Molecular Species Delimitation Research Articles

Application of Modern Approaches to Species Delimitation in Freshwater Erpobdellidae (Hirudinea: Arhynchobdellida) with a New Interpretation of Their Genera

The traditional taxonomy of freshwater invertebrates is a labor-intensive process requiring extensive knowledge and experience. In addition, this science is largely subjective, which makes its digitalization difficult. However, accurate species attribution is becoming increasingly important for environmental surveys and conservation efforts. In the 21st century, molecular biology methods have proven to be a successful tool for objectively determining biodiversity. Achieving objectivity in identifying the taxa of non-parasitic leeches of the family Erpobdellidae was the main goal of this study. To this end, various bioinformatic approaches to DNA sequence analysis have been tested. As a result, the phylogenetic diversity amounted to 47 species versus 29 morphospecies defined traditionally. The use of molecular species delimitation methods made it possible to identify seven complexes of morphologically hidden (cryptic) species and some morphological misidentifications, as well as to discover a new species from Eastern Siberia (Erpobdella sibirica) with a unique molecular diagnosis (DNA barcode). A pioneering attempt to apply the same approach to higher taxa showed that Erpobdellidae probably consists of seven genera, including the unambiguous elimination of the genus Motobdella. Obtaining quantitative and, therefore, objective data is an advantage of the molecular approach, which has proven to be effective in recognizing species and genera, at least in leeches.

Violins we see, species we don’t… Species delimitation of the spider genus Loxosceles Heineken & Lowe (Araneae: Sicariidae) from North America using morphological and molecular evidence

In modern systematics, different sources of evidence are commonly used for the discovery, identification, and delimitation of species, especially when morphology fails to delineate between species or in underestimated species complexes or cryptic species. In this study, morphological data and two DNA barcoding markers—cytochrome c oxidase subunit I (COI) and internal transcribed spacer 2 (ITS2)—were used to delimit species in the spider genus Loxosceles from North America. The molecular species delimitation analyses were carried out using three different methods under the corrected p-distance Neighbor-Joining (NJ) criteria: 1) Assemble Species by Automatic Partitioning (ASAP), 2) General Mixed Yule Coalescent model (GMYC), and 3) Bayesian Poisson Tree Processes (bPTP). The analyses incorporated 192 terminals corresponding to 43 putative species of Loxosceles, of which 15 are newly recognized herein, as putative new species, based on morphology and congruence between molecular methods with COI. The average intraspecific genetic distance (p-distance) was <2%, whereas the average interspecific genetic distance was 15.6%. The GMYC and bPTP molecular methods recovered 65-79 and 69 species respectively, overestimating the diversity in comparison with morphology, whereas the ASAP method delimited 60 species. The morphology of primary sexual structures (males palps and female seminal receptacles) was congruent with most of the molecular methods mainly with COI, showing that they are robust characters for identification at the species level. For species delimitation COI was more informative than ITS2. The diversity of Loxosceles species is still underestimated for North America, particularly in Mexico which holds the highest diversity of this genus worldwide.  

Taxonomic study of four closely-related species of the Pholcus yichengicus species group (Araneae, Pholcidae) from China’s Qinling Mountains: An integrated morphological and molecular approach

Four morphologically similar species of the Pholcus yichengicus species group, occurring in geographic proximity of China’s Qinling Mountains, were recognised, based on morphology and four methods of molecular species delimitation. They comprise two new species, namely Pholcus ankangsp. nov. and P. baojisp. nov. and two previously described species: P. ovatus Yao & Li, 2012 and P. taibaiensis Wang & Zhu, 1992. Their DNA barcodes were obtained to estimate p-distances and K2P distances. In addition, an identification key for the four closely-related species is presented.

Discovery of a new species of Synergus (Hymenoptera, Cynipidae, Synergini) based on morphology and molecular data.

A new species of gall inquiline, Synergusdilatatussp. nov., is described from Hubei Province, China. Morphological descriptions, photographs and biological information are provided. Mitochondrial cytochrome oxidase (COI) sequences of the new species were newly obtained and a molecular species delimitation analysis of 12 species of Synergus performed using the ASAP method recovered 16 molecular operational taxonomic units, providing support for recognition of the new species. The results also highlight a few conflicts between morphological and molecular species delimitations in Synergus.

Six new species of Margattea Shelford, 1911 (Blaberoidea, Pseudophyllodromiidae, Neoblattellini) from China.

Six Margattea species are established and described: three are cryptic species, namely, M.parabisignata Li & Che, sp. nov., M.semicircularis Li & Che, sp. nov., and M.forcipata Li & Che, sp. nov. They are distinguished from known species M.bisignata, M.spinifera, and M.paratransversa by their male genitalia with the aid of molecular species delimitation method (ABGD) using COI as the molecular marker. The other three new species are M.pedata Li & Che, sp. nov., M.undulata Li & Che, sp. nov., and M.bisphaerica Li & Che, sp. nov. Morphological and genitalia photographs of these new species of Margattea, as well as a key to the species of Margattea from China, are provided.

Discovery of five new species of Allacta from Yunnan and Hainan, China (Blattodea, Pseudophyllodromiidae).

We examined new Allacta materials from Yunnan and Hainan Province, China, and discovered new species using both morphological and molecular species delimitation (ABGD) methods. Five new species are described: A.bifolium Li & Wang, sp. nov., A.hemiptera Li & Wang, sp. nov., A.lunulara Li & Wang, sp. nov., A.redacta Li & Wang, sp. nov., and A.unicaudata Li & Wang, sp. nov. All five species are placed under the hamifera species group. An updated key and checklist of Allacta species from China are provided.

Species delimitation and phylogenetic relationships of the Prionospio complex (Annelida, Spionidae) in the Northeast Atlantic

AbstractThe Prionospio complex comprises the most diverse and complex group within the polychaete family Spionidae. The phylogenetic relationships within the group are still poorly understood, and the generic breakdown is unstable. In this study, we assessed the diversity, relationships, and distribution of species of the Prionospio complex occurring in Norwegian waters. We analysed mitochondrial genomes and nuclear ribosomal DNA assembled via whole‐genome shotgun sequencing, and Sanger sequenced fragments of COI and 16S rDNA. Sanger sequencing proved challenging in the group, where COI was only amplified successfully in 14% of specimens. By molecular species delimitation algorithms, our study revealed the presence of four well‐supported but currently undescribed species of Prionospio in Norwegian waters. We observed a novel distribution pattern of polychaetes in coastal waters, where certain species demonstrated distribution ranges spanning over 7000 km. Such wide distribution parallels patterns of deep‐sea Prionospio species, suggesting that factors beyond recent anthropogenic translocations are involved. Our analysis of 38 mitochondrial genomes and ribosomal nuclear DNA enabled us to hypothesise on the phylogenetic relationships of 14 species of the Prionospio complex. The analysis suggested that two characters previously used to designate genera: the beginning of the branchiae from chaetiger 3 and the presence of pinnules on the branchiae, might have evolved more than one time within the complex. We return Aurospio banyulensis to the genus Prionospio according to the diagnosis of Aurospio resulting tree where this species was nested among Prionospio species. Our findings provide new insights into the diversity and distribution patterns of Prionospio species and contribute to a better understanding of marine benthic biodiversity and the importance of taxonomic accuracy in conservation and management practices.

Global species delimitation of the cosmopolitan marine littoral earthworm Pontodrilus litoralis (Grube, 1855)

The marine littoral earthworm Pontodrilus litoralis (Grube, 1855) is widely distributed and is reported as a single species. This study utilized an integrative taxonomic approach based upon morphological examination, phylogenetic reconstruction, and molecular species delimitation, to test whether the taxon is a single species or a species complex. For this, a total of 114 P. litoralis specimens collected from North America, Africa, Australia and Oceania, Europe and Asia were used. The phylogenetic analyses revealed deeply divergent mitochondrial lineages and a high level of genetic diversity among P. litoralis populations. Both single and multi-locus species delimitation analyses yielded several molecular operational taxonomic units. Therefore, due to the homogeneity of morphological characteristics, it is likely that the morphospecies P. litoralis is a complex of four or more cryptic species, suggesting that more sampling is required and that the population structure genetic data and gene flow need to be investigated.

An integrative taxonomy approach evaluates the limits of the widespread tarantula Plesiopelma longisternale (Araneae: Mygalomorphae: Theraphosidae) and reveals a new species from Argentina

Plesiopelma longisternale is a widespread tarantula in Argentina and its limit is difficult to determine due to their morphological variation among populations. Males are distinguishable by the following character combination: a field of spines on the apical half of the palpal tibia, a very long embolus and a field of spines on the reotrolateral face of cymbium; females are characterized by long spiraled ducts distant from each other. Here, we used mitochondrial DNA (cox1) to reconstruct the first phylogeny of P. longisternale, in combination with morphological data and molecular species delimitation, to assess the taxonomic limits of this species. Our phylogenetic results confirm a close relationship among the populations from central Argentina and a new evolutionary independent lineage identified as a new species, Plesiopelma absconditus, which is here diagnose, described and illustrated. We further analyzed the diagnostic characters of P. longisternale through the examination of the types and specimens from many populations and found that their morphological intraspecific variations largely overlap. Altogether, our results demonstrate that some of the variability of P. longisternale in central Argentina represent polymorphisms of a single species supported by molecular data. In addition, we discovered a new species from this genus distinguishable by molecular and morphological data and highlight the need for multiple lines of evidence to solve the taxonomic problems in species of tarantulas.

Molecular and morphometric data provide evidence of intraspecific variation in shape and pigmentation pattern in Otocinclus cocama (Siluriformes: Loricariidae) across major river drainages.

Otocinclus cocama, a uniquely colored species of the loricariid catfish genus Otocinclus described solely from the type locality in the lower Ucayali River in northern Peru, is reported occurring in the Tigre River, a tributary to the Marañón River that drains a different section of the Andean Mountain range in the western Amazon. Both populations differ in the number of dark bars spanning the flanks of the body, and we investigated whether these morphotypes constitute distinct species. The body shapes of populations from the Tigre and Ucayali rivers were compared using geometric morphometrics. Although principal component analysis detected a broad overlap between populations, multivariate analysis of variance and linear driscriminat analysis revealed a subtle differentiation between the populations of the two hydrographic basins. Average body shape of the Ucayali River population tend to be slightly higher than that of the Tigre River, with the caudal peduncle stretched vertically in the Ucayali population. Multivariate regression of shape and centroid size revealed an allometric effect of 10.7% (p < 0.001), suggesting that the variation between Tigre and Ucayali populations was purely shape variation. Molecular data of coI, cytb, nd2, and 16S mitochondrial genes indicated a nucleotide diversity range from 0.001 to 0.003, and haplotypic diversity range from 0.600 ± 0.11 to 0.79 ± 0.07. The median-joining haplotype network for the concatenated matrix exhibited two divergent haplogroups related to the geographic area and separated by <10 mutational steps. The molecular species delimitation methods based on distance (automatic barcode gap discovery and assemble species by automatic partitioning) recovered two molecular lineages evolving independently, being one of the lineages formed by individuals from both populations. Tree-based methods (generalized mixed Yule coalescent and Bayesian implementation of the Poisson tree process) recovered similar topologies and supported single lineage recognition. Methods of molecular delimitation of species disclosed the high similarity between the two populations of Otocinclus cocama, further supported by the presence of old haplotypes common to both groups which could indicate that the populations still maintain gene flow. Although the morphological data reveal a subtle variation between both river basins, the molecular data suggest a weak population structuration based on hydrographic areas, but not different species lineages, therefore Otocinclus cocama is composed of a single lineage with two distinct morphotypes.

A systematic review of the Iberian springsnail subgenus Alzoniella (Navarriella) (Caenogastropoda: Hydrobiidae), with the description of a new potentially relict subfamily

Abstract The threatened springsnail subgenus Alzoniella (Navarriella) in the Iberian Peninsula has been suggested to be an old and relict lineage of the family Hydrobiidae. The subgenus is represented by two morphological species, both endemic to the Pyrenees and their southern foothills. We conducted phylogenetic analyses based on mitochondrial and nuclear gene fragments of topotypes and other populations, four molecular species delimitation methods, and morphological examinations to clarify the uncertain systematic position of the subgenus within the family, assess its species diversity, and understand the population genetic structure of the two geographically restricted species. Our phylogenetic results revealed that Alzoniella (Navarriella) is distantly related to all other species of Alzoniella, even belonging to an independent subfamily-level clade, for which we introduce the new genus Navarriella and the new subfamily Navarriellinae subfam. nov. Molecular methods and geometric morphometric analysis of shell shape identified a single species in the new genus. The significant phylogenetic distance from other hydrobiid taxa, narrow distribution, and limited gene flow among its populations (estimated from mitochondrial cytochrome c oxidase subunit I sequences) highlight Navarriella as an isolated lineage within the family that requires urgent conservation attention. Furthermore, our results cast a new light on the northern Iberian Mountains as a dispersal barrier for ancient spring lineages.

Phylogenomic species delimitation of the twisted‐winged parasite genus Stylops (Strepsiptera)

AbstractThe twisted‐winged parasite genus Stylops has a history of different species concepts with varying host specificity resulting in diverse species diversity estimates in different regions of the Holarctic. The adoption of a supergeneralist species concept in Europe, proposing synonymization of all Western Palaearctic Stylops species, did not facilitate taxonomic clarity and obscured the available life‐history data in the region for decades. Lack of molecular data has allowed divergent opinions on species hypotheses and little opportunity for evaluating them in this morphologically challenging genus. To solve these discrepancies and gain novel information about host associations, we applied whole‐genome sequencing to 163 specimens, representing a significant portion of putative European species. We evaluate the existing and conflicting species hypotheses with molecular species delimitation using Species bOundry Delimitation using Astral (SODA) and use a maximum likelihood phylogeny to investigate host associations of the species. Furthermore, we evaluate the effect of a number of loci used in SODA for the number of inferred species. We find justification for synonymization of multiple species and indications of undescribed species, as well as new host–parasite relationships. We show that the number of inferred species in SODA is exceedingly and positively correlated with the number of loci used, urging for cautious application. The results of our study bring clarity to the Western Palaearctic species diversity of Stylops. Furthermore, the comprehensive molecular dataset generated in this study will be a valuable resource for future studies on Stylops and the evolution of parasites in general.

Molecular species delimitation of marine trematodes over wide geographical ranges: Schikhobalotrema spp. (Digenea: Haplosplanchnidae) in needlefishes (Belonidae) from the Pacific Ocean and Gulf of Mexico.

Geographical distribution plays a major role in our understanding of marine biodiversity. Some marine fish trematodes have been shown to have highly restricted geographical distributions, while some are known to occur over very wide ranges; however, very few of these wide distributions have been demonstrated genetically. Here, we analyse species of the genus Schikhobalotrema (Haplosplanchnidae) parasitizing beloniforms from the tropical west Pacific, the eastern Pacific and the Gulf of Mexico (GoM). We test the boundaries of these trematodes by integrating molecular and morphological data, host association, habitat of the hosts and geographical distribution, following a recently proposed and standardized delineation method for the recognition of marine trematode species. Based on the new collections, Schikhobalotrema huffmani is here synonymized with the type-species of the genus, Schikhobalotrema acutum; Sch. acutum is now considered to be widely distributed, from the GoM to the western Pacific. Additionally, we describe a new species, Schikhobalotrema minutum n. sp., from Strongylura notata and Strongylura marina (Belonidae) from La Carbonera coastal lagoon, northern Yucatán, GoM. We briefly discuss the role of host association and historical biogeography of the hosts as drivers of species diversification of Schikhobalotrema infecting beloniforms.

How complex is the Naineris setosa species complex? First integrative study of a presumed cosmopolitan and invasive annelid (Sedentaria: Orbiniidae).

We performed a comparative study of the specimens from the Naineris setosa complex from the Pacific and the Atlantic Oceans and re-described the syntype of N. setosa, including the selection of the lectotype. Molecular phylogenetic and species delimitation analyses based on two mitochondrial (COI and 16S) and one nuclear (28S) marker revealed the presence of three species. One clade with wide Amphi-Atlantic distribution was attributed as Naineris setosa s. str. The second Atlantic clade restricted to Southern and Southeastern Brazil was described as a new species, Naineris lanai sp. n. The third clade, reported from the Northwestern Pacific, was identified as a new species but was not formally described due to the presence of only juvenile-sized worms in the studied material. Detailed morphological descriptions of several diagnostic characters in the Naineris setosa complex are provided.

Species delimitation and molecular phylogeny of the grasshopper subfamily Gomphocerinae (Orthoptera: Acrididae) from Algeria based on mitochondrial and nuclear DNA markers

Gomphocerinae grasshoppers (Orthoptera: Acrididae) are generally characterised by their polymorphism and cryptic diversity, which can be confusing when relying on morphological identification alone. DNA taxonomy serves as a powerful molecular tool for species identification and biodiversity assessment. In the context of zoogeography and the biodiversity conservation of animal resources in ecosystems, DNA barcoding data for Algerian Gomphocerinae fauna remains limited in global databases, despite their agroeconomic and environmental importance. Therefore, in this study, we collected various species of this subfamily from the Biskra region in Algeria and conducted DNA barcoding analysis, employing different molecular species delimitation methods (the automatic barcode gap discovery, assemble species by automatic partitioning, single-threshold general mixed Yule coalescent model, Bayesian Poisson tree process and multi-rate Poisson tree process methods), as well as phylogenetic analyses (maximum-likelihood and Bayesian inference) based on two molecular markers (COI and ITS). The results of the 163 newly generated sequences demonstrated that DNA barcoding technology is highly efficient and valuable for species identification in the subfamily Gomphocerinae, showing strong congruence with morphological evidence for 12 species, including eight species that were sequenced for the first time. This study also reported a new record of the genus Stenohippus Uvarov, 1926 in Algeria, comprising two species. Additionally, taxonomic revision allowed the species Dociostaurus biskrensis Moussi & Petit, 2014, to be transferred to the genus Stenohippus. Molecular tree analyses revealed the phylogenetic positions of the newly sequenced species within the subfamily Gomphocerinae and provided insights into their evolutionary relationships. These new data serve as a starting point for future research in other geographical areas, enabling a better understanding of the biodiversity of this insect group in Algeria.

Underestimated diversity: Integrative taxonomy of Mesenchytraeus (Enchytraeidae, Clitellata) from Changbai Mountain, China

AbstractThe holarctic genus Mesenchytraeus is one of the species‐rich genera in the family Enchytraeidae. Changbai Mountain supports high diversity of Mesenchytraeus species, making it an ideal area to explore species boundaries. We explored species boundaries of Mesenchytraeus using an integrative approach. Morphological taxonomy recognized 10 species in this region, five of them new to science. In contrast, molecular species delimitation analyses showed that there are at least 16 species, 11 of them new to science. Clear genetic gaps were observed among species with high interspecific distances (10%–21.2%) and low intraspecific distances (0.2%–6.7%) based on uncorrected p‐distance of the COI gene. Morphological species complex M. spermatoglomeratus consists of three species, M. spermatoglomeratus Zhang, Lu &amp; Xie, 2018 sensu stricto, M. rijina sp. n., and M. manchu sp. n. The M. duodiverticulus complex consists of M. duodiverticulus sp. n. and M. similiduodiverticulus sp. n. The M. monodiverticulus complex consists of M. monodiverticulus Shen, Chen &amp; Xie, 2012 sensu stricto, M. ngulen sp. n., M. zhenggulen sp. n., and M. fokulen sp. n. Further new species, distinguishable with morphological as well as molecular methods, are M. similigigachaetus sp. n., M. parvidiverticulus sp. n., M. digitalisdiverticulus sp. n., and M. infradiverticulus sp. n. Finally, we inferred the morphogenetic processes of spermathecae and sperm bundles, and filtrated some morphological characters which are useful to identify species. As the first attempt in this genus, our study provides an opportunity to discuss the currently used taxonomic criteria and acquire new ideas for the taxonomy of enchytraeids.

Phylogenetics, historical biogeography and molecular species delimitation of Chanohirata (Hemiptera: Cicadellidae: Deltocephalinae: Penthimiini)

AbstractThe genus Chanohirata Hayashi &amp; Machida, including the herein synonymized monotypic genus Reticuluma Cheng &amp; Li, is the second most speciose genus in the tribe Penthimiini after Penthimia Germar and is almost exclusively endemic to China. This study presents the first phylogeny of Chanohirata based on three mitochondrial markers (cytochrome c oxidase subunit I and II and 16S ribosomal RNA) and one nuclear fragment (28S ribosomal RNA). Reticuluma syn. n. is recovered nested within Chanohirata with strong support. Reticuluma is, therefore, proposed as a junior synonym of Chanohirata, and a new combination is proposed: Chanohirata citrana (Cheng &amp; Li) comb. n. Molecular species delimitation analyses were conducted by combining six molecular delimitation methods with morphological evidence. The results of the species delimitation analyses confirm the status of eight described species of Chanohirata and support the recognition of three new species: Chanohirata cornicula Wang &amp; Zhang sp. n., Chanohirata elongata Wang &amp; Zhang sp. n. and Chanohirata serrata Wang &amp; Zhang sp. n. Molecular dating and biogeographic analyses suggest that Chanohirata likely originated during the late Eocene in southern China. During the late Miocene to early Pleistocene, the uplift of the Himalayas and Tibetan Plateau and climatic oscillations probably triggered several dispersal and vicariance events in Chanohirata lineages, leading to most speciation events.

A cryptic radiation of Caribbean sea slugs revealed by integrative analysis: Cyerce 'antillensis' (Sacoglossa: Caliphyllidae) is six distinct species.

Integrative studies have revealed cryptic radiations in several Caribbean lineages of heterobranch sea slugs, raising questions about the evolutionary mechanisms that promote speciation within the tropical Western Atlantic. Cyerce Bergh, 1871 is a genus comprising 12 named species in the family Caliphyllidae that lack the photosynthetic ability of other sacoglossans but are noted for vibrant colours on the large cerata (dorsal leaf-like appendages) that characterize many species. Two species are widely reported from the Caribbean: Cyerce cristallina (Trinchese, 1881) and Cyerce antillensis Engel, 1927. Here, we present an integrative assessment of diversity in Caribbean Cyerce. Four methods of molecular species delimitation supported seven species in samples from the Caribbean and adjacent subtropical Western Atlantic. Six delimited species formed a monophyletic lineage in phylogenetic analyses but were > 9% divergent at the barcoding COI locus and could be differentiated using ecological, reproductive and/or morphological traits. We redescribe C. antillensis, a senior synonym for the poorly known Cyerce habanensis Ortea & Templado, 1988, and describe five new species. Evolutionary shifts in algal host use, penial armature and larval life history might have acted synergistically to promote the rapid divergence of endemic species with restricted distributions in this radiation, substantially increasing global diversity of the genus.

Taxonomic revision of the Stenopelmatus talpa species group (Orthoptera: Stenopelmatidae: Stenopelmatini)

We carried out a taxonomic revision of the Stenopelmatus talpa species group (Stenopelmatidae: Stenopelmatini), a poorly studied group of orthopterans whose species are distributed along the Trans-Mexican Volcanic Belt and adjacent areas of the Sierra Madre Oriental and Sierra Madre del Sur in Central Mexico. We examined both features from both the external morphological and male internal genitalia, and also carried out a molecular species delimitation based on DNA barcoding. An integrative approach based on congruence of morphological and molecular evidence helped to delimit 9 species for the S. talpa species group, 7 of which are new to science whereas the remaining 2 are already described, S. talpa and S. typhlops. We described 4 out of the above 7 new species: Stenopelmatus chilango sp. nov., S. purepecha sp. nov., S. sierragordaensis sp. nov. and S. tlaxcalli sp. nov. A key to the described species of the S. talpa species group is provided. Our study highlights the relevance of the male internal genitalia as a valuable character system to delimit species in Stenopelmatus.

Questioning the monophyly of Anthroherponina (Coleoptera: Leiodidae: Cholevinae: Leptodirini) and description of three new, ecologically ultraspecialized subterranean species

Abstract The beetle subtribe Anthroherponina (Coleoptera: Leiodidae), including also ecologically specialized and semi-aquatic hygropetricolous genera, presents one of the most illustrative examples of morphological variability in cave-adapted animals. However, phylogenetic relations within the tribe remain dubious. Recent exploration of deep-phreatic caves of the Western Balkans Dinaric Karst enabled us to collect all known Anthroherponina genera and access new populations of hygropetricolous beetles, representatives of the iconic Hadesia and elusive Nauticiella. We studied the taxonomic structure of newly collected semi-aquatic beetle populations by using traditional morphology and deploying uni- and multilocus molecular species delimitation. In addition, we tested the monophyly of the subtribe Anthroherponina and set the group’s evolutionary development into a palaeogeographic context by reconstructing a multilocus calibrated phylogeny. As a result, three new species of specialized semi-aquatic beetles are described. The morphologically based monophyly of Anthroherponina was shown to be an artefact of evolutionary convergences, while the evolutionary development of the monophylum seems to be paired with the orogeny of the Dinarides. Finally, the new results raise more questions about our understanding of evolution in subterranean beetles and their adaptation to special ecological niches.