A confirmed observation of Oxalis dillenii in Spain

ARTÍCULO

A confirmed observation of Oxalis dillenii in Spain

Q. J. GROOM, I. HOSTE & S. JANSSENS

Botanic Garden Meise, Bouchout Domain, Nieuwelaan, 38, BE-1860 Meise, Belgium

 

ORCID iD: Q. J. GROOM: http://orcid.org/0000-0002-0596-5376, I. HOSTE: http://orcid.org/0000-0003-0603-7587, S. JANSSENS: http://orcid.org/0000-0003-1246-9474

 

E-mail: Q. J. Groom (quentin.groom@plantentuinmeise.be)

 

Editor: A. Susanna

ABSTRACT
A confirmed observation of Oxalis dillenii in Spain.— We confirm the presence of Oxalis dillenii Jacq. in the Catalonian province Lleida (Catalonia, Spain). Identification was confirmed morphologically and through DNA barcode sequencing. A map of the distribution of O. dillenii is supplied, as are notes on its identification.
KEY WORDS: DNA barcode; identification; non-native species.

Observación confirmada de Oxalis dillenii en España

RESUMEN
Observación confirmada de Oxalis dillenii en España.— Confirmamos la presencia de Oxalis dillenii Jacq. en la provincia de Lérida (Cataluña, España). La identidad de la especie fue confirmada tanto por sus características morfológicas como a través de la secuenciación del código de barras del ADN. Se proporciona un mapa de distribución de O. dillenii, así como notas sobre su identificación.
PALABRAS CLAVE: código de barras del ADN; especie no nativa; identificación

Recibido: 16/09/2015 / Aceptado: 14/09/2016 / Publicado on line: 05/04/2017

Cómo citar este artículo / Citation: Groom, Q. J., Hoste, I. & Janssens, S. 2017. A confirmed observation of Oxalis dillenii in Spain. Collectanea Botanica 36: e004. doi: http://dx.doi.org/10.3989/collectbot.2017.v36.004

Copyright: © 2017 Institut Botànic de Barcelona (CSIC). This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) Spain 3.0.

CONTENIDOS

ABSTRACT
RESUMEN
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENTS
REFERENCES

INTRODUCTIONTop

Oxalis is a remarkable genus for many reasons, but one of them is the large number of invasive species in this genus. These are not limited to one section of the genus, but spread within it and include O. pes-caprae L., O. debilis Kunth, O. latifolia Kunth and O. incarnata L. However, the section Corniculatae DC. seems well endowed with invasive members, the most infamous being O. corniculata L. that may be present in every country on the planet (Hassler, 2015Hassler, M. 2015. World Plants: Synonymic Checklists of the Vascular Plants of the World. Species 2000 Secretariat, Leiden. Retrieved March 25, 2015, from http://www.catalogueoflife.org/col/details/database/id/141). Other weedy species in this section are O. dillenii Jacq. and O. stricta L. from North America and O. exilis A. Cunn. and O. perennans Haw. from Australasia (Nesom, 2009Nesom, G. L. 2009. Again: taxonomy of yellow-flowered caulescent Oxalis (Oxalidaceae) in eastern North America. Journal of the Botanical Research Institute of Texas 3: 727–738.; Gray, 2011Gray, A. M. 2011. Oxalidaceae, version 2011:1. In: M. F. Duretto (Ed.), Flora of Tasmania Online. Tasmanian Herbarium, Tasmanian Museum & Art Gallery, Hobart: 8 pp. Retrieved September 3, 2015, from www.tmag.tas.gov.au/floratasmania.).

Oxalis dillenii is native to North America, although 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, 2009Nesom, G. L. 2009. Again: taxonomy of yellow-flowered caulescent Oxalis (Oxalidaceae) in eastern North America. Journal of the Botanical Research Institute of Texas 3: 727–738.). Outside its native range, O. dillenii has been recorded in several countries, including France, Italy, Germany, Belgium, the Netherlands, Great Britain and Austria (Young, 1968Young, D. P. 1968. Oxalis L. In: Tutin T. G., Heywood, V. H., Burges, N. A. Moore, D. M., Valentine, D. H., Walters, S. M. & Webb, D. A. (Eds.), Flora Europaea 2. Cambridge University Press, Cambridge: 192–193.; Moorsel et al., 2000Moorsel, R. C. M. J. van, Beringen, R. & Odé, B. 2000. Een nieuwe klaverzuring in Nederland: Oxalis dillenii Jacq. Gorteria 26: 31–35.; Hoste, 2012Hoste, I. 2012. Een sleutel voor het genus Oxalis in België, met commentaar bij de waargenomen soorten. Dumortiera 101: 9–22.).

MATERIALS AND METHODSTop

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. (1999Tel-Zur, N., Abbo, S., Myslabodski, D. & Mizrahi, Y. 1999. Modified CTAB procedure for DNA isolation from epiphytic cacti of the genera Hylocereus and Selenicereus (Cactaceae). Plant Molecular Biology Reporter 17: 249–254. https://doi.org/10.1023/A:1007656315275) was used for total genomic DNA isolation. Secondary metabolites were removed by washing ground dried leaf material with 1000 µL extraction buffer (100 mM Tris-HCl pH 8, 5 mM EDTA pH 8, 0.35 M sorbitol). After the addition of 800 µL CTAB lysis buffer, as described in Chase & Hills (1991Chase, M. W. & Hills, H. H. 1991. Silica gel: an ideal material for field preservation of leaf samples for DNA studies. Taxon 40: 215–220. https://doi.org/10.2307/1222975) 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% ethanol, air-dried, and dissolved in 50 µL 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 µL). 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. (1990White, T. J., Bruns, T., Lee, S. J. W. T. & Taylor, J. W. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications 18: 315–322.) and Taberlet et al. (1991Taberlet, P., Geilly, L., Pautou, G. & Bouvet, J. 1991. Universal primers for amplification on three non-coding regions of chloroplast DNA. Plant Molecular Biology 17: 1105–1109. https://doi.org/10.1007/BF00037152), respectively. Enzymatically purified PCR products were sequenced by the Macrogen sequencing facilities (Macrogen Europe, Amsterdam, Netherlands).

Contiguous sequences were assembled using Geneious v7.0.6 (Biomatters, New Zealand). Automatic alignments were carried with MAFFT (Katoh et al., 2002Katoh, K., Misawa, K., Kuma, K. I., & Miyata, T. 2002. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research 30: 3059–3066. https://doi.org/10.1093/nar/gkf436) 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-tuning 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 (2000Simmons, M. P. & Ochoterena, H. 2000. Gaps as characters in sequence-based phylogenetic analyses. Systematic Biology 49: 369–381. https://doi.org/10.1093/sysbio/49.2.369).

The global distribution of O. dillenii was created from observations from the Global Biodiversity Information 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).

Table 1. Details of the specimens used in this study. Their accession numbers at the Botanic Garden Meise Herbarium (BR); The GenBank accession numbers of the ITS and trnL-F barcode sequences and the geographic origins of the specimens.
Species Accession number ITS trnL-F Origin
Oxalis corniculata BR0000013236847 KT737723 KT737730 Flanders, Belgium
Oxalis dillenii BR0000009721166 KT737724 KT737731 Louisiana, USA
Oxalis dillenii BR0000013234409 KT737725 KT737732 Lleida, Spain
Oxalis dillenii BR0000013236830 KT737726 KT737733 Flanders, Belgium
Oxalis dillenii BR0000013234225 KT737727 KT737734 Berlin, Germany
Oxalis dillenii BR0000013498894 KT737728 KT737735 Texas, USA
Oxalis stricta BR0000013234621 KT737722 KT737729 Pennsylvania, USA

RESULTSTop

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 septate, 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 (Sánchez-Pedraja, 2015Sánchez-Pedraja, O. 2015 Oxalis L. In: Muñoz Garmendia, F. & Navarro, C. (Eds.), Flora iberica 9. Real Jardín Botánico (CSIC), Madrid: 384–405.), 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 larger flowers with long, very slender pedicels. They have been omitted from the table.

Figure 1. Detail of the stipules of Oxalis dillenii (A) and Oxalis corniculata (B). Both pictures are of dried herbarium material. The Oxalis dillenii photograph is from the Lleida material and the Oxalis corniculata is taken from plants collected in the Botanic Garden Meise, where it grows as a weed.

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Table 2. The Oxalis corniculata group. Underlined text summarizes the key features for identification of Oxalis dillenii.
Oxalis stricta Oxalis dillenii Oxalis corniculata Oxalis exilis
Habit (The habit of each of the four species shows a remarkable range of variation) Stem usually single and erect, but older plants often toppling and decumbent; not rooting at the nodes; at the base often with green or reddish ground-level or white underground rhizomes Stem usually single and erect when young. Older plants often bushy, with several erect stems arising at short distances from either prostrate aboveground or horizontal underground stems that often root Stems few to numerous, radiating from a central taproot. Stems decumbent or, especially in var. atropurpurea, procumbent, sometimes erect in young plants; procumbent stems not, only sparsely or, especially in var. atropurpurea, freely rooting Stems few to numerous, radiating from a central taproot. Stems procumbent and freely rooting at the nodes or, occasionally, decumbent and then more sparsely rooting at the nodes
Stem hairiness (In all four species the young upper part is usually more densely hairy) Sparsely to, less often, densely hairy, sometimes almost glabrous; long, patent, septate hairs usually present (if not, then such hairs typically present on the pedicels); often mixed with some short, curved, whitish, unicellular hairs Usually rather densely, but sometimes only sparsely covered with antrorsely appressed unicellular hairs; these may give the young distal part of the stem a whitish appearance Usually moderately densely covered with whitish nonseptate hairs, some patent, some appressed, most of them curled
Leaf colour and size Green, sometimes with a tinge of purple, especially at the underside. The rarely cultivated var. rufa has dark purple leaves Green, sometimes suffused with purple, especially at the underside of the leaves Green or purple; if purple, leaflets often with a green midrib. Usually smaller in var. atropurpurea than in var. corniculata Usually green; purple-leaved plants very rare among exotics introduced in Europe. Usually smaller than those of the other species
Stipules Seemingly absent (no free membranous part visible) Present but reduced, with a narrow membranous portion (Fig. 1A) Present and well developed; at least some stipules widened into a triangular auricle in the apical part (Fig. 1B)
Leaves Fasciculed or whorled Alternate and clearly distanced, each with an axillary peduncle
Throat of the corolla Usually without markings, but sometimes with fine red stripes (these mostly tiny and few) With or without markings. If present: each petal with 3-4 thin red stripes (var. corniculata) or with a dim to clearly visible orange band (var. atropurpurea) Never (?) with fine red stripes or an orange band
Stamens Typically all stamens (five long and five short) with anthers, but quite often one or more of the short ones with somewhat less well-developed or completely aborted anthers Typically the 5 long stamens with and the 5 short stamens without anthers; not uncommonly, however, 1 or more of the short stamens with a poorly developed anther
Inflorescences Flowers in regular or irregular, often umbelliform cymes Flowers most often in umbelliform cymes Flowers in umbelliform or sometimes irregular cymes 1-flowered, very rarely 2-flowered
Pedicels Erect Erect during flowering, patent or reflexed during maturation of the capsule, eventually upright again when the capsule is ripe and ready for seed dispersal
Capsules (In all four species the short hairs may be inconspicuous) Usually sparsely hairy with long, patent, septate hairs, sometimes mixed with a few short hairs; less often densely hairy or glabrate. Typically most capsules at least 10 mm long Densely hairy with short, whitish, retrorse hairs, usually mixed with at least some long, patent, septate, hyaline hairs. Typically most capsules >12 mm long (often >20 mm) Usually densely hairy with short, whitish, retrorse hairs; long, patent, septate, hyaline hairs zero to numerous. Typically most capsules >10 mm long, sometimes shorter (especially in var. atropurpurea) Usually densely hairy with short retrorse hairs, often mixed with a few to numerous patent, septate hairs. Capsules typically <10 mm long, with <4 seeds per cell
Seeds Usually uniform brown; ridges rarely with ill-defined paler patches or stripes Brown, with sharply defined white dots or lines on the ridges Usually brown, but not infrequently with paler, vaguely defined dots or lines on the ridges Usually uniform brown

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 specimens were compared to an O. corniculata specimen 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 verified 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 perennial 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.

Figure 2. The approximate distribution of Oxalis dillenii in the Northern Hemisphere created from the observations in the Global Biodiversity Information Facility (http://doi.org/10.15468/dl.9imeus). The map uses an Albers equal-area conic projection.

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DISCUSSIONTop

The results demonstrate the presence of O. dillenii in Spain using both morphological and molecular approaches. Aymerich (2013Aymerich, P. 2013. Contribució al coneixement florístic del territorio ausosegàrric (NE de la península Ibèrica). Orsis 27: 209–259.) previously reported O. dillenii from Spain and an image of his specimen was obtained from the Vascular Plant Herbarium of the Real Jardín Botánico (MA specimen number 823538). This specimen is recognisable as O. dillenii and should be considered the first confirmed 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 collected at Fonollet, municipality of Casserres, Barcelona, 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 escombros 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 species is likely to spread further. Indeed, there have been unsubstantiated records from elsewhere in Spain (Bolòs et al., 1990Bolòs, O. de, Vigo, J., Masalles, R. M. & Ninot, J. M. 1990. Flora manual dels Països Catalans. Pòrtic, Barcelona.; Sánchez-Pedraja, 2015Sánchez-Pedraja, O. 2015 Oxalis L. In: Muñoz Garmendia, F. & Navarro, C. (Eds.), Flora iberica 9. Real Jardín Botánico (CSIC), Madrid: 384–405.). 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 invaded more natural habitats. We hope that publishing these observations will make botanists more aware of O. dillenii in Spain.

ACKNOWLEDGMENTSTop

Many thanks to C. Noya Santos and J. L. Fernández of the Real Jardín Botánico de Madrid for supplying an image of the Barcelona specimen. Thanks to I. Van der Beeten for taking photographs of the specimens. The specimen collected in Lleida was done so at a workshop organized by the EU-BON project (http://www.eubon.eu), funded by the EU Framework Programme (FP7/2007-2013) under grant agreement nº 308454.

REFERENCESTop

1. Aymerich, P. 2013. Contribució al coneixement florístic del territorio ausosegàrric (NE de la península Ibèrica). Orsis 27: 209–259.
2. Bolòs, O. de, Vigo, J., Masalles, R. M. & Ninot, J. M. 1990. Flora manual dels Països Catalans. Pòrtic, Barcelona.
3. Chase, M. W. & Hills, H. H. 1991. Silica gel: an ideal material for field preservation of leaf samples for DNA studies. Taxon 40: 215–220. https://doi.org/10.2307/1222975
4. Gray, A. M. 2011. Oxalidaceae, version 2011:1. In: M. F. Duretto (Ed.), Flora of Tasmania Online. Tasmanian Herbarium, Tasmanian Museum & Art Gallery, Hobart: 8 pp. Retrieved September 3, 2015, from www.tmag.tas.gov.au/floratasmania.
5. Hassler, M. 2015. World Plants: Synonymic Checklists of the Vascular Plants of the World. Species 2000 Secretariat, Leiden. Retrieved March 25, 2015, from http://www.catalogueoflife.org/col/details/database/id/141
6. Hoste, I. 2012. Een sleutel voor het genus Oxalis in België, met commentaar bij de waargenomen soorten. Dumortiera 101: 9–22.
7. Katoh, K., Misawa, K., Kuma, K. I., & Miyata, T. 2002. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research 30: 3059–3066. https://doi.org/10.1093/nar/gkf436
8. Moorsel, R. C. M. J. van, Beringen, R. & Odé, B. 2000. Een nieuwe klaverzuring in Nederland: Oxalis dillenii Jacq. Gorteria 26: 31–35.
9. Nesom, G. L. 2009. Again: taxonomy of yellow-flowered caulescent Oxalis (Oxalidaceae) in eastern North America. Journal of the Botanical Research Institute of Texas 3: 727–738.
10. Sánchez-Pedraja, O. 2015 Oxalis L. In: Muñoz Garmendia, F. & Navarro, C. (Eds.), Flora iberica 9. Real Jardín Botánico (CSIC), Madrid: 384–405.
11. Simmons, M. P. & Ochoterena, H. 2000. Gaps as characters in sequence-based phylogenetic analyses. Systematic Biology 49: 369–381. https://doi.org/10.1093/sysbio/49.2.369
12. Taberlet, P., Geilly, L., Pautou, G. & Bouvet, J. 1991. Universal primers for amplification on three non-coding regions of chloroplast DNA. Plant Molecular Biology 17: 1105–1109. https://doi.org/10.1007/BF00037152
13. Tel-Zur, N., Abbo, S., Myslabodski, D. & Mizrahi, Y. 1999. Modified CTAB procedure for DNA isolation from epiphytic cacti of the genera Hylocereus and Selenicereus (Cactaceae). Plant Molecular Biology Reporter 17: 249–254. https://doi.org/10.1023/A:1007656315275
14. White, T. J., Bruns, T., Lee, S. J. W. T. & Taylor, J. W. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications 18: 315–322.
15. Young, D. P. 1968. Oxalis L. In: Tutin T. G., Heywood, V. H., Burges, N. A. Moore, D. M., Valentine, D. H., Walters, S. M. & Webb, D. A. (Eds.), Flora Europaea 2. Cambridge University Press, Cambridge: 192–193.


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