Mitochondrial DNA reveals a mosaic pattern of phylogeographical structure in Atlantic and Mediterranean populations of anchovy (Engraulis encrasicolus)

This study extends the geographic coverage of a previous study of mitochondrial DNA restriction fragment length polymorphism in European anchovy. Both studies together include 24 samples representing 17 localities extending from the Black Sea, through the Mediterranean Sea to the eastern Atlantic as far south as Dakar, Senegal. Eighty-eight haplotypes define two clades (A and B) separated by 3.2% sequence divergence. Clade A has a star-like genealogy indicative of a recent population expansion. Clade B has a more complex genealogy, consisting of several haplotypes at intermediate frequencies. The distributions of these clades consist of a mosaic with abrupt changes between some areas and gradients between other areas. Clade A predominates the Black and Aegean seas, but is present throughout the Mediterranean. Unexpectedly, new data show that clade A is also at a high frequency in the Atlantic, from Portugal to at least Senegal. Overall, the level of genetic differentiation among populations is high (F(ST)=0.148, p<0.0001), with the greatest differences between basins. AMOVA reveals four main geographical groups: Atlantic, central Mediterranean, Aegean Sea, and Black Sea. Mismatch distribution clearly indicates historical bottleneck and population expansion for clade A, while for clade B such evidence is equivocal. This difference may reflect a range expansion for both clades, but with higher gene flow (Nm values) between demes for clade A. Both contemporary and historical processes are important in shaping the complex genetic population structure of European anchovy.     

Genetic structuring of European anchovy (Engraulis encrasicolus) populations through mitochondrial DNA sequences

Mitochondrial DNA sequence variation in 655?bpfragments of the cytochrome oxidase c subunit I gene, known as the DNA barcode, of European anchovy (Engraulis encrasicolus) was evaluated by analyzing 1529 individuals representing 16 populations from the Black Sea, through the Marmara Sea and the Aegean Sea to the Mediterranean Sea. A total of 19 (2.9%) variable sites were found among individuals, and these defined 10 genetically diverged populations with an overall mean distance of 1.2%. The highest nucleotide divergence was found between samples of eastern Mediterranean and northern Aegean (2.2%). Evolutionary history analysis among 16 populations clustered the Mediterranean Sea clades in one main branch and the other clades in another branch. Diverging pattern of the European anchovy populations correlated with geographic dispersion supports the genetic structuring through the Black Sea-Marmara Sea-Aegean Sea-Mediterranean Sea quad.     

Genetic and morphological analyses of tub gurnard Chelidonichthys lucerna populations in Turkish marine waters

Genetic and morphological structure of tub gurnard Chelidonichthys lucerna populations in Turkish marine waters were investigated with mtDNA sequencing of 16S rRNA and morphological characters. C. lucerna samples were collected from the Black Sea, Marmara, Aegean and northeastern Mediterranean coasts of Turkey. The lowest genetic diversity was found in the northeastern Mediterranean (Iskenderun Bay) population, while the highest was in the Marmara population with overall average value of genetic diversity within populations. A total of 14 haplotypes was found, and the highest haplotype diversity was in the Black Sea whereas the lowest was in the northeastern Mediterranean population (Iskenderun Bay). The Black Sea and Iskenderun Bay populations showed the least genetic divergence (0.001081), while the highest was between the Marmara Sea and northeastern Mediterranean (Antalya Bay) populations (0.002067). Pairwise comparisons of genetic distance revealed statistically significant differences (P < 0.05) between the Marmara and both the Aegean and northeastern Mediterranean (Antalya Bay) samples. Neighbour joining tree analyses clustered the northeastern Mediterranean populations (Antalya Bay and Iskenderun Bay) as genetically more interrelated populations, whereas the Aegean Sea population was clustered as most isolated one. Discriminant function analysis of morphological characters showed that only the Black Sea population is differentiated from the other populations.     

Multiple origins for Mediterranean olive (Olea europaea L. ssp. europaea) based upon mitochondrial DNA polymorphisms

A study of nuclear and cytoplasmic genetic diversity of cultivated olive, oleaster and other taxa belonging to the complex O. europaea was performed. Nuclear DNA polymorphism (RAPDs) in oleaster displays a gradient between the east and west of the Mediterranean Basin. In cultivars, the gradient is less visible owing to their diffusion and selection. Furthermore, three mitotypes (ME1, MOM and MCK) were detected in both cultivated olive and oleaster. A fourth mitotype, ME2, was unique to some cultivars. The preponderant mitotype, ME1, marks the Near Eastern origin of olive in oleaster. In the west of the Mediterranean, another mitotype, MOM, was found in most oleaster. and a few cultivars. The third, MCK, was found in a few oleaster from the west and in cultivars originating in Kabylie and Languedoc. We argue that MCK marks an ancestral Mediterranean population. The mitotypes mark independent cultivated olive origins which were not detected with DNA nuclear diversity.     

Genetic differentiation of Mediterranean horse mackerel (Trachurus mediterraneus) populations as revealed by mtDNA PCR-RFLP analysis

The genetic population structure of Mediterranean horse mackerel, Trachurus mediterraneus , from seven locations throughout the Black, Marmara, Aegean and eastern Mediterranean seas was investigated using restriction fragment length polymorphism (RFLP) analysis of the mtDNA 16S rDNA region. An approximately 2000-bp segment was screened in 280 individuals using six restriction enzymes, resulting in 10 composite haplotypes. The most common haplotype was present in 56.42% individuals; the next most frequent haplotype was present in 22.85% individuals. Average haplotype diversity within samples was moderate (0.38), and nucleotide diversity was low (0.00435). Mean nucleotide divergence for the seven sampling sites was 0.0028. Nucleotide divergence among samples was moderate, with the highest value detected between the Aegean Sea (Izmir) and the eastern Black Sea (Trabzon) populations (0.007055), and the lowest (−0.000043) between the Marmara Sea (Adalar) and the western Black Sea (Sile) populations. In Monte Carlo pairwise comparisons of haplotype frequencies, the Sinop from the middle Black Sea, Trabzon from the eastern Black Sea, and Iskenderun Bay from the north-eastern Mediterranean Sea exhibited highly significant (P   <   0.001) geographical differentiation from each other and from all other populations. Mantel's test indicated that the nucleotide divergence among populations of T. mediterraneus was not significantly associated with their geographical isolation ( r  = −0.2963; P   >   0.05). Consequently, the mtDNA 16S rDNA region provided evidence for the existence of three distinct T. mediterraneus populations (Sinop, Trabzon and Iskenderun Bay) in the Black and north-eastern Mediterranean seas.     

Modeling and predicting potential spawning habitat of anchovy (Engraulis encrasicolus) and round sardinella (Sardinella aurita) based on satellite environmental information

Anchovy and round sardinella are two important small pelagic species in the Mediterranean that spawn during the summer period. This is a first attempt to model and predict the two species’ potential spawning habitats in this area. Generalized additive models (GAMs) were constructed based on satellite environmental variables and presence/absence egg data, available from ichthyoplankton surveys conducted in the North Aegean Sea during early summer (June 2003–2006). These models were subsequently used to predict the probability of anchovy and round sardinella spawning in the Greek Seas as well as the entire Mediterranean and Black Sea during the same month of the year. The interaction of bottom depth and chlorophyll explained most of the deviance in the presence/absence GAMs of both species, indicating spawning over continental shelf areas with increased surface chlorophyll values. Round sardinella spawned closer to coast than anchovy. Predicted potential spawning areas for anchovy and round sardinella in unsampled areas of the Greek Seas and the entire Mediterranean and Black Sea were in good agreement with existing information on the distribution and extent of the spawning grounds, especially for anchovy. Modeling the species’ reproductive activity in relation to easily accessible environmental information and applying the models in a predictive way could be an initial, low-cost step to designate potential spawning fish habitats. Guest editor: V. D. Valavanis Essential Fish Habitat Mapping in the Mediterranean     

Modelling the presence of anchovy Engraulis encrasicolus in the Aegean Sea during early summer, based on satellite environmental data

Acoustic and satellite environmental data as well as bathymetry data were used to model the presence of anchovy, Engraulis encrasicolus during early summer in the northern Aegean Sea (Eastern Mediterranean). Generalized Additive Models (GAMs) were used for modelling and subsequently applied in a predictive mode to identify those areas in the Greek Seas and the entire Mediterranean basin that could support species’ presence. Model results were evaluated with the estimation of Receiver Operating Characteristic (ROC)-plots as well as qualitatively, based on (a) acoustic data from concurrent studies in certain areas of the northern Aegean Sea that were not included in the estimation of the GAM model and (b) historical acoustic data from the central Aegean and Ionian Seas. Mapping the estimated environmental conditions in the Mediterranean basin indicated areas that generally agree with the known distribution grounds of anchovy, such as the straits of Sicily and coastal waters of Tunisia, areas in the Tyrrhenian Sea, the Adriatic Sea, the Gulf of Lions and the Catalan Sea. Guest editor: V. D. Valavanis Essential Fish Habitat Mapping in the Mediterranean     

A Second Look at Mitochondrial DNA Variability in European Anchovy (Engraulis encrasicolus): Assessing Models of Population Structure and the Black Sea Isolation Hypothesis

Genetic architectures of marine fishes are generally shallow because of the large potential for gene flow in the sea. European anchovy, however, are unusual among small pelagic fishes in showing large differences among sub-basins and in harbouring two mtDNA phylogroups (‘A’ & ‘B’), representing 1.1–1.85 million years of separation. Here the mtDNA RFLP dataset of Magoulas et al. [1996, Mol. Biol. Evol. 13: 178–190] is re-examined to assess population models accounting for this subdivided population structure and to evaluate the zoogeographical origins of the two major phylogroups. Haplotype and nucleotide diversities are highest in the Ionian Sea and lowest in the Aegean and Black seas. However, this gradient is absent when ‘A’ and ‘B’ haplotypes are examined separately. Neither the self-sustaining nor the basin population models adequately describe anchovy population behaviour. Tests for neutrality, mismatch and nested clade analyses are concordant in depicting recent expansions of both phylogroups. Unimodel mismatch distributions and haplotype coalescences dating to the last (Eemian) interglacial (‘B’) and the Weichselian pleniglacial period (‘A’) indicate separate colonizations of the Mediterranean Basin. Phylogroup ‘A’ is unlikely to have arisen through continuous long-term isolation in the Black Sea because of climate extremes from displaced subpolar weather systems during the ice ages. Ancestors of both groups appear to have colonized the Mediterranean from the Atlantic in the late Pleistocene. Hence, zoogeographic models of anchovy in the Mediterranean must also include the eastern (and possibly southern) Atlantic.     

Contemporary genetic structure and postglacial demographic history of the black scorpionfish,Scorpaena porcus,in the Mediterranean and the Black Seas

Understanding the distribution of genetic diversity in the light of past demographic events linked with climatic shifts will help to forecast evolutionary trajectories of ecosystems within the current context of climate change. In this study, mitochondrial sequences and microsatellite loci were analysed using traditional population genetic approaches together with Bayesian dating and the more recent approximate Bayesian computation scenario testing. The genetic structure and demographic history of a commercial fish, the black scorpionfish, Scorpaena porcus, was investigated throughout the Mediterranean and Black Seas. The results suggest that the species recently underwent population expansions, in both seas, likely concomitant with the warming period following the Last Glacial Maximum, 20 000 years ago. A weak contemporaneous genetic differentiation was identified between the Black Sea and the Mediterranean Sea. However, the genetic diversity was similar for populations of the two seas, suggesting a high number of colonizers entered the Black Sea during the interglacial period and/or the presence of a refugial population in the Black Sea during the glacial period. Finally, within seas, an east/west genetic differentiation in the Adriatic seems to prevail, whereas the Black Sea does not show any structured spatial genetic pattern of its population. Overall, these results suggest that the Black Sea is not that isolated from the Mediterranean, and both seas revealed similar evolutionary patterns related to climate change and changes in sea level.     

Application of a bioenergetics growth model for European anchovy (<Emphasis Type="Italic">Engraulis encrasicolus</Emphasis>) linked with a lower trophic level ecosystem model

A bioenergetics model is implemented for European anchovy (Engraulis encrasicolus) and applied to the north-eastern Aegean Sea (eastern Mediterranean Sea). The model reproduces the growth of anchovy in a one-way linked configuration with a lower trophic level (LTL) ecosystem model. The LTL model provides densities for three zooplankton functional groups (heterotrophic flagellates, microzooplankton and mesozooplankton) which serve as available energy via consumption for the anchovy model. Our model follows the basic structure of NEMURO.FISH type models (North Pacific Ecosystem Model for Understanding Regional Oceanography for Including Saury and Herring). Several model parameters were specific for the Mediterranean or the Black Sea anchovy and some others were adopted from related species and NEMURO.FISH due to lack of biological information on E. encrasicolus. Simulation results showed that the fastest growth rate occurs during spring and the slowest growth rate from August to December. Zooplankton abundance during autumn was low implying that decreased prey density lead to a reduction in anchovy weight, especially for the age-3 class. Feeding parameters were adjusted to adequately fit the model growth estimates to available weight-at-age data. A detailed sensitivity analyses is conducted to evaluate the importance of the biological processes (consumption, respiration, egestion, specific dynamic action, excretion and egg production) and their parameters to fish growth. The most sensitive parameters were the intercept and exponent slope of the weight-dependent consumption and respiration process equations. Fish weight was fairly sensitive to temperature-dependent parameters.     

Microsatellite DNA reveals genetically different populations of Atlantic bonito Sarda sarda in the Mediterranean Basin

Microsatellites were used to investigate population genetic structure of Atlantic bonito Sarda sarda from the Black Sea, Marmara Sea, Aegean Sea, north-eastern Mediterranean Sea and Adriatic Sea. Overall average observed heterozygosity was high (0.93). Average observed heterozygosity per locus ranged from 0.79 to 0.98. Pairwise F_ST estimates for all loci between populations ranged from 0 to 0.07626, and significant F_ST values (P < 0.001) were detected between populations; the Blacks Sea and Marmara Sea samples were not significantly different from each other, but significant different from the other samples, and Aegean Sea and north-eastern Mediterranean Sea samples were also not significantly different from each other, but significantly different from all other samples. The Adriatic Sea sample was significant different from all other samples. The Mantel test revealed a significant (P < 0.001, r = 0.68) isolation-by-distance for these 11 populations. Neighbour-joining analysis clustered the Black Sea and Marmara Sea samples together while collections from Aegean Sea and north-eastern Mediterranean Sea were clustered close to each other and far from the others. On the other hand, the Adriatic Sea collection presented very distinctive relationship from the others.     

The taxonomic status of the Black Sea and Marmara Sea populations of the Broadnosed Pipefish Syngnathus cf. argentatus Pallas (Teleostei: Syngnathidae) based on morphological and molecular characters

As the Black Sea and Marmara Sea population of the Broadnosed Pipefish Syngnathus cf. argentatus show some morphological differences from the Mediterranean Sea populations, some authors regard it as an endemic species Syngnathus argentatus Pallas, 1814, while others consider it as a synonym of S. typhle Linnaeus, 1758. The aim of this study is to compare the populations of the Black Sea and Sea of Marmara with the Aegean Sea population, using a combination of morphological and molecular characters, in order to clarify their taxonomic status. Sampling was carried out at three stations in the Black Sea, two in the Sea of Marmara and three in the Aegean Sea, and a total of 24 morphometric and 6 meristic characters were examined. Metric data were analysed by Principal Component Analysis (PCA) and phylogenetic relationships between the populations were analysed using both cytochrome oxidase subunit 1 (COI) gene and cytochrome b (cyt-b) gene sequences. Although constant differences were observed in snout depths between the Black Sea/Marmara Sea and the Aegean Sea populations, other morphological features and genetic analysis did not enable these populations to be differentiated. These findings indicate that S. argentatus is a synonym of S. typhle.     

Invasion Pathway of the Ctenophore Mnemiopsis leidyi in the Mediterranean Sea

Gelatinous zooplankton outbreaks have increased globally owing to a number of human-mediated factors, including food web alterations and species introductions. The invasive ctenophore Mnemiopsis leidyi entered the Black Sea in the early 1980s. The invasion was followed by the Azov, Caspian, Baltic and North Seas, and, most recently, the Mediterranean Sea. Previous studies identified two distinct invasion pathways of M. leidyi from its native range in the western Atlantic Ocean to Eurasia. However, the source of newly established populations in the Mediterranean Sea remains unclear. Here we build upon our previous study and investigate sequence variation in both mitochondrial (Cytochrome c Oxidase subunit I) and nuclear (Internal Transcribed Spacer) markers in M. leidyi, encompassing five native and 11 introduced populations, including four from the Mediterranean Sea. Extant genetic diversity in Mediterranean populations (n = 8, N _a = 10) preclude the occurrence of a severe genetic bottleneck or founder effects in the initial colonizing population. Our mitochondrial and nuclear marker surveys revealed two possible pathways of introduction into Mediterranean Sea. In total, 17 haplotypes and 18 alleles were recovered from all surveyed populations. Haplotype and allelic diversity of Mediterranean populations were comparable to populations from which they were likely drawn. The distribution of genetic diversity and pattern of genetic differentiation suggest initial colonization of the Mediterranean from the Black-Azov Seas (pairwise F _ST = 0.001–0.028). However, some haplotypes and alleles from the Mediterranean Sea were not detected from the well-sampled Black Sea, although they were found in Gulf of Mexico populations that were also genetically similar to those in the Mediterranean Sea (pairwise F _ST = 0.010–0.032), raising the possibility of multiple invasion sources. Multiple introductions from a combination of Black Sea and native region sources could be facilitated by intense local and transcontinental shipping activity, respectively.     

The use of otolith shape and chemistry to determine stock structure of Mediterranean horse mackerel Trachurus mediterraneus (Steindachner)

Otolith shape and chemistry of Mediterranean horse mackerel Trachurus mediterraneus were simultaneously used to assess the feasibility of using these natural tags to discriminate populations throughout the Black, Marmara, Aegean and eastern Mediterranean Seas. Otolith shape and chemistry analyses showed a similar pattern of differentiation between T. mediterraneus stocks, revealing a clear discreteness of the middle Black Sea (Sinop) and Aegean Sea (Izmir) samples. Otolith upper side length and width, and Na, K, Mg and Ba, were the morphological traits and trace elements, respectively, differing most among groups. Overall assignment of individuals into their original sample was high for both otolith shape and chemistry. Highest reclassification rate was observed for the south-middle Black Sea and Aegean Sea samples for both analyses. Hierarchical cluster analyses also supported high differentiation of the south-middle Black Sea and Aegean Sea samples for both analyses. Mantel's test revealed that the Euclidean distance both for otolith shape ( r =−0·0917, P > 0·05) and chemistry ( r =−0·1248, P > 0·05) between these populations were not significantly associated with their geographical distances.     

The postglacial history of Scots pine (Pinus sylvestris L.) in western Europe: evidence from mitochondrial DNA variation

The geographical structure of mitochondrial (mt)DNA variants (mitotypes) was investigated in 38 western European populations of Scots pine Pinus sylvestris using restriction fragment length polymorphism (RFLP) analysis of total DNA and a homologous cox1 probe. Three major mitotypes (designated a, b and d ) were detected. Within Spain all three major mitotypes were found, gene diversity was high, H_T = 0.586, and this diversity was distributed predominantly among rather than within populations (F_ST(M) = 0.813 for the seven Spanish populations). Mitotype d was present only in the most southerly population from the Sierra Nevada . Elsewhere in Europe, populations showed little or no mtDNA diversity within regions, but there were marked differences between regions. Italian populations were fixed for mitotype b ; populations from northern France, Germany, Poland, Russia and southern Sweden were fixed for mitotype a ; while populations in northern Fennoscandia were fixed for mitotype b . The isolated Scottish populations were predominantly of mitotype a , but mitotype b was present in three of the 20 populations scored. In Scotland, UK gene diversity (H_T = 0.120) and genetic differentiation among populations (F_ST(M) = 0.37) was much lower than in Spain. When interpreted in the light of complementary data from pollen analysis and nuclear genetic markers, the results suggest that present-day populations of P. sylvestris in western Europe have been derived from at least three different sources after glaciation.     

Polymorphism of mitochondrial DNA and distribution of cytoplasmic symbionts in the populations of two-spot ladybird beetle Adalia bipunctata

In geographically distant populations of ladybird beetle Adalia bipunctata from Eurasia mitotypes and infection with symbiotic bacteria Spiroplasma and Rickettsia were determined. All populations examined demonstrated mtDNA polymorphism and striking differences in prevalence of bacteria (from about 50% of individuals infected with Spiroplasma in St.-Petersburg population and 50% of the Rickettsia prevalence in Kem' population to complete absence of bacteria in the population from Archangelsk). In the populations studied a total of 14 mitotypes were discovered, including two mitotypes that were remarkably different from the others in nucleotide composition. Mitotype 10, which was the most different from all the others, was found in all populations from Germany to Transbaikalia, excluding the population from Tashkent. Linkage disequilibrium between mitotype 10 and the Rickettsia infection was confirmed. Infection with the Spiroplasma bacteria was typical of the individuals with haplotype 1 and relative to it. The results obtained supported the conclusion on the association between infection with Spiroplasma and Rickettsia and certain mitotype of A. bipunctata, which was the consequence of either absence or rare horizontal transfer of symbionts and ancientness of the first contact between the bacteria and A. bipunctata ladybird beetles.     

Demosponge distribution in the eastern Mediterranean: a NW–SE gradient

The purpose of this paper was to investigate patterns of demosponge distribution along gradients of environmental conditions in the biogeographical subzones of the eastern Mediterranean (Aegean and Levantine Sea). The Aegean Sea was divided into six major areas on the basis of its geomorphology and bathymetry. Two areas of the Levantine Sea were additionally considered. All available data on demosponge species numbers and abundance in each area, as well as their vertical and general geographical distribution were taken from the literature. Multivariate analysis revealed a NW–SE faunal gradient, showing an apparent dissimilarity among the North Aegean, the South Aegean and the Levantine Sea, which agrees with the differences in the geographical, physicochemical and biological characteristics of the three areas. The majority of demosponge species has been recorded in the North Aegean, while the South Aegean is closer, in terms of demosponge diversity, to the oligotrophic Levantine Sea. The number of studies in the Aegean and Levantine subareas was positively correlated with the number of species recorded within each Aegean subarea. Demosponge species with an Altanto-Mediterranean distribution prevailed in the Aegean and the Levantine. The reduced contribution of the endemic component, as compared to the western Mediterranean, is consistent with the general NW–SE decrease in the number of endemic species in the Mediterranean. Demosponge distribution at the order level showed also a NW–SE gradient, similar to that observed in the entire Atlantic–Mediterranean region, suggesting a warm water affinity. Sublittoral, circalittoral and bathyal zones were clearly distinguishable in the Aegean Sea on the basis of their sponge fauna. The total number of species was an exponential negative function of depth.     

New evidence from mitochondrial DNA of a progenitor-derivative species relationship between black spruce and red spruce (Pinaceae)

Mitochondrial DNA (mtDNA) markers were used to assess the genetic diversity in allopatric populations of black spruce (Picea mariana [Mill.] BSP) and red spruce (P. rubens Sarg.). Patterns of mitochondrial haplotypes (mitotypes) were strikingly different between the two species. All mtDNA markers surveyed were polymorphic in black spruce, revealing four different mitotypes and high levels of mtDNA diversity (P(p) = 100%, A = 2.0, H = 0.496). In contrast, populations of red spruce had only two mitotypes and harbored low levels of ggenetic diversity (P(p) = 13.2%, A = 1.1, H = 0.120). When the southernmost allopatric populations of red spruce were considered, only one mitotype was detected. As previously reported for nuclear gene loci, the diversity observed for mtDNA in red spruce was a subset of that found in black spruce. Comparison of present and previously published data supports the hypothesis of a recent progenitor-derivative relationship between these species, red spruce presumably being derived by allopatric speciation of an isolated population of black spruce during the Pleistocene.     

Biogeographic zonation of the Black Sea and Caspian Sea basin based on Mysid fauna (Crustacea: Mysidacea)

A biogeographic analysis of the Black Sea and Caspian Sea basin and adjacent Aegean Sea and the Sea of Marmara was conducted based on the distribution of 55 mysid species. The Black and Caspian Sea basin proper is inhabited by 35 mysid species belonging to 12 genera, among them 26 species and 3 genera are endemic to the region. Ponto-Caspian and Caspian species are predominant. The explored areas were included into two provinces of the Mediterranean subrealm of the East-Atlantic Subtropical realm and three provinces of the Ponto-Caspian realm.     

DNA barcode discovers two cryptic species and two geographical radiations in the invasive drosophilid Zaprionus indianus

Comparing introduced to ancestral populations within a phylogeographical context is crucial in any study aiming to understand the ecological genetics of an invasive species. Zaprionus indianus is a cosmopolitan drosophilid that has recently succeeded to expand its geographical range upon three continents (Africa, Asia and the Americas). We studied the distribution of mitochondrial DNA (mtDNA) haplotypes for two genes (CO‐I and CO‐II) among 23 geographical populations. mtDNA revealed the presence of two well‐supported phylogenetic lineages (phylads), with bootstrap value of 100%. Phylad I included three African populations, reinforcing the African‐origin hypothesis of the species. Within phylad II, a distinct phylogeographical pattern was discovered: Atlantic populations (from the Americas and Madeira) were closer to the ancestral African populations than to Eastern ones (from Madagascar, Middle East and India). This means that during its passage from endemism to cosmopolitanism, Z. indianus exhibited two independent radiations, the older (the Eastern) to the East, and the younger (the Atlantic) to the West. Discriminant function analysis using 13 morphometrical characters was also able to discriminate between the two molecular phylads (93.34 ± 1.67%), although detailed morphological analysis of male genitalia using scanning electron microscopy showed no significant differences. Finally, crossing experiments revealed the presence of reproductive barrier between populations from the two phylads, and further between populations within phylad I. Hence, a bona species status was assigned to two new, cryptic species: Zaprionus africanus and Zaprionus gabonicus, and both were encompassed along with Z. indianus and Zaprionus megalorchis into the indianus complex. The ecology of these two species reveals that they are forest dwellers, which explains their restricted endemic distribution, in contrast to their relative cosmopolitan Z. indianus, known to be a human‐commensal. Our results reconfirm the great utility of mtDNA at both inter‐ and intraspecific analyses within the frame of an integrated taxonomical project.