An attempt of interspecies mating between Phengaris nausithous and P. teleius (Lepidoptera, Lycaenidae)

. Interspecies mating is rarely observed even in the wild. Such a case in Lycaenidae is reported and photographically documented for the species pairing Phengaris teleius (Bergsträsser, 1779) and P. nausithous (Bergsträsser, 1779), which was observed on 19 July 2016 in northern Bohemia near the village of Hynčice, Czech Republic. Possible causes of this phenomenon are discussed.


Introduction
From time to time, a mating between different species of butterflies is observed.The observation of such behaviour or the occurrence of hybrids in the wild is more common in some groups of butterflies than in others.More such information from the wild is available for Papilionidae (genera Ornithoptera Boisduval, 1832, Papilio Linnaeus, 1758, Troides Hübner, 1819) (Sands andSawyer 1977, Sperling 2011 etc.), Pieridae (genus Colias Fabricius, 1807) (for instance Hovanitz 1949;Taylor 1972), Nymphalidae (genera Danaus Kluk, 1802, Erebia Dalman, 1816, Heliconius Kluk, 1780, Limenitis Fabricius, 1807) (Platt et al. 1978;Baidya et al. 2018 etc.), etc.However, relatively few observations are available for the family Lycaenidae (for instance Downey 1962;Smetacek and Bhakare 2010).The genus Phengaris Doherty, 1891 belonging to this family is the subject of very intensive research due to its complex ecological relationship with ants, yet we are not aware of any similar behaviour being recorded in closely related species of this genus.In the following text, we provide first information and photographic documentation about the interspecific copulation of the sympatric species Phengaris teleius (Bergsträsser, 1799) and P. nausithous (Bergsträsser, 1799), which has been observed in the wild.

Material and methods
Survey of species of the genus Phengaris is mandatory for the conservation of these butterflies protected by the EU Habitat Directive.Our survey of Large Blues of the genus Phengaris took place in the area of the Protected Landscape Area Broumovsko (Northern Bohemia, Czech Republic) in 13 habitat patches in the vicinity of the villages of Hynčice, Ruprechtice and Vižňov which contained the flowering food plant Sanguisorba officinalis L. from 4 July to 5 August, 2016.The location of the patches is shown in Fig. 1.We monitored the species in this northwestern part of Czech Republic to estimate their density using the Mark-Release-Recapture method, calculated by program MARK 8.1 (White and Burnham 1999) with modifications according to Nowicki et al. (2005).Colonies of Phengaris butterflies here form a single metapopulation structure with dispersal occurring between patches.The total number of individuals of Phengaris teleius for this metapopulation was estimated by the Cormack-Jolly-Seber method (see Schwarz and Arnason 1996;Schwarz and Seber 1999) to be 802 individuals while 1487 individuals were estimated for P. nausithous.
We randomly took photographs of individuals throughout the survey, and the sequence of images from the interspecific mating of the aforementioned taxa was revealed later after a thorough review of the photos taken at site Hynčice 2 near the river Stěnava (GPS coordinates roughly in the middle of the territory: 50°36'58"N, 16°18'35"E, elevation about 400 m).The date of the pictures is July 19, 2016, time 13:25 CEST, by one of us (VV).

Results
After the survey in Broumovsko had finished, we encountered an unusual attempt at interspecific mating of a female P. nausithous and male P. teleius during analyses of photo-documentation (Figs 2, 3).At first, we thought it was an interesting photo of both species on a single inflorescence of a food plant, but on closer inspection, we found that the butterflies were attempting to copulate with a visible connection of their abdomens.We thus do not know the exact period of time that they remained in copula.

Discussion
Despite these two species being closely related (see Fric et al. 2007) and that they utilise the same host plant (in Europe exclusively Sanguisorba officinalis), we are not aware of any other similar records of these species mating.
Such intraspecific mating is a prerequisite for hybridization (Descimon and Mallet 2009); however, natural hybridization in Lepidoptera is rare.A famous example is "Erebia serotina Descimon & de Lesse, 1953", which is a natural hybrid of males of E. pronoe (Esper ,1780) and females of E. epiphron (Knoch, 1783) (Michel et al. 2013).The majority of known hybrids are not natural as they are produced in captivity (Wang and Dong 2001), especially between various species of the genus Papilio (for instance Clarke and Sheppard 1955), where even after several generations the hybrids are still fertile (Blanchard and Descimon 1988).Using molecular methods, it has been repeatedly shown that the reproductive barriers in Lepidoptera usually work well (Porter 1989;Elzinga et al. 2014).When natural interspecific mating does occur, it has been found that the willingness is strongly asymmetric between species.For instance, Deering and Scriber (2002) observed high preferences of males of Papilio canadensis (Rothschild & Jordan, 1906) for females of Papilio glaucus Linnaeus, 1758.
Observations of interspecific mating between different members of the family Lycaenidae are reported by Downey (1962) between two species of Callophrys Billberg, 1820 and between two species of Lycaena Fabricius, 1807.Further lycaenid examples are from Smetacek and Bhakare (2010), who reported mating between Castalius rosimon (Fabricius, 1775) and Talicada nyseus (Guerin, 1843) and between Chilades parrhasius (Fabricius 1793) and Zizina otis (Fabricius, 1787).For the genus Phengaris Doherty, 1891, however, we have not found any record of such an observation or information about the presence of hybrids anywhere, and therefore, due to the very frequent sympatry and syntopy of both species, we consider this very interesting.
What are the reasons for inter-specific mating, or even hybridisation?In general, it results from an imperfect reproduction barrier.Such barriers in Lepidoptera usually consist of several levels, many species combining olfactory recognition (Li et al. 2017) with specific behaviour and pheromones, and other signals (Phelan and Baker 1990).However, mating pheromones have frequently similar composition (Ganyard andBrady 1971, 1972) and their content vary not only between species but also within (De Pasqual et al. 2021).Also, the lock-and-key mechanism in lepidopteran genitalia does not always work (see Mikkola 1992Mikkola , 2008)).
Hybridization can have negative impacts when some species newly colonise or are introduced into a landscape with closely related species (Cianchi et al. 2003), but there are cases where it may trigger new speciation (Mavarez et al. 2006;Capblancq et al. 2015).In the case of our observation, a union of butterflies was identified in the photographs, but it is far from certain whether the copulation was successful and whether additional isolation mechanisms prevented the possibility of hybrid offspring.
We cannot be sure why the observed attempted pairing of Phengaris butterflies occurred.As the species in Phengaris vary in number of androconia (see for instance Sibatani et al. 1994), a possible explanation is the confusion of individuals by mixing the pheromones of both species when there is a particularly high density of individuals at the observation point.Alternatively, a suitable conspecific partner might be harder to find if a pheromone is similar.However, neither reason appears particularly likely given that the population density of either species at the observation site was not higher than average, but neither was it at the limit of observability.

Figure 1 .
Figure 1.Distribution of patches where the species Phengaris teleius and P. nausithous were marked in Broumovsko in northern Bohemia of the Czech Republic in 2016.The place of observation is patch H2.Drawn in publicly available document from www.mapy.cz.

Figure 2 .
Figure 2. Individuals of Phengaris teleius and P. nausithous on a flower of either's food plant, Sanguisorba officinalis, while attempting to mate with each other.

Figure 3 .
Figure 3.The mutual connection of individuals of different species in this poorer quality image is evident.