Oxalis dillenii Jacq. 1794

Oxalis dillenii is native to North America, al- though its native distribution in that continent is obscured both by confusion with other species and its modern spread into new areas. It is one of the commonest Oxalis species in the eastern United States, where it usually grows in disturbed habitats (Nesom, 2009). Outside its native range, O. dillenii has been recorded in several countries, including France, Italy, Germany, Belgium, the Netherlands, Great Britain and Austria (Young, 1968; Moorsel et al., 2000; Hoste, 2012). MATERIALS AND METHODS Two small Oxalis plants were collected by the first author the 27 November 2013 from a site north of Solsona, Lleida, Catalonia, Spain (42�� 00��� 40.8��� N, 1�� 31��� 01.5��� E). The plants were growing on bare dry soil under a conifer hedge that surrounds a holiday camp. Molecular protocols and sequence analyses The modified CTAB protocol of Tel-Zur et al. (1999) was used for total genomic DNA isolation. Secondary metabolites were removed by washing ground dried leaf material with 1000 ��L extraction buffer (100 mM Tris-HCl pH 8, 5 mM EDTA pH 8, 0.35 M sorbitol). After the addition of 800 ��L CTAB lysis buffer, as described in Chase & Hills (1991) with addition of 1% PVP-40 and 0.3% 2-mercaptoethanol, the samples were incubated at 60�� C for one hour. Chloroform-isoamylalcohol (24/1 v/v) extraction was done twice, followed by an isopropanol precipitation (0.8 volumes). After centrifugation, the pellet was washed in 70% eth- anol, air-dried, and dissolved in 50 ��L buffer (10 mM Tris-HCl pH 8, 1 mM EDTA pH 8). Amplification reactions of ITS and trnL-F were carried out using a touchdown PCR protocol (25 ��L). Reactions were initiated with a 3-min heating at 95�� C followed by 35 cycles consisting of 95�� C for 30 s, 50-56�� C for 30 s, and 72�� C for 30-90 s. Reactions ended with a 5-min incubation at 72�� C. Amplification and sequencing of ITS and trnL-F was performed using the primers of White et al. (1990) and Taberlet et al. (1991), respectively. En- zymatically purified PCR products were sequenced by the Macrogen sequencing facilities (Macrogen Europe, Amsterdam, Netherlands). Contiguous sequences were assembled using Ge- neious v7.0.6 (Biomatters, New Zealand). Automat- ic alignments were carried with MAFFT (Katoh et al., 2002) under an E-INS-i algorithm, a 100 PAM / k = 2 scoring matrix, a gap open penalty of 1.3 and an offset value of 0.123. Subsequent manual fine-tun- ing of the aligned dataset was done in Geneious v7.0.6. Sequence gaps were treated as missing data, whereas potentially informative base insertions and deletions were coded according to the ���simple indel coding��� method of Simmons & Ochoterena (2000). The global distribution of O. dillenii was created from observations from the Global Biodiversity In- formation Facility on the 5 August 2015 (http://doi. org/10.15468/dl.9imeus). Voucher specimens for O. dillenii, O. stricta and O. corniculata are deposited at the Botanic Garden Meise (BR) (Table 1). RESULTS Compared to O. corniculata the most consistent vegetative characteristics of O. dillenii are the narrow adnate stipules which are fused to the petioles and the antrorse appressed stem hairs, which are usually densest towards the top of the stem (Fig. 1). In O. stricta the stipules have no free margins (and are therefore seemingly lacking); in this species at least part of the stem hairs are long, patent and sep- tate, a feature that separates O. stricta from both O. dillenii and O. corniculata. Since O. dillenii is easily confused with two closely-related species of the Iberian flora (S��nchez-Pedraja, 2015), we have provided a table that compares the distinguishing characteristics of O. dillenii, O. corniculata, O. stricta and O. exilis (Table 2). Very rarely O. conorrhiza Jacq. and O. filiformis Kunth have also been found in Spain, but are easily recognized by their single flowered inflorescences and somewhat larg- er flowers with long, very slender pedicels. They have been omitted from the table. In the specimen from Lleida the narrow stipules and antrorse stem hairs are present as typical for O. dillenii. Other features of O. dillenii are more variable but present in these specimens. The young plants are erect, but with age stems can root at the nodes giving the impression of either a creeping stolon or, if covered by soil, a rhizome. However, the Lleida plants were too young for this to have occurred. Well grown plants in rich soil are usually bright green on both sides of the leaf, but the Lleida specimens have pink-purple leaves apparently due to the nutrient-poor dry soil they were growing in and possibly because they were collected in winter when they frequently experienced cold conditions. Another distinctive character of this species are the fasciculate leaves, whereas other closely related species (with the exception of O. stricta) have more evenly spaced alternate leaves. The DNA barcode sequences of the Lleida spec- imens were compared to an O. corniculata spec- imen from Belgium, an O. stricta specimen from the United States and four specimens of O. dillenii, one from each of Belgium and Germany and two from the United States (Table 1). The chloroplast trnL-F sequence is almost identical for all species. However, for the nuclear ITS sequence the Lleida specimen shows 99.7���100% identity with the ver- ified O. dillenii specimens; but only 93.2% and 95.8% identity with O. corniculata and O. stricta, respectively. Oxalis dillenii is an annual or short lived peren- nial of disturbed ground. It can inhabit waste-places and become a weed of cultivation. Figure 2 shows the distribution of O. dillenii in the Northern Hemisphere. In both Asia and North America it grows at latitudes more southerly than Spain, so there is little reason to think that it is at the boundaries of its potential distribution in Europe. DISCUSSION The results demonstrate the presence of O. dillenii in Spain using both morphological and molecular approaches. Aymerich (2013) previously reported O. dillenii from Spain and an image of his spec- imen was obtained from the Vascular Plant Her- barium of the Real Jard��n Bot��nico (MA specimen number 823538). This specimen is recognisable as O. dillenii and should be considered the first con- firmed observation of O. dillenii in Spain. It is a larger specimen than the specimens from Lleida and is more typical of an older plant, as it has the typical fasciculate leaves. This specimen was col- lected at Fonollet, municipality of Casserres, Bar- celona, at 610 m on the 3 November 2010 by P. Aymerich. The label describes the site as ��� entorno de un camino rural, en taludes con algunos escom- bros y en matorrales de romero ���, which translates to ���in the environment of a country road, on slopes with some debris and rosemary bushes���. However, when he visited the site two years later the species was not refound. It is unlikely that these are the only two sites of O. dillenii in Spain and we suspect that this spe- cies is likely to spread further. Indeed, there have been unsubstantiated records from elsewhere in Spain (Bol��s et al., 1990; S��nchez-Pedraja, 2015). It is an opportunistic invader and has probably not filled its climatic niche in Europe yet. Therefore its current distribution may reflect its introduction pathway rather than suitable habitat availability. Nevertheless, this species is a minor weed where it has become naturalised. It is easy to control with standard horticultural practices and has not yet in- vaded more natural habitats. We hope that publish- ing these observations will make botanists more aware of O. dillenii in Spain.
Published as part of Groom, Q. J., Hoste, I. & Janssens, S., 2017, Observaci��n confirmada de Oxalis dillenii en Espa��a, pp. 1-6 in Collectanea Botanica 36 on pages 1-4, DOI: 10.3989/collectbot.2017.v36.004, http://zenodo.org/record/4266940
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