The shapes of neutral gene genealogies in two species: probabilities of monophyly,paraphyly, and polyphyly in a coalescent model

Abstract.— The genealogies of samples of orthologous regions from multiple species can be classified by their shapes. Using a neutral coalescent model of two species, I give exact probabilities of each of four possible genealogical shapes: reciprocal monophyly, two types of paraphyly, and polyphyly. After the divergence that forms two species, each of which has population size N , polyphyly is the most likely genealogical shape for the lineages of the two species. At ∼ 1.300 N generations after divergence, paraphyly becomes most likely, and reciprocal monophyly becomes most likely at ∼1.665 N generations. For a given species, the time at which 99% of its loci acquire monophyletic genealogies is ∼5.298 N generations, assuming all loci in its sister species are monophyletic. The probability that all lineages of two species are reciprocally monophyletic given that a sample from the two species has a reciprocally monophyletic genealogy increases rapidly with sample size, as does the probability that the most recent common ancestor (MRCA) for a sample is also the MRCA for all lineages from the two species. The results have potential applications for the testing of evolutionary hypotheses.     

Biochemical assessment of evolution and taxonomy of the morphologically poorly diverged geckos, Gekko yakuensis and G. hokouensis (Reptilia: Squamata) in Japan, with special reference to their occasional hybridization

We assessed the validity of two gekkonid species, Gekko yakuensis and G. hokouensis, in southern Japan. We first assigned all 398 specimens into 18 samples merely on the basis of localities. By conducting significance test for deviations of genotype frequencies from Hardy‐Weinberg at 11 allozyme loci, we checked the reproductive unity of constituents in each of those local samples, and where necessary, rearranged them into subsamples on the basis of genetic markers so that we recognized minimum reproductively cohesive units. We then compared allele frequencies among all samples and subsamples examined. Results clearly indicated that all but two can be classified into two groups that can be discriminated from each other by remarkable allele frequency differences at four diagnostic loci, and by large genetic distances even between sympatric subsamples. Observations of morphological features of the samples and subsamples confirmed that the two groups correspond to G. yakuensis and G. hokouensis, supporting validities of these two species. Allele frequency comparisons, however, also revealed that the remaining two samples, both from southern Kyushu, possessed ‘marker alleles' of both G. yakuensis and G. hokouensis at all four diagnostic loci. These samples thus were considered to represent populations that have been derived through hybridization of the two species. Detailed analyses for genetic structures demonstrated that all hybrid genotypes in the two samples are post‐Fi generations with only one individual resulting from the back‐cross with a pure line population of G. yakuensis. This finding negates the possibility that the hybrid populations are maintained by a constant supply of newly produced Fj hybrids, but suggests that the hybrid genotypes constitute stable breeding populations. This implies that the genealogical independence of G. yakuensis and G. hokouensis in several other sympatric areas has been maintained by operations of some isolation mechanisms at a pre‐mating phase. Investigations of the morphological variation in each sample or subsample revealed that although the two species can be externally largely discriminated from each other by slight modifications of the currently used diagnoses, it is difficult to detect their hybrids based solely on the morphological features.     

Genetic relationships and reproductive-isolation mechanisms among the Fejervarya limnocharis complex from Indonesia (Java) and other Asian countries

In order to elucidate the genetic relationships and reproductive-isolation mechanisms among the Fejervarya limnocharis complex from Indonesia and other Asian countries, allozyme analyses and crossing experiments were carried out using 208 individuals from 21 localities in eight Asian countries. The allozyme analyses revealed that 17 enzymes examined were controlled by genes at 27 loci, and that 7.9 phenotypes were produced by 5.2 alleles on average. The two species recognized in F. limnocharis sensu lato from Southeast Asia (i.e., F. limnocharis sensu stricto and F. iskandari) were found to occur sympatrically at three localities (Bogor, Cianjur and Malingping), all on Java, Indonesia. Fejervaya iskandari was dominant at each of these localities and showed substantial geographic genetic variation. Laboratory-produced hybrids between F. limnocharis and F. iskandari from Java became underdeveloped and died at the tadpole stage, suggesting that these species are completely isolated by hybrid inviability. Hybrids between topotypic F. limnocharis and the Malaysian and Japanese conspecific populations developed normally to metamorphosis. Likewise, hybrids between topotypic F. iskandari and the Thailand and Bangladesh conspecific populations also showed normal viability throughout larval development. The present allozyme analyses and crossing experiments strongly suggested the presence of two distinct forms, the large type and the small type, in the F. limnocharis complex from Asia, and further subdivision of the large type into the F. limnocharis assemblage and the F. iskandari assemblage. The small type was found in samples from India, Thailand, Bangladesh and Sri Lanka, and included at least three different species. The sample from Pilok, Thailand, was considered to represent an undescribed species.     

Analysis of allozyme variability in populations of three species of brush-haired mice of species Lophuromys (Rodentia, Muridae) from the Bale Mountains National Park in Ethiopia

Allozyme variability was examined in populations of three endemic species of the species complex Lophuromys flavopunctatus sensu lato: L. chrysopus, L. brevicaudus, and L. melanonyx. These species substitute each other in adjacent latitudinal belts of the Bale Massif in Ethiopia. A deficit of heterozygotes at several loci was found in most samples of all species studied. Moreover, the samples included animals homozygous for two or three minor alleles and heterozygous for alleles that are rare and unique for the given species. It is suggested that the Bale Massif are inhabited by numerous genetically isolated populations of each Lophuromys species, which exchange genes at an extremely low rate. Genotypic disequilibrium observed in most samples is explained by the fact that most sampling localities comprise ranges of two and more micropopulations. In our view, microgeographic subdivision of the populations is caused by recurrent fragmentation of habitats during the Pleistocene glaciation of the Bale Massif and subsequent prolonged isolation of local populations. Gene drift accompanying these processes resulted in high genetic differentiation of the local populations, which probably persisted until the present. Geographical isolation of the Bale Massif, its uniquely diverse ecological conditions, and extraordinary allozyme structure of the Lophuromys populations suggest that these populations represent remnants or direct descendants of relic local populations.     

Natural Selection VS. Random Drift: Evidence from Temporal Variation in Allele Frequencies in Nature

We have obtained monthly samples of two species, Drosophila pseudoobscura and Drosophila persimilis, in a natural population from Napa County, California. In each species, about 300 genes have been assayed by electrophoresis for each of seven enzyme loci in each monthly sample from March 1972 to June 1975. Using statistical methods developed for the purpose, we have examined whether the allele frequencies at different loci vary in a correlated fashion. The methods used do not detect natural selection when it is deterministic (e.g., overdominance or directional selection), but only when alleles at different loci vary simultaneously in response to the same environmental variations. Moreover, only relatively large fitness differences (of the order of 15%) are detectable. We have found strong evidence of correlated allele frequency variation in 13-20% of the cases examined. We interpret this as evidence that natural selection plays a major role in the evolution of protein polymorphisms in nature.     

Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection Specimens

In the recent years, many protocols aimed at reproducibly sequencing reduced-genome subsets in non-model organisms have been published. Among them, RAD-sequencing is one of the most widely used. It relies on digesting DNA with specific restriction enzymes and performing size selection on the resulting fragments. Despite its acknowledged utility, this method is of limited use with degraded DNA samples, such as those isolated from museum specimens, as these samples are less likely to harbor fragments long enough to comprise two restriction sites making possible ligation of the adapter sequences (in the case of double-digest RAD) or performing size selection of the resulting fragments (in the case of single-digest RAD). Here, we address these limitations by presenting a novel method called hybridization RAD (hyRAD). In this approach, biotinylated RAD fragments, covering a random fraction of the genome, are used as baits for capturing homologous fragments from genomic shotgun sequencing libraries. This simple and cost-effective approach allows sequencing of orthologous loci even from highly degraded DNA samples, opening new avenues of research in the field of museum genomics. Not relying on the restriction site presence, it improves among-sample loci coverage. In a trial study, hyRAD allowed us to obtain a large set of orthologous loci from fresh and museum samples from a non-model butterfly species, with a high proportion of single nucleotide polymorphisms present in all eight analyzed specimens, including 58-year-old museum samples. The utility of the method was further validated using 49 museum and fresh samples of a Palearctic grasshopper species for which the spatial genetic structure was previously assessed using mtDNA amplicons. The application of the method is eventually discussed in a wider context. As it does not rely on the restriction site presence, it is therefore not sensitive to among-sample loci polymorphisms in the restriction sites that usually causes loci dropout. This should enable the application of hyRAD to analyses at broader evolutionary scales.     

Development and characterization of nine polymorphic microsatellite markers in the Chilean kelp Lessonia nigrescens

A total of nine microsatellite loci were isolated and characterized in the Chilean kelp Lessonia nigrescens Bory. Using two different enriched libraries, we observed 1-14 alleles per locus in two samples of 21 kelp individuals each. The observed heterozygosities ranged from 0.05 to 0.80 and all loci are in Hardy-Weinberg equilibrium for one or both samples. Seventeen samples collected from different sites showed high allele diversity along the species distribution. The variation detected at these markers is currently being used for the study of populations of Lessonia nigrescens at different geographical scales.     

Local endemism and within‐island diversification of shrews illustrate the importance of speciation in building Sundaland mammal diversity

Island systems are important models for evolutionary biology because they provide convenient, discrete biogeographic units of study. Continental islands with a history of intermittent dry land connections confound the discrete definitions of islands and have led zoologists to predict (i) little differentiation of terrestrial organisms among continental shelf islands and (ii) extinction, rather than speciation, to be the main cause of differences in community composition among islands. However, few continental island systems have been subjected to well‐sampled phylogeographic studies, leaving these biogeographic assumptions of connectivity largely untested. We analysed nine unlinked loci from shrews of the genus Crocidura from seven mountains and two lowland localities on the Sundaic continental shelf islands of Sumatra and Java. Coalescent species delimitation strongly supported all currently recognized Crocidura species from Sumatra (six species) and Java (five species), as well as one undescribed species endemic to each island. We find that nearly all species of Crocidura in the region are endemic to a single island and several of these have their closest relative(s) on the same island. Intra‐island genetic divergence among allopatric, conspecific populations is often substantial, perhaps indicating species‐level diversity remains underestimated. One recent (Pleistocene) speciation event generated two morphologically distinct, syntopic species on Java, further highlighting the prevalence of within‐island diversification. Our results suggest that both between‐ and within‐island speciation processes generated local endemism in Sundaland, supplementing the traditional view that the region's fauna is relictual and primarily governed by extinction.     

Resolution of natural groups using iterative assignment tests: an example from two species of Australian native rats (Rattus)

Sympatric individuals of Rattus fuscipes and Rattus leucopus, two Australian native rats from the tropical wet forests of north Queensland, are difficult to distinguish morphologically and are often confused in the field. When we started a study on fine-scale movements of these species, using microsatellite markers, we found that the species as identified in the field did not form coherent genetic groups. In this study, we examined the potential of an iterative process of genetic assignment to separate specimens from distinct (e.g. species, populations) natural groups. Five loci with extensive overlap in allele distributions between species were used for the iterative process. Samples were randomly distributed into two starting groups of equal size and then subjected to the test. At each iteration, misassigned samples switched groups, and the output groups from a given round of assignment formed the input groups for the next round. All samples were assigned correctly on the 10th iteration, in which two genetic groups were clearly separated. Mitochondrial DNA sequences were obtained from samples from each genetic group identified by assignment, together with those of museum voucher specimens, to assess which species corresponded to which genetic group. The iterative procedure was also used to resolve groups within species, adequately separating the genetically identified R. leucopus from our two sampling sites. These results show that the iterative assignment process can correctly differentiate samples into their appropriate natural groups when diagnostic genetic markers are not available, which allowed us to resolve accurately the two R. leucopus and R. fuscipes species. Our approach provides an analytical tool that may be applicable to a broad variety of situations where genetic groups need to be resolved.     

Species-Specific Allozyme Markers for Appalachian Wood-Feeding Cockroaches (Dictyoptera: Cryptocercidae)

Members of the genus Cryptocercus are wood-feeding cockroaches that live in the temperate forests. Nine species are recognized in the genus worldwide: two in eastern Eurasia, two in China, and five in the United States. Within the United States, one species occurs in the Pacific Northwest and four occur in the Appalachian Mountains. Previous studies have revealed the presence of potential zones of overlap in distribution among the Appalachian species, raising the possibility of hybridization among them. Differences in mitochondrial DNA have previously been identified for the Appalachian species. However, to identify hybrid individuals one or more species-specific, codominant nuclear markers are required. Therefore, our objective was to undertake allozyme analysis of enzymatic loci to identify fixed, species-specific alleles for the four Appalachian species. We assayed a mean of 42 individuals each from 16 sites for allozyme variation for the four species. At 6 of the 33 loci examined, fixed alternate alleles were identified; a combination of 2 loci enabled the identification of all four species. To identify hybrids in the field, we examined 42 individuals each from 13 sites in which two or more of the above species occur in close proximity for presence of heterozygous individuals at one or more of the six fixed loci. No heterozygous individuals were found suggesting the lack of hybridization among the Appalachian species.     

Allozyme Variability among Populations of Three Species of Brush-Furred Mice (Lophuromys,Rodentia, Muridae) from the Bale Mountains National Park (Ethiopia)

Allozyme variability was examined in populations of three endemic species of the species complex Lophuromys flavopunctatussensu lato: L. chrysopus, L. brevicaudus, and L. melanonyx. These species replace each other in adjacent altitudinal belts of the Bale Massif in Ethiopia. A deficit of heterozygotes at several loci was found in most samples of all species studied. Moreover, the samples included animals homozygous for two or three minor alleles and heterozygous for alleles that are rare and unique for the given species. It is suggested that the Bale Massif are inhabited by numerous genetically isolated populations of eachLophuromys species, which exchange genes at an extremely low rate. Genotypic disequilibrium observed in most samples is explained by the fact that most sampling localities comprise ranges of two and more micropopulations. In our view, microgeographic subdivision of the populations is caused by recurrent fragmentation of habitats during the Pleistocene glaciation of the Bale Massif and subsequent prolonged isolation of local populations. Gene drift accompanying these processes resulted in high genetic differentiation of the local populations, which probably persisted until the present. Geographical isolation of the Bale Massif, its uniquely diverse ecological conditions, and extraordinary allozyme structure of the Lophuromys populations suggest that these populations represent remnants or direct descendants of relic local populations.     

Comparing the genetic structure of codling moth Cydia pomonella (L.) from Greece and France: long distance gene-flow in a sedentary pest species

Codling moth Cydia pomonella L. (Lepidoptera: Tortricidae) is the most important insect pest of apple production in Europe. Despite the economic importance of this pest, there is not information about the genetic structure of its population in Greece and the patterns of gene-flow which might affect the success of control programs. In this study, we analysed nine samples from apple, pear and walnut from various regions of mainland Greece using 11 microsatellite loci. Six samples from the aforementioned hosts from southern France were also examined for comparison. Bayesian clustering and genetic distance analyses separated the codling moth samples in two genetic clusters. The first cluster consisted mainly of the individuals from Greece, and the second of those from France, although admixture and miss-classified individuals were also observed. The low genetic differentiation among samples within each country was also revealed by F(ST) statistics (0.009 among Greek samples and 0.0150 among French samples compared to 0.050 global value among all samples and 0.032 the mean of the pair-wise values between the two countries). These F(ST) values suggest little structuring at large geographical scales in agreement with previous published studies. The host species and local factors (climatic conditions, topography, pest control programs) did not affect the genetic structure of codling moth populations within each country. The results are discussed in relation to human-made activities that promote gene-flow even at large geographic distances. Possible factors for the genetic differentiation between the two genetic clusters are also discussed.     

Genetic identification of two sibling species of Lutzomyia longipalpis (Diptera: Psychodidae) that produce distinct male sex pheromones in Sobral,Ceará State,Brazil

Lutzomyia longipalpis, the main sandfly vector for New World visceral leishmaniasis is a complex of an as yet undefined number of sibling species. At present, there is no consensus on the status (single species vs. species complex) of Brazilian populations. We applied five microsatellite loci to test the hypothesis that L. longipalpis occurs as two sympatric cryptic species in Sobral, Ceará State, Brazil as predicted by male sex pheromone chemotypes described previously for field specimens from this site [S-9-methyl-germacrene-B (9MGB) and a cembrene compound]. Abdominal spot morphology corresponds with pheromone type at this locality (9MGB in '1 spot' males and cembrene in '2 spot' males). Genotype data from 190 wild-caught L. longipalpis specimens collected in October 1999 and April 2001 were used to estimate genetic differentiation between the two sex pheromone populations and sampling dates. No significant (P > 0.05) genetic differences were found between the 1999 and 2001 9MGB samples (theta = 0.018; RST = -0.005), and genetic differentiation was low between the cembrene collections (theta = 0.037, P < 0.05; RST = -0.043, P > 0.05). By contrast, highly divergent allelic frequencies (largely at two microsatellite loci) corresponded to significant (P > 0.05) genetic differentiation (theta = 0.221; RST = 0.215) for all comparisons between samples with different pheromones. When pheromone samples were pooled across sample date, genetic differentiation was high (theta = 0.229; P < 0.001; Nem = 0.84). The allele frequency distribution at each of the five microsatellite loci was similar for males and females from the two collection years. Two of these loci showed highly divergent allele frequencies in the two sex pheromone populations. This was reflected in the highly significant genetic differentiation obtained from the male genotypes, between populations producing different pheromones (theta = 0.229-0.268; P < 0.0001 for the 2001 and theta = 0.254-0.558; P < 0.0001 for the 1999 collections, respectively). Similar results were obtained when the females, assigned to a pheromone type, were included in the analysis. Both a Bayesian analysis of the data set and a population assignment test provided strong evidence for two distinct populations corresponding to pheromone type. Given its genotype, the probability of assigning a 9MGB male to the original 9MGB population was 100% once the two years' collections were pooled. For cembrene-producing '2 spot' males this probability although still high, was lower than for 9MGB males, at 86%. This microsatellite data together with previously reported reproductive isolation between the two Sobral populations confirm that premating barriers are important in speciation of L. longipalpis.     

Genetic Structure and Variation of Van Cats

To determine the genetic structure and variation of Van cats and some other cats, seven enzyme loci were examined using horizontal starch gel electrophoresis. ME bands were observed for the first time in cats. For the enzyme loci CA ( 1 ), SOD, GPI, and GOT, neither the individual Van cats nor the specimens of other cat species exhibited any variation. These enzymes presented identical bands, all of which were homozygous. With respect to the PGD, ME, and ESD loci, however, genetic variation was observed in all of the cats. Hence, three of the seven gene-enzyme systems (43%) were polymorphic with two alleles, contributing to an estimate of average heterozygosity of 0.33-0.49 for the Van cats. PGD was the most discriminatory among the three polymorphic loci. The phylogenetic tree indicated that the Van, Persian, Turkish Angora, and Turkish Tekir cats are distinct from Siamese and Bombay cats.     

Electrophoretic investigation of genetic variation in two krill species Euphausia superba and E. crystallorophias (Euphausiidae)

Summary Specimens of Euphausia superba and of E. crystallorophias from different locations were analyzed electrophoretically for protein variation. The present study extends previous genetic studies on E. superba by analyzing samples from the East Wind Drift and repeat samples from the Bransfield Strait, Elephant Island and the Weddell Sea. E. crystallorophias was collected in the Weddell Sea and around the Antarctic Peninsula in order to provide information on the breeding structure of the species in this region. For all loki taking all sampling sites together for both species except GPI in E. crystallorophias no significant deviation of phenotype distributions from random mating expectations was observed. Furthermore, the allele distributions in all polymorphic loci for both species were found to be homogeneous. Estimates of genetic distance between samples within each species are low (0.0001 to 0.0003 in E. superba and 0.0001 to 0.0002 in E. crystallorophias), and are consistent with results expected for samples from a single interbreeding population. Estimate of genetic distance between these two species was 0.9729. These results suggest that for each species specimens from all locations investigated in the Bransfield Strait and Weddell Sea belong to a single genetically homogeneous population. A possible mechanism for maintaining such homogeneity and the implications for fishery management are discussed.     

Two New Computational Methods for Universal DNA Barcoding: A Benchmark Using Barcode Sequences of Bacteria,Archaea, Animals,Fungi, and Land Plants

Taxonomic identification of biological specimens based on DNA sequence information (a.k.a. DNA barcoding) is becoming increasingly common in biodiversity science. Although several methods have been proposed, many of them are not universally applicable due to the need for prerequisite phylogenetic/machine-learning analyses, the need for huge computational resources, or the lack of a firm theoretical background. Here, we propose two new computational methods of DNA barcoding and show a benchmark for bacterial/archeal 16S, animal COX1, fungal internal transcribed spacer, and three plant chloroplast (rbcL, matK, and trnH-psbA) barcode loci that can be used to compare the performance of existing and new methods. The benchmark was performed under two alternative situations: query sequences were available in the corresponding reference sequence databases in one, but were not available in the other. In the former situation, the commonly used “1-nearest-neighbor” (1-NN) method, which assigns the taxonomic information of the most similar sequences in a reference database (i.e., BLAST-top-hit reference sequence) to a query, displays the highest rate and highest precision of successful taxonomic identification. However, in the latter situation, the 1-NN method produced extremely high rates of misidentification for all the barcode loci examined. In contrast, one of our new methods, the query-centric auto-k-nearest-neighbor (QCauto) method, consistently produced low rates of misidentification for all the loci examined in both situations. These results indicate that the 1-NN method is most suitable if the reference sequences of all potentially observable species are available in databases; otherwise, the QCauto method returns the most reliable identification results. The benchmark results also indicated that the taxon coverage of reference sequences is far from complete for genus or species level identification in all the barcode loci examined. Therefore, we need to accelerate the registration of reference barcode sequences to apply high-throughput DNA barcoding to genus or species level identification in biodiversity research.     

Extensive Natural Hybridization Between Two Geckos,Gekko tawaensis and Gekko japonicus (Reptilia: Squamata), Throughout Their Broad Sympatric Area

The status of natural hybridization between the two Japanese geckos, Gekko tawaensis and Gekko japonicus, was surveyed by use of 15 diagnostic allozyme loci. Of 438 specimens examined, 9 were identified as F_l, 1 was a first backcross with G. japonicus, and 15 were identified as more successive generations. Hybridizations were detected at 7 of the 15 localities interspersed throughout a broad sympatric area of the two species, forming a mosaic hybrid zone. A comparison of species–hybrid composition between 2-year samples from a single locality and a 5-year interval showed no evidence for progressive introgression or establishment of a hybrid swarm, despite constant emergences of F_l hybrids. Nonprevalence of the hybrid genotypes was also indicated by the analysis using hybrid index scores for all other localities examined. These results suggest that strong selection acts against hybrid genotypes. Morphological features of hybrid individuals were also provided.     

Microsatellite markers for northern red oak (Fagaceae: Quercus rubra)

We provide primer sequences for 14 (GA) _n microsatellite loci developed from northern red oak, an important timber species. We screened loci using two sets of samples. A parent–offspring set included DNA from seven acorns collected from one mother tree along with maternal DNA, to determine that all progeny carried a maternal allele at each locus. The other set was comprised of 10 adult trees sampled from Indiana old‐growth forest, providing a measure of diversity revealed by each locus.     

A panel of microsatellite loci for population studies of Sakhalin taimen Parahucho perryi (Brevoort)

Using DNA samples of Sakhalin taimen and a set of microsatellite loci, earlier reported for other salmonid fishes (Salmonidae), successful cross-species amplification was performed. A total of 56 Sakhalin taimen (Parahucho perryi) samples from the Daga, Nabil, Poronai, and Agnevo rivers (Sakhalin Island) were examined at 36 microsatellite loci, most of which were described for other species and first tested in taimen. Among the 21 loci first tested in taimen, two loci produced no amplification products. The remaining 19 loci were successfully amplified (for some loci, new primers were generated). Thirteen of these loci were monomorphic, while six loci were polymorphic and used in further population genetic analysis. In addition, with the purpose of modification of the allele sizes and optimization of the research technique, new primers for the already known 12 loci of Sakhalin taimen were designed. Three more loci were included in analysis without changes. As a result, a joint panel consisting of 21 polymorphic microsatellite markers was suggested for analysis of Sakhalin taimen. This panel was tested with four population samples from the rivers of Sakhalin Island.The results showed that this panel of markers could be used in detailed population studies for evaluation of the level of genetic differentiation, inbreeding, and migrations in Sakhalin taimen, an endangered species with a fragmented range. Using this approach in further studies will make it possible to isolate basic populations, which is necessary for conservation of this rare species.     

Comparison of genetic and morphological methods to detect the presence of American lobsters, <Emphasis Type="Italic">Homarus americanus</Emphasis> H. Milne Edwards, 1837 (Astacidea: Nephropidae) in Norwegian waters

American lobsters (Homarus americanus H. Milne Edwards, 1837) are imported live to Europe and should according regulations be kept in land-based tanks until sold. In spite of the strict regulations aimed specifically at preventing the introduction of this species into the NE Atlantic, several specimens of H. americanus have been captured in the wild, especially in Oslofjord, Norway since 1999. One of the great concerns is interbreeding between the introduced American species and the local European lobster, H. gammarus (Linnaeus, 1758). For this reason an awareness campaign was launched in 2000 focusing on morphologically “unusual” lobsters caught in local waters. Morphological characters have been based on colour and sub-ventral spines on the rostrum. Two samples of H. americanus were used for comparisons, as well as samples of European lobster from Oslofjord collected in 1992. Previous genetic analyses (allozymes, mtDNA and microsatellite DNA) have demonstrated that the American lobster is distinct from its European counterpart, with several additional alleles at many loci in addition to different allelic frequency distribution of alleles of “shared” alleles. During the present study, thirteen microsatellite loci were tested in the initial screening, and the three most discriminating loci (Hgam98, Hgam197b and Hgam47b) were used in a detailed comparison between the two species. A total of 45 unusual lobsters were reported captured from Ålesund (west) to Oslofjord (southeast) from 2001 to 2005 and these were analysed for the three microsatellite loci. Nine specimens were identified as American lobsters. Comparisons between morphological and genetic characteristics revealed that morphological differences are not reliable in discrimination the two species, or to identify hybrids. Further, some loci display almost no overlapping in allele frequency distribution for the reference samples analysed, thus providing a reliable tool to identify hybrids.