four species have Hirsutella-like anamorphs with morphological differences between them. By analyzing their molecular phylogeny, inferred from DNA sequences of internal transcribed spacer (ITS) and large subunit (LSU) D1/D2 region of rDNA, they were separated into four close-knit clades. Although C. prolifica and O. ryogamiensis formed their own clades, isolates of C. cuboidea separated into two clades, i.e., a true C. cuboidea clade and one resembling a new species, the O. paracuboidea clade. The latter two species are distinguished by the fruiting region of the stroma. In addition, C. alboperitheciata is regarded as a synonym of C. cuboidea. From the morphology, teleomorph-anamorph relationships, and molecular phylogeny, we concluded these species should be assigned to the genus Ophiocordyceps.