Hybrid origin and differentiation of two tetraploid Achillea species in East Asia: molecular, morphological and ecogeographical evidence

Achillea (Asteraceae-Anthemideae) offers classical models for speciation by hybridization and polyploidy. Here, we test the suspected allotetraploid origin of two species, Achillea alpina and Achillea wilsoniana between phylogenetically distinct lineages in East Asia. A total of 421 AFLP bands from 169 individuals and 19 populations of five 2x- and two 4x-species were obtained. The data set was analysed with a newly developed model that accounts for polyploidy and assumes lack of recombination between the parental chromosome sets (i.e. disomic inheritance). A. alpina and A. wilsoniana then appear to be allotetraploids between Achillea acuminata-2x (sect. Ptarmica) and Achillea asiatica-2x (sect. Achillea). The two 4x-species share 44% and 48% of their AFLP bands with A. acuminata-2x, and 39% and 38% with A. asiatica-2x, respectively. Eight plastid haplotypes (A-H) were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses. A. alpina-4x and A. wilsoniana-4x share haplotype F only with A. asiatica-2x. This is consistent with the hybrid origin(s) involving the latter as the maternal ancestor. This result corroborates our previous DNA sequence data, where A. alpina-4x and A. wilsoniana-4x are also placed close to A. asiatica-2x. Morphology, ecology, and amplified fragment length polymorphism (AFLP) profiles of the two 2x-species are distinct, whereas the two 4x-species, grouped as A. alpina aggregate, form a nearly continuous link between them. Considering all evidence, this 4x-aggregate is regarded as the product of a hybridization between genetically distant 2x-ancestors limited to China and adjacent areas: one A. acuminata-like, and the other A. asiatica-like. The allopolyploid A. alpina agg. exhibits considerable morphological variation and ecological flexibility, and has expanded throughout eastern Asia and to northern North America, far beyond the ranges of their presumed 2x-ancestors.     

Unraveling the nuclear and chloroplast genomes of an agar producing red macroalga, Gracilaria changii (Rhodophyta,Gracilariales)

Agar and agarose have wide applications in food and pharmaceutical industries. Knowledge on the genome of red seaweeds that produce them is still lacking. To fill the gap in genome analyses of these red algae, we have sequenced the nuclear and organellar genomes of an agarophyte, Gracilaria changii. The partial nuclear genome sequence of G. changii has a total length of 35.8 Mb with 10,912 predicted protein coding sequences. Only 39.4% predicted proteins were found to have significant matches to protein sequences in SwissProt. The chloroplast genome of G. changii is 183,855 bp with a total of 201 open reading frames (ORFs), 29 tRNAs and 3 rRNAs predicted. Five genes: ssrA, leuC and leuD CP76_p173 (orf139) and pbsA were absent in the chloroplast genome of G. changii. The genome information is valuable in accelerating functional studies of individual genes and resolving evolutionary relationship of red seaweeds.     

Molecular systematics,GISH and the origin of hybrid taxa in Nicotiana (Solanaceae)

Phylogenetic relationships in the genus Nicotiana were investigated using parsimony analyses of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (nrDNA). In addition, origins of some amphidiploid taxa in Nicotiana were investigated using the techniques of genomic in situ hybridization (GISH), and the results of both sets of analyses were used to evaluate previous hypotheses about the origins of these taxa. Phylogenetic analyses of the ITS nrDNA data were performed on the entire genus (66 of 77 naturally occurring species, plus three artificial hybrids), comprising both diploid and polyploid taxa, and on the diploid taxa only (35 species) to examine the effects of amphidiploids on estimates of relationships. All taxa, regardless of ploidy, produced clean, single copies of the ITS region, even though some taxa are hybrids. Results are compared with a published plastid (matK) phylogeny using fewer, but many of the same, taxa. The patterns of relationships in Nicotiana, as seen in both analyses, are largely congruent with each other and previous evolutionary ideas based on morphology and cytology, but some important differences are apparent. None of the currently recognized subgenera of Nicotiana is monophyletic and, although most of the currently recognized sections are coherent, others are clearly polyphyletic. Relying solely upon ITS nrDNA analysis to reveal phylogenetic patterns in a complex genus such as Nicotiana is insufficient, and it is clear that conventional analysis of single data sets, such as ITS, is likely to be misleading in at least some respects about evolutionary history. ITS sequences of natural and well-documented amphidiploids are similar or identical to one of their two parents-usually, but not always, the maternal parent-and are not in any sense themselves 'hybrid'. Knowing how ITS evolves in artificial amphidiploids gives insight into what ITS analysis might reveal about naturally occurring amphidiploids of unknown origin, and it is in this perspective that analysis of ITS sequences is highly informative.     

The role of climate in the dynamics of a hybrid zone in Appalachian salamanders

I examined the potential influence of climate change on the dynamics of a previously studied hybrid zone between a pair of terrestrial salamanders at the Coweeta Hydrologic Laboratory, U.S. Forest Service, in the Nantahala Mountains of North Carolina, USA. A 16‐year study led by Nelson G. Hairston, Sr. revealed that Plethodon teyahalee and Plethodon shermani hybridized at intermediate elevations, forming a cline between ‘pure’ parental P. teyahalee at lower elevations and ‘pure’ parental P. shermani at higher elevations. From 1974 to 1990 the proportion of salamanders at the higher elevation scored as ‘pure’P. shermani declined significantly, indicating that the hybrid zone was spreading upward. To date there have been no rigorous tests of hypotheses for the movement of this hybrid zone. Using temperature and precipitation data from Coweeta, I re‐analyzed Hairston's data to examine whether the observed elevational shift was correlated with variation in either air temperature or precipitation from the same time period. For temperature, my analysis tracked the results of the original study: the proportion of ‘pure’P. shermani at the higher elevation declined significantly with increasing mean annual temperature, whereas the proportion of ‘pure’P. teyahalee at lower elevations did not. There was no discernable relationship between proportions of ‘pure’ individuals of either species with variation in precipitation. From 1974 to 1990, low‐elevation air temperatures at the Coweeta Laboratory ranged from annual means of 11.8 to 14.2 °C, compared with a 55‐year average (1936–1990) of 12.6 °C. My re‐analyses indicate that the upward spread of the hybrid zone is correlated with increasing air temperatures, but not precipitation, and provide an empirical test of a hypothesis for one factor that may have influenced this movement. My results aid in understanding the potential impact that climate change may have on the ecology and evolution of terrestrial salamanders in montane regions.     

Instar sizes,life cycles,and food habits of five Rhyacophila (Trichoptera: Rhyacophilidae) species from the Appalachian Mountains of South Carolina,U.S.A.

The larval head widths at each instar, life cycles, and food habits of late instars were determined for five species of Rhyacophila from two Appalachian mountain streams in South Carolina, U.S.A. Rhyacophila acutiloba Morse & Ross was univoltine with two cohorts, one emerging in the spring and another presumably emerging in early autumn. Rhyacophila fuscula (Walker), R. nigrita Banks, and R. carolina Banks were apparently multicohort, univoltine species with extended flight periods. Rhyacophila minor Banks was univoltine with a spring emergence. All species were predaceous and consumed mainly Plecoptera nymphs and Trichoptera larvae.     

A new hypothesis for the origin of pentaploid Holcus from diploid Holcus lanatus L. and tetraploid Holcus mollis L. in France

The Holcus complex in France consists of two species, Holcus lanatus L. (Yorkshire fog; 2n= 2x= 14) and Holcus mollis L. (Creeping soft-grass; 2n= 4x= 28) and an interspecific hybrid H.m.×H.l. (2n= 5x= 35), which is morphologically similar to Holcus mollis. A heterologous rDNA probe from wheat was used to detect the corresponding region in Holcus (s. l.) genomic DNA'fragments, for six to eight plants from 13 populations located south-west of Paris. A restriction enzyme map of the ribosomal RNA genes (rDNA) in Holcus (s. l.) was also constructed. The length polymorphism detected in the IGS region was used as a DNA fingerprint for the identification of different cytotypes and species of the Holcus complex and for the typing and delimitation of individuals in populations. In the light of the results we reconsider the assumption that the pentaploid hybrid H.m.×H.l. is purely clonal. New hypotheses concerning the origin of the pentaploid hybrid and its reproduction are proposed, and the consequences for genetic diversity in natural populations discussed.     

The role of hybridization, polyploidization and glaciation in the origin and evolution of the apomictic Ranunculus cassubicus complex

The Ranunculus cassubicus complex, comprising diploids and polyploids, is a good model for studying the role of hybridization and polyploidy in the origin of apomixis. Results from amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) analyses performed on 448 individuals were combined with evidence from morphology, isozymes, karyology and distribution. Our results indicated a unique hybrid origin for the apomictic hexaploid R. carpaticola from north-western Slovakia, involving two sexual parents: autotetraploid R. cassubicifolius from the northern pre-Alps, and diploid R. carpaticola from central Slovakia. The hybrids were intermediate to the parents, but unique alleles have resulted from genomic reorganisation in the allopolyploids, which might also have triggered apomixis. Their distribution patterns and estimated ages suggest that hybridization may be correlated with the last glacial period. Hybridization seems to be the major origination for apomicts in the R. cassubicus complex. Polyploidy creates novel sexual genotypes and acts as a springboard for the production of new hybrids, but it only results in a combination with hybridization in apomixis. In turn, asexuality has permitted the perpetuation and establishment of ecologically divergent hybrid genotypes.     

Distribution,functional impact,and origin mechanisms of copy number variation in the barley genome

Background

There is growing evidence for the prevalence of copy number variation (CNV) and its role in phenotypic variation in many eukaryotic species. Here we use array comparative genomic hybridization to explore the extent of this type of structural variation in domesticated barley cultivars and wild barleys.

Results

A collection of 14 barley genotypes including eight cultivars and six wild barleys were used for comparative genomic hybridization. CNV affects 14.9% of all the sequences that were assessed. Higher levels of CNV diversity are present in the wild accessions relative to cultivated barley. CNVs are enriched near the ends of all chromosomes except 4H, which exhibits the lowest frequency of CNVs. CNV affects 9.5% of the coding sequences represented on the array and the genes affected by CNV are enriched for sequences annotated as disease-resistance proteins and protein kinases. Sequence-based comparisons of CNV between cultivars Barke and Morex provided evidence that DNA repair mechanisms of double-strand breaks via single-stranded annealing and synthesis-dependent strand annealing play an important role in the origin of CNV in barley.

Conclusions

We present the first catalog of CNVs in a diploid Triticeae species, which opens the door for future genome diversity research in a tribe that comprises the economically important cereal species wheat, barley, and rye. Our findings constitute a valuable resource for the identification of CNV affecting genes of agronomic importance. We also identify potential mechanisms that can generate variation in copy number in plant genomes.     

Longitudinal and seasonal patterns of stream acidity in a headwater catchment on the Appalachian Plateau, West Virginia, U.S.A.

The chemical composition during baseflow was used to elucidate the fundamental processes controlling longitudinal and seasonal patterns of stream acidity in Yellow Creek, a chronically acidic headwater (pH range 3.7--4.2) on the Appalachian Plateau in northeastern West Virginia. Sulfate concentrations controlled the variability of stream acidity within the Yellow Creek catchment. Decreases in stream free H⁺ acidity with decreasing elevation likely resulted from SO ₄ ^2– retention in riparian wetland areas as well as spatial variation in dominant tree species. Seasonal variations in free H⁺ and inorganic monomeric aluminum (Alⁿ⁺) concentrations appeared related to seasonal fluctuations in baseflow discharge which was controlled by vegetative activity. Baseflow stream discharge, as well as H⁺ and Alⁿ⁺ acidity, gradually declined during the growing season (June through October), likely reflecting microbial SO ₄ ^2– > reduction in saturated anaerobic environments within riparian wetlands. A marked pulse of stream H⁺, Alⁿ⁺, and SO ₄ ^2– coincided with an abrupt increase in baseflow discharge resulting from the cessation of transpiration after leaf-fall in November. This seasonal pattern suggests that autumn may be a critical period for eastern brook trout in streams draining wetlands on the Appalachian Plateau.     

Genomic origin and organization of the allopolyploid Primula egaliksensis investigated by in situ hybridization

Background and Aims: Earlier studies have suggested that the tetraploid Primula egaliksensis(2n = 40) originated from hybridization between the diploidsP. mistassinica (2n = 18) and P. nutans (2n = 22), which werehypothesized to be the maternal and paternal parent, respectively.The present paper is aimed at verifying the hybrid nature ofP. egaliksensis using cytogenetic tools, and to investigatethe extent to which the parental genomes have undergone genomicreorganization. Methods: Genomic in situ hybridization (GISH) and fluorescent in situhybridization (FISH) with ribosomal DNA (rDNA) probes, togetherwith sequencing of the internal transcribed spacer (ITS) regionof the rDNA, were used to identify the origin of P. egaliksensisand to explore its genomic organization, particularly at rDNAloci. Key Results: GISH showed that P. egaliksensis inherited all chromosomes fromP. mistassinica and P. nutans and did not reveal major intergenomicrearrangements between the parental genomes (e.g. interchromosomaltranslocations). However, karyological comparisons and FISHexperiments suggested small-scale rearrangements, particularlyat rDNA sites. Primula egaliksensis lacked the ITS-bearing heterochromaticknobs characteristic of the maternal parent P. mistassinicaand maintained only the rDNA loci of P. nutans. These resultscorroborated sequence data indicating that most ITS sequencesof P. egaliksensis were of the paternal repeat type. Conclusions: The lack of major rearrangements may be a consequence of theconsiderable genetic divergence between the putative parents,while the rapid elimination of the ITS repeats from the maternalprogenitor may be explained by the subterminal location of ITSloci or a potential role of nucleolar dominance in chromosomestabilization. These small-scale rearrangements may be indicativeof genome diploidization, but further investigations are neededto confirm this assumption.     

Allopolyploidy in the Wintergreen Group of tribe Gaultherieae (Ericaceae) inferred from low‐copy nuclear genes

DNA sequence data from the low‐copy nuclear genes waxy (GBSSI) and leafy were compared with plastid and ITS sequence data from prior studies to reconstruct phylogenetic relationships in the Wintergreen Group of tribe Gaultherieae (Ericaceae). We conducted phylogenetic analysis with 102 species that includes representatives of all 15 major clades previously identified within the Wintergreen Group and that together span its circum‐Pacific distribution. Results yielded two distinct homeologous copies of waxy for two of the clades, each in widely separated parts of the tree. It also yielded two copies of leafy for one of the clades; only one copy of leafy was found for the other clade, but it was placed in the same major clade as its waxy counterpart and well away from its placement in a prior plastid analysis. A combined four‐locus (waxy, leafy, ITS and plastid data) phylogenetic analysis of all available relevant data placed the copies of each of the clades in two distinct positions in the phylogeny with strong overall statistical support. In combination with evidence from morphology, reproductive biology and cytology, the results suggest that these clades arose through allopolyploid hybridization between lineages deep in the phylogeny but relatively close geographically. This finding confirms previous assumptions that hybridization has played an important role in the evolution of the Gaultherieae.     

Geographical variation and systematics of the tetraploid marsh orchid Dactylorhiza majalis subsp. sphagnicola (Orchidaceae) and closely related taxa

Dactylorhiza majalis subsp. sphagnicola is an allotetraploid marsh orchid derived from parents closely similar to present‐day D. incarnata and the western European form of D. maculata subsp. maculata, suggesting that it has a postglacial origin. It extends from northwestern continental Europe into areas formerly covered by the Weichselian ice sheet in mid‐Scandinavia. Here, we studied the variation at both the plastid and nuclear marker systems to describe the geographical variation in subsp. sphagnicola and its evolutionary history. We investigated whether subsp. sphagnicola is affected by secondary hybridization and gene flow from its parental lineages or from other allotetraploid marsh orchids, and we also compared subsp. sphagnicola with other allotetraploids of similar origins. We analysed 492 plants from 50 populations. Thirty‐seven populations were collected as potential Dactylorhiza majalis subsp. sphagnicola, five as subsp. sesquipedalis (D. elata), one as D. elata subsp. brennensis, one as subsp. calcifugiens, one as subsp. occidentalis and the remaining five as populations with some affinity to subsp. lapponica (including D. traunsteineri). All populations were analysed for plastid haplotypes and nuclear internal transcribed spacer (ITS) allele frequencies, and a subset of 43 populations was analysed for five nuclear microsatellite loci. Dactylorhiza majalis subsp. sphagnicola was dominated by a single plastid haplotype that was also dominant in western European D. maculata subsp. maculata, and most of the alternative haplotypes differed by only one mutation from the dominant one. There was more variation in nuclear microsatellites and ITS, and the variation was geographically structured in these markers. Subspecies occidentalis and calcifugiens shared haplotypes with subsp. sphagnicola, whereas subsp. sesquipedalis and brennensis had other haplotypes. Dactylorhiza majalis subsp. sphagnicola may have a postglacial origin within its present continental distribution. It has incorporated genetic material from D. maculata subsp. maculata by secondary hybridization and introgression, and some northern populations have assimilated strongly divergent haplotypes from the northeastern form of D. maculata subsp. maculata. Subspecies sphagnicola has also evolved morphologically divergent local populations in the north that do not differ from the typical populations in genetic markers. It may form mixed populations with other allotetraploid subspecies of D. majalis and, at least at one site, it has become integrated with subsp. lapponica, demonstrating that independently derived allotetraploids may contribute to a common gene pool. Subspecies calcifugiens seems to be derived from subsp. sphagnicola, and further studies based on a larger sample may confirm that it is better recognized as a variety. The so‐called D. elata subsp. brennensis is of hybrid origin and combines markers from subsp. sesquipedalis with markers from the D. majalis core complex, possibly subsp. majalis. The new combination Dactylorhiza majalis subsp. sesquipedalis (Willd.) H.A.Pedersen & Hedrén comb. nov. is provided. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 168 , 174–193.     

AFLP analyses demonstrate genetic divergence, hybridization, and multiple polyploidization in the evolution of Achillea (Asteraceae-Anthemideae)

Achillea, a temperate genus of herbaceous allogamous perennials, is a model for evolutionary radiation through hybridization and polyploidization. AFLP analyses were performed on 300 individuals of 66 populations and 27 taxa/cytotypes, mainly from the polyploid A. millefolium aggregate and its suspected hybrid links with other clades of the genus. The mosaic genetic structure of hybrids and polyploids is revealed by specific AFLP bands shared with their assumed parents. In E Asia, A. alpina-4x and A. wilsoniana-4x are allotetraploids between A. acuminata-2x (sect. Ptarmica) and A. asiatica-2x (sect. Achillea-A. millefolium agg.). A. virescens-4x is a hybrid species linking A. nobilis agg. and A. millefolium agg. in S Europe. The hybrid swarm A. clypeolata-2x yen A. collina-4x recently formed in Bulgaria shows no AFLP bands additive to its parents; by contrast, other more ancient allopolyploids exhibit genetic innovations. Relationships within A. millefolium agg. are complex. Five 2x-taxa, mostly well separated and regressive, are limited to Eurasia; seven 4x- and 6x-taxa are intimately linked by hybridization, are expansive, and through A. asiatica-2x/4x have formed the N American polyploids. All these results from AFLPs correspond well to other evidence, and indicate a long history of reticulate evolution in Achillea.     

Gradient analysis of the distribution of a fungal disease of Cornus florida in the southern Appalachian Mountains,Tennessee

Abstract: Based on 115 samples collected throughout the western portion of Great Smoky Mountains National Park, southern Appalachian Mts., and on spatial data derived in a GIS (Geographical Information System), the distribution of the disease dogwood anthracnose affecting Cornus florida (flowering dogwood), caused by the fungus Discula destructiva in this portion of the Park was assessed, and factors contributing to the disease's severity were identified through correlation and multiple linear regression analysis. The degree of infection varies considerably locally, and is influenced by elevation, slope curvature, slope position, and potential soil moisture. However, the abundance of C. florida (stem density) alone explains 25 % of the variation in disease severity. Factors contributing to disease severity do not change significantly between disturbed and undisturbed sites. The highest mortality rates are restricted to dense stands in damp, sheltered sites at low slope positions, implying that surviving populations of flowering dogwood may represent a biased genetic subset of the original population.     

Barrier to gene flow between two ecologically divergent Populus species, P. alba (white poplar) and P. tremula (European aspen): the role of ecology and life history in gene introgression

The renewed interest in the use of hybrid zones for studying speciation calls for the identification and study of hybrid zones across a wide range of organisms, especially in long-lived taxa for which it is often difficult to generate interpopulation variation through controlled crosses. Here, we report on the extent and direction of introgression between two members of the "model tree" genus Populus: Populus alba (white poplar) and Populus tremula (European aspen), across a large zone of sympatry located in the Danube valley. We genotyped 93 hybrid morphotypes and samples from four parental reference populations from within and outside the zone of sympatry for a genome-wide set of 20 nuclear microsatellites and eight plastid DNA restriction site polymorphisms. Our results indicate that introgression occurs preferentially from P. tremula to P. alba via P. tremula pollen. This unidirectional pattern is facilitated by high levels of pollen vs. seed dispersal in P. tremula (pollen/seed flow = 23.9) and by great ecological opportunity in the lowland floodplain forest in proximity to P. alba seed parents, which maintains gene flow in the direction of P. alba despite smaller effective population sizes (N(e)) in this species (P. alba N(e)c. 500-550; P. tremula N(e)c. 550-700). Our results indicate that hybrid zones will be valuable tools for studying the genetic architecture of the barrier to gene flow between these two ecologically divergent Populus species.     

Genetic admixture supports an ancient hybrid origin of the endangered Hawaiian duck

Speciation is regarded primarily as a bifurcation from an ancestral species into two distinct taxonomic units, but gene flow can create complex signals of phylogenetic relationships, especially among different loci. We evaluated several hypotheses that could account for phylogenetic discord between mitochondrial DNA (mtDNA) and nuclear DNA (nuDNA) within Hawaiian duck (Anas wyvilliana), including stochastic lineage sorting, mtDNA capture and widespread genomic introgression. Our results best support the hypothesis that the contemporary Hawaiian duck is descended from an ancient hybridization event between the mallard (Anas platyrhynchos) and Laysan duck (Anas laysanensis). Whereas mtDNA clearly shows a sister relationship between Hawaiian duck and mallard, nuDNA is consistent with a genetic mosaic with nearly equal contributions from Laysan duck and mallard. In addition, coalescent analyses suggest that gene flow from either mallard or Laysan duck, depending on the predefined tree topology, is necessary to explain contemporary genetic diversity in Hawaiian ducks, and these estimates are more consistent with ancient, rather than contemporary, hybridization. Time since divergence estimates suggest that the genetic admixture event occurred around the Pleistocene–Holocene boundary, which is further supported by circumstantial evidence from the Hawaiian subfossil record. Although the extent of reproductive isolation from either putative parental taxon is not currently known, these species are phenotypically, genetically and ecologically different, and they meet primary criteria used in avian taxonomy for species designation. Thus, the available data are consistent with an admixed origin and support the hypothesis that the Hawaiian duck may represent a young hybrid species.