Molecular phylogeny of Phalaenopsis Blume (Orchidaceae) based on the internal transcribed spacer of the nuclear ribosomal DNA

The internal transcribed spacer (ITS1, 5.8S rDNA, and ITS2) region of nuclear ribosomal DNA (nrDNA) was sequenced from 53 species, which represent most of the living species diversity in the genus Phalaenopsis (Orchidaceae). A phylogeny was developed for the genus based on the neighbor-joining and maximum parsimony analyses of molecular data. Results of these analyses provided support for the monophyly of the genus Phalaenopsis and concurred in that the genera Doritis and Kingidium should be treated as being parts of the genus Phalaenopsis as suggested by Christenson (2001). Within the genus Phalaenopsis, neither subgenera Aphyllae nor Parishianae were monophyletic, and they were highly clustered with subgenus Proboscidioides plus sections Esmeralda and Deliciosae of subgenus Phalaenopsis based on ITS data. Those species also have the same characters of morphology of four pollinia and similar biogeographies. Furthermore, neither subgenus Phalaenopsis nor Polychilos was monophyletic. Within the subgenus Phalaenopsis, only section Phalaenopsis was highly supported as being monophyletic. As for the subgenus Polychilos, only section Polychilos was moderately supported as being monophyletic. In conclusion, the present molecular data obtained from the ITS sequence of nrDNA of the genus Phalaenopsis provide valuable information for elucidating the phylogeny of this genus.     

The ITS region of groundwater amphipods: length,secondary structure and phylogenetic information content in Crangonyctoids and Niphargids

The phylogenetic analysis of groundwater amphipods is challenging due to the lack of suitable morphological characters. However, molecular phylogenies based on the 18S and 28S nuclear genes of two Crangonyctoidea species endemic to Iceland, Crymostygius thingvallensis and Crangonyx islandicus, support the taxonomy of these species on the basis of morphological characters. Molecular analyses suggest that the genus Crangonyx is paraphyletic, with the species that is found in Eurasia being highly divergent genetically from the species present in North America and Iceland. Studies of the phylogenetic relationships within the genus Niphargus also warrant further work. The nuclear ITS2 region has recently been proposed as a barcoding marker for plants and animals. In addition, ITS2 has been used to build phylogenies at high taxonomic levels by including its secondary structure. In this study, we want to evaluate the applicability of the ITS region for this group of species and describe its characteristics. The taxonomy of C. thingvallensis, as well as the paraphyly of the genus Crangonyx, is supported herein by phylogenies based on the ITS2 variation. The secondary structure and the length of the ITS2 sequences of the Crangonyctoidea and the Niphargidae species studied are highly variable and are characterized by duplications. The ITS2 sequence of Niphargus plateaui is the longest metazoan sequence deposited in the ITS2 database so far. Although saturation was observed in the nucleotide variation of this marker, the addition of the secondary structure information for the reconstruction of the phylogeny did not add support to the phylogenetic trees. The ITS1 region, which is known to be more variable than ITS2 and bears a large duplication within C. islandicus, was found to be less useful for phylogenetic reconstruction.     

Radiation of the ,,Drosophila“ subgenus (Drosophilidae,Diptera) in the Neotropics

We present a phylogenetic hypothesis for 72 ,,Drosophila“ species, constructed through analysis from the paralogous alpha methyldopa (amd) and dopa decarboxylase (ddc) nuclear genes (encompassing a total of 2015 base pairs). Our data support the subdivision of the paraphyletic subgenus ,,Drosophila“ into three main radiations (the immigrans‐tripunctata, the virilis‐repleta and the Hawaiian Drosophilidae), each of which is further subdivided to originate monophyletic ‘sub‐radiations’. Moreover, this study raises the possibility that the Zaprionus/Liodrosophila species encompass a fourth radiation within the subgenus ,,Drosophila“ phylogeny and provides temporal estimates for each of the postulated divergence events.     

Diversification of mitogenomes in three sympatric Altica flea beetles (Insecta,Chrysomelidae)

The Asian flea beetles Altica cirsicola, Altica fragariae and Altica viridicyanea are broadly sympatric and morphologically highly similar but feed on distantly related host plants. They have been suggested as a model for ecological speciation studies. However, their phylogeny and species limits remain uncertain. In this study, we added mitochondrial genomes from multiple individuals of each species to the growing database. Phylogenetic analyses based on 15 genes showed clear interspecific divergences of A. fragariae from the other species, but A. cirsicola and A. viridicyanea were not distinguishable by distance‐based or tree‐based methods of species delimitation due to non‐monophyly of mitogenomes relative to the morphologically defined entities, possibly affected by interspecific introgression. This was confirmed by wider sampling of mitochondrial COX1 (58 individuals) and the second internal transcribed spacer of nuclear ribosomal RNA cluster (ITS2; 68 individuals), which showed that ITS2, but not COX1, coincided with the morphological species limits. The full mitochondrial genomes are not able to shed further light on the species status, even with the most sensitive approach based on diagnostic characters, yet the whole mitogenome is useful to get improved estimates of intra‐ and interspecific variation, not affected by the stochastic error seen in individual genes.     

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

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

Phylogenetic analysis of Allium subg. Melanocrommyum infers cryptic species and demands a new sectional classification

Allium subgenus Melanocrommyum (Alliaceae) from Eurasia comprises about 150 mostly diploid species adapted to arid conditions. The group is taxonomically complicated with different and contradictory taxonomic treatments, and was thought to include a considerable number of hybrid species, as the taxa show an admixture of assumed morphological key characters. We studied the phylogeny of the subgenus, covering all existing taxonomic groups and their entire geographic distribution. We analyzed sequences of the nuclear rDNA internal transcribed spacer region (ITS) for multiple individuals of more than 100 species. Phylogenetic analyses of cloned and directly sequenced PCR products confirmed the monophyly of the subgenus, while most sections were either para- or polyphyletic. The splits of the large sections are supported by differences in the anatomy of flower nectaries. ITS data (i) demand a new treatment at sectional level, (ii) do not support the hypotheses of frequent gene flow among species, (iii) indicate that multiple rapid radiations occurred within different monophyletic groups of the subgenus, and (iv) detected separately evolving lineages within three morphologically clearly defined species (cryptic species). In two cases these lineages were close relatives, while in Allium darwasicum they fall in quite different clades in the phylogenetic tree. Fingerprint markers show that this result is not due to ongoing introgression of rDNA (ITS capture) but that genome-wide differences between both lineages exist. Thus, we report one of the rare cases in plants where morphologically indistinguishable diploid species occurring in mixed populations are non-sister cryptic species.     

Lineage-specific variations of congruent evolution among DNA sequences from three genomes,and relaxed selective constraints on <Emphasis Type="Italic">rbc</Emphasis>L in <Emphasis Type="Italic">Cryptomonas</Emphasis> (Cryptophyceae)

Background  

Plastid-bearing cryptophytes like Cryptomonas contain four genomes in a cell, the nucleus, the nucleomorph, the plastid genome and the mitochondrial genome. Comparative phylogenetic analyses encompassing DNA sequences from three different genomes were performed on nineteen photosynthetic and four colorless Cryptomonas strains. Twenty-three rbc L genes and fourteen nuclear SSU rDNA sequences were newly sequenced to examine the impact of photosynthesis loss on codon usage in the rbc L genes, and to compare the rbc L gene phylogeny in terms of tree topology and evolutionary rates with phylogenies inferred from nuclear ribosomal DNA (concatenated SSU rDNA, ITS2 and partial LSU rDNA), and nucleomorph SSU rDNA.     

Molecular Phylogeny of the Chipmunk Genus Tamias Based on the Mitochondrial Cytochrome Oxidase Subunit II Gene

Complete sequences of the mitochondrial cytochrome oxidase subunit II gene were used to construct a phylogeny for 21 of the 25 currently recognized chipmunk species. Phylogenetic analyses indicate that T. striatus (subgenus Tamias, eastern United States) and T. sibiricus (subgenus Eutamias, Asia) are distantly related to the other species (subgenus Neotamias), which constitute a western North American radiation. We discuss and compare our molecular phylogeny to previous taxonomies and present a suggested classification of the species groups for the subgenus Neotamias.     

The evolutionary radiation of Arvicolinae rodents (voles and lemmings): relative contribution of nuclear and mitochondrial DNA phylogenies

Background  

Mitochondrial and nuclear genes have generally been employed for different purposes in molecular systematics, the former to resolve relationships within recently evolved groups and the latter to investigate phylogenies at a deeper level. In the case of rapid and recent evolutionary radiations, mitochondrial genes like cytochrome b (CYB) are often inefficient for resolving phylogenetic relationships. One of the best examples is illustrated by Arvicolinae rodents (Rodentia; Muridae), the most impressive mammalian radiation of the Northern Hemisphere which produced voles, lemmings and muskrats. Here, we compare the relative contribution of a nuclear marker – the exon 10 of the growth hormone receptor (GHR) gene – to the one of the mitochondrial CYB for inferring phylogenetic relationships among the major lineages of arvicoline rodents.     

Phylogeny estimation of the radiation of western North American chipmunks (Tamias) in the face of introgression using reproductive protein genes

The causes and consequences of rapid radiations are major unresolved issues in evolutionary biology. This is in part because phylogeny estimation is confounded by processes such as stochastic lineage sorting and hybridization. Because these processes are expected to be heterogeneous across the genome, comparison among marker classes may provide a means of disentangling these elements. Here we use introns from nuclear-encoded reproductive protein genes expected to be resistant to introgression to estimate the phylogeny of the western chipmunks (Tamias: subgenus: Neotamias), a rapid radiation that has experienced introgressive hybridization of mitochondrial DNA (mtDNA). We analyze the nuclear loci using coalescent-based species-tree estimation methods and concatenation to estimate a species tree and we use parametric bootstraps and coalescent simulations to differentiate between phylogenetic error, coalescent stochasticity and introgressive hybridization. Results indicate that the mtDNA gene tree reflects several introgression events that have occurred between taxa of varying levels of divergence and at different time points in the tree. T. panamintinus and T. speciosus appear to be fixed for ancient mitochondrial introgressions from T. minimus. A southern Rocky Mountains clade appears well sorted (i.e., species are largely monophyletic) at multiple nuclear loci, while five of six taxa are nonmonophyletic based on cytochrome b. Our simulations reject phylogenetic error and coalescent stochasticity as causes. The results represent an advance in our understanding of the processes at work during the radiation of Tamias and suggest that sampling reproductive-protein genes may be a viable strategy for phylogeny estimation of rapid radiations in which reproductive isolation is incomplete. However, a genome-scale survey that can statistically compare heterogeneity of genealogical process at many more loci will be necessary to test this conclusion.     

Sibling species in the Chrysis ignita complex: molecular,morphological and trophic differentiation of Baltic species,with a description of two new cryptic species (Hymenoptera: Chrysididae)

Cryptic species complexes cause major challenges for taxonomists and alter understanding of species diversity. In Northern Europe, the Chrysis ignita species group is one such complex with numerous sympatric sibling species. The objective of this paper is to assess the taxonomy of 15 species from this group using three different approaches: molecular, morphological and trophic differentiation. The analysed set of molecular markers included a 7400‐bp‐long sequence of the mitochondrial genome covering complete sequences of CO1, CO2, ATP8, ATP6, CO3, ND3, 16S and 12S rRNA, nine tRNAs and a partial sequence of CytB, as well as a 3880‐bp‐long sequence of the nuclear DNA covering a part of 18S rRNA, the ITS1, 5.8S rRNA, ITS2 and a part of 28S rRNA. Discrete diagnostic characters of each species sequence were retrieved using the Characteristic Attribute Organisation System algorithm and a molecular identification key was compiled. The study revealed a higher evolutionary rate of the genes ATP8, ATP6, CO3, ND3 and CytB compared to that of CO1, CO2 and 16S; the studied nuclear markers demonstrated a lower evolutionary rate than the mitochondrial markers. A consensus tree compiled based on the combined mtDNA and nuclear markers with a strongly supported topology resolved the position of the C. schencki – C. parietis sp.n. clade as sister to the C. ignita – C. impressa clade and supported the monophyly of the C. angustula – C. longula clade. We compiled a morphometric species identification key applying linear discriminant equations. The trophic differentiation was assessed using data on host preferences of ten Chrysis species reared from trap‐nests; the analysis demonstrated that most of them are specialists exploiting a single or a few taxonomically related host species. In most cases, all three approaches supported the distinct status of the included species. Moreover, two previously undescribed species were consistently supported by the molecular methods. Therefore, we describe these as new, namely C. horridula sp.n. and C. parietis sp.n. Only C. mediata and C. solida were not clearly distinguished using the molecular phylogeny reconstruction methods. However, based on distinctive niche divergence, the presence of molecular characters and morphometric differences, we consider them as phylogenetically young but distinct species. In view of the weak morphological and molecular differentiation, the widely overlapping distribution areas and often similar habitat preferences and the trophic specialization, the C. ignita complex presents a possible model for studies of sympatric cryptic speciation. This published work has been registered in ZooBank, http://zoobank.org/urn:lsid:zoobank.org:pub:1EBAF0E1‐5FB7‐4CF4‐A595‐C11982448360 .     

Phylogenetics,historical biogeography and molecular species delimitation of Gnaptorina Reitter (Coleoptera: Tenebrionidae: Blaptini)

With 38 described species or subspecies, Gnaptorina Reitter is the second‐most species‐rich genus in the darkling beetle subtribe Gnaptorinina (Tenebrionidae: Tenebrioninae). In this study, we reconstructed a phylogeny of the genus based on one nuclear and three mitochondrial genes and used this phylogeny to explore the historical biography and diversification of Gnaptorina species. We implemented multiple molecular species delimitation approaches to reassess the status of Gnaptorina species and taxonomic subdivisions of the genus. Dating and historical biogeography analyses suggest an early Eocene origin of the genus, with the southeastern regions of the Tibetan Plateau most likely as areas of origin. Based on these results, we propose a new classification for Gnaptorina with three major clades identified. Consequently, the monotypic subgenus Boreoptorina is newly synonymized with the more species‐rich subgenus Hesperoptorina, and G. dongdashanensis Shi is transferred from Hesperoptorina to the subgenus Gnaptorina. In addition, G. minxiana Medvedev, formerly treated as a subspecies of G. potanini Reitter, is elevated to species. Results of molecular species delimitation analyses are largely congruent and confirm the status of most morphological species.     

Phylogenetic relationships in genus <Emphasis Type="Italic">Arachis</Emphasis> based on ITS and 5.8S rDNA sequences

Background  

The genus Arachis comprises 80 species and it is subdivided into nine taxonomic sections (Arachis, Caulorrhizae, Erectoides, Extranervosae, Heteranthae, Procumbentes, Rhizomatosae, Trierectoides, and Triseminatae). This genus is naturally confined to South America and most of its species are native to Brazil. In order to provide a better understanding of the evolution of the genus, we reconstructed the phylogeny of 45 species using the variation observed on nucleotide sequences in internal transcribed spacer regions (ITS1 and ITS2) and 5.8 S of nuclear ribosomal DNA.     

Presence of nuclear copies of mitochondrial origin (NUMTs) in two related species of stingless bee genus Melipona (Hymenoptera: Meliponini)

Transferred copies of mitochondrial DNA (mtDNA) into the nuclear genome (numts) have been reported in several Hymenoptera species, even at a high density in the honey bee nuclear genome. The accidental amplification of numts in phylogenetic studies focused on mtDNA highlights the importance of a correct determination of numts and their related mtDNA sequences. We report here the presence of numts derived from a mitochondrial rDNA 16S gene in the genome of the stingless bee species Melipona colimana and M. fasciata (tribe Meliponini) from Western Mexico. PCR products were cloned in both species obtaining thirty paralogous numts. Numts were identified by the presence of insertions and deletions and the disruption of the 16S secondary structure. Further phylogenetic analyses including alternative mitochondrial cox1 and nuclear ITS1 genes have revealed the presence of another numt (cox1) in the nuclear genome of these two species, and place both as sister lineages within the subgenus Michmelia. This is one of the first studies reporting the presence of numts in Meliponini species, and supports previous studies suggesting frequent transfer of mtDNA to the nuclear genome in Hymenoptera.     

Patterns of molecular and morphological differentiation in Fagus (Fagaceae): phylogenetic implications

To study phylogenetic relationships among species of Fagus, the internal transcribed spacer regions ITS1 and ITS2 of the nuclear ribosomal DNA and morphological data were analyzed. Both molecular and morphologically based phylogenies suggest that Eurasian species of Fagus subgenus Fagus are basal to the North American Fagus grandifolia. The subgenus Fagus is a paraphyletic group basal to three East Asian species forming the subgenus Engleriana. Due to a considerably large amount of DNA polymorphism, relationships among basal species of Fagus could not be entirely resolved when analyzing ITS sequences with standard methods. Morphological trees helped to resolve more clearly relationships within the subgenus Fagus. The East Asian F. hayatae is suggested to be basal to the rest of the genus. This hypothesis is further supported by distinctive patterns of nucleotide variability found for ITS regions, allowing for basic and derived types to be distinguished. The high degree of ITS polymorphism within Fagus can be explained by (1) the complex evolutionary behavior of this marker, (2) the stenoecious ecological characteristic of Fagus with respect to its continuous geographic range throughout much of the Cenozoic, and (3) the absence of major radiations into further habitats as occurred in other Fagaceae.     

Phylogeny of korean rubus (rosaceae) based on its (nrDNA) and trnL/F intergenic region (cpdna)

We examined the phylogeny of the genusRubus in Korea using an internal transcribed spacer (ITS) of the nuclear ribosomal DNA and a trnL-trnF (trnL/F) intergenic region of the chloroplast DNA. In all, 21 ingroup species (1.2 kb for each species) were analyzed using parsimony, resulting in 672 aligned sequences from ITS, and 502 bases with trnL/F. Individual and combined analysis of ITS and trnL/F data proved that the genusRubus is a monophyletic group. This phylogeny also substantiated a previous sectional classification scheme rather than a subgerius classification scheme. However, our results did not support the earlier sectional classification by Focke (sect.Corchorifolii), but did support the sectional classification of Nakai: sect.Crataegifolii (R. crataegifolius, R. takesimensis andR. trifidus), and sect.Villosii (R. corchorifolius). Most of these species, which are found in Korea and belong to subg.Idaeobatus, appeared in two different groups in all data sets. This suggests that this subgenus is a polyphyletic group that has gone through at least two independent evolutionary processes. The taxa, when mapped onto the combined tree, showed that the occurrence of their morphological characters of simple and compound leaves was concurrent in KoreanRubus. ITS sequence data were consistent overall with the geographical distribution of each species. Furthermore, the trnL/F sequence data provided phylogenetic information within closely related species.     

A phylogeny of Pacific fiddler crabs of the subgenus Minuca (Crustacea,Brachyura, Ocypodidae: Uca) with the description of a new species from a tropical gulf in Pacific Costa Rica

The phylogeny of all Pacific fiddler crab representatives of the subgenus Minuca Bott, 1954 (sensu Beinlich and von Hagen, 2006) is reconstructed. For the molecular analysis, Cox1 mitochondrial and 28S ribosomal nuclear DNA sequences were used. According to these data, same transisthmian sister species relationships are confirmed and a new species of the genus Uca Leach, 1814, Uca osa sp. n., is described from Golfo Dulce, a tropical gulf in Pacific Costa Rica. Morphological as well as molecular data confirm distinctness of this species compared with all other members of the subgenus Minuca, to which it belongs. Distinctive morphological traits are presented to distinguish Uca osa sp. n. from its congeners in the Eastern Pacific.     

Phylogeny of anopheline (Diptera: Culicidae) species in southern Africa,based on nuclear and mitochondrial genes

A phylogeny of anthropophilic and zoophilic anopheline mosquito species was constructed, using the nuclear internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome oxidase subunit I (COI) genes. The ITS2 alignment, typically difficult due to its noncoding nature and large size variations, was aided by using predicted secondary structure, making this phylogenetically useful gene more amenable to investigation. This phylogeny is unique in explicitly including zoophilic, non‐vector anopheline species in order to illustrate their relationships to malaria vectors. Two new, cryptic species, Anopheles funestus‐like and Anopheles rivulorum‐like, were found to be present in Zambia for the first time. Sequences from the D3 region of the 28S rDNA suggest that the Zambian An. funestus‐like may be a hybrid or geographical variant of An. funestus‐like, previously reported in Malawi. This is the first report of An. rivulorum‐like sympatric with An. rivulorum (Leeson), suggesting that these are separate species rather than geographic variants.     

Evidence of chloroplast capture in South American Nothofagus (subgenus Nothofagus,Nothofagaceae)

Subgenus Nothofagus, although geographically restricted at present to temperate areas of South America, has captured much attention in discussions of plant biogeography due to its widespread distribution through Gondwanan continents during the Tertiary. However, phylogenetic relationships within the subgenus Nothofagus have not yet been resolved. We examined geographic patterns of intraspecific and interspecific genetic variation to detect whether incongruences in nuclear or plastid DNA phylogenies occur, in order to better understand the evolutionary history of the subgenus Nothofagus. We conducted spatially-explicit sampling at 10 distinct locations throughout the range of austral South American forests and sampled all present Nothofagus species. We used ITS and chloroplast DNA sequences to estimate phylogenetic relationships. A phylogeny constructed from nuclear genes resolved the subgenus Nothofagus as monophyletic. We found that N. antarctica was a sister to a clade of evergreen species (N. betuloides, N. dombeyi, and N. nitida), while N. pumilio likely diverged earlier. Nine cpDNA haplotypes were distinguished in the subgenus Nothofagus which were associated to geographic locations rather than to taxonomic relationships. This species-independent cpDNA phylogeographic structures within the subgenus Nothofagus may be related to repeated chloroplast capture events over geological time in Patagonia.