Genetic isolation within the malaria mosquito Anopheles melas

Anopheles melas is a brackish water–breeding member of the Anopheles gambiae complex that is distributed along the coast of West Africa and is a major malaria vector within its range. Because little is known about the population structure of this species, we analysed 15 microsatellite markers and 1161 bp of mtDNA in 11 A. melas populations collected throughout its range. Compared with its sibling species A. gambiae, A. melas populations have a high level of genetic differentiation between them, representing its patchy distribution due to its fragmented larval habitat that is associated with mangroves and salt marsh grass. Populations clustered into three distinct groups representing Western Africa, Southern Africa and Bioko Island populations that appear to be mostly isolated. Fixed differences in the mtDNA are present between all three clusters, and a Bayesian clustering analysis of the microsatellite data found no evidence for migration from mainland to Bioko Island populations, and little migration was evident between the Southern to the Western cluster. Surprisingly, mtDNA divergence between the three A. melas clusters is on par with levels of divergence between other species of the A. gambiae complex, and no support for monophyly was observed in a maximum‐likelihood phylogenetic analysis. Finally, an approximate Bayesian analysis of microsatellite data indicates that Bioko Island A. melas populations were connected to the mainland populations in the past, but became isolated, presumably when sea levels rose after the last glaciation period (≥10 000–11 000 bp ). This study has exposed species‐level genetic divergence within A. melas and also has implications for control of this malaria vector.     

PATERNAL LEAKAGE OF MITOCHONDRIAL DNA IN A FUCUS (PHAEOPHYCEAE) HYBRID ZONE1

Eukaryotic mitochondria are mostly uniparentally (maternally) inherited, although mtDNA heteroplasmy has been reported in all major lineages. Heteroplasmy, the presence of more than one mitochondrial genome in an individual, can arise from recombination, point mutations, or by occasional transmission of the paternal mtDNA (=paternal leakage). Here, we report the first evidence of mtDNA paternal leakage in brown algae. In Denmark, where Fucus serratus L. and Fucus evanescens C. Agardh have hybridized for years, we found eight introgressed individuals that possessed the very distinct haplotypes of each parental species. The finding of heteroplasmy in individuals resulting from several generations of backcrosses suggests that paternal leakage occurred in earlier generations and has persisted through several meiotic bottlenecks.     

Deep intra‐island divergence of a montane forest endemic: phylogeography of the Puerto Rican frog Eleutherodactylus portoricensis (Anura: Eleutherodactylidae)

Aim Hypotheses proposed for lineage diversification of tropical montane species have rarely been tested within oceanic islands. Our goal was to understand how basin barriers and Pleistocene climatic fluctuations shaped the distribution of diversity in Eleutherodactylus portoricensis (Eleutherodactylidae), a frog endemic to the montane rain forests of Puerto Rico. Location The north‐eastern (Luquillo) and south‐eastern (Cayey) mountains of Puerto Rico. Methods We generated mitochondrial DNA (mtDNA) control region sequences (c. 565 bp) from 144 individuals of E. portoricensis representing 16 localities, and sequenced 646 bp of cytochrome b and 596 bp of nuclear DNA (nDNA) rhodopsin exon and intron 1 from a subset of individuals. We conducted a phylogenetic analysis on the mtDNA sequence data and explored population substructure with maximum parsimony networks, a spatial analysis of molecular variance, and pairwise F_ST analysis. Coalescent simulations were performed to test alternative models of population divergence in response to late Pleistocene interglacial periods. Historical demography was assessed through coalescent analyses and Bayesian skyline plots. Results We found: (1) two highly divergent groups associated with the disjunct Luquillo and Cayey Mountains, respectively; (2) a shallow mtDNA genetic discontinuity across the La Plata Basin within the Cayey Mountains; (3) phylogeographic congruence between nDNA and mtDNA markers; (4) divergence dates for both mtDNA and nDNA pre‐dating the Holocene interglacial (c. 10 ka), and nDNA suggesting divergence in the penultimate interglacial (c. 245 ka); and (5) historical demographic stability in both lineages. Main conclusions The low‐elevation Caguas Basin is a long‐term barrier to gene flow between the two montane frog populations. Measures of genetic diversity for mtDNA were similar in both lineages, but lower nDNA diversity in the Luquillo Mountains lineage suggests infrequent dispersal between the two mountain ranges and colonization by a low‐diversity founder population. Population divergence began prior to the Holocene interglacial. Stable population sizes over time indicate a lack of demonstrable demographic response to climatic changes during the last glacial period. This study highlights the importance of topographic complexity in promoting within‐island vicariant speciation in the Greater Antilles, and indicates long‐term persistence and lineage diversification despite late Pleistocene climatic oscillations.     

Simple identification of mitochondrial lineages in contact zones based on lineage-selective primers

A variety of research projects focus on genetic variation among and within maternal lineages as encompassed by mitochondrial DNA (mtDNA). While mtDNA often differs substantially between species, large differences may also be found within species. The evaluation of such divergent lineages, for example in intraspecific contact zones (hybrid zones), commonly involves sequencing numerous individuals. Large‐scale sequencing is both expensive and labour‐intensive. Based on sequences from 15 individuals, we devised a simple and quick polymerase chain reaction assay for identification of divergent mtDNA lineages in a secondary contact zone of the side‐blotched lizard (Uta stansburiana). The application uses lineage‐selective primers to amplify a lineage‐diagnostic product, and is based on each group of mtDNA haplotypes being a monophyletic assemblage of haplotypes sharing the same maternal ancestry, deeply divergent from the other group. The assay was tested on a larger sample (n = 147) of specimens from the contact zone, confirming its usefulness in quick and reliable identification of mtDNA lineages. This approach can be modified for other species, provided diagnostic lineage variation is available, and may also be performed in simple laboratory settings while conducting fieldwork.     

Site heteroplasmy in the mitochondrial cytochrome b gene of the sterlet sturgeon Acipenser ruthenus

Sturgeons are fish species with a complex biology. They are also characterized by complex aspects including polyploidization and easiness of hybridization. As with most of the Ponto-Caspian sturgeons, the populations of Acipenser ruthenus from the Danube have declined drastically during the last decades. This is the first report on mitochondrial point heteroplasmy in the cytochrome b gene of this species. The 1141 bp sequence of the cytb gene in wild sterlet sturgeon individuals from the Lower Danube was determined, and site heteroplasmy evidenced in three of the 30 specimens collected. Two nucleotide sequences were identified in these heteroplasmic individuals. The majority of the heteroplasmic sites are synonymous and do not modify the sequence of amino acids in cytochrome B protein. To date, several cases of point heteroplasmy have been reported in animals, mostly due to paternal leakage of mtDNA. The presence of specific point heteroplasmic sites might be interesting for a possible correlation with genetically distinct groups in the Danube River.     

Fine-scale genetic population structure in a mobile marine mammal: inshore bottlenose dolphins in Moreton Bay, Australia

Highly mobile marine species in areas with no obvious geographic barriers are expected to show low levels of genetic differentiation. However, small‐scale variation in habitat may lead to resource polymorphisms and drive local differentiation by adaptive divergence. Using nuclear microsatellite genotyping at 20 loci, and mitochondrial control region sequencing, we investigated fine‐scale population structuring of inshore bottlenose dolphins (Tursiops aduncus) inhabiting a range of habitats in and around Moreton Bay, Australia. Bayesian structure analysis identified two genetic clusters within Moreton Bay, with evidence of admixture between them (F_ST = 0.05, P = 0.001). There was only weak isolation by distance but one cluster of dolphins was more likely to be found in shallow southern areas and the other in the deeper waters of the central northern bay. In further analysis removing admixed individuals, southern dolphins appeared genetically restricted with lower levels of variation (AR = 3.252, π = 0.003) and high mean relatedness (r = 0.239) between individuals. In contrast, northern dolphins were more diverse (AR = 4.850, π = 0.009) and were mixing with a group of dolphins outside the bay (microsatellite‐based STRUCTURE analysis), which appears to have historically been distinct from the bay dolphins (mtDNA Φ_ST = 0.272, P < 0.001). This study demonstrates the ability of genetic techniques to expose fine‐scale patterns of population structure and explore their origins and mechanisms. A complex variety of inter‐related factors including local habitat variation, differential resource use, social behaviour and learning, and anthropogenic disturbances are likely to have played a role in driving fine‐scale population structure among bottlenose dolphins in Moreton Bay.     

Cryptic speciation in the field vole: a multilocus approach confirms three highly divergent lineages in Eurasia

Species are generally described from morphological features, but there is growing recognition of sister forms that show substantial genetic differentiation without obvious morphological variation and may therefore be considered ‘cryptic species’. Here, we investigate the field vole (Microtus agrestis), a Eurasian mammal with little apparent morphological differentiation but which, on the basis of previous sex‐linked nuclear and mitochondrial DNA (mtDNA) analyses, is subdivided into a Northern and a Southern lineage, sufficiently divergent that they may represent two cryptic species. These earlier studies also provided limited evidence for two major mtDNA lineages within Iberia. In our present study, we extend these findings through a multilocus approach. We sampled 163 individuals from 46 localities, mainly in Iberia, and sequenced seven loci, maternally, paternally and biparentally inherited. Our results show that the mtDNA lineage identified in Portugal is indeed a distinct third lineage on the basis of other markers as well. In fact, multilocus coalescent‐based methods clearly support three separate evolutionary units that may represent cryptic species: Northern, Southern and Portuguese. Divergence among these units was inferred to have occurred during the last glacial period; the Portuguese lineage split occurred first (estimated at c. 70 000 bp ), and the Northern and Southern lineages separated at around the last glacial maximum (estimated at c. 18 500 bp ). Such recent formation of evolutionary units that might be considered species has repercussions in terms of understanding evolutionary processes and the diversity of small mammals in a European context.     

The effects of allospecific mitochondrial genome on the fitness of northern redbelly dace (Chrosomus eos)

Instantaneous mitochondrial introgression events allow the disentangling of the effects of hybridization from those of allospecific mtDNA. Such process frequently occurred in the fish Chrosomus eos, resulting in cybrid individuals composed of a C. eos nuclear genome but with a C. neogaeus mtDNA. This provides a valuable model to address the fundamental question: How well do introgressed individuals perform in their native environment? We infer where de novo production of cybrids occurred to discriminate native environments from those colonized by cybrids in 25 sites from two regions (West‐Qc and East‐Qc) in Quebec (Canada). We then compared the relative abundance of wild types and cybrids as a measure integrating both fitness and de novo production of cybrids. According to mtDNA variation, 12 introgression events are required to explain the diversity of cybrids. Five cybrid lineages could not be associated with in situ introgression events. This includes one haplotype carried by 93% of the cybrids expected to have colonized West‐Qc. These cybrids also displayed a nearly complete allopatric distribution with wild types. We still inferred de novo production of cybrids at seven sites, that accounted for 70% of the cybrids in East‐Qc. Wild‐type and cybrid individuals coexist in all East‐Qc sites while cybrids were less abundant. Allopatry of cybrids restricted to the postglacial expansion suggests the existence of higher fitness for cybrids in specific conditions, allowing for the colonization of different environments and expanding the species’ range. However, allospecific mtDNA does not provide a higher fitness to cybrids in their native environment compared to wild types, making the success of an introgressed lineage uncertain.     

mtDNA evidence for a local northern latitude Pleistocene refugium for the root vole (Microtus oeconomus,Arvicolinae, Rodentia) from Eastern Poland

We analysed the genetic structure of 33 populations of the root vole (tundra vole, Microtus oeconomus, Pallas, 1776) inhabiting their typical habitats, located at different distances from the southern boundary of the species’ range (52°14′–53°56′ N) in eastern Poland. We determined its phylogeographic pattern as well as the possible occurrence of a small, local high‐latitude refugium of this species in southern Poland, previously suggested in palaeontological studies. 908 bp of cytochrome b sequences were analysed from 439 root voles, and 21 mtDNA cytb haplotypes belonging to the Central European (CE) phylogroup were found. Haplotype diversity in the examined populations varied between 0 and 0.872 (mean: 0.425 ± 0.332), while nucleotide diversity ranged between 0 and 0.62% (mean: 0.235% ± 0.217). Within the CE phylogroup of M. oeconomus, we identified with high bootstrap support a newly separated group of M. oeconomus that evolved from CE, denoted CE‐PL S. This group is located in the southern and central part of eastern Poland and most likely diverged from phylogroup CE in a small, cryptic refugium situated in southern Poland, in the Kraków‐Cz?stochowa Upland and/or the Holy Cross Mountains during the LGM and Younger Dryas.     

Heteroplasmy and Ancient Translocation of Mitochondrial DNA to the Nucleus in the Chinese Horseshoe Bat (Rhinolophus sinicus) Complex

The utility and reliability of mitochondrial DNA sequences in phylogenetic and phylogeographic studies may be compromised by widespread and undetected nuclear mitochondrial copies (numts) as well as heteroplasmy within individuals. Both numts and heteroplasmy are likely to be common across diverse taxa yet few studies have characterised their frequencies and variation at the intra-specific level. Here we report the presence of both numts and heteroplasmy in the mitochondrial control region of the Chinese horseshoe bat Rhinolophus sinicus. In total we generated 123 sequences from 18 bats, which contained two different numt clades (i.e. Numt-1 and Numt-2) and one mtDNA clade. The sequence divergence between Numt-1 and Numt-2 was 16.8% and each numt type was found in all four R. sinicus taxa, suggesting either two ancient translocations of mitochondrial DNA into the nucleus from the same source taxon, or a single translocation from different source taxa that occurred before the split of R. sinicus into different lineages. Within the mtDNA clade, phylogenetic relationships among the four taxa of R. sinicus were similar to those seen in previous results. Based on PCR comparisons, heteroplasmy was inferred between almost all individuals of R. sinicus with respect to sequence variation. Consistent with introgression of mtDNA between Central sinicus and septentrionalis, individuals from these two taxa exhibited similar signatures of repeated sequences in the control region. Our study highlights the importance of testing for the presence of numts and heteroplasmy when applying mtDNA markers to phylogenetic studies.     

Drought in an evolutionary context: molecular variability in Flannelmouth Sucker (Catostomus latipinnis) from the Colorado River Basin of western North America

Summary 1. Fishes can often rebound numerically and distributionally from short‐term (i.e. seasonal) drought, yet their capacity to recover from decades or centuries of drought is less apparent. An exceedingly warm and dry period swept the intermontane west of North America ca. 7500 years BP, concomitant with an abrupt extinction of >35 mammal species. Were larger fishes in mainstem rivers also impacted by this drought? 2. The Colorado River Basin encompasses seven states in western North America and drains 600 000 km². Its endemic mainstem fish community is ancient (i.e. Miocene) but depauperate. 3. We evaluated one widely distributed candidate species (flannelmouth sucker, Catostomus latipinnis) for basin‐wide genetic and geographic structure at three fast‐evolving mitochondrial (mt) DNA genes, ND2 with 589 bp and ATPase 8 and 6 with 642 bp. It is hypothesized that a concomitant signature would be present in the mtDNA of this species, if indeed it had been seriously bottlenecked by post‐Pleistocene drought. A total of 352 individuals were sequenced from 24 populations (4–40 individuals/population; average of 14.7). 4. Only 49 unique haplotypes were found, 53% of which represented single individuals. Haplotype diversity was high (0.905 ± 0.007) whereas nucleotide diversity was low (0.002 ± 0.000). 5. A significant and positive geographical cline (P < 0.001) in nucleotide diversity was observed as sampling locations progressed upstream from southwest to northeast. These results divided the Colorado River Basin into three reaches: the lower reach with six populations and 83 individuals; the upper reach with seven populations and 83 individuals; and the middle reach with 11 populations and 186 individuals. An analysis of molecular variance (amova ) revealed that 81.5% of the total genetic variation was within populations, 16% among populations within reaches and 2.5% among reaches. Only the last was significant. Populations from the three reaches diverged from one another by 3400–11 000 years BP. Haplotype distribution suggested populations in the upper Colorado River are expanding. 6. The lack of genetic variation and recent coalescence of lineages in C. latipinnis are unusual given its fossil history, broad geographical sampling, the rapid rate of mtDNA evolution and the number (and evolutionary rate) of the genes examined. The most parsimonious explanation for these data is a rapid expansion following a recent period of low effective population size at the end of the Pleistocene. 7. The intense drought is suggested at the end of the Pleistocene (late‐to‐mid‐Holocene), severely impacted not only large mammals but also larger fishes in western North American rivers. These perspectives have important implications for management of endangered and threatened species in this region.     

Genetic diversity of the dung beetle,Copris tripartitus (Coleoptera: Scarabaeidae), that is endangered in Korea

The dung beetle, Copris tripartitus (Coleoptera: Scarabaeidae), is an endangered insect in Korea. In order to establish a conservation strategy, a preliminary investigation on the genetic diversity of Korean populations was performed using mitochondrial COI (658 bp), CytB (433 bp), and nuclear ITS2 (411 nucleotide positions). Sequencing of 69 individuals collected from five localities showed substantially higher variability (5.02% for COI, 4.62% for CytB, and 8.03% for ITS2). The resulting networks for mitochondrial DNA (mtDNA) haplotypes exhibited two star‐like phylogenies, which might indicate that Korean populations have recently expended from two small populations. The ITS2 network, which was presented in the form of a star‐like phylogeny, confirmed that a recent population expansion occurred. Considering the high genetic diversity and gene flow in C. tripartitus populations, one issue regarding conservation seems to be the recovery of previous habitats.     

Extensive paternal mtDNA leakage in natural populations of Drosophila melanogaster

Strict maternal inheritance is considered a hallmark of animal mtDNA. Although recent reports suggest that paternal leakage occurs in a broad range of species, it is still considered an exceptionally rare event. To evaluate the impact of paternal leakage on the evolution of mtDNA, it is essential to reliably estimate the frequency of paternal leakage in natural populations. Using allele‐specific real‐time quantitative PCR (RT‐qPCR), we show that heteroplasmy is common in natural populations with at least 14% of the individuals carrying multiple mitochondrial haplotypes. However, the average frequency of the minor mtDNA haplotype is low (0.8%), which suggests that this pervasive heteroplasmy has not been noticed before due to a lack of power in sequencing surveys. Based on the distribution of mtDNA haplotypes in the offspring of heteroplasmic mothers, we found no evidence for strong selection against one of the haplotypes. We estimated that the rate of paternal leakage is 6% and that at least 100 generations are required for complete sorting of mtDNA haplotypes. Despite the high proportion of heteroplasmic individuals in natural populations, we found no evidence for recombination between mtDNA molecules, suggesting that either recombination is rare or recombinant haplotypes are counter‐selected. Our results indicate that evolutionary studies using mtDNA as a marker might be biased by paternal leakage in this species.     

Assessing Mitochondrial DNA Variation and Copy Number in Lymphocytes of ~2,000 Sardinians Using Tailored Sequencing Analysis Tools

DNA sequencing identifies common and rare genetic variants for association studies, but studies typically focus on variants in nuclear DNA and ignore the mitochondrial genome. In fact, analyzing variants in mitochondrial DNA (mtDNA) sequences presents special problems, which we resolve here with a general solution for the analysis of mtDNA in next-generation sequencing studies. The new program package comprises 1) an algorithm designed to identify mtDNA variants (i.e., homoplasmies and heteroplasmies), incorporating sequencing error rates at each base in a likelihood calculation and allowing allele fractions at a variant site to differ across individuals; and 2) an estimation of mtDNA copy number in a cell directly from whole-genome sequencing data. We also apply the methods to DNA sequence from lymphocytes of ~2,000 SardiNIA Project participants. As expected, mothers and offspring share all homoplasmies but a lesser proportion of heteroplasmies. Both homoplasmies and heteroplasmies show 5-fold higher transition/transversion ratios than variants in nuclear DNA. Also, heteroplasmy increases with age, though on average only ~1 heteroplasmy reaches the 4% level between ages 20 and 90. In addition, we find that mtDNA copy number averages ~110 copies/lymphocyte and is ~54% heritable, implying substantial genetic regulation of the level of mtDNA. Copy numbers also decrease modestly but significantly with age, and females on average have significantly more copies than males. The mtDNA copy numbers are significantly associated with waist circumference (p-value = 0.0031) and waist-hip ratio (p-value = 2.4×10^-5), but not with body mass index, indicating an association with central fat distribution. To our knowledge, this is the largest population analysis to date of mtDNA dynamics, revealing the age-imposed increase in heteroplasmy, the relatively high heritability of copy number, and the association of copy number with metabolic traits.     

Mutation analysis of the entire mitochondrial genome using denaturing high performance liquid chromatography

In patients with mitochondrial disease a continuously increasing number of mitochondrial DNA (mtDNA) mutations and polymorphisms have been identified. Most pathogenic mtDNA mutations are heteroplasmic, resulting in heteroduplexes after PCR amplification of mtDNA. To detect these heteroduplexes, we used the technique of denaturing high performance liquid chromatography (DHPLC). The complete mitochondrial genome was amplified in 13 fragments of 1–2 kb, digested in fragments of 90–600 bp and resolved at their optimal melting temperature. The sensitivity of the DHPLC system was high with a lowest detection of 0.5% for the A8344G mutation. The muscle mtDNA from six patients with mitochondrial disease was screened and three mutations were identified. The first patient with a limb-girdle-type myopathy carried an A3302G substitution in the tRNA^Leu(UUR) gene (70% heteroplasmy), the second patient with mitochondrial myopathy and cardiomyopathy carried a T3271C mutation in the tRNA^Leu(UUR) gene (80% heteroplasmy) and the third patient with Leigh syndrome carried a T9176C mutation in the ATPase6 gene (93% heteroplasmy). We conclude that DHPLC analysis is a sensitive and specific method to detect heteroplasmic mtDNA mutations. The entire automatic procedure can be completed within 2 days and can also be applied to exclude mtDNA involvement, providing a basis for subsequent investigation of nuclear genes.     

Introgression of mtDNA in Urosaurus lizards: historical and ecological processes

Introgression of mtDNA appears common in animals, but the implications of acquiring a novel mitochondrial genome are not well known. This study investigates mito‐genome introgression between the lizard species Urosaurus graciosus, a thermal specialist, and U. ornatus, a species that occupies a wider range of thermal environments. As ectotherms, their metabolic rate is strongly influenced by the thermal environment; with mitochondria being linked to metabolic rates, overall energy budgets could be impacted by introgression. I use mitochondrial gene trees, inferred from Bayesian analyses of Cyt‐B and ND1 gene sequences, along with morphology and microsatellites from nineteen populations of these two species to address if the direction and location of mito‐nuclear discordance match predictions of introgression resulting from past population expansions. MtDNA is expected to move from resident species into expanding or invading species. Second, does having a heterospecific form of mitochondria impact body size, a trait strongly associated with fitness? Multiple independent introgression events of historic origin were detected. All introgression was unidirectional with U. ornatus‐type mtDNA found in U. graciosus parental type individuals. This result was consistent with population expansions detected in U. graciosus but not U. ornatus. Females with heterospecific mtDNA were significantly smaller than homospecific forms, and heterospecific males had a different relationship of body mass to body length than those with homospecific mtDNA. These changes indicate a potential selective disadvantage for individuals with heterospecific mitochondria and are consistent with the theoretical expectation that deleterious alleles are more likely to persist in expanding populations.     

A 66‐bp deletion in growth hormone releasing hormone gene 5′‐flanking region with largemouth bass recessive embryonic lethal

Growth hormone releasing hormone (GHRH) regulates the secretion of growth hormone (GH) in the pituitary gland. A 66‐bp deletion (c.‐923_‐858del) was detected in the 5′‐flanking sequence of the largemouth bass (Micropterus salmoides) GHRH gene. In two cultured random populations of adult individuals (A: n = 170 and B: n = 150), the genotype ratios of +/+:+/? were 2.5:1 and 2.8:1 respectively. Only one ?/? fish was detected. A Largemouth bass family was constructed with two heterozygous individuals (+/?) as parents. The genotype ratio of +/+:+/?:?/? in the filial generation embryos was 1:1.6:0.1 at the neurula and 1:2:0 at hatched larvae stages. This indicated that the 66‐bp deletion was a recessive lethal site and that homozygous individuals (?/?) died off in embryonic development. The growth traits (body weight, body length and body depth) were measured, and the GHRH mRNA expression levels in brain tissue were detected using real‐time PCR. The effects of genotype (+/?) on growth traits and GHRH mRNA expression were not significant. Although the cause of death was not clear, the results hint that the 66‐bp deletion site in GHRH 5′‐flanking sequence significantly affects the livability in largemouth bass embryonic development.     

A mitochondrial species identification assay for Australian blacktip sharks (Carcharhinus tilstoni, C. limbatus and C. amblyrhynchoides) using real-time PCR and high-resolution melt analysis

Tropical Australian shark fisheries target two morphologically indistinguishable blacktip sharks, the Australian blacktip (Carcharhinus tilstoni) and the common blacktip (C. limbatus). Their relative contributions to northern and eastern Australian coastal fisheries are unclear because of species identification difficulties. The two species differ in their number of precaudal vertebrae, which is difficult and time consuming to obtain in the field. But, the two species can be distinguished genetically with diagnostic mutations in their mitochondrial DNA ND4 gene. A third closely related sister species, the graceful shark C. amblyrhynchoides, can also be distinguished by species‐specific mutations in this gene. DNA sequencing is an effective diagnostic tool, but is relatively expensive and time consuming. In contrast, real‐time high‐resolution melt (HRM) PCR assays are rapid and relatively inexpensive. These assays amplify regions of DNA with species‐specific genetic mutations that result in PCR products with unique melt profiles. A real‐time HRM PCR species‐diagnostic assay (RT‐HRM‐PCR) has been developed based on the mtDNA ND4 gene for rapid typing of C. tilstoni, C. limbatus and C. amblyrhynchoides. The assay was developed using ND4 sequences from 66 C. tilstoni, 33. C. limbatus and five C. amblyrhynchoides collected from Indonesia and Australian states and territories; Western Australia, the Northern Territory, Queensland and New South Wales. The assay was shown to be 100% accurate on 160 unknown blacktip shark tissue samples by full mtDNA ND4 sequencing.     

Frequency and Pattern of Heteroplasmy in the Control Region of Human Mitochondrial DNA

In this work, we present the results of the screening of human mitochondrial DNA (mtDNA) heteroplasmy in the control region of mtDNA from 210 unrelated Spanish individuals. Both hypervariable regions of mtDNA were amplified and sequenced in order to identify and quantify point and length heteroplasmy. Of the 210 individuals analyzed, 30% were fully homoplasmic and the remaining presented point and/or length heteroplasmy. The prevalent form of heteroplasmy was length heteroplasmy in the poly(C) tract of the hypervariable region II (HVRII), followed by length heteroplasmy in the poly(C) tract of hypervariable region I (HVRI) and, finally, point heteroplasmy, which was found in 3.81% of the individuals analyzed. Moreover, no significant differences were found in the proportions of the different kinds of heteroplasmy in the population when blood and buccal cell samples were compared. The pattern of heteroplasmy in HVRI and HVRII presents important differences. Moreover, the mutational profile in heteroplasmy seems to be different from the mutational pattern detected in population. The results suggest that a considerable number of mutations and, particularly, transitions that appear in heteroplasmy are probably eliminated by drift and/or by selection acting at different mtDNA levels of organization. Taking as a whole the results reported in this work, it is mandatory to perform a broad-scale screening of heteroplasmy to better establish the heteroplasmy profile which would be important for medical, evolutionary, and forensic proposes.     

Extracting DNA from museum bird eggs, and whole genome amplification of archive DNA

We present a comprehensive protocol for extracting DNA from egg membranes and other internal debris recovered from the interior of blown museum bird eggs. A variety of commercially available DNA extraction methods were found to be applicable. DNA sequencing of polymerase chain reaction (PCR) products for a 176‐bp fragment of mitochondrial DNA was successful for most egg samples (> 78%) even though the amount of DNA extracted (mean = 14.71 ± 4.55 ng/µL) was significantly less than that obtained for bird skin samples (mean = 67.88 ± 4.77 ng/µL). For PCR and sequencing of snipe (Gallinago) DNA, we provide eight new primers for the ‘DNA barcode’ region of COI mtDNA. In various combinations, the primers target a range of PCR products sized from 72 bp to the full ‘barcode’ of 751 bp. Not all possible combinations were tested with archive snipe DNA, but we found a significantly better success rate of PCR amplification for a shorter 176‐bp target compared with a larger 288‐bp fragment (67% vs. 39%). Finally, we explored the feasibility of whole genome amplification (WGA) for extending the use of archive DNA in PCR and sequencing applications. Of two WGA approaches, a PCR‐based method was found to be able to amplify whole genomic DNA from archive skins and eggs from museum bird collections. After WGA, significantly more archive egg samples produced visible PCR products on agarose (56.9% before WGA vs. 79.0% after WGA). However, overall sequencing success did not improve significantly (78.8% compared with 83.0%).