Aphantopus Hyperantus As a Response to Microhabitat Structures in and Around a Natura-2000 Site in Upper Austria

Investigations on population dynamics of Phengaris teleius, P. nausithous, & Aphantopus hyperantus as a response to microhabitat structures in and around a Natura-2000 site in Upper Austria

Masterarbeit Zur Erlangung des Mastergrades – MSc

An der Naturwissenschaftlichen Fakultät der Paris- Lodron-Universität Salzburg

Eingereicht von Anna Sommer, BSc

GutachterIn: Univ.-Doz. Dr. Andrea Grill Univ.-Prof. Dr. Jan Christian Habel

Fachbereich: Biowissenschaften

Salzburg, September 2020 Abstract Biodiversity is declining worldwide. Insects are among the most threatened groups. Specialist species are in particular negatively affected by habitat loss and deterioration of habitat quality. The occurrence of the two endangered and often sympatrically existing butterfly species Phengaris nausithous and P. teleius is strongly limited by the availability of their host ants (Myrmica) and host plant (Sanguisorba officinalis). Using a mark release recapture approach, this study investigated the dispersal behaviour of these two rare specialist species and one abundant generalist butterfly Aphantopus hyperantus across five meadows from July to August 2019. Based on the obtained data, the following research questions were answered: (1) Does daily abundance of butterflies in the three species differ between the five meadow patches, (2) Is flight distance explained by habitat quality measured in abundance of flowerheads and Myrmica ants, and (3) Does border structure (road, path, forest, bushes, fertilized grassland) as well as the availability of Sanguisorba officinalis flowerheads and nectar beyond the border affect the crossing probability of the butterflies? Statistical analyses revealed that daily abundance of butterflies differed significantly between the five meadows and between species. Flight distances, on the other hand, were most significantly affected by species-membership. Border structure and nectar availability beyond the border affected the crossing probability. Conclusively, the results allow insights into what is hindering or promoting the dispersal of the two endangered lycaenid species between patches.

Biodiversität ist weltweit im Rückgang, wobei Insekten zu den am meistgefährdetsten Tieren zählen. Besonders Spezialisten sind durch Lebensraumverlust und -degradierung stark betroffen. Phengaris teleius und P. nausithous, zwei gefährdete, oft sympatrisch vorkommende Schmetterlinge, sind durch das Vorkommen ihrer Wirtspflanze (Sanguisorba officinalis) und ihrer Wirtsameisen (Myrmica) limitiert. Mittels Fang-Wiederfang Methode untersucht diese Studie das Flugverhalten und die Ausbreitung der zwei spezialisierten Bläulings-Arten und einer generalistischen Art (Aphantopus hyperantus) in fünf Flächen. Im Rahmen dieser Studie werden folgende Fragen beantwortet: (1) Unterscheidet sich die tägliche Abundanz der drei Arten in den fünf untersuchten Flächen?, (2) Ist die Flugdistanz der Schmetterlinge von der Habitatsqualität (gemessen an der Anzahl von S. officinalis Blütenköpfen und Myrmica-Ameisen) abhängig?, (3) Haben verschiedene Grenzstrukturen (Straße, Schotterweg, Wald, Gebüsch, Fettwiese) sowie Nektar- und Wirtspflanzenangebot jenseits der Habitatsgrenze Einfluss auf das Überqueren und somit auf die Ausbreitung der Schmetterlinge in andere Wiesenflächen. Statistische Analysen ergaben signifikante Unterschiede in der täglichen Schmetterlingsabundanz zwischen den fünf Flächen, außerdem unterschieden sich die Arten untereinander in deren Abundanz. Unterschiede in der Flugdistanz wurden nicht durch die Habitatsqualität sondern durch die Artzugehörigkeit erklärt. A. hyperantus legte weitere Strecken zurück als die beiden Bläulings-Arten. Die Art der Grenzstruktur und die Verfügbarkeit von Nektar jenseits der Habitatsgrenze beeinflusste das Überqueren der Grenzen. Basierend auf diesen Ergebnissen bietet diese Studie Einsicht in förderliche und hinderliche Faktoren in Bezug auf das Flugverhalten und somit auf die Ausbreitung der Schmetterlinge. 1. Introduction During the last 50 years, the earth's biodiversity has declined profoundly; furthermore, biodiversity crisis is occurring worldwide (IPBES report on biodiversity and ecosystem services (Díaz et al. 2020)). Further, besides of direct exploitation, climate change, pollution and the invasion of alien species, the authors include land use change into the direct drivers of global biodiversity loss. Moreover, this IPBES report reveals that 75 % of global land surface has been altered due to rapid increase of human population followed by rising human necessities, consumption- and production patterns and an expanding agriculture. Intensified agriculture often leads to monocultures, habitat deterioration and - fragmentation (Crews et al. 2018 and references theirein) mainly affecting terrestrial organisms like insects. Insects are relatively well studied when compared to other invertebrates and heavy declines in both, species richness and abundance have been documented (Wagner 2020, Simmons et al. 2019). Among insects, butterflies belong to the most threatened groups (Habel et al. 2019 and references theirein) being strongly negatively affected by habitat loss and deterioration as a consequence of intensive agriculture (Thomas 2016). They can be divided into two main functional groups: generalists (polyphagous) and specialists (oligo- or monophagous), the latter often are highly specialized feeding and breeding on few or single plant species (Slansky 1976). While generalists can persist and sometimes thrive in homogenous landscapes, specialists mostly are directly negatively affected by habitat homogenisation and the subsequent loss of biodiversity (Melero et al. 2016). Besides habitat loss and -deterioration, habitat fragmentation is a severe threat for butterflies as it bears the risk of genetic isolation. In both, urban and rural areas, high quality habitats (species rich patches, high in biodiversity) often are isolated patches, cut through by buildings, roads etc., or they are embedded into a matrix of intensified agricultural landscape, which may lead to lower dispersal rates and a subsequent decrease of genetic diversity (Rochat et al. 2017 and references theirein, Schtickzelle et al. 2006). Dispersal leads to higher population and metapopulation size, thus to more stability (Hansson 1991) and is therefore detrimental for the persistence of metapopulations (Hanski 1999, Hanski et al. 2000). Although butterflies are highly mobile species, even certain generalist butterflies (e.g. Maniola jurtina) tend to stay within a confined space of 100 m in a non-fragmented area (Grill et al. 2006).

The study species Both large blue butterflies, the dusky large blue (Phengaris nausithous, Bergsträsser 1779) and the scarce large blue (Phengaris teleius, Bergsträsser 1779) (former genus Maculinea), are highly specialized. They often occur sympatrically, inhabiting wetland like fen meadows or heathland, where Sanguisorba officinalis (great burnet) and their host ants of the genus Myrmica occur. P. nausithous is restricted to M. rubra as host ant (Thomas et al. 1989) while P. teleius mainly is confined to M. scabrinodis and M. rubra but also has been reported to parasitize successfully in nests of Aphaenogaster japonica, M. gallienii, M. salina, M. specioides and M. vandeli (Hymenoptera: Formicidae) (Tartally and Varga 2008). For oviposition, females exclusively use the capitulum (flower head) of the great burnet, where the larvae feed on Sanguisorba officinalis inside the flower head until their fourth instar. After these 3-4 weeks (Bubová et al. 2018), once the larva have completed their third molt the caterpillars leave their host plant and drop down onto the ground surface. Using mimicry by imitating certain sounds and pheromones, they lure their host ants, which pick them up and bring them actively into the ant nest, where they live parasitically (Thomas 1984). P. nausithous caterpillars are immediately attractive to Myrmica ants and adopted in less than 6 minutes (on average); in contrast, to become attractive to the host ants, larvae of P. teleius need to perform a certain adoption behaviour by offering secretion droplets from the dorsal nectary organ, which results in an average adoption time of 44 minutes (Fiedler 1990). Inside the nest, P. nausithous lives predatory feeding on ant brood but also is fed actively by worker ants (Thomas and Settele 2004), whereas P. teleius lives predatory only, depleting the ant brood. As a result, several caterpillars of P. nausithous can survive in one ant nest, while only one larva of P. teleius survives per nest (Weidemann 1986). Having finished the metamorphosis, the butterflies fly from July to mid of August and live on average for three days (Bubová et al. 2016, Nowicki et al. 2005). Due to this high level of specialization, both species are regarded as flagship species for insect conservation, they have a high conservation status in Europe (Bubová et al. 2018 and references theirein). The World Conservation Monitoring Centre (IUCN 1996a, IUCN 1996b) lists both species as “Near threatened”. Additionally, they are cited in various red lists of many European countries (Grill et al. 2008). In the red list of Austria both are declared as “Vulnerable” (Höttinger and Pennerstorfer 2005). Moreover, they are listed in Annex II and IV of the European Habitats Directive, which means that conservation areas must be established, and that the species are strictly protected EU wide, inside and outside of Natura 2000 sites (Council of the European Union, EU 1992).

Several studies on the effects of habitat borders on movement and dispersal of butterflies in Central Europe (Grill et al. 2020, Polic et al. 2014) have been conducted so far. Even the flight behaviour of P. nausithous and P. teleius have been investigated (Nowicki et al. 2013, Popović et al. 2017, Skórka et al. 2013a). Grill et al (2008) were the first conducting a field study that investigated different mowing regimes on host ant and host plant abundance, which revealed positive effects of a late mowing regime on Myrmica abundance and thus Phengaris. This study is the first that investigates possible effects of host plant and host ant abundance on their flight distance. Also, studies investigating the effects of boundaries on Phengaris species have already been published; Skórka et al. (2013b) for example investigated how P. teleius responded to boundaries, however studies that include the structure of border types and further possible predictor variables like host plant and nectar availability beyond the border are still missing. Yet, it is still unknown, if both lycaenid species are affected by these variables. Apanthopus hyperantus (L.,1758), abundant in the study area, is included into the study as the generalist counterpart to the specialized lycaenids and the following research questions are addressed: (1) Does daily abundance of butterflies in the three species differ between the five meadow patches, (2) Is flight distance explained by habitat quality measured in abundance of flowerheads and Myrmica ants, and (3) Does border structure (road, path, forest, shrub, fertilized grassland) as well as the availability of Sanguisorba officinalis flowerheads and nectar beyond the border affect the crossing probability of the butterflies?

2. Material and Methods Study regions The study was conducted in the district of Vöcklabruck in Upper Austria, Austria. Five meadows where the three butterfly species occur were investigated. Meadows A (48°00`40,6``N, 13°26`51,1`È) and C (48°02`10,2``N, 13°24`36,1`È) are wet fen meadows, cut annually at the beginning of August. Meadows B (48°00`44,9``N, 13°26`11,0`È), D (48°00`22,9``N, 13°27`05,2`È) and E (48°00`28,0``N, 13°26`41,4`È) are situated in a Natura 2000 area (“Wiesengebiete und Seen im Alpenvorland”). B is an annually mown fen meadow too, D and E are cut more frequently and characterized by a more intensive management like fertilization. Distances between the meadows are under 1 km, except meadow C, its distance to the other meadows ranges from 3 to 4 km (Fig 1.)

Fig. 1 Overview map of the study regions (A-E). The Natura 2000 site is marked in yellow.

Butterfly sampling Butterflies were sampled daily from 04.07.2019 to 10.08.2019 when weather conditions were suitable (no strong wind, warm temperatures, sunny). Meadows were sampled in a random order ranging from 30 to 60 minutes per day. In each meadow a transect of 100 m was walked through at a slow pace, each visible individual of Aphantopus hyperantus, Phengaris nausithous and Phengaris teleius was captured with a hand-held net and marked with consecutive numbers on the hind wing with a permanent marker (Staedtler Lumocolor, fine permanent marker). For each butterfly I noted species, the respective number, sex, estimated age (1 = fresh wing pattern, 2 = slightly damaged wings, 3 = damaged wings), coordinates. When butterflies were spotted and captured close to a habitat edges (< 3 m), their behavior (if they crossed the border or stayed within the meadow, 0/1 binomial data structure) was noted. To avoid disturbance effects due to capture and release procedure subsequently leading to a possible fleeing behavior, butterflies were observed before and after capture. If they approached an edge and crossed, they were followed and captured in the adjacent area. Thus, crossings only were counted when the butterflies crossed before capture and therefore without disturbance. Butterflies that were caught near edges within meadows (mostly sitting on flowerheads or flying along habitat edges) were observed a few minutes after capture, too. If they crossed the border as fleeing behavior after release but returned a few moments later, this was not counted as crossing event. The structure of the respecting habitat edges (border type) as well as if nectar and Sanguisorba flowerheads were available in case of crossing (beyond the border) was noted. Habitat edges were defined as following border types: road, path, forest and fertilized grassland. Meadows A and C directly bordered a paved road with a mean width of 4 m, (regularly frequented by cars) and a forest harbouring large trees, mostly consisting of Picea abies. Meadow B was embedded in a forest; thus, all habitat edges were characterized by shady, low-nectar forest structures. Meadow D additionally was confined by a row of bushes, meadow E by a gravel path with mean width of 2 m. All investigated meadows were directly adjacent to intensively managed fertilized meadows with three to five cuts per year and regular fertilisation. All habitat edges of the investigated meadow patches were dissimilar to the structures of the adjacent area and therefore were defined as borders. For each recaptured butterfly, flight distance from the initial capture to the recapture point based on coordinates was measured as the shortest line using Google Earth.

Ant sampling In each meadow ants were sampled once at the end of August. This is the time, when many larvae of P. nausithous and P. teleius drop down to the ground to be adopted by host ants after three to four weeks of development in the flowerheads. Thus, host ants are expected to be present at this time for representative quantification. On 24 (B, C) and 25 (A, D, E) August 2019 when weather was sunny without precipitation, 30 petri dishes were laid out in a random distribution at the ground of the cut meadows (30 dishes per meadow). In each petri dish one sugar cube was placed to lure ants. Baiting stations were controlled after three hours, the number of ants in the petri dishes was counted and noted in the field. Three individuals (specimens) of each petri dish were taken into the lab for classification, stored in isopropyl alcohol. Because ants are considered territorial, the counted individuals per baiting station were expected to belong to the same species as the collected specimens. In the lab, ants were determined to the genus level using the classification keys for European ants (Seifert 2007, Seifert 2018).

Vegetation Vegetation data was recorded once per meadow in July 2019. Flowering plants except grasses were determined in a representative 2x2 m plot per meadow using classification books (Fischer et al. 2008, Schmeil et al. 2011). Additionally, in each 2x2 m patch, the total amount of Sanguisorba officinalis flowerheads were counted, as comparable measurement of host plant availability per meadow.

Statistical analyses Statistical analyses were conducted in R version 3.4.0 (R Development Core Team 2016). To test for possible differences in daily butterfly abundance between meadows and species, a GLM with quasipoisson error distribution was performed. Flight distances of the observed butterflies as response variables were analysed with two different models. First, a generalized linear model (GLM) with quasipoisson error distribution was applied to test if age of the butterflies, sex and species affiliation affected the flight distance within observed meadows. Second, possible effects of plant species richness (number of angiosperm species per meadow), number of S. officinalis flowerheads and the total number of Myrmica ants per meadow as predictor variables were observed using a generalized linear mixed model (GLMM: lme4 package, (Bates et al. 2015)) with meadow as random factor. To test if the border type, species affiliation and nectar- or S. officinalis availability beyond the border affect the crossing probability of the butterflies, a GLM with binomial error distribution (crossed, not crossed) and a logit link was performed.

Ethics In this study no butterfly was harmed or put to death. The mark release recapture study was conducted in compliance with national and regional laws and only with granted permissions. Handling time was kept as short as possible (< 1 min) and butterflies were released immediately after marking. To minimalize vegetational damage, same paths were walked through during daily observations. Further, ants were taken to the laboratory only in an indispensable amount necessary for classification (three individuals per baiting station).

3. Results

Butterfly population During the mark release recapture study, 296 butterflies (137 females, 150 males) were marked, 105 of them recaptured. 135 of the marked butterflies belong to the genus Phengaris (77 P. nausithous, 58 P. teleius), Aphantopus hyperantus was most abundant with 162 marked individuals. Most butterflies were found in the meadows A and B with highest numbers for P. teleius (48) in A and A. hyperantus (89) in meadow B. In meadow C, D and E mostly P. nausithous, but also A. hyperantus could be found. Phengaris teleius was absent in meadow D (Table 1, Fig. 2).

Daily numbers (total counts) of investigated butterflies differ significantly between species (Fig. 3; F=15,89, df=2,246, p < 0.001) with highest numbers for Aphantopus hyperantus and between meadows (Fig. 4; F=6.38, df= 4,242, p < 0.001). Fig. 5 shows the differences in daily counts between species and between meadows in one graph.

Table 1 Investigated species (Phengaris nausithous, Phengaris teleius and Aphantopus hyperantus) and their mark recapture rates per meadow and in total (M= marked, R= recaptured)

A B C D E Total Species M R M R M R M R M R M R P. nausithous 9 1 1 0 28 20 14 2 25 9 77 32 P. teleius 48 7 8 0 1 0 0 0 1 0 58 7 A. hyperantus 40 15 89 44 18 4 12 3 2 0 161 66 Total 97 23 98 44 47 24 26 5 28 9

Fig. 2 Total butterfly counts per meadow over the whole sampling period. The most abundant species was Aphantopus hyperantus in meadow B with 89 individuals.

Fig. 3 Daily abundance of the investigated butterfly species, based on total counts per day over a sampling period of five weeks. Significantly more individuals of A. hyperantus could be counted daily than of P. nausithous and P. teleius (p < 0.05).

Fig. 4 Differences between the five meadows in daily butterfly abundance (p < 0.05).

Fig. 5 Daily butterfly counts of five weeks of sampling for each investigated species (Aphantopus hyperantus, Phengaris nausithous and Phengaris teleius) per meadow (A,B,C,D,E)

Microhabitat conditions Most Myrmica ants could be sampled in Meadow A; in 30 petri dishes, 493 individuals could be counted, followed by meadows B and C with around 100 individuals. All sampled ants in meadow A and B belonged to the genus Myrmica, Meadows D and E harbored less ants and a smaller proportion of Myrmica ants. Plant species richness of the five meadows ranges from 15 (meadow C) to 31 (meadow B) species. Most S. officinalis flowerheads could be counted in meadows A and D (Table 2). Due to its high numbers of host ants and plants, meadow A represents a good habitat for Phengaris species; consistently, most P. teleius were counted in this meadow, too.

Table 2 Counted numbers of Myrmica ants per meadow (lured with sugar cubes in petri dishes) and their proportion of all sampled ant species in percent; number of classified plant species per meadow and number of Sanguisorba officinalis flowerheads of a representative 2x2 m plot per meadow (A, B, C, D, E)

Meadow Myrmica ants Plant species richness S. officinalis flowerheads A 493 (100 %) 22 85 B 125 (100 %) 31 70 C 100 (79 %) 15 30 D 7 (41 %) 16 30 E 23 (62 %) 19 85

Butterfly flight distances Out of the 296 marked butterfly individuals, 105 could be recaptured. 84 of the recaptured butterflies could be identified by their numbers and allocated to marking- and recapture points with exact coordinates (numbers of the rest were illegible due to wing damage). Thus, flight distances could be calculated for approximately a third of the three butterfly species. 47 of the identified butterflies were Aphantopus hyperantus. Six of them were recaptured in different meadows from where they were marked, they changed plots. Maximal flight distance of them was 3960.0 m. In total, distances for 30 Phengaris nausithous were calculated. Three of them were recaptured in different meadows too, the largest flight distance was 4484.36 m (Table 3). For Phengaris teleius, however, it could not be confirmed, that individuals dispersed across meadows. Only seven of them could be recaptured, all of them in the same meadow as in which they were initially marked. Referring to the three investigated butterfly species, most of the butterflies flew short distances (< 300 m), single individuals reached distances between 3,000 and 5,000 m (Fig. 6A). All seven individuals of Phengaris teleius were recaptured within 50 m (Fig. 6B). Most individuals of P. nausihtous and A. hyperantus preferred short distances too (Fig. 6 C, D).

Table 3 Flight distances of individuals that changed plots and flew long distances across meadows. Butterflies (Aphantopus hyperantus and Phangearis teleius) that were captured in different meadows than in which they were initially captured. Phengaris teleius could not be recaptured only in the same meadows and did not fly large distances.

Species Marking meadow Recapture meadow Flight distance (m) A. hyperantus B A 837.52 A. hyperantus B D 1320.79 A. hyperantus A C 3960.00 A. hyperantus B A 872.74 A. hyperantus D B 1298.36 A. hyperantus C B 3256.21 P. nausithous A C 3931.74 P. nausithous C D 4484.36 P. nausithous E C 4146.76

Age and sex as further possible predictor variables for flight distance (included in the GLM) did not show any significant effects (p > 1). However, species affiliation had a significant effect on the flight distance covered in each meadow. (GLM: F= 3.15, df= 2.72, p < 0.05). The generalist butterfly A. hyperantus differed in its flight distance within meadow patches from P. nausithous and P. teleius with highest values up to 100 m and a mean distance around 30 m. Both Phengaris species flew less far, around 60 m with a mean distance of 20 m (Fig. 7). The conducted GLMM could not reveal any effects of microhabitat conditions (number of Myrmica ants, S. officinalis flowerheads and plant species richness per meadow) on the flight distance. Detailed flight distances per species and per meadow are depicted in Figure 8.

A B

C D

Fig. 6 Histogram of flight distances of the investigated butterflies. Most of the butterflies flew less than 300 m (A). The maximum distance was 4,484 m, recorded for Phengaris nausithous (C).

Fig. 7 Flight distances (m) of the investigated butterflies within meadow patches (excluding single individuals that changed plots). Species affiliation had a significant effect on the flight distance (p < 0.05). Fig. 8 Boxplots of the flight distances per species and per meadow (plot), based on recaptured butterflies within meadows. Missing boxplots indicate that species could not be recaptured in the respective meadow.

Border permeability 198 butterflies could be investigated near habitat edges (borders); a quarter of them 152 crossed the border, three-quarters did not 152 fly across the border and stayed within the same meadow (Fig. 9). Crossing probability was independent of species affiliation. Statistical analysis (GLMs) resulted in significant effects of border type (df= 4,193; X²= 53.78; p < 0.001) and nectar-availability beyond the border (df= 1,192; X²= 11.50; p < 0.001) on the crossing frequency of all three species (species affiliation as predictor 46 variable had no significant effects). Forest and street as border type act as a barrier. No single individual could be observed that entered a forest. Three out of 46 individuals were observed flying across the street. Bushes, a gravel path and a meadow as Fig. 9 Total counts of butterflies’ behaviour (crossed: flew across the border into the adjacent area, not crossed: did adjacent area did not hinder butterflies not fly across the border) at habitat edges. from crossing (Fig. 10). 87 butterflies were observed at borders where nectar was available in the adjacent area, 40 of them crossed, 47 did not cross. 111 butterflies flew near borders where no nectar was available in the contiguous area, 6 of them crossed, 105 did not cross. Summarizing, crossing probability is higher, when nectar is available in neighbor-areas. Detailed crossing frequencies of the butterflies per border type in combination with nectar availability is shown in Fig. 11. Differences of crossing probabilities between species could not be found, neither an effect of Sanguisorba officinalis flowerhead-availability in adjacent areas.

Fig. 10 Total numbers of butterfly individuals that crossed or did not cross a border. Border type has a significant effect on crossing probability (p < 0.001).

Fig. 11 Crossing frequency of the butterflies at different borders when either nectar was available or not available beyond the border 4. Discussion Do the meadows differ in butterfly abundance? Aphantopus hyperantus was expected to be most abundant in all patches among the three investigated species, as the both Phengaris species mostly are considered to be rare. Additionally, the Ringlet is confirmed to occur in a range of habitats, in bushy areas as well as grassland, cleared forest areas and glades; several plants serve as food plants, even larvae feed on a range of plants (Tolman and Lewington 2009), while both lycaenid species depend on the occurrence of their host plant Sanguisorba officinalis. As expected, significant differences in daily counts could be found between species and between meadows. In Fig. 3 it is visible that Aphantopus hyperantus was most abundant with the highest mean, when all counts were analyzed. This result corresponds with the predictions due to its generalist-life history. However, considering single meadows, huge differences in butterfly counts can be seen in Fig. 2 that shows the total numbers of all counts, and in Fig. 3 showing the means and range of daily counts per meadow. Compared to other meadows, patch A harbored a considerably number of P. teleius, the same pattern was found in meadow B for A. hyperantus. Meadow A is a fen meadow funded by the Austrian ÖPUL program (Bundesministerium Landwirtschaft, Regionen und Tourismus 2015) with a single annual cut in August, what results in a suitable, high-quality patch for all three investigated species, especially for P. teleius. However, probably the population numbers of the Phengaris species could be improved in this meadow even more with a later cut, as many studies suggest meadows harboring Phengaris butterflies to be mown in late summer or autumn, definitely not before September (Thomas 1984, Johst et al. 2006, Grill et al. 2008, Popović et al. 2014, Bubová et al. 2018), to ensure larval development inside the flowerheads even of late flying individuals and to be adopted in sufficient time by the host ants. Meadow B differs from meadow A in its location near a marsh and thus in its vegetation, it is neither allowed to be cut more than once a year, leading to a high plant diversity. Embedded in a forest, the Ringlet butterfly profited from this late cut forest glade (August to September) with high individual numbers (89 marked, 44 recaptured). Although S. officinalis and Myrmica ants occurred there, only one individual of the Dusky Large Blue could be found an no single individual of P. teleius. Meadow C, a bog meadow, rich in rare plant species like Menyanthes trifoliata is owned by local conservationists and cut late in summer too. Observations confirm this meadow as ideal habitat for P. nausithous, as well as for A. hyperantus. There, highest numbers (in total 28 individuals) for the Dusky Large Blue could be found. In meadows D and E, both situated in an open mosaic of agricultural used meadows (although embedded in a Natura 2000 area) the lowest amounts of butterflies were observed. Fig. 4 shows the significant differences to the other meadows in daily butterfly counts. Both meadows are cut several times per year, single S. officinalis plants remained at habitat edges after mowing events, where some P. nausithous could be observed, especially in meadow E where an adjacent embankment seemed to serve as refugium. Almost all individuals of P. nausithous were marked within a few meters near or inside this embankment, only single individuals were caught inside the meadow. These two meadows would have a high potential to be high-quality habitats for Phengaris butterflies, if mowing regimes would be adapted. As high-quality habitats in which Phengaris nausithous and P. teleius occur are rare and often cannot be found in large numbers, it was not possible to observe and sample a sufficient number of meadows (five meadows were found in this area) to test statistically for differences in habitat conditions or butterfly communities between the patches. However, daily butterfly counts could be used as replicates, so I had enough statistical power to test for differences between meadows in daily observed butterfly numbers.

Is flight distance explained by habitat quality? Only few butterflies (nine individuals) could be detected to move long distances, six Ringlet and three Dusky Large Blue were recaptured in different meadows, thus could be confirmed to disperse. Those individuals were excluded from the statistical analysis testing if habitat quality affects flight distance within meadows, because microhabitat structures “per meadow” were tested for possible effects on movement inside the patches. Experimentally including these individuals into the statistical analysis did not change the results. Hansson (1991) stated that flight distances are larger in low quality habitats. Moreover, it is a general assumption that non long-distance-migrating butterflies stay within a certain range once a suitable habitat is found. Therefore, flight distances of the marked and recaptured butterflies of this study were assumed to be affected by habitat structures like plant species richness, the amount of Myrmica ants and flowerheads of the Great Burnet. I expected Phengaris butterflies to be more affected than A. hyperantus because of their degree of specialization. However, neither, the number of plant species per meadow, nor the number of ants or flowerheads could be proofed to have a significant effect on the butterflies, neither on the specialists nor on the generalist. Sex and age neither affected their movement. Though, this study could demonstrate that the investigated species differ significantly in their flight distance (n= 84, p < 0.05). In this study A. hyperantus was expected to fly short distances within meadows due to a dense offer of plants for nectaring and ovipositing due to its generalistic lifestyle. In contrast, the study showed highest flight distances within meadows for the Ringlet butterfly, with distances up to 100 m. Both lycaenids, however preferred short flight distances mainly under 50 m. Recorded flight distances for P. teleius coincide with findings of a recent study in Serbia (Popović et al. 2017). Flight distances per species and per meadows are shown in Fig. 8; there is visible, that not all investigated species could be recaptured in all meadows, to account for this (independent data), a generalized linear mixed model with meadow as random factor was used. Additionally, in this graph is visible that butterflies moved maximum distances up to 100 m within meadows A and B, this was not due to the length of the meadows - in all other meadow patches (C, D, E) butterflies had the potential to move around 100 m within the meadow patch (the possible maximum flight length was measured with Google Earth too).

Does border structure as well as the availability of Sanguisorba officinalis flowerheads and nectar beyond the border affect the crossing probability of the butterflies? This study clearly could show that certain types of boundaries act as barriers that hinder the crossing and thus dispersal of butterflies (p < 0.001). It is notable, that only 25 % of butterflies that approached habitat edges, crossed them. Again, Aphantopus hyperantus was expected to be less affected by habitat edges and barriers than the lycaenids, due to its adaption to a range of habitats. However, no species-specific pattern could be found. Habitat edges were equally permeable or non-permeable between species. A swiss study found that even for the Ringlet butterfly connecting structures are indispensable for a successful dispersal between patches (Sutcliffe and Thomas 1996) what supports findings of this study. Nectar availability beyond the border obviously influenced crossing probabilities of the butterflies (p < 0.001, Fig. 11). Few species were observed approaching bushes; butterflies only crossed, flew through the bushes, when nectar was available either between or behind the bushes. Species never approached bushes, when no nectar was available. No single individual entered the forest-matrix, which was without nectar availability at all, inhospitable and shady. Individuals that were observed at habitat edges with forest as adjacent area mostly were observed while flying along the habitat edge inside the patch. When single individuals directly approached the boundary, they reversed or sat down on a plant. The gravel path in contrast was easily permeable for the butterflies. Mostly Phengaris nausithous were observed where a gravel path enclosed the meadow patch at one edge. Here, most individuals (15) crossed the path when nectar was available; however, ten individuals did not cross despite nectar availability beyond the border. An interesting pattern could be found with intensively managed meadows as adjacent area. Every investigated meadow patch except B bordered a frequently cut, plant-species-poor, fertilized meadow. Most butterflies that were observed at the edge near the neighboring meadow (> 20) did not enter the matrix nor fly across, in these cases these meadows either were freshly cut or had low to no nectar. Only three individuals could be observed entering the meadow when no nectar was available. Even though, many butterflies did not enter fertilized meadows when nectar was available (> 15), more individuals dispersed into the adjacent meadow when nectar was present (> 20). This result suggests that even meadows (intensively managed and low in nectar) could seem inhospitable to butterflies and probably hinders dispersal. Nectar availability beyond the paved road could not neutralize the negative effect of the road on butterflies’ dispersal. There was no single observation where nectar was available at the other side of the road and butterflies crossed. In contrary, most observations comprised butterflies at habitat edges where no nectar was available beyond the street and they did not cross, most resided at flowerheads near the edge or flew along the edge. However, a few individuals (< 5) were able to cross the street, even though no nectar was available beyond the street, but the majority of butterflies that were observed near the road did not cross it and stayed within their meadow patch; they did not fly across the road even in cases where rich nectar sources were available at the other side of the street. Similar findings are reported in a study conducted in the Austrian Alps, where a paved road was less likely to be crossed than other boundaries by Ringlet butterflies (genus Erebia) (Grill et al. 2020).

5. Conclusion Flight distances are species-specific, it could not be shown that they are explained by habitat quality. Phengaris teleius and Phengaris nausithous tend to move much lower distances (< 50 m) than Aphantopus hyperantus (< 100 m). Additionally, an adjacent road and forest hindered species from crossing the habitat edge significantly, negatively affecting migration into or colonization of other suitable habitat patches. Bushes, a gravel path and a cut meadow, structures that are more naturally, hindered the butterflies less from crossing. Additionally, it could be shown, that the nectar availability beyond a habitat edge positively influenced dispersal of the three investigated species. These are important findings for conservation issues. Intact, high diversity patches should not be cut through by paved roads to ensure successful dispersal of butterflies. Though, because butterflies react positively to nectar availability, it is indispensable to offer structures rich in nectar to connect habitat patches. Here, a possible suggestion for conservation authorities to connect patches could be to stop regular mowing of roadside verges and habitat edges to enable dispersal, especially for the two endangered lycaenid species. Additionally, it would be important to adapt mowing regimes of intensively managed meadows where Phengaris nausithous and P. teleius occur towards a later cut in autumn to improve habitat quality for Myrmica ants, to enable lycaenid larval development in the flowerheads, subsequently leading to strengthened lycaenid populations. Habel et al. (2019) recommend prioritizing public interest like the persistence of species or high-quality habitats instead of landowner’s rights, of course granting decent recompenses.

6. Acknowledgements I want to thank all persons that were involved in this study, especially to J. Petermann, M. Mayr, M. Affenzeller, J. Eberle, K. Zografou and G. Adamidis for providing material needed for field work and suggestions for statistical analyses. Additionally, many thanks to those family members that provided useful help and support during field work. Further, many thanks to C. Wolkerstorfer, responsible for local conservation areas, who gave useful geographical advice and to local authorities for granting sampling permissions.

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