Local and global multivariate analysis of geographical mitochondrial DNA variation in Leuciscus cephalus L. 1758 (Pisces: Cyprinidae) in the Balkan Peninsula

Geographical variation of cytochrome b mitochondrial DNA (mtDNA) in chub (Leuciscus cephalus L.) was analysed in 31 samples from 20 different river basins in the Balkan Peninsula and Danube catchment. Multivariate methods of ordination were used to analyse variation of the data sets. The results were interpreted in the context of the proposed ichthyogcographic districts separating the Balkan Peninsula into two main ichthyogeographic divisions (Eastern Greece/Ponto-Acgcan and Western Greece/South Adriatic-Ionian). Boundary detection supported these two ichthyogeographic districts for L. cephalus , revealing a boundary that ran from north to south through the Balkan Peninsula and the middle of Greece. The results also revealed the existence of a third division in Central Greece. The results of ordination techniques on homogeneous zones and analysis of the molecular variance confirmed the results obtained in studying local variability (boundaries). They also allowed us to test the existence of possible subdivisions proposed by different authors inside the two main ichthyogeographic districts. These subdivisions were not supported. The multivariate methods used in this study allowed us to propose a coherent picture of chub ichthyogeographic districts in terms of boundary detection and maximal autocorrelation between populations and to explain the patterns of chub mtDNA variation. A complete interpretation of results concerning L. cephalus requires careful consideration of both boundary analysis and autocorrelative approach. Results from an the autocorrclativc approach alone could lead to substantial misinterpretations.     

Identifying main evolutionary mechanisms shaping genetic variation of Leuciscus cephalus L. 1758 (Cyprinidae) in western Greece: discordance between methods.

Numerous methods can be used in intraspecific phylogeographic studies to infer the evolutionary mechanisms that shaped observed genetic variation in populations. However, these methods are scarcely used jointly, and the evolutionary outcomes they could propose are not fully compared. In this study, using a chub (Leuciscus cephalus; Cyprinidae) mitochondrial DNA data set (13 populations in Western Greece, 14 haplotypes), we compare three distinct 'historical' methods that could possibly infer relative importance of basic evolutionary mechanisms (isolation vs migration) shaping genetic variation: the nested clade analysis, the psi-test and the 'mismatch distributions'. Taking together, interpretations of these analyses allow to draw a picture of the evolutionary history of chub in Western Greece based on isolation and genetic drift for higher clades. However, results issued each method can differ for low differentiated clades. We discuss such differences and suggest that methods should be used jointly in phylogeographic studies for a better evaluation of the evolutionary mechanisms that shaped genetic variation.     

Phylogeography and the maternal origin of the tetraploid form of the Japanese spined loach, <Emphasis Type="Italic">Cobitis biwae</Emphasis>, revealed by mitochondrial DNA analysis

The Japanese spined loach Cobitis biwae includes the tetraploid form, which has 96 chromosomes, as well as the diploid form, which has 48 chromosomes. In the present study, we analyzed the mitochondrial DNA (mtDNA) to examine the genetic relationships among 82 populations of diploid–tetraploid complexes of C. biwae. Restriction fragment length polymorphism (RFLP) analysis of the ND1 region on 202 individuals revealed that C. biwae contains three genetically divergent major groups corresponding to geographical proximities (the Eastern, Western, and Kochi groups). Phylogenetic analyses (neighbor-joining, NJ and maximum-parsimony, MP) of a part of the cytochrome b gene sequence (748bp) in 31 individuals supported the three major groups recognized by RFLP, and indicated considerable genetic differentiation between the Western group and the other two groups (average, 15.2%). The genetic relationship and distribution pattern of the three major groups hypothesized two major dispersions of C. biwae during the middle Miocene: first, the ancestor of the Eastern and Kochi groups had spread from the West through wide regions of present-day Honshu and Shikoku Islands, and following that period, the ancestor of the Western group with a different mtDNA composition, probably resulting from the mtDNA introgression from C. takatsuensis, moved into western Japan and pushed the former north eastward. All tetraploid form populations were included in the Western group and treated as a monophyletic cluster with low genetic divergence. It is notable that two diploid populations geographically adjacent to the tetraploid range were genetically closely related to the tetraploid forms. This result suggests that these diploid populations were directly related to the maternal origin of the tetraploid form.     

Phylogeography, genetic structure and diversity in the endangered bearded vulture (Gypaetus barbatus, L) as revealed by mitochondrial DNA

Bearded vulture populations in the Western Palearctic have experienced a severe decline during the last two centuries that has led to the near extinction of the species in Europe. In this study we analyse the sequence variation at the mitochondrial control region throughout the species range to infer its recent evolutionary history and to evaluate the current genetic status of the species. This study became possible through the extensive use of museum specimens to study populations now extinct. Phylogenetic analysis revealed the existence of two divergent mitochondrial lineages, lineage A occurring mainly in Western European populations and lineage B in African, Eastern European and Central Asian populations. The relative frequencies of haplotypes belonging to each lineage in the different populations show a steep East-West clinal distribution with maximal mixture of the two lineages in the Alps and Greece populations. A genealogical signature for population growth was found for lineage B, but not for lineage A; futhermore the Clade B haplotypes in western populations and clade A haplo-types in eastern populations are recently derived, as revealed by their peripheral location in median-joining haplotype networks. This phylogeographical pattern suggests allopatric differentiation of the two lineages in separate Mediterranean and African or Asian glacial refugia, followed by range expansion from the latter leading to two secondary contact suture zones in Central Europe and North Africa. High levels of among-population differentiation were observed, although these were not correlated with geographical distance. Due to the marked genetic structure, extinction of Central European populations in the last century re-sulted in the loss of a major portion of the genetic diversity of the species. We also found direct evidence for the effect of drift altering the genetic composition of the remnant Pyrenean population after the demographic bottleneck of the last century. Our results argue for the management of the species as a single population, given the apparent ecological exchangeability of extant stocks, and support the ongoing reintroduction of mixed ancestry birds in the Alps and planned reintroductions in Southern Spain.     

Genetic characterization of common carp (Cyprinus carpio) populations from Greece using mitochondrial DNA sequences

Wild common carp from two lakes and two rivers in Greece were genetically characterized with sequencing analysis of two mitochondrial DNA segments: cytochrome b (1119 bp) and D-loop (646 bp). A total of 9 variable singleton sites and 7 unique haplotypes were detected. A common haplotype was found in three out of the four populations examined, which seems to be the ancestral one and represents the European origin of common carp from Greece. This haplotype could be also justified by the introductions reported with individuals belonging to the Central European race, into many natural habitats in Greece. Limited genetic variation — in Evros and Aliakmonas populations — could be due to bottleneck effects and small effective population sizes, whereas the different haplotypes found in Lake Volvi could represent different common carp stocks. Values of sequence divergence among Greek haplotypes ranged from 0.0006 to 0.0023. The Neighbour-Joining (NJ) phylogenetic tree constructed based on the combined sequences, reveals that the populations of common carp from Greece belong to the European group of populations — which is highly divergent from the South East-Asia cluster — and to the subspecies Cyprinus carpio carpio.     

New data on the distribution and genetic structure of Greek moles of the genus Talpa (Mammalia,Talpidae)

In this work, we employed molecular markers and confirmed the occurrence of two mole species, Talpa stankovici and Talpa europaea in Greece. For the first species, all analyses revealed three major phylogroups, exhibiting great genetic divergence, possibly due to a vicariant event. This event was probably the Peloponnisos’ insulation during the Pliocene and the following sea level fluctuations. A scenario of stable, large‐sized populations for a long period rather than rapid growth from small‐sized populations or founder events is supported. Additionally, T. stankovici’s distribution area is extended to a southernmost limit. For T. europaea there is evidence of low genetic divergence between Greek and Central and North European populations.     

Biochemical genetic variability in brown hares (Lepus europaeus) from Greece

Allozyme variability of 91 brown hares (Lepus europaeus) from seven regions in Greece was compared to existing data of Bulgarian populations to test the hypothesis of the occurrence of specific alleles in Greece, likely stemming from an isolated Late Pleistocene refugial population in the southern Balkans. This hypothesis is particularly suggested by some subfossil Late Pleistocene hare remains in Greece and the reported high mtDNA diversity in Greek hares. Allozymic diversity could be higher in Greek hares than in hares from neighboring regions as a result of the accumulation of variants in a long-lasting Pleistocene refugium. Conversely, Greek hares could exhibit reduced genetic diversity because of long-lasting low effective population sizes during the Late Glacial Maximum and a lower chance of postglacial gene flow from other populations into this rather marginal part in the southern Balkans. Horizontal starch gel electrophoresis of proteins from 35~loci revealed three alleles (Es-1 ^–162, Pep-2 ¹¹⁴, Mpi ⁸⁸) at low frequencies, which were not found in Bulgarian or any other brown hare population. In contrast, some alleles from the populations from Bulgaria and other regions of Europe were absent in the Greek samples. Population genetic statistics indicated only a slight tendency of increased gene pool diversity in Greek hares, little substructuring in Greek and Bulgarian populations, respectively, as well as an only slightly lower level of gene flow between the two neighboring regions, as compared to the gene flow within each region. The results conform to the hypothesis of a Late Pleistocene refugial population in the southern Balkans, with some few specific nuclear gene pool characteristics, but little effect on the overall genetic differentiation between Greek and Bulgarian hares.     

Assessment of genetic structure of Greek brown hare (Lepus europaeus) populations based on variation in random amplified polymorphic DNA (RAPD)

The RAPD method was used to assess the genetic differentiation of brown hare (Lepus europaeus) populations from Central Greece. Greek wild populations were compared with samples from Austria, Poland, Germany, France, and Bulgaria, as well as with reared/released hares to investigate the impact of the releases on the native populations' genetic structure. The absence of diagnostic bands distinguishing between L. europaeus populations confirmed the high level of gene flow between brown hare populations over long geographic distances reported by other authors. Phylogenetic trees, derived from genetic distances estimated by RAPD band frequencies, suggested one major partitioning event of nuclear DNA lineages found in the samples. The reared individuals clustered with the Austrian, Polish, German, and French populations, whereas the Greek populations clustered apart with the Bulgarian population. Within Greece the distribution of the six wild populations did not follow any geographical trend, since their genetic divergence did not seem to correlate to geographic distances. However, RAPD profiles of some reared and wild specimens were different from the common RAPD pattern observed in the vast majority of sampled hares, probably reflecting an admixture of genetically differentiated individuals. The RAPD analysis indicates that releases might have begun to affect Greek population structure and reinforces the view that appropriate management is needed, adjusted to the local populations' biology and ecology.     

Species diversity,endemism and conservation of the family Caryophyllaceae in Greece

The family Caryophyllaceae includes world-wide 86 genera and approximately 2100 species. Greece is one of its most important centres of diversity and endemism. A total of 428 Caryophyllaceae taxa are distributed in Greece, 161 of them being endemic to the Greek political territory. The endemic element represents approximately 5% of the global diversity of the family at the species level. The aim of this paper is to discuss the distribution patterns of the Greek endemic Caryophyllaceae, as well as those with a limited distribution range to the neighbouring areas of the Balkans and Anatolia, on the basis of the phytogeographical regions of Greece in order to identify the important regions for their conservation. The majority of the Greek endemic Caryophyllaceae (64.6%) are distributed in only one single phytogeographical region, or even a smaller area indicating the extremely restricted distribution ranges of the endemic plants in Greece. Actually 83 Greek endemic Caryophyllaceae can be grouped on cytotaxonomic criteria. Most of them belong to the category of schizoendemics (91.6%), indicating that the endemism of Caryophyllaceae in Greece has mainly originated in an active way. Cluster analysis has been used to classify the phytogeographical regions according to their floristic similarities. Two iterative complementarity methods were used to evaluate the importance of each phytogeographical region in the conservation of the endemic Caryophyllaceae in Greece. Peloponnisos, Kriti-Karpathos and Sterea Ellas are the most important phytogeographical regions in this respect, followed by North Central and North East. When adding the Balkan-Aegean-Anatolian endemics to the analysis, Peloponnisos, North Central, Kriti-Karpathos, North East and Eastern Aegean result as the most important areas. In every case, an elevated number of sites are required for the conservation of Caryophyllaceae in Greece, reflecting the great dissimilarities in the floristic composition of the various phytogeographical regions. The results provided by the different methods are compared. A catalogue of the Greek endemic Caryophyllaceae is appended.     

mtDNA analysis reveals the ongoing speciation on Greek populations of Microtus (Terricola) thomasi (Arvicolidae,Rodentia)

The present article extends our previous work on the phylogenetic history of Microtus (Terricola) thomasi, analysing cytochrome b, 12S rRNA, 16S rRNA and the control region in 65 Greek populations. The analysis revealed three clades: one grouping the populations from Peloponnisos (Southern Greece); the second, the populations from Agios (Ag.) Stefanos and Evvoia island (Central East Greece); and the third, all the remaining populations with no geographical substructure. Genetic distances were low for most populations, with only the populations of Evvoia and Ag. Stefanos being substantially distant. Thus, although this species has a recent colonization history and probably descends from a highly polymorphic ancestor, a monophyletic and highly differentiated lineage is formed in Greece and is distributed in Ag. Stefanos and Evvoia. Molecular differentiation, distinct geographical distribution and restriction of gene flow between this lineage and the rest of the Greek populations provide evidence for its probable subspecific status, Microtus (Tericola) thomasi atticus. A possible mechanism leading the differentiation process of the proposed subspecies is suggested, based on the displacement of this species in central Greece by its congeneric, probably better‐fitted Microtus (Microtus) guentheri and the subsequent separation of Ag. Stefanos and Evvoia from the remaining Greek populations. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 117–130.     

New data on the distribution of Mus spicilegus Petenyi, 1882 (Rodentia, Muridae) and a distinct mtDNA lineage in the southern Balkans

Despite extensive zoogeographical and taxonomical studies in the subgenus Mus, several issues still remain unresolved. In this context, the distribution area of the mound-building mouse, Mus spicilegus, remains undetermined at the southernmost parts of the species’ range, possibly due to low population densities and seasonal character of mound building. The new records from Greece, presented here, significantly expand the knowledge on Mus spicilegus distribution but also reveal eastern Greek populations which are genetically differentiated from all the other Greek and European ones. These results support an additional colonization route to eastern Greece as part of the migration and expansion process of the mound building mouse in Europe.     

Reproductive Biology of Three Commercially Important Hemiramphid Species in South-eastern Australia

Synopsis The reproductive biology of eastern sea garfish Hyporhamphus australis, eastern river garfish H. regularis ardelio, and snub-nosed garfish Arrhamphus sclerolepis were described throughout their respective ranges in the coastal waters of New South Wales (NSW), Australia. Peaks in gonadosomatic indices indicated that spawning of eastern sea garfish occurred in late spring and early summer (November–December) on the south coast of NSW, and in winter and early spring (June–September) on the north coast. Eastern river garfish spawned between July and December in NSW estuaries and snub-nosed garfish spawned between October and January in the Clarence River. The sex ratios in commercial catches of eastern sea garfish from the north coast of NSW were biased toward male fish, but approached equality for fish caught from the south coast. Sex ratios were significantly biased toward female snub-nosed garfish, and female eastern river garfish from all estuaries sampled. Mean (±SE) batch fecundity was 1498 ± 110 (range: 98 – 3449) ripe oocytes per female for eastern sea garfish, 917 ± 36 (range: 102 – 2268) ripe oocytes per female for eastern river garfish, and 716 ± 104 (range: 20 – 2956) ripe oocytes per female for snub-nosed garfish across the range of mature sizes examined. There was a linear relationship between batch fecundity and fish size for all three species of garfish. Eastern sea garfish approached 50% maturity at a larger size than snub-nosed, or eastern river garfish. Size at 50% maturity was similar for male and female eastern river and snub-nosed garfish, but male eastern sea garfish matured at a significantly smaller size than females. All three species appear capable of spawning in the spawning season immediately following the one in which they were born. Mature female fish of all three species had distributions of oocyte diameters consisting of three or four modes, which strongly suggests asynchronous oocyte development and a multiple spawning strategy during the spawning season. Implications for the management of garfish fisheries in NSW are also discussed.     

Gene admixture in ethnic populations in upper part of Silk Road revealed by mtDNA polymorphism

To evaluate the gene admixture on the current genetic landscape in Gansu Corridor (GC) in China, the upper part of the ancient Silk Road which connects the Eastern and Central Asia, we examined mitochondrial DNA (mtDNA) polymorphisms of five ethnic populations in this study. Using PCR-RFLP and sequencing, we analyzed mtDNA haplotypes in 242 unrelated samples in three ethnic populations from the GC region and two ethnic populations from the adjacent Xinjiang Uygur Autonomous Region of China. We analyzed the data in comparison with the previously reported data from Eastern, Central and Western Asia and Europe. We found that both European-specific haplogroups and Eastern Asian-specific haplogroups exist in the Gansu Corridor populations, while a modest matrilineal gene flow from Europeans to this region was revealed. The Gansu Corridor populations are genetically located between Eastern Asians and Central Asians, both of who contributed significantly to the maternal lineages of the GC populations. This study made the landscape of the gene flow and admixture along the Silk Road from Europe, through Central Asia, to the upper part of the Silk Road more complete.     

Seasonal variation of chromophore composition in the eye of the Japanese dace, <Emphasis Type="Italic">Tribolodon hakonensis</Emphasis>

The relationship between seasonal variation and the effect of several different environmental factors on chromophore composition was investigated in the eye of the Japanese dace, Tribolodon hakonensis which lives either in rivers or in the sea. Eyes obtained from river and sea populations had both retinal (A1) and 3,4-didehydroretinal (A2) all through the year but the ratio of these chromophores showed seasonal variation the relative amount of A2 was higher in winter and lower in summer. Besides seasonal variation, A2 showed marked differences depending on habitat: the highest proportion of A2 was 67% in January and the lowest 13% in July, in the river population, whereas in the sea population the highest and the lowest values were only 30 and 6%, respectively, during the same months. The seasonal variation in gonadosomatic index showed no correlation to variations in A2 proportion, and the maximum difference in water temperature between summer and winter was ca. 15°C for both habitats. Because spectral conditions at the locations of capture of both river and sea populations were similar, we conclude that Japanese dace eyes are affected by exogenous factors related to differences between freshwater and seawater environments.     

Mitochondrial DNA phylogeography reveals the existence of an Evolutionarily Significant Unit of the sand goby Pomatoschistus minutus in the Adriatic (Eastern Mediterranean)

The sand goby Pomatoschistus minutus is a major component of marine shelf and estuarine food webs and an important study organism in behavioural research. Yet, despite the sand goby’s significance, its past and present patterns of migration and gene flow are poorly understood. Here we use the mtDNA control region and parts of the flanking tRNA genes of 63 fish from six localities in the Adriatic (Eastern Mediterranean), Western Mediterranean, Atlantic, and North Sea to investigate the phylogeography of this gobiid. Phylogenetic analyses and population genetics statistics reveal the existence of an Evolutionarily Significant Unit, sensu Moritz (1994), in the Adriatic and another in the Western Mediterranean, Atlantic, and North Sea. A possible biogeographical scenario for the separation of the ancestral population is that sand gobies in the Adriatic and Western Mediterranean split between 10,000 and 5000 years ago when due to the rise in sea temperature they migrated northwards and were bisected by the Italian peninsula. A testable prediction of this scenario is that sand gobies from the Western Mediterranean, Adriatic, and Aegean form three reciprocally monophyletic groups which are the descendants of a three-way diversification event.     

ITS 2 sequences heterogeneity in Phlebotomus sergenti and Phlebotomus similis (Diptera,Psychodidae): possible consequences in their ability to transmit Leishmania tropica

An intraspecific study on Phlebotomus sergenti, the main and only proven vector of Leishmania tropica among the members of the subgenus Paraphlebotomus was performed. The internal transcribed spacer 2 (ITS2) sequences of 12 populations from 10 countries (Cyprus, Egypt, Italy, Lebanon, Morocco, Pakistan, Portugal, Spain, Syria, and Turkey) were compared. Samples also included three species closely related to P. sergenti: Phlebotomus similis (three populations from Greece and Malta), Phlebotomus jacusieli and Phlebotomus kazeruni. Our results confirm the validity of the taxa morphologically characterised, and imply the revision of their distribution areas, which are explained through biogeographical events. At the Miocene time, a migration route, north of the Paratethys sea would have been followed by P. similis to colonise the north of the Caucasus, Crimea, Balkans including Greece and its islands, and western Turkey. Phlebotomus sergenti would have followed an Asiatic dispersion as well as a western migration route south of the Tethys sea to colonise North Africa and western Europe. This hypothesis seems to be well supported by high degree of variation observed in the present study, which is not related to colonisation or to intra-populational variation. Two groups can be individualised, one oriental and one western in connection with ecology, host preferences and distribution of L. tropica. We hypothesise that they could be correlated with differences in vectorial capacities.     

Responses to experimental fish manipulations in a shallow,hypereutrophic lake: the relative importance of benthic nutrient recycling and trophic cascade

The genetic polymorphism of twelve enzyme systems in two parthenogenetic tetraploidArtemia populations from N. Greece has been studied, using starch gel electrophoresis. The genetic distance within and among the two Greek populations was calculated. The high degree of genetic identity between the populations indicates that they belong to the same species. Each is composed of electrophoretically identifiable clones; the fitness of these clones under different environmental conditions is discussed.     

Gene admixture in ethnic populations in upper part of Silk Road revealed by mtDNA polymorphism

To evaluate the gene admixture on the current genetic landscape in Gansu Corridor (GC) in China, the upper part of the ancient Silk Road which connects the Eastern and Central Asia, we examined mitochondrial DNA (mtDNA) polymorphisms of five ethnic populations in this study. Using PCR-RFLP and sequencing, we analyzed mtDNA haplotypes in 242 unrelated samples in three ethnic populations from the GC region and two ethnic populations from the adjacent Xinjiang Uygur Autonomous Region of China. We analyzed the data in comparison with the previously reported data from Eastern, Central and Western Asia and Europe. We found that both European-specific haplogroups and Eastern Asian-specific haplogroups exist in the Gansu Corridor populations, while a modest matrilineal gene flow from Europeans to this region was revealed. The Gansu Corridor populations are genetically located between Eastern Asians and Central Asians, both of who contributed significantly to the maternal lineages of the GC populations. This study made the landscape of the gene flow and admixture along the Silk Road from Europe, through Central Asia, to the upper part of the Silk Road more complete.     

Differentiation patterns in the E. MediterraneanScutellaria rubicunda group (Lamiaceae)

Strict sterility barriers where found between theScutellaria populations on Sicily and the Greek populations. The material from Samos is separated by a strict sterility barrier from the other Greek populations. In contrast, the populations on mainland Greece and adjacent islands are all ± interfertile. Three species are recognized,S. rubicunda Hornem., endemic to Sicily, andS. brevibracteata subsp.icarica, endemic to Samos and Ikaria. All other populations are referred toS. rupestris with eight subspecies, most of which are endemic to one island or to one mountain. Three new subspecies are described, viz. subsp.rechingeri and subsp.olympica, endemic to mt Vourinos and mt Olympus in North Greece, respectively, and subsp.caroli-henrici, native to the Malea peninsula of Peloponnisos. The phytogeographical connections and genetic differentiation within and between populations are discussed.Dedicated to Prof. DrK. H. Rechinger on the occasion of his 80th birthday.     

Molecular phylogeny of the Greek populations of the genus Ligidium (Isopoda, Oniscidea) using three mtDNA gene segments

The phylogeny of Greek populations of the terrestrial isopod genus Ligidium is reconstructed based on three mtDNA gene segments: 12S rRNA, 16S rRNA and COI. Two widely distributed European species, as well as three outgroups belonging to different isopod genera, were also included in the analyses. The samples used represent almost all Ligidium species known to occur in Greece, as well as several populations of unknown specific status plus some new records. Phylogenetic analyses of the combined data set were performed using Bayesian inference and maximum parsimony. The two main sister clades with good support indicate the sympatric differentiation of two lineages in southern continental Greece (Peloponnisos), where Ligidium populations exhibit a mosaic distribution of sibling species. The insular populations of the Aegean Islands show increased genetic divergence and form separate clades. The presence of a third lineage of Asiatic origin is strongly suggested by both the molecular phylogeny and morphology. The only presumably valid diagnostic morphological character exhibits only partial correspondence to well supported clades of the molecular phylogeny. Genetic differentiation between populations is very high, a fact that can be attributed to the strict ecological specialization of these animals that leads to increased levels of isolation even between populations that are in close proximity. As a consequence, Greek Ligidium populations, especially those present on islands, are unique genetic pools and extremely vulnerable to extinction.