DNA Sequence Loci Research Articles

ExRec: a python pipeline for generating recombination-filtered multi-locus datasets

Abstract Summary ExRec (Exclusion of Recombined DNA) is a dependency-free Python pipeline that implements the four-gamete test to automatically filter out recombined DNA blocks from thousands of DNA sequence loci. This procedure helps all loci better meet the “no intralocus recombination” assumption common to many coalescent-based analyses in population genomic, phylogeographic, and shallow-scale phylogenomic studies. The user-friendly pipeline contains five standalone applications—four file conversion scripts and one main script that performs the recombination filtering procedures. The pipeline outputs recombination-filtered data in a variety of common formats and a tab-delimited table that displays descriptive statistics for all loci and the analysis results. A novel feature of this software is that the user can select whether to output the longest nonrecombined sequence blocks from recombined loci (current best practice) or randomly select nonrecombined blocks from loci (a newer approach). We tested ExRec with six published phylogenomic datasets that ranged in size from 27 to 2237 loci and came in a variety of input file formats. In all trials the data could be easily analyzed in only seconds for the smaller datasets and <30 min for the largest using a simple laptop computer. Availability and implementation ExRec was written in Python 3 under the MIT license. The program applications, user manual (including step-by-step tutorials), and sample data are freely available at https://github.com/Sammccarthypotter/ExRec.

Phylogeographic reconstructions can be biased by ancestral shared alleles: The case of the polymorphic lichen Bryoria fuscescens in Europe and North Africa.

Large phylogeographic studies on lichens are scarce, and none involves a single species within which different lineages show fixed alternative dispersal strategies. We investigated Bryoria fuscescens (including B.capillaris) in Europe and western North Africa by phenotypically characterizing 1400specimens from 64 populations and genotyping them with 14microsatellites. We studied population structure and genetic diversity at the local and continental scales, discussed the post-glacial phylogeography, and compared dispersal capacities of phenotypes with and without soralia. Our main hypothesis is that the estimated phylogeography, migration routes, and dispersal capacities may be strongly biased by ancestral shared alleles. Scandinavia is genetically the richest area, followed by the Iberian Peninsula, the Carpathians, and the Alps. Three gene pools were detected: two partially linked to phenotypic characteristics, and the third one genetically related to the American sister species B.pseudofuscescens. The comparison of one gene pool producing soredia and one not, suggested both as panmictic, with similar levels of isolation by distance (IBD). The migration routes were estimated to span from north to south, in disagreement with the assessed glacial refugia. The presence of ancestral shared alleles in distant populations can explain the similar IBD levels found in both gene pools while producing a false signal of panmixia, and also biasing the phylogeographic reconstruction. The incomplete lineage sorting recorded for DNA sequence loci also supports this hypothesis. Consequently, the high diversity in Scandinavia may rather come from recent immigration into northern populations than from an in situ diversification. Similar patterns of ancestral shared polymorphism may bias the phylogeographical reconstruction of other lichen species.

Phylogenetics of the freshwater crab (Potamonautes MacLeay, 1838) fauna from ‘sky islands’ in Mozambique with the description of a new species (Brachyura: Potamoidea: Potamonautidae)

Patterns and processes of cladogenesis among taxa living on the Mozambique ‘sky islands’ remain poorly studied. During the present study, we report on a new freshwater crab species from Mount Lico, an inselberg and ‘sky island’ in the Zambezia Province of Mozambique. Phylogenetic analyses using three mitochondrial DNA sequence loci (12S rRNA, 16S rRNA and COI) were used to determine the evolutionary placement of the freshwater crab specimens from Mount Lico. The freshwater crab specimens from Mount Lico were retrieved sister to Potamonautes choloensis. The new species, Potamonautes licoensis sp. nov., is described and compared with other southern African freshwater crab species. Divergence time estimations for the Mozambican freshwater crab species suggest a Miocene / Plio–Pleistocene diversification. Some endemic ‘sky island’ species form an early branching and are sister to other predominantly East African species, while other ‘sky island’ species are more recently derived and nested within a predominantly southern African clade. The present study presents the description of the fourth endemic freshwater crab species from Mozambique and suggests that the species diversity in the country is likely highly underrepresented, reiterating the call for renewed systematic surveys. An argument for the conservation of these mountainous ‘sky islands’ is presented.

Increasing the species diversity in the Aspergillus section Nidulantes: Six novel species mainly from the indoor environment

ABSTRACTAspergillus section Nidulantes encompasses almost 80 homothallic and anamorphic species, mostly isolated from soil, plant material, or the indoor environment. Some species are clinically relevant or produce mycotoxins. This study reevaluated the species boundaries within several clades of section Nidulantes. Five data sets were assembled, each containing presumptive new species and their closest relatives, and phylogenetic and phenotypic analyses were performed. We tested the hypotheses that the newly isolated or reexamined strains constitute separate species (splitting approach) or should be treated as part of broadly defined species (lumping approach). Four DNA sequence loci were amplified, internal transcribed spacer (ITS) and large subunit (LSU) regions of the rDNA and partial sequences of the β-tubulin (benA), calmodulin (CaM), and RNA polymerase II second largest subunit (RPB2) genes. The latter three loci were used for the phylogenetic analysis and served as input for single-locus (GMYC, bGMYC, PTP, and bPTP) and multilocus (STACEY and BP&P) species delimitation analyses. The phenotypic analysis comprised macro- and micromorphology (including scanning electron microscopy) and comparison of cardinal growth temperatures. The phylogenetic analysis supported the splitting hypothesis in all cases, and based on the combined approach, we propose six new species, four that are homothallic and two anamorphic. Four new species were isolated from the indoor environment (Jamaica, Trinidad and Tobago, USA), one originated from soil (Australia), and one from a kangaroo rat cheek pouch (USA).

Allopatric divergence, demographic history, and conservation implications of an endangered conifer Cupressus chengiana in the eastern Qinghai-Tibet Plateau

Isolation and demographic history are key factors that affect lineage divergence of tree species in topographic complex areas, such as the Qinghai-Tibet Plateau (QTP), yet few studies have evaluated these factors in a coalescent-based modeling framework. In the present study, we surveyed ten nuclear DNA sequence loci (nDNA) and six nuclear microsatellite loci (nSSR) for an endangered conifer, Cupressus chengiana, throughout its natural range in the eastern QTP. BARRIER analyses revealed a strong genetic barrier between Gansu and Sichuan populations of C. chengiana, and isolation with migration models detected limited gene flow between them, supporting the division of this species into two evolutionary significant units (ESUs). Two independent coalescent-based approaches suggest a Quaternary divergence between ESUs, their consensus age range ((0.09–) 0.59–1.53 (–2.71) Mya) largely overlaps with the time period when the largest glaciation occurred in the QTP. Both demographic inferences, IMa2 and DIYABC, suggest that both ESUs may have experienced a bottleneck or population contraction event during the late Quaternary. A documented massive recent anthropogenic habitat loss and fragmentation may have led to further decrease of the natural distribution of this conifer. We propose that the conservation and management of both natural stands and plantations of C. chengiana should be reconsidered in the light of our findings.

The Urochloa Foliar Blight and Collar Rot Pathogen Rhizoctonia solani AG-1 IA Emerged in South America Via a Host Shift from Rice.

The fungus Rhizoctonia solani anastomosis group (AG)-1 IA emerged in the early 1990s as an important pathogen causing foliar blight and collar rot on pastures of the genus Urochloa (signalgrass) in South America. We tested the hypothesis that this pathogen emerged following a host shift or jump as a result of geographical overlapping of host species. The genetic structure of host and regional populations of R. solani AG-1 IA infecting signalgrass, rice, and soybean in Colombia and Brazil was analyzed using nine microsatellite loci in 350 isolates to measure population differentiation and infer the pathogen reproductive system. Phylogeographical analyses based on the microsatellite loci and on three DNA sequence loci were used to infer historical migration patterns and test hypotheses about the origin of the current pathogen populations. Cross pathogenicity assays were conducted to measure the degree of host specialization in populations sampled from different hosts. The combined analyses indicate that the pathogen populations currently infecting Urochloa in Colombia and Brazil most likely originated from a population that originally infected rice. R. solani AG-1 IA populations infecting Urochloa exhibit a mixed reproductive system including both sexual reproduction and long-distance dispersal of adapted clones, most likely on infected seed. The pathogen population on Urochloa has a genetic structure consistent with a high evolutionary potential and showed evidence for host specialization.

A new cryptic species in a new cryptic genus in the Caesalpinia group (Leguminosae) from the seasonally dry inter‐Andean valleys of South America

AbstractThe generic affiliation of the Andean species Caesalpinia trichocarpa, C. mimosifolia, and their close relatives has remained uncertain in all recent studies of Caesalpinia s.l. (Leguminosae, subfamily Caesalpinioideae). A new densely sampled phylogeny based on four DNA sequence regions (rps16, trn D‐trnT, ycf6‐ psbM, ITS) strongly supports the monophyly of an Andean clade. We propose that despite the lack of obvious diagnostic morphological synapomorphies, this Andean group should be considered as a distinct genus, here described as the new genus Arquita. Phylogenetic analyses also suggest a problem with species delimitation in this group. Within C. trichocarpa, accessions from disjunct geographic areas in Argentina, Bolivia and Peru each form a robustly supported, unresolved clade that includes C. mimosifolia. The morphological and genetic cohesiveness of the C. trichocarpa complex is investigated using morphometric phenetic analyses of qualitative and quantitative flower and leaf traits, and reconstruction of a densely sampled phylogeny using three plastid and one nuclear ribosomal DNA sequence loci. Our results suggest that the most geographically isolated of these clades, narrowly endemic to two inter‐Andean valleys in central‐north Peru and separated by ∼1350 km, and extensive high Andean cordilleras above 4000 m, from the nearest populations in Bolivia, represents a genetically highly distinct and morphologically cryptic lineage here described as a new species (Arquita grandiflora). A full taxonomic account of the new genus Arquita and its component species is provided, with a distribution map and a key to the species.

New EPIC nuclear DNA sequence markers to improve the resolution of phylogeographic studies of coenagrionids and other odonates

While phylogeographic data provide valuable information to inform conservation plans, there are comparatively few Odonata phylogeographic studies. This lack of research is partially due to a lack of independent DNA markers with appropriate levels of polymorphism that PCR-amplify in a range of species. We followed an exon-primed, intron-crossing (EPIC) PCR strategy to develop five new, polymorphic nuclear DNA sequence loci (six distinct DNA fragments) for the southern damselfly Coenagrion mercuriale. These markers were: cell division cycle 5 protein (CDC5), arginine methyltransferase (PRMT), acetylglucosaminyl-transferase (AgT), myosin light chain (MLC) and phosphoglucose isomerase (PGI). Between three and five of these new markers could be PCR-amplified in five other species from the genus Coenagrion; one locus (PRMT) can be used in 26 other species of odonates that we examined, including three species of Anisoptera belonging to the genus Onychogomphus. These new nuclear genetic markers will be useful for...

A molecular phylogeny of the vataireoid legumes underscores floral evolvability that is general to many early‐branching papilionoid lineages

Flowering traits can sometimes be overemphasized in taxonomic classifications. The fused and completely differentiated papilionate floral organs in the neotropical legume trees Vatairea and Vataireopsis were traditionally used in part to ascribe these genera to the tribe Dalbergieae. In contrast, the free and mostly undifferentiated floral parts of Luetzelburgia and Sweetia fit the circumscription of the "primitive" Sophoreae. Such divergent floral morphologies thought to divide deep phylogenetic lineages indeed may be prone to episodic transformation among close papilionoid relatives. We sampled 26 of 27 known species of Luetzelburgia, Sweetia, Vatairea, and Vataireopsis in parsimony and Bayesian phylogenetic analyses of nuclear ribosomal ITS/5.8S and six plastid (matK, 3'-trnK, psbA-trnH, trnL intron, rps16 intron, and trnD-T) DNA sequence loci. The analyses of individual and combined data sets strongly resolved the monophyly of each of Luetzelburgia, Sweetia, Vatairea, and Vataireopsis. Vataireopsis was resolved as sister to the rest and the morphologically divergent Luetzelburgia and Vatairea were strongly resolved as sister clades. Floral morphology was generally not a good predictor of phylogenetic relatedness. Luetzelburgia, Sweetia, Vatairea, and Vataireopsis are unequivocally resolved as the "vataireoid" clade. Fruit and vegetative traits are found to be more phylogenetically conserved than many floral traits. This explains why the identity of the vataireoids has been overlooked or confused. The evolvability of floral traits may also be a general condition among many of the early-branching papilionoid lineages.

Multi-locus phylogeographic and population genetic analysis of Anolis carolinensis: historical demography of a genomic model species.

The green anole (Anolis carolinensis) has been widely used as an animal model in physiology and neurobiology but has recently emerged as an important genomic model. The recent sequencing of its genome has shed new light on the evolution of vertebrate genomes and on the process that govern species diversification. Surprisingly, the patterns of genetic diversity within natural populations of this widespread and abundant North American lizard remain relatively unknown. In the present study, we use 10 novel nuclear DNA sequence loci (N = 62 to 152) and one mitochondrial locus (N = 226) to delimit green anole populations and infer their historical demography. We uncovered four evolutionarily distinct and geographically restricted lineages of green anoles using phylogenetics, Bayesian clustering, and genetic distance methods. Molecular dating indicates that these lineages last shared a common ancestor ∼2 million years ago. Summary statistics and analysis of the frequency distributions of DNA polymorphisms strongly suggest range-wide expansions in population size. Using Bayesian Skyline Plots, we inferred the timing of population size expansions, which differ across lineages, and found evidence for a relatively recent and rapid westward expansion of green anoles across the Gulf Coastal Plain during the mid-Pleistocene. One surprising result is that the distribution of genetic diversity is not consistent with a latitudinal shift caused by climatic oscillations as is observed for many co-distributed taxa. This suggests that the most recent Pleistocene glacial cycles had a limited impact on the geographic distribution of the green anole at the northern limits of its range.

RAPID SPECIATION FOLLOWING RECENT HOST SHIFTS IN THE PLANT PATHOGENIC FUNGUS RHYNCHOSPORIUM

Agriculture played a significant role in increasing the number of pathogen species and in expanding their geographic range during the last 10,000 years. We tested the hypothesis that a fungal pathogen of cereals and grasses emerged at the time of domestication of cereals in the Fertile Crescent and subsequently speciated after adaptation to its hosts. Rhynchosporium secalis, originally described from rye, causes an important disease on barley called scald, although it also infects other species of Hordeum and Agropyron. Phylogenetic analyses based on four DNA sequence loci identified three host-associated lineages that were confirmed by cross-pathogenicity tests. Bayesian analyses of divergence time suggested that the three lineages emerged between approximately 1200 to 3600 years before present (B.P.) with a 95% highest posterior density ranging from 100 to 12,000 years B.P. depending on the implemented clock models. The coalescent inference of demographic history revealed a very recent population expansion for all three pathogens. We propose that Rhynchosporium on barley, rye, and Agropyron host species represent three cryptic pathogen species that underwent independent evolution and ecological divergence by host-specialization. We postulate that the recent emergence of these pathogens followed host shifts. The subsequent population expansions followed the expansion of the cultivated host populations and accompanying expansion of the weedy Agropyron spp. found in fields of cultivated cereals. Hence, agriculture played a major role in the emergence of the scald diseases, the adaptation of the pathogens to new hosts and their worldwide dissemination.

New Populations of Sclerotinia sclerotiorum from Lettuce in California and Peas and Lentils in Washington

ABSTRACT Four populations of Sclerotinia sclerotiorum in North America were inferred previously, based on analyses of both rapidly evolving markers (DNA fingerprint and mycelial compatiblity), and multilocus DNA sequence spanning the range between fast and slow evolution. Each population was defined as an interbreeding unit of conspecific individuals sharing a common recent ancestor and arising in a unique evolutionary event. The present study applies this standard to extend characterization of S. sclerotiorum populations to the Western United States. Isolates of S. sclerotiorum (N = 294) were determined to represent three genetically differentiated populations: California (CA, lettuce), Washington (WA, pea/lentil), and Ontario (ON, lettuce). CA was the most diverse population yet sampled in North America. Clonality was detected in ON and WA. No DNA fingerprints were common among the populations. The index of association (I(A)), based on fingerprint, was closer to zero (0) for CA than it was for the other populations. High diversity and lack of association of markers in California are consistent either with genetic exchange and recombination, or with large population size and high standing genetic variation. Intra- and interlocus conflict among three DNA sequence loci was consistent with recombination. The coalescent IGS genealogy confirmed subdivision and showed CA to be older than WA or ON. The Nearest Neighbor statistic on combined data confirmed subdivision among all present and previously defined populations. All isolates had both MAT1-1 and MAT1-2, consistent with uniform homothallism.

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.

Phylogenetic analysis of globally distributed Mycosphaerella graminicola populations based on three DNA sequence loci

DNA sequence data from three nuclear loci were collected from 384 isolates representing fourteen globally distributed populations of the plant pathogenic fungus Mycosphaerella graminicola. Gene genealogies were constructed for the actin and β-tubulin loci as well as for the previously characterized RFLP locus STS2. The STS2 and β-tubulin loci showed greater potential for phylogenetic studies than the actin locus. Greater sequence diversity was found in the “Old World” populations (Middle East and Europe) than in the “New World” populations (North and South America and Australia). The gene trees were rooted using homologous DNA sequences of Septoria passerinii, the closest known relative to M. graminicola, as well as coalescent rooting. Based on the rooted trees, a tentative phylogenetic history of these populations was inferred. The Middle East appears to be the most likely center of origin, while European populations are more ancient than New World populations. A test for neutrality indicated that the intron in the actin locus could be under selection, while the other two sequence loci were neutral.