Dipartimento di Biologia Ambientale
ANNALI DI BOTANICA COENOLOGY AND PLANT ECOLOGY
ANNALI DI BOTANICA Volume 11, 2021 V o l u m e
11 Ann. Bot. (Roma), 2021, 11: 00–00 Published in Rome (Italy) 2 ISSN 0365-0812 (print) 0 ISSN 2239-3129 (online) 2 1 Journal homepage: http://annalidibotanica.uniroma1.it Pietro Romualdo Pirotta, founder, 1884
DNA BARCODING SUPPORTS AN EASIER IDENTIFICATION OF ALIEN PLANTS: THE CASE OF THE GENUS PHYSALIS (SOLANACEAE) IN THE IBERIAN PENINSULA (SPAIN)
Terrones A.1, Moreno J.1, Juan A.1*
1 Dpto. Ciencias Ambientales y Recursos Naturales. University of Alicante. P.O. Box 99, E-03080 Alicante (Spain) *Corresponding author, Telephone +34965903400 ext. 3371; e-mail: [email protected]
Received 21 April 2020; received in revised form 6 September 2020; accepted 17 September 2020
Abstract - In the present study, two species of the genus Physalis, P. acutifolia and P. angulata, are reported for the first time as casual aliens for Spain and, even for Europe, in the case of P. acutifolia. Their identification was based ona priori DNA barcoding study to reveal their taxonomic identification, which was followed by a posteriori morphological analysis to support the barcoding identification. As many authors have reported, the reduced and even inexistent information from local or regional Floras makes it difficult to distinguish those unreported taxa from distant geographical areas. Therefore, the successfully obtained results would highlight the importance of using an integrative taxonomic approach for further unidentified alien plant species. Detailed descriptions of both species and a dichotomous key of the alien taxa of Physalis are provided to facilitate their morphological identification.
Keywords: Alien species; European flora; floristic records; genetic markers; morphology; Spain; Solanaceae
Introduction
Most regions of the world have been colonized by species from correct name of an organism are crucial for implementing many other parts of the globe. The accurate identification of effective quarantine measures, monitoring invasions and the allochthonous flora is nowadays a fundamental challenge their invasion pathways, and ensuring that the time to to facilitate the knowledge of the alien species around a given first detection for new invaders is minimized. However, geographical area. Alien plants, and specifically invasive taxonomic identification of alien plants is a hard process that plants, have aroused attention globally for causing negative demands expert taxonomists and time, and it is often difficult impacts on the native biodiversity, among other effects, and to distinguish species only with morphology, especially for the design of adequate management plans for avoiding their those unreported taxa originated in distant geographic areas. uncontrolled dispersal might be crucial. However, it can be Pyšek (2003) already remarked the importance of available extremely challenging to rapidly and accurately identify and updated information about alien flora, which is usually these species only with morphological characters because quite reduced, and even nonexistent from local or regional they are an assemblage of many different families and most published Floras (note the capital letter to denote published plant material lacks enough diagnostic characteristics (Xu work). Moreover, other aspects related to the specific time et al., 2018). Besides, the allochthonous flora is mainly of collection (flowering or fruiting period), the scarcity of composed of a specific number of families (e.g. Asteraceae, plant material and their conservation as preserved material Amaranthaceae, Cactaceae, Fabaceae, Solanaceae, Poaceae) often originate a lack of diagnostic morphological features, whose taxonomy is rather complicated. Pyšek et al. (2013) which also increases the complexity for their satisfactory already stated that taxonomic tasks related to determine the taxonomic identification. doi: 10.13133/2239-3129/16618 Copyright © 2021 2 Terrones A., Moreno J., Juan A. / Ann. Bot. (Roma), 2021, 11: 00–00
DNA barcoding is an effective tool for identifying species observable on fresh plant material (e.g. the number of angles using standard DNA regions or barcodes (Hebert et al., in fruit, color of spots in the throat). However, and depending 2003; CBOL Plant Working Group, 2009). DNA allows on the period of the plant collection, some preserved material a rapid species discovery and identification and has been could not include certain characters that are only well widely used for taxonomic identification by targeting known developed during the flowering or fruiting period (e.g. colour gene regions that permit to discriminate among species. The of the veins of the fruiting calyx for P. philadelphica). In DNA of an unidentified sample is queried against a reference fact, Sullivan (2004) already stated that some specimens of sequence database and the identity of the matching sequence Physalis are difficult to determine from preserved material. is assigned to the sample. Despite the described limitations of Regarding the collected plant material, European and Spanish this molecular tool (Chase & Fay, 2009), it has proved to be Floras were examined to detect previous indications of these functional in the detection of species with high phenotypic species. None of the available literature about Physalis taxa plasticity and in early or incomplete developed specimens around Spain and some close Mediterranean countries concur (Valentini et al., 2008). Among other scopes, DNA barcoding with the observed morphological features of the collected and has been recently successfully applied in plant invasion biology preserved material. Actually, certain morphological aspects (Ghahramanzadeh et al., 2013) and in the identification of detailed by the literature are not fully coincident, which led invasive species of a specific family or a geographical area to certain reservations about their taxonomic identification. (Zhang et al., 2013; Xu et al., 2018). A correct taxonomic Due to this situation, integrative taxonomy would be the identification will lay a solid foundation for further actions (Xu adequate approach to identify the unknown plant species. et al., 2018), and therefore, the application of DNA barcoding Integrative taxonomy is a multidisciplinary discipline that would be crucial to help and support the taxonomists about combines evidence from independent data sources (e.g. the final identification of those new alien species, especially if morphology, DNA, ecology, etc.) in order to reveal biological the available plant material is also somehow incomplete or the features relevant for species delimitation (Dayrat, 2005). preserved material does not show clear diagnostic characters The combination of different characteristics helps to identify (Ghorbani et al., 2017; Xu et al., 2018). taxa from different environmental data at generic and species Based on field surveys along the river Vinalopó (District levels (e.g. De Mattia et al., 2012; Darienko et al., 2015), Medio Vinalopó 38º 28’ 41’’N 0º 47’ 24’’W, southeast Iberian using DNA barcoding analyses together with conventional Peninsula, Spain), two alien specimens belonging to the morphological identifications. Molecular techniques serve as genus Physalis L. (Solanaceae) have been recently found. a complement to classical morphological analyses to give a According to the last phylogenetic approach of the Solanaceae final taxonomic identification especially for non-native plant tribe Physalideae (Deanna et al., 2019), this genus includes species under any stage of their life cycle (e.g. seedlings), about 96 species with a Neotropical distribution, with centres since standard morphological techniques alone are sometimes of diversity in Mexico, United States and Central America. highly laborious and require taxonomic expertise (Whitehurst Physalis is typified by its solitary yellow or white flowers et al., 2020). To our knowledge, this study will comprise with dark maculations and its fruit completely enclosed by an the first approach to identify alien flora based on integrative inflated and accrescent calyx (Pretz & Deanna, 2020). Other taxonomy, and this protocol might be a useful route for further closely related taxa, characterized by their fruiting calyx, have unidentified alien plant species, especially if they belong to also been considered within Physalis according to previous complex taxa. taxonomic treatments, as it happens to the monotypic genus Alkekengi Mill. This genus is well characterized by bright orange to red papery calyx, and its unique species, A. officinarumMoench, appears as P. alkekengi L. in many Floras. Materials and Methods Many Physalis species and close related taxa have largely been cultivated for ornamental or food uses in warm areas worldwide, and therefore, ten alien taxa have been mentioned DNA amplification and molecular analyses for European and North African territories (Valdés, 2012). In the Iberian Peninsula (Spain), Sanz-Elorza & Sobrino (2012) In order to identify the two unknown Physalis samples mentioned the presence of four taxa of the genus Physalis (P. by DNA barcoding, DNA amplifications (PCR) of the ixocarpa Brot., P. peruviana L. and P. philadelphica Lam., nuclear internal transcribed spacer (ITS) were conducted, including A. officinarum as Physalis), which are scattered since this region has recently been used in this genus for around this territory. The species boundaries within Physalis DNA barcoding and phylogenetic studies with notable are sometimes poorly defined (Whitson & Manos, 2005; phylogenetic resolution (Feng et al., 2016; Deanna et al., Deanna et al., 2019) and some taxa are typically defined 2019). Plant material of the two unknown Physalis samples by certain morphological characters, which are adequately was sequenced from the preserved specimens at the ABH BARCODING AND IDENTIFICATION OF ALIEN PLANTS 3
herbarium (University of Alicante, Spain; see Appendix 1) Morphological studies since no new field material was available for this study. The PCR amplifications were directly conducted with the The preserved specimens at the ABH herbarium and some Thermo Scientific Phire Plant Direct PCR Master Mix additional preserved material from the online database of the (Thermo Fisher Scientific Inc, Waltham, MA, USA), following MEXU herbarium from the UNAM-Mexico (datosabiertos. the Dilution and Storage protocol as described by the unam.mx) were also consulted (see Appendix 1). Geocoding manufacturer, using dried plant material (leaves and flowers) of the Spanish localities of the plants was performed using a from the two different specimens (named as Novelda and Elda, portable GPS device Garmin Montana 650t (Garmin, USA), respectively; see Appendixes 1 and 2). The ITS primers used with the geographic system UTM WGS84. The morphological were ITS5 (5’-GGAAGTAAAAGTCGTAACAAGG-3’) and features of the specimens were studied based on regional ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) (White et al., European and American Floras (Hawkes, 1972; Vargas et al., 1990). Cycling protocol was an initial denaturation of 98 ºC 2003; Sanz-Elorza & Sobrino, 2012; Valdés, 2012; Verloove, for 5 min; 35 cycles of 98 ºC for 5 s, 60 ºC for 5 s and 72 ºC for 2019), and specific research papers focused on the genus 20 s; and a final extension of 72 ºC for 1 min. PCR products Physalis (Waterfall, 1958, 1967; D’Arcy, 1973, 1991; Sullivan, were cleaned using UltraClean PCR Clean-up Kit (MoBio 2004; Ward, 2008; Landrum et al., 2013). The given detailed Laboratories Inc, Carlsbad, CA, USA), and sequenced descriptions have been adapted from the above-mentioned bidirectionally by Macrogen Spain (Madrid, Spain). The literature and completed with observations of living plants and obtained sequences were submitted to NCBI GenBank dried specimens at ABH and MEXU (see Appendix 3). (https://www.ncbi.nlm.nih.gov/genbank/) (see Appendix 2). Firstly, the taxonomic identification of the two unknown Physalis sequences was based on the comparison with the reference data deposited in the molecular NCBI GenBank Results database, using the BLAST algorithm (Altschul et al., 1990). The output results yielded a list of the species name showing the top nearest matches (minimum e-value) according to Barcoding data Meiklejohn et al. (2019) guidelines. Identifications were considered reliable if several records with the same taxonomic The first three BLAST top matches are reported for both name showed similar top match statistics. Conversely, if unknown Physalis samples in Table 1. Our results revealed multiple records with different taxonomic names had the that the sequence ITS-Novelda had a clear correspondence same maximum nearest scores, the identification was treated with the taxonomic name P. acutifolia (Miers) Sandwith, as an ambiguous match. since the two first matches showed a similar top score identity Secondly, and following the taxonomic treatment for the (99%). The second sequence (ITS-Elda) showed the same genus Physalis based on Deanna et al. (2019), a search highest identity values with the three first top matches, which for the available ITS sequences of Physalis species always corresponded to the species P. angulata L. Therefore, was performed on NCBI GenBank on 25 February the two outputs provided a reliable taxonomic identification 2020 (Appendix 2). Sequences of the species Alkekengi for both sequenced samples. officinarum and Calliphysalis carpenteri (Riddell) Whitson were used as outgroup according to the phylogenetic treatment of Solanaceae tribe Physalidae (see Zamora- Table 1. BLAST identification of the twoPhysalis samples collected Tavares et al., 2016; Deanna et al., 2019). The sequences using ITS. Identification results were provided as the species name were aligned with ClustalW with default settings and showing the top nearest matches (minimum e-value). The first three posterior manual editing in MEGA X v. 10.1.7 (Kumar maximum nearest matches with the species name, GenBank accession and identity scores are reported for each studied sample. et al., 2018). Those sequences too short, too divergent or with too many ambiguities were finally deleted. The ITS Sample Species GenBank Identity aligned matrix consisted of 128 sequences with a length of 690 bases, that came from 13 different origins according ITS-Novelda Physalis acutifolia KY968916 99% to GenBank records (eight from published articles and Physalis acutifolia AY665876 98% five from unpublished works; see Appendix 2 for details). Neighbour-Joining (NJ) trees were constructed with the Physalis crassifolia AY665889 97% Maximum Composite Likelihood model, uniform rates ITS-Elda Physalis angulata MH050300 100% among sites, pairwise deletion in the gaps, and, similarly Physalis angulata MH763725 100% to Deanna et al. (2019), a bootstrap (BS) based on 1000 Physalis angulata AY665875 100% replications was also conducted to assess nodal support. 4 Terrones A., Moreno J., Juan A. / Ann. Bot. (Roma), 2021, 11: 00–00