Nuclear Protein-coding Markers Research Articles

Multi-locus phylogeny of Bryoria reveals recent diversification and unexpected diversity in section Divaricatae

AbstractIn recent years, the genus Bryoria (Parmeliaceae, Lecanoromycetes) has been the subject of considerable phylogenetic scrutiny. Here we used information on six gene regions, three nuclear protein-coding markers (Mcm7, GAPDH and Tsr1), two nuclear ribosomal markers (ITS and IGS) and a partial mitochondrial small subunit (mtSSU), to examine infrageneric relationships in the genus and to assess species delimitation in the Bryoria bicolor/B. tenuis group in section Divaricatae. For this purpose, phylogenetic analyses and several of the available algorithms for species delimitation (ASAP, GMYC single, GMYC multiple and bPTP) were employed. We also estimated divergence times for the genus using *BEAST. Our phylogenetic analyses based on the combined data set of six gene loci support the monophyly of sections Americanae, Divaricatae and Implexae, while section Bryoria is polyphyletic and groups in two clades. Species from Bryoria clade 1 are placed in an emended section Americanae. Our study reveals that section Divaricatae is young (c. 5 My) and is undergoing diversification, especially in South-East Asia and western North America. Separate phylogenetic analyses of section Divaricatae using ITS produced a topology congruent with the current species concepts. However, the remaining gene regions produced poorly resolved phylogenetic trees and the different species delimitation methods also generated highly inconsistent results, congruent with other studies that highlight the difficulty of species delimitation in groups with recent and rapid radiation. Based on our results, we describe the new species B. ahtiana sp. nov., characterized by its bicolorous, caespitose, widely divergent thallus, conspicuously thickening main stems, well-developed secondary branches, and rather sparse third-order branchlets. Another new lineage, referred to here as B. tenuis s. lat., is restricted to western North America and may represent a new species recently diverged from B. tenuis s. str., though further work is needed.

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 multilocus analysis of Epicopeiidae (Lepidoptera, Geometroidea) provides new insights into their relationships and the evolutionary history of mimicry

The family Epicopeiidae is a small group of day-flying moths, known for mimicking many different groups of butterflies and moths. So far, there still lacks a reliable phylogenetic framework of Epicopeiidae that is necessary to our understanding of the evolutionary process of their mimicry. In this study, we sequenced 94 nuclear protein-coding markers for 56 epicopeiid samples and 11 outgroups, covering all ten genera of Epicopeiidae. We used homemade PCR-generated baits to capture target sequences, which allowed us to utilize old and dried specimens that were difficult to handle by conventional PCR + Sanger sequencing. Maximum likelihood and Bayesian analyses of the newly obtained dataset (86,388 bp) at both DNA and protein levels produced identical phylogenies with strong support. The non-mimicry genus Deuveia is the sister group of other epicopeiid genera. Epicopeia and Nossa are not monophyletic, and these two genera nest together to form a clade. We also estimated divergence times of Epicopeiidae and found that their initial diversification happened in Eocene about 41 million years ago. The ancestral state reconstruction of mimicry type for this family suggested that thelast common ancestor of epicopeiid moths is non-mimetic, and the Riodinidae-mimicry type evolved first. In summary, our work provides a comprehensive and robust time-calibrated phylogeny of Epicopeiidae that provides a sound framework for revising their classification and interpreting character evolution.

Phylogeny and taxonomy of Polyozosia, Sedelnikovaea and Verseghya of the Lecanoraceae (Lecanorales, lichen-forming Ascomycota)

From the combined phylogenetic analysis of multi-locus sequence data of the Lecanoraceae including two nuclear protein-coding markers (RPB2 and RPB1), the internal transcribed spacer and a fragment of the mitochondrial small subunit, found that the originally monotypic eastern Asian genus Verseghya is positioned within the Verseghya-Lecidella-Pyrrhospora clade of the Lecanoraceae and includes one more taxon Verseghya thysanophora widely distributed in Northern Hemisphere. The genus Lecidella forming the Lecidella-Glaucomaria subclade within the same Verseghya-Lecidella-Pyrrhospora clade of the Lecanoraceae found to have tendency to be polyphyletic after including the recently described eastern Asian taxon Lecidella mandshurica into phylogenetic analysis of the Lecanoraceae. It is shown that Lecidella mandshurica was previously recorded from China sub Lecidella aff. elaeochroma. The originally monotypic eastern Asian genus Sedelnikovaea forming a monophyletic branch within the Sedelnikovaea-Lecanoropsis subclade and being in out-position to the Rhizoplaca-Protoparmeliopsis s. str. clade of the Lecanoraceae found to include three more taxa, i.e. Sedelnikovaea marginalis, S. pseudogyrophorica, and S. subdiscrepans. The Eurasian Protoparmeliopsis bolcana, and the eastern Asian P. kopachevskae, are illustrated for the first time as being positioned within the Protopameliopsis branch of the Lecanoraceae, while the South Korean ‘Protoparmeliopsis’ chejuensis found to be positioned in separate monophyletic branch from all other branches of the Rhizoplaca-Protoparmeliopsis s. l. clade of the Lecanoraceae. The genus Polyozosia A. Massal. as earlier name for the former Myriolecis branch of the Lecanoraceae is accepted as far the type species of the latter genus, i.e. P. poliophaea, found to be positioned within this branch. The Polyozosia robust monophyletic branch is positioned in the outermost position in the Rhizoplaca-Protoparmeliopsis s. str. clade of the Lecanoraceae. Position and species content of the accepted genera Glaucomaria, Lecanoropsis, Omphalodina, Polyozosia, and Straminella are discussed in separate nrITS and mtSSU, and combined phylogeny based on concatenated sequences of nrITS, mtSSU, RPB2 and RPB1 genes. Fourty new combinations are proposed: Glaucomaria bicincta, G. carpinea, G. leptyrodes, G. lojkaeana, G. subcarpinea, G. sulphurea, G. swartzii, G. swartzii subsp. caulescens, G. swartzii subsp. nylanderi, Lecanoropsis anopta, L. macleanii, Omphalodina chrysoleuca, O. huashanensis, O. opiniconensis, O. phaedrophthalma, O. pseudistera, Palicella anakeestiicola, Polyozosia albescens, P. andrewii, P. contractula, P. crenulata, P. dispersa, P. hagenii, P. perpruinosa, P. populicola, P. pruinosa, P. reuteri, P. sambuci, P. semipallida, P. straminea, P. thuleana, Sedelnikovaea marginalis, S. pseudogyrophorica, S. subdiscrepans, Straminella bullata, S. burgaziae, S. conizaeoides, S. densa, S. maheui, S. varia, and Verseghya thysanophora. Validation of one name as Polyozosia perpruinosa Fröberg ex S. Y. Kondr. L. Lőkös et Farkas is also proposed.

Highly Resolved Phylogenetic Relationships within Order Acipenseriformes According to Novel Nuclear Markers.

Order Acipenseriformes contains 27 extant species distributed across the northern hemisphere, including so-called “living fossil” species of garfish and sturgeons. Previous studies have focused on their mitochondrial genetics and have rarely used nuclear genetic data, leaving questions as to their phylogenetic relationships. This study aimed to utilize a bioinformatics approach to screen for candidate single-copy nuclear genes, using transcriptomic data from sturgeon species and genomic data from the spotted gar, Lepisosteus oculatus. We utilized nested polymerase chain reaction (PCR) and degenerate primers to identify nuclear protein-coding (NPC) gene markers to determine phylogenetic relationships among the Acipenseriformes. We identified 193 nuclear single-copy genes, selected from 1850 candidate genes with at least one exon larger than 700 bp. Forty-three of these genes were used for primer design and development of 30 NPC markers, which were sequenced for at least 14 Acipenseriformes species. Twenty-seven NPC markers were found completely in 16 species. Gene trees according to Bayesian inference (BI) and maximum likelihood (ML) were calculated based on the 30 NPC markers (20,946 bp total). Both gene and species trees produced very similar topologies. A molecular clock model estimated the divergence time between sturgeon and paddlefish at 204.1 Mya, approximately 10% later than previous estimates based on cytochrome b data (184.4 Mya). The successful development and application of NPC markers provides a new perspective and insight for the phylogenetic relationships of Acipenseriformes. Furthermore, the newly developed nuclear markers may be useful in further studies on the conservation, evolution, and genomic biology of this group.

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.

Who’s getting around? Assessing species diversity and phylogeography in the widely distributed lichen-forming fungal genus Montanelia (Parmeliaceae, Ascomycota)

Brown parmelioid lichens comprise a number of distinct genera in one of the most species-rich families of lichen-forming fungi, Parmeliaceae (Ascomycota). In spite of their superficial similarity, a number of studies of brown parmelioids have provided important insight into diversification in lichen-forming fungi with cosmopolitan distributions. In this study we assess species diversity, biogeography and diversification of the genus Montanelia, which includes alpine to temperate saxicolous species. We sampled each of the five known species, four of which are known from broad, intercontinental distributions. In order to identify potential biogeographical patterns, each broadly distributed species was represented by individuals collected across their intercontinental distributions. Molecular sequence data were generated for six loci, including three nuclear protein-coding markers (MCM7, RPB1, and RPB2), two nuclear ribosomal markers (ITS and nrLSU), and a fragment of the mitochondrial small subunit. We used three sequence-based species delimitations methods to validate traditional, phenotype-based species and circumscribe previously unrecognized species-level lineages in Montanelia. Relationships among putative lineages and divergence times were estimated within a coalescent-based multi-locus species tree framework. Based on the results of the species delimitation analyses, we propose that the genus Montanelia is likely comprised of six to nine species-level lineages, including previously unrecognized species-level diversity in the nominal taxa M. panniformis and M. tominii. In contrast, molecular sequence data suggest that M. predisjuncta may be conspecific with the widespread taxon M. disjuncta in spite of distinct morphological differences. The rate-based age estimation of the most recent common ancestor of Montanelia (ca. 23.1Ma) was similar to previous estimates based on the fossil record. Furthermore, our data suggest that diversification in Montanelia occurred largely during the Neogene. At least three Montanelia species are broadly distributed throughout Asia, Europe, and North America with no evidence of phylogeographic substructure. In contrast to broadly distributed Montanelia species, our study suggests Pleistocene-dominated diversification and complex biogeographic history in the M. tominii group. Our analyses provide additional insight for understanding diversification and uncovering cryptic diversity in cosmopolitan species of lichen-forming fungi.

Phylogeny of the shore crab family Grapsidae (Decapoda: Brachyura: Thoracotremata) based on a multilocus approach

The Grapsidae are a thoracotreme crab family with 40 species in eight genera (in their strict definition), and possess a number of morphological and molecular synapomorphies. Previous phylogenetic studies based on mitochondrial DNA markers established the monophyly of this family, but suggested possible paraphyly or polyphyly of some of the constituent genera. To test the validity of previous hypotheses, the present study reconstructed a molecular phylogeny of the grapsid crabs based on five molecular markers, including mitochondrial DNA markers and the first use of nuclear protein-coding markers to address this issue. Monophyly of Grapsidae was confirmed, with the exception of the position of the monotypic genus Leptograpsodes. The polyphyly of the genus Pachygrapsus is consistent with previous molecular phylogenies, as members from this genus are dispersed throughout our gene tree. Grapsus and Planes were shown to be paraphyletic, with species of Pachygrapsus nested within them. Our study found incongruences between the currently adopted classification of the family, and hence taxonomic revisions will be needed. We hereby demonstrate the use of nuclear protein-coding markers for high confidence reconstruction of decapod phylogenies, resolving most of the early splits that mitochondrial DNA markers alone are unable to tackle.

Speciation history and widespread introgression in the European short-call tree frogs (Hyla arborea sensu lato, H. intermedia and H. sarda)

European tree frogs (Hyla) characterized by short temporal parameters of the advertisement call form six genetically differentiated but morphologically cryptic taxa, H. arborea sensu stricto, H. orientalis and H. molleri from across Europe to western Asia (together referred to as H. arborea sensu lato), two putative taxa within H. intermedia (Northern and Southern) from the Italian Peninsula and Sicily, and H. sarda from Sardinia and Corsica. Here, we assess species limits and phylogenetic relationships within these ‘short-call tree frogs’ based on mitochondrial DNA and nuclear protein-coding markers. The mitochondrial and nuclear genes show partly incongruent phylogeographic patterns, which point to a complex history of gene flow across taxa, particularly in the Balkans. To test the species limits in the short-call tree frogs and to infer the species tree, we used coalescent-based approaches. The monophyly of H. arborea sensu lato is supported by the mtDNA as well as by the all-gene species tree. The Northern and Southern lineages of H. intermedia have been connected by nuclear gene flow (despite their deep mtDNA divergence) and should be treated as conspecific. On the contrary, the parapatric taxa within H. arborea sensu lato should be considered distinct species (H. arborea, H. orientalis, H. molleri) based on the coalescent analysis, although signs of hybridizationwere detected between them (H. arborea×H. orientalis; H. arborea×H. molleri). A mitochondrial capture upon secondary contact appears to explain the close mtDNA relationship between the geographically remote Iberian H. molleri and H. orientalis from around the Black Sea. Introgressive hybridization occurred also between the Balkan H. arborea and northern Italian H. intermedia, and between the Minor Asiatic H. orientalis and Arabian H. felixarabica (the latter belonging to a different acoustic group/clade). Our results shed light on the species limits in the European short-call tree frogs and show that introgression played an important role in the evolutionary history of the short-call tree frogs and occurred even between taxa supported as distinct species.

Identification of 104 rapidly-evolving nuclear protein-coding markers for amplification across scaled reptiles using genomic resources

As the fields of molecular systematics and phylogeography are advancing, it is necessary to incorporate multiple loci in both population and species-level inference. Here, we present primer sets for 104 intronless orthologus exons designed for amplification in all squamates. When comparing the Anolis genome to the Gallus genome, all the markers have less than 67.2% DNA sequence identity, the percent identity of the first third of the commonly used nuclear marker RAG-1. The rate of evolution in these markers is therefore greater than nuclear markers commonly used, and we demonstrate their usefulness for both phylogeographic and phylogenetic studies.