Increasing phylogenetic resolution at low taxonomic levels using massively parallel sequencing of chloroplast genomes

<p>Abstract</p> <p>Background</p> <p>Molecular evolutionary studies share the common goal of elucidating historical relationships, and the common challenge of adequately sampling taxa and characters. Particularly at low taxonomic levels, recent divergence, rapid radiations, and conservative genome evolution yield limited sequence variation, and dense taxon sampling is often desirable. Recent advances in massively parallel sequencing make it possible to rapidly obtain large amounts of sequence data, and multiplexing makes extensive sampling of megabase sequences feasible. Is it possible to efficiently apply massively parallel sequencing to increase phylogenetic resolution at low taxonomic levels?</p> <p>Results</p> <p>We reconstruct the infrageneric phylogeny of <it>Pinus </it>from 37 nearly-complete chloroplast genomes (average 109 kilobases each of an approximately 120 kilobase genome) generated using multiplexed massively parallel sequencing. 30/33 ingroup nodes resolved with ≥ 95% bootstrap support; this is a substantial improvement relative to prior studies, and shows massively parallel sequencing-based strategies can produce sufficient high quality sequence to reach support levels originally proposed for the phylogenetic bootstrap. Resampling simulations show that at least the entire plastome is necessary to fully resolve <it>Pinus</it>, particularly in rapidly radiating clades. Meta-analysis of 99 published infrageneric phylogenies shows that whole plastome analysis should provide similar gains across a range of plant genera. A disproportionate amount of phylogenetic information resides in two loci (<it>ycf</it>1, <it>ycf</it>2), highlighting their unusual evolutionary properties.</p> <p>Conclusion</p> <p>Plastome sequencing is now an efficient option for increasing phylogenetic resolution at lower taxonomic levels in plant phylogenetic and population genetic analyses. With continuing improvements in sequencing capacity, the strategies herein should revolutionize efforts requiring dense taxon and character sampling, such as phylogeographic analyses and species-level DNA barcoding.</p

Morphological Stasis abd Molecular Divergence in the Intercontinental Disjunct Genus Datisca (Datiscaceae)

The genus Datisca comprises two species and has an intercontinentally disjunct distribution: D. cannabina is native to southwest and central Asia, whereas D. glomerata is distributed from northern California to northern Baja California. In 1975, Axelrod proposed a geohistorical scenario to account for such Madrean-Tethyan links, suggesting that these disjunctions resulted from migration across the mid-Atlantic from the Paleogene up to the Neogene, approximately 23 to 65 m.y.a. The two species are quite similar in most phenotypic traits which have been studied to date. The major difference between the two involves their breeding system: D. cannabina is dioecious while D. glomerata is apparently androdioecious. Despite these similarities, Nei\u27s mean genetic identity between the two species is I = 0.142. This is one of the lowest values yet reported for congeneric flowering plants and provides evidence for an ancient origin of the disjunction. Furthermore, the fact that the western populations of D. cannabina have a much higher genetic identity value with D. glomerata than does the eastern population supports the idea that dispersal occurred across the Atlantic. In addition, the population genetic structure of D. glomerata is consistent with an androdioecious breeding system

Mitochondrial genome sequences illuminate maternal lineages of conservation concern in a rare carnivore

<p>Abstract</p> <p>Background</p> <p>Science-based wildlife management relies on genetic information to infer population connectivity and identify conservation units. The most commonly used genetic marker for characterizing animal biodiversity and identifying maternal lineages is the mitochondrial genome. Mitochondrial genotyping figures prominently in conservation and management plans, with much of the attention focused on the non-coding displacement ("D") loop. We used massively parallel multiplexed sequencing to sequence complete mitochondrial genomes from 40 fishers, a threatened carnivore that possesses low mitogenomic diversity. This allowed us to test a key assumption of conservation genetics, specifically, that the D-loop accurately reflects genealogical relationships and variation of the larger mitochondrial genome.</p> <p>Results</p> <p>Overall mitogenomic divergence in fishers is exceedingly low, with 66 segregating sites and an average pairwise distance between genomes of 0.00088 across their aligned length (16,290 bp). Estimates of variation and genealogical relationships from the displacement (<it>D</it>) loop region (299 bp) are contradicted by the complete mitochondrial genome, as well as the protein coding fraction of the mitochondrial genome. The sources of this contradiction trace primarily to the near-absence of mutations marking the D-loop region of one of the most divergent lineages, and secondarily to independent (recurrent) mutations at two nucleotide position in the D-loop amplicon.</p> <p>Conclusions</p> <p>Our study has two important implications. First, inferred genealogical reconstructions based on the fisher D-loop region contradict inferences based on the entire mitogenome to the point that the populations of greatest conservation concern cannot be accurately resolved. Whole-genome analysis identifies Californian haplotypes from the northern-most populations as highly distinctive, with a significant excess of amino acid changes that may be indicative of molecular adaptation; D-loop sequences fail to identify this unique mitochondrial lineage. Second, the impact of recurrent mutation appears most acute in closely related haplotypes, due to the low level of evolutionary signal (unique mutations that mark lineages) relative to evolutionary noise (recurrent, shared mutation in unrelated haplotypes). For wildlife managers, this means that the populations of greatest conservation concern may be at the highest risk of being misidentified by D-loop haplotyping. This message is timely because it highlights the new opportunities for basing conservation decisions on more accurate genetic information.</p

Population structure and genetic diversity of Botrychium pumicola (Ophioglossaceae) based on inter-simple sequence repeats (ISSR)

Species of Botrychium reproduce by spores that form subterranean gametophytes and a few, like B. pumicola, also reproduce asexually with subterranean sporophytic gemmae. The goal of this study was to examine the genetic diversity of B. pumicola populations and to better understand the role of gemmae. Ninety-nine individuals from three monitored populations were sampled. The technique of inter-simple sequence repeats (ISSR) produced 15 polymorphic loci and identified 71 ISSR genotypes. Sixteen of the ISSR genotypes were shared by more that one individual in a population, representing potential clones. Ten of the 16 shared genotypes were not limited to clusters of plants (groups of plants growing from the same point). The ten potential clones were disjunct (separated by other genotypes) and not in patches as might be expected for an underground propagule. There is a high probability that these shared genotypes arose from independent sexual events suggesting they were not clones. These results suggest that the long-distance dispersal of gemmae is at best a rare event

Repeated translocation of a gene cassette drives sex-chromosome turnover in strawberries

Turnovers of sex-determining systems represent important diversifying forces across eukary- otes. Shifts in sex chromosomes—but conservation of the master sex-determining genes— characterize distantly related animal lineages. Yet in plants, in which separate sexes have evolved repeatedly and sex chromosomes are typically homomorphic, we do not know whether such translocations drive sex-chromosome turnovers within closely related taxo- nomic groups. This phenomenon can only be demonstrated by identifying sex-associated nucleotide sequences, still largely unknown in plants. The wild North American octoploid strawberries (Fragaria) exhibit separate sexes (dioecy) with homomorphic, female heteroga- metic (ZW) inheritance, yet sex maps to three different chromosomes in different taxa. To characterize these turnovers, we identified sequences unique to females and assembled their reads into contigs. For most octoploid Fragaria taxa, a short (13 kb) sequence was observed in all females and never in males, implicating it as the sex-determining region (SDR). This female-specific “SDR cassette” contains both a gene with a known role in fruit and pollen production and a novel retrogene absent on Z and autosomal chromosomes. Phy- logenetic comparison of SDR cassettes revealed three clades and a history of repeated translocation. Remarkably, the translocations can be ordered temporally due to the capture of adjacent sequence with each successive move. The accumulation of the “souvenir” sequence—and the resultant expansion of the hemizygous SDR over time—could have been adaptive by locking genes into linkage with sex. Terminal inverted repeats at the inser- tion borders suggest a means of movement. To our knowledge, this is the first plant SDR shown to be translocated, and it suggests a new mechanism (“move-lock-grow”) for expan- sion and diversification of incipient sex chromosomes

Finding a (Pine) Needle in a Haystack: Chloroplast Genome Sequence Divergence in Rare and Widespread Pines

Critical to conservation efforts and other investigations at low taxonomic levels, DNA sequence data offer important insights into the distinctiveness, biogeographic partitioning and evolutionary histories of species. The resolving power of DNA sequences is often limited by insufficient variability at the intraspecific level. This is particularly true of studies involving plant organelles, as the conservative mutation rate of chloroplasts and mitochondria makes it difficult to detect polymorphisms necessary to track genealogical relationships among individuals, populations and closely related taxa, through space and time. Massively parallel sequencing (MPS) makes it possible to acquire entire organelle genome sequences to identify cryptic variation that would be difficult to detect otherwise. We are using MPS to evaluate intraspecific chloroplast-level divergence across biogeographic boundaries in narrowly endemic and widespread species of Pinus. We focus on one of the world\u27s rarest pines - Torrey pine (Pinus torreyana) - due to its conservation interest and because it provides a marked contrast to more widespread pine species. Detailed analysis of nearly 90% ( approximately 105 000 bp each) of these chloroplast genomes shows that mainland and island populations of Torrey pine differ at five sites in their plastome, with the differences fixed between populations. This is an exceptionally low level of divergence (1 polymorphism/ approximately 21 kb), yet it is comparable to intraspecific divergence present in widespread pine species and species complexes. Population-level organelle genome sequencing offers new vistas into the timing and magnitude of divergence within species, and is certain to provide greater insight into pollen dispersal, migration patterns and evolutionary dynamics in plants

An Evaluation of Putative Sympatric Speciation within Limnanthes (Limnanthaceae)

Limnanthes floccosa ssp. floccosa and L. floccosa ssp. grandiflora are two of five subspecies within Limnanthes floccosa endemic to vernal pools in southern Oregon and northern California. Three seasons of monitoring natural populations have quantified that L. floccosa ssp. grandiflora is always found growing sympatrically with L. floccosa ssp. floccosa and that their flowering times overlap considerably. Despite their subspecific rank within the same species crossing experiments have confirmed that their F1 hybrids are sterile. An analysis of twelve microsatellite markers, with unique alleles in each taxon, also shows exceedingly low levels of gene flow between populations of the two subspecies. Due to the lack of previous phylogenetic resolution among L. floccosa subspecies, we used Illumina next generation sequencing to identify single nucleotide polymorphisms from genomic DNA libraries of L. floccosa ssp. floccosa and L. floccosa ssp. grandiflora. These data were used to identify single nucleotide polymorphisms in the chloroplast, mitochondrial, and nuclear genomes. From these variable loci, a total of 2772 bp was obtained using Sanger sequencing of ten individuals representing all subspecies of L. floccosa and an outgroup. The resulting phylogenetic reconstruction was fully resolved. Our results indicate that although L. floccosa ssp. floccosa and L. floccosa ssp. grandiflora are closely related, they are not sister taxa and therefore likely did not diverge as a result of a sympatric speciation event

Genetic diversity of the narrow endemic Astragalus oniciformis (Fabaceae)

Astragalus oniciformis Barneby is a narrow endemic xerophyte of the upper Snake River Plain of central Idaho, USA, where it inhabits stabilized, aeolian sand deposits and previously burned, sandy sites over Quaternary basalt flows. The objective of this study was to determine the levels and distribution of genetic differentiation within and among populations of A. oniciformis. Fifteen individuals from each of eight populations, chosen from throughout the range of the species, were selected based on accessibility, density of individuals, and large population size. Inter-simple sequence repeats were chosen as the marker to assess genetic differentiation. The two primers selected yielded 40 polymorphic loci in A. oniciformis. In an analysis of molecular variance, 88.69% of the variation was significantly attributed to variation within populations. High gene flow (Nm = 3.91–3.93; SD = 0.01) and a low percentage deviation from Hardy-Weinberg equilibrium due to population subdivision (GST = 0.113–0.1134; SD = 0.0002) were found among sampled populations. These results suggest that current threats to this species, including changing fire patterns and habitat loss from grazing disturbance, have not yet affected the genetic diversity of this species. Preservation of large populations and smaller, intervening, dispersed patches will help preserve the genetic integrity and the genetic diversity found in A. oniciformis.Keywords: endemic, conservation, Idaho, USA, genetic diversity, Fabaceae, Astragalus, ISSRGenetic diversity of the narrow endemic… Published version can be found at American Journal of Botany: http://www.amjbot.org/content/vol91/issue12