Low-copy Nuclear Loci Research Articles

Systematics of Libinhania, a new endemic genus of Gnaphalieae (Asteraceae) from the Socotra archipelago (Yemen), inferred from plastid, low-copy nuclear and nuclear ribosomal DNA loci

The Socotra archipelago off the Horn of Africa shelters morphologically diverse endemic species of Asteraceae tribe Gnaphalieae, eight species placed in Helichrysum and five newly discovered. We reconstructed their phylogenetic relationships, analysing nuclear ribosomal DNA (nrDNA) internal transcribed spacer and external transcribed spacer, three plastid DNA and two low-copy nuclear DNA loci with Bayesian inference and maximum parsimony. The plastid DNA and nrDNA trees resolve the endemic helichrysoids as a monophyletic group and agree in that they are not members of Helichrysum or even part of the Helichrysum-Anaphalis-Pseudognaphalium clade, but disagree as to their sister group. In the nrDNA tree, they are nested in the Filago-Leontopodium-Antennaria-Gamochaeta (FLAG) clade, whereas in the plastid DNA tree they are sister to Syncarpha mucronata. The endemic helichrysoids possess two divergent copies of each of the nDNA low-copy markers, similar to those previously found for the FLAG clade. This suggests an ancient allopolyploid origin, with the putative ancestors among the ancestors of Gnaphalium or relatives and of S. mucronata or relatives. The reconstruction of ancestral states of morphological characters suggested hypotheses on the origin and evolution of the morphological diversity of the endemic helichrysoids. We conclude a taxonomic treatment with a key to, and descriptions for, the 13 species accommodated in a new genus, Libinhania.

An expanded nuclear phylogenomic PCR toolkit for Sapindales.

Premise of the study:We tested PCR amplification of 91 low-copy nuclear gene loci in taxa from Sapindales using primers developed for Bursera simaruba (Burseraceae).Methods and Results:Cross-amplification of these markers among 10 taxa tested was related to their phylogenetic distance from B. simaruba. On average, each Sapindalean taxon yielded product for 53 gene regions (range: 16–90). Arabidopsis thaliana (Brassicales), by contrast, yielded product for two. Single representatives of Anacardiaceae and Rutacaeae yielded 34 and 26 products, respectively. Twenty-six primer pairs worked for all Burseraceae species tested if highly divergent Aucoumea klaineana is excluded, and eight of these amplified product in every Sapindalean taxon.Conclusions:Our study demonstrates that customized primers for Bursera can amplify product in a range of Sapindalean taxa. This collection of primer pairs, therefore, is a valuable addition to the toolkit for nuclear phylogenomic analyses of Sapindales and warrants further investigation.

Evolutionary relationships within the lamioid tribe Synandreae (Lamiaceae) based on multiple low-copy nuclear loci.

The subfamily Lamioideae (Lamiaceae) comprises ten tribes, of which only Stachydeae and Synandreae include New World members. Previous studies have investigated the phylogenetic relationships among the members of Synandreae based on plastid and nuclear ribosomal DNA loci. In an effort to re-examine the phylogenetic relationships within Synandreae, the current study incorporates data from four low-copy nuclear loci, PHOT1, PHOT2, COR, and PPR. Our results confirm previous studies based on chloroplast and nuclear ribosomal markers in supporting the monophyly of tribe Synandreae, as well as sister relationships between Brazoria and Warnockia, and between that pair of genera and a monophyletic Physostegia. However, we observe incongruence in the relationships of Macbridea and Synandra. The placement of Synandreae within Lamioideae is poorly resolved and incongruent among different analyses, and the sister group of Synandreae remains enigmatic. Comparison of the colonization and migration patterns corroborates a single colonization of the New World by Synandreae during the Late Miocene/Tortonian age. This is in contrast to the only other lamioid tribe that includes New World members, Stachydeae, which colonized the New World at least twice—during the mid-Miocene and Pliocene. Edaphic conditions and intolerance of soil acidity may be factors that restricted the distribution of most genera of Synandreae to southeastern and south–central North America, whereas polyploidy could have increased the colonizing capability of the more wide-ranging genus, Physostegia.

The Origin of Oranges: A Multi-locus Phylogeny of Rutaceae Subfamily Aurantioideae

Abstract The phylogeny of Rutaceae subfamily Aurantioideae has previously been estimated only using plastid and repetitive nuclear sequences. We added sequences of two low copy nuclear loci to allow further diagnosis of phenomena that may mislead phylogenetic inference. After testing for patterns expected under recombination, positive selection, and hybridization, we excluded data sets or sequences accordingly and then inferred the species tree using the multispecies coalescent. We then reconstructed the ancestral area using parsimony and the dispersal-extinction-cladogenesis model to test the hypothesis that Citrus s. l. may have originated in Australasia and migrated or rafted to Eastern Asia. The ancestral area of Citrus s. l. inferred under either method and several models was west of Wallace's line. Therefore, Citrus s. l. did not appear to have rafted west on what became the Halmahera Islands (Indonesia). Our findings are also consistent with previously reported ages for the origin of this group tha...

Phylogenetic reconstruction using four low-copy nuclear loci strongly supports a polyphyletic origin of the genus Sorghum.

Sorghum is an essential grain crop whose evolutionary placement within the Andropogoneae has been the subject of scrutiny for decades. Early studies using cytogenetic and morphological data point to a poly- or paraphyletic origin of the genus; however, acceptance of poly- or paraphyly has been met with resistance. This study aimed to address the species relationships within Sorghum, in addition to the placement of Sorghum within the tribe, using a phylogenetic approach and employing broad taxon sampling. From 16 diverse Sorghum species, eight low-copy nuclear loci were sequenced that are known to play a role in morphological diversity and have been previously used to study evolutionary relationships in grasses. Further, the data for four of these loci were combined with those from 57 members of the Andropogoneae in order to determine the placement of Sorghum within the tribe. Both maximum likelihood and Bayesian analyses were performed on multilocus concatenated data matrices. The Sorghum-specific topology provides strong support for two major lineages, in alignment with earlier studies employing chloroplast and internal transcribed spacer (ITS) markers. Clade I is composed of the Eu-, Chaeto- and Heterosorghum, while clade II contains the Stipo- and Parasorghum. When combined with data from the Andropogoneae, Clade II resolves as sister to a clade containing Miscanthus and Saccharum with high posterior probability and bootstrap support, and to the exclusion of Clade I. The results provide compelling evidence for a two-lineage polyphyletic ancestry of Sorghum within the larger Andropogoneae, i.e. the derivation of the two major Sorghum clades from a unique common ancestor. Rejection of monophyly in previous molecular studies is probably due to limited taxon sampling outside of the genus. The clade consisting of Para- and Stiposorghum resolves as sister to Miscanthus and Saccharum with strong node support.

Untangling reticulate evolutionary relationships among New World and Hawaiian mints (Stachydeae, Lamiaceae)

The phenomenon of polyploidy and hybridization usually results in novel genetic combinations, leading to complex, reticulate evolution and incongruence among gene trees, which in turn may show different phylogenetic histories than the inherent species tree. The largest tribe within the subfamily Lamioideae (Lamiaceae), Stachydeae, which includes the globally distributed Stachys, and one of the largest Hawaiian angiosperm radiations, the endemic mints, is a widespread and taxonomically challenging lineage displaying a wide spectrum of morphological and chromosomal diversity. Previous molecular phylogenetic studies have showed that while the Hawaiian mints group with Mexican–South American Stachys based on chloroplast DNA sequence data, nuclear ribosomal DNA (nrDNA) sequences suggest that they are most closely related to temperate North American Stachys. Here, we have utilized five independently inherited, low-copy nuclear loci, and a variety of phylogenetic methods, including multi-locus coalescence-based tree reconstructions, to provide insight into the complex origins and evolutionary relationships between the New World Stachys and the Hawaiian mints. Our results demonstrate incongruence between individual gene trees, grouping the Hawaiian mints with both temperate North American and Meso-South American Stachys clades. However, our multi-locus coalescence tree is concurrent with previous nrDNA results placing them within the temperate North American Stachys clade. Our results point toward a possible allopolyploid hybrid origin of the Hawaiian mints arising from temperate North American and Meso-South American ancestors, as well as a reticulate origin for South American Stachys. As such, our study is another significant step toward further understanding the putative parentage and the potential influence of hybridization and incomplete lineage sorting in giving rise to this insular plant lineage, which following colonization underwent rapid morphological and ecological diversification.

Phylogenetic analysis of Saccharum s.l. (Poaceae; Andropogoneae), with emphasis on the circumscription of the South American species.

Polyploidy and reticulate evolution are often a complication for discovering phylogenetic relationships between genera and species. Despite the huge economic importance of sugarcane (Saccharum officinarum-Poaceae, Andropogoneae), the limits of the genus Saccharum and its species are complex and largely unresolved, involving both polyploidy and reticulate evolution. This study aimed to assess the phylogenetic relationships of Saccharum s.l., including Erianthus and Tripidium, as well as investigate the taxonomic circumscription of the South American species of the genus. Molecular cloning and sequencing of five regions of four low-copy nuclear loci were performed, including Aberrant panicle organization1 (apo1), Dwarf8 (d8), two exons of Erect panicle2 (ep2-ex7 and ep2-ex8), and Retarded palea1 (rep1). Concatenated trees were reconstructed using Maximum Parsimony, Maximum Likelihood, and Bayesian Inference analyses. The allopolyploid origin of Saccharum was demonstrated using evidence from nuclear genes. The samples of Saccharum s.l. grouped in two distinct clades, with S. arundinaceum and S. ravennae (= Tripidium, or Erianthus sect. Ripidium) apart from all other species analyzed of the genus. Saccharum angustifolium, S. asperum, and S. villosum correspond to distinct clades (different species). The plants with intermediate morphology between S. angustifolium and S. villosum presented a pattern of paralogues consistent with a hybrid origin. Saccharum s.l. is polyphyletic and Tripidium should be recognized as a distinct genus. However, no strong evidence was found to support the segregation of Erianthus. The taxonomic circumscription of the South American species of the genus was resolved and the occurrence of natural hybrids was documented. Better understanding of the phylogenetic relationships of Saccharum and relatives may be useful for sugarcane breeders to identify potential taxa for interspecific and intergeneric crosses in the genetic improvement of sugarcane.

Phylogeny of Campanuloideae (Campanulaceae) with Emphasis on the Utility of Nuclear Pentatricopeptide Repeat (PPR) Genes

BackgroundThe Campanuloideae (Campanulaceae) are a highly diverse clade of angiosperms found mostly in the Northern Hemisphere, with the highest diversity in temperate areas of the Old World. Chloroplast markers have greatly improved our understanding of this clade but many relationships remain unclear primarily due to low levels of molecular evolution and recent and rapid divergence. Furthermore, focusing solely on maternally inherited markers such as those from the chloroplast genome may obscure processes such as hybridization. In this study we explore the phylogenetic utility of two low-copy nuclear loci from the pentatricopeptide repeat gene family (PPR). Rapidly evolving nuclear loci may provide increased phylogenetic resolution in clades containing recently diverged or closely related taxa. We present results based on both chloroplast and low-copy nuclear loci and discuss the utility of such markers to resolve evolutionary relationships and infer hybridization events within the Campanuloideae clade.ResultsThe inclusion of low-copy nuclear genes into the analyses provides increased phylogenetic resolution in two species-rich clades containing recently diverged taxa. We also obtain support for the placement of two early diverging lineages (Jasione and Musschia-Gadellia clades) that have previously been unresolved. Furthermore, phylogenetic analyses of PPR loci revealed potential hybridization events for a number of taxa (e.g., Campanula pelviformis and Legousia species). These loci offer greater overall topological support than obtained with plastid DNA alone.ConclusionThis study represents the first inclusion of low-copy nuclear genes for phylogenetic reconstruction in Campanuloideae. The two PPR loci were easy to sequence, required no cloning, and the sequence alignments were straightforward across the entire Campanuloideae clade. Although potentially complicated by incomplete lineage sorting, these markers proved useful for understanding the processes of reticulate evolution and resolving relationships at a wide range of phylogenetic levels. Our results stress the importance of including multiple, independent loci in phylogenetic analyses.

Phylogeny and biogeography of New World Stachydeae (Lamiaceae) with emphasis on the origin and diversification of Hawaiian and South American taxa

Due to its unique geological history and isolated location, the Hawaiian Archipelago provides an ideal setting for studies on biogeography, phylogeny and population biology. Species richness in these islands has been attributed to unique colonization events. The Hawaiian mints comprising of three endemic genera represent one of the largest radiations in the island. Previous studies have shown the Hawaiian mints to be nested within the dry-fruited Stachys, probably resulting from one or more hybridization events. Stachydeae, the largest tribe in the subfamily Lamioideae (Lamiaceae), is a taxonomically complex and widespread lineage exhibiting remarkable chromosomal diversity. In this paper we attempted at untangling the relationships between the New World and Hawaiian mint taxa, as well as investigate the origin and diversification of the mints in the New World. There seem to have been at least two independent migration events of Stachys to the New World during the Middle to Late Miocene and towards the beginning of the Pliocene, respectively. Results indicate incongruence between the rDNA and cpDNA phylogenies suggesting a reticulate, New World origin for the Hawaiian mints, although dispersal to Hawaii appears to have happened only once during the Pliocene. South American Stachys diversified from their Mesoamerican relatives around Late Pliocene and may also have arisen from similar reticulate events indicated by their intercalating position among the Mesoamerican Stachys species. Further insights into the phylogenetic relationships between the New World mints may be gathered through the study of low copy nuclear loci.

Molecular Phylogenetics and Taxonomy of the Genus <I>Boechera</I> and Related Genera (Brassicaceae: Boechereae)

Abstract Boechera (Brassicaceae) is a diverse genus of ±70 sexual diploid species and numerous apomictic hybrids concentrated in western North America. It is the largest genus in tribe Boechereae, which also includes seven other small genera. Boechera is closely related to Arabidopsis and is becoming a model genus for diverse studies, focusing particularly on apomixis and hybrid speciation. As part of an ongoing effort to clarify the taxonomy of the group, we present phylogenetic analyses of Boechereae, including all genera of the tribe and most of the sexual diploid species of Boechera. Ten loci are used, including two plastid loci, nrDNA ITS, and seven low-copy nuclear loci. These analyses indicate that Boechera, as currently circumscribed, is polyphyletic, comprising three main clades. Eastern North American species previously assigned to Boechera form a distinct clade with the Asian taxon Borodinia macrophylla and are herein transferred to the genus Borodinia, resulting in seven new combinations (Boro...

Eleven diverse nuclear‐encoded phylogenetic markers for the subfamily Panicoideae (Poaceae)

Polyploidy is common in the grasses and low-copy nuclear loci are needed to further our understanding of phylogenetic relationships. Genetic and genomic resources were combined to identify loci known to influence plant and inflorescence architecture. Degenerate primers were designed and tested to amplify regions of 11 nuclear-encoded loci across the panicoid grasses. The primers designed in this study amplify regions of a diverse set of genes within the panicoid grasses. Properly employed, these markers will allow the identification of allopolyploid taxa and their diploid progenitors.

Integrating different tools to disentangle species complexes: A case study in Epidendrum (Orchidaceae)

AbstractSpecies delimitation remains a central problem in systematic, taxonomic and evolutionary studies. However, the precise delimitation of species depends on the criteria used to identify lineages and the specific species concept that is applied. Recently, multidisciplinary studies using different data sources have significantly improved the delimitation of species within complex taxonomic groups, leading to an integrative taxonomy. To investigate the species delimitation within the Atlantic clade of Epidendrum (subg. Amphyglottium), four different species criteria were examined (phenetic differentiation, mono–phyly, diagnosability, absence of genetic intermediates). Morphometrics, plastid DNA sequences and nuclear microsatellite markers were used to explore the agreement between patterns recovered and species criteria tested. The conflicts among species criteria are discussed in light of pollination ecology, patterns of gene flow, reproductive isolation mechanisms and selective pressures currently acting in deceptive orchid species. Four currently recognized species from the Atlantic clade could be delimitated, including one newly described species, Epidendrum flammeus. Three out of five species satisfied the monophyly criterion, and few diagnostic flower characters were found among species. In contrast, nuclear microsatellite data correctly assigned individuals to their respective species, even in the presence of weak reproductive isolation and extensive hybridization events reported in the literature. One important implication of this finding is that phylogenetic studies in Epidendrum spp. should make use of single– or low–copy nuclear loci instead of plastid markers, which may be true for other plant groups. The results also indicate that the generalized pollination syndrome found among species of the Atlantic clade, the different levels of gene flow observed between nuclear and plastid markers, and hybridization events are commonly observed as the main evolutionary forces within this orchid group. Finally, we discuss evolutionary processes and taxonomic limits among these species, and we highlight the need to increase the inter–disciplinary approach to investigate species limits in complex plant groups.

Five nuclear loci resolve the polyploid history of switchgrass (Panicum virgatum L.) and relatives.

Polyploidy poses challenges for phylogenetic reconstruction because of the need to identify and distinguish between homoeologous loci. This can be addressed by use of low copy nuclear markers. Panicum s.s. is a genus of about 100 species in the grass tribe Paniceae, subfamily Panicoideae, and is divided into five sections. Many of the species are known to be polyploids. The most well-known of the Panicum polyploids are switchgrass (Panicum virgatum) and common or Proso millet (P. miliaceum). Switchgrass is in section Virgata, along with P. tricholaenoides, P. amarum, and P. amarulum, whereas P. miliaceum is in sect. Panicum. We have generated sequence data from five low copy nuclear loci and two chloroplast loci and have clarified the origin of P. virgatum. We find that all members of sects. Virgata and Urvilleana are the result of diversification after a single allopolyploidy event. The closest diploid relatives of switchgrass are in sect. Rudgeana, native to Central and South America. Within sections Virgata and Urvilleana, P. tricholaenoides is sister to the remaining species. Panicum racemosum and P. urvilleanum form a clade, which may be sister to P. chloroleucum. Panicum amarum, P. amarulum, and the lowland and upland ecotypes of P. virgatum together form a clade, within which relationships are complex. Hexaploid and octoploid plants are likely allopolyploids, with P. amarum and P. amarulum sharing genomes with P. virgatum. Octoploid P. virgatum plants are formed via hybridization between disparate tetraploids. We show that polyploidy precedes diversification in a complex set of polyploids; our data thus suggest that polyploidy could provide the raw material for diversification. In addition, we show two rounds of allopolyploidization in the ancestry of switchgrass, and identify additional species that may be part of its broader gene pool. This may be relevant for development of the crop for biofuels.

Navigating the tip of the genomic iceberg: Next‐generation sequencing for plant systematics

Just as Sanger sequencing did more than 20 years ago, next-generation sequencing (NGS) is poised to revolutionize plant systematics. By combining multiplexing approaches with NGS throughput, systematists may no longer need to choose between more taxa or more characters. Here we describe a genome skimming (shallow sequencing) approach for plant systematics. Through simulations, we evaluated optimal sequencing depth and performance of single-end and paired-end short read sequences for assembly of nuclear ribosomal DNA (rDNA) and plastomes and addressed the effect of divergence on reference-guided plastome assembly. We also used simulations to identify potential phylogenetic markers from low-copy nuclear loci at different sequencing depths. We demonstrated the utility of genome skimming through phylogenetic analysis of the Sonoran Desert clade (SDC) of Asclepias (Apocynaceae). Paired-end reads performed better than single-end reads. Minimum sequencing depths for high quality rDNA and plastome assemblies were 40× and 30×, respectively. Divergence from the reference significantly affected plastome assembly, but relatively similar references are available for most seed plants. Deeper rDNA sequencing is necessary to characterize intragenomic polymorphism. The low-copy fraction of the nuclear genome was readily surveyed, even at low sequencing depths. Nearly 160000 bp of sequence from three organelles provided evidence of phylogenetic incongruence in the SDC. Adoption of NGS will facilitate progress in plant systematics, as whole plastome and rDNA cistrons, partial mitochondrial genomes, and low-copy nuclear markers can now be efficiently obtained for molecular phylogenetics studies.

Estimating the species tree for Hawaiian Schiedea (Caryophyllaceae) from multiple loci in the presence of reticulate evolution

Schiedea (Caryophyllaceae) is a monophyletic genus of 34 species, all endemic to the Hawaiian Islands, that arose from a single colonization, providing one of the best examples of adaptive radiation in Hawai‘i. Species utilize a range of habitats and exhibit a variety of growth forms and transitions in breeding systems from hermaphroditism toward dimorphism or autogamy. Our study included the most thorough sampling to date: 2–5 individuals per species and 4 independent genetic partitions: eight plastid and three low-copy nuclear loci (9217 bps), allowing a three-locus BEST species tree. Despite incomplete resolution at the tips, our results support monophyly for each extant species. Gene trees revealed several clear cases of cytonuclear incongruence, likely created by interspecific introgression. Conflict occurs at the divergence of section Alphaschiedea as well as at the tips. Ages inferred from a BEAST analysis allow an original colonization onto either Nihoa or Kauaì and inform some aspects of inter-island migrations. We suggest that several hard polytomies on the species tree are biologically realistic, signifying either nearly simultaneous speciation or historical introgressive hybridization. Based on inferred node ages that exceed expected coalescent times, we propose that undetected nuclear introgression may play a larger role than incomplete lineage sorting in sections Schiedea and Mononeura.

Genetic diversity and structure in semiwild and domesticated chiles (Capsicum annuum; Solanaceae) from Mexico

The chile of Mesoamerica, Capsicum annuum, is one of five domesticated chiles in the Americas. Among the chiles, it varies the most in size, form, and color of its fruits. Together with maize, C. annuum is one of the principal elements of the neotropical diets of Mesoamerican civilizations. Despite the great economic and cultural importance of C. annuum both worldwide and in Mexico, however, very little is known about its geographic origin and number of domestications. Here we sampled a total of 80 accessions from Mexico (58 semiwild and 22 domesticated) and examined nucleotide sequence diversity at three single- or low-copy nuclear loci, Dhn, G3pdh, and Waxy. Across the three loci, we found an average reduction of ca. 10% in the diversity of domesticates relative to semiwild chiles and geographic structure within Mexican populations. The Yucatan Peninsula contained a large number of haplotypes, many of which were unique, suggesting an important region of chile domestication and center of diversity. The present sampling of loci did not conclusively resolve the number and location of domestications, but several lines of evidence suggest multiple independent domestications from widely distributed progenitor populations.

The potential of the WRKY gene family for phylogenetic reconstruction: An example from the Malvaceae

The WRKY gene family of transcription factors is involved in several diverse pathways and includes components of plant-specific, ancient regulatory networks. WRKY genes contain one or two highly conserved DNA binding domains interrupted by an intron. We used partial sequences of five independent WRKY loci to assess their potential for phylogeny reconstruction. Loci were originally isolated from Theobroma cacao L. by PCR with a single pair of degenerate primers; loci-specific primers were subsequently designed. We tested those loci across the sister genera Herrania Goudot and Theobroma L., with Guazuma ulmifolia Lam. as the outgroup. Overall, the combined WRKY matrices performed as well or better than other genes in resolving the intrageneric phylogeny of Herrania and Theobroma. The ease of isolating numerous, independent WRKY loci from diverse plant species with a single pair of degenerate primers designed to the highly conserved WRKY domain, renders them extremely useful tools for generating multiple, single or low copy nuclear loci for molecular phylogenetic studies at lower taxonomic levels. This is the first demonstration of the potential for members of the WRKY gene family for phylogenetic reconstruction.

Intrageneric phylogeny of Capsella (Brassicaceae) and the origin of the tetraploid C. bursa‐pastoris based on chloroplast and nuclear DNA sequences

Polyploidization, often accompanied by hybridization, has been of major importance in flowering plant evolution. Here we investigate the importance of these processes for the evolution of the tetraploid crucifer Capsella bursa-pastoris using DNA sequences from two chloroplast loci as well as from three nuclear low-copy genes. The near-absence of variation at the C. bursa-pastoris chloroplast markers suggests a single and recent origin of the tetraploid. However, despite supporting a single phylogeny, chloroplast data indicate that neither of the extant Capsella diploids is the maternal parent of the tetraploid. Combined with data from the three nuclear loci, our results do not lend support to previous hypotheses on the origin of C. bursa-pastoris as an allopolyploid between the diploids C. grandiflora and C. rubella or an autopolyploid of C. grandiflora. Nevertheless, for each locus, some of the C. bursa-pastoris accessions harbored C. rubella alleles, indicating that C. rubella contributed to the gene pool of C. bursa-pastoris, either through allopolyploid speciation or, more likely, through hybridization and introgression. To our knowledge, this study is the first of a wild, nonmodel plant genus that uses a combination of chloroplast and multiple low-copy nuclear loci for phylogenetic inference of polyploid evolution.

Evolutionary relationships among Pinus (Pinaceae) subsections inferred from multiple low‐copy nuclear loci

Sequence data from nrITS and cpDNA have failed to fully resolve phylogenetic relationships among Pinus species. Four low-copy nuclear genes, developed from the screening of 73 mapped conifer anchor loci, were sequenced from 12 species representing all subsections. Individual loci do not uniformly support either the nrITS or cpDNA hypotheses and in some cases produce unique topologies. Combined analysis of low-copy nuclear loci produces a well-supported subsectional topology of two subgenera, each divided into two sections, congruent with prior hypotheses of deep divergence in Pinus. The placements of P. nelsonii, P. krempfii, and P. contorta have been of continued systematic interest. Results strongly support the placement of P. nelsonii as sister to the remaining members of sect. Parrya, suggest a moderately well-supported and consistent position of P. krempfii as sister to the remaining members of sect. Quinquefoliae, and are ambiguous about the placement of P. contorta. A successful phylogenetic strategy in Pinus will require many low-copy nuclear loci that include a high proportion of silent sites and derive from independent linkage groups. The locus screening and evaluation strategy presented here can be broadly applied to facilitate the development of phylogenetic markers from the increasing number of available genomic resources.

From famine to feast? Selecting nuclear DNA sequence loci for plant species-level phylogeny reconstruction.

Phylogenetic analyses of DNA sequences have prompted spectacular progress in assembling the Tree of Life. However, progress in constructing phylogenies among closely related species, at least for plants, has been less encouraging. We show that for plants, the rapid accumulation of DNA characters at higher taxonomic levels has not been matched by conventional sequence loci at the species level, leaving a lack of well-resolved gene trees that is hindering investigations of many fundamental questions in plant evolutionary biology. The most popular approach to address this problem has been to use low-copy nuclear genes as a source of DNA sequence data. However, this has had limited success because levels of variation among nuclear intron sequences across groups of closely related species are extremely variable and generally lower than conventionally used loci, and because no universally useful low-copy nuclear DNA sequence loci have been developed. This suggests that solutions will, for the most part, be lineage-specific, prompting a move away from 'universal' gene thinking for species-level phylogenetics. The benefits and limitations of alternative approaches to locate more variable nuclear loci are discussed and the potential of anonymous nongenic nuclear loci is highlighted. Given the virtually unlimited number of loci that can be generated using these new approaches, it is clear that effective screening will be critical for efficient selection of the most informative loci. Strategies for screening are outlined.