Exploring the potential of nuclear and mitochondrial sequencing data generated through genome‐skimming for plant phylogenetics: A case study from a clade of neotropical lianas

Few botanical studies have explored the potential of nuclear ribosomal DNA (nrDNA) and mitochondrial DNA (mtDNA) data obtained through genome skimming for phylogeny reconstruction. Here, we analyzed the phylogenetic information included in the nrDNA and mtDNA of 44 species of the “Adenocalymma‐Neojobertia” clade (Bignoniaceae). To deal with intraindividual polymorphisms within the nrDNA, different coding schemes were explored through the analyses of four datasets: (i) “nrDNA contig,” with base call following the majority rule; (ii) “nrDNA ambiguous,” with ambiguous base calls; (iii) “nrDNA informative,” with ambiguities converted to multistate characters; and, (iv) “mitochondrial,” with 39 mitochondrial genes. Combined analyses using the nrDNA and mtDNA data and previously published “plastid” datasets were also conducted. Trees were obtained using Maximum Likelihood and Bayesian criteria. The congruence among genomes was assessed. The nrDNA datasets were shown to be highly polymorphic within individuals, while the “mitochondrial” dataset was the least informative, with 0.36% of informative bases within the ingroup. The topologies inferred using the nrDNA and mtDNA datasets were broadly congruent with the tree derived from the analyses of the “plastid” dataset. The topological differences recovered were generally poorly supported. The topology that resulted from the analyses of the “combined” dataset largely resembles the “plastid” tree. These results highlight limitations of nuclear ribosomal DNA and mitochondrial genes for phylogeny reconstruction obtained through genome skimming and the need to include more data from both genomes. The different topologies observed among genomes also highlight the importance of exploring data from various genomes in plant phylogenetics.     

Sequencing of whole plastid genomes and nuclear ribosomal DNA of Diospyros species (Ebenaceae) endemic to New Caledonia: many species,little divergence

Background and Aims Some plant groups, especially on islands, have been shaped by strong ancestral bottlenecks and rapid, recent radiation of phenotypic characters. Single molecular markers are often not informative enough for phylogenetic reconstruction in such plant groups. Whole plastid genomes and nuclear ribosomal DNA (nrDNA) are viewed by many researchers as sources of information for phylogenetic reconstruction of groups in which expected levels of divergence in standard markers are low. Here we evaluate the usefulness of these data types to resolve phylogenetic relationships among closely related Diospyros species.Methods Twenty-two closely related Diospyros species from New Caledonia were investigated using whole plastid genomes and nrDNA data from low-coverage next-generation sequencing (NGS). Phylogenetic trees were inferred using maximum parsimony, maximum likelihood and Bayesian inference on separate plastid and nrDNA and combined matrices.Key Results The plastid and nrDNA sequences were, singly and together, unable to provide well supported phylogenetic relationships among the closely related New Caledonian Diospyros species. In the nrDNA, a 6-fold greater percentage of parsimony-informative characters compared with plastid DNA was found, but the total number of informative sites was greater for the much larger plastid DNA genomes. Combining the plastid and nuclear data improved resolution. Plastid results showed a trend towards geographical clustering of accessions rather than following taxonomic species.Conclusions In plant groups in which multiple plastid markers are not sufficiently informative, an investigation at the level of the entire plastid genome may also not be sufficient for detailed phylogenetic reconstruction. Sequencing of complete plastid genomes and nrDNA repeats seems to clarify some relationships among the New Caledonian Diospyros species, but the higher percentage of parsimony-informative characters in nrDNA compared with plastid DNA did not help to resolve the phylogenetic tree because the total number of variable sites was much lower than in the entire plastid genome. The geographical clustering of the individuals against a background of overall low sequence divergence could indicate transfer of plastid genomes due to hybridization and introgression following secondary contact.     

植物核DNA含量在全球尺度上的纬度变异式样及其气候适应意义——以菊科植物为例

核DNA含量是重要的生物学概念,涉及DNA C-值和基因组大小。前人有关植物核DNA含量在纬度梯度上的变异规律存在着矛盾的报道,而且多数将核DNA含量与纬度、海拔、气候等因素之间的关系描述成线性关系。核DNA含量是否具有环境适应上的意义,也还存在争议。先前有关核DNA含量与环境因素间关系的矛盾性报道可能与取样过小、地理范围过窄、研究对象遗传背景差异过大有关。如果对一个遗传背景相近的类群在全球范围内进行取样,核DNA含量会呈现有规律的纬度梯度变化,可能与大的气候因素之间存在非线性关系。菊科(Asteraceae)是被子植物的最大科,是一个广泛认可的自然分类群。为揭示全球空间尺度上植物核DNA含量在纬度梯度上的变异规律,以及这种变异是否具有环境适应意义,以菊科为对象开展了核DNA含量与纬度、生物气候因素关系的统计分析。从"植物DNA C-值数据库"检索到822种菊科植物的核DNA含量数据;在全球范围内,沿经度方向上设立10条样带,每条样带横跨15个经度,每条样带又均分成22个样块,每个样块纵跨7.5个纬度;其次,从"世界气候数据网站"下载1950—2000年时间段14个生物气候因子数据,应用Arc GIS 9.3获得每个样块14个生物气候因子的平均值;根据"全球生物多样性信息网站"记录,计算每个样块菊科植物平均的核DNA含量数据。为避免气候变量之间的多重共线性对数据分析的影响,应用主成分分析对数据进行了降维,发现最冷季度平均温度、最干季度雨量分别是第一、二主成分上荷载最大的因子,去除与它们相关性在-0.7至+0.7之间的其他气候因子后获得了最冷季度平均温度、最干季度雨量和最湿月份雨量三个变量用于进一步数据分析。结果发现,菊科植物在全球10个样带上的核DNA含量与纬度关系密切,与最冷季度平均温度、最干季度雨量和最湿月份雨量呈现极显著的单峰型的非线性关系,可以用二项式进行拟合。因此,全球空间尺度上植物核DNA含量沿着纬度梯度有规律性的非线性变化,这种变化具有很强的气候适应意义。     

Genetic variation in chloroplast and nuclear ribosomal DNA in Utah juniper (Juniperus osteosperma, Cupressaceae): evidence for interspecific gene flow

Geographic patterns of genetic variation in chlorolast (cpDNA) and nuclear ribosomal (nrDNA) DNA were examined to test the hypothesis of hybridization between Juniperus osteosperma and Juniperus occidentalis in the Great Basin of western Nevada. Noncoding DNA from the trnL-trnF intergenic spacer and the trnL intron of the chloroplast genome was sequenced from seven populations of J. osteosperma and four populations of J. occidentalis sampled over a large proportion of their respective ranges. An adenine nucleotide at position 436 in the aligned sequence and within a Tru 9I restriction site was found to be present in individuals of J. osteosperma sampled from western Colorado and central Utah, but absent in sequences of J. osteosperma sampled from central and western Nevada and all sequences of J. occidentalis. Two hundred fourteen individuals from 34 populations of J. osteosperma and J. occidentalis were then screened for cpDNA haplotype by Tru 9I digestion of the trnL-trnF polymerase chain reaction (PCR) product. Two cpDNA haplotypes were evident, each consisting of restriction fragment profiles that differed solely with respect to the presence or absence of the Tru 9I site encompassing the adenine nucleotide at position 436. One of these haplotypes was monomorphic in J. occidentalis and exhibited a decreasing frequency in J. osteosperma with increasing geographic distance from J. occidentalis in west-central Nevada. Geographic patterns in nuclear ribosomal DNA (nrDNA) variation were examined by restriction fragment analysis and, although spatially more restricted, exhibited patterns of clinal variation similar to those observed in cpDNA haplotype. Genetic relationships based on DNA sequences and geographic patterns of genetic variation in chloroplast and nuclear ribosomal DNA are consistent with morphology in suggesting interspecific gene flow between J. occidentalis and J. osteosperma.     

Streamlining universal single‐copy orthologue and ultraconserved element design: A case study in Collembola

Genomic data sets are increasingly central to ecological and evolutionary biology, but far fewer resources are available for invertebrates. Powerful new computational tools and the rapidly decreasing cost of Illumina sequencing are beginning to change this, enabling rapid genome assembly and reference marker extraction. We have developed and tested a practical workflow for developing genomic resources in nonmodel groups with real‐world data on Collembola (springtails), one of the most dominant soil animals on Earth. We designed universal molecular marker sets, single‐copy orthologues (BUSCO s) and ultraconserved elements (UCEs), using three existing and 11 newly generated genomes. Both marker types were tested in silico via marker capture success and phylogenetic performance. The new genomes were assembled with Illumina short reads and 9,585?14,743 protein‐coding genes were predicted with ab initio and protein homology evidence. We identified 1,997 benchmarking universal single‐copy orthologues (BUSCO s) across 14 genomes and created and assessed a custom BUSCO data set for extracting single‐copy genes. We also developed a new UCE probe set containing 46,087 baits targeting 1,885 loci. We successfully captured 1,437?1,865 BUSCO s and 975?1,186 UCEs across 14 genomes. Phylogenomic reconstructions using these markers proved robust, giving new insight on deep‐time collembolan relationships. Our study demonstrates the feasibility of generating thousands of universal markers from highly efficient whole‐genome sequencing, providing a valuable resource for genome‐scale investigations in evolutionary biology and ecology.     

Exon-primed, intron-crossing (EPIC) loci for five nuclear genes in deep-sea protobranch bivalves: primer design, PCR protocols and locus utility

We describe PCR primers and amplification protocols developed to obtain introns from conserved nuclear genes in deep-sea protobranch bivalves. Because almost no sequence data for protobranchs are publically available, mollusk and other protostome sequences from GenBank were used to design degenerate primers, making these loci potentially useful in other invertebrate taxa. Amplification and sequencing success varied across the test group of 30 species, and we present five loci spanning this range of outcomes. Intron presence in the targeted regions also varied across genes and species, often within single genera; for instance, the calmodulin and β-tubulin loci contained introns with high frequency, whereas the triose phosphate isomerase locus never contained an intron. In introns for which we were able to obtain preliminary estimates of polymorphism levels in single species, polymorphism was greater than traditional mitochondrial loci. These markers will greatly increase the ability to assess population structure in the ecologically important protobranchs, and may prove useful in other taxa as well.     

Physical mapping of ribosomal RNA genes in peonies (Paeonia,Paeoniaceae) by fluorescent in situ hybridization: implications for phylogeny and concerted evolution

Physical maps of the 18S–5.8S–26S ribosomal RNA genes (rDNA) were generated by fluorescent in situ hybridization for five diploid Paeonia species, P. delavayi and P. rockii of section Moutan, and P. emodi, P. tenuifolia, and P. veitchii of section Paeonia. Of five pairs of mitotic chromosomes, rDNA loci were mapped near the telomeres of chromosomes 3, 4, and 5 of P. rockii and P. tenuifolia, chromosomes 2, 3, 4, and 5 of P. delavayi, and all five pairs of chromosomes of P. emodi and P. veitchii. Combining this information with the previously obtained rDNA maps of P. brownii and P. californica of section Oneapia, we hypothesized that the most recent common ancestor of extant peony species had three rDNA loci located on chromosomes 3, 4, and 5. Increase in number of rDNA loci occurred later in each of the three sections, and the increase from three to four loci represents a parallel gain of an rDNA locus on chromosome 2 in P. delavayi of section Moutan and P. brownii of section Oneapia. The increase in number of rDNA loci likely resulted from the translocation of rDNA repeats from chromosomes bearing rDNA loci to chromosomes without them; such translocation is probably facilitated by the telomeric location of rDNA loci. For allotetraploid peony species lacking polymorphism in sequences of the internal transcribed spacers (ITS) of rDNA, the rDNAs derived from divergent diploid parents may have been homogenized through concerted evolution among at least six rDNA loci in the allotetraploids. Chromosomal location of rDNA loci has a more substantial impact on the tempo of concerted evolution than the number of loci.     

Drosophila mitochondrial DNA: Conserved sequences in the A+T-rich region and supporting evidence for a secondary structure model of the small ribosomal RNA

Summary The sequence of a segment of theDrosophila virilis mitochondrial DNA (mtDNA) molecule that contains the A+T-rich region, the small rRNA gene, the tRNA^f-met, tRNA^gln, and tRNA^ile genes, and portions of the ND2 and tRNA^val genes is presented and compared with the corresponding segment of theD. yakuba mtDNA molecule. The A+T-rich regions ofD. virilis andD. yakuba contain two correspondingly located sequences of 49 and 276/274 nucleotides that appear to have been conserved during evolution. In each species the replication origin of the mtDNA molecule is calculated to lie within a region that overlaps the larger conserved sequence, and within this overlap is found a potential hairpin structure. Substitutions between the larger conserved sequences of the A+T-rich regions, the small mt-rRNA genes, and the ND2 genes are biased in favor of transversions, 71–97% of which are AT changes. There is a 13.8 times higher frequency of nucleotide differences between the 5 halves than between the 3 halves of theD. virilis andD. yakuba small mt-rRNA genes. Considerations of the effects of observed substitutions and deletion/insertions on possible nucleotide pairing within the small mt-rRNA genes ofD. virilis andD. yakuba strongly support the secondary structure model for theDrosophila small mt-rRNA that we previously proposed.     

Comparisons of phylogenetic hypotheses among different data sets in dwarf dandelions (Krigia,Asteraceae): Additional information from internal transcribed spacer sequences of nuclear ribosomal DNA

The internal transcribed spacer (ITS) region of the 18 S–25 S nuclear ribosomal DNA repeat was sequenced from 19 populations of the tribeLactuceae, including all species of dwarf dandelion (Krigia) and five outgroup genera. The incidence of length changes and base substitutions was at least two times higher for ITS 1 than ITS 2. Interspecific sequence divergence withinKrigia averaged 9.62% (1.61%–15.19%) and 4.26% (0%–6.64%) in ITS 1 and ITS 2, respectively. Intergeneric sequence divergence ranged from 15.6% to 44.5% in ITS 1 and from 8.0% to 28.6% in ITS 2. High sequence divergence and homoplasy among genera of tribeLactuceae suggest that the phylogenetic utility of ITS sequence data is limited to interspecific studies or comparisons among closely related genera. Trees generated from ITS sequences are essentially identical to those from restriction site comparisons of the entire nuclear ribosomal (nr) DNA region. The degree of tree resolution differed depending on how gaps were treated in phylogenetic analyses. The ITS trees were congruent with the chloroplast DNA and morphological phylogenies in three major ways: 1) the sister group relationship betweenKrigia andPyrrhopappus; 2) the recognition of two monophyletic sections,Krigia andCymbia, in genusKrigia; and 3) the monophyly of theK. occidentalis-K. cespitosa clade in sect.Cymbia. However, the two nrDNA-based trees are not congruent with morphology/chloroplast DNA-based trees for the interspecific relationships in sect.Krigia. An average of 22.5% incongruence was observed among fourKrigia data sets. The relatively high degree of incongruence among data sets is due primarily to conflict between trees based on nrDNA and morphological/cpDNA data. The incongruence is probably due to the concerted evolution of nrDNA repeating units. The results fromKrigia and theLactuceae suggest that nrDNA data may have limited utility in phylogenetic studies of plants, especially in groups which exhibit high levels of sequence divergence. Our combined phylogenetic analysis as a total evidence shows the least conflict to each of the individual data sets.     

Beyond barcoding: A mitochondrial genomics approach to molecular phylogenetics and diagnostics of blowflies (Diptera: Calliphoridae)

Members of the Calliphoridae (blowflies) are significant for medical and veterinary management, due to the ability of some species to consume living flesh as larvae, and for forensic investigations due to the ability of others to develop in corpses. Due to the difficulty of accurately identifying larval blowflies to species there is a need for DNA-based diagnostics for this family, however the widely used DNA-barcoding marker, cox1, has been shown to fail for several groups within this family. Additionally, many phylogenetic relationships within the Calliphoridae are still unresolved, particularly deeper level relationships. Sequencing whole mt genomes has been demonstrated both as an effective method for identifying the most informative diagnostic markers and for resolving phylogenetic relationships. Twenty-seven complete, or nearly so, mt genomes were sequenced representing 13 species, seven genera and four calliphorid subfamilies and a member of the related family Tachinidae. PCR and sequencing primers developed for sequencing one calliphorid species could be reused to sequence related species within the same superfamily with success rates ranging from 61% to 100%, demonstrating the speed and efficiency with which an mt genome dataset can be assembled. Comparison of molecular divergences for each of the 13 protein-coding genes and 2 ribosomal RNA genes, at a range of taxonomic scales identified novel targets for developing as diagnostic markers which were 117–200% more variable than the markers which have been used previously in calliphorids. Phylogenetic analysis of whole mt genome sequences resulted in much stronger support for family and subfamily-level relationships. The Calliphoridae are polyphyletic, with the Polleninae more closely related to the Tachinidae, and the Sarcophagidae are the sister group of the remaining calliphorids. Within the Calliphoridae, there was strong support for the monophyly of the Chrysomyinae and Luciliinae and for the sister-grouping of Luciliinae with Calliphorinae. Relationships within Chrysomya were not well resolved. Whole mt genome data, supported the previously demonstrated paraphyly of Lucilia cuprina with respect to L. sericata and allowed us to conclude that it is due to hybrid introgression prior to the last common ancestor of modern sericata populations, rather than due to recent hybridisation, nuclear pseudogenes or incomplete lineage sorting.