Nuclear Protein-coding Loci Research Articles

Back together: Over 1000 single-copy nuclear loci and reproductive features support the holoendoparasitic Apodanthaceae and Rafflesiaceae as sister lineages in the order Malpighiales

The systematics of the holoendoparasitic flowering plant families Apodanthaceae and Rafflesiaceae has been discussed for over two centuries. The morphological reduction of roots, shoots and leaves in all members of both families, resulting in a cryptic mycelium-like vegetative body, has been interpreted either as a key common feature, or as a result of convergent evolution due to full dependence upon their hosts. Historically, the two families have been placed together due to similar morphological features, but recent analyses based on few mitochondrial and ribosomal gene markers placed them in the distantly related orders Cucurbitales and Malpighiales. Here we reevaluate the affinities of the Apodanthaceae and the Rafflesiaceae using a phylogenomic approach. We present (1) a historical account on their affinities over the last 200 years; (2) phylogenetic analyses reinstating their sister group relationship as part of the order Malpighiales, based on over 1000 single-copy nuclear protein-coding loci; and (3) a comprehensive list of putative morphoanatomical and developmental synapomorphies in light of the phylogenomic results, with emphasis on shared reproductive traits regardless of dramatic differences in floral size.

Phylogenomics of the major lineages of Bembidion and related ground beetles (Coleoptera: Carabidae: Bembidiini)

Abstract Bembidion Latreille (Coleoptera: Carabidae) is a genus of small ground beetles containing about 1,380 species. To test previous phylogenetic hypotheses about deeper lineages of Bembidion and near relatives, we recover and examine over 1,800 nuclear protein-coding loci from 33 species representing the main lineages of Bembidion, 10 species of other bembidiine genera, and 7 outgroups. We find that Bembidion exclusive of subgenus Phyla Motschulsky is monophyletic, and we reclassify Phyla as a separate genus. Within Bembidion we find 2 dominant clades, the Bembidion superseries (containing about 490 species in the subgenera Eupetedromus Netolitzky and Lindrochthus Maddison, the Philochthus Stephens complex, and the Bembidion series), and the Ocydromus Clairville superseries (containing almost all other Bembidion representing about 840 species). The only known lineages within Bembidion outside of these superseries are subgenus Hoquedela Müller-Motzfeld and the Desarmatocillenus Netolitzky complex, which combined contain less than 30 species. Most clades are insensitive to variations in analyses and hold up under different sets of taxa and loci, analyses at the nucleotide or amino acid levels, and different analytical methods (maximum likelihood, including posterior mean site frequency analyses, Bayesian analyses, invariant-based methods, and those that consider incomplete lineage sorting). Despite the clarity achieved in most aspects of the phylogeny, there are several unresolved regions, notably the relationships of Desarmatocillenus, Hoquedela, and Phyla to other bembidiines. A divergence dating analysis suggests that crown Bembidion is about 48 million years old (95% confidence intervals 40–58 Ma), and that the 2 large superseries are about 38 million years old (95% confidence intervals about 29–47 Ma).

Emergence of multiple Diaporthe species causing kiwifruit rot and occurrence of resistance to a methyl benzimidazole carbamate fungicide in South Korea

In recent years, new fungal diseases in kiwifruits have emerged worldwide. This study is the first report of a postharvest kiwifruit rot in South Korea caused by Diaporthe eres and related fungal pathogens. After single spore isolation of Diaporthe-like fungi isolated from rotten kiwifruits, isolates were identified based on the multi-locus phylogeny using the nuclear rDNA internal transcribed spacer and three nuclear protein-coding loci. Among the 41 single-spore isolates, a majority of the isolates (75%) were identified as D. eres, followed by D. unshiuensis, D. phaseolorum, D. cf. pseudomangiferae and D. caryae. We evaluated fungicide resistance of the 41 isolates and confirmed that a significant fraction of the isolates (15%) was resistant to thiophanate-methyl, raising a concern on overuse of this type of fungicide in kiwi orchards. By sequencing a region of β-tubulin gene, we identified two point mutations altering amino acid sequences correlated with the observed resistance. For effective diagnosis of incidence of the fungicide resistance in the fields, we developed allele-specific PCR analyses detecting the point mutations that confer reduced sensitivity to thiophanate-methyl. Genetic structure of the 41 isolates suggested that there were at least five independent mutational events for acquiring the fungicide resistance. Unexpectedly, there was no clear distinction in the genetic structure between the 41 kiwifruit isolates and Chinese isolates that caused grapevine dieback disease, suggesting these Diaporthe species are non-host-selective pathogens that could damage different horticultural crops of economic importance. This study will help develop a discerned protection program to cope with the ongoing and upcoming threats of these genetically diverse kiwifruit rot pathogens.

Molecular phylogeny of the genus Lasiopogon (Diptera: Asilidae) and a taxonomic revision of the bivittatus section.

Nearctic species of Lasiopogon Loew comprising the bivittatus section (the bivittatus group sensu Cannings 2002) are revised, with the description of 13 new species, elevation of one subspecies to species, and redescriptions of 13 previously described taxa. An updated key to western Nearctic Lasiopogon adults is provided, as are notes on taxonomy, distribution, phylogeny, and ecology. A Bayesian species tree for 67 species of Lasiopogon is estimated from one mitochondrial (COI) and three nuclear protein-coding loci (AATS, PEPCK, Wg), and compared to a previously published morphology-based phylogeny. The following new species of Lasiopogon are described (assigned to the bivittatus section except as noted): L. anaphlecter sp. nov., L. apoecus sp. nov., L. asilomar sp. nov., L. bitumineus sp. nov., L. canningsi sp. nov., L. condylophorus sp. nov., L. esau sp. nov., L. karli sp. nov. (assigned to cinereus group of opaculus section), L. nelsoni sp. nov., L. odontotus sp. nov., L. sierra sp. nov., L. tumulicola sp. nov., L. wilcoxi sp. nov.; L. puyallupi Cole Wilcox 1938 stat. nov. is elevated from subspecies; and the following previously described species are considered valid: L. actius Melander 1923, L. albidus Cole Wilcox 1938, L. arenicola (Osten Sacken 1877), L. bivittatus Loew 1866, L. californicus Cole Wilcox 1938, L. dimicki Cole Wilcox 1938, L. drabicolum Cole 1916, L. gabrieli Cole Wilcox 1938, L. littoris Cole 1924, L. ripicola Melander 1923, L. willametti Cole Wilcox 1938, L. zonatus Cole Wilcox 1938. The species L. martinensis Cole Wilcox 1938 is considered valid but transferred to the tetragrammus group of the opaculus section.

Unioverse: A phylogenomic resource for reconstructing the evolution of freshwater mussels (Bivalvia, Unionoida)

Freshwater mussels (order Unionoida) are a diverse radiation of parasitic bivalves that require temporary larval encystment on vertebrate hosts to complete metamorphosis to free-living juveniles. The freshwater mussel-fish symbiosis represents a useful relationship for understanding eco-evolutionary dynamics in freshwater ecosystems but the practicality of this promising model system is undermined by the absence of a stable freshwater mussel phylogeny. Inadequate character sampling is the primary analytical impediment obfuscating a coherent phylogeny of freshwater mussels, specifically the lack of nuclear molecular markers appropriate for reconstructing supraspecific relationships and testing macroevolutionary hypotheses. The objective of this study is to develop a phylogenomic resource, specifically an anchored hybrid enrichment probe set, capable of capturing hundreds of molecular markers from taxa distributed across the entirety of freshwater mussel biodiversity. Our freshwater mussel specific anchored hybrid enrichment probe set, called Unioverse, successfully captures hundreds of nuclear protein-coding loci from all major lineages of the Unionoida and will facilitate more data-rich and taxonomically inclusive reconstructions of freshwater mussel evolution. We demonstrate the utility of this resource at three disparate evolutionary scales by estimating a backbone phylogeny of the Bivalvia with a focus on the Unionoida, reconstructing the subfamily-level relationships of the Unionidae, and recovering the systematic position of the phylogenetically unstable genus Plectomerus.

The first set of universal nuclear protein-coding loci markers for avian phylogenetic and population genetic studies

Multiple nuclear markers provide genetic polymorphism data for molecular systematics and population genetic studies. They are especially required for the coalescent-based analyses that can be used to accurately estimate species trees and infer population demographic histories. However, in avian evolutionary studies, these powerful coalescent-based methods are hindered by the lack of a sufficient number of markers. In this study, we designed PCR primers to amplify 136 nuclear protein-coding loci (NPCLs) by scanning the published Red Junglefowl (Gallus gallus) and Zebra Finch (Taeniopygia guttata) genomes. To test their utility, we amplified these loci in 41 bird species representing 23 Aves orders. The sixty-three best-performing NPCLs, based on high PCR success rates, were selected which had various mutation rates and were evenly distributed across 17 avian autosomal chromosomes and the Z chromosome. To test phylogenetic resolving power of these markers, we conducted a Neoavian phylogenies analysis using 63 concatenated NPCL markers derived from 48 whole genomes of birds. The resulting phylogenetic topology, to a large extent, is congruence with results resolved by previous whole genome data. To test the level of intraspecific polymorphism in these makers, we examined the genetic diversity in four populations of the Kentish Plover (Charadrius alexandrinus) at 17 of NPCL markers chosen at random. Our results showed that these NPCL markers exhibited a level of polymorphism comparable with mitochondrial loci. Therefore, this set of pan-avian nuclear protein-coding loci has great potential to facilitate studies in avian phylogenetics and population genetics.

Description and phylogenetic classification of Stackelberginia cerberus sp. nov. (Diptera: Asilidae), comprising the first record of this genus from the Nearctic

Stackelberginia cerberus sp. nov. (Diptera: Asilidae) is described from the Amargosa desert (USA: Nevada) and compared to related taxa. This is the first record of the genus in the Western Hemisphere; other species live in the deserts of central Asia. Stackelberginia Lehr is proposed as the sister taxon to Lasiopogon Loew in the subfamily Stichopogoninae based on morphological characters and a Bayesian species tree estimated from one mitochondrial (COI) and three nuclear protein-coding loci (AATS, PEPCK, wingless). Stackelberginia has the medially divided epandrium and rotated hypopygium of Lasiopogon, but the facial gibbosity is flat, macrosetae of thorax, head, and legs are unusually long, and phenology peaks in late autumn.

Ordinal-level phylogenomics of the arthropod class Diplopoda (millipedes) based on an analysis of 221 nuclear protein-coding loci generated using next-generation sequence analyses.

BackgroundThe ancient and diverse, yet understudied arthropod class Diplopoda, the millipedes, has a muddled taxonomic history. Despite having a cosmopolitan distribution and a number of unique and interesting characteristics, the group has received relatively little attention; interest in millipede systematics is low compared to taxa of comparable diversity. The existing classification of the group comprises 16 orders. Past attempts to reconstruct millipede phylogenies have suffered from a paucity of characters and included too few taxa to confidently resolve relationships and make formal nomenclatural changes. Herein, we reconstruct an ordinal-level phylogeny for the class Diplopoda using the largest character set ever assembled for the group.MethodsTranscriptomic sequences were obtained from exemplar taxa representing much of the diversity of millipede orders using second-generation (i.e., next-generation or high-throughput) sequencing. These data were subject to rigorous orthology selection and phylogenetic dataset optimization and then used to reconstruct phylogenies employing Bayesian inference and maximum likelihood optimality criteria. Ancestral reconstructions of sperm transfer appendage development (gonopods), presence of lateral defense secretion pores (ozopores), and presence of spinnerets were considered. The timings of major millipede lineage divergence points were estimated.ResultsThe resulting phylogeny differed from the existing classifications in a number of fundamental ways. Our phylogeny includes a grouping that has never been described (Juliformia+Merocheta+Stemmiulida), and the ancestral reconstructions suggest caution with respect to using spinnerets as a unifying characteristic for the Nematophora. Our results are shown to have significantly stronger support than previous hypotheses given our data. Our efforts represent the first step toward obtaining a well-supported and robust phylogeny of the Diplopoda that can be used to answer many questions concerning the evolution of this ancient and diverse animal group.

Mitochondrial genome primers for Lake Malawi cichlids

Resolving the evolutionary history of rapidly diversifying lineages like the Lake Malawi Cichlid Flock demands powerful phylogenetic tools. Although this clade of over 500 species of fish likely diversified in less than two million years, the availability of extensive sequence data sets, such as complete mitochondrial genomes, could help resolve evolutionary patterns in this group. Using a large number of newly developed primers, we generated whole mitochondrial genome sequences for 14 Lake Malawi cichlids. We compared sequence divergence across protein-coding regions of the mitochondrial genome and also compared divergence in the mitochondrial loci to divergence at two nuclear protein-coding loci, Mitfb and Dlx2. Despite the widespread sharing of haplotypes of identical sequences at individual loci, the combined use of all protein-coding mitochondrial loci provided a bifurcating phylogenetic hypothesis for the exemplars of major lineages within the Lake Malawi cichlid radiation. The primers presented here could have substantial utility for evolutionary analyses of mitochondrial evolution and hybridization within this diverse clade.

Eight independent nuclear genes support monophyly of the plovers: The role of mutational variance in gene trees

Molecular phylogenies of Charadriiformes based on mtDNA genes and one to three nuclear loci do not support the traditional placement of Pluvialis in the plovers (Charadriidae), assigning it instead to oystercatchers, stilts, and avocets (Haematopodidae and Recurvirostridae). To investigate this hypothesis of plover paraphyly, the relationships among Pluvialis and closely related families were revisited by sequencing two individuals of all taxa except Peltohyas for eight independent single copy nuclear protein-coding loci selected for their informativeness at this phylogenetic depth. The species tree estimated jointly with the gene trees in the coalescent programme *BEAST strongly supported plover monophyly, as did Bayesian analysis of the concatenated matrix. The data sets that supported plover paraphyly in Baker et al. (2007) and Fain and Houde (2007) reflect two to four independent gene histories, and thus discordance with the plover monophyly species tree might have arisen by chance through stochastic mutational variance. For the plovers we conclude there is no conclusive evidence of coalescent variance from ancient incomplete lineage sorting across the interior branch leading to Pluvialis in the species tree. Rather, earlier studies seem have been misled by faster evolving mtDNA genes with high mutational variance, and a few nuclear genes that had low resolving power at the Pluvialis sister group level. These findings are of general relevance in avian phylogenetics, as they show that careful attention needs to be paid to the number and the phylogenetic informativeness of genes required to obtain accurate estimates of the species tree, especially where there is mutational heterogeneity in gene trees.

Lizards from the end of the world: Phylogenetic relationships of the Liolaemus lineomaculatus section (Squamata: Iguania: Liolaemini)

The Liolaemus lineomaculatus section is a geographically widely distributed group of lizards from the Patagonian region of southern South America, and includes 18 described species representing the most southerly distributed Liolaemus taxa (the genus includes 228 species and extends from Tierra del Fuego north to south-central Peru). Despite high species diversity, the phylogenetic relationships of this section are unknown. In the present work we sampled all described species in the L. lineomaculatus section as well as currently undescribed candidate species to reconstruct the first complete phylogenetic hypothesis for the clade. Our data set included four anonymous nuclear loci, three nuclear protein-coding loci, and two mitochondrial genes. We compared results obtained with three different phylogenetic methods for the concatenated data set (Maximum Parsimony, Maximum Likelihood and Bayesian Inference) with a coalescent-based species tree approach (BEST), and recovered congruent, strongly-supported topological arrangements across all methods. We identified four main clades within the L. lineomaculatus section: the lineomaculatus, magellanicus, somuncurae, and kingii + archeforus groups, for which we estimated divergence times. We discuss the taxonomic implications of these results and how the future integration of phylogeographic, niche modeling and morphological approaches will allow testing biogeographical hypotheses in this clade.

Slow worm, Anguis fragilis (Reptilia: Anguidae) as a species complex: Genetic structure reveals deep divergences

Phylogenetic relationships of the Western Palearctic legless lizard genus Anguis were inferred based on a fragment of mitochondrial DNA and two nuclear protein-coding loci, C-mos and PRLR. A. cephallonica from the Peloponnese was confirmed as a valid species. It is the sister taxon to a clade comprising all other evolutionary lineages, which were shown to represent three distinct species: (1) A. fragilis sensu stricto occurring in Western and Central Europe, the north-western Balkans, with possibly isolated populations in the eastern Balkans, and presumably also in western Scandinavia and Italy; (2) A. colchica distributed from the eastern Czech Republic and the Baltic region eastward to northern Iran, presumably also in eastern Scandinavia, and the north-eastern Balkans; (3) A. graeca restricted to the southern Balkans, and partially sympatric with A. cephallonica. According to the more variable mitochondrial marker, A. graeca appears to be the sister species to A. colchica, and these taxa together form a sister clade to A. fragilis, whereas the less variable nuclear markers show A. colchica to be closer to A. fragilis. The C-mos gene has not provided substantial variation within this species complex, while the PRLR gene, which was used for the first time in phylogeographic study in a reptile, distinguished all species successfully. Intra-specific differentiation of A. colchica is discussed, and subspecific status of the Caucasian and Caspian populations is proposed. The uncovered genetic differences should be taken into account in all future biogeographical, morphological and ecological studies, as well as in conservation.

Rapid development of multiple nuclear loci for phylogenetic analysis using genomic resources: An example from squamate reptiles

Recently, as genome-scale data have become available for more organisms, the development of phylogenetic markers from nuclear protein-coding loci (NPCL) has become more tractable. However, new methods are needed to efficiently sort the large number of genes from genomic databases into more limited sets appropriate for particular phylogenetic questions, while avoiding introns and paralogs. Here we describe a general methodology for identifying candidate single-copy NPCL from genomic databases. Our method uses information from reference genomes to identify genes with relatively large continuous protein-coding regions (i.e., ⩾700 bp). BLAST comparisons are used to help avoid genes with paralogous copies or close relatives (i.e., gene families) that might confound phylogenetic analyses. Exon boundary information is used to identify appropriately spaced potential priming sites. Using this method, we have developed over 25 novel NPCL, which span a variety of desirable evolutionary rates for phylogenetic analyses. Although targeted for higher-level phylogenetics of squamate reptiles, many of these loci appear to be useful across and within other vertebrate clades (e.g., amphibians), and some are relatively rapidly evolving and may be useful for closely-related species (e.g., within genera). This general method can be used whenever large-scale genomic data are available for an appropriate reference species (not necessarily within the focal clade). The method is also well suited for the development of intron regions for lower-level phylogenetic and phylogeographic studies. We provide an online database of alignments and suggested primers for approximately 85 NPCL that should be useful across vertebrates.

Very large long-term effective population size in the virulent human malaria parasite Plasmodium falciparum.

It has been proposed that the virulent human malaria parasite Plasmodium falciparum underwent a recent severe population bottleneck. In order to test this hypothesis, we estimated the effective population size of this species from the patterns of nucleotide substitution at 23 nuclear protein-coding loci, using a variety of methods based on coalescent theory. Both simple methods and phylogenetically based maximum-likelihood methods yielded the conclusion that the effective population size of this species has been of the order of at least 10(5) for the past 300,000-400,000 years.