Evolution of pre‐zygotic and post‐zygotic barriers to gene flow among three cryptic species within the Anastrepha fraterculus complex

Tropical tephritids are ideally suited for studies on population divergence and speciation because they include species groups undergoing rapid radiation, in which morphologically cryptic species and sister species are abundant. The fraterculus species group in the Neotropical genus Anastrepha is a case in point, as it is composed of a complex of up to seven A. fraterculus morphotypes proposed to be cryptic species. Here, we document pre‐ and post‐zygotic barriers to gene flow among adults of the Mexican A. fraterculus morphotype and three populations (Argentina, Brazil, and Peru) belonging to two separate morphotypes (Brazilian 1 and Peruvian). We unveiled three forms of pre‐zygotic reproductive isolation resulting in strong assortative mating. In field cages, free‐ranging male and female A. fraterculus displayed a strong tendency to form couples with members of the opposite sex belonging to their own morphotype, suggesting that male pheromone emission, courtship displays, or both intervene in shaping female choice before actual contact and coupling. In addition, males and females of the Peruvian morphotype became receptive and mated significantly later than adults of the Mexican and Brazilian 1 morphotypes. After contact, Mexican females exhibited greater mating discrimination than males when facing adults of the opposite sex belonging to either the Peruvian or the Brazilian 1 morphotype as evidenced by vigorous resistance to penetration once they had been forcefully mounted by heterotypic males. Forced copulations resulted in production of F1 hybrids that were either less viable (and partially fertile) than parental crosses or even sterile. Our results suggest that the Mexican morphotype is a distinct biological entity and that pre‐zygotic reproductive isolation through divergence in courtship or male‐produced pheromone and other mechanisms appear to evolve faster than post‐zygotic isolation in the fraterculus species group.     

Reproductive compatibility among Mexican populations of Anastrepha obliqua: theoretical and management implications

The fraterculus species group, composed of 34 species in the genus Anastrepha (Diptera: Tephritidae), includes the fraterculus cryptic species complex formed by eight reproductively isolated morphotypes. A previous study revealed six genetic mitochondrial types of Anastrepha obliqua, suggesting the existence of a second cryptic species complex. However, marked discrepancies between nuclear and mitochondrial loci rather suggest incomplete lineage sorting or introgression between A. obliqua and A. fraterculus. Such hybridization could nevertheless result in reproductive isolation, an outcome that could affect efficacy of area‐wide management for the most important pest of mangos in America. Two mitochondrial types occur in Mexico, and the limits of a third one, encompassing Central American populations, have not been clearly established. Here, we tested reproductive compatibility among three A. obliqua populations from the Pacific and a population from the Gulf of Mexico. No evidence of pre‐zygotic isolation was found. Flies from the Atlantic mated randomly for equal duration with individuals from three Pacific populations. Homotypic and heterotypic crosses produced similar numbers of eggs, with heterotypic crosses of Pacific males and Atlantic females hatching in lower proportions. Larvae of all cross types developed equally in mangos and exhibited no sex ratio distortion of hybrid F1. The three mitochondrial types identified in Mexico and Central America do not appear to be cryptic species and can be managed using one single strain for the sterile insect technique.     

An integrative multidisciplinary approach to understanding cryptic divergence in Brazilian species of the Anastrepha fraterculus complex (Diptera: Tephritidae)

An integrative multidisciplinary approach was used to delimit boundaries among cryptic species within the Anastrepha fraterculus complex in Brazil. Sexual compatibility, courtship and sexual acoustic behaviour, female morphometric variability, variation for the mitochondrial gene COI, and the presence of Wolbachia were compared among A. fraterculus populations from the Southern (Vacaria, Pelotas, Bento Gonçalves, São Joaquim) and Southeastern (Piracicaba) regions of Brazil. Our results suggest full mating compatibility among A. fraterculus populations from the Southern region and partial pre‐zygotic reproductive isolation of these populations when compared with the population from the Southeastern region. A. fraterculus populations from both regions differed in the frequency of courtship displays and aspects of the calling phase and mounting acoustic signal. Morphometric analysis showed differences between Southern region and Southeastern region samples. All populations analyzed were infected with Wolbachia. The trees generated from the COI sequencing data are broadly congruent with the behavioural and morphometric data with the exception of one Southern population. The likely mechanisms by which A. fraterculus populations might have diverged are discussed in detail based on behavioural, morphometric, molecular genetics, and biogeographical studies.     

Characterisation of the chemical profiles of Brazilian and Andean morphotypes belonging to the Anastrepha fraterculus complex (Diptera,Tephritidae)

Fruit fly sexual behaviour is directly influenced by chemical and non-chemical cues that play important roles in reproductive isolation. The chemical profiles of pheromones and cuticular hydrocarbons (CHs) of eight fruit fly populations of the Andean, Brazilian-1 and Brazilian-3 morphotypes of the Anastrepha fraterculus cryptic species complex originating from Colombia (four populations) and Brazil (four populations) were analysed using two-dimensional gas chromatography with mass spectrometric detection. The resulting chemical diversity data were studied using principal component analyses. Andean morphotypes could be discriminated from the Brazilian-1 and Brazilian-3 morphotypes by means of male-borne pheromones and/or male and female CH profiles. The Brazilian-1 and Brazilian-3 morphotypes were found to be monophyletic. The use of chemical profiles as species- and sex-specific signatures for cryptic species separations is discussed.     

Ongoing speciation within the Anastrepha fraterculus cryptic species complex: the case of the Andean morphotype

The Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) cryptic species complex is currently composed of seven taxonomically recognized morphotypes. Both, pre‐ and post‐zygotic isolation has been documented among four of these morphotypes, revealing that in fact they appear to be distinct biological entities. In order to progress in the full delimitation of species within the complex, we examined reproductive isolation between a Colombian population of the Andean morphotype and populations belonging to four other morphotypes spanning from Mexico to Argentina. Flies from the Andean morphotype exhibited strong pre‐zygotic mating isolation through temporal partitioning of mating activity. Post‐zygotic isolation was observed for crosses of males of all morphotypes and Andean morphotype females, yet most of the F1 hybrid ♂ × F1 hybrid ♀ self‐crosses showed normal levels of fertility, a finding suggesting a nuclear–cytoplasmic interaction according to previous studies. Overall, the Andean morphotype within the complex also appears to be a distinct biological entity. We discuss the implications of these findings for the understanding of speciation mechanisms in the Neotropical genus Anastrepha.     

Wolbachia strains in cryptic species of the Anastrepha fraterculus complex (Diptera,Tephritidae) along the Neotropical Region

Infection by Wolbachia was described previously in eleven species of Anastrepha fruit flies some of which are important pests of fruticulture. One such species is the nominal Anastrepha fraterculus, the South American fruit fly, which actually comprises a complex of cryptic species. The suggestions of using Wolbachia for the control of these pest species, make imperative a more precise characterization of the existing strains of the bacteria. In this study, population samples of the A. fraterculus complex from Brazil, Argentina, Peru, Ecuador, Colombia, Guatemala and Mexico were analyzed for Wolbachia infection. The bacteria were genotyped by the MLST and WSP Typing methodologies. All samples were infected with Wolbachia of supergroup “A”. For each of the five MLST genes, unique as well as already known alleles were detected. Nineteen sequence types for the concatenated sequences of the five MLST genes, and twenty wsp alleles were found in the samples. Host-specific haplotypes, shared strains among distinct hosts, and more than one strain of Wolbachia were found in some population samples. Recombination among the MLST genes and intragenic recombination between wsp haplotypes was rare. Phylogenetic analysis showed a great similarity among the Wolbachia strains in the A. fraterculus complex. However, some strains of Wolbachia are found throughout the Neotropical Region and there are specific strains in determined geographical areas.     

Phylogenetic relationships of Andean-Ecuadorian populations of Anastrepha fraterculus (Wiedemann 1830) (Diptera: Tephritidae) inferred from COI and COII gene sequences

Phylogenetic relationships among Andean-Ecuadorian and other Neotropical populations of Anastrepha fraterculus and related species have been studied using two regions of mtDNA : 405 base pairs within Cytochrome Oxidase I ( COI) and 224 base pairs within Cytochrome Oxidase II (COII). Phylogenetic relationships were inferred using Maximun Parsimony (MP) method and haplotype networks. Andean-Ecuadorian populations of A. fraterculus are monomorphic at the COI locus and fall within a clade of South-American lowland populations of A. fraterculus. They appear to be unrelated with populations of northern Andes of Colombia and Venezuela also assigned to A. fraterculus, meaning that this species, as currently circumscribed, is not monophyletic and is composed of different biological entities that are little differentiated morphologically. At the COII locus, Andean-Ecuadorian populations of A. fraterculus show a major haplotype with a few variants, and form a clade with the lowland populations of southern Brazil an Argentina, but are clearly differentiated from them. Andean-Ecuadorian populations of Anastrepha fraterculus appear to be homogeneous with respect to their mitochondrial genome and thus their identity as members of a single gene pool is confirmed by these results.     

Phylogeographic studies on natural populations of the South American fruit fly, <Emphasis Type="Italic">Anastrepha fraterculus</Emphasis> (Diptera: Tephritidae)

Anastrepha fraterculus is an important pest of commercial fruits in South America. The variability observed for morphological and behavioural traits as well as genetic markers suggests that A. fraterculus represents a complex of synmorphic species rather than a single biological species. We studied the correlation between geographical distribution and genetic variation in natural populations from Argentina and south Brazil. Fragments of the mitochondrial gene COII were sequenced in 28 individuals. The matrix of aligned sequences was phylogenetically analysed by parsimony, yielding a cladogram of haplotypes. Based on Templeton’s nested method, no clade showed any geographic pattern for the gene COII, indicating lack of significant association between haplotypic variability and geographic distribution. The analysis of nucleotide substitution distances by Neighbour-Joining algorithm showed that geographically distant populations exhibit low genetic distances. The corresponding trees clustered the populations without showing any geographic pattern. This result suggests that the populations studied are not reproductively isolated.     

Taxonomy and phenotypic relationships of the Anastrepha fraterculus complex in the Mesoamerican and Pacific Neotropical dominions (Diptera,Tephritidae)

Previous morphometric studies based on linear measurements of female structures of the aculeus, mesonotum, and wing revealed the existence of seven morphotypes within the Anastrepha fraterculus cryptic species complex along the Neotropical Region. The current research followed linear and geometric morphometric approaches in 40 population samples of the nominal species Anastrepha fraterculus (Wiedemann) spread throughout the Meso-American and Pacific Neotropical dominions (including Mexico, Central America, Venezuela, Colombia, Ecuador, and Peru). The goals were to explore the phenotypic relationships of the morphotypes in these biogeographical areas; evaluate the reliability of procedures used for delimitation of morphotypes; and describe their current distribution. Findings determined that morphotypes previously recognized via the linear morphometrics were also supported by geometric morphometrics of the wing shape. In addition, we found an eighth morphotype inhabiting the highlands of Ecuador and Peru. Morphotypes are related into three natural phenotypic groups nominated as Mesoamerican-Caribbean lineage, Andean lineage, and Brazilian lineage. The hypothesis that lineages are not directly related to each other is discussed, supported by their large morphological divergence and endemicity in these three well-defined biogeographic areas. In addition, this hypothesis of the non-monophyly of the Anastrepha fraterculus complex is also supported by evidence from other authors based on molecular studies and the strong reproductive isolation between morphs from different lineages.     

Response of two parasitoid species (Hymenoptera: Braconidae,Figitidae) to tephritid host and host food substrate cues

The Neotropical‐native figitid Aganaspis pelleranoi (Brèthes) and the Asian braconid Diachasmimorpha longicaudata (Ashmead) are two parasitoids of Tephritidae fruit flies with long and recent, respectively, evolutionary histories in the Neotropics. Both species experienced a recent range of overlap. In Argentina, A. pelleranoi is a potential species in biological control programs against the pestiferous tephritid species, Anastrepha fraterculus (Wiedemann) and Ceratitis capitata (Wiedemann), whereas D. longicaudata is already used in open‐field releases against Medfly in central‐western Argentina. To characterize the host‐foraging strategies of A. pelleranoi and D. longicaudata, olfactometer experiments were conducted comparing responses to C. capitata and A. fraterculus larvae, in two kinds of food substrate: fruit and artificial larval medium. To control the possible influence of host larvae used for parasitoid rearing on olfactory response, two strains of both parasitoid species, reared on both tephrtid species, were studied. Volatiles directly emanating either from A. fraterculus or C. capitata larvae may be detected by both A. pelleranoi and D. longicaudata, although chemical stimuli originating from the combination of host larvae and the habitat of the host were preferred. However, olfactory cues associated with host larvae probably play a relevant role in host searching behaviour of A. pelleranoi, whereas for D. longicaudata, the host‐habitat olfactory stimuli would be highly essential in short‐range host location. The strain of the parasitoids did not affect host search ability on the two tephritid species evaluated. These evidences are relevant for mass production of both parasitoids and their impact following open‐field augmentative releases.     

Phylogenetic relationships among populations of the Nacobbus aberrans (Nematoda,Pratylenchidae) complex reveal the existence of cryptic species

The plant‐parasitic nematode Nacobbus aberrans sensu lato is an agricultural pest of quarantine importance. Due to the morphometric, physiological and genetic variability observed within the species, there is no agreement on the taxonomy of this nematode. The objective of this study was to analyse the ITS rDNA region and the D2–D3 expansion segments of 28S rDNA in 10 Argentine populations and one from Ecuador and to establish their phylogenetic relationship with other known sequences from South and North America. Phylogenetic trees of the ITS gene showed seven statistically well‐supported clades; the high and significant F_st values obtained among these groups confirmed this partitioning. The Argentine populations here considered were separated into three clades: one comprising a population from the Andean region and two grouping nematodes from lower altitudes. Three other clades were distinguished for South American populations, which included known sequences of individuals from Peru, Bolivia and north of Argentina. The other clade included sequences from Mexico, Ecuador and two Argentine populations of unknown origin. The important degree of genetic divergence observed among Andean populations suggests that the Andes may have played a crucial role in speciation of Nacobbus, which would have originated in this region. Although D2–D3 segments exhibited lower variation, they were useful for establishing phylogenetic relationships among the Argentine populations considered in this work. As there are no other GenBank sequences available for these segments, it was not possible to make comparisons with other populations from South and North America. The considerable genetic differentiation observed in ITS rDNA region among Nacobbus populations showed evidence of cryptic species within the N. aberrans s.l. complex. Integration of morphological and morphometric studies and molecular analyses considering other genes may aid in the identification of species and their phylogenetic relationships within this genus.     

Multispectral imaging for quality control of laboratory‐reared Anastrepha fraterculus (Diptera: Tephritidae) pupae

The sterile insect technique (SIT) has been widely used to suppress several fruit fly species. In southern Brazil, millions of sterile flies of the South American fruit fly, Anastrepha fraterculus Wiedemann (Dipetra: Tephritidae), will be produced in a mass‐rearing facility called MOSCASUL to suppress wild populations from commercial apple orchards. In spite of standard rearing conditions, the quality of pupal batches can be inconsistent due to various factors. The quantification of poor quality material (e.g. empty pupae, dead pupae or larvae) is necessary to track down rearing issues, and pupal samples must be taken randomly and evaluated individually. To speed up the inspection of pupal samples by replacing the manual testing with the mechanized one, this study assessed a multispectral imaging (MSI) system to distinguish the variations in quality of A. fraterculus pupae and to quantify the variations based on reflectance patterns. Image acquisition and analyses were performed by the VideometerLab4 system on 7‐d‐old pupae by using 19 wavelengths ranging from 375 to 970 nm. The image representing the near infrared wavelength of 880 nm clearly distinguished among high‐quality pupae and the other four classes (i.e. low‐quality pupae, empty pupae, dead pupae and larvae). The blind validation test indicated that the MSI system can classify the fruit fly pupae with high accuracy. Therefore, MSI‐based classification of A. fraterculus pupae can be used for future pupal quality assessments of fruit flies in mass‐rearing facilities.     

Molecular circumscription of the blackfly Simulium oyapockense from South America

Problems related to the identity of Simuliidae species are impediments to effective disease control in Amazonia. Some of these species, such as Simulium oyapockense Floch & Abonnenc, 1946 (Diptera: Simuliidae), are vectors of the organisms that cause onchocerciasis and mansonellosis diseases. This blackfly species has a wide distribution in South America, and it is suspected of being a complex of cryptic species. The aim of this study is to characterize the nominal species S. oyapockense using partial COI gene sequences. Seven populations of S. oyapockense (morphologically identified) were analysed, including one from its type-locality. The other six populations were collected in Brazil and in Argentina. A taxon collected in Amazonas state, Brazil, with adults similar to S. oyapockense but with distinct pupae, was also included in the analysis (Simulium ‘S’). The nominal species S. oyapockense is circumscribed, and its geographical distribution is restricted to areas north of the Amazon River. Populations of S. oyapockense s.l. collected south of the Amazon River comprise a species complex that needs to be evaluated using integrative taxonomy. Simulium ‘S’ represents a species with unique morphological and molecular characteristics. Distinguishing cryptic species is a prerequisite for reducing the taxonomic impediment, especially in medically important taxa.     

Phylogeography and population genetics of the cryptic bonnethead shark Sphyrna aff. tiburo in Brazil and the Caribbean inferred from mtDNA markers

Resolving the identity, phylogeny and distribution of cryptic species within species complexes is an essential precursor to management. The bonnethead shark, Sphyrna tiburo, is a small coastal shark distributed in the Western Atlantic from North Carolina (U.S.A.) to southern Brazil. Genetic analyses based on mitochondrial markers revealed that bonnethead sharks comprise a species complex with at least two lineages in the Northwestern Atlantic and the Caribbean (S. tiburo and Sphyrna aff. tiburo, respectively). The phylogeographic and phylogenetic analysis of two mitochondrial markers [control region (mtCR) and cytochrome oxidase I (COI)] showed that bonnethead sharks from southeastern Brazil correspond to S. aff. tiburo, extending the distribution of this cryptic species >5000 km. Bonnethead shark populations are only managed in the U.S.A. and in the 2000s were considered to be regionally extinct or collapsed in southeast Brazil. The results indicate that there is significant genetic differentiation between S. aff. tiburo from Brazil and other populations from the Caribbean (Φ_ST = 0.9053, P < 0.000), which means that collapsed populations in the former are unlikely to be replenished from Caribbean immigration. The species identity of bonnethead sharks in the Southwest Atlantic and their relationship to North Atlantic and Caribbean populations still remains unresolved. Taxonomic revision and further sampling are required to reevaluate the status of the bonnethead shark complex through its distribution range.     

Genetic identification of members of the Bemisia tabaci cryptic species complex from South Africa reveals native and introduced haplotypes

The whitefly Bemisia tabaci cryptic species complex contains some important agricultural pest and virus vectors. Members of the complex have become serious pests in South Africa (SA) because of their feeding habit and their ability to transmit begomovirus species. Despite their economic importance, studies on the biology and distribution of B. tabaci in SA are limited. To this end, a survey was made to investigate the diversity and distribution of B. tabaci cryptic species in eight geographical locations (provinces) in SA, between 2002 and 2009, using the mitochondrial cytochrome oxidase I (mtCOI) sequences. Phylogenetic analysis revealed the presence of members from two endemic sub‐Saharan Africa (SSAF) subclades coexisting with two introduced putative species. The SSAF‐1 subclade includes cassava host‐adapted B. tabaci populations, whereas the whiteflies collected from cassava and non‐cassava hosts formed a distinct subclade, referred to as SSAF‐5, and represent a new subclade among previously recognized southern Africa clades. Two introduced cryptic species, belonging to the Mediterranean and Middle East–Asia minor 1 clades, were identified and include the B and Q types. The B type showed the widest distribution, being present in five of the eight provinces explored in SA, infesting several host plants and predominating over the indigenous haplotypes. This is the first report of the occurrence of the exotic Q type in SA alongside the more widely distributed B type. Furthermore, mtCOI PCR‐RFLP was developed for the SA context to allow rapid discrimination between the B, Q and SSAF putative species. The capacity to manage pests and disease effectively relies on knowledge of the identity of the agents causing the damage. Therefore, this study contributes to the understanding of South African B. tabaci species diversity, information needed for the development of knowledge‐based disease management practices.     

Current knowledge of the species complex Anastrepha fraterculus (Diptera,Tephritidae) in Brazil

The study of the species complex Anastrepha fraterculus (Af complex) in Brazil is especially important in a taxonomical, evolutionary and pest management context, because there are evidences that some of them may occur in sympatry. In this review, we analyzed the main results supporting evidences that three cryptic species occur in Brazil. The taxonomical and phylogenetic relationships based on eggshell morphology, adult morphometrics, as well as cytotaxonomy and genetic differentiations are discussed. We also review available information on sexual behavior including acoustic communication of males during courtship and sexual incompatibility; and chemical signals involved in the communication between sexes, with a special focus on sex pheromones. We examined the role of long- and short-range pheromones (male-produced volatiles and cuticular hydrocarbons, respectively), their implications in sexual isolation, and their possible use for chemotaxonomic differentiation of the putative species of the Af complex.     

The true identity of Obama (Platyhelminthes: Geoplanidae) flatworm spreading across Europe

Since 2008 there have been many records in Europe (British Isles, Spain, France, Italy) of a large terrestrial planarian morphologically very similar to the Brazilian species Obama marmorata. Sequences of mitochondrial (Cox1) and nuclear (18S, 28S, ITS‐1 and EF) genes from European specimens and some from Brazil indicate that they belong to a species different from that of other specimens also collected in Brazil. Moreover, the phylogenetic results show that they are not sister‐species. Histological sections of both Brazilian and European specimens reveal subtle morphological differences between the two species. Obama marmorata is confined to Brazil, and the second, herein described new species, O bama nungara sp. nov. , is found in Brazil and Europe. These cryptic species may be syntopic in areas in Brazil. The new species occurs in human‐modified environments both in Brazil and in Europe. We also conclude that the specimens from Spain and Argentina identified previously as Obama marmorata belong to the new species.     

Genetic variability and interpopulational differentiation of Artemia strains from South America

Seven Artemia samples from three South American countries (Chile, Brazil, Peru) were studied by starch electrophoresis with the aim of comparing levels of genetic variation and genetic similarity to representative populations of A. franciscana (San Francisco Bay, California, USA) and A. persimilis (Buenos Aires, Argentina), which are species endemic to the New World. Based on the analysis of 22 loci, parameters measuring genetic variability were, for some populations, found to be among the highest reported for Artemia so far. The percentage of polymorphic loci varied from 31.8% (Piura, Peru; Buenos Aires) to 50% (Los Vilos and Salar de Atacama, Chile), while the observed heterozygosity varied from 0.025 (Piura) to 0.165 (Los Vilos, Chile). A dendrogram based on Nei's genetic distance (D) produced four major groups. The Argentinian form, A. persimilis; the San Francisco Bay strain together with samples from Brazil (Macau and Rio Grande do Norte) and Chile (Pichilemu and Salar de Atacama); two coastal populations from Chile (Los Vilos and Iquique) and the sample from Peru (Piura). These four groups have inter-group D values that are, in some cases, far above those normally associated with conspecific populations.     

Sperm dispersal distances estimated by parentage analysis in a brooding scleractinian coral

Within populations of brooding sessile corals, sperm dispersal constitutes the mechanism by which gametes interact and mating occurs, and forms the first link in the network of processes that determine specieswide connectivity patterns. However, almost nothing is known about sperm dispersal for any internally fertilizing coral. In this study, we conducted a parentage analysis on coral larvae collected from an area of mapped colonies, to measure the distance sperm disperses for the first time in a reef‐building coral and estimated the mating system characteristics of a recently identified putative cryptic species within the Seriatopora hystrix complex (ShA; Warner et al. 2015). We defined consensus criteria among several replicated methods (colony 2.0, cervus 3.0, mltr v3.2) to maximize accuracy in paternity assignments. Thirteen progeny arrays indicated that this putative species produces exclusively sexually derived, primarily outcrossed larvae (mean t_m = 0.999) in multiple paternity broods (mean r_p = 0.119). Self‐fertilization was directly detected at low frequency for all broods combined (2.8%), but comprised 23% of matings in one brood. Although over 82% of mating occurred between colonies within 10 m of each other (mean sperm dispersal = 5.5 m ± 4.37 SD), we found no evidence of inbreeding in the established population. Restricted dispersal of sperm compared to slightly greater larval dispersal appears to limit inbreeding among close relatives in this cryptic species. Our findings establish a good basis for further work on sperm dispersal in brooding corals and provide the first information about the mating system of a newly identified and abundant cryptic species.     

Cryptic speciation in the narrow‐headed vole Lasiopodomys (Stenocranius) gregalis (Rodentia: Cricetidae)

Recent study on Lasiopodomys (Stenocranius) gregalis has revealed four clear mitochondrial lineages with differences exceeding the species level. The most divergent is the clade from south‐eastern Transbaikalia. Multilocus (six nuclear genes), morphological (dental characters of m1 and M3) and behavioural (breeding experiments) analyses were conducted to test the hypothesis that L. gregalis is a complex of two cryptic species. All of the applied methods distinguish the independent status of voles from south‐eastern Transbaikalia. This clade occupies a portion of the distribution range of the currently valid subspecies L. g. raddei including its terra typica. According to the results of cyt b analysis, the lectotype of L. g. raddei belongs to the Transbaikal clade. We re‐establish the species status for the taxon Lasiopodomys raddei including populations from south‐eastern Transbaikalia, as a cryptic species of the narrow‐headed vole complex.