Evolution of cox2 introns in angiosperm mitochondria and efficient splicing of an elongated cox2i691 intron.

In angiosperms, the mitochondrial cox2 gene harbors up to two introns, commonly referred to as cox2i373 and cox2i691. We studied the cox2 from 222 fully-sequenced mitogenomes from 30 angiosperm orders and analyzed the evolution of their introns. Unlike cox2i373, cox2i691 shows a distribution among plants that is shaped by frequent intron loss events driven by localized retroprocessing. In addition, cox2i691 exhibits sporadic elongations, frequently in domain IV of introns. Such elongations are poorly related to repeat content and two of them showed the presence of LINE transposons, suggesting that increasing intron size is very likely due to nuclear intracelular DNA transfer followed by incorporation into the mitochondrial DNA. Surprisingly, we found that cox2i691 is erroneously annotated as absent in 30 mitogenomes deposited in public databases. Although each of the cox2 introns is âˆ¼1.5 kb in length, a cox2i691 of 4.2 kb has been reported in Acacia ligulata (Fabaceae). It is still unclear whether its unusual length is due to a trans-splicing arrangement or the loss of functionality of the interrupted cox2. Through analyzing short-read RNA sequencing of Acacia with a multi-step computational strategy, we found that the Acacia cox2 is functional and its long intron is spliced in cis in a very efficient manner despite its length.

Large scale genome skimming from herbarium material for accurate plant identification and phylogenomics.

BACKGROUND: Herbaria are valuable sources of extensive curated plant material that are now accessible to genetic studies because of advances in high-throughput, next-generation sequencing methods. As an applied assessment of large-scale recovery of plastid and ribosomal genome sequences from herbarium material for plant identification and phylogenomics, we sequenced 672 samples covering 21 families, 142 genera and 530 named and proposed named species. We explored the impact of parameters such as sample age, DNA concentration and quality, read depth and fragment length on plastid assembly error. We also tested the efficacy of DNA sequence information for identifying plant samples using 45 specimens recently collected in the Pilbara. RESULTS: Genome skimming was effective at producing genomic information at large scale. Substantial sequence information on the chloroplast genome was obtained from 96.1% of samples, and complete or near-complete sequences of the nuclear ribosomal RNA gene repeat were obtained from 93.3% of samples. We were able to extract sequences for the core DNA barcode regions rbcL and matK from 96 to 93.3% of samples, respectively. Read quality and DNA fragment length had significant effects on sequencing outcomes and error correction of reads proved essential. Assembly problems were specific to certain taxa with low GC and high repeat content (Goodenia, Scaevola, Cyperus, Bulbostylis, Fimbristylis) suggesting biological rather than technical explanations. The structure of related genomes was needed to guide the assembly of repeats that exceeded the read length. DNA-based matching proved highly effective and showed that the efficacy for species identification declined in the order cpDNA >> rDNA > matK >> rbcL. CONCLUSIONS: We showed that a large-scale approach to genome sequencing using herbarium specimens produces high-quality complete cpDNA and rDNA sequences as a source of data for DNA barcoding and phylogenomics.

Recent range expansion in Australian hummock grasses (Triodia) inferred using genotyping-by-sequencing.

The Australian arid zone (AAZ) has undergone aridification and the formation of vast sandy deserts since the mid-Miocene. Studies on AAZ organisms, particularly animals, have shown patterns of mesic ancestry, persistence in rocky refugia and range expansions in arid lineages. There has been limited molecular investigation of plants in the AAZ, particularly of taxa that arrived in Australia after the onset of aridification. Here we investigate populations of the widespread AAZ grass Triodia basedowii to determine whether there is evidence for a recent range expansion, and if so, its source and direction. We also undertake a dating analysis for the species complex to which T. basedowii belongs, in order to place its diversification in relation to changes in AAZ climate and landscapes. We analyse a genomic single nucleotide polymorphism data set from 17 populations of T. basedowii in a recently developed approach for detecting the signal and likely origin of a range expansion. We also use alignments from existing and newly sequenced plastomes from across Poaceae for analysis in BEAST to construct fossil-calibrated phylogenies. Across a range of sampling parameters and outgroups, we detected a consistent signal of westward expansion for T. basedowii, originating in central or eastern Australia. Divergence time estimation indicates that Triodia began to diversify in the late Miocene (crown 7.0-8.8 million years (Ma)), and the T. basedowii complex began to radiate during the Pleistocene (crown 1.4-2.0 Ma). This evidence for range expansion in an arid-adapted plant is consistent with similar patterns in AAZ animals and likely reflects a general response to the opening of new habitat during aridification. Radiation of the T. basedowii complex through the Pleistocene has been associated with preferences for different substrates, providing an explanation why only one lineage is widespread across sandy deserts.

Wide outcrossing provides functional connectivity for new and old Banksia populations within a fragmented landscape.

Habitat fragmentation affects landscape connectivity, the extent of which is influenced by the movement capacity of the vectors of seed and pollen dispersal for plants. Negative impacts of reduced connectivity can include reduced fecundity, increased inbreeding, genetic erosion and decreased long-term viability. These are issues for not only old (remnant) populations, but also new (restored) populations. We assessed reproductive and connective functionality within and among remnant and restored populations of a common tree, Banksia menziesii R.Br. (Proteaceae), in a fragmented urban landscape, utilising a genetic and graph theoretical approach. Adult trees and seed cohorts from five remnants and two restored populations were genotyped using microsatellite markers. Genetic variation and pollen dispersal were assessed using direct (paternity assignment) and indirect (pollination graphs and mating system characterisation) methods. Restored populations had greater allelic diversity (Ar = 8.08; 8.34) than remnant populations (Ar range = 6.49-7.41). Genetic differentiation was greater between restored and adjacent remnants (FST = 0.03 and 0.10) than all other pairwise comparisons of remnant populations (mean FST = 0.01 ± 0.01; n = 16 P = 0.001). All populations displayed low correlated paternity (rp = 0.06-0.16) with wide-ranging realised pollen dispersal distances (< 1.7 km) and well-connected pollen networks. Here, we demonstrate reproductive and connective functionality of old and new populations of B. menziesii within a fragmented landscape. Due to long-distance pollination events, the physical size of these sites underestimates their effective population size. Thus, they are functionally equivalent to large populations, integrated into a larger landscape matrix.

Plastome-Wide Rearrangements and Gene Losses in Carnivorous Droseraceae.

The plastid genomes of four related carnivorous plants (Drosera regia, Drosera erythrorhiza, Aldrovanda vesiculosa, and Dionaea muscipula) were sequenced to examine changes potentially induced by the transition to carnivory. The plastid genomes of the Droseraceae show multiple rearrangements, gene losses, and large expansions or contractions of the inverted repeat. All the ndh genes are lost or nonfunctional, as well as in some of the species, clpP1, ycf1, ycf2 and some tRNA genes. Uniquely, among land plants, the trnK gene has no intron. Carnivory in the Droseraceae coincides with changes in plastid gene content similar to those induced by parasitism and mycoheterotrophy, suggesting parallel changes in chloroplast function due to the similar switch from autotrophy to (mixo-) heterotrophy. A molecular phylogeny of the taxa based on all shared plastid genes indicates that the "snap-traps" of Aldrovanda and Dionaea have a common origin.

Genome-scale transfer of mitochondrial DNA from legume hosts to the holoparasite Lophophytum mirabile (Balanophoraceae).

Angiosperm mitochondrial horizontal gene transfer (HGT) has been widely reported during the past decades. With a few exceptions, foreign sequences are mitochondrial genes or intronic regions from other plants, indicating that HGT has played a major role in shaping mitochondrial genome evolution. Host-parasite relationships are a valuable system to study this phenomenon due to the high frequency of HGT. In particular, the interaction between mimosoid legumes and holoparasites of the genus Lophophytum represents an outstanding opportunity to discern HGT events. The mitochondrial genome of the holoparasite L. mirabile has remarkable properties, the most extraordinary of which is the presence of 34 out of 43 mitochondrial protein genes acquired from its legume host, with the stunning replacement of up to 26 native homologs. However, the origin of the intergenic sequences that represent the majority (>90%) of the L. mirabile mtDNA remains largely unknown. The lack of mitochondrial sequences available from the donor angiosperm lineage (mimosoid legumes) precluded a large-scale evolutionary study. We sequenced and assembled the mitochondrial genome of the mimosoid Acacia ligulata and performed genome wide comparisons with L. mirabile. The A. ligulata mitochondrial genome is almost 700 kb in size, encoding 60 genes. About 60% of the L. mirabile mtDNA had greatest affinity to members of the family Fabaceae (∼49% to mimosoids in particular) with an average sequence identity of ∼96%, including genes but mostly intergenic regions. These findings strengthen the mitochondrial fusion compatibility model for angiosperm mitochondrion-to-mitochondrion HGT.

Ethical seed sourcing is a key issue in meeting global restoration targets.

The global demand for restoration has increased orders of magnitude in the last decade, and hundreds of thousands of tonnes of native seed are required to feed this restoration engine [1] (Figure 1). But where are all the seeds required by restoration going to come from? Wild seed resources continue to be depleted by habitat loss, land degradation and climatic change, and over-collection of seed from wild populations threatens to erode these resources further. Ethical seed sourcing for restoration now represents a core issue in responsible restoration practice. Solutions include the introduction of regulatory frameworks controlling seed sourcing from wild populations, the development of seed farming capacity and advancement of seed enhancement technologies and precision delivery systems reducing seed wastage.

What can we do with 1000 plastid genomes?

The plastid genome of plants is the smallest and most gene-rich of the three genomes in each cell and the one generally present in the highest copy number. As a result, obtaining plastid DNA sequence is a particularly cost-effective way of discovering genetic information about a plant. Until recently, the sequence information gathered in this way was generally limited to small portions of the genome amplified by polymerase chain reaction, but recent advances in sequencing technology have stimulated a substantial rate of increase in the sequencing of complete plastid genomes. Within the last year, the number of complete plastid genomes accessible in public sequence repositories has exceeded 1000. This sudden flood of data raises numerous challenges in data analysis and interpretation, but also offers the keys to potential insights across large swathes of plant biology. We examine what has been learnt so far, what more could be learnt if we look at the data in the right way, and what we might gain from the tens of thousands more genome sequences that will surely arrive in the next few years. The most exciting new discoveries are likely to be made at the interdisciplinary interfaces between molecular biology and ecology.

Microsatellite primers for the rare sedge Lepidosperma bungalbin (Cyperaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for the rare sedge Lepidosperma bungalbin (Cyperaceae) to assess genetic variation and its spatial structuring. METHODS AND RESULTS: We conducted shotgun sequencing on an Illumina MiSeq and produced 6,215,872 sequence reads. The QDD pipeline was used to design 60 primer pairs that were screened using PCR. We developed 17 loci, of which 12 loci were identified that were polymorphic, amplified reliably, and could be consistently scored. We then screened these loci for variation in individuals from three populations. The number of alleles observed for these 12 loci across the three populations ranged from nine to 19 and expected heterozygosity ranged from 0.41 to 0.89. CONCLUSIONS: These markers will enable the quantification of the potential impact of mining on genetic variation within L. bungalbin and establish a baseline for future management of genetic variation of the rare sedge.

Microsatellite primers for the rare shrub Acacia adinophylla (Fabaceae).

PREMISE OF THE STUDY: Microsatellite primers were developed for the rare shrub Acacia adinophylla (Fabaceae) to assess genetic diversity and its spatial structuring. METHODS AND RESULTS: Shotgun sequencing on an Illumina MiSeq produced 6,372,575 reads. Using the QDD pipeline, we designed 60 primer pairs, which were screened using PCR. Seventeen loci were developed, of which 12 loci were identified that were polymorphic, amplified reliably, and could be consistently scored. These loci were then screened for variation in individuals from three populations. The number of alleles observed for these 12 loci ranged from three to 18 and expected heterozygosity ranged from 0.13 to 0.85. CONCLUSIONS: These markers will enable the quantification of genetic impact of proposed mining activities on the short-range endemic Acacia adinophylla.

Integration of complete chloroplast genome sequences with small amplicon datasets improves phylogenetic resolution in Acacia.

Combining whole genome data with previously obtained amplicon sequences has the potential to increase the resolution of phylogenetic analyses, particularly at low taxonomic levels or where recent divergence, rapid speciation or slow genome evolution has resulted in limited sequence variation. However, the integration of these types of data for large scale phylogenetic studies has rarely been investigated. Here we conduct a phylogenetic analysis of the whole chloroplast genome and two nuclear ribosomal loci for 65 Acacia species from across the most recent Acacia phylogeny. We then combine this data with previously generated amplicon sequences (four chloroplast loci and two nuclear ribosomal loci) for 508 Acacia species. We use several phylogenetic methods, including maximum likelihood bootstrapping (with and without constraint) and ExaBayes, in order to determine the success of combining a dataset of 4000bp with one of 189,000bp. The results of our study indicate that the inclusion of whole genome data gave a far better resolved and well supported representation of the phylogenetic relationships within Acacia than using only amplicon sequences, with the greatest support observed when using a whole genome phylogeny as a constraint on the amplicon sequences. Our study therefore provides methods for optimal integration of genomic and amplicon sequences.

Seed production areas for the global restoration challenge.

Wild-collected seed can no longer meet global demand in restoration. Dedicated Seed Production Areas (SPA) for restoration are needed and these require application of ecological, economic, and population-genetic science. SPA design and construction must embrace the ecological sustainability principles of restoration.

The critical role of ants in the extensive dispersal of Acacia seeds revealed by genetic parentage assignment.

Ants are prominent seed dispersal agents in many ecosystems, and dispersal distances are small in comparison with vertebrate dispersal agents. However, the distance and distribution of ant-mediated dispersal in arid/semi-arid environments remains poorly explored. We used microsatellite markers and parentage assignment to quantify the distance and distribution of dispersed seeds of Acacia karina, retrieved from the middens of Iridomyrmex agilis and Melophorus turneri perthensis. From parentage assignment, we could not distinguish the maternal from each parent pair assigned to each seed, so we applied two approaches to estimate dispersal distances, one conservative (CONS), where the parent closest to the ant midden was considered to be maternal, and the second where both parents were deemed equally likely (EL) to be maternal, and used both distances. Parentage was assigned to 124 seeds from eight middens. Maximum seed dispersal distances detected were 417 m (CONS) and 423 m (EL), more than double the estimated global maximum. Mean seed dispersal distances of 40 m (±5.8 SE) (CONS) and 79 m (±6.4 SE) (EL) exceeded the published global average of 2.24 m (±7.19 SD) by at least one order of magnitude. For both approaches and both ant species, seed dispersal was predominantly (44-84% of all seeds) within 50 m from the maternal source, with fewer dispersal events at longer distances. Ants in this semi-arid environment have demonstrated a greater capacity to disperse seeds than estimated elsewhere, which highlights their important role in this system, and suggests significant novel ecological and evolutionary consequences for myrmecochorous species in arid/semi-arid Australia.

The Complete Sequence of the Acacia ligulata Chloroplast Genome Reveals a Highly Divergent clpP1 Gene.

Legumes are a highly diverse angiosperm family that include many agriculturally important species. To date, 21 complete chloroplast genomes have been sequenced from legume crops confined to the Papilionoideae subfamily. Here we report the first chloroplast genome from the Mimosoideae, Acacia ligulata, and compare it to the previously sequenced legume genomes. The A. ligulata chloroplast genome is 174,233 bp in size, comprising inverted repeats of 38,225 bp and single-copy regions of 92,798 bp and 4,985 bp [corrected]. Acacia ligulata lacks the inversion present in many of the Papilionoideae, but is not otherwise significantly different in terms of gene and repeat content. The key feature is its highly divergent clpP1 gene, normally considered essential in chloroplast genomes. In A. ligulata, although transcribed and spliced, it probably encodes a catalytically inactive protein. This study provides a significant resource for further genetic research into Acacia and the Mimosoideae. The divergent clpP1 gene suggests that Acacia will provide an interesting source of information on the evolution and functional diversity of the chloroplast Clp protease complex.

Next generation restoration genetics: applications and opportunities.

Restoration ecology is a young scientific discipline underpinning improvements in the rapid global expansion of ecological restoration. The application of molecular tools over the past 20 years has made an important contribution to understanding genetic factors influencing ecological restoration success. Here we illustrate how recent advances in next generation sequencing (NGS) methods are revolutionising the practical contribution of genetics to restoration. Novel applications include a dramatically enhanced capacity to measure adaptive variation for optimal seed sourcing, high-throughput assessment and monitoring of natural and restored biological communities aboveground and belowground, and gene expression analysis as a measure of genetic resilience of restored populations. Challenges remain in data generation, handling and analysis, and how best to apply NGS for practical outcomes in restoration.

Genetic and palaeo-climatic evidence for widespread persistence of the coastal tree species Eucalyptus gomphocephala (Myrtaceae) during the Last Glacial Maximum.

BACKGROUND AND AIMS: Few phylogeographic studies have been undertaken of species confined to narrow, linear coastal systems where past sea level and geomorphological changes may have had a profound effect on species population sizes and distributions. In this study, a phylogeographic analysis was conducted of Eucalyptus gomphocephala (tuart), a tree species restricted to a 400 × 10 km band of coastal sand-plain in south west Australia. Here, there is little known about the response of coastal vegetation to glacial/interglacial climate change, and a test was made as to whether this species was likely to have persisted widely through the Last Glacial Maximum (LGM), or conforms to a post-LGM dispersal model of recovery from few refugia. METHODS: The genetic structure over the entire range of tuart was assessed using seven nuclear (21 populations; n = 595) and four chloroplast (24 populations; n = 238) microsatellite markers designed for eucalypt species. Correlative palaeodistribution modelling was also conducted based on five climatic variables, within two LGM models. KEY RESULTS: The chloroplast markers generated six haplotypes, which were strongly geographically structured (GST = 0·86 and RST = 0·75). Nuclear microsatellite diversity was high (overall mean HE 0·75) and uniformly distributed (FST = 0·05), with a strong pattern of isolation by distance (r(2) = 0·362, P = 0·001). Distribution models of E. gomphocephala during the LGM showed a wide distribution that extended at least 30 km westward from the current distribution to the palaeo-coastline. CONCLUSIONS: The chloroplast and nuclear data suggest wide persistence of E. gomphocephala during the LGM. Palaeodistribution modelling supports the conclusions drawn from genetic data and indicates a widespread westward shift of E. gomphocephala onto the exposed continental shelf during the LGM. This study highlights the importance of the inclusion of complementary, non-genetic data (information on geomorphology and palaeoclimate) to interpret phylogeographic patterns.

DNA barcoding for conservation, seed banking and ecological restoration of Acacia in the Midwest of Western Australia.

We used DNA barcoding to address an important conservation issue in the Midwest of Western Australia, working on Australia's largest genus of flowering plant. We tested whether or not currently recommended plant DNA barcoding regions (matK and rbcL) were able to discriminate Acacia taxa of varying phylogenetic distances, and ultimately identify an ambiguously labelled seed collection from a mine-site restoration project. Although matK successfully identified the unknown seed as the rare and conservation priority listed A. karina, and was able to resolve six of the eleven study species, this region was difficult to amplify and sequence. In contrast, rbcL was straightforward to recover and align, but could not determine the origin of the seed and only resolved 3 of the 11 species. Other chloroplast regions (rpl32-trnL, psbA-trnH, trnL-F and trnK) had mixed success resolving the studied taxa. In general, species were better resolved in multilocus data sets compared to single-locus data sets. We recommend using the formal barcoding regions supplemented with data from other plastid regions, particularly rpl32-trnL, for barcoding in Acacia. Our study demonstrates the novel use of DNA barcoding for seed identification and illustrates the practical potential of DNA barcoding for the growing discipline of restoration ecology.

Development of microsatellite markers for two Australian Persoonia (Proteaceae) species using two different techniques.

PREMISE OF THE STUDY: Microsatellite markers were developed and cross-species transferability assessed for two Persoonia species to evaluate genetic diversity and population genetic structure of these broadly distributed southwest Australian tree species. • METHODS AND RESULTS: Microsatellite-enriched libraries and 454 GS-FLX shotgun sequencing were used to identity nine microsatellite loci for P. elliptica (one 454; eight cloning) and six for P. longifolia (three 454; three cloning). These loci were screened for variation in individuals from populations in southwestern Australia. In P. elliptica, observed and expected heterozygosities ranged from 0.46 to 0.93 and 0.42 to 0.88, respectively. For P. longifolia, observed and expected heterozygosities ranged from 0.04 to 0.88 and 0.04 to 0.84, respectively. • CONCLUSIONS: The microsatellites identified in this study will enable the examination of population and spatial structuring of genetic diversity in P. elliptica and P. longifolia, two priority species for mine site restoration in southwestern Australia.

Microsatellite primers identified by 454 sequencing in the floodplain tree species Eucalyptus victrix (Myrtaceae).

PREMISE OF THE STUDY: Microsatellite primers were developed for Eucalyptus victrix (Myrtaceae) to evaluate the population and spatial genetic structure of this widespread northwestern Australian riparian tree species, which may be impacted by hydrological changes associated with mining activity. • METHODS AND RESULTS: 454 GS-FLX shotgun sequencing was used to obtain 1895 sequences containing putative microsatellite motifs. Ten polymorphic microsatellite loci were identified and screened for variation in individuals from two populations in the Pilbara region. Observed heterozygosities ranged from 0.44 to 0.91 (mean: 0.66) and the number of alleles per locus ranged from five to 25 (average: 11). • CONCLUSIONS: These microsatellite loci will be useful in future studies of population and spatial genetic structure in E. victrix, and inform the development of seed sourcing strategies for the species.