The Brazilian elodea ( Egeria densa Planch . ) invasion reaches Southeast Europe

Egeria densa is a South American aquatic plant species considered highly invasive outside of its original range, especially in temperate and warm climates and artificially heated waters in colder regions. We report the first occurrence and the spread of E. densa in Southeast Europe, along with physicochemical and phytosociological characteristics of its habitats. Flowering male populations were observed and monitored in limnocrene springs and rivers in the Mediterranean part of Croatia from 2013 to 2017. The populations inhabited clear, slow flowing, oligohaline water with high alkalinity and conductivity. Over the course of our research, the species displayed its invasive potential forming monospecific stands and surpassing the native flora in particular localities, while being obviously codominant with indigenous species in others. Although we cannot specify the exact path of introduction, we presume two possible scenarios: release from aquaria or epizoochory through water birds. The current situation in SE Europe, including Croatia, seems satisfactory regarding the number of aquatic alien species; nevertheless a careful and systematic approach to research into the management of invasive alien species is needed.


Introduction
Egeria densa Planch. is a submerged, freshwater perennial plant of the family Hydrocharitaceae.It is native to South America, specifically Brazil, Uruguay, and Argentina.The original distribution range of the species includes SE Brazil, extending from the regions Minas Gerais and Espirito Santo, following the coast southwards through Uruguay and reaching Buenos Aires.In Argentina, the species is also present along the Parana and Uruguay Rivers almost to the border with Paraguay and occupies the region around Cordoba (Cook and Urmi-König 1984).In its native range, it is commonly found in still, 1-2 m deep water and is less common in shallower and flowing water (Cook and Urmi-König 1984).
Nowadays, this species is considered a threat to aquatic ecosystems outside of its original range.It has shown major invasive potential in countries where it has been introduced, mainly through the ornamental trade, as it is very practical and attractive aquarium plant (Hussner 2012).This species has been proved to be a highly efficient invader due to its high biomass production, fast vegetative reproduction and efficient dispersal (Yarrow et al. 2009).Egeria densa is considered invasive or a pest in South America (Yarrow et al. 2009), North America (Les and Mehrhoff 1999), Asia (Haramoto and Ikusima 1988;Alfasane et al. 2010), the Pacific islands (Wagner et al. 1999), Australia (Roberts et al. 1999), New Zealand (Mccullough 1997), Africa (Coetzee et al. 2011) and Europe (Hussner 2012).
The species is included in the List of Invasive Alien Plants proposed by the European and Mediterranean Plant Protection Organization (EPPO) and is reported from 17 European countries (Seregin 2008;Kikodze et al. 2010;Hussner 2012;Medvecká et al. 2012;Wasowicz et al. 2014;Fominykh et al. 2016).Originally a subtropical species, in colder regions of Europe E. densa often forms self-sustaining populations in water bodies with artificially increased water temperature (Hussner and Lösch 2005;Wasowicz et al. 2014;Fominykh et al. 2016).It is found in both lentic and lotic environments (Yarrow et al. 2009) and appears to be truly naturalized in warm-temperate and cool subtropical conditions.Within subtropical and tropical areas, E. densa is limited to high altitudes or cold-water springs (Cook and Urmi-König 1984).
This paper presents new localities for E. densa, and is the first to document its spread to the Balkan Peninsula and Southeast Europe.Along with distribution data, we present data on habitat characteristics and the observed trend of its spread.Furthermore, we aim to emphasize the importance and benefits of national monitoring of surface waters, which has been carried out according to the Water Framework Directive (European Community 2000), not only as a tool for water quality assessment but also as a way in which alien aquatic taxa may be rapidly detected.

Study area
The field survey was performed in over 600 locations covering most of the Croatian territory, including 277 rivers and 46 lakes (including reservoirs).
All sites at which Egeria densa occurred are situated within the alluvial delta that extends along the final 30 km of the course of the Neretva River, before its mouth into the Adriatic Sea.The Neretva River and its tributaries comprise the largest complex of wetland habitats in the Croatian coastal zone.The Delta is rich in underground water, which supplies numerous springs, streams and lakes, and is characterized by well-developed aquatic, wetland and coastal vegetation.It is one of a few preserved wetland areas in the Mediterranean region of Europe and consequently a Ramsar site of great importance for the breeding, migration and wintering of numerous wetland bird species.Furthermore, it is protected on a national level and includes four Special Reserves and two Significant Landscapes.The area is also included in the Natura 2000 network as an SPA (Special Protection Area) and a pSCI (proposed Site of Community Importance).
The whole Delta area has been strongly anthropologically influenced since the 19 th century, when reclamation, construction of irrigation channels, river bed regulation and hydrological regulation in general started to alter the landscape.The marshes, lagoons and lakes that once marked the lower course of the Neretva River have been drastically reduced or have disappeared, and what was a delta swamp became a predominantly agricultural area within the last fifty years.Only fragments of the old Mediterranean wetlands have survived (Margeta and Fistanić 2000;Kralj et al. 2016).To gain more arable land, people used a method of digging and depositing soil and created a unique landscape of dense networks of channels and arable fields which can be cultivated from boats (Curić 1994;Kralj et al. 2016).Due to the above mentioned interventions and changes, the majority of the water bodies in the Delta are now very well interconnected.
Along the left and right edge of the Delta numerous big karst springs, such as Bađula, Bili Vir, Mislina, Mlinište, Modro Oko, Prud appear, as well as smaller ones.These are ascending springs in which water upwells through cavernous karst formations and forms small lakes (limnocrene springs) on the surface.

Description of the sites
The Norin River is a tributary of the Neretva River, and is a medium sized, lowland river on limestone bedrock (European Community 2000; Mihaljević 2011) (Figure 1).The Mislina River is also a medium sized watercourse on limestone bedrock (European Community 2000; Mihaljević 2011), strongly channelized and connected both to the Neretva River and to the Adriatic Sea.The Prud Spring and the Modro Oko Spring supply the Norin River and the Neretva River, respectively.The Prud Spring is protected as an ornithological Special Reserve and is situated close to the ornithological reserve Pod Gredom.The Modro Oko Spring is a limnocrene karst spring, situated at the right bank of the Neretva River, in the immediate vicinity of the Significant Landscape Modro Oko and Lake Desne (Figure 1).

Macrophyte survey
Data on the distribution of E. densa, as well as physicochemical and phytosociological characteristics of its habitats, were collected within the national system for monitoring surface waters and the scientific project MULTISEC (Multimetric System of Evaluating Crenobiocoenosis).Over 600 locations throughout Croatia have been periodically visited within the monitoring scheme, while the particular occurrence sites for E. densa were visited 1-4 times within the study period.Monitoring of macrophytes was carried out during the vegetation seasons (June-September) from 2010 to 2017.Watercourses were surveyed for macrophytes along 100 m-long transects, while 6 × 100 m transects were used when surveying macrophytes in lakes.In less-accessible areas, the river/lake bottom was raked to reach the macrophytes, with the rake either on a long pole or at the end of a rope.Cover and abundance of each plant species were estimated according to the five degree Kohler scale (1 -very rare, 2 -rare, 3 -common, 4 -frequent, 5 -abundant, predominant), which estimates the frequency of each species and takes into account the three-dimensional development of the plant stands (Kohler 1978).
All macrophyte species were recorded and identified to species level.Egeria densa was identified based on morphological characteristics using several identification keys (Pignatti 1982;Cook and Urmi-König 1984;Koehler and Bove 2001;Dandy 2010;van de Weyer et al. 2011).Collected specimens of E. densa were conserved in 50% ethanol with 10% glycerol added, or desiccated in a herbarium press and deposited in herbarium ZA (Thiers 2018) under the ID numbers: ZA5686, ZA5687, ZA5688, ZA5689.
All localities within the national surface water monitoring system were also sampled for basic water physicochemical parameters and nutrients, while localities outside national monitoring were sampled only for basic water physicochemical parameters.
Conductivity, salinity and pH were measured in situ with a SevenMulti Modular Meter System (Mettler Toledo) under standard conditions.Dissolved oxygen was measured in situ using a Hach HQ40D Portable Multi Meter using an optical sensor.Water samples for alkalinity measurement were stored at 4-5 °C and alkalinity was determined by titration within 24 h of sampling.Water samples for total phosphorus analysis were preserved in sulphuric acid and analysed with a UV-VIS spectrometer (Perkin Elmer Lambda 25).Water samples for total nitrogen and total organic carbon (TOC) were collected in dark bottles, filled to the top, stored at 4-8 °C and analysed within 24 h using a Shimadzu TOC-VCPH equipped with an analyser for total nitrogen and TOC.Water samples for ammonium, nitrate and orthophosphate analyses were immediately filtered through glass filters with 0.45 µm pores, stored at 4-8 °C and analysed within 24 hours using an ionic chromatographer (Dionex 3000).The values of nutrients were used to assess water quality status according to the Regulation on the Water Quality Standard (Official Gazette 73/2013, 151/2014, 78/2015), with predefined thresholds delimiting between the categories: very good, good, moderate, poor, very poor.
Table 1.A summary of the establishment and spread of Egeria densa in the Neretva Delta.Presence and abundance of E. densa at surveyed localities according to Kohler's scale: 0 -absent, 1 -very rare, 2 -rare, 3 -common, 4 -frequent, 5 -abundant, predominant (Kohler 1978), X -longitude and Y -latitude coordinates in WGS84 coordinate system.*Location was not surveyed in that particular year.

Results
Egeria densa was first recorded in the lower reach of the Norin River in 2013, near the settlement Romići.
After the initial discovery, a detailed search of the location was conducted and E. densa was subsequently recorded in nearby sites (Table 1, Table S4).Periodical searches of the same sites have indicated its invasive potential, whereby the species tended to increase its abundance in particular localities (Table 1).Naturalized populations of E. densa were observed in five locations (Figure 1), located in the Mediterranean subecoregion of the Dinaric ecoregion of Croatia, in rivers and springs of the Neretva River Basin and include two limnocrene karst springs (Prud and Modro Oko), as well as two rivers (Norin and Mislina).

Habitat characterization of the sites
The Norin River Water at this site is clear, slightly basic, oligohaline and characterized by high conductivity and alkalinity (Table S2).Vegetation is typical of slow-flowing water, characterized by a high abundance of Sparganium emersum Rehmann, Berula erecta (Huds.)Coville and Nuphar lutea (L.) Sm. and a welldeveloped belt of Phragmites australis (Cav.)Trin.ex.Steud.along the watercourse (Table S1).Three stable populations of E. densa were found and monitored in the Norin River over the course of our research, distributed in the Prud Spring, and in the middle and lower reach of the river (Figure 1).Based on the data collected from 2010 to 2017, we can assume that the spread in the Norin River began in the lower section and moved upstream (Table 1).

The Prud Spring
Although the site was visited in 2013, dense stands of E. densa were first recorded in 2014, already relatively abundant at the beginning of the vegetation season (Table 1).In shallow water next to the bank, E. densa was associated with B. erecta and Alisma lanceolatum With., while in deeper water it was associated with Potamogeton nodosus Poir.(Table S1).The species was absent from fast-flowing water.During the vegetation season, the abundance of E. densa increased and it finally formed dense, submerged monospecific stands in up to 1 m deep slowflowing water (Figure 2).In deeper water, up to 2.2 m, it was present in lower abundance.The predominance of E. densa remained constant in subsequent years.
The levels of nitrates, total nitrogen, orthophosphates and total phosphorous were slightly elevated (Table S3), and water status was assessed as good regarding nutrients.

Middle reach
In the middle reach, E. densa was first recorded in 2014 when it was frequent but codominant with the indigenous B. erecta, Hydrocharis morsus-ranae L., N. lutea, Potamogeton lucens L., S. emersum, Sparganium erectum L. and Ceratophyllum demersum L. (Table S1).In 2017, the abundance of E. densa was higher than in 2014, although no major change in indigenous vegetation was observed (Table 1).Although E. densa was clearly self-sustaining here, in 2017 it was still associated, and codominant, with native vegetation (Table S1).
Physicochemical analysis of water showed only slightly elevated levels of ammonium, nitrates and total nitrogen (Table S3), and water status was assessed as good.

Lower reach
In the lower reach of the river, the population of E. densa gradually declined from 2013, when it was recorded for the first time and estimated as frequent (Table 1).In surveys carried out in 2014 and 2017, only individual plants were present.Native aquatic vegetation was abundantly developed in the lower reach, dominated by N. lutea, Nymphaea alba L., Oenanthe aquatica (L.) Poir.and S. emersum, all characteristic for slow and medium flowing waters (Table S1).
Water status was assessed as good due to only slightly elevated levels of nitrates and total nitrogen (Table S3).

The Modro Oko Spring
The population of E. densa was well established and abundant in the Modro Oko Spring.Male plants were recorded for the first time in 2014, when abundance scored 3 (medium) on Kohler's scale (Table 1).In the following years the species was more abundant, surpassing indigenous vegetation (Table 1).It was rooting in 0.3-2.2m deep, slowflowing water forming monospecific stands, or was associated with the species N. lutea, which can access light above the dense canopy of E. densa.In deeper water, E. densa was associated with P. lucens, while in shallower water it was more abundant than Hyppuris vulgaris L. and B. erecta.Notably, at the peak of the vegetation season, E. densa was completely dominant in the area inhabited by aquatic vegetation (Figure 2), while flowering occurred at the end of the September.
Water on this locality is clear, oligohaline, with high alkalinity and conductivity (Table S2).

The Mislina River
Flowering male plants of E. densa were recorded at the beginning of the vegetation season, in March 2016, forming monospecific stands in the lower reach of the river (Table 1).In these stands, the predominance of E. densa over the indigenous flora was evident as no other species were observed.Notably, this population extended to the nearby Mlinište Spring, which is situated on the left bank of the river and is connected to the main course (Figure 2).
The water in the Mislina River is clear, slightly basic, and characterized by high conductivity and alkalinity (Table S2).Values of ammonium, nitrates and total nitrogen were only slightly elevated (Table S3), and water status was assessed as good regarding nutrients.

Discussion
Potential invasion of E. densa poses a serious threat to aquatic ecosystems by affecting water-flow, sedimentation, water quality and hydrochemistry, light penetration and native species (Yarrow et al. 2009).Aquatic alien species can outcompete and displace native aquatic species, as well as negatively affect populations and assemblages of aquatic fauna (Boylen et al. 1999;Stiers et al. 2011;Hussner 2012).Furthermore, for invasive clonal aquatic plants, such as E. densa, high phenotypic plasticity enables relatively fast adaptation to a wide range of habitats in introduced areas (Riis et al. 2010) and augments their invasive potential.In the Neretva Delta, high biomass production of E. densa could have a negative impact on irrigation systems and recreational activities such as fishing, boating and swimming.Moreover, it could have a serious negative impact on the biodiversity of protected areas and Natura 2000 sites located in the Delta.
In the Neretva Delta, E. densa grows in clear water with low flow velocity.In all localities the water is characterized by high alkalinity and is slightly basic, due to the limestone bedrock.The water is oligohaline because of the intrusion of the sea water.Nutrient content suggests moderate eutrophication, at least during some months, which can be explained by the land use of the surrounding area which is composed mainly of arable land.
Water turbidity is often a limiting factor for aquatic plants, reducing light availability which, in more plastic and adaptable plants like E. densa, can cause a shift in plant growth.In waters with high turbidity, E. densa shows an increase in shoot elongation, which improves light harvesting capability, while in clear water and suitable light conditions it has greater productivity and branching, thus having a greater invasive potential (Tanner et al. 1993;Riis et al. 2012).Consequently, E. densa is frequently dominant and appears to perform best in clear water conditions (Bini et al. 1999;Carrillo et al. 2006).Such conditions are present not only in our study sites but also in the majority of watercourses and lakes in the surrounding area.Occurrence sites in the Neretva Delta also provide favourable temperature conditions for the species, since E. densa shows relatively constant growth between 16 and 28 °C (Barko and Smart 1981).Although E. densa is more competitive in warm waters (Riis et al. 2012), and the summer temperatures in most of our study sites do not exceed 20 °C, E. densa was still dominant over native vegetation in some localities.
Salinity measured at E. densa occurrence sites ranges between 0.3 and 0.4‰.These oligohaline conditions support the growth of the species, while salinity around 1.2‰ is the most favourable for its growth and productivity.Development and distribution of E. densa are limited at salinities higher than 5‰ (Hauenstein and Ramirez 1986).Therefore, we can expect the further spread of E. densa in water bodies in the Neretva Delta, but we presume it will be somewhat restricted since the salinity in parts of the area exceeds the level for optimum growth (Romić et al. 2008).Regarding alkalinity, the measured values are high in all occurrence sites and are expected to be similar in the whole area due to the limestone bedrock.This may have a positive impact on the spread and potential invasion of E. densa since the species can efficiently use HCO 3 − as an alternative source of inorganic carbon (Browse et al. 1979;Pierini and Thomaz 2004).
Although concentrations of nutrients in the occurrence sites are moderately elevated and thus provide suitable conditions for macrophytes, we did not observe any specific pattern that would provide a link between E. densa abundance or dominance and the concentration of nutrients.
During our study, we recorded only male E. densa plants, which is in accordance with previous records of flowering populations in Europe (Medina and Cirujano 1995;Gros et al. 2009;Aymerich 2012;Fominykh et al. 2016).Male flowers have generally been observed across the whole introduced range (Mccullough 1997;DiTomaso et al. 2013; but see Coetzee et al. 2011), while the plant reproduces and spreads very efficiently via vegetative shoot fragments that contain "double nodes"-a special nodal region that consists of two single nodes separated by a greatly shortened internode which can produce lateral buds, branches and adventitious roots (Cook and Urmi-König 1984).Dispersal via fragments is an efficient strategy in aquatic plants and is particularly effective in areas such as the Neretva Delta.This Mediterranean wetland area is characterized by a vast net of very well interconnected springs, rivers, small lakes, melioration channels and ditches, representing a suitable environment for the establishment and growth of E. densa.Additionally, this area is subject to human activities such as boating and fishing, which could greatly contribute to the dispersal of E. densa fragments (Brundu 2015).Furthermore, the whole area hosts a large number of wetland bird species, which can also play a significant role in the dispersal of fragments (Reynolds et al. 2015;Green 2016;Coughlan et al. 2017).
Since E. densa is a widely used aquarium plant, it is presumed that the most common route of introduction is the ornamental trade and release from aquaria (Mccullough 1997;Les and Mehrhoff 1999;Hussner and Lösch 2005;Wasowicz et al. 2014;Fominykh et al. 2016).This is the most probable scenario for the introduction of the plant into the Neretva Delta as well.On the other hand, migratory water birds also play an important role in the dispersal of aquatic alien species, through both epizoochory and endozoochory, thus enabling transport across considerable distances (Reynolds et al. 2015;Green 2016;Coughlan et al. 2017).Although endozoochory is considered a dominant form of dispersal of aquatic organisms and enables transport over larger distances, ectozoochory by birds has also been documented as an efficient dispersal mechanism of aquatic plants.In the case of E. densa, transport of vegetative fragments could be the only possible form of transport by birds, since the species does not produce fruits and seeds outside the native range.The Neretva Delta is a Ramsar site with over 300 registered bird species, representing an important stop-over site during migration of birds from middle and northeast Europe to Africa.Since the area is situated on the route of the Central European (Black Sea/Mediterranean) Flyway, it is possible that water birds have been transporting E. densa fragments.Although we cannot ascertain the exact vector by which E. densa was introduced into the studied area, we believe that introduction via watercourses is unlikely, as the species has not yet been recorded from neighbouring countries, or from Southeast Europe (Hussner 2012).
The total number of alien aquatic plant species in Croatian freshwater ecosystems is relatively low, especially in comparison with Central European countries (Hussner 2012).There are only seven alien aquatic plant species recorded from Croatian watercourses so far, including E. densa.Of these, only Elodea canadensis Michx. is considered invasive (Boršić et al. 2008).In the Dinaric ecoregion, including the Mediterranean part of the country, the situation appears to be even more benign, since there are few records of alien aquatic plant species so far (Nikolić 2018).This is quite surprising considering the warm Mediterranean climate, which should be very suitable for the establishment of most alien aquatic plant species.The majority of these plants are thermophilous subtropical and tropical species, negatively affected primarily by low temperatures (Chytrý et al. 2009).Nonetheless, prior to our record of E. densa, only one alien aquatic species was recorded from the Mediterranean part of Croatia -M.heterophyllum Michx (Starmühler 2009).Similarly, in other SE European countries the climate is suitable but the available distributional data shows a low number of alien aquatic plants (Hussner 2012).The main reason may be insufficient botanical research within these countries (Lambdon et al. 2008).We believe, however, that this is not the case with the Neretva Delta as a considerable amount of floristic data is available for the area (Topić 1995;Glasnović et al. 2015;Nikolić 2018); yet E. densa was not recorded until 2013 as the first alien aquatic plant species in the area, followed by M. heterophyllum in 2016 (Jasprica et al. 2017).
Implementation of the Water Framework Directive (European Community 2000), which includes monitoring of the ecological status of inland waters, provides the most comprehensive data on aquatic flora in Croatia but also confirms the low number and limited distribution of alien aquatic species, especially within the Dinaric ecoregion.Since the ornamental trade is the most common path for the introduction of aquatic species worldwide (Kay and Hoyle 2001;Brundu 2015), the present situation might be a consequence of the almost non-existent water garden industry in Croatia, and the low intensity of both intentional and unintentional introduction by humans.Another reason might be the features of the Adriatic Sea Basin in Croatia, which consists of several larger, unconnected karst river basins, with no possibility for the exchange of taxa.
In invasion management, the benefits of preventative measures far exceed the potential economic and ecological costs (Leung et al. 2002) and are much more effective than eradication.Preventative management requires international coordination of early-warning systems, immediate access to critical information, specialized training of personnel, and rapid-response strategies (Ricciardi et al. 2011).Although the current situation in Croatian watercourses seems favourable in comparison to Central European countries, a careful and systematic approach to the research and management of invasive alien species is still necessary.In addition to the regular national monitoring conducted in Croatia, we have started a project of detailed mapping of E. densa in the Neretva Delta, with a plan to establish periodical monitoring of the present populations in order to estimate their trend and promptly detect the possible threat to resident species and habitats.

Figure 1 .
Figure 1.Study area and occurrence sites for Egeria densa in Croatia: 1 -the Prud Spring, 2 -middle reach of the Norin River, 3 -lower reach of the Norin River, 4 -the Modro Oko Spring, 5 -the Mislina River.

Figure 2 .
Figure 2. Egeria densa habitat in the Neretva Delta.A -monospecific stand at the Prud Spring, B -submerged stand at the Prud Spring, C and D -invasion at Modro Oko Spring, E -codominance with native species at the Norin River, F -monospecific stand at Mlinište Spring.Photographs by Sanja Gottstein and Igor Stanković.