A multiple PCR‐LDR assay for identification of two invasive cryptic species of whiteflies Bemisia tabaci

The MEAM1 and MED species of the cryptic species complex Bemisia tabaci are important invasive pests that cause tremendous crop losses worldwide. A rapid and highly reliable molecular technique is necessary to identify these species because they are morphologically indistinguishable. Therefore, a multiple polymerase chain reaction coupled with a ligase detection reaction (PCR‐LDR) that was based on polymorphisms in the mitochondrial cytochrome oxidase I (mtCOI) gene of B. tabaci was developed to distinguish the two cryptic species. An assessment of the method indicated that PCR‐LDR provided high specificity and sensitivity in discriminating MEAM1 (SHB) and MED (SHQ) whiteflies. In field tests, PCR‐LDR genotyping was performed in one 96‐well plate to identify 93 individuals collected from 8 districts in the suburbs of Shanghai. Complete concordance was observed between PCR‐LDR and sequencing methods. The method was used to confirm that MEAM1 and MED were found in two districts, but only the MED was found in the other six districts. PCR‐LDR, which is a transplantable platform, provides an alternative method for species identification of B. tabaci at low cost.     

DNA mini‐barcoding of leporids using noninvasive fecal DNA samples and its significance for monitoring an invasive species

Introduced in South America at the end of the 19th century, the European hare population has expanded dramatically and now represents a risk to native Brazilian forest rabbits. Monitoring the invasive Lepus europaeus and its coexistence with native Sylvilagus brasiliensis is a challenge that can be efficiently addressed by the use of molecular tools. This work describes a set of primers useful for amplifying three mini‐barcodes for the molecular identification of both invasive and native leporid species using degraded fecal DNA. In addition, tests in silico indicate that these mini‐barcodes can successfully amplify the DNA sequences of a number of leporids. These mini‐barcodes constitute a powerful tool for the monitoring and management of the invasive L. europaeus and the conservation of native rabbits.     

Presence of Anopheles culicifacies B in Cambodia established by the PCR-RFLP assay developed for the identification of Anopheles minimus species A and C and four related species

Abstract A polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) assay developed for identification of five species of the Anopheles minimus Theobald group and a related mosquito species of the Myzomyia Series (Diptera: Culicidae) was applied to morphologically identified adult female specimens collected in Ratanakiri Province, north‐eastern Cambodia. In addition to finding An. aconitus Dönitz, An. minimus species A and An. pampanai Büttiker & Beales, some specimens showed a new restriction banding pattern. Siblings of specimens that exhibited this new PCR‐RFLP pattern were morphologically identified as An. culicifacies James sensu lato. Based on nucleotide sequences of the ribonuclear DNA internal transcribed spacer 2 region (ITS2) and the mitochondrial cytochrome oxidase I gene (COI), these specimens were recognized as An. culicifacies species B (sensu Green & Miles, 1980 ), the first confirmed record of the An. culicifacies complex from Cambodia. This study shows that the PCR‐RFLP assay can detect species not included in the initial set‐up and is capable of identifying at least seven species of the Myzomyia Series, allowing better definition of those malaria vector and non‐vector anophelines in South‐east Asia.     

Common species affects the utility of non‐invasive genetic monitoring of a cryptic endangered mammal: The bridled nailtail wallaby

Non‐invasive methods of monitoring wild populations (such as genotyping faeces or hair) are now widely used and advocated. The potential advantages of such methods over traditional direct monitoring (such as live capture) are that accuracy improves because sampling of non‐trappable individuals may be possible, species in difficult and remote terrain can be surveyed more efficiently, and disturbance to animals is minimal. Few studies have assessed the effects of interactions between species on remote sampling success. We test the use of non‐invasive monitoring for the cryptic, forest‐dwelling, solitary and endangered bridled nailtail wallaby (Onychogalea fraenata) that is sympatric with the ecologically similar and more common black‐striped wallaby (Macropus dorsalis). Six types of hair traps were tested for 3668 trap days, and hairs were caught with about a 10% success rate. Camera traps showed that baited hair traps targeted both wallaby species. We microscopically identified hair as bridled nailtail wallaby or black‐striped wallaby. We compared these hairs and their genotypes (using seven microsatellite loci) with known bridled nailtail wallaby hairs and genotypes derived from animal trapping. Trapped bridled nailtail wallaby hairs had characteristics that could be mistaken for black‐stripe wallaby hairs; characteristics were not diagnostic. Genetic assignment tests consistently differentiated the known bridled nailtail wallaby samples from identified black‐striped wallaby samples, however genetic overlap between most of the microsatellite markers means that they are not suitable for species identification of single samples, with the possible exception of the microsatellite locus B151. With similar trapping effort and within the same area, live‐capture mark‐recapture techniques estimated 40–60 individuals and non‐invasive methods only detected 14 genotypes. A species‐specific genetic marker would allow more efficient targeting of bridled nailtail wallaby samples and increase capture rates.     

Identification of forensically important blow fly species (Diptera: Calliphoridae) in China by mitochondrial cytochrome oxidase I gene differentiation

Abstract Unambiguous and rapid sarcosaphagous insect species identification is an essential requirement for forensic investigations. Although some insect species are difficult to classify morphologically, they can be effectively identified using molecular methods based on similarity with abundant authenticated reference DNA sequences in local databases. However, local databases are still relatively incomplete in China because of the large land area with distinct regional conditions. In this study, 75 forensically important blow flies were collected from 23 locations in 16 Chinese provinces, and a 278‐bp segment of the cytochrome oxidase subunit I gene of all specimens was successfully sequenced. Phylogenetic analysis of the sequenced segments showed that all Calliphorid specimens were properly assigned into nine species with relatively strong supporting values, thus indicating that the 278‐bp cytochrome oxidase subunit one region is suitable for identification of Calliphorid species. The clear difference between intraspecific threshold and interspecific divergence confirmed the potential of this region for Calliphorid species identification, especially for distinguishing between morphologically similar species. Intraspecific geographic variations were observed in Lucilia sericata (Meigen, 1826) and Lucilia caesar (Linnaeus, 1758).     

A real‐time PCR toolbox for accurate identification of invasive fruit fly species

Significant plant pests such as fruit flies that travel with fresh produce between countries as eggs or larvae pose a great economic threat to the agriculture and fruit industry worldwide. Time‐limited and expensive quarantine decisions require accurate identification of such pests. Immature stages are often impossible to identify, making them a serious concern for biosecurity agencies. Use of COI barcoding PCR, often the only molecular identification resource, is time‐consuming. We assess the suitability of the COI barcoding region for real‐time PCR assays to identify four pest fruit fly species (Family: Tephritidae), in a diagnostic framework. These species, namely Mediterranean fruit fly (Ceratitis capitata), Queensland fruit fly (Bactrocera tryoni), African invader fly (Bactrocera invadens) and Island fly (Dirioxa pornia) each provide a different set of genetic species delimitation problems. We discuss the benefits and limitations of using a single‐gene TaqMan^? real‐time approach for such species. Our results indicate that COI‐based TaqMan^? real‐time PCR assays, in particular for genetically distinct species, provide an accurate, sensitive and rapid diagnostic tool.     

An allele-specific polymerase chain reaction assay for the differentiation of members of the<Emphasis Type="Italic">Anopheles culicifacies</Emphasis> complex

Anopheles culicifacies, the principal vector of malaria in India, is a complex of five cryptic species which are morphologically indistinguishable at any stage of life. In view of the practical difficulties associated with classical cytotaxonomic method for the identification of members of the complex, an allele-specific polymerase chain reaction (ASPCR) assay targeted to the D3 domain of 28S ribosomal DNA was developed. The assay discriminatesAn. culicifacies species A and D from species B, C and E. The assay was validated using chromosomally-identified specimens ofAn. culicifacies from different geographical regions of India representing different sympatric associations. The assay correctly differentiates species A and D from species B, C and E. The possible use of this diagnostic assay in disease vector control programmes is discussed.     

Quantitative assessment of species identification in aerial transect surveys for ice‐associated seals

Technological advances have facilitated collection of vast quantities of photographic data from aerial surveys of marine mammals. However, when it is difficult to distinguish species from a distance, reliable identification from aerial images can often be challenging. This is the case for ice‐associated seals, species for which global climate change has motivated intensive monitoring efforts in recent years. We assess species and age class identification from aerial images of four ice seal species (bearded seals, Erignathus barbatus; ribbon seals, Histriophoca fasciata; ringed seals, Pusa hispida; spotted seals, Phoca largha) in the Bering Sea. We also investigate the specific phenomenological and behavioral traits commonly associated with species identification and observer confidence. We generally found species and age class misidentification occurred at relatively low levels, but only 83% of spotted seals tended to be correctly identified (with 11% mistaken as ribbon seals). We also found certain traits were strong predictors for observed species, age class, or observer confidence. Our findings add to the growing body of evidence that species misidentification is pervasive in passive sampling of animal populations. Even low levels of misidentification have been demonstrated to induce substantial biases in estimators of species distribution and abundance, and it is important that statistical models account for such errors.     

Testing the divergent adaptation of two congeneric tree species on a rainfall gradient using eco‐physio‐morphological traits

In tropical Africa, evidence of widely distributed genera transcending biomes or habitat boundaries has been reported. The evolutionary processes that allowed these lineages to disperse and adapt into new environments are far from being resolved. To better understand these processes, we propose an integrated approach, based on the eco‐physio‐morphological traits of two sister species with adjacent distributions along a rainfall gradient. We used wood anatomical traits, plant hydraulics (vulnerability to cavitation, wood volumetric water content, and hydraulic capacitance), and growth data from the natural habitat, in a common garden, to compare species with known phylogeny, very similar morphologically, but occupying contrasting habitats: Erythrophleum ivorense (wet forest) and Erythrophleum suaveolens (moist forest and forest gallery). We identified some slight differences in wood anatomical traits between the two species associated with strong differences in hydraulics, growth, and overall species distribution. The moist forest species, E. suaveolens, had narrower vessels and intervessel pits, and higher vessel cell‐wall reinforcement than E. ivorense. These traits allow a high resistance to cavitation and a continuous internal water supply of the xylem during water shortage, allowing a higher fitness during drought periods, but limiting growth. Our results confirm a trade‐off between drought tolerance and growth, controlled by subtle adaptations in wood traits, as a key mechanism leading to the niche partitioning between the two Erythrophleum species. The generality of this trade‐off and its importance in the diversification of the African tree flora remains to be tested. Our integrated eco‐physio‐morpho approach could be the way forward.     

A novel approach to field identification of cryptic Apodemus wood mice: calls differ more than morphology

Field identification of European wood mice Apodemus spp. is challenging due to their morphological resemblance and frequent sympatry. We developed discriminant functions based on body mass and acoustic variables of distress calls to identify three cryptic species of wood mice (Apodemus alpicola, Apodemus flavicollis and Apodemus sylvaticus) in Italy. We achieved an overall correct classification rate of 86–98%; the best results (100% correct classification) were obtained for Apodemus sylvaticus calls. This minimally invasive, effective and low‐cost method highlights the potential role of bioacoustics as a powerful tool for field discrimination of cryptic species of terrestrial mammals.     

Integrative species delimitation reveals cryptic diversity in the southern Appalachian Antrodiaetus unicolor (Araneae: Antrodiaetidae) species complex

Although species delimitation can be highly contentious, the development of reliable methods to accurately ascertain species boundaries is an imperative step in cataloguing and describing Earth's quickly disappearing biodiversity. Spider species delimitation remains largely based on morphological characters; however, many mygalomorph spider populations are morphologically indistinguishable from each other yet have considerable molecular divergence. The focus of our study, the Antrodiaetus unicolor species complex containing two sympatric species, exhibits this pattern of relative morphological stasis with considerable genetic divergence across its distribution. A past study using two molecular markers, COI and 28S, revealed that A. unicolor is paraphyletic with respect to A. microunicolor. To better investigate species boundaries in the complex, we implement the cohesion species concept and use multiple lines of evidence for testing genetic exchangeability and ecological interchangeability. Our integrative approach includes extensively sampling homologous loci across the genome using a RADseq approach (3RAD), assessing population structure across their geographic range using multiple genetic clustering analyses that include structure , principal components analysis and a recently developed unsupervised machine learning approach (Variational Autoencoder). We evaluate ecological similarity by using large‐scale ecological data for niche‐based distribution modelling. Based on our analyses, we conclude that this complex has at least one additional species as well as confirm species delimitations based on previous less comprehensive approaches. Our study demonstrates the efficacy of genomic‐scale data for recognizing cryptic species, suggesting that species delimitation with one data type, whether one mitochondrial gene or morphology, may underestimate true species diversity in morphologically homogenous taxa with low vagility.     

新疆林果外来入侵生物的发生及检测监测和控制策略

近10年来,新疆林果业快速发展。随着林果种苗等繁殖材料的运输,林果有害生物由境外、省外及在区内传播的风险加剧,外来有害生物入侵成为当下影响新疆林果业健康发展的重要问题。在鉴定工作方面,目前新疆林果外来入侵生物的鉴定更多地采用了分子生物学鉴定的方法;在监测工作层面,新疆林果有害生物普查以人工地面调查为主;在防控方面,主要以药剂防治的方法为主,辅以生物防治技术及栽培措施等多种方法。全疆的林业主管部门成功控制了多种林果外来入侵生物引起的疫情,包括枣实蝇和扶桑绵粉蚧这2种极具危害性的林果外来入侵生物。新疆林果外来入侵生物的鉴定、监测及防控工作的开展,可将林果外来入侵生物所带来的危险降到最低程度,促进全疆林业的发展。     

A high‐throughput detection method for invasive fruit fly (Diptera: Tephritidae) species based on microfluidic dynamic array

Invasive species can be detrimental to a nation's ecology, economy and human health. Rapid and accurate diagnostics are critical to limit the establishment and spread of exotic organisms. The increasing rate of biological invasions relative to the taxonomic expertise available generates a demand for high‐throughput, DNA‐based diagnostics methods for identification. We designed species‐specific qPCR primer and probe combinations for 27 economically important tephritidae species in six genera (Anastrepha, Bactrocera, Carpomya, Ceratitis, Dacus and Rhagoletis) based on 935 COI DNA barcode haplotypes from 181 fruit fly species publically available in BOLD, and then tested the specificity for each primer pair and probe through qPCR of 35 of those species. We then developed a standardization reaction system for detecting the 27 target species based on a microfluidic dynamic array and also applied the method to identify unknown immature samples from port interceptions and field monitoring. This method led to a specific and simultaneous detection for all 27 species in 7.5 h, using only 0.2 μL of reaction system in each reaction chamber. The approach successfully discriminated among species within complexes that had genetic similarities of up to 98.48%, while it also identified all immature samples consistent with the subsequent results of morphological examination of adults which were reared from larvae of cohorts from the same samples. We present an accurate, rapid and high‐throughput innovative approach for detecting fruit flies of quarantine concern. This is a new method which has broad potential to be one of international standards for plant quarantine and invasive species detection.     

Direct PCR of indigenous and invasive mosquito species: a time‐ and cost‐effective technique of mosquito barcoding

Millions of people die each year as a result of pathogens transmitted by mosquitoes. However, the morphological identification of mosquito species can be difficult even for experts. The identification of morphologically indistinguishable species, such as members of the Anopheles maculipennis complex (Diptera: Culicidae), and possible hybrids, such as Culex pipiens pipiens/Culex pipiens molestus (Diptera: Culicidae), presents a major problem. In addition, the detection and discrimination of newly introduced species can be challenging, particularly to researchers without previous experience. Because of their medical importance, the clear identification of all relevant mosquito species is essential. Using the direct polymerase chain reaction (PCR) method described here, DNA amplification without prior DNA extraction is possible and thus species identification after sequencing can be achieved. Different amounts of tissue (leg, head; larvae or adult) as well as different storage conditions (dry, ethanol, ?20 and ?80 °C) and storage times were successfully applied and showed positive results after amplification and gel electrophoresis. Overall, 28 different indigenous and non‐indigenous mosquito species were analysed using a gene fragment of the COX1 gene for species differentiation and identification by sequencing this 658‐bp fragment. Compared with standard PCR, this method is time‐ and cost‐effective and could thus improve existing surveillance and control programmes.     

Marine invasive species: validation of citizen science and implications for national monitoring networks

Approximately 1,000 volunteers assessed the presence of invasive (Carcinus maenas and Hemigrapsus sanguineus) and native crabs within the intertidal zone of seven coastal states of the US, from New Jersey to Maine. Identification of crab species and determination of the gender of the observed crabs was documented at all 52 sites across a 725-km coastal transect. Using quantitative measures of accuracy of data collected by citizen scientists, a significant predictor of a volunteer’s ability was determined and eligibility criteria were set. Students in grade three and seven had the ability to differentiate between species of crabs with over 80% and 95% accuracy, respectively. Determination of gender of the crabs was more challenging and accuracy exceeded 80% for seventh grade students, while 95% accuracy was found for students with at least 2 years of university education. We used the data collected by citizen scientists to create a large-scale standardized database of the distribution and abundance of the native and invasive crabs. Hemigrapsus sanguineus dominated the rocky intertidal zone from Sandy Hook, New Jersey to Boston Harbor, Massachusetts while C. maenas dominated the northern extent of the sampled coastline. A citizen scientist of this monitoring network detected a range expansion of H. sanguineus. We identified obstacles to creating a national monitoring network and proposed recommendations that addressed these issues.     

Distinguishing Anuran species by high‐resolution melting analysis of the COI barcode (COI‐HRM)

Taxonomic identification can be difficult when two or more species appear morphologically similar. DNA barcoding based on the sequence of the mitochondrial cytochrome c oxidase 1 gene (COI) is now widely used in identifying animal species. High‐resolution melting analysis (HRM) provides an alternative method for detecting sequence variations among amplicons without having to perform DNA sequencing. The purpose of this study was to determine whether HRM of the COI barcode can be used to distinguish animal species. Using anurans as a model, we found distinct COI melting profiles among three congeners of both Lithobates spp. and Hyla spp. Sequence variations within species shifted the melting temperature of one or more melting domains slightly but do not affect the distinctness of the melting profiles for each species. An NMDS ordination plot comparing melting peak profiles among eight Anuran species showed overlapping profiles for Lithobates sphenocephala and Gastrophryne carolinensis. The COI amplicon for both species contained two melting domains with melting temperatures that were similar between the two species. The two species belong to two different families, highlighting the fact that COI melting profiles do not reveal phylogenetic relationships but simply reflect DNA sequence differences among stretches of DNA within amplicons. This study suggests that high‐resolution melting analysis of COI barcodes (COI‐HRM) may be useful as a simple and rapid method to distinguish animal species that appear morphologically similar.     

New insight into the identification and molecular phylogeny of dagger nematodes of the genus Xiphinema (Nematoda: Longidoridae) with description of two new species

The genus Xiphinema constitutes a large group of about 260 species of plant‐ectoparasitic nematodes. The group is polyphagous and distributed almost worldwide. Some of the species of this genus damage agricultural crops by direct feeding on root cells as well as by transmitting nepoviruses. Species discrimination in Xiphinema is complicated by phenotypic plasticity leading to potential misidentification. We conducted nematode surveys in cultivated and natural environments in Spain from 2009 to 2012, from which we identified 20 populations of Xiphinema species morphologically close to the virus‐vector nematode species Xiphinema diversicaudatum, three apomictic populations tentatively identified as species from the complex Xiphinema aceri‐pyrenaicum group, and one population morphologically different from all others that is characterized by a female tail elongate to conical and absence of uterine differentiation. We developed comparative multivariate analyses for these related species by using morphological and morphometrical features together with molecular data from nuclear ribosomal DNA genes [D2‐D3 expansion segments of large ribosomal subunit 28S, internal transcribed spacer 1 (ITS1), and partial small ribosomal subunit (18S)]. The results of multivariate, molecular, and phylogenetic analysis confirmed the morphological hypotheses and allowed the delimitation and discrimination of two new species in the genus described herein as Xiphinema baetica sp. nov. and Xiphinema turdetanensis sp. nov. , and ten known species: Xiphinema adenohystherum, Xiphinema belmontense, Xiphinema cohni, Xiphinema coxi europaeum, Xiphinema gersoni, Xiphinema hispidum, Xiphinema italiae, Xiphinema lupini, Xiphinema nuragicum, and Xiphinema turcicum. Multivariate analyses based on quantitative and qualitative characters and phylogenetic relationships of Xiphinema spp. based on the three molecular ribosomal markers resulted in a partial consensus of these species grouping as nematode populations were maintained for the majority of morphospecies groups (e.g. morphospecies groups 5 and 6), but not in some others (e.g. position of Xiphinema granatum), demonstrating the usefulness of these analyses for helping in the diagnosis and identification of Xiphinema spp. The clade topology of phylogenetic trees of D2‐D3 and partial 18S regions in this study were congruent in supporting the polyphyletic status of some characters, such as the female tail shape and the degree of development of the genital system in species with both genital branches equally developed. This is the most complete phylogenetic study for Xiphinema non‐americanum‐group species. Agreement between phylogenetic trees and some morphological characters (uterine spines, pseudo‐Z organ, and tail shape) was tested by reconstruction of their histories on rDNA‐based trees using parsimony and Bayesian approaches. Thus, integrative taxonomy, based on the combination of multivariate, molecular analyses with morphology, constitutes a new insight into the identification of Xiphinema species. © 2013 The Linnean Society of London     

Multiplex PCR assay for malaria vector Anopheles minimus and four related species in the Myzomyia Series from Southeast Asia

Abstract. Mosquitoes (Diptera: Culicidae) of the Anopheles (Cellia) Myzomyia Series are important malaria vectors in Africa, India and Southeast Asia. Among 10 named species of Myzomyia known from the Oriental Region, seven form the An. minimus group. Even for expert taxonomists, the adults of these species remain difficult to identify morphologically. For technical staff of malaria control programmes, confusion may extend to misidentification of species that are not formally within the minimus group. For identification of specimens from Indochina (Cambodia, Laos, Vietnam), we describe a multiplex polymerase chain reaction (PCR) assay, based on rDNA internal transcribed spacer 2 (ITS2) sequences, that employs a cocktail of primers to identify An. minimus Theobald sibling species A and C (sensu; Green et al., 1990) and three other species in the An. minimus group (An. aconitus Dönitz, An. pampanai Büttiker & Beales, An. varuna Iyengar), as well as An. jeyporiensis James, also belonging to the Myzomyia Series. As the test is DNA‐based, it can be applied to all life stages of these mosquitoes for ecological investigations and vector incrimination studies. This PCR assay is simpler, quicker, cheaper and more readily interpreted than previous assays.     

Identifying potentially invasive non‐native marine and brackish water species for the Arabian Gulf and Sea of Oman

Invasive non‐native species (NNS) are internationally recognized as posing a serious threat to global biodiversity, economies and human health. The identification of invasive NNS is already established, those that may arrive in the future, their vectors and pathways of introduction and spread, and hotspots of invasion are important for a targeted approach to managing introductions and impacts at local, regional and global scales. The aim of this study was to identify which marine and brackish NNS are already present in marine systems of the northeastern Arabia area (Arabian Gulf and Sea of Oman) and of these which ones are potentially invasive, and which species have a high likelihood of being introduced in the future and negatively affect biodiversity. Overall, 136 NNS were identified, of which 56 are already present in the region and a further 80 were identified as likely to arrive in the future, including fish, tunicates, invertebrates, plants and protists. The Aquatic Species Invasiveness Screening Kit (AS‐ISK) was used to identify the risk of NNS being (or becoming) invasive within the region. Based on the AS‐ISK basic risk assessment (BRA) thresholds, 36 extant and 37 horizon species (53.7% of all species) were identified as high risk. When the impact of climate change on the overall assessment was considered, the combined risk score (BRA+CCA) increased for 38.2% of all species, suggesting higher risk under warmer conditions, including the highest‐risk horizon NNS the green crab Carcinus maenas, and the extant macro‐alga Hypnea musciformis. This is the first horizon‐scanning exercise for NNS in the region, thus providing a vital baseline for future management. The outcome of this study is the prioritization of NNS to inform decision‐making for the targeted monitoring and management in the region to prevent new bio‐invasions and to control existing species, including their potential for spread.     

A sensitive and reliable restriction enzyme assay to distinguish between the mosquitoes Culex torrentium and Culex pipiens

Culex pipiens pipiens Linnaeus and Culex torrentium Martini (Diptera: Culicidae) are closely related vector species that exist sympatrically in Europe. The two species are morphologically almost identical and can only be distinguished with certainty by characters of the male genitalia. Hence, correct species identification and conclusions on distribution and vector status are very difficult and often neglected. Therefore, we developed a reliable and simple mitochondrial cytochrome c oxidase subunit I (COI) gene restriction enzyme assay to discriminate between Cx. pipiens and Cx. torrentium, based on the analysis of morphologically identified male specimens. We sequenced approximately 830 bp in the 3′ region of the mitochondrial COI gene of 18 morphologically identified males of Cx. pipiens and Cx. torrentium. Two restriction enzymes (FspBI and SspI) that could distinguish between the two species according to species‐specific differences in these sequences were chosen. The restriction enzymes were tested on 227 samples from Sweden and verified by sequencing 44 of them. The enzyme FspBI correctly identified all investigated samples; the enzyme SspI identified all samples except one Cx. torrentium. We hope the method and the findings presented here will help to shed light on the true distribution and relative proportions of the two species in Europe.