Effect of larval brain extracts derived from three swallowtail butterflies,Papilio helenus L., P. machaon L. and P. memnon L., on seasonal morph development of P. xuthus L. (Lepidoptera: Papilionidae)

Adults of the three papilionid butterflies, Papilio helenus L., Papilio machaon L. and Papilio memnon L., exhibit seasonal diphenism comprising spring and summer morphs. To elucidate the physiological mechanism underlying seasonal morph development in papilionid butterflies, we investigated whether a cerebral factor showing summer‐morph‐producing hormone (SMPH) activity is present in the brain of three Papilio species using an assay system with chilled male short‐day pupae of P. xuthus L. When 2% NaCl extracts derived from 20 larval brains of the three species were injected into abdomens of chilled male short‐day pupae of P. xuthus, all recipients destined to develop into spring‐morph adults developed into summer‐ and intermediate‐morph adults. On the other hand, all recipients injected with distilled water as a control developed into spring‐morph adults. These results indicate that a cerebral factor showing SMPH activity is present in the larval brain of the three Papilio species. Additionally, all recipients injected with 2% NaCl extracts derived from 20 adult brains of Bombyx mori L. also developed into summer‐ and intermediate‐morph adults. The results revealed that SMPH or a cerebral factor showing SMPH activity is widely distributed among lepidopteran insects.     

DNA barcoding of genus Toxoptera Koch (Hemiptera: Aphididae): Identification and molecular phylogeny inferred from mitochondrial COI sequences

Abstract Identification of aphid species is always difficult due to the shortage of easily distinguishable morphological characters. Aphid genus Toxoptera consists of species with similar morphology and similar to Aphis in most morphological characters except the stridulatory apparatus. DNA barcodes with 1 145 bp sequences of partial mitochondrial cytochrome‐coxidase I (COI) genes were used for accurate identification of Toxoptera. Results indicated mean intraspecific sequence divergences were 1.33%, whereas mean interspecific divergences were greater at 8.29% (0.13% and 7.79% if T. aurantii 3 and T. aurantii 4 are cryptic species). Sixteen samples were distinguished to four species correctly by COI barcodes, which implied that DNA barcoding was successful in discrimination of aphid species with similar morphology. Phylogenetic relationships among species of this genus were tested based on this portion of COI sequences. Four species of Toxoptera assembled a clade with low support in maximum‐parsimony (MP) analysis, maximum‐likelihood (ML) analysis and Bayesian phylogenetic trees, the genus Toxoptera was not monophyletic, and there were two sister groups, such as T. citricidus and T. victoriae, and two clades of T. aurantii which probably presented cryptic species in the genus.     

Molecular Cloning and Characterization of a Microsatellite Locus Found in an RAPD Marker of a Spider Mite, Panonychus Citri (Acari: Tetranychidae)

Genetic markers were searched using PCR with 40 kinds of decanucleotide primers to investigate DNA polymorphism in Panonychuscitri. A region consisting of a variable number of CT tandem repeats (microsatellite) was found in a fragment amplified with the OPB10 primer. The microsatellite differed in size by ca. 100bp among several P. citri populations screened and was derived from at least seven alleles. This region was characteristic of P. mori and P. osmanthi, but was lacking in P. ulmi. The flanking regions were highly conserved among these species.     

Genetic relationships between Oeneis urda and O. mongolica (Nymphalidae: Lepidoptera)

The species status of Oeneis urda (Eversmann) and O. mongolica (Oberthür) has been argued based on morphological characters. Reexamination of their major morphological characters has shown a slight differentiation in the two species. Sequences of three mitochondrial genes (COI, ND6, and ND1) and one nuclear region (internal transcribed spacer 2, ITS2) from two O. urda populations (Yangyang and Mt. Hanla) and one O. mongolica population (Uljin) were performed for phylogenetic and population genetic inferences. Sharing of identical sequences in the ND6 gene and ITS2, minimal sequence divergence in the COI and ND1 genes, and phylogenetically undividable sequence types in all mitochondrial genes and ITS2 suggest genetic continuity between the two species. Nevertheless, significant F_ST estimates (P < 0.05) were found for the COI gene in comparisons between Yangyang (O. urda) and Uljin (O. mongolica), between Yangyang (O. urda) and Mt. Hanla (O. urda), and between Uljin (O. mongolica) and Mt. Hanla (O. urda) populations. These F_ST estimates, along with other gene‐based analyses collectively suggest isolation of the two species at some point in the past, but incomplete separation between the two species on the mainland (Yangyang and Uljin) and biogeographically forced isolation of the O. urda population on Mt. Hanla collectively appear to complicate species status of these two species that were once further clearly separated.     

Development of species‐specific primers for rapid diagnosis of Tetranychus urticae,T. kanzawai,T. phaselus and T. truncatus (Acari: Tetranychidae)

Species diagnosis is of the utmost importance to both pest management and plant quarantine services. Because of difficulties in the morphological diagnosis of spider mites, molecular techniques are of great value to rapidly and accurately diagnose closely related species. We examined four species of genus Tetranychus (the green and red forms of T. urticae, and T. kanzawai, T. phaselus and T. truncatus), which are found in Korea and are of significance to plant quarantine services. DNA samples isolated from a single egg, larva or adult weighed 64–188 ng. We designed species‐specific primers by performing sequence alignment for 107 sequences of the ribosomal internal transcribed spacer 2 (ITS2) region, which we obtained from GenBank, and sequences generated in this study. Specific nucleotides of each species were selected for designing primers specific for each species. Each species‐specific primer pair, when used to perform PCR analyses, detected only the species from which it originated. However, a T. urticae‐specific primer pair did not discriminate between the green and red forms of this species. These species‐specific primers can be applied in practice for the rapid and accurate diagnosis of spider mite species in plant quarantine and in agricultural fields.     

<Emphasis Type="Italic">Rhodotorula oryzae</Emphasis> sp. nov., a novel basidiomycetous yeast species isolated from paddy rice

A basidiomyetous yeast strain RO-203, which formed orange-red colored colonies, was isolated from a sample of paddy rice crops at the ripe stage in Japan. Morphological, physiological and biochemical characterization indicated that this strain belonged to the genus Rhodotorula. Molecular taxonomic analysis based on the 26S rDNA D1/D2 domain and internal transcribed spacer (ITS) region sequences showed that RO-203 represents an undescribed yeast species, for which the name Rhodotorula oryzae sp. nov. is proposed (type strain: AS 2.2363^T = MAFF 516128^T). The new species clustered in a branch together with Sakaguchia dacryoidea in phylogenetic trees based on the D1/D2 and ITS sequences. These two species differed by 2.3% and 12% nucleotide divergences in the D1/D2 and ITS regions, respectively.     

Assessing the potential of candidate DNA barcodes for identifying non‐flowering seed plants

In plants, matK and rbcL have been selected as core barcodes by the Consortium for the Barcode of Life (CBOL) Plant Working Group (PWG), and ITS/ITS2 and psbA‐trnH were suggested as supplementary loci. Yet, research on DNA barcoding of non‐flowering seed plants has been less extensive, and the evaluation of DNA barcodes in this division has been limited thus far. Here, we evaluated seven markers (psbA‐trnH, matK, rbcL, rpoB, rpoC1, ITS and ITS2) from non‐flowering seed plants. The usefulness of each region was assessed using four criteria: the success rate of PCR amplification, the differential intra‐ and inter‐specific divergences, the DNA barcoding gap and the ability to discriminate species. Among the seven loci tested, ITS2 produced the best results in the barcoding of non‐flowering seed plants. In addition, we compared the abilities of the five most‐recommended markers (psbA‐trnH, matK, rbcL, ITS and ITS2) to identify additional species using a large database of gymnosperms from GenBank. ITS2 remained effective for species identification in a wide range of non‐flowering seed plants: for the 1531 samples from 608 species of 80 diverse genera, ITS2 correctly authenticated 66% of them at the species level. In conclusion, the ITS2 region can serve as a useful barcode to discriminate non‐flowering seed plants, and this study will contribute valuable information for the barcoding of plant species.     

Intra‐specific variability in Rhinoleucophenga punctulata populations (Diptera: Drosophilidae) from Neotropical biomes: Combined analyses of morphological and molecular data

Despite the fact that Drosophilidae is a very diverse and well‐studied taxon, the New World genus Rhinoleucophenga is yet poorly understood even in regard to species distribution and morphological variability pattern. In this sense, R. punctulata is a species widely distributed in the Neotropical region. Specimens of R. punctulata were collected from different biomes in Brazil: Pampa, Cerrado and Caatinga sensu strictu, and a southern Amazonian savannah enclave area. Geographical variations in the external body morphology and in the morphology of spermatheca were noticed among the different populations. The hypothesis that each population could be a different species was tested through molecular data. A fragment of the mitochondrial cytochrome c oxydase subunit I (COI) gene was sequenced to perform phylogenetic analyses through neighbor‐joining and Bayesian inferences. Pairwise genetic divergences of COI sequences were calculated using DNA barcode premises. The analyzed populations presented different variation levels in both morphology and molecular traits. However, new species were not proposed because the intra‐population nucleotide variations exceeded the inter‐population ones. The noticeable morphological and genetic variations revealed among the four studied populations of R. punctulata in different biomes of Brazil suggest the necessity that morphological, distributional and molecular data at the population level should be integrated into complementary datasets to better understand the biological diversity of Rhinoleucophenga through Neotropical environments.     

On polyphyly of the former section Ochlopoa and the hybridogenic section Acroleucae (Poa,Poaceae): insights from molecular phylogenetic analyses

In this study, sequence data from the inert nuclear region ITS1‐5.8S rDNA‐ITS2 and the chloroplast region trnL–F, as well as a few morphological characters, are analysed to the relationships among known annual Poa (bluegrasses). It is shown that all taxa from the Poa annua aggregate distinguished by lemma characters and growth form have identical ITS and trnL–trnF sequences, all ITS sequences of this aggregate are the same as thethose of P. supina, and all trnL–trnF sequences are homologous with those of P. infirma. Furthermore, no differences were found between unusual morphotypes of P. supina with short spinules on their panicle branches and typical plants, but Siberian samples were found to have a slightly differentiated trnL–trnF region. These results suggest a hybrid origin of the Asian annual bluegrasses. Their maternal genome is close to that of P. sect. Homalopoa, but their ITS sequences are different. Some ITS sequences from annual Asian bluegrasses are resolved among representatives of P. sect. Stenopoa while for other (morphologically closely similar) species they fall in a clade with representatives of P. sect. Malacanthae. The latter group is distant from P. sect. Ochlopoa and is better treated as a separate section, viz P. sect. Acroleucae. The American annual bluegrasses are heterogeneous and also rather distant from P. sect. Ochlopoa. Poa chapmaniana, a species with cleistogamic flowers, is nested among the basal Subantarctic sections, far away from the taxa with which it has previously been considered related. It is indeed closer to P. sect. Ochlopoa than to other annual American bluegrasses. Thus, the studied annual species in fact belong to four independent evolutionary lines (or six including the separate genus Eremopoa and the Turkish Poa jubata), one of which, Acroleucae, has gone through three reticulation events. As in previous studies, our analysis did not support the generic status of P. sect. Ochlopoa.     

Developing a new molecular marker for aphid species identification: Evaluation of eleven candidate genes with species-level sampling

The mitochondrial cytochrome c oxidase subunit I (COI) gene has been utilized as a molecular marker for aphid species identification. However, this gene has sometimes resulted in misidentification because of low interspecific genetic divergences between some species pairs. In this study, to propose new molecular markers for the family Aphididae, we first screened 2289 sequences of 11 genes (COI, COII, CytB, ATP6, lrRNA, srRNA, ITS1, ITS2, EF1a, 18S, and 28S) collected from the GenBank. Among the 11 genes, ATP6 gene revealed the largest genetic divergence among congeneric species with the smallest divergence among conspecific individuals; in contrast, species pairs with low genetic divergences (< 1%) were not observed. Secondly, for statistically testing the usefulness of ATP6 gene in species identification, we analyzed genetic distances between all of the combinations of 32 individuals of 20 species for both COI and ATP6 genes. The ATP6 gene showed lower intraspecific (on average 0.08%) and higher interspecific (on average 8.28%) genetic distances than the COI gene (on average 0.19% and 6.24%, respectively) for the same pairs of individuals. This study corroborates the usefulness of the ATP6 gene as a new molecular marker that could improve the misidentification problems that are inherent with the COI gene.     

Review of the Halieutichthys aculeatus species complex (Lophiiformes: Ogcocephalidae), with descriptions of two new species

The Halieutichthys aculeatus species complex is reviewed. Members of this clade are distinguished by the presence of tubercles on the tail and a reticulate dorsal pigmentation pattern. Three species are recognized, including two species new to science. A neotype is chosen for H. aculeatus. Halieutichthys bispinosus n. sp. is characterized by having relatively strong tubercles on the dorsal surface, a row of tubercles almost always present dorsal to the orbit, both sphenotic tubercles well developed and sharp, trifid principal tubercles on the disk margin with anterior spinelet enlarged, dense arrangement of tubercles on the tail and a comparatively large adult body size. Halieutichthys intermedius n. sp. can be distinguished from congeners by having both sphenotic tubercles strongly reduced, the tubercles almost always absent dorsal to the orbit, principal tubercles on the disk margin uniformly short and blunt tubercles, with all spinelets generally reduced.     

Small snails in a big place: a radiation in the semi‐arid rangelands in northern Australia (Eupulmonata,Camaenidae, Nanotrachia gen. nov.)

Continuing the revision of the Camaenidae in the Australian Monsoon Tropics, we employed comparative analyses of morphological features (with a focus on shell and penial anatomy) and genetic markers (with a focus on mitochondrial COI and 16S sequences) to address the systematic relationships of land snails from the Victoria River District, Northern Territory, and adjacent East Kimberley (Western Australia). These analyses revealed that the species under study represented the previously undescribed genus Nanotrachia. This genus differs from all other camaenid genera known from north‐western Australia most conspicuously by its small, flat, and ribbed shell. Six species are identified as members of the new genus, four of them new species ( Nanotrachia costulata sp. nov. , Nanotrachia carinata sp. nov. , Nanotrachia coronata sp. nov. , Nanotrachia levis sp. nov. ). Two further species have already been described previously but assigned to different genera. These species, Ordtrachia intermedia (as the type species of Nanotrachia) and Mouldingia orientalis, are here transferred to Nanotrachia. Like other camaenids from the Australian Monsoon Tropics, species of Nanotrachia are characterized by essentially allopatric distributions, regional endemism, and a patchy distribution across their range. © 2013 The Linnean Society of London     

A critical review on some closely related species of <Emphasis Type="Italic">Tetranychus</Emphasis> sensu stricto (Acari: Tetranychidae) in the public DNA sequences databases

Taxonomic misidentification of the specimens used to obtain DNA sequences is a growing problem reported for different groups of organisms, which threatens the utility of the deposited sequences in public DNA databases. This paper provides new evidence of misidentifications in molecular DNA public databases in phytophagous mites of the Tetranychidae family belonging to the group Tetranychus (Tetranychus). Several species in this group are of economic and quarantine importance in agriculture and among them Tetranychus urticae, a highly polyphagous mite causing outbreaks in many crops worldwide, is certainly the most studied. We analyzed and evaluated the identity of 105 GenBank accessions of ITS2 rDNA and 138 COI mtDNA sequences which were deposited as T. urticae and those of 14 other taxa morphologically closely related to Tetranychus sensu stricto. In addition, ITS2 and COI sequences of 18 T. urticae samples collected for this study and identified by morphological criteria, were generated and included in the analyzed dataset. Among the deposited sequences in the GenBank database, numerous cases of apparently mistaken identities were identified in the group Tetranychus s. str., especially between T. urticae, T. cinnabarinus, T. kanzawai and T. truncatus. Unreliable sequences (misidentified or dubious) were estimated at nearly 30%. In particular the analysis supports the invalidity of the controversial species status of T. cinnabarinus. More generally, it highlights the need of using combined morphological and molecular approaches to guarantee solid species diagnostics for reliable sequence accessions in public databases.     

DNA barcoding of Gaultheria L. in China (Ericaceae: Vaccinioideae)

Abstract Four DNA barcoding loci, chloroplast loci rbcL, matK, trnH‐psbA, and nuclear locus internal transcribed spacer (ITS), were tested for the accurate discrimination of the Chinese species of Gaultheria by using intraspecific and interspecific pairwise P‐distance, Wilcoxon signed rank test, and tree‐based analyses. This study included 186 individuals from 89 populations representing 30 species. For all individuals, single locus markers showed high levels of sequencing universality but were ineffective for species resolvability. Polymerase chain reaction amplification and sequencing were successful for all four loci. Both ITS and matK showed significantly higher levels of interspecific species delimitation than rbcL and trnH‐psbA. A combination of matK and ITS was the most efficient DNA barcode among all studied regions, however, they do not represent an appropriate candidate barcode for Chinese Gaultheria, by which only 11 out of 30 species can be separated. Loci rbcL, matK, and trnH‐psbA, which were recently proposed as universal plant barcodes, have a very poor capacity for species separation for Chinese Gaultheria. DNA barcodes may be reliable tools to identify the evolutionary units of this group, so further studies are needed to develop more efficient DNA barcodes for Gaultheria and other genera with complicated evolutionary histories.     

Analysis of mitochondrial COI sequences of the Diachasmimorpha longicaudata (Hymenoptera: Braconidae) species complex in Thailand

Diachasmimorpha longicaudata is an Opiinae parasitoid used to control tephritid fruit flies, which cause tremendous economic losses of fruits worldwide. In Thailand, D. longicaudata is classified as three sibling species, DLA, DLB and DLBB, based on the morphological and biological species concepts but their genetic variation has not been studied. Therefore, we investigated the genetic differentiation of the mitochondrial COI gene to clarify the ambiguous taxonomy of this species complex. The 603‐bp COI region was sequenced from laboratory‐bred colonies and field‐collected specimens from seven locations representing five geographical regions in Thailand. DLA was associated with the host Bactrocera correcta while DLB and DLBB were associated with Bactrocera dorsalis. The interspecific nucleotide differences of COI sequences among the three groups ranged from 6.70% to 7.62% (Kimura 2‐parameter distance), which adequately separates species complexes within the order Hymenoptera and supports the current sibling species classification. The neighbor joining, maximum likelihood and consensus Bayesian phylogenetic trees constructed from COI sequences revealed that the three sibling species of laboratory and field‐collected D. longicaudata are monophyletic with 100% support. The high genetic variation and molecular phylogeny of the COI sequences were shown to discriminate between the D. longicaudata species examined in this study.     

A DNA barcoding approach for identification of hidden diversity in Parmeliaceae (Ascomycota): Parmelia sensu stricto as a case study

Accurate specimen identification is challenging in groups with subtle or scarce taxonomically diagnostic characters, and the use of DNA barcodes can provide an effective means for consistent identification. Here, we investigate the utility of DNA barcode identification of species in a cosmopolitan genus of lichen‐forming fungi, Parmelia (Parmeliaceae). Two hundred and two internal transcribed spacer (ITS) sequences generated from specimens collected from all continents, including Antarctica, were analysed, and DNA barcodes of 14 species of Parmelia s.s. are reported. Almost all species show a barcode gap. Overall, intraspecific divergence values were lower than the threshold previously established for Parmeliaceae. However, the mean and range were elevated by deep barcode divergences in three species, indicating the likely occurrence of overlooked species‐level lineages. Here, we provide a DNA barcode reference library with well‐identified specimens sampled worldwide and sequences from most of the type material to enable easy and fast accurate sample identification and to assist in uncovering overlooked species in Parmelia s.s. Further, our results confirm the efficiency of the ITS region in the identification of species of Parmelia s.s. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180 , 21–29.