Eutrema salsugineum (Cruciferae) new to Mexico: a surprising generic record for the flora of Middle America

Abstract The paper reports Eutrema salsugineum as a novelty to the flora of Mexico and Middle America in general. The finding stands ca. 1600 km apart from the closest known locality in the Rocky Mountains of Colorado, USA. The species is considered native to NW Mexico and its late discovery in the region is presumably explained by its tiny habit, early flowering time, and subephemeral life cycle. The phylogenetic position of this Mexican population in a haplotype network based on the chloroplast DNA fragment psbA-trnH confirms this hypothesis and also suggests, in contrast to the previously held viewpoint, multiple colonizations of North American continent from Asia.


Introduction
Eutrema salsugineum (Pall.) Al-Shehbaz & Warwick, long known as Th ellungiella salsuginea (Pall.) O.E. Schulz, is, along with E. edwardsii R. Br., the most widely distributed representative of the genus Eutrema R. Br., occuring in temperate and, to a lesser extent, the Arctic zone of Eurasia and North America. Being an obligatory halophytic annual related to Arabidopsis thaliana (L.) Heynh., it has become a new model for studying abiotic stress (salt, cold, and draught) tolerance in plants (Bressan et al. 2001;Amtmann 2009). As a result, Eutrema salsugineum became one of the fi rst representatives of Brassicaceae to have its genome sequenced (Wu et al. 2012). Nevertheless, fundamental (e. g., geographic) information on the species is incomplete (Koch and German 2013). Due to the peculiar ecological requirements of the species, E. salsu gineum has a highly disjunct distribution in both continents (Korobkov 1975;Ovchinnikova 1994;Rollins 1993;. Despite being described in the 18th century (Pallas 1773) and being noted for its geographic disposition (Schulz 1924), the exact distribution of this species remained unknown, until recently. One of the reasons for the uncertainty was the misidentifi cation of multiple specimens from Russia and Kazakhstan incorrectly referred to as "Th ellungiella salsuginea". Of these, all specimens from the western-most part of the range belong to another species, E. botschantzevii (D.A. German) Al-Shehbaz & Warwick [Th ellungiella botschantzevii D.A. German] (German 2002(German , 2006(German , 2008. Th is fi nding now means that E. salsugineum is completely absent from Europe being bounded at the west by the lower reaches of Amu Darya river, Aral Sea, and the Turgai Valley. Our knowledge on the distribution of the species in America had also undergone improvements, and nowadays E. salsugineum's range is known to extend discontinuously from boreal and Arctic Canada (Alberta, Manitoba, Northwest Territories, Saskatchewan, Yukon) down through the Rocky Mountains within British Columbia, and south to Montana and Colorado. Previous reports for other states such as Idaho (Rydberg 1917) and Ontario (Scoggan 1957) were not confi rmed by subsequent studies (Al-Shehbaz 2010; Brouillet et al. 2010). Here we report of Eutrema salsugineum specimens collected by the second author in 2010 from arid regions in north-eastern Mexico, revealing another highly disjunct and we believe indigenous population. Our fi ndings are supported with additional genetic analyses from which we draw further biogeographic conclusions.

Materials and methods
Th e specimen documenting the occurrence of E. salsugineum in Middle America is deposited in HEID. For estimation of the newly revealed disjunction in the distribution area of the species, available information regarding the closest occurence has been used (Weber 1966). Results were interpreted in light of the most recent phylogeographic study of E. salsugineum by Wang et al. (2015). In this study various plastid genes (ndhF1, ndhF2, psbA-trnH, rpoC1, rbcL, trnD-trnT, trnL, trnL-trnF, trnV) have been sequenced and DNA polymorphisms have been detected in one marker only. In order to fi nd the inter-species affi nity of the Mexican population, its position in the haplotype network based on the respective variable chloroplast DNA fragment, psbA-trnH, was determined. For this purpose, representatives of the two geographically closest populations (from Montana and Colorado) not studied molecularly before and one accession from Canada, as internal control, were also sequenced.
Total DNA was extracted from 100 mg of herbarium tissue using the Invisorb Spin Plant Mini Kit (Stratec Biomedical AG, Birkenfeld, Germany). PCR ampli-fi cation was performed in a volume of 25 μL, using 10 μM of each primer, respectively, a total of 2.0 mM MgCl 2 and 0.5 U of Mango-Taq polymerase (Bioline, Luckenwalde, Germany). Th e primers used for amplifi cation were psbA-for: 5'-GTT ATG CAT GAA CGT AAT GCT C-3', and trnH-rev: 5'-CGC GCA TGG TGG ATT CAC AAT CC-3'). All primers were extended by the M13 sequence for subsequent sequencing using M13 universal sequencing primers. Th e amplifi cations were run on a PTC 200 Peltier Th ermal Cycler (MJ Research, Waltham, Massachusetts, USA) under the following conditions: 3 min initial denaturation at 95°C; 30 cycles of amplifi cation with 30s at 95°C, 30s at 50°C, and 1 min at 72°C; and 5 min of fi nal elongation at 72°C. PCR success was checked with electrophoresis in a 1% agarose gel in TAE-buff er. PCR product clean-up was executed using the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, USA). Custom Sanger-sequencing was performed with GATC-Biotech (Konstanz, Germany). Th e electropherograms were checked and trimmed to the borders of the analysed markers using the program SeqMan DNA-Star Lasergene software package (DNASTAR, Madison, Wisconsin, USA).
DNA sequence variation was compared with cpDNA haplotypes detected by Wang et al. (2015). Haplotype network analysis was conducted with SplitsTree4 vers. 4.14.4 (Huson and Bryant 2006) with gaps treated as additional binary character.
Apart from the Mexican one, the newly sequenced specimens are: 1) USA, Colorado, Park Co. As taxonomy/nomenclature is not the focus of the present study, only most frequently used synonyms are given here. For detailed synonymy, Schulz (1924: 252) and Tropicos (tropicos.org) can be consulted. Th is is the fi rst record of Eutrema salsugineum (and the genus) from Middle America (specifi cally Mexico) which shifts the southern limit of the distribution of the species ca. 1600 km to south-southeast from the closest, isolated fragment of the distribution area of the species, confi ned to Park County, Colorado (Weber 1966). Th is introduces a hitherto unrecognized disjunction within the New World part of Eutrema salsugineum's range. In Sierra Madre Oriental the plants are found at elevations of ca. 1880 m a.s.l. in a semi-open habitat among halophytic, oak/Juniperus (J. fl accida)-and grassesdominated vegetation on alkaline soil which fi ts well the ecological requirements of the species (Fig. 1). Th e habitat at the Mexican location of E. salsugineum is apparently natural and shows no obvious signs of anthropogenic modifi cation. Having in mind the naturally disjunct biogeography of this species we consider it as a natural element of the fl ora of Sierra Madre Oriental which was overlooked by previous studies due to its tiny habit, trivial appearance, early fl owering time, and short, almost ephemeral life cycle. Results of the haplotype analysis (below) apparently confi rm this conclusion.
DNA sequence data revealed a new plastid DNA haplotype of Mexican E. salsugineum indicating its genetic distinctiveness. Th e haplotype code is indicated in Table  1 summarizing also haplotypes detected earlier (Wang et al. 2015). Th e geographic distribution of the various accessions and their plastid haplotypes are given in Fig. 2. SplitTree analysis indicates that the Mexican plastid haplotype is most closely related to haplotype H5 (numbering following Wang et al. 2015), which was originally found only once in Tuva (South Central Siberia) ( Fig. 2; Wang et al. 2015). However, the same H5 haplotype was found here in Montana and Colorado, largely representing the mountain regions of the western United States. As expected, the Canadian sample from Saskatchewan analyzed herein carries haplotype H2, which is widely distributed in NE China and the amphi-Beringian region (Fig. 2c).
In their recent phylogeographic study Wang et al. (2015) discovered extremely low level of genetic diversity throughout the whole distribution area of E. salsugineum, especially in NE Asia/America, apparently refl ecting a very recent (ultimately Late-Pleistocene/very Early Holocene) range expansion. Th ese authors suggested wind to be the main agent mediating the fast and long-distance dispersal of E. salsugineum and a single colonization of the New World by the species (Wang et al. 2015). However, a present-day fragmentary sublongitudinal distribution of E. salsugineum in the Rocky Mountains along with our fi nding that relevant populations carry a haplotype (H5) which is identical to a unique haplotype found in South Siberia, suggests that the North American continent has been colonized independently two times. Th is conclusion is supported by the fact that the newly described accession occurring in Mexico carries a haplotype which most likely derived directly from haplotype H5. Th is overlapping distribution pattern of an amphi-Beringian group carrying H1/H2 and a disjunct group (South Siberia versus western North America) carrying H5 plus the Mexican type can also be best explained by a fi rst and older immigration of E. salsugineum Table 1. Variable sites of the polymorphic cpDNA fragment psbA-trnH in Eutrema salsugineum and closely related species (modifi ed from Wang et al. 2015). GenBank code of the Mexican haplotype is KY073435.  from Asia predating the last glaciation. During the Last Glaciation Maximum the northern populations in America apparently went extinct and/or migrated southwards. If this scenario is correct, the last latitudinal shift of vegetation belts could enable the species to reach the Middle Americas and subsequently survive there in appropriate habitats at higher elevation. Th e wide amphi-Beringian distribution of H1 and H2 might refl ect postglacial expansion as demonstrated by the phylogeography inferred by Wang et al. (2015) based on nuclear genes. Th is multiple immigration pattern of various temperate-cold and often draught adapted Brassicaceae taxa between East Asia and the North America has been documented e.g. also for Arabidopsis and Arabis L. (Schmickl et al. 2008(Schmickl et al. , 2010Koch et al. 2010;Koch and Grosser 2017), highlighting the general signifi cance of this spatio-temporal pattern.

Conclusion
As shown herein, Asian-North American halophyte Eutrema salsugineum has a far more southern distribution in the New World than previously considered almost reaching the Northern Tropic. Th e character of the discovered habitat suggests native rather than human-mediated occurrence of the species in Mexico, a conclusion supported by molecular footprints. A psbA-trnH-based haplotype network demonstrates more complicated infraspecifi c structure and biogeographic history of E. salsugineum than previously thought and suggests its multiple invasions to the New World.