Artificial Wetlands As Breeding Habitats for Colonial Waterbirds

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Article Artiﬁcial Wetlands as Breeding Habitats for Colonial Waterbirds within Central Romania

Dan Traian Ionescu 1 ,Călin Vasile Hodor 2 and Ion Cătălin Petritan 1,*

1 Department of Forest Engineering, Transilvania University of Bra¸sov, 500123 Bras, ov, Romania; [email protected] 2 Wildlife Management Consulting Company, 500295 Bras, ov, Romania; [email protected] * Correspondence: [email protected]

Received: 18 August 2020; Accepted: 22 September 2020; Published: 25 September 2020

Abstract: The historical loss of natural wetlands in Romania is significant. The building of lakes, ponds, and dams has compensated for some of these losses, as was the case in the study area, the artificial wetlands designated as the Natura 2000 (special protection area, SPA) and Ramsar sites. In this context, to evaluate the conservation status of colonial waterbirds (cormorants, herons, and egrets), we analyzed actual distributions, habitat selection, features, and population sizes. Standardized counting of colonial species was used, supplemented by nest counting and colony-area estimation during winter, and drone footage. All species and their colonies were built in artificial or anthropically influenced habitats. Concerning distribution and population sizes, Phalacrocorax carbo and Microcarbo pygmeus were found to nest only in central Romania. The pair numbers of Ardea alba, Ardea purpurea, Ardea cinerea, and Nycticorax nycticorax in the area exceeded 1% of the national minimum breeding population. There was a statistically significant positive trend of breeding populations for Ardea alba, Nycticorax nycticorax, and Egretta garzetta. In conclusion, these artificial wetlands offer optimal breeding conditions for a relatively large diversity of colonial bird species over areas that are not very large.

Keywords: cormorant; heron; egret distribution; population dynamics; Natura 2000; Ramsar

1. Introduction Historically, biodiversity, including waterbirds, in Romania has suffered significant losses of populations and habitats, especially through drainage and dam works mainly along the Danube floodplain, where over 530,000 hectares of wetlands with diverse habitats have been transformed or lost [1]. The creation of artificial wetlands such as fish farms, lakes, and reservoirs with various functions has partially compensated for these losses, and bird populations adapted to conditions which are more or less different from natural ones [2]. This was found to be the case for the colonial birds in this study, with some being new breeding species for the reference area (Transylvania or the Bras, ov depression). Integrated management can be performed within fish farms, targeting the interests of both aquaculture and biodiversity conservation. The main study aim was the inventory and monitoring of eight colonial waterbird species concerning the actual occurrence, distribution, habitat selection, colony characteristics and relation with human constructions, and population size in the study area, all in interdependence with aquaculture management and other human-related activities over a decade (2010–2020). Distribution and population size were compared with those on the regional (central Romania, Transylvania, or inside the Carpathian chain) and national levels to establish the importance of the study area. This research focused on relevant artificial wetlands southeast of Transylvania: Dumbrăvi¸ta(including

Diversity 2020, 12, 371; doi:10.3390/d12100371 www.mdpi.com/journal/diversity Diversity 2020, 12, 371 2 of 17 Diversity 2020, 12, 371 2 of 16 the Dumbrăvița fishing-complex Ramsar site) and the Rotbav Natura 2000 Site, special protection area (SPA; ROSPA0037 Dumbrăviţa-Rotbav-Măgura Codlei). the Dumbrăvita ﬁshing-complex Ramsar site) and the Rotbav Natura 2000 Site, special protection area These aspects, are relevant for reasons such as the extension of breeding areas for some species; (SPA; ROSPA0037 Dumbrăvi¸ta-Rotbav-Măgura Codlei). the existence or creation of new or recent wetlands such as fish farms, lakes, gravel pits, and dams; These aspects are relevant for reasons such as the extension of breeding areas for some species; the and the lack of or poor knowledge on the distribution, population size, and their trends over various existence or creation of new or recent wetlands such as ﬁsh farms, lakes, gravel pits, and dams; and the periods on the local, regional, or national levels [3–6]. In addition, these data can be used in lack of or poor knowledge on the distribution, population size, and their trends over various periods conservation and sustainable and integrated management, including the assessment of species on the local, regional, or national levels [3–6]. In addition, these data can be used in conservation and conservation status and preparing conservation measures within the future Natura 2000 site- sustainable and integrated management, including the assessment of species conservation status and management plan (2021–2026). preparing conservation measures within the future Natura 2000 site-management plan (2021–2026).

2. Materials and Methods

2.1. Study Area

Research waswas carried carried out out on on two two main main artificial artificial wetlands wetlands within within the Dumbr the ăDumbrvit, a–Rotbav–Măvița–Rotbav–ăgura CodleiMăgura Natura Codlei 2000 Natura site (N2000 45 ◦site43010” (N and45°43 E′ 2510◦″24 and038”). E 25°24 These′38 are″). situatedThese are at thesituated internal at curvaturethe internal of thecurvature Carpathians, of the Carpathians, a territory in centrala territory Romania in central named Romania Transylvania named provinceTransylvania (Figure province1). The (Figure total area 1). ofThe Transylvania total area of is Transylvania 57,000 km2. Theis 57,000 study km². area The is an st artificialudy area wetland is an artificial and one wetland of the most and importantone of the concentrationmost important sites concentration for water birds sites withinfor water Transylvania birds within (Figure Transylvania2). The Dumbr (Figureăvi¸tafishing 2). The Dumbr complexăviţa coversfishing ancomplex area of covers over 450 an ha, area and of is over a large 450 system ha, and of is fishponds a large system and reservoirs. of fishponds It was and also reservoirs. designated It aswas the also Ramsar designated site—wetland as the Ramsar of international site—wetland importance of international (the only suchimportance site in Transylvania) (the only such with site the in proposalTransylvania) to expand with thethe siteproposal to 2282 to ha expand (including the bothsite to wetlands 2282 ha and (including the adjacent both areas).wetlands The and Rotbav the lakesadjacent and areas). fishponds The coverRotbav over lakes 230 ha.and There,fishponds there cover is another over anthropically230 ha. There, influenced there is wetland:another aanthropically marsh covered influenced by emergent wetland: vegetation a marsh resulting covered from by the emergent clogging of vegetation drainage channels.resulting The from marsh the wasclogging formed of drainage over 30 years channels. ago by Th abandoninge marsh was the formed works over of unclogging 30 years ago the by canals abandoning and a nonfunctioning the works of water-pumpingunclogging the canals station. and The a mainnonfunctioning human activities water-pumping in the whole station. wetland The main area arehuman aquaculture activities and in angling.the whole Asian wetland fish speciesarea are are aquaculture mainly cultivated, and angling. such as AsianHypophthalmichthys fish species are nobilis mainly, Hypophthalmichthys cultivated, such molitrixas Hypophthalmichthys, and Ctenopharyngodon nobilis, idellaHypophthalmichthys, but also Eurasian molitrix, species andCyprinus Ctenopharyngodon carpio. In addition, idella, therebut also are otherEurasian small species fish, some Cyprinus invasive, carpio such. In as addition,Pseudorasbora there parva are ,other which small are food fish, for some many invasive, fish-eating such birds as (personalPseudorasbora observation). parva, which Without are food a qualitative for many and fish-eating quantitative birds study (personal of the foodobservation). available Without to birds ina thequalitative concerned and area, quantitative the appearance, study of the maintenance, food available and to growth birds in of the bird concerned populations area, can the be appearance, attributed tomaintenance, the existence and of growth various of and bird rich populations trophic sources can be in attributed the conditions to the of existence artificial of foraging various and and large rich trophic sources in the conditions of artificial foraging and large yields per hectare (more than 1–1.5 yields per hectare (more than 1–1.5 t/ha) of farmed fish (Crizbăs, an D., general manager of the fish farm, verbalt/ha) of communication). farmed fish (Crizbășan D., general manager of the fish farm, verbal communication).

Figure 1.1. StudyStudy area. area. White White circle circle (Bra (Bras, ovș depression),ov depression), part ofpart Transylvania of Transylvania province; province; black oval, black Romania. oval, Romania. Diversity 2020, 12, 371 3 of 16 Diversity 2020, 12, 371 3 of 17

FigureFigure 2. Detail 2. Detail of studyof study area. area. DumbrDumbrăăvițt,a (bottom) and and Rotbav Rotbav wetlands wetlands (white (white ovals) ovals) within within ROSPA0037ROSPA0037 (red (red border). border).

DependingDepending on on the the type type of of Ramsar Ramsar site site wetland,wetland, the the main main habitats habitats within within the thestudy study area areawere were permanentpermanent freshwater freshwater marshes marshes (Tp), (Tp), freshwater-tree-dominated freshwater-tree-dominated wetlands wetlands (Xf), (Xf), aquaculture aquaculture ponds, ponds, reservoirs,reservoirs, and and seasonal seasonal/intermittent/intermittent freshwater freshwater marshes marshes (Ts) (Ts) [ 7[7].]. TheseThese kindskinds of artificial artificial wetlands wetlands are are recognized as very important or even “key” sites for many vulnerable, rare, or endangered bird recognized as very important or even “key” sites for many vulnerable, rare, or endangered bird species; species; because of that, Dumbrăviţa and Rotbav were designated as Important Bird Areas [8]. The because of that, Dumbrăvi¸taand Rotbav were designated as Important Bird Areas [8]. The Natura Natura 2000 Site has an official management plan comprising several concrete measures for the long- 2000term Site hasconservation an official and management maintenance of plan bird comprising species and populations several concrete in balanc measurese with sustainable for the long-term fish conservationfarming. and maintenance of bird species and populations in balance with sustainable fish farming. TransylvaniaTransylvania has has wetlands, wetlands, especially especially inin itsits central and and southern southern parts, parts, represented represented mainly mainly by by artificialartificial types types such such as lakes,as lakes, reservoirs, reservoirs, and and fishponds.fishponds. We We chose chose the the two two areas areas for forstudy study considering considering theirtheir importance, importance, recognized recognized on on the the European European andand in internationalternational level, level, being being the theonly only Ramsar Ramsar site in site in Transylvania.Transylvania. Studies Studies and and monitoring monitoring havehave taken pl placeace here here for for 25 25years years for forsome some groups groups of bird of bird species.species. The The number number of coloniesof colonies and and component component species was was also also a criterion a criterion in this in this regard. regard.

2.2. Methods2.2. Methods For bird, nest, and colony identification, only standardized, noninvasive methods were applied. For bird, nest, and colony identification, only standardized, noninvasive methods were Concerning the monitoring of species populations/colonies, one main method was used [9]: applied.counting/monitoring Concerning the colonial monitoring species of speciesof herons populations and cormorants./colonies, This one method main methodis based was on usedthe [9]: countingestimation/monitoring of apparently colonial occupied species ofnests, herons in our and case, cormorants. by choosing This observation method ispoints based which on the avoided estimation of apparentlydisturbing occupied the colony nests, but from in our where case, the by entire choosing colony observation could best be points observed. which All avoided entrances disturbing and the colonyexits of butthe birds from from where the the colony entire were colony noted, could and observable best be observed. nests were Allcounted. entrances Thus, andwe counted exits of the birdsand from noted the colonythe breeding were pairs. noted, The and relative observable position nests of the were colonies counted. was Thus,drawn weon countedmaps and and then noted the breedingcompared pairs. with Thethat relative identified position by drone of the footage colonies and was satellite drawn images on maps (Google and thenEarth). compared Several with that identifiedsuccessive byobservations drone footage during and the satellite breeding images period (Google(1 April–31 Earth). July) Severalwere made. successive To complete observations the counting of the nests and to precisely establish the position of the colonies and their surface, they during the breeding period (1 April–31 July) were made. To complete the counting of the nests and to were accessed during winter on ice (using a global positioning system (GPS) with points and track precisely establish the position of the colonies and their surface, they were accessed during winter on functions). ice (usingCounting/estimation a global positioning of systembreeding (GPS) pairs withwas also points done and by trackfilming functions). the colonies with a drone. We Countingused a DJI/ estimationPhantom 4 ofPro breeding drone with pairs a mobile was also camera done byand filming a noise the level colonies of 81 dB. with The a drone.drone was We used a DJIflown Phantom out at4 20–30 Pro dronem from with the reedbeds a mobile or camerawoody vegetation. and a noise The level drone’s of 81flight dB. comprised The drone one was pass flown out at 20–30 m from the reedbeds or woody vegetation. The drone’s flight comprised one pass over the colonies, usually lasting 10–15 min. The only observed effects on the birds in the colony were their attention to the drone and, at most, the lifting of the adults from the nest without leaving it. Diversity 2020, 12, 371 4 of 16

We also estimated breeding populations for all waterbirds using the point-count transect method of 2 2 squares [9]. Binoculars (10 50), a scope ( 20–60), and a professional camera were used. × × × The monitoring period was 2010–2020. To calculate diﬀerent types of distances and the colony areas, Google Earth was used with speciﬁc tools. We calculated some measurable elements of the colonies, i.e., the total or smallest areas occupied by the colonies during the monitoring period, and the nearest distance from the edge of the colony to terrestrial habitats—in our case, to dams, roads, or buildings. Regarding the total area occupied by the colonies, we separately overlapped and summed the areas for the monitoring period of the two areas—Dumbrăvit, a and Rotbav. The minimal area occupied by various colonies (e.g., built on islands) was also calculated.

2.3. Statistical Analysis Linear-regression analysis was applied to investigate if there was a significant relation between colony area and breeding pairs or the nearest distance to the dam, but to also test if there was a significant population trend over the last 10 years for different species or on the colony level. One-way ANOVA was performed to test if the substrate nest types affected the population trend of two species, Nycticorax nycticorax and Egretta garzetta. All statistical analyses was done using STATISTICA 10 (StatSoft 2012, Inc., Tulsa, OK, USA).

3. Results A total of eight colonial waterbird species were recorded within the study area during the observation period: great cormorant (Phalacrocorax carbo), pygmy cormorant (Microcarbo pygmaeus), gray heron (Ardea cinerea), purple heron (Ardea purpurea), great white egret (Ardea alba), little egret (Egretta garzetta), black-crowned night heron (Nycticorax nycticorax) and squacco heron (Ardeola ralloides). In terms of conservation, six of the species are included in Annex I of the Birds Directive (2009/147/EC), as they are species of community interest (Table1).

Table 1. Breeding habitats and nest-building substrates.

Nests on No. Species Habitat Type Emergent Woody Vegetation Vegetation Trees in wetlands; artificial land Salix babylonica (Rotbav) and S. 1 Phalacrocorax carbo - islands with woody vegetation. fragilis (dead trees, Dumbrăvit, a). Microcarbo 2 Shrubs in wetlands. - Salix cinerea. pygmaeus * Reed/reedmace beds; shrubs or Phragmites australis, Salix cinerea, S. fragilis, Quercus 3 Ardea cinerea trees in wetlands; mature pure Typha sp. robur. oak forest. Phragmites australis, 4 Ardea purpurea * Reed/reedmace beds. - Typha sp. Reedbed; shrubs or trees in 5 Ardea alba * Phragmites australis. Salix cinerea. wetlands. Reedbed; shrubs or trees in 6 Egretta garzetta * wetlands; artificial land islands Phragmites australis. Salix cinerea. with woody vegetation. Reedbed; shrubs or trees in Nycticorax Salix cinerea, Prunus cerasifera, 7 wetlands; artificial land islands Phragmites australis. nycticorax * Sambucus nigra. with woody vegetation. Reedbed; shrubs or trees in 8 Ardeola ralloides * wetlands; artificial land islands Phragmites australis. Salix cinerea. with woody vegetation. * species of community interest included in Annex I of the Birds Directive (2009/147/EC) considered “key” to the conservation of the Natura 2000 site [10]. Diversity 2020, 12, 371 5 of 16

3.1. Colony Types Two types of colonies were formed:

— Monospeciﬁc, for the following species: Phalacrocorax carbo, Ardea cinerea, Ardea purpurea, and Ardea alba. All of them can also form mixed colonies, and some can also breed in isolated pairs (Ardea cinerea, Ardea purpurea, Ardea alba). — Only mixed for the following species: Microcarbo pygmaeus, Egretta garzetta, Nycticorax nycticorax, and Ardeola ralloides. The most stable type of mixed colony was formed by all four of these species,

with Ardea cinerea at Dumbrăvit, a (since 2012) and the other three species at Rotbav (since 2016).

3.2. Breeding Habitat Types/Nest Locations Regarding the habitats where the colonies were located, there were three major types: reedbed, shrubs or trees in wetlands, and artificial land islands with various woody species (Table1). The first habitat was, according to CORINE classification 53.11, common reed (Phragmites australis) bed, and to EUNIS classification C3.21, Phragmites australis beds. In some cases, two of the colonial species, Ardea cinerea and A. purpurea, also bred inside reedmace. Thus, the habitat according to CORINE was 53.13, i.e., reedmace (Typha) beds, and, according to EUNIS, was C3.231/232 Typha latifolia/T. angustifolia beds. For colonies inside scrubs and trees, the most relevant CORINE habitat was 44.921, i.e., grey willow scrub; for EUNIS, it was F9.213, i.e., intra-Carpathian grey willow carrs, where the dominant woody species is Salix cinerea [11]. One of the oldest monospecific colonies (Ardea cinerea) was built in a mature oak forest (Quercus robur) near the Dumbrăvit, a wetland, but it has been extinct since 2011. In this case, it was a colony formed in a terrestrial habitat (nonwetland). We assumed that the birds that had formed this colony occupied fish-farm wetlands to breed in the following years. Concerning the construction substrate of the nests, there are two distinct situations: emerged vegetation (reed, reedmace) and woody vegetation. In woody vegetation, nests are located mainly on Salix cinerea, but also on S. fragilis and S. babylonica. In addition to willows, other species were used in one location, such as Prunus cerasifera and Sambucus nigra. In the first two years of the decade (2010–2011), a colony of Ardea cinerea bred in mature oaks (Quercus robur) from a pure mature-oak forest. The only species that built nests exclusively on emergent vegetation (especially on reedbed) was Ardea purpurea, and the only species that built their nests only on woody vegetation were Phalacrocorax carbo and Microcarbo pygmaeus (Table1). Regarding the frequency of use of the two major habitat types, as a substrate for breeding by the mixed colony (Egretta garzetta, Nycticorax nycticorax, Ardeola ralloides), woody vegetation was used

(100%) in Dumbrăvit, a in all years, while the reedbed was used only in 3 years (37.5%).

3.3. Colony Distribution within Study Area Six fishponds and lakes were used as breeding sites by the studied colonies (Figures3–8). The area of he six artificial water bodies had a range of 36–44 ha, with an average of 39 ha, but not all species and types of colonies used all these lakes and ponds. In addition, three of the species bred colonially in a marsh formed between drainage channels, in flooded reeds (Figure4). The positioning of the colonies may differ from year to year, with the birds either being able to keep their location for a number of years or changing it to somewhere else in the same basin or elsewhere. Diversity 2020, 12, 371 6 of 17 DiversityDiversity 20202020,, 1212,, 371371 66 of of 1617 Diversity 2020, 12, 371 6 of 17

Figure 3. Colony location on Lake 3 Rotbav, 2010–2020 (black circle, Phalacrocorax carbo; white oval, mixedFigure consisting 3. Colony oflocation Nycticorax on Lake nycticorax 3 Rotbav,, Egretta 2010–2020 garzetta ,(black Ardeola circle, ralloides Phalacrocorax). carbo; white oval, Figure 3. Colony location on Lake 3 Rotbav, 2010–2020 (black circle, Phalacrocorax carbo; white oval, Figuremixed consisting3. Colony locationof Nycticorax on Lake nycticorax 3 Rotbav,, Egretta 2010–2020 garzetta (black, Ardeola circle, ralloides Phalacrocorax). carbo; white oval, mixed consisting of Nycticorax nycticorax,, Egretta garzettagarzetta,, ArdeolaArdeola ralloidesralloides).).

Figure 4. Colony location on fishponds and in marsh from Rotbav, 2010–2020 (yellow oval, Ardea alba; Figure 4. Colony location on ﬁshponds and in marsh from Rotbav, 2010–2020 (yellow oval, Ardea alba ; whiteFigure oval, 4. Colony A. cinerea location, A. purpurea on fishponds). and in marsh from Rotbav, 2010–2020 (yellow oval, Ardea alba; whiteFigurewhite oval,oval, 4. Colony A.A. cinereacinerea location,, A.A. purpureapurpurea on fishponds).). and in marsh from Rotbav, 2010–2020 (yellow oval, Ardea alba; white oval, A. cinerea, A. purpurea).

Figure 5. Colony location on ﬁshponds from Dumbrăvita, 2010–2020 (black oval, Phalacrocorax carbo ; Figure 5. Colony location on fishponds from Dumbrăviț,a, 2010–2020 (black oval, Phalacrocorax carbo ; whiteFigure oval,oval, 5. Colony mixed, mixed, location consisting consisting on offishponds Microcarboof Microcarbo from pygmaeus Dumbrpygmaeus, Ardeaăvi, ța,Ardea cinerea 2010–2020 cinerea, Nycticorax ,(black Nycticorax nycticorax oval, Phalacrocoraxnycticorax, Egretta, garzettaEgretta carbo;, Figure 5. Colony location on fishponds from Dumbrăvița, 2010–2020 (black oval, Phalacrocorax carbo; garzettaandwhiteArdeola oval,, and ralloides mixed,Ardeola). ralloidesconsisting). of Microcarbo pygmaeus, Ardea cinerea, Nycticorax nycticorax, Egretta whitegarzetta oval,, and mixed, Ardeola consistingralloides). of Microcarbo pygmaeus, Ardea cinerea, Nycticorax nycticorax, Egretta garzetta, and Ardeola ralloides). Diversity 2020, 12, 371 7 of 17 DiversityDiversity 20202020,, 1212,, 371371 77 of of 1617 Diversity 2020, 12, 371 7 of 17

Figure 6. Colony location on fishponds from Dumbrăvița, 2010–2020 (yellow oval/circle, Ardea alba ). Figure 6. Colony location on fishponds from Dumbrăvița, 2010–2020 (yellow oval/circle, Ardea alba). Figure 6. Colony location on ﬁshpondsfishponds from Dumbrăvitviț, a, 2010–2020 (yellow(yellow ovaloval/circle,/circle, ArdeaArdea albaalba).).

Figure 7. Colony location on fishponds from Dumbrăvița, 2010–2020 (white oval, A. cinerea; black Figure 7. Colony location on ﬁshponds from Dumbrăvit, a, 2010–2020 (white oval, A. cinerea; black oval, oval,Figure A. 7.purpurea Colony). location on fishponds from Dumbrăvița, 2010–2020 (white oval, A. cinerea; black A.oval,Figure purpurea A. 7. purpurea Colony). ). location on fishponds from Dumbrăvița, 2010–2020 (white oval, A. cinerea; black oval, A. purpurea).

Figure 8. Location of old Ardea cinerea colony (white oval) within mature-oak ( Quercus robur )) forest Figure 8. Location of old Ardea cinerea colony (white oval) within mature-oak (Quercus robur) forest from Dumbr ăvivitț, a; 2011 was the last year when this species bred within thethe forest. fromFigure Dumbr 8. Locationăvița; 2011of old was Ardea the lastcinerea year colony when (white this species oval) bredwithin within mature-oak the forest. (Quercus robur) forest from Dumbrăvița; 2011 was the last year when this species bred within the forest. Diversity 2020, 12, 371 8 of 16

3.4. Colony Characteristics/Elements

3.4.1. Areas Occupied by Colonies during Monitoring Period Within the Rotbav area, the total area occupied by the mixed colony (Nycticorax nycticorax, Egretta garzetta, Ardeola ralloides) during the monitoring period (5 years) was 1380 m2. This represents 62% of the total potential nesting area (islands with woody vegetation). The total area of the mixed colony from

Dumbrăvit, a (Nycticorax nycticorax, Egretta garzetta, Ardeola ralloides, Microcarbo pygmaeus) amounted to 12,080 m2 (1.2 ha) during 9 years. Of this total area, the colony formed only on woody vegetation totaled 7560 m2, which means that 62.6% of the total area was occupied and, for the one formed on reeds, 4520 m2 (37.4% of the total area occupied). The area occupied by the colony on reeds represents 3.5% of the total area of reeds and 23.8% of the total area of reed islands and peninsulas. Regarding

Ardea alba at Dumbrăvit, a, two breeding nuclei (grouping of nests of over 70% of the total nests in that year) totaled 7000 m2, the smaller of the two being 1500 m2 (formed by 12 nesting pairs). Concerning the minimal area occupied by an isolated colony (island type), for Rotbav, it was only 320 m2, and for 2 Dumbrăvit, a (reedbed island) 1350 m . The total areas occupied by the mixed colony at Dumbrăvit, a therefore mostly overlapped with woody vegetation. Ardea alba, A. cinerea, and A. purpurea could also form fairly small colonies in terms of area, even if some pairs bred isolated or in groups of 2–3 nests.

3.4.2. Nearest Distance from Colony Edge to Terrestrial Habitats and Human Constructions (Dams, Roads, or Buildings) The smallest distances between the edge of the colonies and dams were of the order of tens of meters, and even distances to the nearest, very crowded road were around 100 m. Two of the breeding colonies in the Rotbav marsh were also built at a distance of just over 200 m from an active and noisy ballast- and cement-processing plant (Table2).

Table 2. Nearest distance (m) from colony edge to terrestrial habitats and human constructions.

Location Nearest Distance (m) from Edge of Colony to: Colony Type National Local Road Cement R D Dam Building Road (DN13) (DJ 112C) Factory Mixed x 25 130 260 Ardea purpurea (ﬁsh farm) x 30 350 480 Ardea purpurea (marsh) x 90 225 Ardea alba (marsh) x 110 250 Mixed x 50 780 800 Ardea alba x 40 880 Ardea cinerea (oak forest) x 330 60 Phalacrocorax carbo x 105 175 300 Phalacrocorax carbo x 150 880

R, Rotbav area; D, Dumbrăvit, a area.

The breeding pairs increased significantly with the increase in colony area for the Dumbrăvit, a mixed colony, formed by Egretta garzetta, Nycticorax nycticorax, and Ardeola ralloides (p < 0.05, Figure9); for the Rotbav mixed colony (same species as those of the Dumbrăvit, a mixed colony), we did not find significant correlation between breeding pairs and occupied area (p > 0.05, Figure9), most probably due to the very low number of samples (n = 5) and very weak variability within the colony surface variable. Diversity 2020, 12, 371 9 of 17

Diversity 2020, 12, 371 9 of 17 Diversity 2020, 12, 371 9 of 16

Figure 9. Relation between breeding pairs and colony area separated by (left) both colonies and Figure(right) 9.betweenRelation nearest between distance breeding to dam pairs and and colony colony area. area separated by (left) both colonies and (right) Figure 9. Relation between breeding pairs and colony area separated by (left) both colonies and between nearest distance to dam and colony area. (right) between nearest distance to dam and colony area. Regarding the relation between the nearest distance to the dam and the Dumbrăvița mixed colonyRegarding area formed the relation by Egretta between garzetta the nearest, Nycticorax distance nycticorax to the dam, Ardeola and theralloides Dumbr, weăvi tfound, a mixed negative colony Regarding the relation between the nearest distance to the dam and the Dumbrăvița mixed areacorrelation formed (r by = −Egretta0.58, Figure garzetta 9, ,right),Nycticorax but this nycticorax was only, Ardeola marginally ralloides significant, we found (p negative= 0.099). correlation colony area formed by Egretta garzetta, Nycticorax nycticorax, Ardeola ralloides, we found negative (r = No0.58, effect Figure of substrate-type9, right), but this change was onlyon the marginally population signiﬁcant trend could ( p = be0.099). detected for the two species correlation− (r = −0.58, Figure 9, right), but this was only marginally significant (p = 0.099). of NycticoraxNo eﬀect nycticorax of substrate-type and Egretta change garzetta on the (Figure population 10, p > trend0.05, one-way could be ANOVA, detectedfor F-test). the two species of NycticoraxNo effect nycticorax of substrate-typeand Egretta change garzetta on the(Figure popu 10lation, p > trend0.05, one-waycould be ANOVA,detected for F-test). the two species of Nycticorax nycticorax and Egretta garzetta (Figure 10, p > 0.05, one-way ANOVA, F-test).

Figure 10. Inﬂuence of substrate type (woody vegetation vs. reed) on (left) Nycticorax nycticorax and

(Figureright) Egretta10. Influence garzetta ofpopulation substrate type trends. (woody vegetation vs. reed) on (left) Nycticorax nycticorax and (right) Egretta garzetta population trends. Figure 10. Influence of substrate type (woody vegetation vs. reed) on (left) Nycticorax nycticorax and 3.5. Population Size/Number of Pairs and Trends (right) Egretta garzetta population trends. 3.5. Population Size/Number of Pairs and Trends The largest number of pairs belonged to the Ardea cinerea and Nycticorax nycticorax species, and the 3.5.fewest PopulationThe pairs largest to Size/Number the numberArdeola of ralloidesof pairs Pairs belonged, andPhalacrocorax Trends to the carbo Ardea, and cinereaMicrocarbo and Nycticorax pygmaeus nycticoraxspecies. Although species, and the breedingthe fewest populations pairs to the ofArdeola the analyzed ralloides, species Phalacrocorax in the studiedcarbo, and area Microcarbo were much pygmaeus smaller species. than those Although of the The largest number of pairs belonged to the Ardea cinerea and Nycticorax nycticorax species, and nationalthe breeding ones, populations for some species, of the analyzed the minimal species or maximal in the studied estimated area number were much was smaller equal to than or higher those the fewest pairs to the Ardeola ralloides, Phalacrocorax carbo, and Microcarbo pygmaeus species. Although thanof the 1% national compared ones, to for the some national species, one, especiallythe minimal for theor maximal 2008–2013 estimated period (Table number3). was equal to or the breeding populations of the analyzed species in the studied area were much smaller than those higher than 1% compared to the national one, especially for the 2008–2013 period (Table 3). of the national ones, for some species, the minimal or maximal estimated number was equal to or higher than 1% compared to the national one, especially for the 2008–2013 period (Table 3).

Diversity 2020, 12, 371 10 of 16 Diversity 2020, 12, 371 10 of 17

TableTable 3. PopulationPopulation size size of of analyzed analyzed species species within within stud studyy area area and and their their comparison comparison with with the the national national populationpopulation [5,6]. [5,6].

Percentage of No. No.No. of Pairs of Pairs on on Percentage of of No.No. of of PairsPairs on Percentage of No. of Pairs/Decade National of Pairs/Decade NationalNational Level Level [5] NationalNational Population National Level Level [6] National Species within Study Area Population Species within Study (Period:[5 2008–2013)] (Period: Population(%) [5] (%) (Period:[6] (Period: 2013–2018) Population (%, 2020) [6] Area 2008–2013) [5] 2013–2018) (%, 2020) [6] Min Max Min Max Min Max Min Max Min Max Phalacrocorax Min Max Min Max Min Max Min Max Min Max 10 10 12,000 20,000 0.08 0.05 12,000 20,000 0.08 0.05 carbo Phalacrocorax Microcarbo 10 10 12,000 20,000 0.08 0.05 12,000 20,000 0.08 0.05 carbo 5 15 9400 10,500 0.05 0.14 9400 10,500 0.05 0.14 pygmaeus Microcarbo Ardea cinerea 40 560 154500 9400 6000 10,500 0.88 0.05 1.00 0.14 94004500 10,50010,000 0.050.88 0.14 0.60 pygmaeus Ardea purpurea 16 24 850 1500 1.88 1.60 1797 7830 0.89 0.30 ArdeaArdea alba cinerea 1 4021 60210 4500 370 6000 0.47 0.88 5.671.00 4500400 10,0001000 0.880.25 0.602.10 EgrettaArdea garzetta purpurea 4 16 40 24 4000 850 8000 1500 0.101.88 0.50 1.60 1797 4000 78308000 0.890.10 0.300.50 NycticoraxArdea alba 1 21 210 370 0.47 5.67 400 1000 0.25 2.10 8 70 4000 8000 0.20 0.87 4000 8000 0.20 0.87 nycticoraxEgretta garzetta 4 40 4000 8000 0.10 0.50 4000 8000 0.10 0.50 ArdeolaNycticorax 2 8 6 70 2500 4000 5500 8000 0.08 0.20 0.11 0.87 4000 2700 80006000 0.200.07 0.870.11 ralloidesnycticorax ArdeolaPercentages ralloides equal2 to or greater 6 than 2500 1% of 5500national minimal 0.08 or 0.11 maximal 2700 population 6000 noted 0.07 in bold 0.11. Percentages equal to or greater than 1% of national minimal or maximal population noted in bold. A significant increase in breeding pairs over the last 10 years was found for four species (Ardea alba, EgrettaA significant garzetta increase, Nycticorax in breeding nycticorax, pairs and over Ardeola the last ralloides 10 years; p was< 0.001, found Figure for four 11), speciesbut no significant (Ardea alba , tendencyEgretta garzetta in the, lastNycticorax decade nycticoraxwas confirmed, and Ardeolafor the two ralloides other; p species< 0.001, (Ardea Figure purpurea 11), but and no Microcarbo significant pygmaeustendency; inp > the 0.05, last Figure decade 11). was confirmed for the two other species (Ardea purpurea and Microcarbo pygmaeus; p > 0.05, Figure 11).

Figure 11. Cont. Diversity 2020, 12, 371 11 of 17

Diversity 2020, 12, 371 1111 of of 17 16

Figure 11. Population trend over last 10 years for Ardea alba, Egretta garzetta, Nycticorax nycticorax, Figure 11. Population trend over last 10 years for Ardea alba, Egretta garzetta, Nycticorax nycticorax, ArdeolaFigureArdeola ralloides 11. ralloides Population, Ardea, Ardea purpurea trend purpurea over, and, and last MicrocarboMicrocarbo 10 years pygmaeusfor pygmaeus Ardea. Linear.alba Linear, Egretta regression regression garzetta (continuous (continuous, Nycticorax black nycticorax black line) line) , andArdeolaand associated associated ralloides 95% 95%, Ardea confidence confidence purpurea interval interval, and Microcarbo(dotted (dotted blac black pygmaeusk line). line). Gray. GrayLinear lines lines regression represent represent minimal(continuous minimal and and blackmaximal maximal line) estimatedandestimated associated breeding breeding 95% pairs confidence pairs in in the the field. interval field. (dotted black line). Gray lines represent minimal and maximal estimated breeding pairs in the field. AtAt the the mixed-colony mixed-colony level level formed formed by by EgrettaEgretta garzetta garzetta, Nycticorax, Nycticorax nycticorax nycticorax, and, and ArdeolaArdeola ralloides ralloides, , At the mixed-colony level formed by Egretta garzetta, Nycticorax nycticorax, and Ardeola ralloides, FigureFigure 12 12 shows shows the the population population trends trends over over the thelast lastdecade. decade. In the In Dumbr the Dumbrăvița ămixedvit, a mixed colony, colony, the breedingFigurethe breeding 12 pairs shows pairs increased the increased population significantly significantly trends in theover in thelast the last10 last years; 10 decade. years; for forthe In the Rotbavthe Rotbav Dumbr colony, colony,ăvița this mixed this tendency tendency colony, was wasthe confirmedbreedingconfirmed pairsbut but not notincreased significantly significantly significantly tested. tested. in the last 10 years; for the Rotbav colony, this tendency was confirmed but not significantly tested.

FigureFigure 12. 12. PopulationPopulation trends trends over over the the last last 10 10 years. years. Figure 12. Population trends over the last 10 years. 4. Discussion 4. Discussion 4.4.1. Discussion Use of Habitat Type or Nesting Location in Relation to Natural and Human Factors 4.1. Use of Habitat Type or Nesting Location in Relation to Natural and Human Factors 4.1. UseWithin of Habitat the species Type or range,Nesting heronries Location in can Relation be located to Natural in wetlands and Human or terrestrialFactors habitats. In dry Within the species range, heronries can be located in wetlands or terrestrial habitats. In dry forests, heronries can be located in natural habitats (e.g., oak [12]) or in various plantations, such as forests,Within heronries the speciescan be locatedrange, inheronries natural canhabitats be located (e.g., oak in [12])wetlands or in orvarious terrestrial plantations, habitats. such In dryas pine [13], false acacia groves, or poplar [14]. In wetlands, heronries can be located in woody vegetation, pineforests, [13], heronries false acacia can begroves, located or in poplar natural [14]. habitats In wetlands, (e.g., oak [12])heronries or in variouscan be plantations,located in woody such as such as alder and willow [14], tamarisks, alder and willow [15], various willow species [16], or on vegetation,pine [13], suchfalse asacacia alder groves,and willow or poplar[14], tamarisks, [14]. In alderwetlands, and willowheronries ([15]), can various be located willow in species woody reedbeds [14,16–21]. Nycticorax nycticorax, Egretta garzetta, and Ardeola ralloides are known to breed [16],vegetation, or on reedbeds such as [14,16–21].alder and willowNycticorax [14], nycticorax tamarisks,, Egretta alder andgarzetta, willow and ([15]), Ardeola various ralloides willow are known species only on woody vegetation within central Romania–Transylvania [4,12,13] (Daróczi Sz., David A., to[16], breed or ononly reedbeds on woody [14,16–21]. vegetation Nycticorax within nycticorax central ,Romania–Transylvania Egretta garzetta, and Ardeola [4,12,13] ralloides (Daróczi are known Sz., Sándor D. A., Szabó Z., Stermin A., Kósa F., verbal communication), but in recent years, Nycticorax Davidto breed A., Sándoronly on D. woody A., Szabó vegetation Z., Stermin within A., centraKósa F.,l Romania–Transylvania verbal communication), [4,12,13] but in recent(Daróczi years, Sz., nycticorax has started breeding on reedbeds at the Sânpaul ﬁshponds (Harghita county, 35 km from NycticoraxDavid A., nycticorax Sándor D. has A., started Szabó breedingZ., Stermin on reedbedsA., Kósa F.,at theverbal Sânpaul communication), fishponds (Harghita but in recent county, years, 35 our area) (dr. Szabo J., verbal communication). Therefore, the breeding of Egretta garzetta and Ardeola kmNycticorax from our nycticorax area) (dr. has Szabo started J., verbalbreeding communication). on reedbeds at Therefore,the Sânpaul the fishponds breeding (Harghita of Egretta county, garzetta 35 km from our area) (dr. Szabo J., verbal communication). Therefore, the breeding of Egretta garzetta Diversity 2020, 12, 371 12 of 16

ralloides at Dumbrăvit, a reeds is a unique case for Transylvania, and Nycticorax nycticorax rarely nests in reeds in the center of the country. Concerning the relation between the mixed colonies’ occupied area and population size, it is normal for Dumbrăvit, a to increase the occupied area of the colony with the increase in breeding pairs, especially if the reedbed is also used. In this case, the colony extends horizontally, compared to the woody vegetation where the extension can be done vertically. For reed-breeding species such as Ardea purpurea, water depth is a major factor determining reedbed occupancy [17,18]. Sometimes, the use of a habitat type or nesting location is forced by various factors, such as insufficient or sudden fluctuations of water level [16]. Reed-nesting herons may select flooded reedbeds for breeding, which reduce the access of terrestrial predators [12,22]. The water level in all ponds within the studied artificial wetlands is artificially adjusted according to the needs of the aquaculture management, and, during April–July in normal conditions, it is approximately 60–80 cm in the reedbeds with colonies of Ardea purpurea and A. alba. In some years (e.g., 2019 and 2020), when the water level was 0–10 cm in the reeds during April–May on the main breeding pond at Rotbav (12 ha of reedbed), these species had to form colonies in the flooded marsh in the vicinity

(Figure4). At the same time, the decrease by over 25–30 cm in water level in a pond from Dumbr ăvit, a (e.g., April 2018) facilitated the access of wild boar Sus scrofa. This led to the relocation of the Ardea alba colony formed in the reeds to another pond. The new colony was built on Salix with other species of herons and cormorants, and it was the only breeding case of great white egret on woody species in the study area. For the mixed colony on the woody vegetation of the islands, water level may be variable or even zero in the nesting willows (e.g., in 2020 in the Rotbav colony, there was no water in April–May, but the island was surrounded by water, with no access land). Such islands also provide nesting opportunities by protecting birds from terrestrial predators [23,24]. For example, in the Czech Republic, artificial pond islands seem to be essential for the survival of several waterbird species [24]. A similar case, however, regarding the increase in water level can lead to the flooding of the nests and thus to the abandonment of the nesting place (e.g., in the end of April 2020, a colony of Ardea purpurea in formation on a reedbed within a main pond from Dumbrăvit, a left the location after the water level increased by 20–30 cm). However, in the two studied wetlands, there are sufficient areas for breeding in good conditions for a constant or increasing number of pairs. Thus, for Ardea purpurea and A. alba, there are currently about 26 ha of flooded reedbeds (including reedmace) as a potential habitat for these species. The species that form mixed colonies (herons, egrets), in addition to the surface occupied by woody vegetation, can also use part of the flooded reeds, which happened in 3 years of the decade. Moreover, the management plan of the Natura 2000 site provides for the maintenance of a sufficient water level in the ponds preferred by the colonies during the breeding season. This measure favors both fishery management and the life of fish raised by a high level of water in the warm period of the year, as well as all species of breeding birds in various types of flooded vegetation that require sufficient water during nesting. In addition, the plan requires the conservation of the whole habitat consisting of reedbeds and willow clumps [25]. We are, therefore, talking about the sustainable management of the aquatic resource and the entire artificial wetland.

4.2. Nearest Distance from Colony Edge to Terrestrial Habitats and Human Contructions The nearest distance from the edge of the colonies to terrestrial habitats could reflect predator accessibility to the colonies [17,26,27]. At least in the case of the Dumbrăvit, a fish farm, potential terrestrial predators (e.g., wild boars) can penetrate the vegetation of fishponds anywhere, and there is a network of paths on some of them (own observations). Thus, the distance from the colony to the dams is probably not important in protecting the nests from wild boars. At the same time, it is relevant that the whole area is subject to anthropogenic activities and pressure (aquaculture, angling, car and rail traffic, human habitation, leisure activities). Considering the calculated distances, there is a fairly high degree of tolerance of the colonies to humans and some human activities.

Regarding the relation between the nearest distance to the dam and the Dumbrăvit, a mixed colony area formed by Egretta garzetta, Nycticorax nycticorax, and Ardeola ralloides, the negative correlation that Diversity 2020, 12, 371 13 of 16 we found can be explained by the fact that, with its expansion, the colony approaches at least one of the dams. Below are some general considerations on the trend of breeding populations. For none of the species was there an obvious negative trend in nesting population during the 10-year study period. The causes of fluctuations in the annual number of pairs in the case of some species, such as Microcarbo pygmaeus and Ardea purpurea, are not known; these may be local, but also may not be related to the studied area. Maintaining or significantly increasing the number of pairs depending on the species is due to habitat conditions and food supply. Subsequent studies can show to what extent the type and amount of food available on the fish farm and its surroundings influence the presence and dynamics of waterbird breeding populations. Until then, we can only assume that, at least inside the fish ponds, there is a rich trophic source that can maintain a certain number of pairs of colonial waterbirds, such as cormorants, herons, and egrets. Commercial fish in particular reach stocks that are much higher than those in natural ecosystems (1–1.5 t/ha). At the same time, fish from all fattening ponds are fed throughout the warm season, and some of the food is probably eaten by economically unimportant wild fish upon which birds feed (e.g., Pseudorasbora parva, Carassius gibelio).

4.3. Species Occurrence, and Their Distribution and Population Size These aspects are discussed in comparison with the reference area (Transylvania) and with the situation on the national level. Regarding the distribution of species, data published in monographic books and atlases were considered, [3,4,28–32]. The most recent and official publication of national breeding and nonbreeding birds is the atlas of bird species of community interest in Romania [5]. This atlas presents distribution maps of species using grids (squares) of 10 10 km as in other × monographic books [4]. The assessed population size for each of the eight species is compared with the national one according to two official and current sources [5,6]. In Transylvania, some former or recent heronries are known. One of the largest has been known since 1986 with three species (Nycticorax nycticorax, Ardea cinerea, and Egretta garzetta). Nycticorax nycticorax was most abundant in 1995, with about 300 pairs, compared with only 25 pairs of Ardea cinerea [13,33]. Another colony where Nycticorax nycticorax was also the dominant breeding species is located in the Iernut-Cipău fishponds [34]. Without making a comparison, recent data (2015–2018) from the largest natural wetland in Romania and Europe, the Danube Delta Biosphere Reserve, showed higher or lower fluctuations in the considered colonial species; the only species with an obvious growth trend was Nycticorax nycticorax [35].

4.3.1. Great Cormorant (Phalacrocorax carbo) Our observations are the ﬁrst published evidence of Phalacrocorax carbo breeding in Transylvania [3,

5,28–32], but there is an observation regarding this from 2000–2001 in a colony on the Mures, river at Acmariu (N 45.955271, E 23.382908), i.e., 8–14 pairs (Sándor D.A., verbal communication). Moreover, the great cormorant does not currently appear as a breeding species anywhere within the interior of the Carpathian chain [5], but there are reports of it as a nesting species (Bărbos L., verbal communication) within a mixed colony at Suplacu de Barcău, Bihor county.

4.3.2. Pygmy Cormorant (Microcarbo pygmaeus) So far, the studied area is the only known one in Transylvania where Microcarbo pygmaeus nests [5,36]. This location is also among the few breeding places of the pygmy cormorant inside the Carpathian chain.

4.3.3. Grey Heron (Ardea cinerea) In the central part of Romania (Transylvania) there are another 9 squares of distribution of Ardea cinerea compared to that of the studied area [5]. Munteanu et al. [4] present it as a breeding species in two locations in Transylvania. The Grey Heron bred in a monospeciﬁc colony in an oak Diversity 2020, 12, 371 14 of 16

forest from Dumbrăvit, a, and in 2004, breeding began in the emergent vegetation from a ﬁsh farm. It is important to point out that the breeding population in our study area exceeds 1% of the national minimum population.

4.3.4. Purple Heron (Ardea purpurea) In central Romania, Ardea purpurea was found in 4 other squares of distribution in addition to our locations. In 6 other squares, the species breeds inside the Carpathian chain [5]. According to [4], in addition to our area, there is only one breeding location in Transylvania. From a breeding-population point of view, it is clear that the breeding pairs there exceeded 1% of the minimal or maximal values of the breeding population in Romania for the period of 2008–2013. The values that we found were also higher than 1% of the minimal population in 2013–2018. The breeding population of the species in the studied area was close to the threshold (C6—30 pairs) used in Romania as a criterion for selecting the area as an Important Birds Area [8].

4.3.5. Black-Crowned Night Heron (Nycticorax nycticorax) In central Romania, Nycticorax nycticorax was found in 3 other squares of distribution in addition to our locations. The latest data also showed 9 squares inside the Carpathian chain [5]. Another monographic paper [4] presented only three locations of known, current, and recent colonies in Transylvania. The breeding population in recent years in our study area exceeded 1% of the national minimal population.

4.3.6. Squacco Heron (Ardeola ralloides) In central Romania, Ardeola ralloides breeds only in one square of distribution beside the area described by us, and in the entire area of the interior of the Carpathian chain in two other squares [5]. According to [4], it does not appear as a breeding species in Transylvania.

4.3.7. Little Egret (Egretta garzetta) In central Romania, Egretta garzetta appears in only 2 squares of distribution (outside the area described by us) [5]. According to [4], there is only one location of a known, current, and recent colony in Transylvania. The breeding population within the study area in recent years has reached 1% of the national minimal population.

4.3.8. Great White Egret (Ardea alba) Ardea alba was registered in only 3 distribution squares in the central part of Romania and in 3 more in the interior of the Carpathian chain [5]. According to [4], outside the area described by us, there is only one breeding location in Transylvania. The species was ﬁrst documented as a breeding bird in the central part of Romania (Transylvania) in 2006 [37] through a nest found on a pond at Dumbrăvit, a. In relation to breeding-population size, the species reached almost 6% of the maximal population of Romania for the period of 2008–2013. At least in recent years, their numbers of pairs in the studied area exceeded 1% of the national minimal or maximal population. At the same time, the breeding population of the species in the studied area reached and exceeded the threshold (C6—15 pairs) used in Romania as a selection criterion for an Important Birds Area [8].

5. Conclusions As a general conclusion, fish farms and other types of artificial wetlands within protected areas (e.g., the Natura 2000 and Ramsar sites) where integrated management is applied can support many bird species, including some of conservation concern, sometimes with relatively large populations [25]. These wetlands could provide suitable habitats, such as those described by us, which would allow waterbird colonies to settle and be maintained. Some of these species have expanded their nesting areas Diversity 2020, 12, 371 15 of 16 in such artificial wetlands, while others bred for the first time in certain national territories, such as the center of Romania. In these farms, only the long-term continuation of economic activity (sustainable fish farming in our case) can maintain or increase the number of bird species and populations, as well as overall biodiversity. Sustainable inland fisheries must be maintained and encouraged in the European Union and beyond, especially in the countries of central, eastern, and southeastern Europe, where such farms support significant species and populations of waterbirds which are recognized on the national or international levels [8,21,24,38,39].

Author Contributions: Conceptualization, D.T.I. and C.V.H.; methodology, D.T.I.; statistical analysis, I.C.P.; data curation, D.T.I.; writing—original-draft preparation, D.T.I. and I.C.P.; writing—review and editing, D.T.I., C.V.H., and I.C.P. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by Wildlife Management Consulting through technical assistance and equipment. Acknowledgments: We are grateful for constructive comments and suggestions of the three anonymous reviewers, which were really helpful to improve the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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