Local host ant usage of scarce large blue Phengaris teleius and dusky large blue P. nausithous (Lepidoptera: Lycaenidae) at Goričko Nature Park (NE Slovenia)

. In our contribution we report on the local host ants of two threatened and protected butterfly species, the scarce large blue (Phengaris teleius) and the dusky large blue (P. nausithous) at Goričko (NE Slovenia), which hosts one of the largest and the most important metapopulation for both species of large blues in Slovenia. Larvae of both species are developing in Myrmica ant nests and different ant species may be used as larval hosts in different parts of the species range. During our study, seven species of potential host ants of the genus Myrmica were found at 12 selected sampling sites. A total of 142 ant nests were examined for the presence of Phengaris larvae. Larvae of P. teleius were found in 25 nests of three ant species: M. scabrinodis, M. rubra and M. gallienii, while larvae of P. nausithous were found in 9 nests, all of M. rubra. The results of the present study provide the first insight into the local host ants of the two Phengaris species in Slovenia and could serve as an important source for active conservation of both species in Slovenia.


Introduction
Butterflies of the genus Phengaris Doherty, 1891(syn. Maculinea van Ecke, 1915) are among the most studied insects in Europe, mainly due to their highly specific myrmecophilous life cycle, vulnerability and endangerment (e.g.Settele et al. 2005).All four European Phengaris species, i.e.P. arion (Linnaeus, 1758), P. alcon (Denis & Schiffermüller, 1775), P. teleius (Bergsträsser, 1779) and P. nausithous (Bergsträsser, 1779), live in Slovenia (Verovnik et al. 2012).The scarce large blue (P.teleius) and the dusky large blue (P.nausithous) occur sympatricaly in northeastern Slovenia, where they often live syntopically.The range of the P. nausithous in Slovenia is limited to the northeastern part of the country, while the range of the P. teleius also extends to the western parts.Both species live on extensively used humid meadows that are among the most threatened habitats, mainly due to agricultural intensification, fragmentation and abandonment of traditional management (e.g.van Swaay et al. 2012).As such, they have become flagship species for nature conservation and both are protected by the Habitats' Directive (Annex II, IV) as a key instrument for biodiversity conservation in Europe (OJ EC 1992).
Although both species use the same host plant, the great burnet (Sanguisorba officinalis L.), and share some general characteristics of their habitat, there are differences in their ecology, including lifecycle, microhabitat preferences and use of host ants (e.g.Nowicki et al. 2005).The females lay their eggs only on the S. officinalis and the early instar of larvae feed on host plant's flowers and seeds.When the larvae reach the fourth instar, they leave the host plant.On the ground, they have to be adopted by ants of the genus Myrmica Latreille, 1804, which take the larvae into the ant nest.There they predate on ant brood and/or mimic ant larvae and are fed by ant workers (Elmes et al. 1992).They overwinter (one or two winters) in ant nests and pupate in late spring (Thomas et al. 1998).
Pioneering work on the adoption by Myrmica ants had suggested that each Phengaris species parasitizes a specific Myrmica species (Thomas et al. 1989).Later, it was shown that larvae can be adopted by different Myrmica species (the so-called primary and secondary host: Elmes et al. 1998) and host species and colony size have an important impact on larval survival (Witek et al. 2010).However, several recent studies raise doubts on a high host ant specialization of Phengaris species across Europe (e.g.Pech et al. 2007, Tartally et al. 2019).The most recent and comprehensive survey across Europe (Tartally et al. 2019) showed that each Phengaris species is predominantly specialised to a single, basically the most abundant Myrmica species found on Phengaris site.However, there is a geographic mosaic as a difference; populations of the same Phengaris species, sometimes in close proximity, often use different host ant species.
Most data on the host specificity of Phengaris were obtained in Central and Eastern Europe, especially in Poland, Hungary and Romania (e.g.Stankiewicz et al. 2005, Tartally & Varga 2008, Witek et al. 2008, Pech & Sedlachek 2016).The term host specificity usually refers to the ability of Phengaris butterflies to develop within the nests of particular host ant species.To quantify specificity, ideally the number of Phengaris larvae adopted by each Myrmica ant species, should be known, as well as the number that survive in ant nests and develop into adults (Thomas et al. 2005).Although the level of specialisation and the character of the Phengaris-Myrmica host system is now better understood, local data on hosts are still needed, and are useful for site management and species conservation at the local scale.Knowledge of host ant specificity is essential for the conservation of these two butterfly species.
Although the host ant specificity of Phengaris species (especially for P. teleius and P. nausithous) is relatively well known in Europe, there are no published data on this topic for Slovenia.Only a basic survey of Myrmica ants in the habitat of P. teleius and P. nausithous was conducted in the central area of Slovenske Gorice in 2003 and 2004 (Zakšek 2004).The study revealed that M. scabrinodis and M. rubra are the commonest species in the vicinity of S. officinalis, although none of the ant nests were opened and examined for the presence of Phengaris larvae.To fill this gap, we studied host ant specificity of P. teleius and P. nausithous in the Goričko region and the pattern of their local variability.Our results are of high importance for establishing site-specific management of the habitat of the two threatened butterfly species in the region.

Study area and study sites
Goričko is a predominantly rural hilly countryside in NE Slovenia and most of it is included at Goričko Nature Park and the Goričko Natura 2000 site (SI3000221) designated to maintain a favourable conservation status of P. teleius and P. nausithous and other selected species and habitat types (Ur.l.RS 2004).Humid meadows in the valleys are home to the largest populations of P. teleius and P. nausithous in Slovenia (Zakšek et al. 2005).Twelve sampling sites were selected in the eastern part of Goričko NP (Fig. 1), where the population density of both species is the highest (Zakšek et al. 2012).All sampling sites hosted P. teleius, while P. nausithous was present at ten of the selected sites (see Tab. 1 for details).

Field work
Field sampling was carried out in 2011 and 2012, from mid-May to mid-June when Phengaris larvae were at the pre-pupal stage.At selected sites, we searched for Myrmica ant nests within one-metre radius of S. officinalis, which is likely within a foraging distance of Myrmica ants (Elmes et al. 1998).All Myrmica ant nests found were marked and carefully excavated.The nest material was placed on a white sheet, where the nests were opened to check for the presence of Phengaris larvae.After excavation, the nest material and vegetation were restored to their original condition as much as possible.Five to ten ant workers were collected from each nest and preserved in 70% ethanol for later identification.In addition, a random sample of foraging NATURA SLOVENIAE 23(1): 21-33 Myrmica ant species was collected on sampling sites when searching for ant nests using the hand collection method.Myrmica species were identified according to Seifert (2018) and Radchenko & Elmes (2010).Phengaris larvae were identified at the site using a 20× magnifier lens and determination key by Śliwińska et al. (2006) and afterward returned to the nests.

Ant species on the study sites
A total of 142 Myrmica nests were surveyed revealing presence of seven species at twelve sites hosting P. teleius and/or P. nausithous in the eastern Goričko NP: M. curvithorax Bondroit, 1920, M. gallienii Bondroit, 1920, M. rubra (Linnaeus, 1758), M. ruginodis Nylander, 1846, M. sabuleti, Meinert, 1861, M. scabrinodis Nylander, 1846 and M. schencki Viereck, 1903 (Tab. 2).The total number of Myrmica nests found on each locality varied from 4 at MOT 2 to 35 at KOB (Tab.3).Myrmica scabrinodis was the commonest species of the genus found on ten sites with 105 recorded nests.It was followed by M. rubra, found on eight sites (22 nests), and M. gallienii, found on four sites (6 nests) (Tab 3).On all sites where it occurred, M. scabrinodis had the highest proportion of the total nests found (Fig. 1).

Host ants
P. teleius larvae were found in 25 Myrmica ant nests and P. nausithous in 9 nests (Tab.3).Altogether, 56 larvae of P. teleius and 87 larvae of P. nausithous were found (Tab.3).P. nausithous larvae were found exclusively in M. rubra nests, while P. teleius larvae were recorded in ant nests of three Myrmica species: M. scabrinodis, M. rubra and M. gallienii.The highest proportion of all infested nests with P. teleius was in M. scabrinodis (64%), while the proportion of infestation with P. teleius per investigated ant nest and species was the highest in M. rubra.A different species of host ants were found on three locations.The co-occurrence of both butterfly species larvae in the same ant nest was observed in six ant nests, in all cases M. rubra was a host species.
In general, P. teleius was recorded in a larger number of ant nests compared to P. nausithous, while P. nausithous larvae were found in a larger number per ant nest.The median value of P. teleius larvae found per ant nest is 1, while the median value of P. nausithous larvae number found per ant nest is 3.In three M. rubra nests, larger numbers of larvae (more than 20) per nest were found.The largest number of larvae in a single nest was counted in M. rubra nest in the locality MOT2 -a total of 35 larvae, 11 larvae of P. teleius and 24 larvae of P. nausithous.In the locality NER2, 26 and 22 larvae of P. nausithous were found, respectively, accompanied by a single P. teleius larva in each nest.Altogether, more than half of all larvae of both species were found in the three M. rubra nests mentioned above.

Discussion
During our study, a total of seven species of Myrmica ants were recorded in meadows with P. teleius and/or P. nausithous at Goričko Nature Park, which is half of all Myrmica ant species known for Slovenia (Bračko 2007).
The fact that 19.7% of all Myrmica nests examined contained Phengaris larvae and most of these contained only a few larvae, follows the pattern of ant nests infestation on Phengaris sites across Europe (e.g.Witek et al. 2010).As ant nests vary greatly in their susceptibility to larval parasitism, typical Phengaris sites include many uninfested nests, several with moderate infestation, and a few with high infestation (Tartally et al. 2008).According to our results, we can consider M. rubra as the primary and most important ant host of P. nausithous at Goričko.A high specificity in relationship of P. nausithous with M. rubra has also been reported across Europe (Witek et al. 2008(Witek et al. , 2010)), including western Hungary, which is closest to our Goričko study region (Tartally & Varga 2005, Tartally et al. 2019).For P. teleius, M. rubra and M. scabrinodis can be considered as primary hosts at Goričko.P. teleius has the most diverse hosts among European Phengaris species and often uses the locally most abundant Myrmica species (e.g.Tartally & Varga 2008, Witek et al. 2010, Tartally et al. 2019).This is in line with our observations, as the two primary host Myrmica species were also the most abundant in our study area, and with observations at the Őrség National Park in Hungary (Tartally & Varga 2008).Among the other five Myrmica species recorded, we found only one larva of P. teleius in an ant nest of M. gallienii.Larvae of P. teleius were found in nests of M. gallienii also in Poland (Stankiewicz & Sielezniew 2002).
NATURA SLOVENIAE 23(1): 21-33 Although the number of M. scabrinodis nests discovered was much higher than that of M. rubra (105 vs. 22), the percentage of nests parasitized by Phengaris was higher in M. rubra (41% vs. 24%), as was the total number of larvae detected (111 in M. rubra vs. 31 in M. scabrinodis).Three nests of M. rubra contained more than 20 Phengaris larvae.Moreover, co-occurrence of both Phengaris species larvae was detected exclusively in M. rubra nests.We found six cases of co-occurrence out of total 8 or 9 nests found infected by P. teleius or P. nausithous, respectively.The large number of larvae and co-occurrence of the two Phengaris species in M. rubra nests was previously reported by Tartally & Varga from western Hungary (2005).In their study, they found a M. rubra colony with 36 larvae of both Phengaris species, which is comparable to our colony from MOT2 (35 larvae).Since M. rubra forms the most populous colonies of all Central European Myrmica species (Seifert 2018), the highest numbers of parasitizing larvae in their colonies is not surprising.Such large colonies have a higher carrying capacity to host and survive a large number of Phengaris larvae.Although M. scabrinodis nests were parasitized in lower percentage and contained lower total number of larvae, this species still represents an important host for Phengaris larvae at Goričko, mostly as it is the most frequent ant species in Phengaris sites.It is particularly important host for P. teleius, which as mentioned above, often uses locally most abundant Myrmica species.

Conservation implications
The nest of Myrmica rubra hosting the largest number of Phengaris larvae was found at Motvarjevci (MOT2), along an abandoned road verge.This finding indicates and confirms the importance of road verges and grassland margins as larval habitat for P. teleius and, in particular, for P. nausithous (e.g.Wynhoff et al. 2011).Myrmica rubra, as the main host ant of P. nausithous, is known to be less thermophilic than M. scabrinodis, preferring sheltered and more overgrown habitat for its nests (Wynhoff et al. 2011).According to Seifert (2018), M. rubra gains optimum in mesophilic to moist conditions, and is absent only from most xerothermic and sparsely-vegetated habitats.It is often the only ant in very high-grassy lowland meadows and tall herb communities.Therefore, it is important to maintain parts of more dense vegetation with the larval host plant along meadow margins, which could be beneficial for M. rubra and both target butterfly species (see also Tartally & Varga 2005).Myrmica scabrinodis is a hygrophilous to moderately thermophilic species and reaches highest densities in meadows or ecotones with moderate height of grasses (Seifert 2018).Each host ant species has its own niche and, according to current knowledge, the two Myrmica species require different mowing regimes (Wynhoff et al. 2011), which should be taken into consideration in practical management, especially on smaller, isolated sites where both ant species occur together.
Myrmica scabrinodis seems to benefit from mowing in early June, while M. rubra benefits from mowing in late autumn which should be combined with some annually unmown parts/verges that are alternated among years to prevent overgrowing especially by invasive plants (Wynhoff et al. 2011).The general recommendation for habitat management should thus consider mosaic mowing offering many different microhabitat opportunities.However, at least in the last decade, the habitat and populations of both species in NE Slovenia have been declining (Zakšek et al. 2020), so moving towards more active conservation of both species is essential for their long term survival.With basic knowledge on their larval ecology in hand, potential restoration of new habitats or focusing on a conservation of the most important parts of the larval habitat of both species will enable a first step to halt their decline.It should be emphasised that there is no active habitat restoration possible without knowledge and monitoring of ant communities (e.g.Wynhoff et al. 2017).

Figure 1 .
Figure 1.Map of Goričko Nature Park (dark line represents its southern border) showing the position of the sampling sites of our study in 2011 and 2012.The proportion of Myrmica species ant nests at each sampling site is shown with pie charts.The size of the circle corresponds to the number of examined Myrmica ant nests (see Tab. 3 for details).

Table 2 .
All Myrmica ant species recorded on selected sampling sites at Goričko Nature Park during this study in years 2011 and 2012.For codes of sampling sites see Tab. 1.In species marked with asterisk only individual ant workers were recorded on the study site (no nests were found and excavated).