Invasion history of Harmonia axyridis (Pallas, 1773) (Coleoptera: Coccinellidae) in Ecuador

Harmonia axyridis is a ladybird extensively used around the world for biological control of agricultural pests. However, it has become invasive in several countries, producing negative ecological and socio-economic impacts. Herein, we review the invasion history of the Harlequin Ladybird Harmonia axyridis (Pallas, 1773) in Ecuador. Although first reported in Ecuador in 2012, museum specimens date back to 2004 and it is currently established across the country, especially along the Andean region. Due to its invasive nature, further studies are urgently needed to evaluate possible impacts of H. axyridis on the Ecuadorian biodiversity and agroindustry.

Harmonia axyridis (Pallas, 1773), commonly referred to as Harlequin Ladybird or Asian Multicolored Ladybeetle, is a member of the family Coccinellidae native to East Asia (Orlova-Bienkowskaja, Ukrainsky & Brown, 2015). It has been deliberately translocated as a control agent in America, Europe and Africa since the beginning of the 20th century, establishing naturalized and expanding populations in the three continents, becoming invasive (Lombaert et al., 2010;Brown et al., 2011). It is a successful invader due to its wide dietary range, ability to establish and disperse, and robustness and descend from eastern USA stock. It is probable that unrecorded international shipments were sent to Colombia, Argentina, and Chile-possibly to private farmers, thus the absence of public records. Subsequent South American records come from Ecuador (2004, see below), Paraguay ( , Silvie et al., 2007, Uruguay (2007, Nedvěd & Krejčík, 2010Serra, González & Greco-Spíngola, 2013), Peru (ca. 2010, Grez et al., 2010), and Venezuela (ca. 2014, Solano & Arcaya, 2014. It has not been formally reported from Guyana, Suriname and Bolivia (Camacho-Cervantes, Ortega-Iturriaga & Del-Val, 2017;Hiller & Haelewaters, 2019), but a recent citizen-science record shows that it is already present in Bolivia (Maslowski, 2020 (2015) reported the species from mangroves on Santay Island (at sea level), province of Guayas, southwestern Ecuador. González (2015) reported H. axyridis from the provinces of Azuay, Guayas and Loja, but without referencing any voucher specimen from Azuay. Guamán Montaño (2017) presented photographs of H. axyridis from El Pangui (830 m elevation), province of Zamora-Chinchipe, providing the first reports on the south-eastern slopes of the Andes of Ecuador. Geographic and ecological data of H. axyridis in Ecuador are scarce. Herein, we discuss the distribution, natural history, and introduction history of Harmonia axyridis in Ecuador, showing that it has been present at least since 2004 and is currently widespread across the country.

MATERIALS AND METHODS
Coccinellid beetles were opportunistically collected since 2015 during field surveys of the Universidad San Francisco de Quito USFQ at 17 localities across northern Ecuador, (Table S1; Figs. 1 and 2). Field surveys were conducted by the authors, usually with 8-15 undergraduate students of the USFQ Biology program. All specimens were found by searching vegetation to look for adults and larvae. Collected specimens were euthanized by immersion in 70% ethanol or by placing in a killing jar and stored. An Olympus Research Stereomicroscope System SZX16 outfitted with an Olympus DP73 digital colour camera was used to examine specimens. Voucher specimens collected during our surveys are deposited at the Museo de Zoología (ZSFQ), Universidad San Francisco de Quito USFQ, Ecuador. We reviewed the entomological collections of Museo de Zoología, Pontificia Universidad Católica del Ecuador, Quito (QCAZ), and Instituto Nacional de Biodiversidad INABIO, Quito (MECN). Published information on Harmonia axyridis in Ecuador was synthesised based on a literature review using the library systems of King's College London and Universidad San Francisco de Quito USFQ and Google Scholar TM scholarly text search (https://scholar.google.com). Relevant references were gathered using the search terms 'Coccinellidae,' 'Harmonia,' 'Harmonia axyridis,' 'Mariquita,' 'Ladybird,' 'Ladybug' and 'Lady beetle', each one combined with 'Ecuador' by the Boolean operator 'AND'. Since H. axyridis has a diagnostic colouration pattern that allows its identification in photographs, we assembled data from photographic vouchers using the search engines of Flickr TM (https://www.flickr.com, by Yahoo!) and iNaturalist.org TM (http://www.inaturalist.org, by California Academy of Sciences and the National Geographic Society) through GeoCat (Bachman et al., 2011; http://geocat.kew.org/) using the same search terms used for text searches. All searches were run on 10 February 2019 using on-site search engines and were not limited by study type, study design, or language. iNaturalist searches were rerun on 09 August 2019 and on 01 April 2020.
All localities, based on field surveys, literature, museum and photographic records, were georeferenced manually in Google Earth TM mapping service (7.3.1.5491 release by Google, Inc. on July 2018) based on direct information (coordinates and altitudinal data) when available, and additional data relevant to obtain an accurate and precise positioning, including catalogue and field notes, following recommendations by Wieczorek, Guo &  Table S1) Full-size  DOI: 10.7717/peerj.10461/ fig-1 Hijmans (2004). All localities were reviewed and validated individually, and coordinates were amended when incorrectly georeferenced in the source (Table S1). We determined the position most closely related with the locality description using toponymic information based on the Geographic Names Database, containing official standard names approved by the United States Board on Geographic Names and  Table S1) Full-size  DOI: 10.7717/peerj.10461/ fig-2 maintained by the National Geospatial-Intelligence Agency (http://geonames.nga.mil/gns/ html/), OpenMapStreet data available under the Open Database Licence (http://www. openstreetmap.org), and gazetteers for Ecuador (Brown, 1941;Peters, 1955;Lynch & Duellman, 1997).

RESULTS
In total, we collected information for 294 records of Harmonia axyridis from 53 localities in Ecuador (Table S1; Figs. 1 and 2), including: 106 specimens collected during field surveys and deposited at ZSFQ, 11 individuals recorded during field surveys but uncollected, 37 museum specimens (37 at QCAZ, none at MECN), 118 individuals recorded in iNaturalist, five photographic records from Flickr, and 17 literature records (González & Kondo, 2012;Cornejo & González, 2015;Guamán Montaño, 2017). Most records presented herein correspond to adult individuals, but larvae and pupa were recorded across the Andes (Table S1). Specimens were identified as Harmonia axyridis by its characteristic morphology (Fig. 3), including: upper surfaces of elytra not hairy, distinct transverse fold at rear of elytra, underside of abdomen at least partially orange, brown to orange legs (Koch, 2003;Roy et al., 2016). One phenotypic colour form was found: f. succinea, with ground colour of pronotum white to light brown with M-shaped black marks, ground colour of elytra bright orange, usually with nine black elytral spots (2-3-3-1) on each elytra, and a scutellary spot (Dobzhansky, 1933;Tan & Li, 1934;Koch, 2003;Brown et al., 2008a;Roy et al., 2013;Roy et al., 2016). The first specimens of H. axyridis in Ecuador were collected on both geographical extremes of the country: in 2004 at Mindo, northwestern Ecuador, and in 2007 at Loja, in southwestern Ecuador (Table S1). Both localities are separated by about 450 km and now have established populations. Our study reveals that H. axyridis is now established in all main biogeographic regions of Ecuador, from sea level to at least 4,020 m elevation, across 16 provinces (administrative geopolitical divisions of Ecuador) (Table S1; Fig. 1). At least one record (at Estación Científica Yasuní) may correspond to a hitchhiking individual, since no established population in the Amazonian lowlands has been confirmed. The only regions where we did not find records are the southern Amazonian lowlands and the Galapagos Archipelago.
Almost two-thirds of the localities where we found records of axyridis are anthropogenic habitats (51% are urban green spaces in mid-size towns and large cities, and 20% are agricultural lands; Table S1). Most of the records of H. axyridis in Ecuador come from the Andes, a region with significant agricultural and urban areas, including the capital city, Quito, and its metropolitan district. In urban green spaces, H. axyridis usually occupies gardens and parks dominated by non-native plants. Harmonia axyridis has been found also in 12 different ecosystems with native vegetation, usually collected along road borders and near human settlements.
During our surveys, H. axyridis was associated with the following plants ( Almost 30% of our field records of H. axyridis come from the Cumbayá-Tumbaco valley, an inter-Andean valley near Quito (Fig. 2), in northern Ecuador, where we had a higher sampling effort and were able to observe the coccinellid community in more detail. We found six coccinellid species in sympatry in gardens, parks and agricultural areas of the Cumbayá-Tumbaco valley: Harmonia axyridis, Cheilomenes sexmaculata, Hippodamia convergens, Mulsantina mexicana, Rodolia cardinalis and Cycloneda sanguinea. Harmonia axyridis was the most common species in green urban areas, but is uncommon in agricultural areas, where H. convergens was dominant. Rodolia cardinalis and C. sexmaculata were found in syntopy with H. axyridis. By 2017, H. convergens was almost absent in most urban green areas of the Cumbayá-Tumbaco valley, remaining common only in agricultural areas. Cheilomenes sexmaculata, an apparently recent arrival-first recorded in the area on 2017, is nowadays becoming the second most common coccinellid in urban green areas of Quito-Cumbayá-Tumbaco, although still with a patchy distribution. Introduction of ladybirds has a long history in Ecuador; for example in 1978, official national authorities released 24 million individuals of Hippodamia convergens in the city of Quito and surroundings, in an attempt to control Icerya purchasi (Molineros Andrade, 1984). However, it is also possible that Ecuadorian populations spread from southern Colombia, since the oldest Colombian records occurred very close to the Ecuadorian border (Kondo & González, 2013). The presence of earliest Ecuadorian localities on opposite sides of the country (Mindo and Loja) and the absence of geographically intermediate records could suggest that Ecuadorian populations had two independent origins. However, museum records are biased due to limited collection efforts in the central provinces of Ecuador. Furthermore, spread rate of H. axyridis may be extremely fast and compensate for the distance between the localities (58-144.5 km/year in the UK, Brown et al., 2008b;200 km/year in Slovakia, Roy et al., 2016;185 km/year in Chile, Grez et al., 2016;442 km/year in USA-Canada, McCorquodale, 1998;500 km/year in South Africa, Stals, 2010). If the southern Ecuadorian population is demonstrated to have an independent origin, they may have been the source of the northern Peru populations, that remained unrecorded during extensive surveys in 2006 (Miró-Agurto & Castillo-Carrillo, 2010) and became established around 2010 (Grez et al., 2010).

DISCUSSION
The highest record of H. axyridis in Ecuador, at 4,020 m at Mojanda, is also the highest record worldwide, 500 m higher than the upper elevational ranges reported by Grez et al. (2017) and González, Bustamante & Grez (2019). Unfortunately, no ecological information was associated with that specimen. Lowland records mainly come from the Pacific lowlands and western Andean slopes, but also from the Amazonian foothills and lowlands. The Andean region was predicted as suitable for the expansion of H. axyridis by distribution models analysed by Koch, Venette & Hutchison (2006) and Poutsma et al. (2008), and although they predicted the expansion of H. axyridis across different habitats of America, their models did not show lowland forest. Interestingly, Ecuadorian records come from a variety of habitats, including forest and shrublands, evergreen and semideciduous vegetation, and across the urban-agricultural matrix. However, the most extensive and dense populations were found in urban areas.
It is likely that H. axyridis will keep spreading across most of Ecuador, especially in urban and agricultural environments, having effects on the diverse Ecuadorian fauna of coccinellids due to competition, exclusion, and intraguild predation. Harmonia axyridis may significantly impact predatory arthropod guilds, interfering with invertebrate population dynamics, potentially producing impacts on native aphidophage groups and agricultural pests (Lucas, Gagné & Coderre, 2002;Koch, 2003;Pervez & Omkar, 2006;Koch & Galvan, 2008;Ducatti, Ugine & Losey, 2017). In particular, the arrival of H. axyridis to the Galapagos Archipelago could be problematic, due to the vulnerability of island ecosystems to impacts on endemic and native invertebrates and profound irruptions on trophic interactions (Causton et al., 2006).
Harmonia axyridis has attained the status of agricultural pest in North America because it feeds opportunistically on fruit when prey is scarce and acts as a wine contaminant (Koch et al., 2004;Koch & Galvan, 2008). Grapes, apples, peaches, plums, pears, raspberries, among other fruits, have been reported to be consumed by H. axyridis, blemishing the fruits and reducing their value (Majerus, Strawson & Roy, 2006;Koch & Galvan, 2008;Guedes & Almeida, 2013). All these fruits are produced in Ecuador, usually for local consumption but, in recent decades, have become important exportation products. Fruit crops occupy over 1600 km 2 in Ecuador (excluding bananas), are produced by more than 120,000 farmers, and represent a small but growing sector in non-traditional agricultural exports in the country-contributing to ca. 4.4% of all non-traditional exportations (Viera et al., 2016;Verdugo-Morales & Andrade-Díaz, 2018;Banco Central del Ecuador (BCE), 2020). Most Ecuadorian fruit crops are located across the highlands and western lowlands of the country (Niegel, 1992;Huttel, Zebrowski & Gondard, 1999), coinciding with areas where H. axyridis is expanding.
Wine contamination has been described as the most important agricultural impact of H. axyridis. Adults aggregate on injured grapes and can be disturbed or crushed during harvesting or pressing, releasing haemolymph that affects wine quality by causing unpleasant odour and taste-that is ladybug taint (Pickering et al., 2004(Pickering et al., , 2008Koch & Galvan, 2008). Although table and wine grapes have been grown in Ecuador for local consumption since the 16th century (Popenoe, 1924), commercial production has only been fostered in recent decades (El Comercio, 2017;Revista Líderes, 2012, 2013Viera et al., 2016). Vineyards in Ecuador have increased from 0.6 km 2 in 1985 to more than 2 km 2 today-and are expected to reach 10 km 2 in the near future (Niegel, 1992;El Comercio, 2017;Revista Líderes, 2012, 2013. Established populations of H. axyridis have been reported in all areas where Ecuadorian wineries are situated (i.e. provinces of Guayas, Pichincha, Azuay and El Oro). Although Ecuadorian wine production is still modest, its presence in national and international markets is expanding (ProEcuador, 2017) and ladybug taint could negatively impact this growing industry.

CONCLUSIONS
Harlequin Ladybird Harmonia axyridis currently holds established populations across Ecuador. It was introduced to the country at the beginning of the 21st century and, with a fast spread rate, nowadays occupy most Andean highlands (including the highest elevation worldwide at 4,020 m), and it is expanding across the Pacific and Amazonian lowlands. Information on the coccinellids of Ecuador is limited and fragmentary. It is important to increase research on the diversity, distribution, natural history, ecology and socio-economic effects of coccinellids in the country. Information is needed across urban-agricultural-natural matrices, in order to evaluate the impacts of H. axyridis and other non-native species. Research on the impacts of H. axyridis in Ecuadorian agribusiness, especially fruit and wine production, is urgently needed. Carlos Ruales for helping us to find some key literature about the first introductions of ladybirds in Ecuador and to all citizens scientist that continuously contribute to iNaturalist. We thank Peter Brown and Lucia Almeida for their comments on a previous version of this article.

ADDITIONAL INFORMATION AND DECLARATIONS Funding
This study was supported by Universidad San Francisco de Quito USFQ (Research Funds for projects ID 35 "Biodiversity of urban and rural areas of Ecuador", and ID 1057 "Impacts of habitat changes on the biological diversity of the northern tropical Andes", Outreach project "Celebrando la Naturaleza" 2017-2020, and Publication Fund to Diego F. Cisneros-Heredia) and operative funds assigned to Instituto de Diversidad Biológica Tropical iBIOTROP, Museo de Zoología & Laboratorio de Zoología Terrestre, Colegio de Ciencias Biológicas y Ambientales COCIBA and by Programa "Becas de Excelencia" of Secretaría de Educación Superior, Ciencia, Tecnología e Innovación SENESCYT, Ecuador. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Grant Disclosures
The following grant information was disclosed by the authors: Universidad San Francisco de Quito USFQ.

Competing Interests
The authors declare that they have no competing interests.

Author Contributions
Diego F. Cisneros-Heredia conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft. Emilia Peñaherrera-Romero conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

Field Study Permissions
The following information was supplied relating to field study approvals (i.e. approving body and any reference numbers): Research permits were issued by

Data Availability
The following information was supplied regarding data availability: Raw data are available as a Supplemental File.

Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.10461#supplemental-information.