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.

The role of mitochondrial derived peptides (MDPs) in metabolism

The term "mitokines" refers to signals derived from mitochondria that have an impact on other cells or tissues (Durieux et al., 2011). Rather than being simply a set of DNA composed by 37 genes, the mitochondrial DNA (mtDNA) is quite complex and includes small RNAs (Mercer et al., 2011). Mitochondrial-derived peptides (MDPs) are encoded by functional short open reading frames (sORFs) in the mtDNA.

DNA barcoding: an effective tool to overcome morphological identification constraints in the assessment of the ecological quality

DNA barcoding has the potential to overcome taxonomic challenges in biological community assessments. However, fulfilling that potential requires successful amplification of a large and unbiased portion of the community. In this study, we attempted to identify mitochondrial gene cytochrome c oxidase I (COI) barcodes from 1024 benthic invertebrate specimens belonging to 54 taxa from low salinity environments of the Mira estuary and Torgal riverside (SW Portugal). Up to 17 primer pairs and several reaction conditions were attempted among specimens from all taxa, with amplification success defined as a single band of approximately 658 bp visualized on a pre-cast agarose gel, starting near the 5' end of the COI gene and suitable for sequencing. Amplification success was achieved for 99.6% of the 54 taxa, though no single primer was successful for more than 88.9% of the taxa. However, only 68.5% of the specimens within these taxa successfully amplified. Inhibition factors resulting from a non-purified DNA extracted and inexistence of species-specific primers for COI were pointed as the main reasons for an unsuccessful amplification. These results suggest that DNA barcoding can be an effective tool for application in low salinity environments where taxa such as chironomids and oligochaetes are challenging for morphological identification. Nevertheless, its implementation is not simple, as methods are still being standardized and multiple species

Mitochondrial demographic history of the Egyptian mongoose (Herpestes ichneumon), an expanding carnivore in the Iberian Peninsula

Describing the genetic patterns and the demographic history of expanding species is essential for providing insights into the processes linked with range dynamics. We analysed the mitochondrial diversity of the Egyptian mongoose (Herpestes ichneumon) across the Iberian Peninsula, where the species is currently expanding northwest. A total of 242 individuals were analysed, together with nine representatives from the North African dispersal source. Haplotype segregation and strong differentiation between Iberian and North African populations confirmed the longterm presence of the species in the Iberian Peninsula. The distribution of mitochondrial diversity fitted the pattern of a historically diversified population in southern Iberia, from which the recent dispersals into northern areas may have occurred. Higher levels of haplotype and nucleotide diversities in the northern areas, together with the heterogeneous distribution of pairwise population differentiations and the weak signal for isolationbydistance suggest the existence of longdispersal migrants across the Iberian Peninsula. Sudden and spatial expansion scenarios of H. ichneumon in the Iberian Peninsula were supported by mismatch analysis and marginally supported by neutrality tests. However, the precise time of occurrence of the detected expansion remains unclear. Future studies should incorporate additional markers in order to further clarify the population dynamics of the Egyptian mongoose in its Iberian range.

Differentiation of Melipona quadrifasciata L. (Hymenoptera, Apidae, Meliponini) subspecies using cytochrome b PCR-RFLP patterns

Melipona quadrifasciata quadrifasciata and M. quadrifasciata anthidioides are subspecies of M. quadrifasciata, a stingless bee species common in coastal Brazil. These subspecies are discriminated by the yellow stripe pattern of the abdominal tergites. We found Vsp I restriction patterns in the cytochrome b region closely associated to each subspecies in 155 M. quadrifasciata colonies of different geographical origin. This mitochondrial DNA molecular marker facilitates diagnosis of M. quadrifasciata subspecies matrilines and can be used to establish their natural distribution and identify hybrid colonies.

Morphometric and genetic changes in a population of Apis mellifera after 34 years of Africanization

Though the replacement of European bees by Africanized honey bees in tropical America has attracted considerable attention, little is known about the temporal changes in morphological and genetic characteristics in these bee populations. We examined the changes in the morphometric and genetic profiles of an Africanized honey bee population collected near where the original African swarms escaped, after 34 years of Africanization. Workers from colonies sampled in 1968 and in 2002 were morphometrically analyzed using relative warps analysis and an Automatic Bee Identification System (ABIS). All the colonies had their mitochondrial DNA identified. The subspecies that mixed to form the Africanized honey bees were used as a comparison for the morphometric analysis. The two morphometric approaches showed great similarity of Africanized bees with the African subspecies, Apis mellifera scutellata, corroborating with other markers. We also found the population of 1968 to have the pattern of wing venation to be more similar to A. m. scutellata than the current population. The mitochondrial DNA of European origin, which was very common in the 1968 population, was not found in the current population, indicating selective pressure replacing the European with the African genome in this tropical region. Both morphometric methodologies were very effective in discriminating the A. mellifera groups; the non-linear analysis of ABIS was the most successful in identifying the bees, with more than 94% correct classifications.

Genetic variability of the Amazon River prawn Macrobrachium amazonicum (Decapoda, Caridea, Palaemonidae)

The freshwater prawn Macrobrachium amazonicum is widely distributed in South America, and occupies habitats with a wide range of salinities. Several investigations have revealed the existence of wide intraspecific variability among different populations, although the understanding of this variability is still fragmentary and incomplete. We compared and characterized inland and coastal populations of M. amazonicum from Brazil, using molecular data (16S and COI mtDNA) to describe the degree of variability, structure, and relationships among them. Genetic divergence rates among populations showed variability at the intraspecific level. All the analyses evidenced significant genetic divergence among populations, structuring them in three groups: I-inland waters of the Amazonian Hydrographic Region (HR); II-Parana/Paraguay HR; and III-coastal systems of northern and northeastern Brazil. Phylogenetic reconstructions revealed that the populations form a single monophyletic clade, which supports their characterization as a single species. Clade I was a sister clade of that formed by clades II and III, which were themselves sister clades. Populations from Sertaozinho/Miguelopolis and Avare, introduced into the state of Sao Paulo, may have originated from natural populations in the states of Mato Grosso do Sul and Para, respectively. Geographical isolation probably contributed to the observed variation, and if this isolation continues. M. amazonicum may undergo speciation within its broad geographical distribution. The sequences obtained here can be used as name-tags for population identification, and the DNA barcodes are useful to identify the origin of specimens used in different freshwater-prawn cultures or introduced populations of unknown origin.

Lineage divergence detected in the malaria vector Anopheles marajoara (Diptera: Culicidae) in Amazonian Brazil

Background: Cryptic species complexes are common among anophelines. Previous phylogenetic analysis based on the complete mtDNA COI gene sequences detected paraphyly in the Neotropical malaria vector Anopheles marajoara. The ""Folmer region"" detects a single taxon using a 3% divergence threshold. Methods: To test the paraphyletic hypothesis and examine the utility of the Folmer region, genealogical trees based on a concatenated (white + 3' COI sequences) dataset and pairwise differentiation of COI fragments were examined. The population structure and demographic history were based on partial COI sequences for 294 individuals from 14 localities in Amazonian Brazil. 109 individuals from 12 localities were sequenced for the nDNA white gene, and 57 individuals from 11 localities were sequenced for the ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2). Results: Distinct A. marajoara lineages were detected by combined genealogical analysis and were also supported among COI haplotypes using a median joining network and AMOVA, with time since divergence during the Pleistocene (< 100,000 ya). COI sequences at the 3' end were more variable, demonstrating significant pairwise differentiation (3.82%) compared to the more moderate 2.92% detected by the Folmer region. Lineage 1 was present in all localities, whereas lineage 2 was restricted mainly to the west. Mismatch distributions for both lineages were bimodal, likely due to multiple colonization events and spatial expansion (similar to 798 - 81,045 ya). There appears to be gene flow within, not between lineages, and a partial barrier was detected near Rio Jari in Amapa state, separating western and eastern populations. In contrast, both nDNA data sets (white gene sequences with or without the retention of the 4th intron, and ITS2 sequences and length) detected a single A. marajoara lineage. Conclusions: Strong support for combined data with significant differentiation detected in the COI and absent in the nDNA suggest that the divergence is recent, and detectable only by the faster evolving mtDNA. A within subgenus threshold of >2% may be more appropriate among sister taxa in cryptic anopheline complexes than the standard 3%. Differences in demographic history and climatic changes may have contributed to mtDNA lineage divergence in A. marajoara.

Inferences of evolutionary and ecological events that influenced the population structure of Plebeia remota, a stingless bee from Brazil

The present study characterised the population genetic structure of Plebeia remota through mitochondrial DNA (mtDNA) analysis and evaluated evolutionary and ecological processes that may have contributed to the species current genetic scenario. Seventy feral nests were sampled representing four geographic regions (Cunha, Curitiba, Prudentopolis, and Blumenau). Fifteen composite mtDNA haplotypes were determined and a high genetic structure was detected among all populations. The current population structure may be a result of queen philopatry and vegetation shifts caused by palaeoclimatic changes and uplift of Brazilian coastal ranges. Finally, this study strongly suggests a revision of the taxonomic status of P. remota from the Prudentopolis region.

Detection of polymorphisms of the mtDNA control region of Caretta caretta (Testudines: Cheloniidae) by PCR-SSCP

Marine turtles are increasingly being threatened worldwide by anthropogenic activities. Better understanding of their life cycle, behavior and population structure is imperative for the design of adequate conservation strategies. The mtDNA control region is a fast-evolving matrilineal marker that has been employed in the study of marine turtle populations. We developed and tested a simple molecular tracing system for Caretta caretta mtDNA haplotypes by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Using this technique, we were able to distinguish the SSCP patterns of 18 individuals of the haplotypes CC-A4, CC-A24 and CCxLO, which are commonly found in turtles sampled on the Brazilian coast. When we analyzed 15 turtles with previously unknown sequences, we detected two other haplotypes, in addition to the other four. Based on DNA sequencing, they were identified as the CC-A17 and CC-A1 haplotypes. Further analyses were made with the sea turtles, Chelonia mydas (N = 8), Lepidochelys olivacea (N = 3) and Eretmochelys imbricata (N = 1), demonstrating that the PCR-SSCP technique is able to distinguish intra-and interspecific variation in the family Cheloniidae. We found that this technique can be useful for identifying sea turtle mtDNA haplotypes, reducing the need for sequencing.

Phylogeography of Chelonus insularis (Hymenoptera: Braconidae) and Campoletis sonorensis (Hymenoptera: Ichneumonidae), Two Primary Neotropical Parasitoids of the Fall Armyworm (Lepidoptera: Noctuidae)

In a previous study, we observed no spatial genetic structure in Mexican populations of the parasitoids Chelonus insularis Cresson (Hymenoptera: Braconidae) and Campoletis sonorensis Cameron (Hymenoptera: Ichneumonidae) by using microsatellite markers In the current study, we Investigated whether for these important parasitoids of the fall armyworm (Lepidoptera: Noctuidae) there is any genetic structure at a larger scale Insects of both species were collected across the American continent and their phylogeography was Investigated using both nuclear and mitochondria] markers Our results suggest an ancient north-south migration of C insularis, whereas no clear pattern] could be determined for C sonorensis. Nonetheless, the resulting topology indicated the existence of a cryptic taxon within this later species. a few Canadian specimens determined as C. sonorensis branch outside a clack composed of the Argentinean Chelonus grioti Blanchard, the Brazilian Chelonus flavicincta Ashmead, and the rest of the C sonorensis individuals The individuals revealing the cryptic taxon were collected from Thichoplusia in (Hubner) (Lepidoptera. Noctuidae) on tomato (Lycopersicon spp) and may represent a biotype that has adapted to the early season phenology of its host. Overall, the loosely defined spatial genetic structure previously shown at a local fine scale also was found at the larger scale, for both species Dispersal of these insects may be partly driven by wind as suggested by genetic similarities between Individuals coming from very distant locations.

Geographic variation and positive selection on M7 lysin, an acrosomal sperm protein in mussels (Mytilus spp.)

Successful fertilization in free-spawning marine organisms depends on the interactions between genes expressed on the surfaces of eggs and sperm. Positive selection frequently characterizes the molecular evolution of such genes, raising the possibility that some common deterministic process drives the evolution of gamete recognition genes and may even be important for understanding the evolution of prezygotic isolation and speciation in the marine realm. One hypothesis is that gamete recognition genes are subject to selection for prezygotic isolation, namely reinforcement. In a previous study, positive selection on the gene coding for the acrosomal sperm protein M7 lysin was demonstrated among allopatric populations of mussels in the Mytilus edulis species group (M. edulis, M. galloprovincialis, and M. trossulus). Here, we expand sampling to include M7 lysin haplotypes from populations where mussel species are sympatric and hybridize to determine whether there is a pattern of reproductive character displacement, which would be consistent with reinforcement driving selection on this gene. We do not detect a strong pattern of reproductive character displacement; there are no unique haplotypes in sympatry nor is there consistently greater population structure in comparisons involving sympatric populations. One distinct group of haplotypes, however, is strongly affected by natural selection and this group of haplotypes is found within M. galloprovincialis populations throughout the Northern Hemisphere concurrent with haplotypes common to M. galloprovincialis and M. edulis. We suggest that balancing selection, perhaps resulting from sexual conflicts between sperm and eggs, maintains old allelic diversity within M. galloprovincialis.

Cryptic vicariance in Gulf of California fishes parallels vicariant patterns found in Baja, California mammals and reptiles

Comparisons across multiple taxa can often clarify the histories of biogeographic regions. In particular, historic barriers to movement should affect multiple species and, thus, result in a pattern of concordant intraspecific genetic divisions among species. A striking example of such comparative phylogeography is the recent observation that populations of many small mammals and reptiles living on the Baja, California peninsula have a large genetic break between northern and southern peninsular populations. In the present study, I demonstrate that five species of near-shore fishes living on the Baja coastline of the Gulf of California share this genetic pattern. The simplest explanation for this concordant genetic division within both terrestrial and marine vertebrates is that the Baja peninsula was fragmented by a Plio-Pleistocene marine seaway and that this seaway posed a substantial barrier to movement for near-shore fishes. The genetic divisions within Gulf of California fishes also coincide with recognized biogeographic regions based on fish community composition and several environmental factors. It is likely that adaptation to regional environments and present-day oceanographic circulation limits gene exchange between biogeographic regions and helps maintain evidence of past vicariance.

Differential patterns of Male and Female mtDNA exchange across the Atlantic Ocean in the blue mussel, Mytilus edulis

Comparisons among loci with differing modes of inheritance can reveal unexpected aspects of population history. We employ a multilocus approach to ask whether two types of independently assorting mitochondrial DNAs (maternally and paternally inherited: F- and M-mtDNA) and a nuclear locus (ITS) yield concordant estimates of gene flow and population divergence. The blue mussel, Mytilus edulis, is distributed on both North American and European coastlines and these populations are separated by the waters of the Atlantic Ocean. Gene flow across the Atlantic Ocean differs among loci, with F-mtDNA and ITS showing an imprint of some genetic interchange and M-mtDNA showing no evidence for gene flow. Gene flow of F-mtDNA and ITS causes trans-Atlantic population divergence times to be greatly underestimated for these loci, although a single trans-Atlantic population divergence time (1.2 MYA) can be accommodated by considering all three loci in combination in a coalescent framework. The apparent lack of gene flow for M-mtDNA is not readily explained by different dispersal capacities of male and female mussels. A genetic barrier to M-mtDNA exchange between North American and European mussel populations is likely to explain the observed pattern, perhaps associated with the double uniparental system of mitochondrial DNA inheritance.

Positive selection on an acrosomal sperm protein, M7 lysin, in three species of the mussel genus Mytilus

Marine invertebrate sperm proteins are particularly interesting because they are characterized by positive selection and are likely to be involved in prezyogotic isolation and, thus, speciation. Here, we present the first survey of inter and intraspecific variation of a bivalve sperm protein among a group of species that regularly hybridize in nature. M7 lysin is found in sperm acrosomes of mussels and dissolves the egg vitelline coat, permitting fertilization. We sequenced multiple alleles of the mature protein-coding region of M7 lysin from allopatric populations of mussels in the Mytilus edulis species group (M. edulis, M. galloprovincialis, and M. trossulus). A significant McDonald-Kreitman test showed an excess of fixed amino acid replacing substitutions between species, consistent with positive selection. In addition, Kolmogorov-Smirnov tests showed significant heterogeneity in polymorphism to divergence ratios for both synonymous variation and combined synonymous and non-synonymous variation within M. galloprovincialis. These results indicate that there has been adaptive evolution at M7 lysin and, furthermore, shows that positive selection on sperm proteins can occur even when post-zygotic reproductive isolation is incomplete.