The origin and characterization of new nuclear genes originating from a cytoplasmic organellar genome

Endosymbiotic transfer of DNA and functional genes from the cytoplasmic organelles (mitochondria and chloroplasts) to the nucleus has been a major factor driving the origin of new nuclear genes, a process central to eukaryote evolution. Although organelle DNA transfers very frequently to the nucleus, most is quickly deleted, decays, or is alternatively scrapped. However, a very small proportion of it gives rise, immediately or eventually, to functional genes. To simulate the process of functional transfer, we screened for nuclear activation of a chloroplast reporter gene aadA, which had been transferred from the chloroplast to independent nuclear loci in 16 different plant lines. Cryptic nuclear activity of the chloroplast promoter was revealed, which became conspicuous when present in multiple nuclear copies. We screened ～50 million cells of each line and retrieved three plants in which aadA showed strong nuclear activation. Activation occurred by acquisition of the CaMV 35S nuclear promoter or by nuclear activation of the native chloroplast promoter. Two fortuitous sites within the 3' UTR of aadA mRNA both promoted polyadenylation without any sequence change. Complete characterization of one nuclear sequence before and after gene transfer demonstrated integration by nonhomologous end joining involving simultaneous insertion of multiple chloroplast DNA fragments. The real-time observation of three different means by which a chloroplast gene can become expressed in the nucleus suggests that the process, though rare, may be more readily achieved than previously envisaged.     

Phylogeny of Decapoda using two nuclear protein-coding genes: origin and evolution of the Reptantia

The phylogeny of Decapoda is contentious and many hypotheses have been proposed based on morphological cladistic analyses. Recent molecular studies, however, yielded contrasting results despite their use of similar data (nuclear and mitochondrial rDNA). Here we present the first application of two nuclear protein-coding genes, phosphoenolpyruvate carboxykinase and sodium-potassium ATPase alpha-subunit, to reconstruct the phylogeny of major infraorders within Decapoda. A total of 64 species representing all infraorders of Pleocyemata were analyzed with five species from Dendrobranchiata as outgroups. Maximum likelihood and Bayesian inference reveal that the Reptantia and all but one infraorder are monophyletic. Thalassinidea, however, is polyphyletic. The nodal support for most of the infraordinal and inter-familial relationships is high. Stenopodidea and Caridea form a clade sister to Reptantia, which comprises two major clades. The first clade, consisting of Astacidea, Achelata, Polychelida and three thalassinidean families (Axiidae, Calocarididae and Eiconaxiidae), corresponds essentially to the old taxon suborder Macrura Reptantia. Polychelida nests within Macrura Reptantia instead of being the most basal reptant as suggested in previous studies. The high level of morphological and genetic divergence of Polychelida from Achelata and Astacidea justifies its infraorder status. The second major reptant clade consists of Anomura, Brachyura and two thalassindean families (Thalassinidae and Upogebiidae). Anomura and Brachyura form Meiura, with moderate support. Notably thalassinidean families are sister to both major reptant clades, suggesting that the stem lineage reptants were thalassinidean-like. Moreover, some families (e.g. Nephropidae, Diogenidae, Paguridae) are paraphyletic, warranting further studies to evaluate their status. The present study ably demonstrates the utility of nuclear protein-coding genes in phylogenetic inference in decapods. The topologies obtained are robust and the two molecular markers are informative across a wide range of taxonomic levels. We propose that nuclear protein-coding genes should constitute core markers for future phylogenetic studies of decapods, especially for higher systematics.     

Molecular phylogeny of RPB2 gene reveals multiple origin, geographic differentiation of H genome, and the relationship of the Y genome to other genomes in Elymus species

It has been hypothesized from isozymic and cytological studies of Elymus species that the Old and New World taxa may be of separate origin of the H genome in the StH genome species. To test this hypothesis, and estimate the phylogenetic relationships of polyploid Elymus species within the Triticeae, the second largest subunit of RNA polymerase II (RPB2) sequence of 36 Elymus accessions containing StH or StY genomes was analyzed with those of Pseudoroegneria (St), Hordeum (H), Agropyron (P), Australopyrum (W), Lophopyrum(Ee), Thinopyrum(Eb) and Dasypyrum (V). Our data indicated that the H genome in Elymus species is differentiated in accordance with geographical origin, and that the Eurasian and American StH genome species have independent alloploid origins with different H-genome donors. Phylogenetic analysis of Y genome sequences with other genome donors (St, H, P, W) of Elymus revealed that W and P genomes are sister to Y genome with a 87% bootstrap support, and that StY and StH species group might have acquired their RPB2 St sequences from distinct Pseudoroegneria gene pools. Our data did not support the suggestion that the St and Y genomes have the same origin as put forward in a previous study using ITS data. Our result provides some insight on the origin of Y genome and its relationship to other genomes in Elymus.     

The origin of the H, St, W, and Y genomes in allotetraploid species of Elymus L. and Stenostachys Turcz. (Poaceae: Triticeae)

Based on sequences from two single-copy nuclear genes (DMC1 and EF-G), four plastid genes (rbcL, rpoA, matK, and ndhF), and one mitochondrial gene (coxII), we investigate the origin of the H, St, W, and Y genomes in four allotetraploid species of Elymus and two allotetraploid species of Stenostachys. Despite significant incongruence between the two nuclear genes and between the nuclear and organelle data partitions, individual and combined analyses of the data partitions unequivocally show that the St and H genomes of the tetraploid American species of Elymus are derived from Pseudoroegneria and Hordeum, respectively, with Pseudoroegneria serving as the female parent, and that the H and W genomes of Stenostachys are derived from Hordeum and Australopyrum, respectively, with Hordeum serving as the female parent. The analyses equally clearly demonstrate that the St genome of the tetraploid Asiatic Elymus species is derived from Pseudoroegneria, with the latter serving as the female parent, but the relationship of the Y genome is less clear. Individual analyses of the nuclear genes provide conflicting results, but combined analysis of all data suggests a sister group relationship to Heteranthelium, albeit without any jackknife support.     

Origin of the H genome in StH-genomic Elymus species based on the single-copy nuclear gene DMC1

Previous studies have suggested that the H haplome in Elymus could originate from different diploid Hordeum species, however, which diploid species best represent the parental species remains unanswered. The focus of this study seeks to pinpoint the origin of the H genome in Elymus. Allopolyploid Elymus species that contain the StH genome were analyzed together with diploid Hordeum species and a broad sample of diploid genera in the tribe Triticeae using DMC1 sequences. Both parsimony and maximum likelihood analyses well separated the American Hordeum species, except Hordeum brachyantherum subsp. californicum, from the H genome of polyploid Elymus species. The Elymus H-genomic sequences were formed into different groups. Our data suggested that the American Horedeum species, except H. brachyantherum subsp. californicum, are not the H-genomic donor to the Elymus species. Hordeum brevisubulatum subsp. violaceum was the progenitor species to Elymus virescens, Elymus confusus, Elymus lanceolatus, Elymus wawawaiensis, and Elymus caninus. Furthermore, North American H. brachyantherum subsp. californicum was a progenitor of the H genome to Elymus hystrix and Elymus cordilleranus. The H genomes in Elymus canadensis, Elymus sibiricus, and Elymus multisetus were highly differentiated from the H genome in Hordeum and other Elymus species. The H genome in both North American and Eurasian Elymus species was contributed by different Hordeum species.     

Evolutionary analysis of a large mtDNA translocation (numt) into the nuclear genome of the Panthera genus species

Translocation of cymtDNA into the nuclear genome, also referred to as numt, has been reported in many species, including several closely related to the domestic cat (Felis catus). We describe the recent transposition of 12,536 bp of the 17 kb mitochondrial genome into the nucleus of the common ancestor of the five Panthera genus species: tiger, P. tigris; snow leopard, P. uncia; jaguar, P. onca; leopard, P. pardus; and lion, P. leo. This nuclear integration, representing 74% of the mitochondrial genome, is one of the largest to be reported in eukaryotes. The Panthera genus numt differs from the numt previously described in the Felis genus in: (1) chromosomal location (F2-telomeric region vs. D2-centromeric region), (2) gene make up (from the ND5 to the ATP8 vs. from the CR to the COII), (3) size (12.5 vs. 7.9 kb), and (4) structure (single monomer vs. tandemly repeated in Felis). These distinctions indicate that the origin of this large numt fragment in the nuclear genome of the Panthera species is an independent insertion from that of the domestic cat lineage, which has been further supported by phylogenetic analyses. The tiger cymtDNA shared around 90% sequence identity with the homologous numt sequence, suggesting an origin for the Panthera numt at around 3.5 million years ago, prior to the radiation of the five extant Panthera species.     

Phylogeny of the treehoppers (Insecta: Hemiptera: Membracidae): evidence from two nuclear genes

We present a molecular systematic investigation of relationships among family-group taxa of Membracidae, comprising nearly 3.5 kb of nucleotide sequence data from the nuclear genes elongation factor-1alpha (EF-1alpha: 958 bp) and 28S ribosomal DNA (28S rDNA: 2363 bp); data partitions are analyzed separately and in combination for 79 taxa. Analysis of the combined sequence data provided a better-resolved and more robust hypothesis of membracid phylogeny than did separate analyses of the individual genes. Results support the monophyly of the family Membracidae and indicate the presence of two major lineages (Centrotinae + Stegaspidinae + Centrodontinae and Darninae + Membracinae + Smiliinae). Within Membracidae, molecular data support the following assertions: (1) the previously unplaced genera Antillotolania and Deiroderes form a monophyletic group with Microcentrini; (2) Centrodontini and Nessorhinini are monophyletic clades that arise independently from within the Centrotinae; (3) Centrotinae is paraphyletic with respect to Centrodontinae; (4) the subfamily Membracinae is monophyletic and possibly allied with the darnine tribe Cymbomorphini; (5) the subfamily Darninae is paraphyletic; (6) the subfamily Smiliinae is paraphyletic, with molecular evidence indicating the exclusion of Micrutalini and perhaps Acutalini and Ceresini; and (7) Membracidae arose and diversified in the New World with multiple subsequent colonizations of the Old World. Our phylogenetic results suggest that morphology-based classifications of the Membracidae need to be reevaluated in light of emerging molecular evidence.     

Snake phylogeny: evidence from nuclear and mitochondrial genes

We constructed phylogenies of snakes from the c-mos and cytochrome b genes using conventional phylogenetic methods as well as the relatively new method of Bayesian inference. For all methods, there was excellent congruence between the c-mos and cytochrome b genes, implying a high level of support for the shared clades. Our results agree with previous studies in two important respects: first, that the scolecophidians and alethinophidians are monophyletic sister clades; and second, that the Colubroidea is a monophyletic group with the Acrochordidae as its sister clade. However, our results differ from previous studies in the finding that Loxocemus and Xenopeltis cluster with pythons. An additional noteworthy result from our data is that the genera Exiliboa and Ungaliophis, often placed with Tropidophis (and Trachyboa, not included in the present study) in the Tropidophiidae, are in reality boids.     

Phylogeography of Eurasian Larix species inferred from nucleotide variation in two nuclear genes

Larch (Larix Mill.) is one of the most widely distributed tree genera in Eurasia. To determine population structure and to verify classification of five species and three varieties of the Eurasian Larix species, we investigated levels and patterns of nucleotide variation of two nuclear gene regions: the 4-coumarate coenzyme A ligase (4CL) and the coumarate 3-hydroxylase (C3H). In the 4CL region nucleotide diversity at silent sites (pi(sil)) varied between 0.0020 in L. gmelinii to 0.0116 in L. gmelinii var. japonica and in the C3H region between 0.0019 in L. kaempferi to 0.0066 in L. gmelinii var. japonica. In both gene regions statistically significant population differentiation (F(ST)) was detected among adjacent refugial populations of some species suggesting limited gene flow and/or long time isolation of some refugial populations. On the other hand, populations of L. sukaczewii from northwestern Russia, which was glaciated 20,000 years ago showed no differentiation. This result is consistent with recent postglacial origin of these populations. Haplotype composition of some of the investigated Eurasian Larix species suggested that they are considerably diverged. Some haplotypes were unique to individual species. Our results indicate that more intensive sampling especially from known refugial regions is necessary for inferring correct classification of Eurasian Larix species and inferring their postglacial migration.     

Physical mapping of repetitive sequences and genome analysis in six Elymus species by in situ hybridization

Genome constitution and genetic relationships between six Elymus species were assessed by physical mapping of different repetitive sequences using a technique of sequential fluorescence in situ hybridization and genomic in situ hybridization.The six Elymus species are all naturally growing species in northwest China,namely,E.sibiricus,E.nutans,E.barystachyus,E.xiningensis,E.excelsus,and E.dahuricus.An StStHH genome constitution was revealed for E.sibiricus and StStHHYY for the remainder species.Each chromosome could be clearly characterized by physical mapping with 18S-26S rDNA,5S rDNA,Afa-family,and AAG repeats,and be allocated to a certain genome by genomic in situ hybridization.Two 5S rDNA sites,each in the H and St genomes,and three 18S-26S rDNA sites,two in the St genome and one in the Y genome,were uncovered in most of the species.The strong Afa-family hybridization signals discriminated the H genome from the St and Y genomes.The H and Y genome carried more AAG repeats than St.A common non-Robertsonian reciprocal translocation between the H and Y genomes was revealed in E.barystachyus,E.xiningensis,E.excelsus and E.dahuricus.Comparison of molecular karyotypes strongly suggests that they can be classified into three groups,namely,E.sibiricus,E.nutans,and others.     

Untangling the hybrid origin of the Chinese tea roses: evidence from DNA sequences of single-copy nuclear and chloroplast genes

The tea-scented China roses largely correspond to the three recognized double-petaled Rosa odorata (Andrews) Sweet (Rosoideae, Rosaceae) varieties, which are the ancestors of modern hybrid tea roses and had a definite and permanent influence on the evolution of modern garden roses. Here the hypothesis of a hybrid origin of the tea-scented China roses between R. odorata var. gigantea and R. chinensis was tested. Two single-copy nuclear genes of the cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and the chloroplast-expressed glutamine synthetase (ncpGS) together with two plastid loci (trnL-F and psbA-trnH) were sequenced for representative accessions of four R. odorata varieties, R. chinensis, and 28 other Rosa species. Phylogenetic relationships were estimated from two nuclear loci using maximum parsimony and Bayesian analyses, and a haplotype network was constructed on the combined plastid data using NETWORK. For GAPDH and ncpGS loci, the clonal sequences of the three double-petaled varieties were clustered into two clades, one clade with R. odorata var. gigantea, and the other with partial sequences of R. chinensis, which suggested that the tea-scented China roses were hybrids between R. odorata var. gigantea and R. chinensis. Two plastid loci suggested that R. odorata var. gigantea could be the maternal parent and R. chinensis the paternal parent.     

Systematics and evolution of the cutworm moths (Lepidoptera: Noctuidae): evidence from two protein-coding nuclear genes

Abstract. A broad molecular systematic survey of Noctuidae was undertaken to test recent hypotheses on the problematic definitions and relationships of the subfamilies, with special emphasis on the ‘trifines.’ An initial hypothesis of noctuid classification to the subtribal level was synthesized from recent reviews, and then sampled as broadly as possible. Concatenated sequences for the nuclear genes elongation factor‐1α (EF‐1α; 1200 bp) and dopa decarboxylase (DDC; 700–1100 bp) were analysed for a total of 146 exemplar species, twice that of a previous study. Trees were estimated using likelihood, distance, and both equally weighted and ‘six‐parameter’ parsimony. Of the 144 possible nodes, bootstrap support (BP) was ≥ 50% for ～80, and ≥ 80% for ～60. There was very strong support (BP ≥ 90%) for an ‘L.A.Q.’ clade encompassing nearly all quadrifine noctuids plus Arctiidae and Lymantriidae, decisively rendering Noctuidae paraphyletic. We present a new classification for Noctuoidea in which Noctuidae sensu stricto is restricted to trifines; most quadrifine subfamilies are raised to full families. Within the ‘L.A.Q.’ clade, Aganainae and Herminiinae were strongly grouped, but other relationships were weakly supported, probably due to limited taxon sampling. Nolidae and Euteliinae + Stictopterinae are generally grouped with the ‘L.A.Q.’ clade, but with weak support. All analyses favour the broadest definitions proposed for the trifine clade (our Noctuidae sensu stricto) although support is not strong, except that the exemplar of Eustrotiinae: Eublemmini is placed securely in the ‘L.A.Q.’ clade. Numerous recent proposals for dismantling and recombining the ‘Hampsonian’ traditional trifine subfamilies are strongly supported, most notably a broadly defined ‘true cutworm’ clade (Noctuinae s.l.), encompassing the greater part of the traditional subfamilies Amphipyrinae, Cuculliinae, Hadeninae and Noctuinae s.s. (BP ≥ 95%). Within this clade there is strong support for Apameini s.s.+ Xylenini s.l. and for Noctuinae s.s. and divisions thereof, but little support for monophyly or subdivision of Hadeninae. Noctuinae s.l. invariably are allied with Heliothinae, scattered remnants of the traditional Amphipyrinae, and several smaller groups in a broader ‘pest clade’, albeit with weak support. Relationships among the remaining ‘lower’ trifines are not strongly resolved. Mapping of a preliminary synopsis of species diversities, host use patterns and latitudinal distributions on the phylogeny suggests that the diversification of trifines may have been promoted, to a degree unique among Macrolepidoptera, by the Tertiary expansion of seasonal, open habitats and their associated herbaceous floras.     

Re: Phylogenetic relationships in Sphingidae (Insecta: Lepidoptera): initial evidence from two nuclear genes

Partial DNA sequences from two mitochondrial (mt) and one nuclear gene (cytochrome b, 12S rRNA, and C-mos) were used to estimate the phylogenetic relationships among the six extant species of skinks endemic to the Cape Verde Archipelago. The species form a monophyletic unit, indicating a single colonization of the islands, probably from West Africa. Mabuya vaillanti and M. delalandii are sister taxa, as indicated by morphological characters. Mabuya fogoensis and M. stangeri are closely related, but the former is probably paraphyletic. Mabuya spinalis and M. salensis are also probably paraphyletic. Within species, samples from separate islands always form monophyletic groups. Some colonization events can be hypothesized, which are in line with the age of the islands. C-mos variation is concordant with the topology derived from mtDNA.     

Use of mitochondrial and nuclear genes to infer the origin of two endemic pigeons from the Canary Islands

DNA nucleotide sequences from two mitochondrial genes (cytochrome b and NADH dehydrogenase subunit 2) and the nuclear intron 7 of β-fibrinogen were obtained to infer the phylogenetic origin of the two endemic Canarian pigeons: Bolle’s Pigeon (Columba bollii) and Laurel Pigeon (C. junoniae). Phylogenetic analyses of mitochondrial and nuclear genes based on maximum parsimony, maximum likelihood and Bayesian inference all converged into a congruent topology: C. bollii clusters together with the Wood Pigeon (C. palumbus) which is common in Europe and Asia, while C. junoniae was found near the base of the clade that includes other species of the genus Columba from the Old World. Laurel Pigeon probably represents an old lineage that might have colonized the Canary Islands a long time ago (20 My) while Bolle’s Pigeon might have arrived on the archipelago much later during the Upper Miocene (5 My). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation and characterization of Glu-1 genes from the St genome of Pseudoroegneria libanotica

The St genome, which is present in nearly half of all Triticeae species, originates from the genus Pseudoroegneria. However, very little is known about the high molecular weight (HMW) subunits of glutenin which are encoded by the St genome. In this paper, we report the isolation from Pd. libanotica of four sequences encoding HMW subunits of glutenin. The four genes were all small compared to standard glutenin genes. All four sequences resemble y-type glutenins rather than x-types. However, their N-terminal domains contain a glutamine residue which is present in all x-type, but very few y-type subunits, and their central repetitive domains included some irregular motifs. The indication is therefore that the Glu-1St genes evolved earlier than other modern day homoeologues, so that they represent an intermediate state in the divergence between x- and y-type subunits. No x-type Glu-1St subunit genes were identified.     

Phylogenetic analysis of arthropods using two nuclear protein-encoding genes supports a crustacean + hexapod clade

Recent phylogenetic analyses using molecular data suggest that hexapods are more closely related to crustaceans than to myriapods, a result that conflicts with long-held morphology-based hypotheses. Here we contribute additional information to this debate by conducting phylogenetic analyses on two nuclear protein-encoding genes, elongation factor-1 alpha (EF-1 alpha) and the largest subunit of RNA polymerase II (Pol II), from an extensive sample of arthropod taxa. Results were obtained from two data sets. One data set comprised 1092 nucleotides (364 amino acids) of EF-1 alpha and 372 nucleotides (124 amino acids) of Pol II from 30 arthropods and three lobopods. The other data set contained the same EF-1 alpha fragment and an expanded 1038-nucleotide (346-amino-acid) sample of Pol II from 17 arthropod taxa. Results from maximum-parsimony and maximum-likelihood analyses strongly supported the existence of a Crustacea + Hexapoda clade (Pancrustacea) over a Myriapoda + Hexapoda clade (Atelocerata). The apparent incompatibility between the molecule-based Pancrustacea hypothesis and morphology-based Atelocerata hypothesis is discussed.     

Phylogeny and molecular evolution of the rbcL gene of St genome in Elymus sensu lato (Poaceae: Triticeae)

Analysis of the patterns and levels of diversity in duplicate gene not only traces evolutionary history of polyploids, but also provides insight into how the evolutionary process differs between lineages and between homoeologous loci within lineages. Elymus sensu lato is a group of allopolyploid species, which share a common St genome and with the different combinations of H, Y, P, and W genomes. To estimate the evolutionary process of the rbcL gene in species of Elymus s. l. and its putative dioploid relatives, 74 sequences were obtained from 21 species of Elymus s. l. together with 24 diploid taxa representing 19 basic genomes in Triticeae. Phylogeny and sequence diversity pattern analysis suggested that (1) species of Pseudoroegneria (Nevski) Á. Löve might serve as the maternal donor of the species of Elymus s. l; (2) differentiation of St genome were shown in the species of Elymus s. l. following polyploidy event; (3) divergences within the species might associate with geographic diversity and morphological variability; (4) differences in the levels and patterns of nucleotide diversity of the rbcL gene implied that the St genome lineages in the species of Elymus s. l. have differently evolutionary potentials.     

Rodent phylogeny and a timescale for the evolution of Glires: evidence from an extensive taxon sampling using three nuclear genes

Rodentia is the largest order of placental mammals, with approximately 2,050 species divided into 28 families. It is also one of the most controversial with respect to its monophyly, relationships between families, and divergence dates. Here, we have analyzed and compared the performance of three nuclear genes (von Willebrand Factor, interphotoreceptor retinoid-binding protein, and Alpha 2B adrenergic receptor) for a large taxonomic sampling, covering the whole rodent and placental diversity. The phylogenetic results significantly support rodent monophyly, the association of Rodentia with Lagomorpha (the Glires clade), and a Glires + Euarchonta (Primates, Dermoptera, and Scandentia) clade. The resolution of relationships among rodents is also greatly improved. The currently recognized families are divided here into seven well-defined clades (Anomaluromorpha, Castoridae, Ctenohystrica, Geomyoidea, Gliridae, Myodonta, and Sciuroidea) that can be grouped into three major clades: Ctenohystrica, Gliridae + Sciuroidea, and a mouse-related clade (Anomaluromorpha, Castoridae + Geomyoidea, and Myodonta). Molecular datings based on these three genes suggest that the rodent radiation took place at the transition between Paleocene and Eocene. The divergence between rodents and lagomorphs is placed just at the K-T boundary and the first splits among placentals in the Late Cretaceous. Our results thus tend to reconcile molecular and morphological-paleontological insights.     

A phylogenetic analysis of Myriapoda (Arthropoda) using two nuclear protein-encoding genes

Nucleotide and inferred amino acid sequences from two nuclear protein-encoding genes, elongation factor-aα and RNA polymerase II, were obtained from 34 myriapods and 14 other arthropods to determine phylogenetic relationships among and within the myriapod classes. Phylogenetic analyses using maximum parsimony and maximum likelihood methods recovered all three represented myriapod classes (Chilopoda, Diplopoda, Symphyla) and all multiply sampled chilopod and diplopod orders, often with high node support. In contrast, relationships between classes and between orders were recovered less consistently and node support was typically lower. The temporal structure of phylogenetic diversification in Myriapoda may explain this apparent pattern of the phylogenetic recovery.