Stoneflies are important ecological indicators of aquatic ecosystems, yet poorly explored due to their difficult species-level taxonomy and cryptic larvae forms. Nowadays, barcoding is widely used in biodiversity assessments, complementing morphological identification and requiring comprehensive reference DNA sequence databases. The DNA barcode region exhibits high interspecific and low intraspecific variation, making it a suitable tool for species delimitation, detecting cryptic biodiversity and species-level identification of morphologically cryptic life stages. In Romania, there are 132 (of which 24 are dubious) known species of stoneflies, of which 74 (57%) have standard barcode sequences in databases generated by European countries. Here we present a DNA barcode initiative based on local populations of Plecoptera, which aims to create molecular data of stoneflies from a well-known aquatic biodiversity hotspot in the Romanian Carpathians, the Apuseni Mountains. 175 specimens were collected between 2020 and 2021 and analyzed using the standard barcode region (mtCOI). The 97 larvae and 62 adult sequences represented 29 known species, of which six lacked a Barcode Index Number (BIN). We further identified six previously unknown new BINs, indicating overlooked diversity or providing new molecular data for existing taxa, and suggesting the presence of cryptic or undescribed species. In total, we added 12 new BINs to the Barcode of Life Data System. We managed to associate 86 larval stage Plecoptera specimens with 24 known species using barcode sequences. For the first time, we generated DNA barcode data for one endemic and two sub-endemic species of the Carpathians: Brachyptera starmachi Sowa, 1966, Leuctra carpathica Kis, 1966, and Protonemura aestiva Kis, 1965. Our results provide a more realistic estimate of the aquatic biodiversity in the Apuseni Mountains and underline the importance of local datasets to support regional aspects of the larvae-taxonomy for Plecoptera from Romania.

Genus Mochlonyx Loew, 1844 is reported for the first time from the Carpathians, with two species, Mochlonyx fuliginosus (Felt, 1905) and Mochlonyx velutinus (Ruthe 1831). Additionally, Chaoborus obscuripes (Wulp, 1859) is also newly recorded in Romania, marking new occurrences for all three species, and bringing the number of Chaoboridae species from the country from three to six. A total number of 525 larvae, pupae, and adults were morphologically analysed, and standard mtCOI DNA sequences were generated for 20 specimens in order to help species level identifications, and association of pupae and larvae with corresponding adults. DNA barcode data delimits well the six species identified from Romania in all life stages. The observed genetic distances are low, with an average p-distance of 0.9%, compared to the conspecific sequences that are available in public genetic databases. Specimens from Romania are grouped into seven different Barcode Index Number (BIN) System clusters. Only Ch. flavicans was represented by two different BINs from Romania, with a genetic distance of only 1.67%, while each of the specimens of the other five species undergoes genetic grouping into a single BIN. To the best of our knowledge, this is the first integrative study of Chaoboridae from Romania, and it contributes to a better understanding of the still largely overlooked aquatic Diptera biodiversity of the region.

Here we present the first reliable record of the rare Hyperoscelis veternosa Mamaev and Krivosheina, 1969 from the headwaters area of the Someșul River, Apuseni Natural Park, Romania. Species level identification was made by detailed examination of the single female genitalia, supported by DNA barcode data. The new finding confirms the presence of the species in Romania, whereas a very old, single and doubtful record from the Bucharest area dates back to 1900 needs further confirmation. The presence of the second species of genus Hyperoscelis from Romania is of high conservation value, and confirm the persistence of a relict biodiversity of an old-growth forests-spot in the Carpathians.

The Mediterranean Region of the West Palearctic is one of the most species-rich biomes in the world, hosting a high level of endemism and relict species with important conservation value. The North Africa Atlas Mountains (spanning Morocco, Algeria and Tunisia) belong to a poorly-investigated region of the Mediterranean area, with overlooked aquatic biodiversity; hence, a number of species still remain to be discovered. The subgenus Antocha (Antocha) Osten Sacken, 1860 is recorded for the first time from Africa, with a description of A. (A.) staryi Keresztes & Mabrouki sp. nov. from hilly regions of the Middle Atlas region, Morocco. The unique design of the male terminalia differentiates well the newly-discovered species from its closely-related and range-restricted A. (A.) phoenicia Thomas and Dia, 1982. This is in contrast with the high intraspecific and geographically poorly-defined variability of the widespread A. (A.) vitripennis (Meigen, 1830), for which morphological variability of male genital structures is discussed. Illustrations of male genital parts, distribution data and key to the species from the West Palearctic area are also provided.

This paper attempts to fill the gaps in knowledge about the biodiversity of some winter-active fly families from snowfields and caves in Vârghiș, Romania. A total of 15 fly species were recorded from caves and 9 species from snowfields. Exechiopsis (Exechiopsis) pseudindecisa Lastovka et Matile, 1974 and Rymosia placida Winnertz, 1863 from caves and Mycetophila mitis (Johannsen, 1912) (all Mycetophilidae) from snow represent the first records for Romania. Habitus photographs of these three species are provided.

The identity and distribution of a neglected fungus gnat species, Rymosia tolleti Burghele-Balacesco, 1965, in Europe is reviewed based on examination of newly collected specimens as well as available museum mate-rials. Rymosia tolleti is widespread but rather rare in Central Europe, with confirmed records from Romania, Slo-vakia, Germany, and France. All the specimens with known collection details originate from cave environments. Detailed photographs of the male terminalia are provided for the first time, along with two unique DNA barcodes for the species.

Dicranomyia (Idiopyga) nigristigma Nielsen, 1919 (Diptera, Limoniidae) is recorded for the first time from Romania. The species was collected in some calcareous springs in the Apuseni Mountains and highlight an overlooked Limoniidae diversity in similar habitats from the karstic area from here.

Zwicknia acuta is reported for the first time from the South-Eastern Carpathians (Romania and Ukraine). Additionally, another stonefly species, Zwicknia kovacsi, previously described from Romania, is added to the Plecoptera fauna of Ukraine. New faunistic data are presented for two other winter-active species, Capnia vidua rilensis and Zwicknia bifrons. Based on a combination of personal and literature data we present a checklist of Capniidae from Romania and provide a key for them.