Low guanine content and biased nucleotide distribution in vertebrate mtDNA can cause overestimation of non-CpG methylation

ABSTRACT Mitochondrial DNA (mtDNA) methylation in vertebrates has been hotly debated for over 40 years. Most contrasting results have been reported following bisulfite sequencing (BS-seq) analyses. We addressed whether BS-seq experimental and analysis conditions influenced the estimation of the levels of methylation in specific mtDNA sequences. We found false positive non-CpG methylation in the CHH context (fpCHH) using unmethylated Sus scrofa mtDNA BS-seq data. fpCHH methylation was detected on the top/plus strand of mtDNA within low guanine content regions. These top/plus strand sequences of fpCHH regions would become extremely AT-rich sequences after BS-conversion, whilst bottom/minus strand sequences remained almost unchanged. These unique sequences caused BS-seq aligners to falsely assign the origin of each strand in fpCHH regions, resulting in false methylation calls. fpCHH methylation detection was enhanced by short sequence reads, short library inserts, skewed top/bottom read ratios and non-directional read mapping modes. We confirmed no detectable CHH methylation in fpCHH regions by BS-amplicon sequencing. The fpCHH peaks were located in the D-loop, ATP6, ND2, ND4L, ND5 and ND6 regions and identified in our S. scrofa ovary and oocyte data and human BS-seq data sets. We conclude that non-CpG methylation could potentially be overestimated in specific sequence regions by BS-seq analysis.

Embryonic Development of Grasshopper Populations Along Latitudinal Gradients Reveal Differential Thermoaccumulation for Adaptation to Climate Warming

Climate warming has a remarkable effect on the distribution, phenology, and development of insects. Although the embryonic development and phenology of non-diapause grasshopper species are more susceptible to warming than those of diapause species, the responses of developmental traits in conspecifically different populations to climate warming remain unknown. Here, we compared the mtDNA sequences and embryonic development of eight populations of grasshopper species (Chorthippus dubius) in field-based manipulated warming and laboratory experiments. The mtDNA sequences showed a significant genetic differentiation of the southernmost population from the other seven populations on the Mongolian Plateau. The embryonic development of the southernmost population was significantly slower than those of the northern populations at the same incubation temperatures. Interestingly, laboratory experiments showed that a significant difference exists in the effective accumulated degree days (EADD) but not in the lower development threshold temperatures (LDTT) among the different populations. The high-latitude populations required less EADD than the low-latitude populations. The warming treatments significantly accelerated the embryonic development in the field and decreased duration from embryos to hatchlings of all eight populations in the incubation. In addition, warming treatments in field significantly increased EADD requirement per stage in the incubation. Linear regression model confirmed that the embryonic development characteristics of eight populations were correlated with the annual mean temperature and total precipitation of embryonic development duration. The results indicated that grasshopper species have evolved a strategy of adjusting their EADD but not their LDTT to adapt to temperature changes. The variations in the EADD among the different populations enabled the grasshopper eggs to buffer the influences of higher temperatures on development and preserve their univoltine nature in temperate regions while encountering warmer climatic conditions. Thus, the findings of this study is valuable for our understanding species variation and evolution, and as such has direct implication for modeling biological response to climate warming.

A population genetic analysis of the New Zealand spotty (Notolabrus celidotus) using mitochondrial DNA and microsatellite DNA markers

<p>Notolabrus celidotus (the New Zealand spotty) is a common rocky reef species that is endemic to New Zealand. This species is the most abundant demersal reef fish in New Zealand, and is distributed throughout the North and South Islands, and Stewart Island. Notolabrus celidotus consumes a wide variety of small invertebrates, and juveniles are reliant on coastal kelp forests as nursery habitats. Because N. celidotus is such a common species on New Zealand rocky reefs it is a good model species for population genetic studies. The primary goal of this research was to investigate new genetic markers and add new sample locations to bolster previous genetic population data from N. celidotus. The thesis research utilised DNA sequences obtained from a 454 massively parallel DNA sequencer and reports six new microsatellite loci for N. celidotus. These loci are the first microsatellite DNA markers to be developed for this species. Additional mitochondrial DNA (mtDNA) control region sequences were obtained from new samples of N. celidotus and combined with previously reported mtDNA sequences. Increasing the sample size improved the genetic coverage of N. celidotus populations around coastal New Zealand. The mtDNA sequences were analysed to examine the population connectivity and demographic history of N. celidotus. The microsatellite DNA loci reported in this study were also used to examine the levels of genetic diversity and population structure in N. celidotus. Results of the combined genetic analyses revealed extremely high levels of genetic diversity among the population sample of the mtDNA control region. Both the mitochondrial DNA and microsatellite DNA analyses showed a distinct lack of population genetic structuring, which suggests there is constant mixing of N. celidotus among sites. The results of this study have the potential to inform the expectations about the genetic structure of closely related wrasse species, such as Notolabrus fucicola, as well as other coastal species that have a similar life history, dispersal power, and New Zealand-wide distribution.</p>

A population genetic analysis of the New Zealand spotty (Notolabrus celidotus) using mitochondrial DNA and microsatellite DNA markers

<p>Notolabrus celidotus (the New Zealand spotty) is a common rocky reef species that is endemic to New Zealand. This species is the most abundant demersal reef fish in New Zealand, and is distributed throughout the North and South Islands, and Stewart Island. Notolabrus celidotus consumes a wide variety of small invertebrates, and juveniles are reliant on coastal kelp forests as nursery habitats. Because N. celidotus is such a common species on New Zealand rocky reefs it is a good model species for population genetic studies. The primary goal of this research was to investigate new genetic markers and add new sample locations to bolster previous genetic population data from N. celidotus. The thesis research utilised DNA sequences obtained from a 454 massively parallel DNA sequencer and reports six new microsatellite loci for N. celidotus. These loci are the first microsatellite DNA markers to be developed for this species. Additional mitochondrial DNA (mtDNA) control region sequences were obtained from new samples of N. celidotus and combined with previously reported mtDNA sequences. Increasing the sample size improved the genetic coverage of N. celidotus populations around coastal New Zealand. The mtDNA sequences were analysed to examine the population connectivity and demographic history of N. celidotus. The microsatellite DNA loci reported in this study were also used to examine the levels of genetic diversity and population structure in N. celidotus. Results of the combined genetic analyses revealed extremely high levels of genetic diversity among the population sample of the mtDNA control region. Both the mitochondrial DNA and microsatellite DNA analyses showed a distinct lack of population genetic structuring, which suggests there is constant mixing of N. celidotus among sites. The results of this study have the potential to inform the expectations about the genetic structure of closely related wrasse species, such as Notolabrus fucicola, as well as other coastal species that have a similar life history, dispersal power, and New Zealand-wide distribution.</p>

Mitochondrial Genome Variation and Metabolic Traits in a Maori Community

<p>The mitochondrion is the energy producing factory of cells and it has long been thought that disruption to mitochondrial systems is linked to energy metabolism dysfunction. Sequence variants in the mitochondrial genome are plausible candidate risk factors for numerous human diseases, and research has identified specific mitochondrial DNA (mtDNA) variants associated with metabolic disorders such as obesity and type-2 diabetes. As part of the Rakaipaaka Health and Ancestry Study (RHAS) it has been observed that the Maori community of Nuhaka (Ngati Rakaipaaka) have a high incidence of certain metabolic diseases, namely obesity and diabetes. The reason for this is not well understood, but is likely to be a combination of both current lifestyle (e.g. dietary) and ancestral genetic factors. This study set out to sequence the entire mitochondrial genome in a sample of RHAS Maori participants. The aim was to discover genetic variation that might be specific to this Maori community and test whether such variants are associated with diabetes and other metabolic traits. This study used a novel RFLP assay to screen the mtDNA control region for Polynesian mtDNA ancestry. This established an initial group (n=30) with high levels of Maori mtDNA. Hypervariable (HVRI) sequencing was then used to generate a large dataset of sequences (n=94). This dataset was representative of individuals showing high Maori ancestry and aided the selection of 20 mtDNA's for Mitochip analysis. Combining the RHAS Maori HVRI sequences with those from previous studies indicated elevated variation in Maori mtDNA. Haplotype analysis identified 17 unique Maori haplotypes, 10 more than previously recorded. Mitochip resequencing has provided the first complete Maori mtDNA sequences to date. When compared to other mtDNA sequences it was identified that RHAS Maori share similar haplotype markers with Polynesians. Seven novel undocumented variants were found, as well as four variants that had previously been associated with various metabolic disorders. Mitochip analysis of mtDNA sequences revealed three variants which created a RHAS Maori specific signature; C1185T, G4769A, and T16126C. These variants also defined 3 unique mtDNA haplotypes within RHAS Maori, which are the first Maori specific haplotypes reported. Variant genotyping and correlation with metabolic traits identified significant associations within the wider RHAS Maori community. It was identified that RHAS Maori with T16189C showed elevation in both vitamin B12 levels and mean diastolic blood pressure. Individuals with the G4769A variant were shown to have significant increases in specific metabolic risk factors for cardiovascular disease. Conversely individuals with the more common A4769G variant were 2 times less likely to be diagnosed with diabetes. The findings from this study have identified a series of potential markers of metabolic disease within the RHAS Maori community. The goal now is to understand how these markers interact with environmental variables to increase the risk of metabolic syndrome. Such an outcome may open the way to designing personalised intervention strategies (e.g. dietary) to increase the health and well-being of at risk individuals.</p>

Mitochondrial Genome Variation and Metabolic Traits in a Maori Community

<p>The mitochondrion is the energy producing factory of cells and it has long been thought that disruption to mitochondrial systems is linked to energy metabolism dysfunction. Sequence variants in the mitochondrial genome are plausible candidate risk factors for numerous human diseases, and research has identified specific mitochondrial DNA (mtDNA) variants associated with metabolic disorders such as obesity and type-2 diabetes. As part of the Rakaipaaka Health and Ancestry Study (RHAS) it has been observed that the Maori community of Nuhaka (Ngati Rakaipaaka) have a high incidence of certain metabolic diseases, namely obesity and diabetes. The reason for this is not well understood, but is likely to be a combination of both current lifestyle (e.g. dietary) and ancestral genetic factors. This study set out to sequence the entire mitochondrial genome in a sample of RHAS Maori participants. The aim was to discover genetic variation that might be specific to this Maori community and test whether such variants are associated with diabetes and other metabolic traits. This study used a novel RFLP assay to screen the mtDNA control region for Polynesian mtDNA ancestry. This established an initial group (n=30) with high levels of Maori mtDNA. Hypervariable (HVRI) sequencing was then used to generate a large dataset of sequences (n=94). This dataset was representative of individuals showing high Maori ancestry and aided the selection of 20 mtDNA's for Mitochip analysis. Combining the RHAS Maori HVRI sequences with those from previous studies indicated elevated variation in Maori mtDNA. Haplotype analysis identified 17 unique Maori haplotypes, 10 more than previously recorded. Mitochip resequencing has provided the first complete Maori mtDNA sequences to date. When compared to other mtDNA sequences it was identified that RHAS Maori share similar haplotype markers with Polynesians. Seven novel undocumented variants were found, as well as four variants that had previously been associated with various metabolic disorders. Mitochip analysis of mtDNA sequences revealed three variants which created a RHAS Maori specific signature; C1185T, G4769A, and T16126C. These variants also defined 3 unique mtDNA haplotypes within RHAS Maori, which are the first Maori specific haplotypes reported. Variant genotyping and correlation with metabolic traits identified significant associations within the wider RHAS Maori community. It was identified that RHAS Maori with T16189C showed elevation in both vitamin B12 levels and mean diastolic blood pressure. Individuals with the G4769A variant were shown to have significant increases in specific metabolic risk factors for cardiovascular disease. Conversely individuals with the more common A4769G variant were 2 times less likely to be diagnosed with diabetes. The findings from this study have identified a series of potential markers of metabolic disease within the RHAS Maori community. The goal now is to understand how these markers interact with environmental variables to increase the risk of metabolic syndrome. Such an outcome may open the way to designing personalised intervention strategies (e.g. dietary) to increase the health and well-being of at risk individuals.</p>

Integrative taxonomic revision of the New Caledonian endemic genus Taophila Heller (Coleoptera: Chrysomelidae, Eumolpinae)

There are 96 endemic species of Eumolpinae (Coleoptera: Chrysomelidae) described from New Caledonia, but some estimates propose that the actual number could be at least twice this figure. Not surprisingly, when a particular species assemblage has been revised, the number of species in that group increases significantly. Here, we revise the New Caledonian endemic genus Taophila Heller, 1916, the best studied in this fauna and currently known to include eleven species, one in the subgenus Jolivetiana Gómez-Zurita & Cardoso, 2014, and ten in the nominal subgenus. The analysis of morphological differences in a large sample of Taophila and the validation of the resulting species hypotheses in an integrative fashion based on a phylogenetic analysis of partial mtDNA sequences (cox1 and rrnS) resulted in the addition of eleven more taxa. Taxonomic splits mainly reinterpreted the previous observation of mtDNA paraphyly affecting T. subsericea Heller, 1916, shown to represent a complex of species mostly distinguishable by diagnostic differences among females. The new species described are: T. bituberculata n. sp., T. carinata n. sp., T. dapportoi n. sp., T. davincii n. sp., T. draco n. sp., T. goa n. sp., T. hackae n. sp., T. samuelsoni n. sp., T. sideralis n. sp., T. taaluny n. sp. and T. wanati n. sp. These additions and the synonymy T. subsericea Heller = Stethotes mandjeliae Jolivet, Verma & Mille, 2010 n. syn., bring to 21 the total number of species in Taophila. Moreover, we also found the first evidence of mtDNA introgression between species of New Caledonian Eumolpinae, resulting from putative recent hybridization of T. subsericea and T. dapportoi where these species coexist. We describe a model incorporating the mtDNA genealogy of T. subsericea about the conditions that may have favored the secondary geographic encounter required for the hybridization of these species.

Origin and establishment of the introduced Cuban Blue Anole, Anolis allisoni, in Florida

In the state of Florida, USA, lizards of the genus Anolis are well represented with at least nine established non-native species and a single native species, A. carolinensis. The most recently introduced species is A. allisoni, a close relative to both the native A. carolinensis and one of the introduced species (A. porcatus). Anolis allisoni is thought to have been present in two locations in Florida since at least 2013 based on photographic evidence. Here, we analyzed mitochondrial DNA (mtDNA) sequences from these three closely related Anolis species to infer the most likely region of origin in the native range and confirm the establishment of the recent invader in Tampa, Florida. We found a single haplotype belonging to A. allisoni, which was closely related to native sequences from east-central Cuba. The most likely geographic origin is a tourist destination in the province of Sancti Spiritus, suggesting the potential for human-mediated introduction of A. allisoni to Florida. Given the evidence of hybridization within the carolinensis subgroup, the presence and establishment of the phylogenetically related and ecomorphologically similar A. allisoni may create novel opportunities for interspecific genetic exchange.

Origin and establishment of the introduced Cuban Blue Anole, Anolis allisoni, in Florida

In the state of Florida, USA, lizards of the genus Anolis are well represented with at least nine established non-native species and a single native species, A. carolinensis. The most recently introduced species is A. allisoni, a close relative to both the native A. carolinensis and one of the introduced species (A. porcatus). Anolis allisoni is thought to have been present in two locations in Florida since at least 2013 based on photographic evidence. Here, we analyzed mitochondrial DNA (mtDNA) sequences from these three closely related Anolis species to infer the most likely region of origin in the native range and confirm the establishment of the recent invader in Tampa, Florida. We found a single haplotype belonging to A. allisoni, which was closely related to native sequences from east-central Cuba. The most likely geographic origin is a tourist destination in the province of Sancti Spiritus, suggesting the potential for human-mediated introduction of A. allisoni to Florida. Given the evidence of hybridization within the carolinensis subgroup, the presence and establishment of the phylogenetically related and ecomorphologically similar A. allisoni may create novel opportunities for interspecific genetic exchange.

Molecular phylogenetic analysis of the Paguristes tortugae Schmitt, 1933 complex and selected other Paguroidea (Crustacea: Decapoda: Anomura)

Morphological characters, as presently applied to describe members of the Paguristes tortugae Schmitt, 1933 species complex, appear to be of limited value in inferring phylogenetic relationships within the genus, and may have similarly misinformed understanding of relationships between members of this complex and those presently assigned to the related genera Areopaguristes Rahayu & McLaughlin, 2010 and Pseudopaguristes McLaughlin, 2002. Previously undocumented observations of similarities and differences in color patterns among populations additionally suggest genetic divergences within some species, or alternatively seem to support phylogenetic groupings of some species. In the present study, a Maximum Likelihood (ML) phylogenetic analysis was undertaken based on the H3, 12S mtDNA, and 16S mtDNA sequences of 148 individuals, primarily representatives of paguroid species from the western Atlantic. This molecular analysis supported a polyphyletic Diogenidae Ortmann, 1892, although incomplete taxonomic sampling among the genera of Diogenidae limits the utility of this finding for resolving family level relationships. Several hypotheses regarding the evolutionary relationships among hermit crab genera were refuted by the Kishino-Hasegawa (KH). Shimodaira-Hasegawa (SH) and Approximately Unbiased (AU) tree topology tests, among them the hypothesis that Areopaguristes is monophyletic. A lack of support for the monophyly of Areopaguristes calls into question the phylogenetic validity of gill number for the differentiation of Paguristes, Areopaguristes, and Pseudopaguristes. The study was inconclusive with regard to the relationships among these three genera, but previously unknown diversity within both Paguristes and Areopaguristes was demonstrated. Existence of an undescribed species confounded under the name Paguristes tortugae Schmitt, 1933 was supported by genetics, morphology, and coloration. A second undescribed species with remarkable similarity to Areopaguristes hummi Wass, 1955 was discovered based on genetics and coloration.