Elongation factor-1 alpha occurs as two copies in bees: implications for phylogenetic analysis of EF-1 alpha sequences in insects

We report the complete sequence of a paralogous copy of elongation factor-1 alpha (EF-1 alpha) in the honeybee, Apis mellifera (Hymenoptera: Apidae). This copy differs from a previously described copy in the positions of five introns and in 25% of the nucleotide sites in the coding regions. The existence of two paralogous copies of EF-1 alpha in Drosophila and Apis suggests that two copies of EF-1 alpha may be widespread in the holometabolous insect orders. To distinguish between a single, ancient gene duplication and parallel, independent fly and bee gene duplications, we performed a phylogenetic analysis of hexapod EF-1 alpha sequences. Unweighted parsimony analysis of nucleotide sequences suggests an ancient gene duplication event, whereas weighted parsimony analysis of nucleotides and unweighted parsimony analysis of amino acids suggests the contrary: that EF-1 alpha underwent parallel gene duplications in the Diptera and the Hymenoptera. The hypothesis of parallel gene duplication is supported both by congruence among nucleotide and amino acid data sets and by topology-dependent permutation tail probability (T-PTP) tests. The resulting tree topologies are also congruent with current views on the relationships among the holometabolous orders included in this study (Diptera, Hymenoptera, and Lepidoptera). More sequences, from diverse orders of holometabolous insects, will be needed to more accurately assess the historical patterns of gene duplication in EF-1 alpha.      

Phylogenetic analysis of Myriapoda using three nuclear protein-coding genes

We assessed the ability of three nuclear protein-encoding genes-elongation factor-1alpha (EF-1alpha), RNA polymerase II (Pol II), and elongation factor-2 (EF-2)-from 59 myriapod and 12 non-myriapod species to resolve phylogenetic relationships among myriapod classes and orders. In a previous study using EF-1alpha and Pol II (2134 nt combined) from 34 myriapod taxa, Regier and Shultz recovered widely accepted classes, orders, and families but failed to resolve interclass and interordinal relationships. The result was attributed to heterogenous rates of cladogenesis (specifically, the inability of the slowly evolving sequences to capture phylogenetic signal during rapid phylogenetic diversification) but the possibility of inadequate taxon sampling or limited sequence information could not be excluded. In the present study, the myriapod taxon sample was increased by 25 taxa (73%) and sequence length per taxon was effectively doubled through addition of EF-2 (4318 nt combined). Parsimony and Bayesian analyses of the expanded data set recovered a monophyletic Myriapoda, all four myriapod classes and all multiply sampled orders, often with high node support. However, except for three diplopod clades (Colobognatha, Helminothomorpha, and a subgroup of Pentazonia), few interordinal relationships and no interclass relationships were well supported. These results are similar to those of the earlier study by Regier and Shultz, which indicates that taxon sample and sequence length alone do not readily explain the weakly supported resolution in the earlier study. We review recent paleontological evidence to further develop our proposal that heterogeneity in phylogenetic signal provided by our slowly evolving sequences is due to heterogeneity in the temporal structure of myriapod diversification.     

Single-copy nuclear genes recover cretaceous-age divergences in bees

We analyzed the higher level phylogeny of the bee family Halictidae based on the coding regions of three single-copy nuclear genes (long-wavelength [LW] opsin, wingless, and elongation factor 1-alpha [EF-1 alpha]). Our combined data set consisted of 2,234 aligned nucleotide sites (702 base pairs [bp] for LW opsin, 405 bp for wingless, and 1,127 bp for EF-1 alpha) and 779 parsimony-informative sites. We included 58 species of halictid bees from 33 genera, representing all subfamilies and tribes, and rooted the trees using seven outgroups from other bee families: Colletidae, Andrenidae, Melittidae, and Apidae. We analyzed the separate and combined data sets by a variety of methods, including equal weights parsimony, maximum likelihood, and Bayesian methods. Analysis of the combined data set produced a strong phylogenetic signal with high bootstrap and Bremer support and high posterior probability well into the base of the tree. The phylogeny recovered the monophyly of the Halictidae and of all four subfamilies and both tribes, recovered relationships among the subfamilies and tribes congruent with morphology, and provided robust support for the relationships among the numerous genera in the tribe Halictini, sensu Michener (2000). Using our combined nucleotide data set, several recently described halictid fossils from the Oligocene and Eocene, and recently developed Bayesian methods, we estimated the antiquity of major clades within the family. Our results indicate that each of the four subfamilies arose well before the Cretaceous-Tertiary boundary and suggest that the early radiation of halictid bees involved substantial African-South American interchange roughly coincident with the separation of these two continents in the late Cretaceous. This combination of single-copy nuclear genes is capable of recovering Cretaceous-age divergences in bees with high levels of support. We propose that LW opsin, wingless, and EF-1 alpha(F2 copy) may be useful in resolving relationships among bee families and other Cretaceous-age insect lineages.     

Synergistic effects of combining morphological and molecular data in resolving the phylogeny of butterflies and skippers

Phylogenetic relationships among major clades of butterflies and skippers have long been controversial, with no general consensus even today. Such lack of resolution is a substantial impediment to using the otherwise well studied butterflies as a model group in biology. Here we report the results of a combined analysis of DNA sequences from three genes and a morphological data matrix for 57 taxa (3258 characters, 1290 parsimony informative) representing all major lineages from the three putative butterfly super-families (Hedyloidea, Hesperioidea and Papilionoidea), plus out-groups representing other ditrysian Lepidoptera families. Recently, the utility of morphological data as a source of phylogenetic evidence has been debated. We present the first well supported phylogenetic hypothesis for the butterflies and skippers based on a total-evidence analysis of both traditional morphological characters and new molecular characters from three gene regions (COI, EF-1alpha and wingless). All four data partitions show substantial hidden support for the deeper nodes, which emerges only in a combined analysis in which the addition of morphological data plays a crucial role. With the exception of Nymphalidae, the traditionally recognized families are found to be strongly supported monophyletic clades with the following relationships: (Hesperiidae+(Papilionidae+(Pieridae+(Nymphalidae+(Lycaenidae+Riodinidae))))). Nymphalidae is recovered as a monophyletic clade but this clade does not have strong support. Lycaenidae and Riodinidae are sister groups with strong support and we suggest that the latter be given family rank. The position of Pieridae as the sister taxon to nymphalids, lycaenids and riodinids is supported by morphology and the EF-1alpha data but conflicted by the COI and wingless data. Hedylidae are more likely to be related to butterflies and skippers than geometrid moths and appear to be the sister group to Papilionoidea+Hesperioidea.     

Phylogenetic relationships of butterflies of the tribe Acraeini (Lepidoptera, Nymphalidae, Heliconiinae) and the evolution of host plant use

The tribe Acraeini (Nymphalidae, Heliconiinae) is believed to comprise between one and seven genera, with the greatest diversity in Africa. The genera Abananote, Altinote, and Actinote (s. str.) are distributed in the Neotropics, while the genera Acraea, Bematistes, Miyana, and Pardopsis have a Palaeotropical distribution. The monotypic Pardopsis use herbaceous plants of the family Violaceae, Acraea and Bematistes feed selectively on plants with cyanoglycosides belonging to many plant families, but preferentially to Passifloraceae, and all Neotropical species with a known life cycle feed on Asteraceae only. Here, a molecular phylogeny is proposed for the butterflies of the tribe Acraeini based on sequences of COI, EF-1alpha and wgl. Both Maximum Parsimony and Bayesian analyses showed that the tribe is monophyletic, once the genus Pardopsis is excluded, since it appears to be related to Argynnini. The existing genus Acraea is a paraphyletic group with regard to the South American genera, and the species of Acraea belonging to the group of "Old World Actinote" is the sister group of the Neotropical genera. The monophyly of South American clade is strongly supported, suggesting a single colonization event of South America. The New World Actinote (s. str.) is monophyletic, and sister to Abananote+Altinote (polyphyletic). Based on the present results it was possible to propose a scenario for the evolution in host plant use within Acraeini, mainly concerning the use of Asteraceae by the South American genera.     

Apis mellifera cytoplasmic elongation factor 1 alpha (EF-1 alpha) is closely related to Drosophila melanogaster EF-1 alpha

Using low stringency hybridisation with a Drosophila melanogaster EF-1 alpha gene fragment we have isolated a genomic DNA clone encoding elongation factor 1 alpha (EF-1 alpha) from Apis mellifera. The hybridising Apis mellifera sequence could be delineated to two small EcoRI fragments that were also revealed by genomic Southern hybridisation. By comparison with the corresponding Drosophila melanogaster data the complete translational reading frame has been deduced. It is interrupted by two intervening sequences of 220 and about 790 nucleotides. Comparison with known eucaryotic EF-1 alpha sequences further confirms that certain amino acid sequences seem to be invariable within the EF-1 alpha protein family.     

Phylogeny, historical biogeography, and taxonomic ranking of Parnassiinae (Lepidoptera, Papilionidae) based on morphology and seven genes

We tested the taxonomic utility of morphology and seven mitochondrial or nuclear genes in a phylogenetic reconstruction of swallowtail butterflies in the subfamily Parnassiinae. Our data included 236 morphological characters and DNA sequences for seven genes that are commonly used to infer lepidopteran relationships (COI+COII, ND5, ND1, 16S, EF-1alpha, and wg; total 5775 bp). Nuclear genes performed best for inferring phylogenies, particularly at higher taxonomic levels, while there was substantial variation in performance among mitochondrial genes. Multiple analyses of molecular data (MP, ML and Bayesian) consistently produced a tree topology different from that obtained by morphology alone. Based on molecular evidence, sister-group relationships were confirmed between the genera Hypermnestra and Parnassius, as well as between Archon and Luehdorfia, while the monophyly of the subfamily was weakly supported. We recognize three tribes within Parnassiinae, with Archon and Luehdorfia forming the tribe Luehdorfiini Tutt, 1896 [stat. rev.]. Three fossil taxa were incorporated into a molecular clock analysis with biogeographic time constraints. Based on dispersal-vicariance (DIVA) analysis, the most recent common ancestor of Parnassiinae occurred in the Iranian Plateau and Central Asia to China. Early diversification of Parnassiinae took place at the same time that India collided into Eurasia, 65-42 million years ago.     

Developmental analysis of elongation factor-1 alpha expression in transgenic tobacco.

The developmental regulation of the translational elongation factor EF-1 alpha has been analyzed in tobacco. A gene fusion was constructed consisting of the 5' and 3' regions of the tomato genomic clone LeEF-A from the EF-1 alpha gene family and the beta-glucuronidase coding region. Analysis of the transgenic plants containing this chimeric gene demonstrated that the tomato LeEF-A flanking sequences were sufficient to confer expression patterns similar to those of the endogenous tobacco EF-1 alpha gene. The patterns of beta-glucuronidase activity in this system indicated that during plant growth and development EF-1 alpha is regulated with increased expression corresponding to regions of high protein synthesis, including meristems, rapidly growing tissues, and developing gametophytes. In addition, EF-1 alpha expression responds rapidly to changes in growth patterns induced by hormone treatment. Our results are in agreement with studies in animals indicating that EF-1 alpha expression may be rate limiting for protein synthesis and demonstrate that the analysis of EF-1 alpha is of value for studying interrelationships between protein synthesis and developmental control.     

Interaction of process partitions in phylogenetic analysis: an example from the swallowtail butterfly genus Papilio

In this study, we explored how the concept of the process partition may be applied to phylogenetic analysis. Sequence data were gathered from 23 species and subspecies of the swallowtail butterfly genus Papilio, as well as from two outgroup species from the genera Eurytides and Pachliopta. Sequence data consisted of 1,010 bp of the nuclear protein-coding gene elongation factor-1 alpha (EF-1 alpha) as well as the entire sequences (a total of 2,211 bp) of the mitochondrial protein-coding genes cytochrome oxidase I and cytochrome oxidase II (COI and COII). In order to examine the interaction between the nuclear and mitochondrial partitions in a combined analysis, we used a method of visualizing branch support as a function of partition weight ratios. We demonstrated how this method may be used to diagnose error at different levels of a tree in a combined maximum-parsimony analysis. Further, we assessed patterns of evolution within and between subsets of the data by implementing a multipartition maximum-likelihood model to estimate evolutionary parameters for various putative process partitions. COI third positions have an estimated average substitution rate more than 15 times that of EF-1 alpha, while COII third positions have an estimated average substitution rate more than 22 times that of EF-1 alpha. Ultimately, we found that although the mitochondrial and nuclear data were not significantly incongruent, homoplasy in the fast-evolving mitochondrial data confounded the resolution of basal relationships in the combined unweighted parsimony analysis despite the fact that there was relatively strong support for the relationships in the nuclear data. We conclude that there may be shortcomings to the methods of "total evidence" and "conditional combination" because they may fail to detect or accommodate the type of confounding bias we found in our data.     

Towards a better understanding of the higher systematics of Nymphalidae (Lepidoptera: Papilionoidea)

Research on the molecular systematics of higher taxa in the butterfly family Nymphalidae (Lepidoptera) is only just beginning. Outgroup selection is difficult at the moment due to the lack of consensus on the basal relationships of the major groups in Nymphalidae. We identify four major clades in the Nymphalidae based on a cladistic analysis of one mitochondrial gene sequence (COI, 1450 bp) and two nuclear gene sequences (EF-1alpha, 1064 bp, and wingless, 412-415 bp) from 54 exemplar species sampled from all currently recognized subfamilies. The COI data set was found to be highly incongruent with the nuclear data sets and a Partitioned Bremer Support analysis shows that the COI data set largely undermines support for most clades. Transitions at the third codon positions of the COI data set were highly saturated, but analyzing the combined data set with the COI third positions removed did not change the results. The major clades we found are termed the danaine clade (including Danainae), the satyrine clade (including Charaxinae, Satyrinae, Calinaginae, and Morphinae), the heliconiine clade (including Heliconiinae and Limenitidinae excluding Biblidini, Cyrestini, Pseudergolini, and Coeini) and the nymphaline clade (including Nymphalinae, Apaturinae, and Coeini, Cyrestini, Pseudergolini, and Biblidini from Limenitidinae). The heliconiine and nymphaline clades are sister groups, while the most parsimonious explanation for the combined data set places the danaine clade as the most basal large group of Nymphalidae. Our results give one of the strongest hypotheses for the subfamilial relationships within Nymphalidae. We were able to resolve the polyphyletic nature of Limenitidinae, which we recommend to be split into three subfamilies: Limenitidinae, Biblidinae, and Cyrestinae. The tribe Coeini belongs in Nymphalinae.     

LDL binds to surface-expressed human T-cadherin in transfected HEK293 cells and influences homophilic adhesive interactions

The interaction between elongation factor 1alpha (EF-1alpha) and alpha/beta-tubulins has been analyzed in vivo and in vitro. An association of both alpha- and beta-tubulins with EF-1alpha in the lysate of Tetrahymena pyriformis was detected by co-immunoprecipitation analysis. In contrast, in vitro biomolecular interaction analysis with glutathione S-transferase (GST) fusion proteins revealed that GST-beta-tubulin, but not GST-alpha-tubulin, can bind to GST-EF-1alpha. Two beta-tubulin binding sites have been identified to reside in the domains I and III of EF-1alpha. In addition, beta-tubulin itself seems to have two distinct interaction sites for each of the domains. Since domain II of EF-1alpha did not interact with beta-tubulin, we have re-evaluated the phylogenetic status of ciliates using EF-1alpha sequences devoid of domain II. The phylogenetic tree thus obtained was significantly different from that inferred from the whole sequence of EF-1alpha, suggesting the presence of functional constraints on the molecular evolution of EF-1alpha.     

EVOLUTION IN A PUTATTVELY ANCIENT ASEXUAL APHID LINEAGE: RECOMBINATION AND RAPID KARYOTYPE CHANGE

Ancient asexual lineages are of great potential significance for understanding the evolutionary biology of sex, but their existence is controversial. In part, this is because claims of ancient asexuality have rested on negative evidence—a mere absence of evidence for sexuality in a taxon. M. Meselson has suggested a method, discussed by Judson and Normark (1996) and by Birky (1996), that has the potential to uncover positive evidence of ancient asexuality. Phylogenetic relationships between alleles and interallelic divergences are predicted to be very different in diploid lineages that lack recombination from those in diploid lineages that undergo recombination. I have applied Meselson's method to the putatively ancient asexual aphid tribe Tramini (Homoptera: Aphidoidea: Lachnidae), using the intron-bearing nuclear protein-coding gene elongation factor 1α (EF-1α). I found heterozygosities much lower than intraspecific divergences, indicating that some recombination has occurred, but not discriminating between recombination within an asexual lineage (automixis or mitotic recombination) and outcrossing sex. Species of Tramini (especially in the genus Trama) typically have highly structurally heterozygous karyotypes that appear to be incompatible with regular successful meiosis, and have very high levels of karyotype variability within species. I found very high levels of karyotype variability within lineages with identical EF-1α and mitochondrial (cytochrome oxidase 1 and 2) genotypes, indicating a high rate of karyotype evolution compared to the rate of nucleotide substitution.     

Retropseudogenes constitute the major part of the human elongation factor 1 alpha gene family.

The elongation factor 1 alpha (EF-1 alpha) is a protein which promotes the GTP-dependent binding of aminoacyl-tRNA to ribosomes in the protein synthesis process. A human gene coding for EF-1 alpha has previously been cloned and sequenced along with a pseudo-gene. Here, we have further analyzed the family of human EF-1 alpha genes. Using an EF-1 alpha cDNA as probe twelve genomic EF-1 alpha-like clones were isolated and analyzed. Four of these were sequenced and found to contain EF-1 alpha retropseudogenes. A Southern blot analysis indicated that the remaining eight clones also contained retropseudogenes. Genomic Southern blot analysis revealed at least twenty loci in the human genome with sequence homology to the EF-1 alpha cDNA. Besides the already described active gene only one potentially active locus was found. The others appeared to be retropseudogenes. EF-1 alpha retropseudogenes were also found to be abundant in the mammalian species mouse and pig, while the chicken contained only one presumably active EF-1 alpha gene.     

Phylogeny and evolution of butterflies of the genus Parnassius: inferences from mitochondrial 16S and ND1 sequences

Phylogenetic relationships among species of the genus Parnassius and its related taxa were analyzed by comparing nucleotide sequences of mitochondrial 16S ribosomal RNA (504 sites) and NADH-dehydrogenase subunit 1 (469 sites). In the phylogenetic trees, Parnassius was found to be most closely related to Hypermnestra helios, whereas Archon apollinus, which has been classified in the tribe Parnassiini together with Parnassius and Hypermnestra, was more closely related to members of the tribe Zerynthiini. Within the Parnassius clade, six major clades corresponding to species groups were well supported, although the phylogenetic relationships among them were not clear. Although the results of the present study were in agreement with those of a previous phylogenetic study based on mitochondrial NADH-dehydrogenase subunit 5 sequences, our study strongly supported a close relationship between Parnassius and Hypermnestra, which was not well supported in the previous study.     

中国黄粉蝶亚科六属间基于COⅡ和EF-1α基因部分序列的系统发育关系（鳞翅目：粉蝶科）

为了探讨中国黄粉蝶亚科属间的系统发育关系,我们对其中6属9种的细胞色素氧化酶Ⅱ（COⅡ）的部分序列和延伸因子基因（EF-1α）部分序列进行了分析。分别采用最大简约法（maximum parsimony, MP）、最大似然法（maximum likelihood, ML）和贝叶斯推论法（bayesian inference, BI）构建黄粉蝶亚科分子系统树。结果表明：在测得的COⅡ基因的648 bp序列和EF-1α基因的504 bp序列中,有261个变异位点,151个简约信息位点,黄粉蝶亚科内各属COⅡ基因A+T含量（77.3%）均明显偏高。系统发育分析显示黄粉蝶属为亚科中较为原始的类群,分化较早,豆粉蝶属和迁粉蝶属亲缘关系较近,但钩粉蝶属与豆粉蝶属、迁粉蝶属之间的亲缘关系还不能确定。本研究结果和传统的基于形态学的黄粉蝶亚科的分类体系有所不同,最显著的分歧是本研究支持内群中分化最早的属应为黄粉蝶属,而不是豆粉蝶属和迁粉蝶属。     

A new nuclear gene for insect phylogenetics: dopa decarboxylase is informative of relationships within Heliothinae (Lepidoptera: Noctuidae)

The lack of a readily accessible roster of nuclear genes informative at various taxonomic levels is a bottleneck for molecular systematics. In this report, we describe the first phylogenetic application of the sequence that encodes the enzyme dopa decarboxylase (DDC). For 14 test species within the noctuid moth subfamily Heliothinae that represent the previously best-supported groupings, a 690-bp fragment of DDC resolved relationships that are largely concordant with prior evidence from elongation factor-1 alpha (EF-1 alpha), morphology, and allozymes. Although both synonymous and nonsynonymous changes occur in DDC substantially more rapidly than they do in EF-1 alpha, DDC divergences within Heliothinae are below saturation at all codon positions. Analysis of DDC and EF-1 alpha in combination resulted in increased bootstrap support for several groupings. As a first estimate of previously unresolved relationships, DDC sequences were analyzed from 16 additional heliothines, for a total of 30 heliothine species plus outgroups. Previous relationships based on DDC were generally stable with increased taxon sampling, although a two- to eightfold downweighting of codon position 3 was required for complete concordance with the 14-species result. The weighted strict consensus trees were largely resolved and were congruent with most although not all previous hypotheses based on either morphology or EF-1 alpha. The proposed phylogeny suggests that the major agricultural pest heliothines belong to a single clade, characterized by polyphagy and associated life history traits, within this largely host-specific moth subfamily. DDC holds much promise for phylogenetic analysis of Tertiary-age animal groups.     

Phylogenetic resolution within the tribe Episcieae (Gesneriaceae): congruence of ITS and NDHF sequences from parsimony and maximum-likelihood analyses

Generic relationships within Episcieae were assessed using ITS and ndhF sequences. Previous analyses of this tribe have focussed only on ndhF data and have excluded two genera, Rhoogeton and Oerstedina, which are included in this analysis. Data were analyzed using both parsimony and maximum-likelihood methods. Results from partition homogeneity tests imply that the two data sets are significantly incongruent, but when Rhoogeton is removed from the analysis, the data sets are not significantly different. The combined data sets reveal greater strength of relationships within the tribe with the exception of the position of Rhoogeton. Poorly or unresolved relationships based exclusively on ndhF data are more fully resolved with ITS data. These resolved clades include the monophyly of the genera Columnea and Paradrymonia and the sister-group relationship of Nematanthus and Codonanthe. A closer affinity between Neomortonia nummularia and N. rosea than has previously been seen is apparent from these data, although these two species are not monophyletic in any tree. Lastly, Capanea appears to be a member of Gloxinieae, although C. grandiflora remains within Episcieae. Evolution of fruit type, epiphytic habit, and presence of tubers is re-examined with the new data presented here.     

Biological activities of homologous loop regions in the laminin alpha chain G domains

Laminin alpha chains (alpha1-alpha5 chains) have diverse chain-specific biological functions. The LG4 modules of laminin alpha chains consist of a 14-stranded beta-sheet (A-N) sandwich structure. Several biologically active sequences have been identified in the connecting loop regions. Here, we evaluated the biological activities of the loop regions of the E and F strands in the LG4 modules using five homologous peptides from each of the mouse alpha chains (EF-1: DYATLQLQEGRLHFMFDLG, alpha1 chain 2747-2765; EF-2: DFGTVQLRNGFPFFSYDLG, alpha2 chain 2808-2826; EF-3: RDSFVALYLSEGHVIFALG, alpha3 chain 2266-2284; EF-4: DFMTLFLAHGRLVFMFNVG, alpha4 chain 1511-1529; EF-5: SPSLVLFLNHGHFVAQTEGP, alpha5 chain 3304-3323). These homologous peptides showed chain-specific cell attachment and neurite outgrowth activities. Well organized actin stress fibers and focal contacts with vinculin accumulation were observed in fibroblasts attached on EF-1, whereas fibroblasts on EF-2 and EF-4 showed filopodia with ruffling. Fibroblast attachment to EF-2 and EF-4 was mediated by syndecan-2. In contrast, EF-1 promoted alpha2beta1 integrin-mediated fibroblast attachment and inhibited fibroblast attachment to a recombinant laminin alpha1 chain LG4-5. The receptors for EF-3 and EF-5 are unknown. Further, when the active core sequence of EF-1 was cyclized, utilizing two additional cysteine residues at both the N and C termini through a disulfide bridge, the cyclic peptide significantly enhanced integrin-mediated cell attachment. These results indicate that integrin-mediated cell attachment to the EF-1 sequence is conformation-dependent and that the loop structure is important for the activity. The homologous peptides, which promote either integrin- or syndecan-mediated cell attachment, may be useful for understanding the cell type- and chain-specific biological activities of the laminins.