Volume 101, Issue 10 p. 1686-1699
Special Invited Paper
Free Access

Fragaria: A genus with deep historical roots and ripe for evolutionary and ecological insights

Aaron Liston

Corresponding Author

Aaron Liston

Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331 USA

Author for correspondence (e-mail: [email protected])Search for more papers by this author
Richard Cronn

Richard Cronn

Pacific Northwest Research Station, USDA Forest Service, Corvallis, Oregon 97331 USA

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Tia-Lynn Ashman

Tia-Lynn Ashman

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 USA

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First published: 01 October 2014
Citations: 119

Freely available online through the AJB open access option.

The authors thank Vivian Lee Bowden, Michel Chauvet, Michelle Coyne, and Alice Telka for strawberry information; Michel Chauvet and Ariadne Luh for assistance with French translation; Branko Bakan, Gerald Carr, Luxi Chen, and Shiro Tsuyuzaki for permission to reproduce their photographs; and Matt Koski, Jacob Tennessen, Dan Potter, and an anonymous reviewer for comments on a draft of the manuscript. Our strawberry research is supported by the National Science Foundation (DEB 1020523 and DEB 1241006 to T.L.A., DEB 1020271 to A.L., and DEB 1241217 to A.L. and R.C.).

Abstract

The cultivated strawberry, Fragaria ×ananassa, is one of the youngest domesticated plants. Its 18th century origin via hybridization in Europe between the North American F. virginiana and the South American F. chiloensis was documented by the botanist Antoine Nicolas Duchesne. His 1766 “Natural History of Strawberries” is an extraordinary work that integrates fundamental discoveries on the biology, ecology, and phylogeny of Fragaria with applied information on cultivation and ethnobotanical uses, serving as an inspiration for current research in the genus. Fragaria species exhibit the full range of sexual systems in the gynodioecy pathway from hermaphroditism to dioecy (and back again), as well as variation in self-compatibility, and evidence of sex chromosomes with female heterogamety. The genus is also characterized by interspecific hybridization and polyploidy, with a natural range of ploidy levels from diploids to decaploids. This biological diversity, combined with the availability of genomic resources and the ease of growing and experimenting with the plants, makes Fragaria a very attractive system for ecological and evolutionary genomics. The goal of this review is to introduce Fragaria as a model genus and to provide a roadmap for future integrative research. These research directions will deepen our understanding of the ecological and evolutionary context that shaped the ancestors of the cultivated strawberry, not only providing information that can be applied to efforts to shape the future of this important fruit crop but also our understanding of key transitions in plant evolution.

Strawberries are an important food plant. Global strawberry production is twice the amount of all other berry crops combined (Stewart, 2011) and U.S. per capita consumption doubled between 1970 and 2004 (Boriss et al., 2006). Today, fresh consumption of strawberries trails only banana, apple, watermelon, and grape (USDA Economic Research Service, 2012). While primarily valued for their flavor, strawberries also have potential health benefits. Medicinal claims for strawberries have been made for centuries (Duchesne, 1766). Strawberries are high in the nutrients vitamin C, folate, and manganese (Giampieri et al., 2012). They are also rich in phenolic compounds, including anthocyanins, hydrolyzable tannins, and phenolic acids (Giampieri et al., 2012). Vitamin C and phenolic compounds are efficient antioxidants, and the potential role of strawberry consumption in human health and disease prevention is an active research area (Romandini et al., 2013). Tempering the enthusiasm for strawberry consumption is their prominent position on the list of foods with the highest pesticide residues (Environmental Working Group, 2013). Resistance to a Fragaria-specific powdery mildew (Podosphaera macularis) has been demonstrated in F. ×ananassa transformed with a peach locus (Jiwan et al., 2013), and the cultivation of such transgenic plants could reduce pesticide usage in strawberry. While transgenic strawberries are an important research tool (Folta, 2013), current prospects for their commercialization are limited, due to public resistance, a lack of industry support, and concerns over gene flow to the wild species of Fragaria (Mezzetti, 2009; Hummer et al., 2011).

An educationally important use of strawberries is for demonstrating DNA extraction (Gonda et al., in press). An advantage of using strawberries in this application is that they can be placed in a plastic bag and easily liquefied by hand. The cells are lysed with detergent (dish washing liquid or shampoo), filtered through a coffee filter, and DNA is precipitated in the presence of salt and ethanol (GenomeTV, 2010). The octoploid genome of the cultivated strawberry likely contributes to the abundant amounts of DNA obtained from this simple procedure.

Wild Fragaria (Rosaceae) species occur across the northern hemisphere and disjunctly in southern South America (Fig. 1, Table 1). Humans have likely consumed the fruits of wild Fragaria species for millennia. While archaeobotanical evidence is limited (Walshaw, 2009), Fragaria achenes have been documented from two pre-Columbian sites in eastern North America (Gremillion and Sobolik, 1996; Pauketat et al., 2002). Fragaria chiloensis was domesticated by the Picunche and Mapuche people of Chile over 1000 yr ago (Finn et al., 2013). In Europe, F. vesca has been grown in gardens at least since the time of the Romans, and F. moschata since the 16th century (Wilhelm & Sagen, 1974). The modern cultivated strawberry, Fragaria ×ananassa originated in the 18th century in Europe from hybridization between two species imported from North and South America (Duchesne, 1766). The parental species, F. virginiana and F. chiloensis, also hybridize naturally in northwestern North America (Luby et al., 1992; Salamone et al., 2013), but there is no evidence that they were ever cultivated by the native Americans in this area. However, these hybrids represent an untapped opportunity to study the genetic equivalent of the domesticated strawberry in a natural ecological context (Table 2).

Details are in the caption following the image

Approximate geographic distribution of Fragaria species and ploidy. Due to uncertainty over species boundaries, the six endemic Chinese species are designated as diploids (F. chinensis, F. pentaphylla) or tetraploids (F. corymbosa, F. gracilis, F. moupinensis, F. tibetica). Data sources include the GBIF data portal (GBIF, 2014), the “Wild Strawberry” Dimensions of Biodiversity US-China project website (Ashman et al., 2014), published distribution maps (Staudt, 1999a, 1999b, 2003b, 2005, 2006, 2008; Staudt and Dickoré, 2001; Chukhina, 2008; Staudt and Olbricht, 2008; Rousseau-Gueutin et al., 2009), and base map (Shorthouse, 2010).

Table 1. Fragaria species and their ploidy level, mating system and geographic distribution.
Taxon Ploidy Mating system Distribution
Vesca clade
F. ×ananassa Duchesne subsp. ananassa 8x Subdioecious (modern cultivars: hermaphroditic) cultivated
F. ×ananassa Duchesne subsp. cuneifolia (Nutt. ex Howell) Staudt 8x Subdioecious NW N. America
F. bucharica Losinsk. 2x Hermaphrodite SI W Himalayas
F. cascadensis Hummer 10x Subdioecious Oregon, USA
F. chiloensis (L.) Duchesne 8x Subdioecious Alaska-California; Hawaii; Chile, Argentina
F. iturupensis Staudt 8x, 10x Subdioecious Iturup Island
F. mandshurica Staudt 2x Hermaphrodite SI NE Asia
F. moschata Duchesne 6x Dioecious W Eurasia
F. orientalis Losinsk. 4x Dioecious NE Asia
F. vesca L. subsp. americana (Porter) Staudt 2x Hermaphrodite SC NE N. America
F. vesca L. subsp. bracteata (A. Heller) Staudt 2x Gynodioecious or Hermaphrodite SC W N. America
F. vesca L. subsp. californica (Cham. & Schldl.) Staudt 2x Gynodioecious or Hermaphrodite SC SW N. America
F. vesca L. subsp. vesca L. 2x Hermaphrodite SC W Eurasia, also introduced (see Fig. 1)
F. virginiana Duchesne 8x Subdioecious N. America
China clade
F. chinensis Losinsk. 2x Hermaphrodite SI China
F. corymbosa Losinsk. 4x Dioecious China
F. daltoniana J. Gay 2x Hermaphrodite SC Nepal, adjacent China
F. gracilis Losinsk. 4x Dioecious China
F. moupinensis (Franch.) Cardot 4x Dioecious China
F. nipponica Makino 2x Hermaphrodite SI Japan
F. nubicola Lindl. 2x Hermaphrodite SI Himalayas
F. pentaphylla Losinsk. 2x Hermaphrodite SI China
F. tibetica Staudt & Dickore 4x Dioecious China
Unresolved phylogenetic position
F. hayatai Makino 2x Hermaphrodite SC? Taiwan
F. iinumae Makino 2x Hermaphrodite SC Japan
F. nilgerrensis Schltdl. ex J. Gay 2x Hermaphrodite SC SE Asia
F. viridis Duchesne 2x Hermaphrodite SI W Eurasia
Table 2. Potential research areas that can be addressed in Fragaria through a combination of phylogenetic and population genomics/genetic approaches, and ecological, physiological or developmental biology.
Research Area/Ecological question Example of Fragaria species that can be used to address issue Current understanding in Fragaria Exemplar references
A) Sexual and mating system evolution
 How do sex chromosomes evolve? Octoploid clade Evidence for proto-sex chromosomes; dynamic sex determining regions. Spigler et al., 2008, 2010; Goldberg et al., 2010; Tennessen et al., 2013
 How do separate sexes evolve from hermaphroditism? Genus-wide Ecological drivers include inbreeding depression, stressful habitats and antagonists. Ashman, 1999, 2005; Ashman and Penet, 2007
 What genetic mechanisms underlie male (female) sterility? Gynodioecious F. vesca subsp. bracteata Novel form of nuclear dominant male sterility. Tennessen et al., 2013
 How does the polyploidy–dioecy association arise? Diploid–polyploid ancestor–descendent pairs Dioecy is not associated with loss of SI, but polyploidization can alter sex allocation. Njuguna et al., 2013; Ashman et al., 2013; Kwok, 2013
 Is self incompatibility reversible? Is selfing a dead end? SI species/SC species Both loss and gain are possible in Fragaria; genetic dissection suggests multiple S-loci. Bošković et al., 2010; Njuguna et al., 2013
B) Speciation, hybridization, polyploidy and range expansion
 How does polyploidy contribute to ecological and evolutionary amplitude? Diploid–polyploid ancestor–descendent pairs Based on current understanding range size is related to species age but not ploidy. Johnson et al., 2014
 Are polyploids the result of single or multiple origins? Diploid–polyploid ancestor–descendent pairs Two octoploid ancestors identified, ancestry of other polyploids. uncertain; single or multiple origins untested for all polyploids. Rousseau-Gueutin et al., 2009; Njuguna et al., 2013
 To what degree are polyploid subgenomes repatterned? Diploid–polyploid ancestor–descendent pairs Sex locus is in different locations in F. chiloensis and F. virginiana. Spigler et al., 2008, 2010; Goldberg et al., 2010; Tennessen et al., 2013
 How reproductively isolated are species? Is the hybrid zone stable, or does hybridization contribute to adaptation? Natural hybrids
F. ×ananassa subsp. cuneifolia
F. ×bifera
Pattern of introgression in subsp. cuneifolia suggests geographic limitations that may be reinforced by selection via bioclimate. Salamone et al., 2013
 How do speciation genes arise? What are the molecular mechanisms of reproductive isolation? Diploid species Variation in interfertility among diploid species, yet unrelated to genetic distance. Bors and Sullivan, 2005; Nosrati et al., 2011b
 What contributes to successful invasion? What are the molecular mechanisms of weediness? F. vesca subsp. vesca established outside of native range Evidence of establishment in multiple independent locations (Hawaii, Andes, Azores, La Réunion, Madagascar, New Zealand). Staudt, 1999a; Plantwise Knowledge Bank, 2014
C) Life-history transitions
 How does flowering time evolve? Wide latitudinal range in F. vesca subsp. vesca Central genetic components of flowering time pathways, but with novel regulatory mechanisms, have been identified. Mouhu et al., 2009
 How does drought tolerance evolve and by what genetic or functional pathways? F. chiloensis vs. F. virginiana Potential candidate genes in control of plant responses to drought stress identified. Razavi et al., 2011; Šurbanovski et al., 2013
 How does frost tolerance evolve and by what genetic or functional pathways? Wide latitudinal range in F. vesca subsp. vesca Clinal variation in frost hardiness with latitude of population origin. Sønsteby and Heide, 2011
 What genetic or functional pathways control allocation between sexual/asexual reproduction or alternative strategies for clonal growth? Sympodial and monopodial species and runnerless variants of F. vesca Genetic dissection of regulation of flowering time in response to temperature and photoperiod. Clonal growth strategies enhance fitness via resource sharing. Alpert, 1991; Hytönen et al., 2009; Koskela et al., 2012
 How do fleshy fruits evolve from dry fruits? Fragaria vs. other members of Potentilleae Transcriptomic profiling of early fruit development. Hollender et al., 2014
 How does woodiness evolve from herbaceousness (or vice versa)? Fragaria vs. Dasiphora fruticosa Transcriptome comparison. T. Poulson and D. Hearn, Towson University, unpublished manuscript

The sequencing and annotation of a genome provides access to the genes, their putative functions, and their genomic locations, as well as a reference that can be used to assay nucleotide variation and gene expression across individuals and conditions (Bevan and Uauy, 2013). The genome sequence of Fragaria vesca was a scientific milestone, representing the first published plant genome obtained solely with next-generation sequencing technology (Shulaev et al., 2011). Fragaria vesca has several attributes that made it an attractive target for genome sequencing. It is a diploid with a relatively small 206-Mbp genome (Longhi et al., 2014). Compared with most other cultivated perennials, the plants are compact, easily grown under controlled conditions, and can be propagated both sexually and asexually (Slovin and Michael, 2011). An efficient transformation system for F. vesca has been developed (Oosumi et al., 2006) and transposon-tagged mutants are available for reverse genetics applications (Veilleux et al., 2012). The genome sequence of F. vesca has already facilitated significant progress in functional genomics of flowering (Mouhu et al., 2013; Hollender et al., 2014) and fruiting (Kang et al., 2013), and these advances will ultimately be applied to crop improvement efforts (Folta, 2013; Longhi et al., 2014). More immediate progress is anticipated from the vast numbers of genetic markers developed from the genome sequence of F. vesca that can be used in genetic linkage mapping (Tennessen et al., 2013) and high-throughput assays for marker-assisted breeding (Chambers et al., 2013). The cultivated strawberry F. ×ananassa (which, like F. vesca, is readily transformed genetically; Shulaev et al., 2008) has also been used as a “taxonomic surrogate” for peach (a species not amenable to tissue culture) in a recent study of a mildew resistance locus isolated from peach and shown to be functional in strawberry (Jiwan et al., 2013). These results illustrate the kind of “cross-fertilization” that can occur with the development of genomic resources across Rosaceae (Shulaev et al., 2008; Longhi et al., 2014).

The small genome size in Fragaria and the availability of a reference sequence also facilitate comparative genomics, and a recent study has included whole genome sequencing of the cultivated strawberry and four wild relatives (Hirakawa et al., 2014). The biological diversity of wild Fragaria (Table 1) has been regularly used in breeding efforts as a source of novel genetic variation that can be introgressed into the cultivated strawberry (Hancock, 1999; Chambers et al., 2013). It is less appreciated that the biological diversity of Fragaria, especially with respect to sexual system and polyploidy, also makes it a very attractive system for ecological and evolutionary genomics. The goal of this review is to introduce Fragaria as a model genus and to provide a roadmap for future integrative research in these and other areas.

MORPHOLOGY, SPECIES, AND PHYLOGENY OF FRAGARIA

Technically, the strawberry is an aggregate accessory fruit. The botanical fruits are dry achenes that are embedded in a fleshy receptacle. This morphological combination is shared by all species of Fragaria and has apparently evolved in parallel three times in Rosaceae tribe Potentilleae (Eriksson et al., 1998). The other occurrences are in Comarum palustre and in two species of Potentilla that were formerly segregated as the genus Duchesnea. The coordinated development of seed maturation in the achene with the softening and expansion of the receptacle in Fragaria provides a unique opportunity to investigate signaling between plant organs (Kang et al., 2013). It would be instructive to extend this study of gene expression in a developmental context to the other two cases of this unusual fruit morphology to determine whether the same gene expression patterns occur.

The same basic life history is shared by all Fragaria species: they are insect-pollinated, low-growing, herbaceous perennials capable of clonal growth, and have animal-dispersed fleshy accessory fruits (Johnson et al., 2014). The floral and vegetative morphology of Fragaria is also relatively uniform. The leaves are usually evergreen (F. iinumae is deciduous), and generally trifolioliate. Five leaflets are typical of some Chinese species, while 4–5 leaflets are rarely observed in F. virginiana (Kellerman, 1892) and F. cascadensis (Hummer, 2012). Flowers are always actinomorphic, white (sometimes tinged with pink), and usually 5-petalled (F. iinumae has 6–8 petals, Fig. 2D). In some species, staminate and pistillate flowers are readily distinguished (Fig. 2A, B), but in others (e.g., gynodioecious Fragaria vesca subsp. bracteata) the pistillate flowers have anthers and are very similar to the bisexual ones (Li et al., 2012; Ashman et al., 2012). The mature receptacles (“fruits”) are more diverse and can be used to differentiate species (Fig. 2E–I). However, they are also quite variable within a species, and because their characteristic features (color, shape, and achene and calyx positions at maturity) are “inevitably destroyed” (Staudt, 1999a) in herbarium specimens, they are not well documented in the botanical literature. For this reason, identification keys tend to focus on details of leaf texture, dentation, venation, and pubescence. Further complicating identification is extensive phenotypic plasticity within species (Chabot and Chabot, 1977; Jurik et al., 1982). The morphology of the stolons is also a reliable diagnostic character (Staudt, 2008), with some species having monopodial stolons (after the first sterile internode, a plantlet can develop at every internode) and others sympodial (plantlets and new stolons develop at alternate internodes, Fig. 2C). Like fruits and flower sexuality, this trait is best observed in living specimens. Achene size is known to differ between diploid and tetraploid plants of F. mandshurica and F. orientalis (Staudt, 2003b), and between diploids of F. vesca and the North American octoploids (Staudt, 1999a). However, its reliability for ploidy estimation across a diversity of species and populations remains to be determined.

Details are in the caption following the image

Morphological diversity of Fragaria. (A) F. chiloensis, staminate flower. (B) F. chiloensis, pistillate flower. (C) F. chiloensis, sympodial stolons, with sterile node (red arrow) alternating with fertile node (yellow arrow). (D) F. iinumae, flower with 7 petals, 6–9 petals are characteristic of this species. (E) F. iinumae, achenes in shallow pits on the receptacle, also found in the octoploid clade. Also note the glaucous leaflets, a characeteristic shared with F. virginiana. (F) F. vesca, achenes raised above the surface of the receptacle. (G) Tetraploid F. moupinensis (back) and diploid F. pentaphylla (front). Note that F. moupinensis is larger, and the central leaflet overlaps the two lateral leaflets. (H) F. daltoniana, elongated mature receptacle and small, coriaceous leaves. One of the most distinctive species of the China clade. (I) F. viridis, the reflexed calyx and mature receptacle that detaches from the calyx with an audible “click” are characteristics of this species. Photographer and Location: (A, B) Gerald Carr (Oregon, USA); (C) Aaron Liston (Oregon, USA); (D, E) public domain (Japan); (F) Oxfordian Kissuth (Germany); (G) Aaron Liston (Sichuan, China); (H) Luxi Chen (Tibet, China); (I) Branko Bakan (Slovenia).

Current species concepts in Fragaria are predominantly based on the systematic studies of Günter Staudt (1926–2008). His research incorporated crossing studies, cytogenetics, and the taxonomic revision of most species (Table 1). With one exception (F. iturupensis), intraspecific variation in ploidy is not known, and thus chromosome number is a reliable way to distinguish Fragaria species. The use of flow cytometry in conjunction with an 8× standard for F. ×ananassa is an efficient method for determining ploidy (R. Cronn, unpublished data).

Molecular phylogenetic analyses based on two nuclear genes (Rousseau-Gueutin et al., 2009) and chloroplast genomes (Njuguna et al., 2013) consistently resolve two clades: the “China clade” comprising nine diploid and tetraploid species—eight of which are distributed in China and adjacent Himalayan countries, one of which is endemic to Japan; and the “vesca clade” comprising 11 species ranging from diploid to decaploid and distributed in northern Eurasia, North and South America, and Hawaii. Monopodial stolons are characteristic of all species in the China clade plus F. viridis (Staudt, 2008). The phylogenetic positions of three diploid species, Eurasian F. viridis, southeast Asian F. nilgerrensis, and Japanese F. iinumae, currently remain unresolved. Fragaria hayatai, endemic to Taiwan, was recently recognized as distinct from F. nilgerrensis based on the presence of spinules on the pollen exine (Staudt, 2008). It has not been included in any genetic or phylogenetic studies of the genus. The vesca clade includes Fragaria chiloensis and F. virginiana, the octoploid ancestors of the cultivated strawberry, F. ×ananassa subsp. ananassa. Phylogenetic analyses of nuclear genes (Rousseau-Gueutin et al., 2009; DiMeglio et al., 2014) implicate F. iinumae and F. vesca s.l. (including F. mandshurica) as progenitors of these two octoploids as well as the octoploid/decaploid F. iturupensis. Plastome phylogenetics (Njuguna et al., 2013) resolve these three species as monophyletic and sister to F. vesca subsp. bracteata, suggesting a shared cytoplasm from a western North American ancestor.

Conflicting evidence exists for the origin of the tetraploid F. orientalis and hexaploid F. moschata. Nuclear genes are consistent with an allotetraploid origin of F. orientalis from F. vesca and F. mandshurica (Rousseau-Gueutin et al., 2009), while these nuclear data and two chloroplast intergenic spacers (Lin and Davis, 2000) support F. viridis as the maternal progenitor of F. moschata. In contrast, plastome evidence supported a common cytoplasmic ancestry for F. moschata and tetraploid F. orientalis, and no evidence for F. viridis maternal ancestry in F. moschata (Njuguna et al., 2013). An important caveat is that these phylogenetic studies included a single accession of each species and very few genes; thus, increased sampling of both should resolve these discrepancies.

Two different decaploids are known in the vesca clade. Fragaria iturupensis is only known from Iturup Island (in the southern part of the Kuril archipelago) and octoploidy has been observed in two accessions (Staudt and Olbricht, 2008). Subsequently, a decaploid accession of F. iturupensis was described (Hummer et al., 2009). Thus, it seems likely that both 8x and 10x individuals exist in the narrow geographic range of this species. The decaploid Fragaria cascadensis was recently described from populations in the Cascade range of western Oregon (Hummer, 2012). This taxon is morphologically similar to F. virginiana and was previously confused with this taxon. The phylogenetic origins of these two decaploids have not been determined.

The China clade includes four tetraploids; however, only one species, F. tibetica, has been the subject of a taxonomic revision (Staudt and Dickoré, 2001), and the taxonomic status of the other three (F. corymbosa, F. gracilis, F. moupinensis) remains uncertain. Likewise, published phylogenetic analyses (Rousseau-Gueutin et al., 2009; Njuguna et al., 2013; DiMeglio et al., 2014) have not resolved the diploid ancestors of these species, nor whether they have single or multiple origins.

The extant species of Fragaria are estimated to have last shared a common ancestor between 1.0 and 4.1 million years ago (mya), while the octoploid clade originated between 0.370 and 2.05 mya (Njuguna et al., 2013). These estimates are consistent with the only fossil evidence of the genus, a single achene (A. Telka, retired, Geological Survey of Canada, personal communication) dating to 2.9 ± 0.4 mya (Matthews and Ovenden, 1990; Töpel et al., 2012). The fossil was collected on Prince Patrick Island, Canada at 76°–77° N latitude, representing a remnant of Pliocene-Pleistocene Beringia. Although this sample predates the estimated age of the octoploid clade, the current distribution of their known ancestors (F. iinumae in Japan and F. vesca subsp. bracteata in Northwest North America) suggests a possible octoploid origin in Beringia. Unfortunately, the specimen is “not currently discoverable” (M. Coyne, Canada Centre for Mapping and Earth Observation, Ottawa, ON, personal communication) and thus unavailable for further study of its morphology and species identity.

HISTORICAL BACKGROUND

Two major topics of Fragaria research, sex and polyploidy, can be traced back to the extraordinary work of Antoine Nicolas Duchesne (1747–1827). In 1766, he published Histoire Naturelle des Fraisiers (Natural History of Strawberries). The book's subtitle, “les vues d'économie réunies à la botanique”, can be translated as “an integration of economic and botanical perspectives.” This aim of 250 yr ago resonates with the theme of this special issue (plus ça change…). The fact that the author was 19 yr old when his book was published is only one of its several remarkable features. The historical significance of Duchesne's work has been previously described (Paris, 2000; Chauvet, 2003; Ratcliff, 2007). In addition to precise botanical descriptions, the history, cultivation, flowering and fruiting seasons, and effects of climate on each species are described. In “a truly unusual attempt for this century” (Lee, 1966), Duchesne also describes his ideas on the relationships of the species, arranges them by this system, and even includes a drawing of a genealogical tree. This diagram is considered the second published phylogenetic network (Morrison, 2014) and shows his novel (and subsequently proven) hypothesis that Fragaria ×ananassa originated as a hybrid between F. chiloensis and F. virginiana. The first section concludes with a discussion of strawberry cultivation and utilization for food, medicine, and other uses (e.g., stolons for dental floss!).

Part two of his book, titled Remarques Particulières, includes the first description of separate sexes in strawberry. Not only did Duchesne observe the somewhat cryptic floral differences, but he also conducted experiments that proved both male and female plants are required for successful fruit production in the dioecious, cultivated species F. moschata and F. chiloensis. Duchesne's discovery of strawberry sex had immediate practical benefits: it explained why the common practice of culling “sterile” male plants from plantings of F. moschata was a bad idea. He also discovered the key to fruit production in F. chiloensis. Only female plants were introduced to Europe from South America, and fruit production was very sporadic. In 1764, Duchesne successfully pollinated female plants of F. chiloensis with pollen from F. moschata. The large, beautiful strawberries produced led to an audience with King Louis XV and served as the impetus for the production of his book (Lee, 1966)6.

Duchesne also attempted to cross the European F. vesca with the South American F. chiloensis, without success. A century and a half later, the discovery of a polyploid series in Fragaria (Ichijima, 1926; Longley, 1926) would explain why this cross failed. Differences in chromosome number between the diploid F. vesca and the octoploid F. chiloensis is the cause of the reproductive isolation observed by Duchesne. Likewise, the vigorous F1 hybrid that Duchesne made between hexaploid F. moschata and octoploid F. chiloensis is also completely sterile (Mangelsdorf and East, 1927). Duchesne was apparently aware that interspecific hybrids were often sterile, as he wrote in 1766 that he was still waiting to see if his hybrid plants would produce fertile seed (Lee, 1966).

Duchesne's interest in strawberry can be traced to his 1761 discovery in the gardens of Versaille (where his father was superintendent of the king's buildings) of a unifoliolate plant in a bed of the typical, trifolioliate Fragaria vesca. He sent specimens to Linnaeus, who published it as the species Fragaria monophylla. This experience, and his observations of variable reproductive isolation among Fragaria, led Duchesne to ponder the nature of plant species, and the final section of his book includes a thorough exposition of what became known two centuries later as the biological species concept (Paris, 2000). In fact, this is why Duchesne used the term “race” instead of species in his 1766 treatment of Fragaria.

Fragaria vesca was used in some of the earliest plant genetic studies following the rediscovery of Mendel's laws. Richardson (1914) cited Duchesne's (1766) observations of “F. monophylla” and reported Mendelian segregation of these leaf traits (as well as red vs. white fruit color and stolon presence/absence) in F. vesca. Likewise, the polyploid series in Fragaria was discovered in the early years of cytogenetics (Ichijima, 1926; Longley, 1926). In general, polyploid species of Fragaria are larger, more vigorous, and more variable than the diploids (Schiemann, 1932). Octoploidy has likely contributed to the large increases in fruit size (Sherman et al., 1966) and probably other aspects of yield (fruit number, plant vigor, disease resistance, Hancock, 1999) obtained in the cultivated strawberry.

From an applied perspective, the presence of different ploidy levels among Fragaria species has functioned as an impediment to the incorporation of desirable traits from the diploid F. vesca and hexaploid F. moschata into the the octoploid F. ×ananassa (Evans, 1977). However, efforts to overcome this barrier have provided a rich data set on chromosome behavior in interspecific crosses (Senanayake and Bringhurst, 1967), the occurrence of unreduced gametes (Bringhurst and Gill, 1970) and varying levels of fertility among ploidy levels in Fragaria (Bors, 2000). The presence of separate sexes in F. ×ananassa was also treated as an undesirable trait in early cultivars (Darrow, 1966), and their absence in modern cultivars apparently represents “unconscious artificial selection” by strawberry breeders (Schiemann, 1932). However, both of these features, sexual system diversity and polyploidy, are what make Fragaria a unique system for answering basic questions about two fundamental and universal evolutionary transitions in flowering plants.

SEXUAL AND MATING SYSTEM EVOLUTION

While cultivated Fragaria ×ananassa is a self-compatible hermaphrodite, it is derived from the hybridization of two wild species that show gender dimorphism, dioecious (males and females) F. chiloensis and subdioecious (hermaphrodites, males, and females) F. virginiana. Gender dimorphism is common among Fragaria species (Fig. 2A, B; Table 1), and the genus is proving to be an exceptional model system for understanding sexual system and sex chromosome evolution, not only because it hosts the full range of sexual systems in the gynodioecy pathway from hermaphroditism to dioecy (and back again), but also because it has variation in self-compatibility, and evidence of sex chromosomes with female heterogamety (Kihara, 1930). Knowledge of the genetics of sex determination in Fragaria is also an important tool for crop development as wild species continue to be resources for genetic improvement of the cultivar (reviewed in Hancock, 1999). In addition, this knowledge will provide genetic insight into a key transition in plant evolution.

Even though dioecy is rare in angiosperms (6%), its evolution from hermaphroditism has occurred in over half of all plant families (Renner and Ricklefs, 1995) suggesting strong convergent evolution. On the basis of character-state mapping on the chloroplast phylogeny of Fragaria, male sterility evolved in both major clades independently, and there may have been as many as six transitions from hermaphroditism to male sterility (Njuguna et al., 2013). Interestingly, female function was also lost several times, and female sterility was associated with increases in ploidy. Comparisons between closely related taxa that differ in sexual system offer powerful means for understanding the origin of sex chromosomes from autosomes, as well as provide evidence for the initial steps in sex chromosome evolution, including linkage of and recombination suppression between sex function “loci” (Charlesworth et al., 2005).

Sex determination in octoploid strawberries was previously thought to be determined by a single Mendelian locus consisting of either a single gene or a gene complex with femaleness dominant to maleness, the latter of which was recessive to hermaphroditism (Ahmadi and Bringhurst, 1991). However, a reassessment via genetic mapping in F. virginiana led to the discovery of the earliest stage of a sex chromosome, the linkage of two gene regions (one for male function and one for female function), but with recombination between them (Spigler et al., 2008, 2010). Moreover, comparative mapping in the sibling species F. chiloensis (Goldberg et al., 2010) also showed dominant male sterility was linked in coupling to female fertility, but here there was no evidence of recombination between them, suggestive of the next stage of the stepwise evolution of a sex chromosome (Charlesworth et al., 2005). However, the location of the sex determining region (SDR) was not syntenous in the two species, the SDR mapped to the bottom of chromosome VI-A in F. chiloensis but the top of Vl-C in F. virginiana. This result suggests that either the two do not share the same sex determining locus as previously thought (Ahmadi and Bringhurst, 1991) or that the SDR has been translocated between chromosomes in the same homeologous group. Both dynamics have been documented in other taxa (e.g., sticklebacks, Ross et al., 2009). The development of genomic resources in Fragaria is providing ingress to tests of these alternatives as well as determining whether the SDR show the hallmarks of evolving sex chromosomes (sex-linkage of sexually antagonistic genes, degeneration of genes, the accumulation of repetitive elements, and low density of genes [Ming and Moore, 2007; Otto et al., 2011; Spigler et al., 2011]). Extending these approaches to F. moschata, where ZW sex chromosomes have been observed (Kihara, 1930) will also make Fragaria an ideal system for answering the open question of whether ZW sex determination systems follow the same dynamics as XY systems (Bachtrog et al., 2011).

In addition to the underlying genetics of sex expression, population genetic models identify the central role of selfing rate, inbreeding depression, and relative seed and pollen fitness gain curves in affecting sex ratio and sex system evolution (reviewed by Webb, 1999). Empirical work in a number of systems has demonstrated the importance of plant interactions with both abiotic (resources) and biotic (pollinators and enemies) factors in modifying these (reviewed by Ashman, 2002, 2006; Spigler and Ashman, 2012). The combination of observational and experimental studies of F. virginiana, in particular, have provided the most complete evaluation of interplay of ecological and genetic forces determining the location of a population along the hermaphroditism–dioecy continuum. For instance, despite strong resource-based selection on female frequency, i.e., the sex ratio (Ashman, 1999), the evolution of full dioecy in this subdioecious species is retarded by selection against males via florivore damage (Ashman et al., 2004; Ashman and Penet, 2007), and gene flow from other populations where sex allocation plasticity and/or the colonizing ability in hermaphrodites is beneficial (Spigler and Ashman, 2011). Furthermore, studies of phenotypic selection combined with a growing understanding of the genetic architecture has provided unparalleled insight into how and when sexual dimorphism evolves during the evolution of separate sexes (Morgan and Ashman, 2003; Ashman, 2005; Spigler and Ashman, 2011, 2012). Moreover, comparative analyses across species has confirmed the existence of greater sexual dimorphism in dioecious species compared with subdioecious and gynodioecious species (Ashman et al., 2013), though hybridization can lead to the breakdown of these associations (Govindarajulu et al., 2013). Hybridization may in fact fuel transitions in sex determining regions in this genus (Govindarajulu et al., 2013). Developing molecular tools will be used to confirm this and to add a mechanistic understanding of the control of sexual dimorphism (Spigler et al., 2011).

Finally, although many ecological and genetic factors have been associated with the transition to separate sexes in plants (Givnish, 1980; Vamosi et al., 2003; Ashman, 2006; Spigler and Ashman, 2012), one of the most bewildering relationships is that between dioecy and an increase in ploidy (Baker, 1984; Miller and Venable, 2000; Ashman et al., 2013). The genus Fragaria is amenable to answering the question of how genome doubling leads to the initiation of dimorphic sexual systems (Ashman et al., 2013). The prominent hypothesis that invokes loss of self-incompatibility and concomitant increased selfing and inbreeding depression following whole genome duplication (Miller and Venable, 2000) is not supported in Fragaria because although polyploid dioecious taxa are often sister to diploid hermaphroditic taxa, these diploids are not always self-incompatible (Njuguna et al., 2013). As a result, the door is open for tests of other possible pathways in Fragaria given its high rate of unreduced gamete formation, ongoing natural polyploid formation, and ease of synthetic polyploid creation (Ashman et al., 2013; Kwok, 2013).

SPECIATION, HYBRIDIZATION, POLYPLOIDY, AND RANGE EXPANSION

Fragaria species occur across a broad range of temperate habitats and elevations (Fig. 1) from sea level sand dunes to moist, productive meadows to high, dry mountain summits (Johnson et al., 2014). In addition, considerable ecological amplitude is found within and between many of the species (Hancock and Bringhurst, 1978, 1979, 1981; Hancock and Luby, 1993). This variation represents a potential source of genetic variation for climatic tolerance, disease/pest resistance, and yield-associated traits (Hancock and Luby, 1993; Stewart and Folta, 2010). Furthermore, this natural variation (Table 2) provides the opportunity to investigate the ecological genomics of traits such as flowering time (Mouhu et al., 2009), drought (Razavi etal., 2011; Šurbanovski et al., 2013), and frost tolerance (Sønsteby and Heide, 2011).

Despite sharing the same life history traits, only one species, F. vesca subsp. vesca, is known to have become established in natural environments outside of its native range (Staudt, 1999a; Plantwise Knowledge Bank, 2014). In fact, the sequenced genome (Shulaev et al., 2011) was obtained from a U.S. Department of Agriculture (USDA) accession “Hawaii 4” originally collected as a naturalized plant near Volcanoes National Park in Hawaii. It is also established on other oceanic islands and in the South American Andes (Table 2). The available genomic resources for this subspecies make it a promising candidate for characterizing the molecular mechanisms of weediness, as well as the impact of isolation on genetic drift.

Most, if not all, flowering plants are polyploids, having undergone one or more whole genome duplications, making polyploidy the single most important mechanism of speciation in land plants (Otto and Whitton, 2000; Wood et al., 2009; Jiao et al., 2011). Polyploidy is a revolutionary event that induces drastic changes in both genome organization and ecological properties that can lead not only to speciation, but biological adaptation and range expansion (Stebbins, 1985; Vamosi and Dickinson, 2006; Pandit et al., 2011; Parisod, 2012)—perhaps especially in response to rapid environmental change (Dynesius and Jansson, 2000; Fawcett et al., 2009).

Despite long-held views that polyploids are more successful because they have enhanced tolerance to abiotic stress and greater ecological amplitude, this issue remains controversial (Martin and Husband, 2009; Soltis et al., 2010; Madlung, 2013). A recent phylogenetically controlled analysis of bioclimatic niche and range size for 21 Fragaria species (Johnson et al., 2014) shows high levels of divergence in bioclimatic niches, and even significant overdispersion along some bioclimatic gradients, suggesting extensive evolutionary lability in physiology and climate tolerance among species. Indeed, the absence of niche conservativism and/or high selfing ability could contribute to wide range breadth and even weediness of the diploid F. vesca subsp. vesca (Table 2). Moreover, although polyploids had larger mean area and more northerly position of their ranges than diploids, there was no significant difference between the two groups, possibly due to the opposing forces of breeding system and polyploidy or the complicating factors of species age and geographic barriers (Johnson et al., 2014).

While bioclimatic studies can provide a snapshot of interspecific distributions at a global scale, only integrated genetic, functional, and population studies can determine whether the proposed mechanisms that follow polyploidization/hybridity contribute to ecological amplitude and stress tolerance of polyploids independent of the contributions of multiple origins or age of species on range size. The potential role of the proposed mechanisms for polyploid fitness (e.g., increased gene dosage, biochemical activity, cell and organ size, increased gene or biochemical diversity or plasticity, genetic novelty arising from transgressive segregation or genome restructuring; reviewed by Soltis et al. [2010]) can all be readily evaluated in Fragaria (see following section and Table 2).

Genomic repatterning that follows whole genome duplication is also thought to be an important source of genetic diversity that fuels adaptive evolution in polyploids (Hegarty and Hiscock, 2008). As a result, intensive focus has been placed on characterizing the effects of allopolyploidy on genome structure or gene expression in a handful of taxa (e.g., Brassica, Glycine [soy], Gossypium [cotton], Tragopogon, Triticum [wheat]). This effort has revealed significant structural changes, including deletions, translocations, and transpositions in both paleo- and neopolyploids (Song et al., 1995; Ozkan et al., 2001; Chen and Ni, 2006; Gaeta and Pires, 2010). Linkage mapping results to date in Fragaria suggests a high degree of synteny among the octoploid subgenomes in F. virginiana (Spigler et al., 2010) and F. ×ananassa (Isobe et al., 2013). However, evidence for inversions confined to single homoeologs has been reported in F. ×ananassa (Sargent et al., 2012; van Dijk et al., 2014). These linkage maps were all created with microsatellite markers. The utilization of targeted sequence capture (Tennessen et al., 2013) for genetic linkage mapping has the advantage of also providing sequence data from each mapped locus. These sequences can be used to discriminate among octoploid subgenomes, providing for the first time the opportunity to quantify aspects of genome repatterning in high polyploid Fragaria (J. A. Tennessen et al., Oregon State University, unpublished manuscript).

Diploid and polyploid Fragaria species often grow sympatrically (Hancock and Bringhurst, 1981; Fig. 2G); thus, the potential for interspecific hybridization exists (Table 2). Naturally occurring pentaploids have been reported from California (F. vesca × F. chiloensis, Bringhurst and Senanayake, 1966) and Northeast China (presumably F. mandshurica × F. orientalis; Lei et al., 2005). In Europe, both 5x and 7x individuals of F. vesca × F. moschata been reported (Nosrati et al., 2011a, 2013), while a single 9x individual of F. vesca × F. chiloensis has been observed in California (Bringhurst and Senanayake, 1966). In each of these cases, the odd polyploid hybrids co-occur with the parental species, and their persistence is likely attributable to clonal reproduction. For this reason, they are not included as “species” in Table 1.

Interspecific hybridization also occurs within a ploidy level. In Europe, hybrids of diploid F. vesca and F. viridis have been named F. ×bifera, and probably can occur wherever the two parents are present (Staudt et al., 2003). In northwestern North America, a large zone of introgression occurs between the octoploids F. chiloensis and F. virginiana (Luby et al., 1992; Salamone et al., 2013). In the case of F. ×bifera, the frequency and ecological context of hybridization has not been quantified. In the case of the octoploid hybrids (F. ×ananassa subsp. cuneifolia), the stability and potential adaptive significance of the introgression zone remains unexplored. In general, varying degrees of reproductive isolation within and among ploidy levels (Bors and Sullivan, 2005; Nosrati et al., 2011b) provides a rich opportunity to investigate the molecular mechanisms of reproductive isolation and the potential role of speciation genes (Table 2).

An additional feature of Fragaria biology is the ability for clonal growth. Ecological (Alpert, 1991) and molecular genetic (Hytönen et al., 2009; Koskela et al., 2012) studies have provided insights into the functional pathways that control allocation between sexual and asexual reproduction. Resource transfer among ramets has also been demonstrated to have a genetic basis (Alpert et al., 2003). Clonality may also play a role in facilitating the survival and establishment of new polyploids in Fragaria. An initially sterile hybrid of F. chiloensis × F. virginiana (no flowers between 1923 and 1939 and male-sterile flowers from 1939 to 1951) produced its first fertile flowers after nearly 30 yr (Schiemann, 1958). Not mentioned in the paper is the miraculous survival of this clone in Berlin throughout the Second World War! The responsible genetic mechanism remains unknown, and the experiment would be a challenge to replicate.

GENE EXPRESSION AND TRANSCRIPTOMICS

With the publication of the genome of F. vesca (Shulaev et al., 2011), a comprehensive catalog of expressed genes are available for Fragaria. Through a combination of gene prediction models and transcriptome evidence, the diploid F. vesca genome is estimated to contain 34809 predicted protein coding genes, and annotations are available for 25050 genes (Shulaev et al., 2011). Over 90% of predicted genes are supported by transcript evidence, and extensive efforts to define organ- and tissue-specific expression have drawn from a large number of independent libraries (Bombarely et al., 2010; Folta et al., 2010; Darwish et al., 2013). Nearly 1125 different non-translated RNAs have also been characterized, including transfer RNAs, ribosomal RNAs, small RNAs, and microRNAs, with most efforts focused on diploid F. vesca (Shulaev et al., 2011) and octoploid Fragaria ×ananassa (Ge et al., 2013). Short RNAs are exceedingly diverse in F. × ananassa (Ge et al., 2013), and they far outnumber the known classes of small RNAs, accounting for 96.4% of the sRNA pool. Given the recent discovery of diverse phenotypes ascribed to small ORFs in Arabidopsis (Hanada et al., 2013), this uncharacterized pool of RNAs may be an untapped resource for understanding the genetic control of agronomic traits, leaf and flower morphogenesis, and different attributes of fitness.

The vast majority of gene expression and transcriptomic studies in Fragaria have focused on agronomic traits, although increasing attention is being given to the transcriptomic (and related proteomic and metabolomics) components of climate sensing and climatic adaptation. Fruit traits are an important focal point in domesticated strawberry, and several transcriptomic studies have focused on aspects of fruit development, including the role of hormone signaling in receptacle enlargement (Kang et al., 2013), the role of MYBs transcription factors and phenylpropanoids in fruit development and ripening (Muñoz et al., 2011; Medina-Puche et al., 2014), and transcript coexpression during receptacle ripening (Hollender et al., 2014). Stress-related expression responses have been measured under a number of agronomically relevant treatments (osmotic stress, water saturation, high temperature, cold temperature), and a number of pathways have been implicated in these different responses (Rivarola et al., 2011).

The process of cold acclimation, winter hardiness and desiccation tolerance, and the timing of spring growth and fall dormancy are all key components of yield and fitness, and they are likely to show strong gradients in natural populations of widespread Fragaria species. To date, transcriptomic approaches have not been used to study gene expression over the span of a complete growing cycle. Focused studies on diploid F. vesca have dissected the role of key seasonal “integration” genes (e.g., SOC1; Mouhu et al., 2013) that regulate yearly cycles of vegetative and reproductive development. Detailed studies of gene expression during the induction of dormancy show that phytohormones promote changes in DNA methylation (Zhang et al., 2012), and these types of transcriptomic responses are amenable to transcriptomic dissection. Recent examinations of metabolite (Rohloff et al., 2012) and proteomic changes (Koehler et al., 2012) during cold acclimation have identified the proteins and metabolites that participate in cold acclimation, and genotypic differences between populations and cultivars in these gene products. These types of quantitative phenotypes are easily merged with transcriptomic studies, and they have the potential to reveal the order of steps behind the orchestration of perennial cycles of dormancy and growth.

With the ability to create synthetic polyploids (Kwok, 2013; Zhang et al., 2014) and to measure environmental responses with genetically identical (clonal) individuals across diverse environments, combined with the availability of genetic and genomic resources, Fragaria is an ideal system for exploring the relationship between ploidy formation, ploidy level, and the coordination of transcriptomic control. The processes and rules that coordinate gene expression in Fragaria polyploid genomes remain largely unexplored, and the contribution of multiple genomes and multiplicative sets of genes—perhaps as many as 140000 protein coding genes in the octoploid species—to reproductive fitness and climatic adaptation are equally obscure. The myriad of genetic and epigenetic processes known to modify gene expression patterns in polyploids is diverse and includes structural rearrangement and gene conversion (Ozkan et al., 2001; Salmon et al., 2010; Buggs et al., 2012), epigenetic repatterning (Madlung et al., 2002), subgenomic expression dominance (Adams et al., 2003; Grover et al., 2012; Xu et al., 2014), and possible interactions with small RNAs (Ha et al., 2009). The extent to which these mechanisms operate in polyploid Fragaria is unknown, but the availability of synthetic and natural polyploids offers the ability to examine the importance of these mechanisms to Fragaria polyploids overall, and to determine whether specific mechanisms are correlated with successive ploidy levels. In light of the young age of the genus, high sequence similarity across the genomes of Fragaria species (and subgenomes of polyploid species) will make it difficult to discriminate among “alloalleles” and identify the subgenomic origin for specific transcripts. However, even if only one subgenome can be reliably identified in a polyploid, it still provides a testable scenario for the null hypothesis of equal contribution of subgenomes to the transcript pool. In this context, experiments focused on highly divergent sequence regions, such as 3′ untranslatable regions (UTRs) (Beck et al., 2010) or known alloallelic single nucleotide polymorphisms (SNPs) (Tennessen et al., 2013), seem the most likely to identify transcripts to a genome of origin.

CONCLUSION

In contrast to more established “model plants” in ecology and evolution, such as Mimulus (Wu et al., 2008), Silene (Bernasconi et al., 2009), Aquilegia (Kramer, 2009), and Boechera (Rushworth et al., 2011), Fragaria includes a species of agricultural importance. Research involving wild strawberry species is far from unique in being able to capitalize on the genetic and genomic resources obtained for plants of economic importance. Studies of the diverse wild relatives of cultivated Helianthus (sunflower) have made enormous contributions to our knowledge of hybrid speciation (Rieseberg et al., 2003), while our understanding of polyploidy has been greatly illuminated by research on wild species of Glycine and Gossypium (Doyle et al., 2004; Adams and Wendel, 2005). Ecological and evolutionary genomics in Fragaria has the potential to provide further insights into both hybridization and polyploidy, while uniquely presenting the opportunity to investigate sexual system evolution. In addition to benefiting from the efforts of a large research community focused on the cultivated strawberry, the familiarity of strawberries provides an opportunity to engage and educate the public about botanical research (e.g., Gonda et al., in press). Ultimately, the increased understanding of plant evolution in an ecological context obtained through the study of wild relatives of crop species can contribute essential information that will be needed to adapt agriculture to a changing global environment.

  • 6 A collection of 73 drawings of Fragaria prepared by Duchesne was published in 2003 (Staudt, 2003a). The originals were discovered in the Library of the Paris Natural History Museum in 1962 by Vivian Lee, an undergraduate at Stanford University (Lee, 1964, V. L. Bowden, personal communication). She wrote four chapters on the history of strawberry (Lee in Darrow, 1966). Her contribution is clearly acknowledged in the preface, but not on the title page, and most subsequent authors have misattributed the chapters to George M. Darrow, a USDA strawberry geneticist.