Factors affecting avian cross-species microsatellite amplification

Compilation and analysis of information from the literature regarding cross-species microsatellite amplification and polymorphism success, and relating this to source-target species genetic distance as estimated by pairwise cytochrome b ( cytb ) divergence, enabled an in-depth investigation of factors affecting avian cross-species microsatellite amplification. Source-target species cytb distances provided accurate estimates of cross-species microsatellite amplification/polymorphism success rates not only in birds, but also in taxa where microsatellites cross-amplify across contrasting levels of taxonomic classification (frogs and cetaceans). As cytb is one of the most commonly sequenced DNA regions, pairwise cytb genetic distances should therefore be useful for predicting cross-species microsatellite success across a range of taxonomic groups. While the most important factor affecting cross-species microsatellite amplification/polymorphism success was a negative association with source-target species genetic distance, associations with additional features affecting cross-species amplification/polymorphism success included: decreasing PCR annealing temperature significantly increasing the chance of successful cross-species amplification, and a significant positive association between source species polymorphism and the proportion of target species in which a locus revealed polymorphism. No association between cross-species amplification and repeat motif (di-, tri-, or tetranucelotide) or repeat structure (perfect, imperfect, or compound) was observed. A set of nine loci which cross-amplified across an unusually broad range of passerine bird species were also identified, and could serve as a good starting point for cross-species amplification testing in passerine species for which insufficient loci are available.     

Parasite fauna of bream Abramis brama and roach Rutilus rutilus from a man-made waterway and a freshwater habitat in northern Germany

Fifty specimens each of bream Abramis brama and roach Rutilus rutilus were examined for metazoan parasite fauna and trichodinid ciliates; 25 specimens of each species were collected from the Kiel Canal, a man-made waterway, and a nearby freshwater lake, the Dieksee. This is the first detailed parasitological examination of A. brama and R. rutilus at these locations: 30 parasite species were found, comprising 4 protozoans, 4 myxozoans, 5 digeneans, 3 monogeneans, 2 cestodes, 6 nematodes, 2 acanthocephalans, 3 crustaceans and 1 hirudinean. The crustacean Caligus lacustris occurred in both habitats while 2 other crustacean species, 2 acanthocephalans and 1 hirudinean were recorded exclusively for the lake habitat. Larval as well as adult stages of the different parasite species were found, indicating that both fish species act as intermediate and final hosts in both habitats. The Kiel Canal (total of 17 parasite species) showed a lower parasite species richness for A. brama and R. rutilus (14 and 10 parasite species, respectively) than the lake (25 parasite species). A. brama had a higher parasite richness (22 species) than R. rutilus (16 species) in the lake habitat. Most parasites collected were of freshwater origin. Consequently, the observed infection pattern of both fish species in the waterway is mainly influenced by the limited salinity tolerance of freshwater parasites, which are negatively affected even by a salinity of 2.3 to 4.5. In the central Kiel Canal, neither fish species was infected with marine parasites of low host specifity. These parasites are either limited by the low salinity at this sampling site (<4.5 to 6.0) or they cannot enter the canal due to the environmental conditions prevailing in this artificial brackish water habitat. Thus, the canal may comprise a natural barrier preventing the distribution of North Sea parasites into the Baltic Sea. However, the brackish water Baltic Sea nematodes Paracuaria adunca and Cosmocephalus obvelatus were found in R. rutilus from the canal, demonstrating the ability of some parasite species to invade and extend their range of distribution through this man-made shipping route from the Baltic to the North Sea.     

RAPD fingerprinting of common bream Abramis brama L., roach Rutilus rutilus L., and their F1 hybrids

The polymerase chain reaction with arbitrary primers (RAPD-PCR) was used to study and to evaluate the genetic variation in the hybrid progeny of two Cyprinidae species, common bream Abramis brama and roach Rutilus rutilus. Genetic polymorphism was studied in 20 fishes (young of the current year) obtained in four individual crosses: R. rutilus x R. rutilus (RR), A. brama x A. brama (AA), R. rutilus x A. brama (RA), and A. brama x R. rutilus (AR). Amplification spectra obtained with eight primers contained 288 fragments, 97.6% of which proved to be polymorphic. The proportion of polymorphic fragments was 75.0% in the RR progeny, 58.1% in the AA progeny, 84.9% in the AR progeny, and 77.8% in the RA progeny. Classification analysis in the space of principal components was performed with the first four components, which together accounted for 64% of the total variance of the character under study. The individual contributions of components I, II, III, and IV were 26.8, 16.8, 11.5, and 8.9%, respectively. Fishes of the two pure species and the hybrid progeny (direct and reverse hybrids together) were clearly differentiated in the space of principal components I and II. The best differentiation of the four samples (RR, AA, RA, and AR) was observed in the space of principal components II and IV. Possible causes of high genetic variation in interspecific hybrids are discussed.     

Cytochrome b phylogeography of chamois (Rupicapra spp.). Population contractions, expansions and hybridizations governed the diversification of the genus

The chamois provides an excellent model for exploring the effect of historical and evolutionary events on diversification. We investigate cytochrome b (cytb) sequences in the 10 recognized subspecies of Rupicapra classified within 2 species: Rupicapra pyrenaica, with the subspecies parva, pyrenaica, and ornata, and Rupicapra rupicapra, with cartusiana, rupicapra, tatrica, carpatica, balcanica, asiatica, and caucasica. A fragment of 349 bp of the cytb was sequenced in 189 individuals. We identified 3 cytb lineages: Clade West in Iberia and Western Alps; Clade Central in the Apennines and the Massif of Chartreuse; and Clade East present in populations to the east of the Alps. The 2 proposed species were polyphyletic; the clades West and Central are represented in both, whereas the Clade East is restricted to R. rupicapra. In contrast to the current systematic, cytb phylogenies suggest the classification of the 10 subspecies of chamois into a single species, R. rupicapra. Phylogeny and geographical distribution of the 3 lineages show the effects of limited latitudinal range expansions, contractions, and hybridizations among highly divergent lineages, along with a major role of the glacial ice sheets of the Alps and the Pyrenees as barriers to gene flow, on the diversification of extant taxa.     

How does heme axial ligand deletion affect the structure and the function of cytochrome b(562)?

We have recently generated a new mutant of cytochrome b(562) (cytb(562)) in which Met7, one of the axial heme ligands, is replaced by Ala (M7A cytb(562)). The M7A cytb(562) can bind heme and the UV-visible absorption spectrum is of a typical high-spin ferric heme. To investigate the effect of the lack of Met7 ligation on the structural integrity of cytb(562), thermal transition analyses of M7A cytb(562) were conducted. From the thermodynamic parameters obtained, it is concluded that the folding of M7A cytb(562) is comparable to the apoprotein despite the presence of heme. On the other hand, exogenous ligands such as cyanide and azide ions are readily bound to the heme iron, indicating that the axial coordination site is available for substrate binding. The peroxidase activity of this mutant is thus examined to evaluate new enzymatic function at this site and M7A cytb(562) was found to catalyze an oxidation reaction of aromatic substrates with hydrogen peroxide. These observations demonstrate that the Met7/His102 bis-ligation to the heme iron is crucial for the stable folding of cytb(562), whereas the functional conversion of cytb(562) is successfully achieved by the loose folding together with the open coordination site.     

Success of embryonic development of reciprocal hybrids of bream Abramis brama (L.) and white bream Blicca bjoerkna (L.)

Success of embryonic development of reciprocal hybrids of bream Abramis brama and white bream Blicca bjoerkna was tested under laboratory conditions. Fertilization and embryonic development success of hybrids until hatching was high and comparable to that of pure species.     

Evidence for the intertwined dimer of the cytochrome bc(1) complex in solution

To confirm that the cytochrome bc(1) complex exists as a dimer with intertwining Rieske iron-sulfur proteins in solution, four Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes containing two pairs of cysteine substitutions, one in the interface between the head domain of iron-sulfur protein (ISP) and cytochrome b and the other between the tail domain of ISP and cytochrome b, were generated and characterized. They are: K70C(ISP)/A185C(cytb).P33C(ISP)/G89C(cytb), K70C(ISP)/A185C(cytb).P33C(ISP)/M92C (cytb), K70C (ISP)/A185C(cytb).L34C(ISP)/V64C(cytb), and K70C(ISP)/A185C(cytb).N36C(ISP)/G89C(cytb). The K70C(ISP)/A185C(cytb) cysteine pair cross-links the head domain of ISP and cytochrome b; the P33C(ISP)/G89C(cytb), P33C(ISP)/M92C (cytb), L34C(ISP)/V64C(cytb), and N36C(ISP)/G89C(cytb) cysteine pairs cross-link the tail domain of ISP and cytochrome b. An adduct protein with an apparent molecular mass of 128 kDa containing two cytochrome b and two ISP proteins is detected in the K70C(ISP)/A185C(cytb).P33C(ISP)/G89C(cytb) and K70C(ISP)/A185C(cytb).N36C(ISP)/G89C(cytb) mutant complexes, confirming that the bc(1) complex exists as a dimer with intertwining ISPs. The loss of activity in these two double-cysteine-pair mutant complexes was attributed to the disulfide bond between the head domain of ISP and cytochrome b and not the one between the tail domain of ISP and cytochrome b.     

A comparative summary of genetic distances in the vertebrates from the mitochondrial cytochrome b gene

Mitochondrial cytochrome b (cytb) is among the most extensively sequenced genes to date across the vertebrates. Here, we employ nearly 2,000 cytb gene sequences from GenBank to calculate and compare levels of genetic distance between sister species, congeneric species, and confamilial genera within and across the major vertebrate taxonomic classes. The results of these analyses parallel and reinforce some of the principal trends in genetic distance estimates previously reported in a summary of the multilocus allozyme literature. In particular, surveyed avian taxa on average show significantly less genetic divergence than do same-rank taxa surveyed in other vertebrate groups, notably amphibians and reptiles. Various biological possibilities and taxonomic "artifacts" are considered that might account for this pattern. Regardless of the explanation, by the yardstick of genetic divergence in this mtDNA gene, as well as genetic distances in allozymes, there is rather poor equivalency of taxonomic rank across some of the vertebrates.     

Assessing phylogenetic resolution among mitochondrial, nuclear, and morphological datasets in Nothonotus darters (Teleostei: Percidae)

External morphological characters are the basis of our understanding of diversity and species relationships in many darter clades. The past decade has seen the publication of many studies utilizing mtDNA sequence data to investigate darter phylogenetics, but only recently have nuclear genes been used to investigate darter relationships. Despite a long tradition of use in darter systematics few studies have examined the phylogenetic utility of external morphological characters in estimating relationships among species in darter clades. We present DNA sequence data from the mitochondrial cytochrome b (cytb) gene, the nuclear encoded S7 intron 1, and discretely coded external morphological characters for all 20 species in the darter clade Nothonotus. Bayesian phylogenetic analyses result in phylogenies that are in broad agreement with previous studies. The cytb gene tree is well resolved, while the nuclear S7 gene tree lacks phylogenetic resolution, node support, and is characterized by a lack of reciprocal monophyly for many of the Nothonotus species. The phylogenies resulting from analysis of the morphological dataset lack resolution, but nodes present are found in the cytb and S7 gene trees. The highest resolution and node support is found in the Bayesian combined data phylogeny. Based on our results we propose continued exploration of the phylogenetic utility of external morphological characters in other darter clades. Given the extensive lack of reciprocal monophyly of species observed in the S7 gene tree we predict that nuclear gene sequences may have limited utility in intraspecific phylogeographic studies of Nothonotus darters.     

Cytochrome b pseudogene originated from a highly divergent mitochondrial lineage in genus Rupicapra

We have identified a nuclear pseudogene (numt) of cytochrome b (cytb) in chamois. The comparison of a fragment of 402 nucleotides of cytb and the pseudogene between the 2 species Rupicapra rupicapra and Rupicapra pyrenaica allowed direct measurement of relative rates and patterns of evolution. Mitochondrial genes evolved 7 to 12 times faster than their nuclear counterparts. Substitutions in the nucleus include a frameshift and a stop codon. Phylogenetic analysis of nuclear and mitochondrial lineages on Rupicapra and related species showed that the nuclear branch evolved as a functional mitochondrial gene until the split of the 2 species of chamois and as a typical pseudogene later on. We propose that the pseudogene originated from a highly divergent mitochondrial lineage that did not persist in the mitochondrion and transposed to the nucleus in a time close to speciation. The concurrence of highly differentiated lineages at speciation points to hybridization between highly divergent populations.     

Phylogeography of the White-Toothed Shrews Crocidura suaveolens and Crocidura sibirica: Searching for the Geographical Homeland

Biology Bulletin - We studied the polymorphism of the cytb gene in two forms of the Lesser White-toothed Shrew species complex: Crocidura suaveolens s. stricto and C. sibirica. The haplotypes of C....     

以线粒体DNA Cytb确立红喉雉鹑和黄喉雉鹑的分类地位

采用PCR技术获得红喉雉鹑Tetraophasis obscurus和黄喉雉鹑Tetraophasis szechenyii等物种的828 bp Cytb基因片段,雉鹑属Tetraophasis可变位点为26个,并用MEGA3和K-Estimator 6.1软件进行了序列分析.研究发现红喉雉鹑和黄喉雉鹑之间序列差异为3.0%～3.1%,与其它属种间序列差异相比较,两种雉鹑之间的序列差异达到了种的水平;它们地理分布互相替代;形态差异稳定.地理分布、形态特征和遗传特征均验证了它们应为两个独立的种.依据分子进化速率推测,两种雉鹑分歧时间距今2.0Myr左右,与早更新世冰期发生的时间一致.雉鹑起源于青藏高原,随着青藏高原的抬升,早更新世冰期气候变冷,寒温性针叶林的林线下移,大面积高原草甸出现,雉鹑的祖先种群被隔离进化,而形成了两种雉鹑.     

Rapid Rate of Control-Region Evolution in Pacific Butterflyfishes (Chaetodontidae)

Sequence differences in the tRNA-proline (tRNA^pro) end of the mitochondrial control-region of three species of Pacific butterflyfishes accumulated 33–43 times more rapidly than did changes within the mitochondrial cytochrome b gene (cytb). Rapid evolution in this region was accompanied by strong transition/transversion bias and large variation in the probability of a DNA substitution among sites. These substitution constraints placed an absolute ceiling on the magnitude of sequence divergence that could be detected between individuals. This divergence ``ceiling' was reached rapidly and led to a decay in the relative rate of control-region/cytb b evolution. A high rate of evolution in this section of the control-region of butterflyfishes stands in marked contrast to the patterns reported in some other fish lineages. Although the mechanism underlying rate variation remains unclear, all taxa with rapid evolution in the 5′-end of the control-region showed extreme transition biases. By contrast, in taxa with slower control-region evolution, transitions accumulated at nearly the same rate as transversions. More information is needed to understand the relationship between nucleotide bias and the rate of evolution in the 5′-end of the control-region. Despite strong constraints on sequence change, phylogenetic information was preserved in the group of recently differentiated species and supported the clustering of sequences into three major mtDNA groupings. Within these groups, very similar control-region sequences were widely distributed across the Pacific Ocean and were shared between recognized species, indicating a lack of mitochondrial sequence monophyly among species. Received: 30 June 1996 / Accepted: 15 May 1997     

Karyotypes divergence in superspecies complex Cricetulus barabensis sensu lato and their interrelationships in natural contact zones

Interrelationships between three chromosomal forms of striped hamsters belonging to the superspecies complex Cricetulus barabensis sensu lato (Cricetidae, Rodentia), namely 'barabensis', 'pseudogriseus', and 'griseus', are examined from the standpoint of different biological/genetic species concepts. Craniometric data suggest that differences between the karyotypes are rather of subspecies level. Cytogenetic differences between the karyotypes are also not great and correspond to the level of chromosomal races within a single species. The analysis of mitochondrial gene for cytochrome b (cytb) indicates the level of differences between forms of about 2.3-4.1%. Such a level may correspond to intraspecific differences as well as to differences between allied species. Experiments on hybridization resulted in hybrid litters obtained in different mating combinations of karyotypes. Both hybrid males and females fertility was confirmed, too. In nature, there exist several zones where contact between karyotypes 'barabensis' and 'pseudogriseus' is possible. Within one of such zones, in Central Mongolia, two males with atypical karyotype were detected by use of chromosome analysis. With chromosomes number and form, this karyotype may correspond to the hybrid between second and subsequent generations. However, typing of gene cytb and the intron localized in Y-chromosome (DBY1) did not reveal any traces of recent or ancient hybridization. In the other zone where contact between the karyotypes is possible, in South Buryatia, 'barabensis' and 'pseudogriseus' populations are separated by the River Chika and the distance between them does not exceed 5 km. Nevertheless, no hamsters with non-standard karyotypes and no traces of gene flow are detected by karyological and molecular analyses of these populations. The results of Tajima's test, analysis of pairwise differences distribution and indices of nucleotide diversity obtained in the course of examining gene cytb full nucleotide sequence all suggest that both contact zones studied have formed long ago and the absence of gene flow cannot be explained by their forming in the recent past. Thus, the karyotypes studied appear to be evolutionary lines developing independently from each other.     

Understanding the colonization history of the Galápagos flycatcher (Myiarchus magnirostris)

The Galápagos archipelago has never been connected to any continental land masses, so it is of interest to know the colonization and diversification history of its endemic species. We analyzed the phylogenetic placement of the endemic Galápagos flycatcher, M. magnirostris, within Myiarchus by using the genes ND2 and cytb (1970 bp) to compare 16 of the 22 species that comprise this genus. We also analyzed variability in cytb sequences from 154 M. magnirostris individuals captured on seven Galápagos islands. Our phylogenetic analyses recovered the two main Myiarchus clades that had been described by previous genetic, morphological, and vocal analyses. M. magnirostris is monophyletic and its closest living relative is M. tyrannulus from Mexico and Central America. The average age for the split node between these two groups was approximately 850,000 years (95% C.I. 630,735-1,087,557). M. tyrannulus, M. nugator, M. nuttingi, M. sagrae, and M. stolidus are not monophyletic species. Within M. magnirostris itself, we found low nucleotide and haplotype diversities (π=0.0009 and h=0.4913, respectively) and a high genetic structure among populations. We also detected a star-shaped haplotype network and significantly negative values for Tajima's D and Fu's Fs for this species. Our results suggest that M. magnirostris originated from a single colonization event and had a recent population expansion in the Galápagos archipelago.     

Identification of blood meal sources of Lutzomyia longipalpis?using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.     

Genetic composition of a population of natural common bream Abramis brama × roach Rutilus rutilus hybrids and their morphological characteristics in comparison with parent species

Common bream Abramis brama , roach Rutilus rutilus and their hybrids were collected in the Dobczyce Reservoir in southern Poland in 2006–2013 to study whether it is better for a hybrid individual to resemble and compete with one of its parents, or to minimize competition by having a distinctive phenotype. All hybrids were F₁ crosses and originated predominantly (93·2%) from matings between female A. brama and male R. rutilus parents. In morphometric analyses, a newly defined coefficient, L ₃ = 2·5 (body mass) (L _S × body depth)^?1, which enables forms with similar length–depth proportions but different length–mass relationships to be distinguished was used. Morphometric and meristic characteristics of the hybrids were intermediate in comparison with the parental species, with small but significant deviation towards R. rutilus in longitudinal body dimensions (trunk and tail length) and towards A. brama in body cross‐sectional shape (body depth and L ₃ coefficient). This may result in a more R. rutilus like propulsion in hybrids and a more A. brama like ability to manoeuvre.     

Mitochondrial gene diversity in the common vole Microtus arvalis shaped by historical divergence and local adaptations

The phylogeography of the common vole (Microtus arvalis) was examined by analysing mitochondrial DNA (mtDNA) sequence variation in 1044 base pairs (bp) of the cytochrome b (cytb) gene and in 322 bp of the control region (ctr) among 106 individuals from 58 locations. The geographical distribution of four previously recognized cytb evolutionary lineages in Europe was refined and a new lineage was found in southern Germany. All lineages were distributed allopatrically, except in one sample that was probably located in a contact zone. The occurrence of several lineages in the Alps is in keeping with their recent recolonization from distinct sources. The translation of 84 cytb DNA sequences produced 33 distinct proteins with relationships that differed from those of the DNA haplotypes, suggesting that the mtDNA lineages did not diverge in response to selection. In comparison with M. agrestis, a neutrality test detected no overall evidence for selection in the cytb gene, but a closer examination of a structural model showed that evolutionarily conserved and functionally important positions were often affected. A new phylogeographical test of random accumulation of nonsynonymous mutations generated significant results in three lineages. We therefore conclude that the molecular diversity of cytb in M. arvalis is overall the result of the demographic history of the populations, but that there have been several episodes of local adaptation to peculiar environments.     

Confirmation of the involvement of protein domain movement during the catalytic cycle of the cytochrome bc1 complex by the formation of an intersubunit disulfide bond between cytochrome b and the iron-sulfur protein

To study the essentiality of head domain movement of the Rieske iron-sulfur protein (ISP) during bc(1) catalysis, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with three pairs of cysteines engineered (one cysteine each) on the interface between cytochrome b and ISP, A185C(cytb)/K70C(ISP), I326C(cytb)/G165C(ISP), and T386C(cytb)/K164C(ISP), were generated and characterized. Formation of an intersubunit disulfide bond between cytochrome b and ISP is detected in membrane (intracytoplasmic membrane and air-aged chromatophore), and purified bc(1) complex was prepared from the A185C(cytb)/K70C(ISP) mutant cells. Formation of the intersubunit disulfide bond in this cysteine pair mutant complex is concurrent with the loss of its bc(1) activity. Reduction of this disulfide bond by beta-mercaptoethanol restores activity, indicating that mobility of the head domain of ISP is functionally important in the cytochrome bc(1) complex. The rate of intramolecular electron transfer, between 2Fe2S and heme c(1), in the A185C(cytb)/K70C(ISP) mutant complex is much lower than that in the wild type or in their respective single cysteine mutant complexes, indicating that formation of an intersubunit disulfide bond between cytochrome b and ISP arrests the head domain of ISP in the "fixed state" position, which is too far for electron transfer to heme c(1).