Plant Syst Evol (2018) 304:133–141 https://doi.org/10.1007/s00606-017-1447-2 SHORT COMMUNICATION Evidence for genetic allopolyploidy in Eutrema edwardsii (Brassicaceae): implications for conservation Jared Mastin1 · Neil Luebke2 · Peter Anthamatten3 · Leo P. Bruederle1 Received: 20 December 2016 / Accepted: 19 July 2017 / Published online: 5 September 2017 © Springer-Verlag GmbH Austria 2017 Abstract Eutrema edwardsii R.Br. (Brassicaceae) is an progenitor(s) cannot be elucidated from these data. The data arctic-alpine mustard with a circumpolar distribution. Its reported herein support the recognition of E. penlandii as closest relative, Eutrema penlandii Rollins, is a federally taxonomically distinct, which has implications for conserva- listed, threatened species that is endemic to the Mosquito tion, and reveal cryptic variation within E. edwardsii. Range in the Southern Rocky Mountains of Colorado, USA. As part of a larger project addressing the systematics of this Keywords Allopolyploidy · Allozyme · Cryptic species complex in North America, we conducted chromo- variation · Eutrema · Fixed heterozygosity · Flow some counts, flow cytometry, and allozyme analysis to test cytometry the hypothesis that these taxa comprise an autopolyploid complex. Within that context, it should be noted that a chromosome count has not been reported previously for E. Introduction penlandii. Results obtained from mitotic counts obtained for two populations of E. penlandii reveal this taxon to be Although flowering plants are under-represented in the liter- diploid. Diploidy was confirmed using flow cytometry for ature addressing cryptic species (Bickford et al. 2006), there an additional 15 individuals representing four populations. is a growing body of research addressing these species in the Previously published chromosome counts for E. edwardsii flora of the arctic, in particular (e.g., Grundt et al. 2006). The reveal a polyploid complex of tetraploid, hexaploid, and underlying explanations proposed to explain this phenom- octaploid populations for which an autopolyploid origin enon include recent speciation, incomplete divergence, and has been presumed. However, allozyme analysis revealed reticulate evolution, with selection, inbreeding, and genetic an allopolyploid origin for E. edwardsii, as evidenced from drift proposed as mechanisms leading to cryptic variation. fixed heterozygosity at six loci. Although our data suggest It is also clear that polyploidy—particularly autopolyploidy that E. penlandii is a close relative of one of the progeni- in plants—is an important and overlooked mechanism lead- tors of E. edwardsii, the taxonomic identity of the other ing to the formation of cryptic species (Soltis et al. 2007). However, there has been a resistance to recognize multiple Handling editor: Martin Lysak. cytotypes as distinct—even when they confer reproductive isolation—as well as a reliance upon the morphological spe- * Jared Mastin cies concept, or some variant thereof (Soltis et al. 2007). [email protected] Herein, we report the results of multidisciplinary research addressing the systematics of the Eutrema edwardsii R.Br. 1 Department of Integrative Biology, University of Colorado Denver, Denver, CO 80217-3364, USA species complex (Brassicaceae), with a goal of contributing to a better understanding of E. penlandii Rollins, a federally 2 Botany, Milwaukee Public Museum, Milwaukee, WI 53233-1478, USA listed Colorado endemic. Until 1985, two Eutrema species were recognized from 3 Department of Geography and Environmental Sciences, University of Colorado Denver, Denver, CO 80217-3364, North America: E. edwardsii and E. penlandii, when Weber USA (1985) published the combination E. edwardsii R.Br. subsp. Vol.:(0123456789)1 3 134 J. Mastin et al. penlandii (Rollins) W.A.Weber, thereby reducing the rank Wrangell-St. Elias Mountains, and the Mackenzie Mountains of this taxon to subspecies, although no rationale was pro- of the northern Rocky Mountains in western North America vided. More recently, E. penlandii was subsumed into E. (Fig. 1) and has been reported from the Altai Mountains, edwardsii, as part of a larger revision to the genus following Tian Shan Mountains, Pamir Mountains, and Himalayas in a critical evaluation of Neomartinella Pilg., Platycraspedum Asia. In contrast, E. penlandii is a narrow endemic that is O.E.Schulz, Taphrospermum C.A.Mey., and Thellungiella restricted in distribution to the Mosquito Range in the South- O.E. Schulz; this resulted in the aforementioned genera ern Rocky Mountains of Colorado (USA), where typically it being subsumed into Eutrema (Al-Shehbaz and Warwick occupies alpine peatlands overlying calcareous substrates. 2005; Warwick et al. 2006). Therein and subsequently, Al- Relatively little is known about the natural history (e.g., Shehbaz (2010) claimed that the variation exhibited by E. reproductive biology) of either species. Although chro- edwardsii (e.g., plant height, leaf base shape, petal shape) mosome counts conducted for E. edwardsii have revealed could easily accommodate E. penlandii. As part of a larger tetraploid (2n = 4x = 28), hexaploid (2n = 6x = 42), and study using allozyme analysis to address conservation genet- octaploid (2n = 8x = 56) populations, where n = 7, no ics in Colorado populations of E. penlandii (Hardwick counts have been reported for E. penlandii (Warwick and 1997), as well as the systematic relationship of E. penlan- Al-Shehbaz 2006). dii to E. edwardsii, fixed heterozygosity was observed in populations of E. edwardsii suggesting to us an allopolyploid Field collections origin for the latter (e.g., Brochmann et al. 2004 and refer- ences therein). Plant material, including leaf tissue for allozyme analysis Although it has long been assumed that the aforemen- and flow cytometry and seeds for chromosome counts, was tioned two species form a polyploid complex, with autopoly- collected between 1995 and 2014 (Table 1). As part of a ploidy presupposed for E. edwardsii (e.g., Jørgensen, et al. larger study using allozyme analysis to characterize popu- 1958), no chromosome counts have been reported for E. pen- lation genetic diversity and structure in E. penlandii, Hard- landii, in particular. Herein, we take multiple approaches wick (1997) collected leaf tissue from seven populations using chromosome counts, flow cytometry, and allozyme of the narrowly distributed Colorado endemic and three analysis to better understand the systematics of E. edward- populations of its widespread congener E. edwardsii from sii s.l., and to test the hypothesis that E. penlandii and E. Alaska. Bruederle subsequently collected leaf tissue from an edwardsii form an autopolyploid complex. Whereas chro- additional five populations of E. penlandii and seven popu- mosome counts and flow cytometry are used to document lations of E. edwardsii from Alaska and Yukon Territory. ploidy for E. penlandii, allozyme analysis is used to eluci- Population samples were collected from 7 to 34 individuals date the polyploid origin of E. edwardsii. per population, depending on the size of the population, and maintained as such—samples were not bulked. Approval for tissue collection from E. penlandii was granted by the U.S. Materials and methods Fish and Wildlife Service under the authority of permits PRT-704930 and TE12513A-0. Natural history Chromosome counts Al-Shehbaz and Warwick (2005) recognized 26 species comprising Eutrema, the majority of which occur in Cen- Counts were obtained from squashes of root tips harvested tral and East Asia. Several additional species have recently from seedlings germinated specifically for this purpose. In been described, also from Asia (Hao et al. 2015, 2016, Xiao the process of collecting tissue for population genetic and et al. 2015). Only Eutrema salsugineum (Pall.) Al-Shebhaz molecular systematics research, a small number of fruits & Warwick and E. edwardsii extend from Asia into North were harvested. Seeds were germinated in 0.005 M gibber- America where, like E. salsugineum, E. penlandii is highly ellic acid in a Percival environmental chamber set at 15 °C; disjunct in the Southern Rocky Mountains (Al-Shehbaz attempts were made to cultivate those seedlings that were 2010; German and Koch 2017). not harvested in a research greenhouse for subsequent use Eutrema edwardsii and E. penlandii are perennial herbs for these purposes. As such, either whole seedlings or root that occupy a variety of imperfectly drained arctic and alpine tips were harvested within 2–4 h after light conditions were tundra habitats, such as meadows, margins of ponds and restored and placed in a saturated solution of paradichlo- stream banks, and solifluction slopes (Aiken et al. 2007). robenzene for 2 h, after which they were fixed in Farmer’s Eutrema edwardsii is widespread, with a circumpolar dis- solution, in which they were stored. Squashes were prepared tribution (Aiken et al. 2007); it also occurs in the alpine of by macerating the root tip in a drop of 1% aceto-orcein on the White Mountains, Alaska Range, Chugach Mountains, a microscope slide cleaned with 95% ethanol, heating for 1 3 Allopolyploidy in Eutrema edwardsii 135 Fig. 1 Distribution of Eutrema edwardsii s.l. in North America 2 min at 50 °C, and squashing. Squashes were scanned Relative genome size was obtained for three individu- at 100× and 400× for meiotic figures and photographed als from each sampled population, depending upon the at 1000×. The chromosome count for E. penlandii was amount of dried leaf tissue that was available. Standards