Palaeoclimatic changes explain Anatolian mountain frog evolution: a test for alternating vicariance and dispersal events

Holarctic biodiversity has been influenced by climatic fluctuations since the Pliocene. Asia Minor was one of the major corridors for postglacial invasions in the Palearctic. Today this area is characterized by an extraordinarily rich fauna with close affiliation to European, Asian and Indo-African biota. However, exact scenarios of range expansion and contraction are lacking. Using a phylogeographical approach we (i). identify monophyletic lineages among Anatolian mountain frogs and (ii). derive a spatio-temporal hypothesis for the invasion process in Anatolia. We sequenced 540 bp of the mitochondrial 16S rRNA gene from 40 populations of mountain frogs from Anatolia, the Elburz Mountains and the Caucasus. Our samples comprise all known species and subspecies: Rana macrocnemis macrocnemis, R. m. tavasensis, R. m. pseudodalmatina, R. camerani and R. holtzi. They include the type localities of four of these taxa. We used a nested clade analysis (NCA) to infer historical and recurrent events that account for the observed geographical distribution of haplotypes. None of the extant species is monophyletic. Based on a molecular clock calibration using homologous sequences of Western Palearctic water frogs of the same genus, we estimated that a basic radiation into three lineages c. 2 Mya was followed by several dispersal and fragmentation events. The geographical distribution of resident and widespread haplotypes allows us to infer and date scenarios of range expansion and fragmentation that are aligned with dramatic climatic oscillations that have occurred during the last 600000 years. Consequently, Pliocene and Pleistocene climatic oscillations triggered the evolution of Anatolian mountain frogs through an interplay of vicariance and dispersal events.     

Phylogeographic patterns of genetic diversity in eastern Mediterranean water frogs have been determined by geological processes and climate change in the Late Cenozoic

AIM: Our aims were to assess the phylogeographic patterns of genetic diversity in eastern Mediterranean water frogs and to estimate divergence times using different geological scenarios. We related divergence times to past geological events and discuss the relevance of our data for the systematics of eastern Mediterranean water frogs. LOCATION: The eastern Mediterranean region. METHODS: Genetic diversity and divergence were calculated using sequences of two protein-coding mitochondrial (mt) genes: ND2 (1038 bp, 119 sequences) and ND3 (340 bp, 612 sequences). Divergence times were estimated in a Bayesian framework under four geological scenarios representing alternative possible geological histories for the eastern Mediterranean. We then compared the different scenarios using Bayes factors and additional geological data. RESULTS: Extensive genetic diversity in mtDNA divides eastern Mediterranean water frogs into six main haplogroups (MHG). Three MHGs were identified on the Anatolian mainland; the most widespread MHG with the highest diversity is distributed from western Anatolia to the northern shore of the Caspian Sea, including the type locality of Pelophylax ridibundus. The other two Anatolian MHGs are restricted to south-eastern Turkey, occupying localities west and east of the Amanos mountain range. One of the remaining three MHGs is restricted to Cyprus; a second to the Levant; the third was found in the distribution area of European lake frogs (P. ridibundus group), including the Balkans. MAIN CONCLUSIONS: Based on geological evidence and estimates of genetic divergence we hypothesize that the water frogs of Cyprus have been isolated from the Anatolian mainland populations since the end of the Messinian salinity crisis (MSC), i.e. since c. 5.5-5.3 Ma, while our divergence time estimates indicate that the isolation of Crete from the mainland populations (Peloponnese, Anatolia) most likely pre-dates the MSC. The observed rates of divergence imply a time window of c. 1.6-1.1 million years for diversification of the largest Anatolian MHG; divergence between the two other Anatolian MHGs may have begun about 3.0 Ma, apparently as a result of uplift of the Amanos Mountains. Our mtDNA data suggest that the Anatolian water frogs and frogs from Cyprus represent several undescribed species.     

Population genetic characterization of the endangered silver skipper,Leptalina unicolor (Lepidoptera: Hesperiidae), using novel microsatellite markers and mitochondrial DNA gene sequences

The silver stripped skipper, Leptalina unicolor, is listed as an endangered species with a paucity of leftover populations in Korea. Prior population genetic analysis is essential for the establishment of effective conservation strategies. In the present study, we investigated the genetic diversity and the relationships of L. unicolor populations in Korea using 12 newly developed L. unicolor-specific microsatellite markers and two mitochondrial DNA (mtDNA) sequences (a total of 1283 bp from COI and CytB). Due to the endangered status and rarity, a total of 33 individuals from two localities in Gangwon-do Province (Seohwa and Girin) and one in Gyeongsangnam-do Province (Danjang) were sampled. The concatenated mtDNA sequences revealed very low genetic diversity in each population, with only one to three independent haplotypes per population. F_ST, principal coordinate analysis, and independent structural analysis of the concatenated mtDNA sequences and the 12 microsatellite loci developed in this study showed that each L. unicolor population in Korea is nearly completely isolated, although inbreeding has not yet occurred. Long-term conservation habitat recovery that increases the available population appears to be important in the prevention of genetic drift and inbreeding, which occur in small isolated populations and reduce the viability of populations under fluctuating environmental conditions.     

Complete mitochondrial genomes of three sea basses Lateolabrax (Perciformes,Lateolabracidae) species: Genome description and phylogenetic considerations

Genus Lateolabrax consists of three species, Japanese sea bass Lateolabrax japonicus, spotted sea bass Lateolabrax maculatus and blackfin sea bass Lateolabrax latus. The complete mitochondrial DNA (mtDNA) of the three sea basses were amplified and sequenced to characterize and discuss their phylogenetic relationships. The length of mitogenomes was 16,593 bp, 16,479 bp and 16,600 bp, respectively, and all of them consisted of 13 protein-coding genes, 2 ribosomal RNA (rRNA), 22 transfer RNA (tRNA) and a control region, which are typical for mtDNA of vertebrate. Most genes were encoded on the H-strand, except for the ND6 and eight tRNA genes encoding on the L-strand. A significant variation among the three species was detected in length of the control region. Phylogenetic relationship among the three species was constructed based on the datasets, including the 12 protein-coding genes (except ND6 gene), 22 tRNA and 2 rRNA sequences. The results supported the sister taxon between L. japonicus and L. maculatus. The genetic resources reported here are useful for further studies in taxonomy and phylogeny of the three sea basses and related species.     

The mitochondrial genome and ribosomal operon of Brachycladium goliath (Digenea: Brachycladiidae) recovered from a stranded minke whale

Members of the Brachycladiidae are known to cause pathologies implicated in cetacean strandings and it is important to develop accurate diagnostic markers to differentiate these and other helminths found in cetaceans. Brachycladium goliath (van Beneden, 1858) is a large trematode found, as adults, usually in the hepatic (bile) and pancreatic ducts of various cetaceans. Complete sequences were determined for the entire mitochondrial genome, and phylogenetically informative nuclear genes contained within the ribosomal operon, from a small piece of an individual worm taken from a common minke whale Balaenoptera acutorostrata Lacépède, 1804. Genomic DNA was sequenced using an Illumina MiSeq platform. The mtDNA is 15,229 bp in length consisting of 12 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 2 non-coding regions of which the larger is comprised of 4 tandemly repeated units (260 bp each). The ribosomal RNA operon is 9297 bp long. These data provide a rich resource of molecular markers for diagnostics, phylogenetics and population genetics in order to better understand the role, and associated pathology of helminth infections in cetaceans.     

Exploring phylogenetic informativeness and nuclear copies of mitochondrial DNA (numts) in three commonly used mitochondrial genes: mitochondrial phylogeny of peppermint,cleaner, and semi‐terrestrial shrimps (Caridea: Lysmata,Exhippolysmata, and Merguia)

Of paramount importance to studies that profit from molecular trees is the accuracy and robustness of the reconstructed phylogenies. Causes of systematic error that can mislead phylogenetic methods include nuclear copies of mitochondrial DNA (numts) and low phylogenetic informativeness (PI). Herein, numts and PI were explored in three mitochondrial genes commonly used for phylogenetic reconstruction: 16S, 12S, and cytochrome c oxidase I (COI). Shrimps from the genera Lysmata, Exhippolysmata, and Merguia were used as a model system. The existence of: (1) multiple bands on gels of COI and 12S polymerase chain reaction (PCR) products from various species; (2) double peaks, background noise, and ambiguity in sequence chromatograms of COI and 12S PCR products that produced a single clear band in other species; and (3) indels, stop codons, and considerable composition bias in COI‐like cloned sequences of one problematic species (Lysmata seticaudata), was interpreted as evidence of pervasive non‐functional nuclear copies of mitochondrial DNA (numts) of the targeted COI (and probably 12S) mtDNA fragment. The information content of the three mtDNA markers studied was investigated using PI profiling, spectral analysis, and neighbour‐nets. Marker‐specific PI profiles suggested that the COI marker has the highest information content and greatest power for resolving both shallow and deep nodes in trees depicting the phylogenetic relationship among the species studied. Nonetheless, spectral analysis of splits and neighbour‐nets suggested that the 16S and 12S markers were equally or even more powerful than the COI marker for resolving nodes at all phylogenetic levels. Altogether, these analyses suggest that all three mtDNA markers are equally useful for resolving phylogenetic relationships in the shrimps studied, and that PI profiling is not necessarily useful to estimate overall gene utility. A ‘total‐evidence’ phylogenetic analysis that included 34 species and used a concatenated data set of 1403 characters (from reliable 16S, 12S and COI sequences), demonstrated that the genus Lysmata is paraphyletic, and that the monophyletic clade comprising species of Lysmata and Exhippolysmata can be divided into four well‐supported subclades (Neotropical, Cleaner, Cosmopolitan, and Morphovariable). © 2013 The Linnean Society of London     

Polymorphic population genetic markers for the Australian wood cockroach Panesthia australis

Primers for five polymorphic nuclear DNA (nDNA) markers and one mitochondrial DNA (mtDNA) gene (COI) were developed for the Australian wood cockroach Panesthia australis using a range of techniques. Eight mitochondrial haplotypes and four to 36 alleles per nuclear locus were detected in 744 cockroaches. Observed heterozygosity ranged from 0.117 to 0.816 in a sample of 30 animals from one population. The markers will be useful for population biology and for the measurement of the effects of habitat fragmentation on this ecologically important forest‐dependent species.     

Resurrecting an extinct species: archival DNA, taxonomy, and conservation of the Vegas Valley leopard frog

Suggestions that the extinct Vegas Valley leopard frog (Rana fisheri = Lithobates fisheri) may have been synonymous with one of several declining species have complicated recovery planning for imperiled leopard frogs in southwestern United States. To address this concern, we reconstructed the phylogenetic position of R. fisheri from mitochondrial and nuclear sequence data obtained from century-old museum specimens. Analyses incorporating representative North American Rana species placed archival specimens within the clade comprising federally Threatened Chiricahua leopard frogs (Rana chiricahuensis = Lithobates chiricahuensis). Further analysis of Chiricahua leopard frogs recovered two diagnosable lineages. One lineage is composed of R. fisheri specimens and R. chiricahuensis near the Mogollon Rim in central Arizona, while the other encompasses R. chiricahuensis populations to the south and east. These findings ascribe R. chiricahuensis populations from the northwestern most portion of its range to a resurrected R. fisheri, demonstrating how phylogenetic placement of archival specimens can inform recovery and conservation plans, especially those that call for translocation, re-introduction, or population augmentation of imperiled species.     

Fast assembly of the mitochondrial genome of a plant parasitic nematode (Meloidogyne graminicola) using next generation sequencing

Little is known about the variations of nematode mitogenomes (mtDNA). Sequencing a complete mtDNA using a PCR approach remains a challenge due to frequent genome reorganizations and low sequence similarities between divergent nematode lineages. Here, a genome skimming approach based on HiSeq sequencing (shotgun) was used to assemble de novo the first complete mtDNA sequence of a root-knot nematode (Meloidogyne graminicola). An AT-rich genome (84.3%) of 20,030 bp was obtained with a mean sequencing depth superior to 300. Thirty-six genes were identified with a semi-automated approach. A comparison with a gene map of the M. javanica mitochondrial genome indicates that the gene order is conserved within this nematode lineage. However, deep genome rearrangements were observed when comparing with other species of the superfamily Hoplolaimoidea. Repeat elements of 111 bp and 94 bp were found in a long non-coding region of 7.5 kb, as similarly reported in M. javanica and M. hapla. This study points out the power of next generation sequencing to produce complete mitochondrial genomes, even without a reference sequence, and possibly opening new avenues for species/race identification, phylogenetics and population genetics of nematodes.     

Regional variation in drivers of connectivity for two frog species (Rana pretiosa and R. luteiventris) from the U.S. Pacific Northwest

Comparative landscape genetics has uncovered high levels of variability in which landscape factors affect connectivity among species and regions. However, the relative importance of species traits versus environmental variation for predicting landscape patterns of connectivity is unresolved. We provide evidence from a landscape genetics study of two sister taxa of frogs, the Oregon spotted frog (Rana pretiosa) and the Columbia spotted frog (Rana luteiventris) in Oregon and Idaho, USA. Rana pretiosa is relatively more dependent on moisture for dispersal than R. luteiventris, so if species traits influence connectivity, we predicted that connectivity among R. pretiosa populations would be more positively associated with moisture than R. luteiventris. However, if environmental differences are important drivers of gene flow, we predicted that connectivity would be more positively related to moisture in arid regions. We tested these predictions using eight microsatellite loci and gravity models in two R. pretiosa regions and four R. luteiventris regions (n = 1,168 frogs). In R. pretiosa, but not R. luteiventris, connectivity was positively related to mean annual precipitation, supporting our first prediction. In contrast, connectivity was not more positively related to moisture in more arid regions. Various temperature metrics were important predictors for both species and in all regions, but the directionality of their effects varied. Therefore, the pattern of variation in drivers of connectivity was consistent with predictions based on species traits rather than on environmental variation.     

Genetic diversity in mitochondrial 12S rDNA of western Palearctic water frogs (Anura, Ranidae) and implications for their systematics

The nucleotide sequence of a part of the mitochondrial 12S rRNA gene of eight western Palearctic water frog species was analysed. The results are consistent with the species status of Rana bedriagae, Rana bergeri, Rana epeirotica, Rana lessonae, Rana perezi, Rana ridibunda, Rana saharica and Rana shqiperica . The obtained DNA data suggest that lake frogs from Greece and Yugoslavia on the one hand and lake frogs from Georgia, Uzbekistan and Turkmenistan on the other hand represent two distinct species. However, it is not yet clear whether lake frogs from Georgia, Uzbekistan and Turkmenistan belong to R. ridibunda or represent a new species. The very high similarity between the analysed 12S rDNA segments of German R. ridibunda and R. lessonae confirm the finding that mtDNA of R. lessonae was transmitted into the mitochondrial gene pool of R. ridibunda probably as a result of backcrosses with the hybridogenetic hybrid R. kl. esculenta . The results of parsimony analyses speak in favour of very close phylogenetic relations between R. perezi and R. saharica ; with a high probability these species represent an adelphotaxon. Furthermore, the clades ( R. lessonae + R. shqiperica + R. bergeri ) and ( R. ridibunda + R. bedriagae ) are considered to be sister groups. According to the mt 12S rDNA data R. epeirotica seems to be more closely related to the supraspecific taxon ( R. ridibunda + R. bedriagae ) than to ( R. lessonae + R. shqiperica + R. bergeri ). Thus, it can be excluded that R. shqiperica and R. epeirotica represent sister species.     

Polymerase γ efficiently replicates through many natural template barriers but stalls at the HSP1 quadruplex

Faithful replication of the mitochondrial genome is carried out by a set of key nuclear-encoded proteins. DNA polymerase γ is a core component of the mtDNA replisome and the only replicative DNA polymerase localized to mitochondria. The asynchronous mechanism of mtDNA replication predicts that the replication machinery encounters dsDNA and unique physical barriers such as structured genes, G-quadruplexes, and other obstacles. In vitro experiments here provide evidence that the polymerase γ heterotrimer is well-adapted to efficiently synthesize DNA, despite the presence of many naturally occurring roadblocks. However, we identified a specific G-quadruplex–forming sequence at the heavy-strand promoter (HSP1) that has the potential to cause significant stalling of mtDNA replication. Furthermore, this structured region of DNA corresponds to the break site for a large (3,895 bp) deletion observed in mitochondrial disease patients. The presence of this deletion in humans correlates with UV exposure, and we have found that efficiency of polymerase γ DNA synthesis is reduced after this quadruplex is exposed to UV in vitro.     

The complete mitochondrial genome of Gomphocerus tibetanus Uvarov, 1935 (Orthoptera: Acrididae: Gomphocerinae)

The complete nucleotide sequence of the mitochondrial genome (mitogenome) of Gomphocerus tibetanus Uvarov, 1935 (Orthoptera: Acrididae: Gomphocerinae) was determined. It is 15,571 bp in length and contains 74.8% A + T. All Gomphocerus tibetanus protein-coding sequences start with a typical ATN codon. The usual termination codons (TAA and TAG) were found from 13 PCGs except COI and COII which took incomplete codon T as termination codons. All tRNA genes could be folded into the typical cloverleaf secondary structure, except tRNA^Ser(AGN) lacking of dihydrouridine (D) arm. The sizes of the large and small ribosomal RNA genes are 1313 and 822 bp, respectively. The A + T content of the A + T-rich region is 82.3%. A preliminary analysis on characteristics of Gomphocerinae mitogenome was made by comparision among three Gomphocerinae mitogenomes and Locusta migratoria.     

Sequence variation and structural conservation in the D-loop region and flanking genes of mitochondrial DNA from Japanese pond frogs

The nucleotide sequences of the D-loop region and its flanking genes of the mitochondrial DNA (mtDNA) from Japanese pond frogs were determined by the methods of PCR, cloning, and sequencing. The frogs belonged to two species, one subspecies, and one local race. The gene arrangements adjacent to the D-loop region were analyzed. The frogs shared a unique mitochondrial gene order that was found in Rana catesbeiana; i.e., cyt b--D-loop region--tRNA(Leu(CUN))--tRNA(Thr)--tRNA(Pro)--tRNA(Phe)--12S rRNA. The arrangements of the three tRNA genes of these frogs were different from those of X. laevis, a species which has the same overall structure as in mammals. Highly repetitive sequences with repeat units (16-bp or 17-bp sequence specific for each taxon) were found in the D-loop region. The length of repetitive sequences varied from 0.6 kbp to 1.2 kbp, and caused the extensive size variation in mtDNA. Several short sequence elements such as putative TAS, OH, CSB-1, and CSB-2 were found in the D-loop region of these frogs. The sequences of these short regulatory elements were conserved in R. catesbeiana, X. laevis, and also in human. The comparison of sequence divergences of the D-loop region and its adjacent genes among various taxa revealed that the rates of nucleotide substitutions depend on genes. The nucleotide sequences of the 3'-side segment of the D-loop region were the most variable among taxa, whereas those of the tRNA and 12S rRNA genes were the most conservative.     

The mitochondrial genome of the stramenopile alga Chrysodidymus synuroideus. Complete sequence, gene content and genome organization

This is the first report of a complete mitochondrial genome sequence from a photosynthetic member of the stramenopiles, the chrysophyte alga Chrysodidymus synuroideus. The circular-mapping mitochondrial DNA (mtDNA) of 34 119 bp contains 58 densely packed genes (all without introns) and five unique open reading frames (ORFs). Protein genes code for components of respiratory chain complexes, ATP synthase and the mitoribosome, as well as one product of unknown function, encoded in many other protist mtDNAs (YMF16). In addition to small and large subunit ribosomal RNAs, 23 tRNAs are mtDNA-encoded, permitting translation of all codons present in protein-coding genes except ACN (Thr) and CGN (Arg). The missing tRNAs are assumed to be imported from the cytosol. Comparison of the C.synuroideus mtDNA with that of other stramenopiles allowed us to draw conclusions about mitochondrial genome organization, expression and evolution. First, we provide evidence that mitochondrial ORFs code for highly derived, unrecognizable versions of ribosomal or respiratory genes otherwise ‘missing’ in a particular mtDNA. Secondly, the observed constraints in mitochondrial genome rearrangements suggest operon-based, co-ordinated expression of genes functioning in common biological processes. Finally, stramenopile mtDNAs reveal an unexpectedly low variability in genome size and gene complement, testifying to substantial differences in the tempo of mtDNA evolution between major eukaryotic lineages.     

Phylogenetic relationships among four species of Mullidae (Perciformes) inferred from DNA sequences of mitochondrial cytochrome b and 16S rRNA genes

DNA sequence comparisons of two mitochondrial DNA genes were used to infer phylogenetic relationships among four species of mullids. Approximately 238 bp of the mitochondrial 16S ribosomal RNA (rRNA) and 261 bp of the cytochrome b (cytb) genes were sequenced from representatives of three mullid genera (Mullus, Upeneus, Pseudopeneus), present in the Mediterranean Sea. Trees were constructed using three methods: maximum likelihood (ML), neighbor joining (NJ) and parsimony (MP). The results of the analyses of these data together with published data of the same mtDNA segments of two other perciform species (Sparus aurata, Perca fluviatilis), support the previous taxonomic classification of the three genera examined, as well as the classification of the two red mullet species in the same genus.     

Complete mitochondrial genome of the Seoul frog Rana chosenica (Amphibia, Ranidae): comparison of R. chosenica and R. plancyi

Here, we have sequenced the complete mitochondrial genome of the Seoul frog Rana chosenica (Amphibia, Ranidae), which is known as a Korean endemic species. It is listed as a vulnerable species by IUCN Red List and also an endangered species in South Korea. The complete mitochondrial genome of R. chosenica consists of 18,357?bp. Its gene arrangement pattern was identical with those of other Rana frogs. We compared the mitochondrial genome of R. chosenica with that of the Peking frog Rana plancyi that has been known closely related to R. chosenica. Nucleotide sequence similarity between the two whole mitochondrial genomes was 95.7%, and the relatively low similarity seems to indicate that the two species are distinctly separated on the species level. The information of mitochondrial genome comparison of the two species was discussed in detail.     

The complete mitochondrial genome of <Emphasis Type="Italic">Thrinchus schrenkii</Emphasis> (Orthoptera: Caelifera,Acridoidea, Pamphagidae)

Complete nucleotide sequences of mitochondrial genome (mitogenome) of Thrinchus schrenkii (Orthoptera: Acridoidea: Pamphagidae) were determined. It is 15672 bp in length and contains 71.2% A + T. All T. schrenkii protein-coding sequences except for the cytochrome oxidase subunit I (COI) start with a typical ATN codon. Instead, CCG, which is a rare but possible initiation codon, is located at the initiation context of COI. The usual termination codons (TAA and TAG) were found from 12 PCGs. However, the ND5 had incomplete termination codon (T). All tRNA genes could be folded into the typical cloverleaf secondary structure, excluding tRNA ^Ser(AGN) which forms another structure according to the Steinberg–Cedergren tertiary structure. The sizes of the large and small ribosomal RNA genes are 1319 and 848 bp, respectively. The A + T content of the A + T-rich region is 78.7%, which is the lowest among the known mitogenome of Acridoidea.     

Mitogenomic analysis of a 50-generation chicken pedigree reveals a rapid rate of mitochondrial evolution and evidence for paternal mtDNA inheritance

Mitochondrial genomes represent a valuable source of data for evolutionary research, but studies of their short-term evolution have typically been limited to invertebrates, humans and laboratory organisms. Here we present a detailed study of 12 mitochondrial genomes that span a total of 385 transmissions in a well-documented 50-generation pedigree in which two lineages of chickens were selected for low and high juvenile body weight. These data allowed us to test the hypothesis of time-dependent evolutionary rates and the assumption of strict maternal mitochondrial transmission, and to investigate the role of mitochondrial mutations in determining phenotype. The identification of a non-synonymous mutation in ND4L and a synonymous mutation in CYTB, both novel mutations in Gallus, allowed us to estimate a molecular rate of 3.13 × 10⁻⁷ mutations/site/year (95% confidence interval 3.75 × 10⁻⁸–1.12 × 10⁻⁶). This is substantially higher than avian rate estimates based upon fossil calibrations. Ascertaining which of the two novel mutations was present in an additional 49 individuals also revealed an instance of paternal inheritance of mtDNA. Lastly, an association analysis demonstrated that neither of the point mutations was strongly associated with the phenotypic differences between the two selection lines. Together, these observations reveal the highly dynamic nature of mitochondrial evolution over short time periods.