Optimizing the widely used nuclear protein‐coding gene primers in beetle phylogenies and their application in the genus Sasajiscymnus Vandenberg (Coleoptera: Coccinellidae)

Advances in genomic biology and the increasing availability of genomic resources allow developing hundreds of nuclear protein‐coding (NPC) markers, which can be used in phylogenetic research. However, for low taxonomic levels, it may be more practical to select a handful of suitable molecular loci for phylogenetic inference. Unfortunately, the presence of degenerate primers of NPC markers can be a major impediment, as the amplification success rate is low and they tend to amplify nontargeted regions. In this study, we optimized five NPC fragments widely used in beetle phylogenetics (i.e., two parts of carbamoyl‐phosphate synthetase: CADXM and CADMC, Topoisomerase, Wingless and Pepck) by reducing the degenerate site of primers and the length of target genes slightly. These five NPC fragments and 6 other molecular loci were amplified to test the monophyly of the coccinellid genus Sasajiscymnus Vandenberg. The analysis of our molecular data set clearly supported the genus Sasajiscymnus may be monophyletic but confirmation with an extended sampling is required. A fossil‐calibrated chronogram was generated by BEAST, indicating an origin of the genus at the end of the Cretaceous (77.87 Myr). Furthermore, a phylogenetic informativeness profile was generated to compare the phylogenetic properties of each gene more explicitly. The results showed that COI provides the strongest phylogenetic signal among all the genes, but Pepck, Topoisomerase, CADXM and CADMC are also relatively informative. Our results provide insight into the evolution of the genus Sasajiscymnus, and also enrich the molecular data resources for further study.     

A PCR survey of <Emphasis Type="Italic">Hox</Emphasis> genes in the myzostomid <Emphasis Type="Italic">Myzostoma cirriferum</Emphasis>

Using degenerate primers, we were able to identify seven Hox genes for the myzostomid Myzostoma cirriferum. The recovered fragments belong to anterior class (Mci_lab, Mci_pb), central class (Mci_Dfd, Mci_Lox5, Mci_Antp, Mci_Lox4), and posterior class (Mci_Post2) paralog groups. Orthology assignment was verified by phylogenetic analyses and presence of diagnostic regions in the homeodomain as well as flanking regions. The presence of Lox5, Lox4, and Post2 supports the inclusion of Myzostomida within Lophotrochozoa. We found signature residues within flanking regions of Lox5, which are also found in annelids, but not in Platyhelminthes. As such the available Hox genes data of myzostomids support an annelid relationship. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Inheritance of mitochondrial DNA in the rotifer <Emphasis Type="Italic">Brachionus plicatilis</Emphasis>

By crossing Brachionus plicatilis s.s. NH1L strain and German strain, we obtained two types of hybrids, NH1L female × German male designated as NXG and German female × NH1L male designated as GXN. To confirm the crossing of the two hybrid strains at the genetic level, random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis using 10 kinds of primers (10 and 12 mers) was carried out. Some amplified DNA fragments from RAPD of the hybrid strain showed mixed patterns of both parental strains, thus confirming that both hybrids were crossbreeds of the NH1L and German strains. Using these hybrids, we investigated the mode of mitochondrial inheritance in B. plicatilis. Full-length mtDNA of the four strains was amplified by PCR, and digested with restriction enzymes to obtain restriction fragment length polymorphism (RFLP) patterns. Both hybrid strains had the same RFLP patterns as their female parents. This result shows that mitochondrial inheritance in rotifers is maternal. Guest editors: S. S. S. Sarma, R. D. Gulati, R. L. Wallace, S. Nandini, H. J. Dumont and R. Rico-Martínez Advances in Rotifer Research     

Prospects on the evolutionary mitogenomics of plants: A case study on the olive family (Oleaceae)

The mitogenome is rarely used to reconstruct the evolutionary history of plants, contrary to nuclear and plastid markers. Here, we evaluate the usefulness of mitochondrial DNA for molecular evolutionary studies in Oleaceae, in which cases of cytoplasmic male sterility (CMS) and of potentially contrasted organelle inheritance are known. We compare the diversity and the evolution of mitochondrial and chloroplast genomes by focusing on the olive complex and related genera. Using high‐throughput techniques, we reconstructed complete mitogenomes (ca. 0.7 Mb) and plastomes (ca. 156 kb) for six olive accessions and one Chionanthus. A highly variable organization of mitogenomes was observed at the species level. In olive, two specific chimeric genes were identified in the mitogenome of lineage E3 and may be involved in CMS. Plastid‐derived regions (mtpt) were observed in all reconstructed mitogenomes. Through phylogenetic reconstruction, we demonstrate that multiple integrations of mtpt regions have occurred in Oleaceae, but mtpt regions shared by all members of the olive complex derive from a common ancestor. We then assembled 52 conserved mitochondrial gene regions and complete plastomes of ten additional accessions belonging to tribes Oleeae, Fontanesieae and Forsythieae. Phylogenetic congruence between topologies based on mitochondrial regions and plastomes suggests a strong disequilibrium linkage between both organellar genomes. Finally, while phylogenetic reconstruction based on plastomes fails to resolve the evolutionary history of maternal olive lineages in the Mediterranean area, their phylogenetic relationships were successfully resolved with complete mitogenomes. Overall, our study demonstrates the great potential of using mitochondrial DNA in plant phylogeographic and metagenomic studies.     

Use of Comparative Genomics to Develop EST-SSRs for Red Drum (Sciaenops ocellatus)

Microsatellites physically linked to expressed sequence tags (EST-SSRs) are an important resource for linkage mapping and comparative genomics, and data mining in publicly available EST databases is a common strategy for EST-SSR discovery. At present, many species lack species-specific EST sequence data needed for the efficient characterization of EST-SSRs. This paper describes the discovery and development of EST-SSRs for red drum (Sciaenops ocellatus), an estuarine-dependent sciaenid species of economic importance in the USA and elsewhere, using a phylogenetically informed, comparative genomics approach to primer design. The approach entailed comparing existing genomic resources from species closely allied phylogenetically to red drum, with resources from more distantly related outgroup species. By taking into account the degree to which flanking regions are conserved across taxa, the efficiency of PCR primer design was increased greatly. The amplification success rate for primers designed for red drum was 100?% when using EST libraries from confamilial species and 92?% when using an EST library from a species in the same suborder. The primers developed also amplified EST-SSRs in a wide range of perciform fishes, suggesting potential use in comparative genomics. This study demonstrates that EST-SSRs can be efficiently developed for an organism when limited species-specific data are available by exploiting genomic resources from well-studied species, even those at extended phylogenetic distances.     

The mitochondrial genome of the water spider Argyroneta aquatica (Araneae: Cybaeidae)

We sequenced nearly the entire mitochondrial genome of Argyroneta aquatica, a wholly underwater‐living spider, thereby enhancing the available genomic information for Arachnida. The confirmed sequences contained the complete set of known genes present in other metazoan mitochondrial genomes. However, the mitochondrial gene order of A. aquatica was distinctly different from that of the most distant Chelicerata Limulus polyphemus (Xiphosura), probably because of a series of gene translocations and/or inversions. Comparison of arachnid mitochondrial gene orders for the purpose of phylogenetic inference is only minimally useful, but provides a strong signal in closely related lineages. To test the basal relationships and the evolutionary pattern of tRNA gene rearrangements among Arachnida, phylogenetic analyses using amino acid sequences of the 13 protein‐coding genes were performed. An interesting feature, the five 135‐bp tandem repeats and two 363‐bp tandem repeats, was identified in the putative control region. Although control region tandem repeats have been reported in many other arachnid and metazoan species, this is the first time it has been described in spiders.     

Protein–protein interactions of the cytochrome c oxidase DNA barcoding region

Enzymatic amplification of homologous regions of DNA using ‘universal’ polymerase chain reaction primers has provided insight into insect systematics, phylogeography, molecular evolution and species identification. One of the more commonly amplified and sequenced regions is a short region of the cytochrome c oxidase subunit I gene (COI), commonly called the barcoding region. COI is one of three mitochondrial‐encoded subunits of complex IV (Cox) of the electron transport chain. In addition to the mitochondrial subunits there are nine nuclear‐encoded subunits of the complex in Drosophila. Whereas a number of phylogenetic biases associated with this region have been examined and the quaternary structure of Cox has been modelled, the influence of protein–protein interactions on the observed patterns of evolution in this barcoding region of insects has never been examined critically. Using a well‐resolved independently derived phylogeny of 38 Diptera species, we examined the homogeneity of the substitution processes within the barcoding region. We show that, within Diptera, amino acid residues interacting with nuclear‐encoded subunits of Cox are evolving at elevated rates across the phylogeny. Furthermore, we show that codon position two is biased by protein–protein interactions. In contrast, third codon positions provide a less biased estimate of genetic variation in the region. This study highlights the need to examine the potential for systematic bias in DNA barcoding regions as part of the critical assessment of evidence in systematics and in biodiversity assessments.     

PCR with degenerate primers amplifies a subgenomic DNA fragment from the endoglucanase gene(s) of Torula thermophila, a thermophilic fungus

The aim of this study was to enable the polymerase chain reaction (PCR) amplification of DNA fragments within endoglucanase gene(s) of Torula thermophila, by using degenerate primers so that the amplified fragment(s) could be used as homologous probe(s) for cloning of full-length endoglucanase gene(s). The design of the degenerate PCR primers was mainly based on the endoglucanase sequences of other fungi. The endoglucanase gene sequence of Humicola insolens was the only sequence from a thermophilic fungus publicly available in the literature. Therefore, the endoglucanase sequences of the two Trichoderma species, Trichoderma reesei and Trichoderma longibrachiatum, were used to generalize the primers. PCR amplification of T. thermophila genomic DNA with these primers resilied in a specific amplification. The specificity of the amplified fragment was shown by Southern hybridization analysis using egl3 gene of T. reesei as probe. This result suggested that the degenerate primers used in this study may be of value for studies aimed at cloning of endoglucanase genes from a range of related fungi.     

Genome‐wide survey of nuclear protein‐coding markers for beetle phylogenetics and their application in resolving both deep and shallow‐level divergences

Beetles (Coleoptera) are the most diverse and species‐rich insect group, representing an impressive explosive radiation in the evolutionary history of insects, and their evolutionary relationships are often difficult to resolve. The amount of ‘traditional markers’ (e.g. mitochondrial genes and nuclear rDNAs) for beetle phylogenetics is small, and these markers often lack sufficient signals in resolving relationships for such a rapidly radiating lineage. Here, based on the available genome data of beetles and other related insect species, we performed a genome‐wide survey to search nuclear protein‐coding (NPC) genes suitable for research on beetle phylogenetics. As a result, we identified 1470 candidate loci, which provided a valuable data resource to the beetle evolutionary research community for NPC marker development. We randomly chose 180 candidate loci from the database to design primers and successfully developed 95 NPC markers which can be PCR amplified from standard genomic DNA extracts. These new nuclear markers are universally applicable across Coleoptera, with an average amplification success rate of 90%. To test the phylogenetic utility, we used them to investigate the backbone phylogeny of Coleoptera (18 families sampled) and the family Coccinellidae (39 species sampled). Both phylogenies are well resolved (average bootstrap support >95%), showing that our markers can be used to address phylogenetic questions of various evolutionary depth (from species level to family level). In general, the newly developed nuclear markers are much easier to use and more phylogenetically informative than the ‘traditional markers’, and show great potential to expedite resolution of many parts in the Beetle Tree of Life.     

Evolutionary history of the fish genus <Emphasis Type="Italic">Astyanax</Emphasis> Baird &; Girard (1854) (Actinopterygii,Characidae) in Mesoamerica reveals multiple morphological homoplasies

Background  

Mesoamerica is one of the world's most complex biogeographical regions, mostly due to its complex geological history. This complexity has led to interesting biogeographical processes that have resulted in the current diversity and distribution of fauna in the region. The fish genus Astyanax represents a useful model to assess biogeographical hypotheses due to it being one of the most diverse and widely distributed freshwater fish species in the New World. We used mitochondrial and nuclear DNA to evaluate phylogenetic relationships within the genus in Mesoamerica, and to develop historical biogeographical hypotheses to explain its current distribution.     

Genomic outposts serve the phylogenomic pioneers: designing novel nuclear markers for genomic DNA extractions of lepidoptera

Increasing the number of characters used in phylogenetic studies is the next crucial step towards generating robust and stable phylogenetic hypotheses - i.e., strongly supported and consistent across reconstruction method. Here we describe a genomic approach to finding new protein-coding genes for systematics in nonmodel taxa, which can be PCR amplified from standard, slightly degraded genomic DNA extracts. We test this approach on Lepidoptera, searching the draft genomic sequence of the silk moth Bombyx mori, for exons > 500 bp in length, removing annotated gene families, and compared remaining exons with butterfly EST databases to identify conserved regions for primer design. These primers were tested on a set of 65 taxa primarily in the butterfly family Nymphalidae. We were able to identify and amplify six previously unused gene regions (Arginine Kinase, GAPDH, IDH, MDH, RpS2, and RpS5) and two rarely used gene regions (CAD and DDC) that when added to the three traditional gene regions (COI, EF-1alpha and wingless) gave a data set of 8114 bp. Phylogenetic robustness and stability increased with increasing numbers of genes. Smaller taxanomic subsets were also robust when using the full gene data set. The full 11-gene data set was robust and stable across reconstruction methods, recovering the major lineages and strongly supporting relationships within them. Our methods and insights should be applicable to taxonomic groups having a single genomic reference species and several EST databases from taxa that diverged less than 100 million years ago.     

Dynamics of Mhc evolution in birds and crocodilians: amplification of class II genes with degenerate primers

Genes of the major histocompatibility complex (Mhc) are the most polymorphic functional loci in mammalian populations, but little is known of Mhc variability in natural populations of nonmammalian vertebrates. To help extend such studies to birds and relatives, we present a pair of degenerate primers that amplify polymorphic segments of one chain (the β chain) of the class II genes from the major histocompatibility complex (Mhc) of archosaurs (birds + crocodilians). The primers target two conserved regions lying within portions of the antigen-binding site (ABS) encoded by the second exon and amplify multiple genes from both genomic DNA and cDNA. The pattern of nucleotide substitution in ABS codons of 51 sequences amplified and cloned from five species of passerine birds and an alligator (Alligator mississippiensis) indicates that archosaurian class II β genes are subject to selective forces similar to those operating in mammalian populations. Hybridization of a genomic clone generated by the primers revealed highly polymorphic bands in a sample of Florida scrub jays (Aphelocoma coerulescens coerulescens). Because the primers amplify only part of the ABS from multiple class II genes, they will be useful primarily for generating species specific clones, thereby providing a critical inroad to more detailed structural and evolutionary studies.     

Complete mitochondrial DNA sequences of the Nile tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>) and Blue tilapia (<Emphasis Type="Italic">Oreochromis aureus</Emphasis>): genome characterization and phylogeny applications

Cichlid fishes have played an important role in evolutionary biology and aquaculture industry. Nile tilapia (Oreochromis niloticus), blue tilapia (Oreochromis aureus) and Mozambique tilapia (Oreochromis mossambicus), the useful models in studying evolutionary biology within Cichlid fishes, are also mainly cultured species in aquaculture with great economic importance. In this paper, the complete nucleotide sequence of the mitochondrial genome for O. niloticus and O. aureus were determined and phylogenetic analyses from mitochondrial protein-coding genes were conducted to explore their phylogenetic relationship within Cichlids. The mitogenome is 16,625 bp for O. niloticus and 16,628 bp for O. aureus, containing the same gene order and an identical number of genes or regions with the other Cichlid fishes, including 13 protein-coding genes, two rRNA genes, 22 tRNA genes and one putative control region. Phylogenetic analyses using three different computational algorithms (maximum parsimony, maximum likelihood and Bayesian method) show O. niloticus and O. mossambicus are closely related, and O. aureus has remotely phylogenetic relationship from above two fishes.     

Development of genomic resources for Nothofagus species using next‐generation sequencing data

Using next‐generation sequencing, we developed the first whole‐genome resources for two hybridizing Nothofagus species of the Patagonian forests that crucially lack genomic data, despite their ecological and industrial value. A de novo assembly strategy combining base quality control and optimization of the putative chloroplast gene map yielded ~32 000 contigs from 43% of the reads produced. With 12.5% of assembled reads, we covered ~96% of the chloroplast genome and ~70% of the mitochondrial gene content, providing functional and structural annotations for 112 and 52 genes, respectively. Functional annotation was possible on 15% of the contigs, with ~1750 potentially novel nuclear genes identified for Nothofagus species. We estimated that the new resources (13.41 Mb in total) included ~4000 gene regions representing ~6.5% of the expected genic partition of the genome, the remaining contigs potentially being nongenic DNA. A high‐quality single nucleotide polymorphisms resource was developed by comparing various filtering methods, and preliminary results indicate a strong conservation of cpDNA genomes in contrast to numerous exclusive nuclear polymorphisms in both species. Finally, we characterized 2274 potential simple sequence repeat (SSR) loci, designed primers for 769 of them and validated nine of 29 loci in 42 individuals per species. Nothofagus obliqua had more alleles (4.89) on average than N. nervosa (2.89), 8 SSRs were efficient to discriminate species, and three were successfully transferred in three other Nothofagus species. These resources will greatly help for future inferences of demographic, adaptive and hybridizing events in Nothofagus species, and for conserving and managing natural populations.     

Fifteen species in one: deciphering the <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> species complex (Rotifera,Monogononta) through DNA taxonomy

Understanding patterns and processes in biological diversity is a critical task given current and rapid environmental change. Such knowledge is even more essential when the taxa under consideration are important ecological and evolutionary models. One of these cases is the monogonont rotifer cryptic species complex Brachionus plicatilis, which is by far the most extensively studied group of rotifers, is widely used in aquaculture, and is known to host a large amount of unresolved diversity. Here we collate a dataset of previously available and newly generated sequences of COI and ITS1 for 1273 isolates of the B. plicatilis complex and apply three approaches in DNA taxonomy (i.e. ABGD, PTP, and GMYC) to identify and provide support for the existence of 15 species within the complex. We used these results to explore phylogenetic signal in morphometric and ecological traits, and to understand correlation among the traits using phylogenetic comparative models. Our results support niche conservatism for some traits (e.g. body length) and phylogenetic plasticity for others (e.g. genome size).     

Rapid Diagnosis of Soya Bean Root Rot Caused by Fusarium culmorum Using a Loop‐Mediated Isothermal Amplification Assay

We report a rapid diagnosis of soya bean (Glycine max L.) root rot caused by Fusarium culmorum, using a loop‐mediated isothermal amplification (LAMP) assay. We used the CYP51C gene sequence to design LAMP assay primers specific for F. culmorum. The LAMP assay amplified the target gene efficiently in 60 min at 63°C. The sensitivity of the assay was 100 pg/μl of genomic DNA. Among the tested soya bean pathogens, a positive colour (sky blue) was only observed in the presence of F. culmorum with the addition of hydroxynaphthol blue (HNB) dye prior to amplification, whereas other species isolates showed no colour change. Suspected diseased soya bean samples collected in the field from Jiangsu, Shandong and Anhui provinces and Beijing were diagnosed successfully using the LAMP assay reported here. This study provides a new and readily available method for rapid diagnosis of soya bean root rot caused by F. culmorum.     

Amplification of multiple copies of mitochondrial Cytochrome b gene fragments in the Australian freshwater crayfish,Cherax destructor Clark (Parastacidae: Decapoda)

Non-coding copies of fragments of the mitochondrial genome translocated to the nucleus or pseudogenes are being found with increasing frequency in a diversity of organisms. As part of a study to evaluate the utility of a range of mitochondrial gene regions for population genetic and systematic studies of the Australian freshwater crayfish, Cherax destructor (the yabby), we report the first detection of Cytochrome b (Cyt b) pseudogenes in crustaceans. We amplified and sequenced fragments of the mitochondrial Cyt b gene from 14 individuals of C. destructor using polymerase chain reaction (PCR) with primers designed from conserved regions of Penaeus monodon and Drosophila melanogaster mitochondrial genomes. The phylogenetic tree produced from the amplified fragments using these primers showed a very different topology to the trees obtained from sequences from three other mitochondrial genes, suggesting one or more nuclear pseudogenes have been amplified. Supporting this conclusion, two highly divergent sequences were isolated from each of two single individuals, and a 2 base pair (bp) deletion in one sequence was observed. There was no evidence to support inadvertent amplification of parasite DNA or contamination of samples from other sources. These results add to other recent observations of pseudogenes suggesting the frequent transfer of mitochondrial DNA (mtDNA) genes to the nucleus and reinforces the necessity of great care in interpreting PCR-generated Cyt b sequences used in population or evolutionary studies in freshwater crayfish and crustaceans more generally.     

Nematode‐specific PCR primers for the 18S small subunit rRNA gene

A set of polymerase chain reaction primers were designed, which amplify a c. 1 kb fragment of the 18S ribosomal DNA gene, and are specific to the phylum Nematoda. These have proven useful in isolating nematode genes from samples mixed with other biological material, particularly with application to DNA barcoding. Optimal reaction conditions are described. These primers have successfully amplified the correct fragment from a wide phylogenetic range of nematodes, and have so far generated no sequences from any other organismal group.