Testing conflicting taxonomic hypotheses in myrmecophilous Oochrotus Lucas, 1852 (Coleoptera, Tenebrionidae)

159 Testing conflicting taxonomic hypotheses in myrmecophilous Oochrotus Lucas, 1852 (Coleoptera, Tenebrionidae) Julene Gómez-Vicioso1, Álvaro Conca-Esquembre1,2 , Pilar Jurado-Angulo1,3,4 , Mario García-París1 1 Department of Biodiversity and Evolutionary Biology, Museo Nacional de Ciencias Naturales (MNCN-CSIC), c/ José Gutiérrez Abascal, 2. 28006, Madrid, Spain 2 Division of Ecology and Evolution (E&E), Research School of Biology (RSB), Australian National University, Canberra, ACT, Australia 3 CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, InBIO Laboratório Associado e Faculdade de Ciências da Universidade do Porto, Vairão, Portugal 4 Universidade Técnica do Atlântico, UTA – Instituto de Engenharias e Ciências do Mar (ISECMAR), Mindelo, Cabo Verde Corresponding author: Julene Gómez-Vicioso ([email protected]) Copyright: © Julene Gómez-Vicioso et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract Cryptic and pseudocryptic species are common in myrmecophilous insects, making their taxonomic classification complex when based solely on morphology. This is the case for the beetles of the genus Oochrotus Lucas, 1852, a group of small tenebrionids inhabiting ant nests. In 1961, Canzoneri described one new species and eight subspecies based on the morphology of the aedeagus and ovipositor. However, in 2000, Soldati and Soldati synonymised most of these taxa, arguing that the differences found by Canzoneri were not significant. The aim of our study was to test these two competing hypotheses using a molecular approach. For this purpose, partial sequences of the nuclear gene ITS2 and the mitochondrial gene cytb were obtained from individuals from North Africa, Italy, and the Iberian Peninsula, followed by phylogenetic analyses based on Bayesian inference. The results show that specimens from these three territories are in separate lineages corresponding to three different species: 1) O. unicolor Lucas, 1852; 2) O. laurae Canzoneri, 1961, stat. rev., and 3) O. lusitanicus Canzoneri, 1961, stat. nov. (= O. u. espagnoli Canzoneri, 1961, syn. nov.; = O. u. hispanus Canzoneri, 1961, syn. nov.; = O. u. meridionalis Canzoneri, 1961, syn. nov.). This new proposal diverges from both preceding hypotheses, showing an intermediate level of diversity between the two. This reflects that species of the genus Oochrotus are probably pseudocryptic, whose morphological examination may lead to misidentification in the absence of molecular data. Key words: Beetles, evolution, mitochondrial DNA, molecular analysis, nuclear DNA, phylogeography, western Mediterranean Introduction Myrmecophilous species are those species that live in association with ants. The term myrmecophilous encompasses a total of about 10,000 species of arthropods (Elmes 1996), although not all of them are related to ants in the same way. The classical concept involves a mutualistic type of relationship, usually found in the order Hemiptera (Ivens, 2015). However, other insects have a parasitoid or social parasitism association with ants. The latter case occurs in the Academic editor: Patrice Bouchard Received: 12 April 2025 Accepted: 10 October 2025 Published: 3 November 2025 ZooBank: https://zoobank. org/38FC3BA3-7114-489D-9AD54FDA150EB046 Citation: Gómez-Vicioso J, ConcaEsquembre Á, Jurado-Angulo P, García-París M (2025) Testing conflicting taxonomic hypotheses in myrmecophilous Oochrotus Lucas, 1852 (Coleoptera, Tenebrionidae). ZooKeys 1258: 159–174. https://doi. org/10.3897/zookeys.1258.155620 ZooKeys 1258: 159–174 (2025) DOI: 10.3897/zookeys.1258.155620 160 ZooKeys 1258: 159–174 (2025), DOI: 10.3897/zookeys.1258.155620 Julene Gómez-Vicioso et al.: Resolving Oochrotus taxonomy with a molecular approach order Coleoptera, in which beetles exploit the resources and social structure of the ants, inflicting a cost on their communities, but without increasing direct mortality (Parker 2016). Myrmecophilous beetles are currently known to occur in at least 33 families within the order Coleoptera (Hölldobler and Wilson 1990; Parker 2016). In general, this interaction has been most studied in the subfamilies Aleocharinae (Kistner 1993) and Pselaphinae (Chandler 2001) within Staphylinidae, in the subfamily Paussinae (Geiselhardt et al. 2007) within the family Carabidae (Di Giulio et al. 2003), and in some species of the family Coccinellidae (Vantaux et al. 2012) and Scarabaeidae (Vaz-de-Mello et al. 1998). Through the study of the different lineages, it has been observed that myrmecophilous beetles tend to have common adaptations that allow them to access ant nests, integrate into colonies and exploit their resources. These include, among others, the ability to camouflage themselves chemically (Vander Meer and Wojcik 1982; Akino 2002; Lenoir et al. 2013) and to present morphological structures that make them difficult for ants to attack (Parker 2016). In addition to converging on similar morphological features, these beetles tend to share common traits across closely related species, a pattern also observed in other groups of myrmecophilous insects (Thomas et al. 1989; Elmes et al. 1994, 1999; Schönrogge et al. 2002). This morphological homogeneity is a source of conflict for taxonomic studies, often rendering the definition of taxa complicated. One example of taxonomic complexity is provided by the genus Oochrotus Lucas, 1852. Oochrotus is a genus of small myrmecophilous tenebrionid beetles included within the tribe Crypticini in the subfamily Diaperinae (Iwan et al. 2020). They are small organisms (2–3 mm), with an ovoid convex body. As regards their colouring, they present an earthy orange-brown testaceous colour, somewhat similar to that of the arid substrates they inhabit. It is noteworthy that they lack posterior wings and that they have no eyes, the latter character being used to separate them from the rest of the Palaearctic Crypticini (Español 1955). Their distribution is limited to the Mediterranean region (Canzoneri 1961; Español 1963; Cabon and Soldati 2024), where they are found inhabiting the nests of the ants of the genus Messor Forel, 1890 (Español 1949, 1955, 1963). They feed on the remains of seeds, flour, or other debris in the waste pits of the ants’ nests (Parmentier et al. 2019). The taxonomy of the genus Oochrotus is still unresolved, and the authors who have worked with the group have presented very different perspectives on the internal diversity within it. After the description of the genus Oochrotus based on its type species, O. unicolor Lucas, 1852, little further work was done on the diversity of the group during the next century, the most notable being the description of the Eastern Mediterranean O. glaber Demaison, 1905 (Lucas 1852; Demaison 1905; Lokay 1907; Koch 1935). It was not until Canzoneri (1961) that an intensive effort was made to describe the diversity within the genus, based mainly on the morphology of the aedeagus and the distal end of the ovipositor. Canzoneri (1961) retained O. glaber and O. unicolor, and defined six additional subspecies within O. unicolor, four of them distributed in different regions of the Iberian Peninsula, and two in Italy. He also described a new species, O. laurae Canzoneri, 1961, with two subspecies distributed in Italy. Subsequently, Rallo (1974) disregarded O. laurae, including its two subspecies within O. unicolor, and he also described a new Italian subspecies. Soldati and Solda- 161 ZooKeys 1258: 159–174 (2025), DOI: 10.3897/zookeys.1258.155620 Julene Gómez-Vicioso et al.: Resolving Oochrotus taxonomy with a molecular approach ti (2000) presented a totally different perspective, and after reviewing several populations of O. unicolor, they considered that the morphological differences reported by Canzoneri (1961) were not significant, synonymising six subspecies with O. u. unicolor. Soldati and Soldati (2000) additionally remarked that, despite their inability to study all the subspecies proposed by Canzoneri (1961), future revisionary efforts will most likely be able to generalize the synonymy of all the remaining subspecies. The aim of our study was to test the conflicting hypotheses proposed by Canzoneri (1961) and Soldati and Soldati (2000) with respect to the internal subdivision of O. unicolor by using a molecular approach. The first hypothesis suggests that two species with nine subspecies can be distinguished within the group, whereas the second considers one species including four not studied subspecies (see Table 1 for details). These contrasting hypotheses are based on the relative importance given to certain morphological characters as taxonomically diagnostic, more particularly to the genital structures. To test them, we performed phylogenetic analyses with partial sequences of two widely used DNA markers, mtDNA cytochrome b gene (cytb) and nuclear internal transcribed spacer 2 (ITS2) and revised previous statements of morphological diversification. Methods Taxon sampling Specimens were collected opportunistically at 10 localities in the Iberian Peninsula, North Africa, and Italy with the aim of covering part of the known distribution of the group. Sampling was initially directed towards type localities, but most of the specimens were found in non-selected areas. Identification was primarily based on geography, with a review of morphological characters, Table 1. Taxonomic hypotheses proposed by Canzoneri (1961), Soldati and Soldati (2000) and the present study with respect to the internal subdivision of Oochrotus unicolor. The different taxonomic positions of the described (sub)species are shown according to each proposal, together with their type localities. Taxa regarded as valid under each hypothesis are shown in bold and, when synonymised, the taxon in which they are included is shown in regular font. Taxonomic decisions newly proposed in this study are indicated with light gray shading. Taxa not examined in this study and whose taxonomic assignments were based on Canzoneri (1961) or Soldati and Soldati (2000) are marked with an asterisk (*). Note that O. unicolor chilivanii does not appear in Canzoneri’s original hypothesis (indicated by ‘–’ in the table), as it was described later by Rallo (1974); it is nevertheless included here for clarity. Canzoneri (1961) Soldati and Soldati (2000) This study Type locality O. laurae laurae Canzoneri, 1961 O. unicolor unicolor O. laurae stat. rev. “Moscona (Grossetto)” O. laurae sardous Canzoneri, 1961 O. unicolor unicolor O. unicolor unicolor*“Flumentorgiu (Sardegna)” O. unicolor unicolor Lucas, 1852 O. unicolor unicolor O. unicolor unicolor “plateaux de Médéah et de Boghar” O. unicolor ardoini Canzoneri, 1961 O. unicolor ardoini O. unicolor ardoini* “Roma dintorni” O. unicolor chilivanii Rallo, 1974 (–) O. unicolor unicolor O. unicolor unicolor*“Chilivani (Sassari)” O. unicolor espagnoli Canzoneri, 1961 O. unicolor unicolor O. lusitanicus syn. nov. “Tiana, prov. Barcellona” O. unicolor hispanus Canzoneri, 1961 O. unicolor unicolor O. lusitanicus syn. nov. “Robledo (Madrid)” O. unicolor lusitanicus Canzoneri, 1961 O. unicolor lusitanicus O. lusitanicus stat. nov. “Evora (Portogallo)” O. unicolor meridionalis Canzoneri, 1961 O. unicolor unicolor O. lusitanicus syn. nov. “Algeciras” O. unicolor moltonii Canzoneri, 1961 O. unicolor moltonii O. unicolor moltonii* “Ficuzza (Palermo)” 162 ZooKeys 1258: 159–174 (2025), DOI: 10.3897/zookeys.1258.155620 Julene Gómez-Vicioso et al.: Resolving Oochrotus taxonomy with a molecular approach especially in the case of the Italian population (see Results). Specimens were located under stones, along tunnels occupied by ants of the genus Messor or walking on the underside of the stones, generally in grassland areas, at the edge of Quercus rotundifolia Lam. patches. At each locality individuals were visually searched, hand-collected, and georeferenced. All were preserved in 96% to absolute ethanol. In each population, 1–15 individuals were included in the molecular analysis, and at least one individual was retained for future morphological studies. All specimens are stored at the Museo Nacional de Ciencias Naturales (MNCN-CSIC) (Madrid, Spain). DNA extraction and sequencing DNA was extracted from a total of 57 individuals of the genus Oochrotus (Table 2). For this purpose, the specimens were punctured in the upper abdomen and the entire individuals were included in the extraction buffer. Total genomic DNA was extracted using the Qiagen DNeasy extraction kit (Qiagen) and following the protocol indicated by the manufacturer. PCRs were performed to amplify the sequences of the cytb and ITS2 markers. For cytb, a 25 μl mix was used, which included 17.55 μl of H20, 2.5 μl of Nzytech reaction buffer (10×), 1.75 μl of MgCl2 (50 mM), 1 μl of dNTP (10 mM), 0.5 μl of both forward and reverse primers (10 μM), 0.2 μl of Taq polymerase (Nzytech, 5 U/μL) and 1 μl of sample DNA. The primers used were CB-J-10933 (Simon et al. 1994) as forward and CB4 (Pons 2006) as reverse. PCR conditions were as follows: 5 min at 96 °C for initial denaturation, 35–40 cycles of 1 min of denaturation at 94 °C, 1 min of annealing at 40–41 °C and 1 min of extension at 72 °C, with a final extension at 72 °C for 5 min. In the case of ITS2, a 25 μl mix was used, which included 17.8 μl of H20, 2.5 μl of Nzytech reaction buffer (10×), 1.5 μl of MgCl2 (50 mM), 1 μl of dNTP (10 mM), 0.5 μl of both forward and reverse primers (10 μM), 0.2 μl of Taq polymerase (Nzytech, 5 U/μL) and 1 μl of sample DNA. The primers used were Cas5p8sFc as forward and CAS28sB1d as reverse (Ji et al. 2003). PCR conditions were as follows: 5 min at 96 °C for initial denaturation, 40 cycles of 30 s of denaturation at 94 °C, 45 s of annealing at 45 °C and 1 min of extension at 72 °C, with a final extension at 72 °C for 5 min. The amplification products were verified via electrophoresis on 0.8% agarose gels and then sent for Sanger sequencing to Macrogen Spain Inc. (Macrogen Europe, Amsterdam, The Netherlands). The chromatograms and their sequences were individually checked and then aligned using the ClustalW Multiple Alignment tool (BioEdit Sequence Alignment Editor v. 7.7.1.0). The sequences of cytb for 12 specimens and ITS2 for three specimens were not obtained due to problems in the amplification or sequencing processes. As a result, a dataset of 45 specimens and 349 base pairs for cytb and another of 54 specimens and 510 base pairs for ITS2 were obtained. Phylogenetic analysis Independent phylogenetic analyses were performed for cytb and ITS2. For both markers, sequences of phylogenetically close taxa were searched in GenBank Data Libraries and selected as outgroups. For cytb a sequence of 163 ZooKeys 1258: 159–174 (2025), DOI: 10.3897/zookeys.1258.155620 Julene Gómez-Vicioso et al.: Resolving Oochrotus taxonomy with a molecular approach Table 2. List of Oochrotus specimens included in the study. Species identity, collection localities and coordinates, voucher numbers, MNCN-CSIC identification codes and GenBank accession numbers are shown. Species Locality GPS Coordinates Voucher MNCN Genbank cytb Genbank ITS2 O. laurae Lazio: Ciudad metropolitana de Roma Capital: Allumiere 42°08'00"N; 11°54'07"E jgv22001a 366745 PQ376647 PQ348543 Lazio: Ciudad metropolitana de Roma Capital: Allumiere 42°08'00"N; 11°54'07"E jgv22002a 366746 PQ376648 Lazio: Ciudad metropolitana de Roma Capital: Allumiere 42°08'00"N; 11°54'07"E jgv22003a 366747 PQ376649 PQ348544 Lazio: Ciudad metropolitana de Roma Capital: Allumiere 42°08'00"N; 11°54'07"E jgv22005a 389968 PQ376650 PQ348545 O. lusitanicus Andalucía: Huelva: Santa Olalla del Cala 37°54'25"N; 6°14'09"W jgv22007b 389969 PQ376651 PQ348546 O. lusitanicus Andalucía: Huelva: Santa Olalla del Cala 37°54'25"N; 6°14'09"W jgv22008b 389970 PQ376652 PQ348547 O. lusitanicus Andalucía: Huelva: Santa Olalla del Cala 37°54'25"N; 6°14'09"W jgv22009b 389971 PQ376653 PQ348548 O. lusitanicus Andalucía: Huelva: Santa Olalla del Cala 37°54'25"N; 6°14'09"W jgv22010b 389972 PQ376654 PQ348549 O. lusitanicus Andalucía: Huelva: Santa Olalla del Cala 37°54'25"N; 6°14'09"W jgv22011b 389973 PQ376655 PQ348550 O. lusitanicus Andalucía: Sevilla: El Coronil 37°03'02"N; 5°37'40"W jgv22064j 390007 PQ376681 PQ348584 O. lusitanicus Andalucía: Sevilla: El Coronil 37°03'02"N; 5°37'40"W jgv22065j 390008 PQ376682 PQ348585 O. lusitanicus Andalucía: Sevilla: El Coronil 37°03'02"N; 5°37'40"W jgv22066j 390009 PQ376683 PQ348586 O. lusitanicus Andalucía: Sevilla: El Coronil 37°03'02"N; 5°37'40"W jgv22067j 390010 PQ376684 O. lusitanicus Andalucía: Sevilla: El Coronil 37°03'02"N; 5°37'40"W jgv22068j 390011 PQ348587 O. lusitanicus Andalucía: Sevilla: El Coronil 37°03'02"N; 5°37'40"W jgv22069j 390012 PQ376685 PQ348588 O. lusitanicus Andalucía: Sevilla: El Coronil 37°03'02"N; 5°37'40"W jgv22071j 390013 PQ348589 O. lusitanicus Andalucía: Sevilla: El Coronil 37°03'02"N; 5°37'40"W jgv22072j 390014 PQ376686 PQ348590 O. lusitanicus Andalucía: Sevilla: El Coronil 37°03'02"N; 5°37'40"W jgv22073j 390015 PQ376687 O. lusitanicus Andalucía: Sevilla: El Coronil 37°03'02"N; 5°37'40"W jgv22074j 390016 PQ348591 O. lusitanicus Andalucía: Sevilla: El Coronil 37°03'02"N; 5°37'40"W jgv22075j 390017 PQ376688 PQ348592 O. lusitanicus Andalucía: Sevilla: El Coronil 37°03'02"N; 5°37'40"W jgv22076j 390018 PQ376689 PQ348593 O. lusitanicus Andalucía: Sevilla: El Coronil 37°03'02"N; 5°37'40"W jgv22077j 390019 PQ376690 PQ348594 O. lusitanicus Andalucía: Sevilla: El Coronil 37°03'02"N; 5°37'40"W jgv22078j 390020 PQ348595 O. lusitanicus Andalucía: Sevilla: El Coronil 37°03'02"N; 5°37'40"W jgv22079j 390021 PQ376691 PQ348596 O. lusitanicus Castilla-La Mancha: Albacete: Chinchilla de Montearagón 38°55'23"N; 1°43'43"W jgv22013c 389974 PQ376656 PQ348551 O. lusitanicus Castilla-La Mancha: Albacete: Chinchilla de Montearagón 38°55'23"N; 1°43'43"W jgv22014c 389975 PQ348552 O. lusitanicus Castilla-La Mancha: Albacete: Chinchilla de Montearagón 38°55'23"N; 1°43'43"W jgv22015c 389976 PQ376657 PQ348553 O. lusitanicus Castilla-La Mancha: Albacete: Chinchilla de Montearagón 38°55'23"N; 1°43'43"W jgv22016c 389977 PQ376658 PQ348554 O. lusitanicus Castilla-La Mancha: Albacete: Chinchilla de Montearagón 38°55'23"N; 1°43'43"W jgv22017c 389978 PQ376659 PQ348555 O. lusitanicus Castilla-La Mancha: Albacete: Chinchilla de Montearagón 38°55'23"N; 1°43'43"W jgv22018c 389979 PQ376660 PQ348556 O. lusitanicus Castilla-La Mancha: Albacete: Chinchilla de Montearagón 38°55'23"N; 1°43'43"W jgv22019c 389980 PQ376661 PQ348557 O. lusitanicus Castilla-La Mancha: Albacete: Chinchilla de Montearagón 38°55'23"N; 1°43'43"W jgv22020c 389981 PQ376662 PQ348558 O. lusitanicus Castilla-La Mancha: Albacete: Chinchilla de Montearagón 38°55'23"N; 1°43'43"W jgv22021c 389982 PQ376663 PQ348559 O. lusitanicus Castilla-La Mancha: Albacete: Chinchilla de Montearagón 38°55'23"N; 1°43'43"W jgv22022c 389983 PQ376664 PQ348560 164 ZooKeys 1258: 159–174 (2025), DOI: 10.3897/zookeys.1258.155620 Julene Gómez-Vicioso et al.: Resolving Oochrotus taxonomy with a molecular approach Species Locality GPS Coordinates Voucher MNCN Genbank cytb Genbank ITS2 O. lusitanicus Castilla-La Mancha: Albacete: Chinchilla de Montearagón 38°55'23"N; 1°43'43"W jgv22023c 389984 PQ376665 PQ348561 O. lusitanicus Castilla-La Mancha: Ciudad Real: Fontanosas 38°45'48"N; 4°32'34"W jgv22059i 390003 PQ376677 PQ348580 O. lusitanicus Castilla-La Mancha: Ciudad Real: Fontanosas 38°45'48"N; 4°32'34"W jgv22060i 390004 PQ376678 PQ348581 O. lusitanicus Castilla-La Mancha: Ciudad Real: Fontanosas 38°45'48"N; 4°32'34"W jgv22061i 390005 PQ376679 PQ348582 O. lusitanicus Castilla-La Mancha: Ciudad Real: Fontanosas 38°45'48"N; 4°32'34"W jgv22062i 390006 PQ376680 PQ348583 O. lusitanicus Castilla-La Mancha: Ciudad Real: Poblete 38°54'14"N; 4°00'31"W jgv22058h 390002 PQ376676 PQ348579 O. lusitanicus Castilla-La Mancha: Toledo: Fuentes 39°40'05"N; 5°03'55"W jgv22051f 389999 PQ376673 PQ348576 O. lusitanicus Castilla-La Mancha: Toledo: Fuentes 39°40'05"N; 5°03'55"W jgv22052f 390000 PQ376674 PQ348577 O. lusitanicus Extremadura: Badajoz: Montemolín 38°09'15"N; 6°12'28"W jgv22057g 390001 PQ376675 PQ348578 O. lusitanicus Extremadura: Badajoz: Villanueva del Fresno 38°22'27"N; 7°09'01"W jgv22042e 389997 PQ348574 O. lusitanicus Extremadura: Badajoz: Villanueva del Fresno 38°22'27"N; 7°09'01"W jgv22043e 389998 PQ376672 PQ348575 O. unicolor Tánger-Tetuán-Alhucemas: Fahs-Anyera: Ksar Sghir 35°46'20"N; 5°31'36"W jgv22026d 389985 PQ376666 PQ348562 O. unicolor Tánger-Tetuán-Alhucemas: Fahs-Anyera: Ksar Sghir 35°46'20"N; 5°31'36"W jgv22027d 389986 PQ376667 PQ348563 O. unicolor Tánger-Tetuán-Alhucemas: Fahs-Anyera: Ksar Sghir 35°46'20"N; 5°31'36"W jgv22028d 389987 PQ376668 PQ348565 O. unicolor Tánger-Tetuán-Alhucemas: Fahs-Anyera: Ksar Sghir 35°46'20"N; 5°31'36"W jgv22029d 389988 PQ348564 O. unicolor Tánger-Tetuán-Alhucemas: Fahs-Anyera: Ksar Sghir 35°46'20"N; 5°31'36"W jgv22030d 389989 PQ376669 PQ348566 O. unicolor Tánger-Tetuán-Alhucemas: Fahs-Anyera: Ksar Sghir 35°46'20"N; 5°31'36"W jgv22031d 389990 PQ348567 O. unicolor Tánger-Tetuán-Alhucemas: Fahs-Anyera: Ksar Sghir 35°46'20"N; 5°31'36"W jgv22032d 389991 PQ348568 O. unicolor Tánger-Tetuán-Alhucemas: Fahs-Anyera: Ksar Sghir 35°46'20"N; 5°31'36"W jgv22033d 389992 PQ376670 PQ348569 O. unicolor Tánger-Tetuán-Alhucemas: Fahs-Anyera: Ksar Sghir 35°46'20"N; 5°31'36"W jgv22034d 389993 PQ348570 O. unicolor Tánger-Tetuán-Alhucemas: Fahs-Anyera: Ksar Sghir 35°46'20"N; 5°31'36"W jgv22035d 389994 PQ376671 PQ348571 O. unicolor Tánger-Tetuán-Alhucemas: Fahs-Anyera: Ksar Sghir 35°46'20"N; 5°31'36"W jgv22036d 389995 PQ348572 O. unicolor Tánger-Tetuán-Alhucemas: Fahs-Anyera: Ksar Sghir 35°46'20"N; 5°31'36"W jgv22037d 389996 PQ348573 Strongylium cf. indignum Gebien, 1920 (Accession KX461872.1, Soldati et al. 2016) was used, whereas for ITS2 a sequence of Tenebrio molitor Linnaeus, 1758 (Accession AJ635266.1, Bologna et al. 2008) was used. For both markers, phylogenetic reconstruction was carried out by Bayesian inference using the MrBayes program (Huelsenbeck and Ronquist 2001; Ronquist et al. 2012). Two runs were programmed with four chains that ran over 10 million generations, sampling trees every 100 generations. The program was instructed to calculate and use the best model of substitution and best codon partition scheme. In the case of cytb it was the model M142 = 111234 and in the case of ITS2 the model M15 = 121121 (known as HKY model). The convergence 165 ZooKeys 1258: 159–174 (2025), DOI: 10.3897/zookeys.1258.155620 Julene Gómez-Vicioso et al.: Resolving Oochrotus taxonomy with a molecular approach between both chains was evaluated taking into account the maximum-likelihood value. After obtaining all the trees, a consensus tree was generated with all of them, previously eliminating the first 25,000 trees as burn-in. Phylogeographic analyses were carried out by network reconstruction. For this purpose, allele networks were constructed using Population Analysis with Reticulate Trees (PopART) (Leigh and Bryant 2015), applying the TCS algorithm (Clement et al. 2002). In the occasional cases where nucleotide ambiguity was observed, the allele that was common to the rest of the specimens in the region was selected, following a maximum parsimony criterion. Results Bayesian reconstruction for cytb (Fig. 1a) showed that specimens are clustered in three main groups. Two of them are supported groups, corresponding to the Italian (Clade A, PP = 1) and Spanish (Clade B, PP = 0.92) populations. In the case of the Moroccan population (Clade C), although most individuals are genetically identical, they appear to form a polytomy with the Spanish populations (a common artefact in Bayesian phylogenetic analyses when identical sequences are included, due to the lack of informative sites for resolving branching order). In the case of the ITS2 marker (Fig. 1b), the same three groups are also differentiated. In this case it is the Moroccan (Clade C, PP = 0.95) and Italian (Clade A, PP = 0.99) populations which form well-supported groups, whereas the sequences from the Spanish populations (Clade B) are artefactually represented as a polytomy. The individual jgv22035d from Morocco, which clustered with other Moroccan individuals in the ITS2 tree, showed an ambiguous position in the cytb tree. This particular specimen showed a high level of heteroplasmy in the mitochondrial marker cytb, producing alleles that cluster in different positions of the tree (Fig. 1). The haplotypic network obtained for cytb (Fig. 2a) showed a total of 10 haplotypes, separated into three main haplogroups. The first one corresponds to the Italian individuals (Clade A), where a single haplotype is found, clearly differentiated in 35 positions from the next closest haplogroup, the one from Morocco. In Morocco (Clade C), three haplotypes are found, separated by only one position between them. The last haplogroup, that of the Iberian Peninsula (Clade B), is the most complex of them. Here there are a total of six haplotypes, which can be grouped into two subgroups. On the one hand there is a group of specimens from the central-eastern peninsula (Clade B.1), and on the other, those from the southwest (Clade B.2). One haplotype from the south of Badajoz was found in between, close to both groups. It is noteworthy that both subgroups are notably different from each other (seven positions), even more than with respect to the Moroccan population (three and four positions, respectively). In the case of the ITS2 marker (Fig. 2b), three nuclear alleles were found, each corresponding to one of the three main regions studied: Italy (Clade A), the Iberian Peninsula (Clade B), and Morocco (Clade C). The three are closely related, with a difference of two positions between the Iberian Peninsula and Morocco and four positions between these two territories and Italy. Geographical structuring, and congruence between nuclear and mtDNA markers, support a well-defined taxonomic structure in our sampling reflected in the existence of three evolutionary units, that represent independent species, 166 ZooKeys 1258: 159–174 (2025), DOI: 10.3897/zookeys.1258.155620 Julene Gómez-Vicioso et al.: Resolving Oochrotus taxonomy with a molecular approach jgv22007b_Huelva jgv22008b_Huelva jgv22009b_Huelva jgv22010b_Huelva jgv22011b_Huelva jgv22013c_Albacete jgv22014c_Albacete jgv22015c_Albacete jgv22016c_Albacete jgv22017c_Albacete jgv22018c_Albacete jgv22019c_Albacete jgv22020c_Albacete jgv22021c_Albacete jgv22022c_Albacete jgv22023c_Albacete jgv22042e_Badajoz jgv22043e_Badajoz jgv22051f_Toledo jgv22052f_Toledo jgv22057g_Badajoz jgv22058h_Ciudad Real jgv22059i_Ciudad Real jgv22060i_Ciudad Real jgv22061i_Ciudad Real jgv22062i_Ciudad Real jgv22064j_Sevilla jgv22065j_Sevilla jgv22066j_Sevilla jgv22068j_Sevilla jgv22069j_Sevilla jgv22071j_Sevilla jgv22072j_Sevilla jgv22074j_Sevilla jgv22075j_Sevilla jgv22076j_Sevilla jgv22077j_Sevilla jgv22078j_Sevilla jgv22079j_Sevilla jgv22001a_Italy jgv22003a_Italy jgv22005a_Italy jgv22026d_Morocco jgv22027d_Morocco jgv22028d_Morocco jgv22029d_Morocco jgv22030d_Morocco jgv22031d_Morocco jgv22032d_Morocco jgv22033d_Morocco jgv22034d_Morocco jgv22035d_Morocco * jgv22036d_Morocco jgv22037d_Morocco Tenebrio 1 0.99 0.67 0.95 O. laurae O. unicolor O. lusitanicus (Clade A) (Clade B) (Clade C) jgv22001a_Italy jgv22002a_Italy jgv22003a_Italy jgv22005a_Italy jgv22007b_Huelva jgv22008b_Huelva jgv22009b_Huelva jgv22010b_Huelva jgv22011b_Huelva jgv22035d_Morocco* jgv22064j_Sevilla jgv22065j_Sevilla jgv22066j_Sevilla jgv22067j_Sevilla jgv22069j_Sevilla jgv22072j_Sevilla jgv22073j_Sevilla jgv22075j_Sevilla jgv22076j_Sevilla jgv22077j_Sevilla jgv22079j_Sevilla jgv22057g_Badajoz jgv22013c_Albacete jgv22015c_Albacete jgv22016c_Albacete jgv22017c_Albacete jgv22018c_Albacete jgv22019c_Albacete jgv22020c_Albacete jgv22021c_Albacete jgv22022c_Albacete jgv22023c_Albacete jgv22043e_Badajoz jgv22051f_Toledo jgv22052f_Toledo jgv22058h_Ciudad Real jgv22059i_Ciudad Real jgv22060i_Ciudad Real jgv22061i_Ciudad Real jgv22062i_Ciudad Real jgv22026d_Morocco jgv22027d_Morocco jgv22028d_Morocco jgv22030d_Morocco jgv22033d_Morocco Strongylium 1 0.78 0.78 0.92 0.89 0.98 0.98 1 O. laurae O. unicolor O. lusitanicus jgv22035d_Morocco 0.87 (Clade A) (Clade B) (Clade C) a) cytb b) ITS2 Figure 1. Bayesian phylogenetic hypothesis based on a. cytb mitochondrial data and b. ITS2 nuclear data. The colours represent the different lineages recovered in the study and their distribution on the map: Oochrotus lusitanicus in the Iberian Peninsula (orange), O. laurae in Italy (pink) and O. unicolor in North Africa. Numbers near the nodes indicate Posterior Probabilities values (PP). The position in the tree of the alleles of a possible heteroplasmic individual for the cytb marker, jgv22035d, is reflected based on two different assumptions. The first is assuming ambiguity at loci with more than one allele (with *) and the second is choosing the most parsimonious alleles according to its population of origin (without *). In both trees there are populations with individuals with identical sequences (the Moroccan in the case of cytb and the Iberian Peninsular in the case of ITS2) forming polytomies with phylogenetically close groups. This is an artefact in Bayesian inference when several identical sequences are included in the analyses. 167 ZooKeys 1258: 159–174 (2025), DOI: 10.3897/zookeys.1258.155620 Julene Gómez-Vicioso et al.: Resolving Oochrotus taxonomy with a molecular approach Figure 2. TCS network of Oochrotus based on a) the mitochondrial marker cytb and b) the nuclear marker ITS2. The size of the circles indicates the relative frequency of sequences belonging to a particular allele and the colours correspond to the geographic origin of the specimens. Inferred intermediate haplotypes are represented by small black circles. The North African, Iberian Peninsula and Italian populations differed from each other in both markers, with no alleles shared between them. In the case of the Iberian Peninsula, some genetic structuring appeared in the cytb marker, but it was absent for the ITS2 marker. !"#!"#$ $"#%&'( Badajoz (South) Albacete Ciudad Real Toledo Huelva Sevilla Morocco Italy 1 sample 10 samples Clade A Clade C Clade B Clade A Clade C Clade B Clade B.1 Clade B.2 a) cytb b) ITS2 Badajoz (Southwest) corresponding to the three main lineages found: North African, Italian, and Iberian. Our sampling is quite representative of the Iberian taxon, that might include all previously proposed names within the region (O. u. lusitanicus Canzoneri, 1961; O. u. espagnoli Canzoneri, 1961; O. u. hispanus Canzoneri, 1961; O. u. meridionalis Canzoneri, 1961). Since all the available names were proposed in the same work by Canzoneri (1961), they all have equal priority. We choose as the specific epithet the name with the type locality geographically closest to any of our studied populations (Évora to Villanueva del Fresno), which is O. lusitanicus Canzoneri, 1961. The only available name for northwestern Africa is O. unicolor, which is therefore retained for the African lineage represented by our Moroccan sample. The Italian name is more problematic since our single sample is almost equally distant from O. u. ardoini Canzoneri, 1961 and O. laurae Canzoneri, 1961. 174 ZooKeys 1258: 159–174 (2025), DOI: 10.3897/zookeys.1258.155620 Julene Gómez-Vicioso et al.: Resolving Oochrotus taxonomy with a molecular approach polymerase chain reaction primers. 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