Re-evaluation of the genus Phonochorion ( Orthoptera : Tettigoniidae : Phaneropterinae )

Phonochorion Uvarov (Orthoptera: Tettigoniidae: Phaneropterinae) is a little known genus consisting of three species: Ph. satunini, Ph. artvinensis and Ph. uvarovi. The objective of this study is to conduct a thorough distributional, taxonomic and systematic revision of the genus Phonochorion using both bioacustic and external morphological characters. Field surveys indicate that the genus is distributed from the Trabzon region of Turkey to the Khulo province of Georgia however the exact limit of the eastern distribution of the genus remains unknown. Phonochorion species occur only on the northern slopes of the East Black Sea and Lesser Caucasus Mountains. The Coruh Valley, which seprates the East Black Sea and Lesser Caucasus Mountain ranges, seems to be an effective physical and climatic barrier and determines the distribution of these species. Ph. uvarovi can clearly be distinguished from Ph. satunini and Ph. artvinensis by the calling songs of males and external morphological characters. Ph. artvinensis and Ph. satunini differ in several taxonomic characters but the males have virtually identical calling songs. From a character evolution perspective, although geographically more distant, Ph. satunini is more closely related to Ph. uvarovi than Ph. artvinensis. Morphological similarities in several characters indicate Phonochorion to be most closely related to Polysarcus zacharovi and the Poecilimon heroicus-group. Within the genus Phonochorion, song structure and morphological characters clearly indicate Ph. uvarovi to be the basal taxon. 631 * Corresponding author; e-mail: iksaglam@hacettepe.edu.tr MATERIAL AND METHODS


Field studies
Field surveys were undertaken in the years 2003-2008, during the months of June, July and August, which are the months when these species are active.The total area sampled was 23,342 km 2 and included parts of the Trabzon, Rize and Artvin provinces of Turkey and the Khulo province of Georgia.In order to determine the presence or absence of species belonging to this genus, approximately 300 patches of characteristic habitat for this genus were visited in the study area.The presence of Phonochorion spp. was determined by listening for their characteristic calling songs and finding adults and nymphs.When no specimens were observed and no calling songs heard, the localities where rechecked the following year in order to prevent any bias that might arise due to seasonal changes in population abundance.

Species delimitation: Taxonomic key and song analysis
Species were delimitated using both morphological characters and recordings of males stridulating.A key to species is presented based on external morphological characters, which are illustrated in Figs 2-10.All characters were studied under a Leica MZ-7.5 stereoscopic zoom dissection microscope and photographed using a DC-300 digital camera system attached to the microscope.Sounds were recorded in the field using a portable stereo audio recorder (TASCAM HD P2) fitted with a bat detector (TD 15-2 with HF-output via Mini-XLR, Laartech) or a M Audio Transit (mobile 24-bit/96kHz USB audio upgrade) and microphone (kondensator ansteckmicrophone, 30-30000 Hz, distance to microphone about 10-20 cm) attached to a notebook computer.Sounds were digitized at a sampling rate of 96 KHz and 24 bit resolution with Adobe Audition and Spectraplus software.
Oscillograms were generated using the above software after filtering out background noise.The following qualitative and quantitative parameters were analyzed: duration of the calling song, micro and macro syllable duration, syllable period, syllable group duration (echeme), number of syllables in each group, number of impulses in each syllable and amplitude modulation of syllables.Analyses were carried on a total of 32 calls recorded from 10 different populations of each of the three species.Terminology used in song descriptions are as follows (Ragge & Reynolds, 1998); Calling song: song produced by an isolated male; Syllable: the sound produced by one complete up (opening) and down (closing) stroke of the wing; Syllable period: time between the start of two consecutive syllables; Echeme: a first-order assemblage of syllables; Impulse: a simple, undivided, transient train of sound waves.

Defining features
In males the pronotum distinctly rises vertically and the metazona widens anteriorly; in males the tegmina are well developed and highly chitinized; males have distinct and large dorsal glands on the first tergum; in males the subgenital plate is long and curls vertically upwards; in both sexes there is a dorsal cavity on the fastigium; in males the coloration is aposematic.

Distribution and habitat characteristics
Results of the field surveys indicate that the genus is distributed from the Ya murdere in the Gümü hane province of Turkey to the Djvari Mindori plateau in the Khulo province of Georgia.The distributional range of the genus and species distributions are depicted in Fig. 1.Within this range species are found only on the northern slopes of the East Black Sea and Lesser Caucasus Mountains associated with local stands of various herbaceous plants and shrubs (mostly Urtica dioca, Sambucus nigra and Rumex tuberosum) and pasture vegetation forming discrete local populations in areas with a rainy, humid and cold climate.From the Ya murdere to the Fırtına Valley species are patchily distributed between 1400-2600 m.However, in the Fırtına Valley species can be found in every suitable habitat patch (from shrubs to meadows) nearly forming a continuous population from 1000 to 2600 m.From east of the Fırtına Valley to Georgia, populations of Phonochorion species occur locally in discrete patches usually at altitudes above 1800 m and exclusively on Rumex tuberosum.
The evaluation of the distribution of individual species indicates that the ranges of Ph. uvarovi and Ph.artvinensis overlap from Ya murdere to Arde en in Rize (Fig. 1).The distributional range of Ph. uvarovi ends at Arde en, but that of Ph. artvinensis extends all the way to Meydancık in Artvin, near the Georgian border.Where the ranges of Ph. uvarovi and Ph.artvinensis overlap their populations do not mix as Ph.uvarovi is found mainly in habitats at high altitudes (1800-2500 m), whereas Ph. artvinensis occurs mainly at low altitudes (1200-1800 m).The distribution of Ph. satunini seems to be limited to the Khulo province of Georgia and does not extend into Turkey (Fig. 1).However the eastern limit of the distribution of this species is unknown.

Phonochorion uvarovi
Song.Songs were recorded at night at various temperatures (22, 25, 27 and 35°C).However the species is also bioacoustically active during the day.Sounds produced by males of Ph. uvarovi consist of orderly permutations of a single powerful crescendo type syllable (Fig. 11A-C).Duration of the crescendo type syllables produced by males is variable with a mean of 0.353 ms ± 0.133 SD (n = 83, min-max.0.076-0.639ms) and are repeated at 1.021 ms ± 0.442 SD (n = 81, min-max: 0.426-2.925ms).On average the syllables consist of 111 impulses (n = 83, min-max.33-172).Some individuals produce syllables that are a crescendo from start to finish, while in those produced by others only the last 16-68 (n = 46, mean 35.21 ± 14 SD) impulses are powerful crescendos.Uvarov, 1916 References.Uvarov (1916): 48-49 (description); Kartsivadze (1949): 144 (distribution); Bey-Bienko (1954): 364 (key), 365-366 (re-description).Type locality.Georgia, Southwestern Georgia, Adzhar Range.Defining features.Body size is relatively small; when the first tergum of the male is viewed from above the fold between the metanotal glands rises upwards forming a curled bracket or brace shape (Fig. 4D); in males the subgenital plate is red-lilac, and narrows gradually in the middle and neck narrows down towards apex (Fig. 5D); body coloring of males consists mainly of a pattern of black and lilac.

Phonochorion satunini
Song.Songs were recorded at high temperatures (35°C).Song consists of major (first group) and minor (second group) syllables (Fig. 12A-B).Mean duration of the first group is 9.63 s ± 3.24 SD, (n = 49) and is made up of powerful crescendo type major syllables (Fig. 12C-D).Number of major syllables in the fist group ranges from 7-23.Mean duration and period of major syllables are 0.375 s ± 0.082 and 1.050 s ± 0.114 SD, respectively.Number of impulses within major syllables ranges from 102-153 (mean: 135, n = 18).Mean duration of the second group is 5.33 ± 1.62 SD (n = 16) and consists of minor syllables.Number of minor syllables in the second group ranges from 7-23, and 3-6 minor syllables are produced per second.Mean duration and period of minor syllables are 0.114 s ± 0.027 and 0.367 s ± 0.030 SD (n = 35), respectively.Defining features.Body size is relatively large; when the fastigium is viewed from above, the dorsal cavity is concave at the sides, shallow and mainly located at the base (Fig. 7B and C); in females viewed from above the tegmina clearly protrudes from under the pronotum (Fig. 8B and C); in males when the first tergum is viewed from above the metanotal gland distinctly folds and rises upwards forming a curved bracket or brace shape (Fig. 4B and C); in males the subgenital plate is red-lilac, narrows gradually at the middle, the neck is long (3.00-4.30mm) and narrows down towards apex (Fig. 5B) except in individuals from populations east of the Canyon, in which the neck is relatively short (2.10-3.00mm) (Fig. 5C); in males the cerci are relatively thick (0.80-1.10 mm) and long (3.50-4.45mm) (Fig. 9B and C); in females the protrusion at the anterior end of the subgenital plate is missing or very blunt (Fig. 6B and C); body coloring of males is mainly black and lilac.

Phonochorion artvinensis
Song.Songs were recorded at night at 24-27°C.The song pattern of Ph. artvinensis is similar to that of Ph. satunini and composed of two groups of echmes with the first consisting of major syllables and a second of minor syllables (Fig. 13A and B).Mean duration of the first group is 5.55 s ± 1.44 SD (n = 25, min-max: 3.05-8.47s) and is composed of powerful crescendo type major syllables.The main group is repeated at intervals of on average 41.30 ± 10.14 SD seconds (min-max: 20.73-58.17s, n = 20).Number of major syllables within the fist group ranges from 3-9.Mean duration and period of major syllables are 0.358 s ± 0.086 SD and 1.27 s ± 0.15 SD (n = 99), respectively.Number of impulses within the major syllables ranges from 57-135 (mean: 126.5 ± 15.91 SD, n = 16).Impulses within major syllables start at low intensities and finish strongly.From time to time minor syllables similar to the ones recorded from the second group can be produced within the major syllables (Fig. 13C and D).On average these minor syllables are produced 0.280 ± 0.038 s after the main syllables.This first group is followed by a second group composed of one section of crescendo type or two of minor syllables (Fig. 13A and B).If there are two types of syllables in the second group the first is a crescendo and the second a decrescendo.However, from time to time individuals produce only the second group and remain silent during the interval (Fig. 14A-C).Mean duration of the second group is 20.85 s ± 2.34 SD, (n = 17).Numbers of minor syllables in the second group ranges from 29-184 and 2-5 minor syllables are produced per second.Mean duration and period of minor syllables are 0.057 ± 0.007 SD and 0.309 s ± 0.089 SD, respectively.Numbers of pulses in minor syllables are quite variable ranging from 7-46 with a mean of 23.82 ± 8.03 SD.

Key to species
Based on the above descriptions and characters given in Table 1 the following key to species was constructed: 1 In both sexes the pronotum is smooth with no distinct tubercles on the sides (Figs 2B-D, 3B-D); when the first tergum is viewed from above (especially in males) the metanotal gland distinctly folds and rises upwards forming a curved bracket or brace shape (Fig. 4B-D); in males the subgenital plate is red-lilac, narrowing in the middle and neck is gradual (Fig. 5B-D); body coloring of males is mainly black and lilac. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 -In both sexes the pronotum has distinct tubercles on the sides (especially on the mesozone) (Fig. 2A and 3A); when the first tergum is viewed from above the region near the metanotal gland distinctly folds and rises up forming an inverted v shape (Fig. 4A); in males the base of the subgenital plate is quite wide and the narrowing in the middle is acute, neck is relatively short and stretches parallel towards apex (1.60-3.00mm) (Fig. 5A); in females there is always a clear projection on the middle section of the subgenital plate (Fig. 6A); ovipositor length can range between 8.50-13.40mm (Fig. 10A); body coloring of males is mainly black and yellow. .East Anatolian Mountains resulting in heavy orographic rain fall (Akçar & Schluchter, 2005;Atalay, 2006).In addition, the high altitude of these mountain ranges also creates a natural barrier between coastal and internal areas resulting in a negative precipitation gradient towards inner Anatolia (Akçar & Schluchter, 2005).In the East Black Sea region a dramatic decrease in rainfall occurs as one moves 130 km inland from Rize (annual mean 2200 mm) to Erzurum (annual mean 400 mm) (Akçar & Schluchter, 2005).Therefore, due to the rain shadow and intense solar radiation (Atalay, 2006) the south facing slopes of these mountains are dominated by dry forest and scrub vegetation, which is an unsuitable habitat for Phonochorion species.This would also explain why Ph. artvinensis was not found at its type locality: the Yalnızçam Mountains and the Opiza and Porta Monastries.These localities lie well inland of the North East Anatolian Mountains where both the vegetation and climate are unsuitable for species of this genus.
Another important geographic feature affecting the distribution of species in this region would seem to be the Çoruh Valley.Like most orogenic mountains within Anatolia the North East Anatolian Mountains are dissected by rivers as a result of antecendent and superimposition events (Atalay, 2006).The largest of these river systems within the region is the Çoruh River, which by cutting through the North East Anatolian Mountains in a northsouth direction creates a deep valley separating the Lesser Caucausus Mountains from the East Black Sea Mountains.The altitudinal difference between the river bed and the upper parts of the mountains is nearly 3000 m in the Çoruh Valley (Atalay, 2006).This deep valley has important implications for both the micro and macro climatic characteristics of the region.The north facing slopes of the East Black Sea Mountains are covered by humid forests and montane habitats as a result of abundant rainfall and reduced radiation (Atalay, 2006).On the other hand along the River Çoruh corridor humid-640 semiarid conditions and Mediterranean maquis vegetation prevail as a result of the absence of moisture and increased solar radiation (Atalay, 2006;Zengin et al., 2010).
Therefore, the Çoruh Valley would seem to be an important physical and climatic barrier to the dispersal and spread of species.If this is the case, then this valley effectively limits the westward distribution of Ph. artvinensis to east of the Çoruh Valley and its eastward distribution to west of the Çoruh Valley.Hence, the Çoruh Valley effectively separates the distribution of Ph. satunini and Ph.artvinensis in the Artvin region from that of Ph. uvarovi and Ph.artvinensis in the Rize and Trabzon regions.

Bioacustics
The Phaneropterinae Phonochorion spp., Polysarcus spp.and Poecilimon brunneri species-groups produce the most complicated acoustic signals in terms of the temporal structure of their songs (Korsunovskaya, 2008).The songs of these bush-crickets usually contain several distinct components to which the females respond only to one (Korsunovskaya, 2008).Oscillograms of the songs of two of the three species (Ph.uvarovi and Ph.artvinensis) were published earlier by Heller (1990).However Heller's (1990) recordings of Ph. satunini are of Ph. artvinensis.Based on similarities in the acoustic signatures (sequential repeats of structurally similar, simple, crescendo type syllables) and wing stroke patterns Heller (1990) classified Ph. uvarovi song as similar to that of the ancestral Barbitistini tribe.The Barbitistini tribe can be divided into two distinct phylogenetic groups depending on duration of the male call and male-female duet patterns: a clade where couples perform duets and a clade where male-female duets are absent (Heller, 1990;Bailey & Hammond, 2003).According to this classification it is hypothesized that short calling songs of males and short responses by females is the ancestral state and that species with long calling songs have evolved from this ancestral state.In this study the songs of females were not recorded.However, the structure of the wings of the females of Phonochorion indicates they can produce sounds.If as stated by Heller (1990) Phonochorion species performs duets, we would expect females to give short responses to male calling songs, which would place Phonochorion in the ancestral clade of the Barbitistini 641 tribe.Short responses of this kind are also quite common in closely related groups, such as Poecilimon and Isophya (Heller, 1990;Bailey & Hammond, 2003;H. Sevgili, unpubl. data), which gives some support to this conclusion.
In the present study male calling songs of Ph. satunini, Ph. artvinensis and Ph.uvarovi were recorded at many different localities.When the calling songs of males of the three species are compared, those of Ph. artvinensis and Ph.satunini are clearly different from that of Ph. uvarovi.In all three species the main part of the calling song is composed of a powerful crescendo type syllable repeated at regular intervals.However in contrast to Ph. uvarovi, in Ph. artvinensis and Ph.satunini this main group is interrupted by a second group composed of rapid repeats of a minor two-piece syllable.In spite of this clear difference, divergence in calling songs between species does not seem to be totally complete as both Ph.artvinensis and Ph.satunini individuals may not produce this second group but remain silent during this period.In this form the calling songs produced by Ph. artvinensis and Ph.satunini are virtually identical to that of Ph. uvarovi.Therefore, based on acoustic data Ph.uvarovi has a simple song and seems to be the ancestral species, and Ph.artvinensis and Ph.satunini have complex songs and have diverged and evolved from this ancestral state.
Although calling songs enable us to discriminate Ph. uvarovi from the other two species the same cannot be said for discriminating between Ph. artvinensis and Ph.satunini.Songs produced by Ph. satunini and Ph.artvinensis are very similar with no significant differences between the structure of their songs.Although Ph. satunini and Ph.artvinensis show some differences in song structure in relation to duration and number of syllables these differences could easily be attributed to differences in the temperature at which they were recorded.Therefore, it is not possible to clearly differentiate between the two species using bioacoustic data.

External morphology
Although it is not possible to discriminate between Ph. artvinensis and Ph.satunini using bioacoustic data all three species are easily distinguishable using external morphological characters.Ph. satunini and Ph.artvinensis differ from Ph. uvarovi in the structure of its pronotum and metanotal gland, and in the colour and shape of the male subgenital plates.From a character evolution perspective, although geographically more distant, Ph. satunini is more closely related to Ph. uvarovi than Ph.artvinensis since the female tegmen, female subgenital plate, fastigium, male cerci and female ovipositor in Ph. satunini are identical with those in Ph. uvarovi.Retention of these characters in Ph. satunini also facilitates the differentiation between Ph. satunini and Ph.artvinensis as Ph.artvinensis differs in all the above characters.
Ph. artvinensis also shows substantial within species variation in the neck of male subgenital plates.The Ph. artvinensis populations east of the Çoruh Valley do not differ in this character from either Ph.satunini or Ph.uvarovi, but in the populations west of the Çoruh Valley the neck is very elongated.The male subgenital plate has proved a very useful character for delimitating Barbitistine species (e. g.Heller et al., 2004;Sevgili et al., 2006) and given its importance in mating and spermatophore transfer (Helversen & Helversen, 1991); it is likely to be species specific.This along with the allopatric distribution of eastern and western Ph.artvinensis populations (in relation to the Çoruh Valley) might warrant the splitting of Ph. artvinensis into two sub species.However, since eastern and western populations of Ph. artvinensis do not differ in any of the remaining diagnostic characters the east and west populations of Ph. artvinensis are not classified here as two separate sub species.In addition there are numerous empirical studies showing the widespread occurrence of intra-specific variation in genitalia across insect taxa (Garnier et al., 2005;Mutanen et al., 2006;Polihronakis, 2006;Song & Wenzel, 2008).There- fore, we believe great care should be taken when assigning sub species status based on a single character.

Systematics
Based on morphological characteristics and bioacoustics Phonochorion spp.can be placed in the tribe Barbitistini, a group of short winged species within the subfamily Phaneropterinae (Tettigoniidae: Orthoptera) (Heller, 1990).However, its relation to other Barbitistine species is unclear.Morphologically Phonochorion spp.are most closely related to Polysarcus zacharovi and to members of the Poecilimon heroicus-group (Poecilimon heroicus Stshelkanovtzev, 1911;Poecilimon bifenestratus Miram, 1929;Poecilimon tricuspis Miram, 1926 andPoecilimon tschorochensis Adelung, 1907).Both P. zacharovi and the Poecilimon heroicus-group occur in north east Turkey and the latter is also widespread throughout the larger Caucasus region (Heller et al., 2006;Salman, 1978).They share the following features: general form of the pronotum (relatively wide, distinct rise in the metazona), relatively large tegmina, the presence of dorsal glands in males and the long upward curling subgenital plate in males.
Acoustically Phonochorion spp.and P. zacharovi are quite distinct from the Poecilimon heroicus-group.Acoustic signals of Phonochorion spp.and P. zacharovi are very complicated in their temporal structure (Korsunovskaya, 2008) and both groups produce a continuous song (Heller, 1990;Heller et al., 2006), whereas the temporal structure of the acoustic signals of the Poecilimon heroicus-group is less complicated (Korsunovskaya, 2008) and consist of isolated pairs of syllables (Heller et al., 2006).Due to the complexity of their song patterns Phonochorion spp.and P. zacharovi can be classified as more derived clades to the Poecilimon heroicus-group.
Interestingly Phonochorion spp. shares several distinct features with the most basal taxon of the Poecilimon heroicus-group: P. tschorochensis (Heller et al., 2006).Females of Phonochorion species and P. tschorochensis have overlapping tegmina and are capable of responding acoustically to males whereas those of the other species in the Poecilimon heroicus-group have highly reduced nonoverlapping tegmina and do not respond acoustically to males (Heller et al., 2006).Overlapping female tegmina and acoustic response of females to males also occurs in the genus Polysarcus (Korsunovskaya, 2008).In addition, Phonochorion spp.and P. tschorochensis share the ancestral cercal shape in which the cerci of males end in one simple tooth.P. zacharovi and all other species within the Poecilimon heroicus-group possess cerci with at least two teeth (Heller et al., 2006;Salman, 1978).
The similarity in distribution of Phonochorion spp., P. zacharovi and the Poecilimon heroicus-group and the closeness of Phonochorion spp., P. zacharovi and P. tschorochensis (the most basal taxon of the Poecilimon heroicus-group) suggests that these three groups might have diverged from a common ancestor distributed somewhere around the Circum-Caucasion region.
Within the genus Phonochorion, song structure clearly indicates Ph. uvarovi to be the basal taxon.Additional support can be inferred from morphological characteristics such as colour and shape of the male subgenital plate and the relatively small dorsal glands of Ph. uvarovi, all of which are relatively simple characters compared with 643  the more derived characters of Ph. satunini and Ph.artvinensis.
The songs of Ph. satunini and Ph.artvinensis are not significantly different.This similarity suggests that morphological differentiation of the two species occurred after the initial split from Ph. uvarovi.The similarity in song raises the question of whether the two groups should be synonomyzed.However, although the two species look quite similar they differ significantly in important diagnostic traits such as male cerci and male and female subgenital plates.Therefore, the species status of Ph. artvinensis and Ph.satunini is retained until further information becomes available.The basic problem is that of sample size, as we have only one population of Ph. satunini to base our conclusions on.Further sampling, most notably in Georgia, needed to clarify whether differences in traits truly reflect different species or intraspecies variation.
Based on the acoustic and morphological data presented the following systematic relationship between Phonochorion spp is proposed.Ph. uvarovi (or its ancestral stock) is the most basal clade and gave rise to the (Ph.satunini + Ph. artvinensis) clade as a result of diversification in male calling song and overall male coloration.Later the ancestral (Ph.satunini + Ph. artvinensis) clade gave rise to Ph. satunini and Ph.artvinensis, most probably as a result of further character divergence in Ph. artvinensis.

Conclusion
Both the bioacoustic and morphological data indicate that at the species level none of the characters of all of the extant taxa clearly differ.Therefore, it is likely that divergence within this group is quite recent and not yet complete.Furthermore the level and nature of the divergence between and within species suggests that both the level of bioacoustic and morphological divergence is stronger between species with overlapping ranges than those that are allopatric.Therefore divergence within the genus might be the result of ecological or reproductive character displacement.However, empirical tests are needed to substantiate the validity of this assumption.

Fig. 1 .
Fig. 1.Distribution of the species of the genus Phonochorion.

Fig. 13 .
Fig. 13.Oscillograms of the song of Ph. artvinensis when two echmes were produced each containing a major and minor syllable.

Fig. 14 .
Fig. 14.Oscillograms of the song of Ph. artvinensis when only the first echme containing a major syllable was produced.