A new species of cave cricket, Dolichopoda balrogi sp. nov., is described from the Greek island of Kastellorizo (Dodecanese, Levantine Sea). The species is documented from artificial subterranean habitats, as no accessible natural caves are known on the island. Detailed morphological analysis of both sexes reveals a unique combination of characters, including the structure of the male epiphallus, the shape of the tenth tergite, the presence of styli on the male subgenital plate, and the morphology of the female ovipositor, which clearly differentiate it from all known congeners. Comparative morphology indicates strong affinities with Anatolian species of Dolichopoda, particularly D. sbordonii and D. lycia, and no close relationship with the Aegean species complex occurring on islands of East Aegean and the adjacent Anatolian coast. These affinities are further supported by molecular phylogenetic analyses inferred from DNA barcoding. Both Bayesian inference and maximum likelihood analyses consistently recover the Kastellorizo lineage as a distinct and well-supported clade nested within the southwestern Anatolian lineage of the genus. The discovery of D. balrogi sp. nov. represents the first documented occurrence of an Anatolian lineage of Dolichopoda within European territory, underscoring the unique biogeographical character of Kastellorizo as a Greek territory with pronounced Anatolian biogeographical affinities. This finding emphasizes the importance of integrated morphological and molecular approaches in resolving species boundaries and evolutionary relationships and contributes to the growing evidence of underestimated biodiversity in the eastern Mediterranean region.

Anatolia, artificial tunnel, DNA barcoding, endemic species, Megisti, molecular phylogeny, new species, subterranean fauna

Kastellorizo, also known as Megisti (Dodecanese, Greece), is the largest island of a small island complex located about 2.2 km off the coast of SW Anatolia in the Levantine basin. The island covers an area of 9.1 km², and its coastline extends for approximately 19 km (Stamatelatos and Vamva 1996). Its shape is almost triangular, measuring about 6 km in length and 3 km in width. On the northern side of the island lies a bay where the traditional settlement of Megisti is built. At the entrance to the bay are the rocky islets of Psoradia, Agios Georgios, Agrielia, Polyphados, Psomi, Mavro Poini, and Mavropoinaki (Nezis 2010). Rising above the settlement is Vigla (273 m), the highest point on the island (Stamatelatos and Vamva 1996). Other notable elevations include Hatzilias (230 m), Munda (203 m), Kiolia (193 m), and Avgo (177 m). The island’s special features include its steep coastline, which forms a plateau across much of the island, as well as its rugged, rocky morphology with pronounced karstification resulting from the limestone formations (Greuter 1979, Kalaentzis et al. 2018a). These characteristics have led to the development of numerous caves, mainly at sea, namely the famous Blue Cave, the Parastas Hole, the Katrantzis Cave, the Arnaoutis Cave, and the Kolones Cave (Gerovasileiou et al. 2016). On land, only artificial tunnels have been identified and explored on the island, while caves on vertical cliffs remain inaccessible and underexplored (Strachinis et al. 2018). The vegetation of Kastellorizo is typical of the Eastern Mediterranean, featuring low shrubs, aromatic herbs and scattered maquis. Small patches of pine and olive trees can also be found in sheltered areas. The island’s flora is well adapted to the rocky terrain and dry climate (Greuter 1979, Constantinidis 2013). The island experiences a typical Eastern Mediterranean climate, characterized by warm, dry summers and mild, wet winters, which strongly influences its vegetation and wildlife (Bisia et al. 2024).

The entire island of Kastellorizo is included within the Natura 2000 network (GR4210004), reflecting its ecological importance and the presence of rare and protected species of flora and fauna. Perhaps the most remarkable faunistic and floristic aspect of the island is the abundance of West Anatolian or local East Mediterranean elements that are otherwise very rare in the Greek flora. For many of these, the Kastellorizo island group remains the only known Greek locality (Constantinidis 2013). Of particular interest is the presence of many rare invertebrates, such as Zonites caricus (J. R. Roth, 1839) (Gastropoda: Zonitidae) (Mylonas et al. 2019), the scorpions Neocalchas gruberi (Fet, Soleglad & Kovarik, 2009) (Arachnida: Iuridae) and Anatoliurus kraepelini (von Ubisch, 1922) (Arachnida: Buthidae) (Fet et al. 2018, Parmakelis et al. 2022), the isopod Armadillidium ameglioi Arcangeli, 1914 (Crustacea: Armadillidiidae) (Alexiou and Sfenthourakis 2013), the bush-cricket Poecilimon inflatus Brunner von Wattenwyl, 1891 (Orthoptera: Tettigoniidae) (Kazilas et al. 2023), and the beetle Psallidium difficile Faust, 1889 (Germann et al. 2026) (Coleoptera: Curculionidae). Additionally, unusual color morphotypes have been documented in the island’s herpetofauna, further highlighting the biological singularity of Kastellorizo (Kalaentzis et al. 2018b, Kazilas et al. 2018).

The genus Dolichopoda Bolívar, 1880 (Orthoptera: Rhaphidophoridae) comprises 67 mostly cave-dwelling species of crickets (Cigliano et al. 2026), scattered across southern Europe and Anatolia to Caucasus. Greece is a major center of diversity for the genus, hosting 40 species, all but one endemic to insular and peninsular Greece (Alexiou et al. 2024). Most species of Dolichopoda are strictly dependent upon caves and are therefore highly isolated, with limited gene flow between populations, which can lead to reduced genetic diversity and increased extinction risk. This has resulted in high levels of endemism, making the genus particularly interesting for studies on evolutionary biology and biogeography (Allegrucci et al. 2009, 2021). The frequent discovery of new species over the last two decades reflects this endemic differentiation as well as the more intensified field research; specifically, 20 species of Dolichopoda were known from Greece by 2008 (Rampini et al. 2008). Since then, this number has doubled to 40 (Alexiou et al. 2013, 2024, Allegrucci et al. 2021, Di Russo et al. 2014, 2017, 2018, Rampini et al. 2008, 2012).

The islands of the Aegean Archipelago hold 13 species of Dolichopoda (Rampini et al. 2012, Di Russo et al. 2018). Except for the species closer to the Greek mainland (i.e. Andros, Evia, and Thasos), the rest belong to a morphologically homogenous complex (Rampini et al. 2012), including Dolichopoda sutini Rampini & Taylan, 2012, endemic to the Aegean coast of Turkish mainland (West Anatolia: Izmir) (Rampini et al. 2012). Notably, the insular-Aegean group shows no close affinities to the Anatolian species.

In the East Aegean, five species are currently known: Dolichopoda giulianae Rampini & Di Russo, 2012 and Dolichopoda kalithea Di Russo & Rampini, 2012 from Samos; Dolichopoda calidnae Rampini & Di Russo, 2012 from Kalymnos; and Dolichopoda margiolis Di Russo & Rampini, 2018 from Kinaros D. sutini (Rampini et al. 2012, Di Russo et al. 2018) (Fig. 1).

Fig. 1. 

Geographic distribution of Eastern Mediterranean Dolichopoda species. Colored symbols indicate known localities of previously described species of the Aegean (depicted in circles) and the SW Anatolian (depicted in triangles) groups. The yellow star marks the type locality of Dolichopoda balrogi sp. nov. on Kastellorizo (Megisti) Island, Greece (scale bar 100 km). Inset: detailed view of the Kastellorizo island and the seven surrounding Greek islets (missing the two larger islands of Ro and Strongyli) with the precise sampling site (scale bar: 1 km). Base map: ESRI Satellite (© Environmental Systems Research Institute, Inc., CC BY 3.0).

In this study, we present and describe a new cave cricket species of the genus Dolichopoda from the tiny island of Kastellorizo, Greece, based on both morphological and molecular data.

Study area.—The cave crickets were discovered inside an artificial tunnel located at an elevation of approximately 242 meters on Mt. Vigla (Fig. 1) in a central area of Kastellorizo Island, independently by two research groups: KK on 17 October 2025 (Fig. 2) and DK, CK, and AC on 15–16 November 2025. The tunnel, situated in a rocky calcareous area with low maquis vegetation and phrygana, measures about 25 m in depth, 1.5 m in width, and between 1.80 and 2.5 m in height, with an internal temperature of approximately 17 °C and natural light reaching only the entrance of the tunnel. In the tunnel, many cave crickets of Dolichopoda were detected scattered along the walls in various points, with 37 individuals—7 males, 10 females, and 20 juveniles—detected during the final visit on 16 November 2025. Adult individuals were collected by hand for further study, preserved in 97% ethanol and stored at -18 °C. The specimens are deposited at the Korinthian Museum of Natural History (KMNH), Greece and the Museum of Zoology of the University ‘La Sapienza’ of Rome (MZUR), Italy.

Fig. 2. 

Individuals of the newly discovered Dolichopoda balrogi sp. nov. in the artificial tunnel of Kastellorizo on 17 October 2025. A. Female individual. B. Male individual. C. Juvenile. Photo credit: K. Kalaentzis.

Morphological analysis.—For the morphological analysis, six external body characters were utilized: shape of tergum X of the male; median process of the epiphallus; shape of the epiphallus; amount of spinulation of the hind tibia; shape of the female subgenital plate; and ovipositor. Photographs of morphological characteristics were made using a BMS 133 Zoom Trino stereomicroscope.

Molecular analysis.—Genomic DNA was extracted from the femur of four adult specimens (two males and two females) preserved in ethanol using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. A partial fragment of the mitochondrial cytochrome c oxidase I (COI) gene was amplified using the primers LCO1490 and HCO2198 (Folmer et al. 1994). PCR reactions were performed in a final volume of 30 μL containing 1× PCR buffer, 2.5 mM MgCl₂, 0.2 mM dNTPs, 0.4 μM of each primer, 1 U Taq DNA polymerase, and 50–100 ng of template DNA. Thermal cycling conditions consisted of an initial denaturation at 94 °C for 3 min, followed by 35 cycles of 95 °C for 40 s, 50 °C for 40 s, and 72 °C for 40 s, with a final extension at 72 °C for 5 min. PCR products were visualized on 2% agarose gels and sequenced by GENEWIZ Germany GmbH (part of Azenta Life Sciences).

A total of 53 nucleotide sequences of the mitochondrial COI marker were accessed and downloaded from GenBank belonging to 20 species of Dolichopoda from Greece and Turkey and the Western Mediterranean Dolichopoda linderii, which was used as an outgroup. All sequences, including the four sequences generated in this study, were aligned in MEGA (Kumar et al. 2024) and inspected manually to ensure positional homology.

Maximum likelihood (ML) phylogenetic inference was performed using the general time reversible with gamma distribution and invariant sites model (GTR+G+I), which was selected as the optimal nucleotide substitution model based on information criteria in MEGA; Bayesian information criterion (BIC) = 13994.07, Akaike information criterion (AIC) = 12921.8 and log-likelihood (lnL) = -6339.62. Node support was assessed using nonparametric bootstrap analysis with 1,000 replicates.

Bayesian phylogenetic inference was conducted using a Markov chain Monte Carlo (MCMC) approach using MrBayes (Ronquist et al. 2012). Analyses were run for 1,000,000 generations, sampling trees every 100 generations and calculating diagnostics every 5,000 generations. Two independent runs with four chains each were performed. The first 25% of sampled trees were discarded as burn-in, and posterior probabilities were estimated from the remaining trees to generate a majority-rule consensus tree. Branch lengths represent estimated substitutions per site under the GTR+G+I model.

The Greek Island of Kastellorizo is separated from the Turkish coast only by 2–3 km. Its recent separation from the Anatolian mainland has resulted in well documented strong zoogeographical and phytogeographical connection to Anatolia (Greuter 1979, Constantinidis 2013, Benda and Uhrin 2017, Kalaentzis et al. 2018a, Strachinis et al. 2018). Greece is a hotspot for the genus, but Turkey holds its own share of cave crickets, with nine species of the genus Dolichopoda known so far, all in Anatolia (Taylan et al. 2023). The area opposite of Kastellorizo is dominated by the Taurus Mountains, on the western slopes of which three species have been described: Dolichopoda sbordonii Di Russo & Rampini, 2006, Dolichopoda lycia (Galvagni 2006), and Dolichopoda fortuita Gorochov & Ünal, 2015. Dolichopoda aranea Bolívar,1899, Dolichopoda pusilla Bolívar, 1899, and Dolichopoda rampinii Taylan & Şirin, 2023 are recorded from the Eastern Taurus (Gorochov and Ünal 2015, Taylan et al. 2011, 2023).

Dolichopoda balrogi sp. nov. belongs to the group of species that occupy Anatolia and shows no affinities with the Aegean species complex (Fig. 1). Specifically, the new species shows strong affinities to D. sbordonii, a species occupying caves in the adjacent Anatolian area. Both species have styli at the subgenital plate of the male, and both have short, moderately sclerotized and strongly curved apically median process of epiphallus. The subgenital plate of the females is bifurcated in both species. The new species clearly differ from D. sbordonii in having only traces of lateral lobes posteriorly on the X tergite of the male (instead of distinct lateral lobes) and a trapezoidal (instead of triangular) median process of epiphallus. The female of D. balrogi differs mainly by having a longer ovipositor, with more denticles.

Dolichopoda lycia and D. fortuita, although geographically and morphologically very close to D. sbordonii and D. balrogi sp. nov., present clear differences especially in the shape of the epiphallus (clearly bilobed median process) and absence of styli and are most similar to each other (Galvagni 2006, Rampini and Di Russo 2008, Taylan et al. 2011, Gorochov and Ünal 2015). On the other hand, D. balrogi sp. nov. shares with both D. lycia and D. fortuita the shape of the tenth male tergite, with only inconspicuous lateral lobes (Rampini and Di Russo 2008, Taylan et al. 2011, Gorochov and Ünal 2015). The plica dorsalis could not be assessed because it was accidentally damaged during stereoscopic examination of the available material, and no additional well-preserved specimens were available; however, species delimitation is robustly supported by multiple independent diagnostic characters in both sexes and strong molecular support.

Because of all of the above, D. balrogi sp. nov. is unique within European entomofauna, being the first representative of an Anatolian lineage of cave crickets. The new species does not share similarities with the Aegean species (the Greek species from Cyclades, Crete and, East Aegean and the Turkish D. sutini from West Anatolia), which, based mainly on characters such as shape of the epiphallus and tenth tergite of the male, form a homogenous group (Rampini et al. 2012, Di Russo et al. 2014). Dolichopoda balrogi sp. nov. clearly belongs to a group of Anatolian species with a short and rounded distally median process of the epiphallus, in which belongs D. sbordonii from south Anatolia and the north Anatolian Dolichopoda noctivaga Di Russo & Rampini, 2007. The two trans-Caucasian species Dolichopoda euxina Semenov, 1901 and Dolichopoda hyrcana Bey-Bienko, 1969 also present similarly shaped epiphallus (Di Russo et al. 2007).

The phylogenetic reconstruction (Fig. 4) provides further support for the placement of Dolichopoda balrogi sp. nov. within the Anatolian lineage of the genus. Both Bayesian inference and ML analyses unambiguously recovered the samples from Kastellorizo as a well-supported clade, sister to D. sbordonii and closely related to D. lycia, clearly separating them from the Aegean, the mainland Greek, and the Caucasian species groups. This topology corroborates the morphological evidence and reinforces the strong biogeographical link between Kastellorizo and southwestern Anatolia. Similar Anatolian affinities have been documented for other invertebrate taxa from the island group, reinforcing the interpretation of Kastellorizo as a biogeographical outlier within the Greek fauna (Kazilas et al. 2023).

Nevertheless, the interpretation of phylogenetic relationships within the broader Anatolian–Aegean group is inevitably limited by the incomplete taxon sampling currently available. Unfortunately, several already-described species from both Anatolia and the eastern Aegean remain unsequenced, precluding a more comprehensive assessment of lineage diversification and historical biogeography. Expanded molecular sampling, particularly through standardized DNA barcoding of additional Dolichopoda species across Anatolia and the Aegean region, would greatly improve resolution within the genus and allow a more robust evaluation of evolutionary relationships, dispersal patterns, and the origin of insular endemism. Nevertheless, noctivaga is recovered as paraphyletic in the mitochondrial phylogeny (Fig. 4), with other Caucasian taxa nested within the broader noctivaga lineage, corroborating the phylogeny retrieved by Taylan and Şirin (2016). This pattern suggests that D. noctivaga may represent a species complex shaped by long-term isolation in the Pontic–Caucasus region, rather than a single cohesive evolutionary unit, and is in need of further resolution.

The discovery of the new species exclusively in artificial subterranean structures is particularly noteworthy. Kastellorizo lacks extensive natural cave systems, and the occurrence of a cave-adapted Dolichopoda on the island would likely have remained undetected in the absence of deep human excavation. This finding highlights the role of anthropogenic underground habitats as refuges or secondary habitats for subterranean organisms as well as their potential to reveal previously hidden subterranean biodiversity.

A more detailed and specialized survey of the island’s caves should be conducted to determine whether additional populations of the species exist on the island. Furthermore, it would be valuable to consider possible management strategies to ensure the conservation of the population. The importance also lies in the fact that new species of the genus are constantly being discovered, highlighting how little was known about it until a few years ago. This discovery increases the number of Dolichopoda species to 68 and the number of them present in Greece to 41.

We are grateful to Elias Tzoras, Michail Manonas, and Alexios Panagiotopoulos for providing the locality of the artificial tunnel and to Kostantinos Tsoleridis and Giorgos Chatzikallinikidis for their support during the lab work. We are indebted to Claudio Di Russo and Mauro Rampini for their thoughtful input on the manuscript. We are also thankful to the anonymous reviewers for their review and constructive comments on the manuscript. KK was the recipient of a Bodossaki Foundation PhD scholarship.