The unexpected finding of Parapholidoptera castaneoviridis in south-eastern Romania (Insecta, Orthoptera, Tettigoniidae)

Abstract The Balkano-Anatolian genus Parapholidoptera comprises 21 species and the westernmost one, Parapholidoptera castaneoviridis, previously recognized to occur in western Turkey, north-eastern Greece and south-eastern Bulgaria is recorded for the first time from south-eastern Romania, almost 300 km away from the closest known locality. Illustrations and measurements of morphological characters are given and the male calling song from this new, northernmost population is described.


Introduction
With 21 described species, the genus Parapholidoptera Mařan is distributed from the Caucasus in the east, to the Balkans in the west, across the Anatolian Plateau and reaching Israel and Jordan in the south (Çiplak 2000, Katbeh Bader and Massa 2001, Çiplak 2004, Eades et al. 2016. Within the genus, Çiplak (2000) defines two clades: P. castaneoviridis group with a south-western distribution and P. distincta group, occurring in north-eastern Anatolia and the Caucasus. It seems that the Anatolian mountain ranges played an important role in the speciation and distribution of the genus (Çiplak 2004), 16 species being endemic to various localities and mountains in Turkey, Georgia, Armenia and Jordan.
Parapholidoptera castaneoviridis (Brunner von Wattenwyl, 1882) is one of the most widely distributed species of this genus, known to occur from the north Aegean region of Turkey, Samothrace Island, Macedonia and Thrace regions in Greece, Strandzha Mountains and the territory eastwards from Eastern Rhodopes in Bulgaria (Ingrisch and Pavićević 1985, Çiplak 2000, Popov and Chobanov 2004. The species is characterized by a cylindrical pronotum, extended metazona, male with last tergum lightcolored, with two small, straight processes and females with a long, straight ovipositor and subgenital plate narrowly rounded, with a deep groove (Çiplak 2000).
The finding of Parapholidoptera castaneoviridis in Romania, the northernmost known location in the distribution of this species, is discussed.

Material and methods
The first specimens of Parapholidoptera castaneoviridis were found while actively searching for orthopterans in the forest clearings near Ciucurova village (Tulcea county, south-eastern Romania), in the summer of 2016. The bush-crickets were collected at daytime, in xerophytic Quercus forest clearings with scrub, at altitudes of approximately 200 m ASL. Individuals were identified according to genital morphology and calling song oscillographic structure. Photos were taken with a Canon EOS 6D DSLR camera and a Canon EF 180 mm f3.5 macro lens. Acoustic recordings were made at night, in laboratory conditions with an Edirol R-09HR digital recorder (sampling rate 96 kHz, 24-bit amplitude resolution, microphone response frequency up to 45 kHz) and a Knowles electret condenser microphone connected to a PC through a TransitUSB external sound card (48 kHz, 16-bit) and the sound analysis was run with Audacity 2.1.2 and Batsound 4 software. As song element durations are usually temperature dependent, the ambient air temperature was measured during these recordings.
The bioacoustic terminology is adopted mainly from Ragge and Reynolds (1998): calling song -song produced by an isolated male; syllable -the song produced by one to-and-fro movement of the tegmina; hemisyllable -the song produced by one unidirectional movement of the tegmina (opening or closing); echeme -a first order assemblage of syllables; echeme sequence -a first order assemblage of echemes. The following oscillographic characters were measured in the songs of three males from Ciucurova and compared to previously known results (Heller 1988, Sevgili et al. 2011: duration of an echeme (DE) -the time elapsed from the beginning of first syllable to the end of last syllable of an echeme; duration of a syllable (DS) -the time elapsed from the beginning of first impulse to the end of last impulse of a syllable; duration of an opening hemisyllable (DOH) -the time elapsed from the beginning of first impulse to the end of last impulse of an opening hemisyllable; duration of a closing hemisyllable (DCH) -the time elapsed from the beginning of first impulse to the end of last impulse of a closing hemisyllable; echeme repetition period (ERP) -the time elapsed from the beginning of an echeme to the beginning of the next echeme (Fig. 1); echeme recurrence rate -density of echemes repetition in a specific time period.

Calling song
Typically, the males produce their calling song in the evening and at night. In the recorded males, the calling song consists of a long series of echemes, each echeme lasting for 426-583 ms (mean ± SD 503.87 ± 46.81 ms) and containing 8-10 syllables (mean ± SD 8.49 ± 0.52) (Fig. 4). The recurrence rate is fairly 20-30 / minute, the echeme repetition period being 2122-2760 ms (mean ± SD 2408.28 ± 197.71 ms). Syllables are quite short (39-54 ms, mean ± SD 47.28 ± 4.1 ms) and consist of two distinct parts: a shorter, lower amplitude opening hemisyllable (15-20 ms, mean ± SD 17.55 ± 1.64 ms) and a longer, higher amplitude closing hemisyllable (20-39 ms, mean ± SD 29.73 ± 4.49 ms). Acoustic signal amplitude modulation pattern is increasing and de-    creasing in both the opening and closing hemisyllables. The detailed descriptive statistics of the song characters are presented in Table 1. The calling song has the dominant frequency components between 7 kHz and up to more than 45 kHz (45 kHz being the upper limit of microphone frequency response in our recordings), with a main peak at about 12 kHz in both opening and closing hemisyllables (Fig. 5).

Discussion
Although recently Parapholidoptera was divided in two groups relying exclusively on morphological traits (Çiplak 2000), the genus is characterized by a more or less uniform calling song, especially among the Parapholidoptera castaneoviridis group -multisyllabic echemes separated by silent intervals lasting for several seconds, with differences in the syllable number per echeme (Heller 2006). The males discovered in Romania are characterized by a homogeneous song pattern with the specimens from Strandzha Mountains and Turkey: variable echemes consisting of 5-8 (Sevgili et al. 2011), 10-12 or 11-13 syllables (Heller 1988(Heller , 2006, lasting for 268-420 ms (Sevgili et al. 2011) or more than 500 ms (see oscillogram in Heller 1988) and repeated at 1.5-3 s (Sevgili et al. 2011) or up to 2.78-4.20 s (Heller 1988) are produced by individuals from the core area of species distribution (south-eastern Balkans and western Asia Minor). Having 8-10 syllables/echeme and lasting for 426-583 ms, with a repetition period of 2.1-2.7 s, the song of Romanian specimens fits well within this description. Moreover, the morphological structures and measurements of specimens from the newly found population (Fig. 6) are very similar with the ones described by Çiplak (2000) (for a complete numeric comparison, see Table 2).   Parapholidoptera castaneoviridis is a micropterous insect with limited dispersal abilities. The newly discovered population from the northern area of Dobrogea most likely persisted as relict from a previously wider distribution when the forest habitat was continuous, rather than being the result of a recent expansion. This hypothesis may be supported by the isolated former findings of this species in northern Bulgaria, while recently it has not been found in this area, regardless of the significant collecting efforts that have been made in the northeastern part of this country. Currently, the species is common in south-eastern Bulgaria (D. Chobanov, pers. obs.; fig. 3), where it can be found both in natural and agricultural areas, mostly tolerating dry warm microclimate in scrub-grass associations or thermophilic sparse forests.
Numerous recent studies targeting the Balkan orthopteran fauna confirmed the need of exploring unknown territories for obtaining a complete picture of the species geographic ranges (eg. Çiplak et al. 2007, Chobanov and Heller 2010, Chobanov et al. 2013, Chobanov 2014, Kaya et al. 2015, Iorgu et al. 2016. The finding of the only Balkan Parapholidoptera species in Romania is just another example of how exhaustive field work in a previously poorly studied area could produce unexpected results, providing a better understanding of the currently interrupted distribution area of a formerly wider spread species.