A new species of Isoperla (Insecta, Plecoptera) from the Karawanken, with considerations on the Southern Limestone Alps as centers of endemism

Abstract A new species of the genus Isoperla (Plecoptera, Perlodidae), belonging to the oxylepis species-group is described, and the male mating call is characterized. Its range falls within a small region of the Southern Limestone Alps which is well known to be one endemism-centre of aquatic insects.


Introduction
The genus Isoperla consists of about 150 species (DeWalt et al. 2011, Baumann and Lee 2009, Murányi 2011, Szczytko and Stewart 1979, Zwick and Surenkhorloo 2005 and covers the Holarctic and Oriental regions. In Europe 56 species are known so far (Graf et al. 2009, Murányi 2011, of which ten occur in Austria (Graf 2010). Isoperla is a morphologically difficult genus, especially the grammatica and tripartita species groups that both exhibit high variability, and requires further resolution. A synthesis of zoogeographical, morphological, molecular, and possibly behavioural data will be required to get full knowledge on the diversity of this highly interesting genus.
Recently a series of specimens were collected from the Karawanken Alps in southern Austria and the nearby Kamnik Alps in northern Slovenia deviating from all hitherto known species. In this paper we provide morphological descriptions of males, females and the larva. Additionally we illustrate drumming signals of one male.

Material and methods
Adult specimens were collected using sweep nets, larvae were collected by handpicking from cobbles (mesolithal), the dominant substrate type. Collected specimens were stored in 70% ethanol. Morphological characteristics of male terminalia were examined in KOH-treated, cleared specimens. Comparative material from the authors' collections enabled the identification of the new species.
Vibratory signal recordings were made using a small, dynamic speaker (SAL YD78) as a vibration transducer. The speaker was connected to the microphone input socket of a solid state, digital recorder (Zoom H4n). The examined specimen was placed on the diaphragm of the speaker. To prevent the specimen from escaping the speaker was covered by a sheet of hobby glass. During the recordings ambient air temperature was measured using a P 300W thermometer. Vibration recordings were analysed and oscillograms produced using the software Adobe Audition 1.5 (Adobe Systems Incorporated, San Jose, California, USA). Drumming signal terminology follows Stewart and Sandberg (2006) and Murányi et al. (2014). Diagnosis. An Isoperla exhibiting the following combination of characters:

Isoperla claudiae
(1) a small medial penial armature in the form of an equilateral triangle, lacking lateral penial armatures; (2) yellow head and pronotum with a small horseshoe-like brown marking connecting the occelli.
Description. Medium-sized species, macropterous. Body length: holotype 10.5 mm, allotypes 11-12 mm; forewing length: holotype 12 mm, paratypes 12-14 mm. Primary colouration yellow, head and pronotum mostly yellow with dark brown markings; pilosity short. Primary colouration of the head yellow, with a dark horseshoe-like brown patch connecting the three ocelli (Fig. 1A, B). Occiput with indistinct rugosities but with brown patches laterally. Eyes normal sized. Scape brown, pedicel and the following antennomeres brown; palpi greyish to light brown. Pronotum yellow with a delicate brownish marking at the posterior margin, trapezoidal, edges angled; rugosities hardly visible and yellow. Anterior part of the mesonotum yellow, remaining portions brown; metanotum medially dark brown, laterally and anterior of the insertion of wings whitish. Wings yellowish, particularly the anterior half; venation mostly whitish to yellow, costa and apical part of radii brown. Ventral surface of thorax pale, meso-and metabasisternum inconspicous, furcasternites and furcal pits pale. Femora brown dorsally and yellow ventrally. Tibiae brownish dorsally, pale ventrally; tarsi brown.
Male abdomen (Fig. 1C): 1 st to 7 th tergite dorsally brown (with some tiny pale spots) with increasing laterally whitish areas towards the apex, 8 th to 10 th tergite mostly yellowish with small brown medial patches and medially interrupted anterior stripes up to T9, T10 pale without markings. Laterally and ventrally all segments whitish to yellow, lacking dark markings. Pilosity on segment posterior ends short and inconspicuous. Ventral lobe of sternite VIII yellow, slightly longer than wide, its posterior margin strongly convex with long marginal pilosity. Sternite IX yellowish. Paraprocts brown, regularly curved in caudal with with blunt tips; cerci light brown, apically dark brown. Penis (Fig. 2): Divided into four lobes and a basal section in extruded position. Medial penial armature located on the medial lobe adjacent to the ventral lobe, lateral penial armatures lacking. The medial penial armature resembles an equilateral triangle of 130 μm width and 97 μm length formed by slightly brownish coloured scales that are relatively blunt and short and vary in length (4.98-6.26 μm). The median basal area is sparsely covered by shorter scales. The medial penial armatures are connected distally by a narrow band of colourless scales with an area densely covered by smaller triangular scales. Similar scales are located proximal to the medial armatures. Their length varies between 4.4 and 7.2 μm. With the exception of the medial armatures the central area of the ventral penis is bald. Lateral portions of lateral lobes covered by dense scales similar to the ones on medial and ventral lobes, being denser at the connection to the ventral lobe.
Female abdomen (Fig. 1D): 1 st to 7 th tergite dorsally brown with increasing laterally whitish areas towards the apex, 8 th to 10 th tergite mostly yellowish with small brown medial patches. Laterally and ventrally all segments entirely whitish with dark markings reduced to delicate brownish lines at the posterior end of sternites. Subgenital plate covers most of sternite VIII width and half of sternite IX length, posterior margin rounded semicircularly. Sternite X and paraprocts yellowish; cerci generally brownish, the first segment being pale. Larva (Fig. 3): Body length of the matured larva: 13 mm. General colour brown but with pale markings. Pilosity dense, pronotal, posterior tergal and cercal fringes short and acute; swimming hairs lacking. Head dark brown with two yellow spots anterior to the M-line, two posterior to the M-line, one around the median occellus and one laterally to the each posterior ocellus. Two large pale spots laterally on the occipit (Fig. 3A). M-line distinct, tentorial callosities hardly visible; eyes normal sized. Scape and pedicel pale, the following antennomeres light brown; palpi yellowish, mouthparts light brown. Lacinia triangular, with 6 strong setae beneath the two apical teeth, thin hairs present all along the inner margin; galea with scattered setae on the whole surface (Fig. 3C). Pronotum rectangular with rounded corners, twice as wide as long, brown but with a narrow medial pale stripe along the medial suture and a marbled impression due to several medial pale areas, lateral parts uniformly brown, margins laterally pale. Mesonotum and metanotum mostly brown but with a pale, marmoreal pattern; wingpads brownish. Ventral surface of thorax pale, furcasternites and furcal pits inconspicuous. Legs uniformly pale. Abdominal tergites brown with a pair of roundish pale spots laterally to a median, darker area. The spots are increasing in size towards the entirely pale last tergite. Ventral surface of abdomen pale brown, the distal segments darker. Paraprocts brown; cerci light brown with dense circumferential rows of bristles of varying length at the end of each segment. Ecology and distribution. The species was collected in a small spring-brook at 535 m a.s.l. in the Karawanken, and a small forest brook at 980 m a.s.l. in the Kamnik Alps (Southern Limestone Alps).
Preliminary description of the male drumming call. Since only one signal from a single male could be recorded we cannot give any information on the variation range of the signal parameters in this species. The aim of this preliminary description is only to report the basic features of the signal, but even that should be treated with some caution since we cannot be sure whether or not the recorded signal shows some deviant features.
As it is observable in (Fig. 4A) the male call is a sequence of bi-beats. After an initial crescendo the peak amplitude of bi-beats fluctuate around a constant value. In bi-beats the first beat is of lower amplitude (missing in the low amplitude initial part of the call and sometimes hardly detectable even in the main part of the signal), the second one is of higher amplitude and followed by a long, decaying wave train (Fig.  4B). Inter beat interval within bi-beats varied between 8-20 ms during the call. The interval between bi-beats (or single beats at the initial part) gradually increased during the sequence (except for a short initial part of the sequence where inter beat interval decreases) varying between 230-350 ms (Fig. 4C). Inter-beat intervals were measured from the amplitude peak of one bi-beat to the amplitude peak of the next bi-beat (measured on the same call presented in A).
Isoperla claudiae sp. n. is most similar to I. orobica, a species restricted to the southwestern Alps, but can be easily distinguished from the latter species as the scales of the medial penial armature are shorter in I. claudiae sp. n., a higher density of uncoloured scales on the penis in I. claudiae sp. n., as well as yellow, hardly visible rugosities of the pronotum in I. claudiae sp. n.
The male drumming call of I. claudiae sp. n. is clearly different from the drumming call of I. oxylepis, which is the only species of the I. oxylepis species-group, where published information regarding the vibratory signals is available (Rupprecht 1969(Rupprecht , 1983. Amongst the European species of Isoperla the male call of I. claudiae sp. n. is most similar to that of I. rivulorum (Pictet) (Rupprecht 1969, Tierno de Figueroa and Sánchez-Ortega 1999, Tierno de Figueroa et al. 2000, 2011, Luzón-Ortega et al. 2010), but the beat group repetition period seems to be longer in this species (230-350 ms, 24.2 °C, Fig. 4C) than in I. rivulorum (Luzón-Ortega et al. 2010) reported 103-163 ms at 20 °C), and I. rivulorum frequently produces 3 beats per beat group.

The Southern Limestone Alps as centers of endemism
The southern slopes of the Alps from the Ligurian Prealps in the southwest to the Pohorje Mountains in the east are densely covered by microendemic species. Concentrations of endemic species in the south and south-eastern Alps are well known among Trichoptera species (Malicky 1983(Malicky , 2000; regarding Plecoptera Leuctra dylani Graf, L. juliettae Vinçon & Graf, L. muranyii Vinçon & Graf and Protonemura bipartita Consiglio are restricted to small areas from the Bergamo prealps to the Lessinian Alps; the apterous L. istenicae Sivec and Siphonoperla ottomoogi Graf are nested as microendemics in southeastern refugia referred to as the Steirische Randgebirge.
The western alpine slopes (Biellese, Graian and Cottian Alps) are another area of alpine endemism where a high diversity within the genus Leuctra is found (Ravizza Dematteis and Ravizza 1988;Vinçon and Ravizza 1998), and their distribution patterns are associated with the presence of nunataks during the Würm glaciation Ravizza Dematteis 1993, 1994). The hitherto known range of Isoperla claudiae sp. n. fits well in this hot-spot of biodiversity and supports the Dinodal theory of Malicky (1983Malicky ( , 2000, which suggests glacial species-specific refugia within the Alps based on distribution patterns of endemic caddisfly species. Most of these microendemic species are stenoecious elements of springs and small streams in medium altitudes.