Thomas J. Parsons

Diversification of Neoaves: integration of molecular sequence data and fossils

Patterns of diversification and timing of evolution within Neoaves, which includes almost 95% of all bird species, are virtually unknown. On the other hand, molecular data consistently indicate a Cretaceous origin of many neoavian lineages and the fossil record seems to support an Early Tertiary diversification. Here, we present the first well-resolved molecular phylogeny for Neoaves, together with divergence time estimates calibrated with a large number of stratigraphically and phylogenetically well-documented fossils. Our study defines several well-supported clades within Neoaves. The calibration results suggest that Neoaves, after an initial split from Galloanseres in Mid-Cretaceous, diversified around or soon after the K/T boundary. Our results thus do not contradict palaeontological data and show that there is no solid molecular evidence for an extensive pre-Tertiary radiation of Neoaves.

Population data for 101 Austrian Caucasian mitochondrial DNA d-loop sequences: application of mtDNA sequence analysis to a forensic case.

Abstract The sequence of the two hypervariable segments of the mitochondrial DNA (mtDNA) control region was generated for 101 random Austrian Caucasians. A total of 86 different mtDNA sequences was observed, where 11 sequences were shared by more than 1 individual, 7 sequences were shared by 2 individuals and 4 sequences were shared by 3 individuals. One of the four most common mtDNA sequences in Austrians is also the most common sequence in both U.S. and British Caucasians, found in approximately 3.0% of Austrians, 4.0% of British, and 3.9% of U.S. Caucasians. Of the remaining three common Austrian sequences, one was not observed in either U.S. or British Caucasians. However, three British Caucasians exhibited a similar sequence type. Therefore, this particular cluster of sequence polymorphisms may represent a common “European” mtDNA sequence type. In general, Austrian Caucasians show little deviation from other Caucasian databases of European descent. Finally, mtDNA sequence analysis was applied to a forensic case, where hairs found at a crime scene matched the control hairs from the suspect.

Single nucleotide polymorphisms over the entire mtDNA genome that increase the power of forensic testing in Caucasians

We have sequenced the entire mtDNA genome (mtGenome) of 241 individuals who match 1 of 18 common European Caucasian HV1/HV2 types, to identify sites that permit additional forensic discrimination. We found that over the entire mtGenome even individuals with the same HV1/HV2 type rarely match. Restricting attention to sites that are neutral with respect to phenotypic expression, we have selected eight panels of single nucleotide polymorphism (SNP) sites that are useful for additional discrimination. These panels were selected to be suitable for multiplex SNP typing assays, with 7–11 sites per panel. The panels are specific for one or more of the common HV1/HV2 types (or closely related types), permitting a directed approach that conserves limiting case specimen extracts while providing a maximal chance for additional discrimination. Discrimination provided by the panels reduces the frequency of the most common type in the European Caucasian population from ~7% to ~2%, and the 18 common types we analyzed are resolved to 105 different types, 55 of which are seen only once.

Rapid screening of mtDNA coding region SNPs for the identification of west European Caucasian haplogroups

This work presents a selection of 16 SNPs from the coding region of the human mitochondrial DNA. The selected markers are used for the assignment of individuals to one of the nine major European Caucasian mitochondrial haplogroups. The selected SNPs are targeted in two multiplex systems, via the application of the SNaPshot kit, a multiplex method based on the dideoxy single-base extension of unlabeled oligonucleotide primers. The method is conceived as a rapid screening technique prior to sequencing analysis, in order to eliminate multiple suspects from an inquiry or to discriminate between stains in a high volume casework example. Moreover, the ability to assign an unknown sample to an mtDNA type of known Caucasian origin could be of probative value in some investigations. A database of 277 Austrian Caucasians has been constructed, and the probability of a chance match between two unrelated individuals is calculated as 11.4%. This novel multiplex PCR amplification and typing system for mtDNA coding region SNPs promises to be a convenient and informative new DNA profiling system in the forensic field.

A multiplex allele-specific primer extension assay for forensically informative SNPs distributed throughout the mitochondrial genome

The typing of single nucleotide polymorphisms (SNPs) located throughout the mitochondrial genome (mtGenome) can help resolve individuals with an identical HV1/HV2 mitotype. A set of 11 SNPs selected for distinguishing individuals of the most common Caucasian HV1/HV2 mitotype were incorporated in an allele specific primer extension assay. The assay was optimized for multiplex detection of SNPs at positions 3010, 4793, 10211, 5004, 7028, 7202, 16519, 12858, 4580, 477 and 14470 in the mtGenome. Primers were designed to allow for simultaneous PCR amplification of 11 unique regions in the mtGenome and subsequent primer extension. By enzymatically incorporating fluorescently labeled dideoxynucleotides (ddNTPs) onto the 3’ end of the extension primer, detection can be accomplished with a capillary-based electrophoresis (CE) platform common in most forensic laboratories. The electrophoretic mobility for the extension primers was compared in denaturing POP4 and POP6 CE running buffers. Empirical adjustment of extension primer concentrations resulted in even signal intensity for the 11 loci probed. We demonstrate that the assay performs well for heteroplasmy and mixture detection, and for typical mtDNA casework samples with highly degraded DNA.

Unidirectional Spread of Secondary Sexual Plumage Traits Across an Avian Hybrid Zone

Theory predicts that traits under positive selection can rapidly cross a hybrid zone in spite of a substantial barrier to neutral gene flow between hybridizing taxa. An avian hybrid zone between Manacus candei (white-collared manakin) and M. vitellinus (golden-collared manakin) is reported here that displays an unusual pattern of noncoincident clines. Male secondary sexual traits of M. vitellinus have spread into populations that are genetically and morphometrically like M. candei. These birds have a lek breeding system in which male mating success is highly skewed, suggesting that sexual selection is driving male sexual traits across the zone.

Mitochondrial DNA control region sequences from Nairobi (Kenya): inferring phylogenetic parameters for the establishment of a forensic database

Large forensic mtDNA databases which adhere to strict guidelines for generation and maintenance, are not available for many populations outside of the United States and western Europe. We have established a high quality mtDNA control region sequence database for urban Nairobi as both a reference database for forensic investigations, and as a tool to examine the genetic variation of Kenyan sequences in the context of known African variation. The Nairobi sequences exhibited high variation and a low random match probability, indicating utility for forensic testing. Haplogroup identification and frequencies were compared with those reported from other published studies on African, or African-origin populations from Mozambique, Sierra Leone, and the United States, and suggest significant differences in the mtDNA compositions of the various populations. The quality of the sequence data in our study was investigated and supported using phylogenetic measures. Our data demonstrate the diversity and distinctiveness of African populations, and underline the importance of establishing additional forensic mtDNA databases of indigenous African populations.

Investigation of Heteroplasmy in the Human Mitochondrial DNA Control Region: A Synthesis of Observations from More Than 5000 Global Population Samples

Instances of point and length heteroplasmy in the mitochondrial DNA control region were compiled and analyzed from over 5,000 global human population samples. These data represent observations from a large and broad population sample, representing nearly 20 global populations. As expected, length heteroplasmy was frequently observed in the HVI, HVII and HVIII C-stretches. Length heteroplasmy was also observed in the AC dinucleotide repeat region, as well as other locations. Point heteroplasmy was detected in approximately 6% of all samples, and while the vast majority of heteroplasmic samples comprised two molecules differing at a single position, samples exhibiting two and three mixed positions were also observed in this data set. In general, the sites at which heteroplasmy was most commonly observed correlated with reported control region mutational hotspots. However, for some sites, observations of heteroplasmy did not mirror established mutation rate data, suggesting the action of other mechanisms, both selective and neutral. Interestingly, these data indicate that the frequency of heteroplasmy differs between particular populations, perhaps reflecting variable mutation rates among different mtDNA lineages and/or artifacts of particular population groups. The results presented here contribute to our general understanding of mitochondrial DNA control region heteroplasmy and provide additional empirical information on the mechanisms contributing to mtDNA control region mutation and evolution.

Systemically wound-responsive genes in poplar trees encode proteins similar to sweet potato sporamins and legume Kunitz trypsin inhibitors

When the lower leaves of hybrid poplar trees are mechanically wounded, several novel mRNAs accumulate in the unwounded upper leaves (Parsons TJ, Bradshaw HD, Gordon MP: Systemic accumulation of specific mRNAs in response to wounding in poplar trees, Proc Natl Acad Sci USA, in press). A partial cDNA clone corresponding to a transcript from the wound-responsive gene designated win 3 (wound-inducible) has been cloned by differential hybridization to 32P-labelled cDNA from the leaves of wounded trees. Northern blots show a large accumulation of win 3 transcripts in the unwounded leaves of wounded trees. Southern blot analysis of poplar DNA suggests that win 3 is a member of a multigene family. The nucleotide sequences of several win 3 cDNA clones have been determined, indicating that at least three win 3 gene family members are transcribed. A genomic clone of a win 3 gene family member has been isolated and a 1.5 kb Hind III fragment containing the predicted protein-coding and 5′ upstream regions has been sequenced. The putative win 3 gene product is similar to the major soluble proteins of sweet potato tubers, sporamin A and sporamin B. Both Win3 and the sporamins share significant amino acid sequence identity with Kunitz-type trypsin inhibitors from legume seeds. The Kunitz family of proteinase inhibitors thus joins three other proteinase inhibitor families which are systemically responsive to wounding.

DNA Commission of the International Society for Forensic Genetics: Revised and extended guidelines for mitochondrial DNA typing

The DNA Commission of the International Society of Forensic Genetics (ISFG) regularly publishes guidelines and recommendations concerning the application of DNA polymorphisms to the question of human identification. Previous recommendations published in 2000 addressed the analysis and interpretation of mitochondrial DNA (mtDNA) in forensic casework. While the foundations set forth in the earlier recommendations still apply, new approaches to the quality control, alignment and nomenclature of mitochondrial sequences, as well as the establishment of mtDNA reference population databases, have been developed. Here, we describe these developments and discuss their application to both mtDNA casework and mtDNA reference population databasing applications. While the generation of mtDNA for forensic casework has always been guided by specific standards, it is now well-established that data of the same quality are required for the mtDNA reference population data used to assess the statistical weight of the evidence. As a result, we introduce guidelines regarding sequence generation, as well as quality control measures based on the known worldwide mtDNA phylogeny, that can be applied to ensure the highest quality population data possible. For both casework and reference population databasing applications, the alignment and nomenclature of haplotypes is revised here and the phylogenetic alignment proffered as acceptable standard. In addition, the interpretation of heteroplasmy in the forensic context is updated, and the utility of alignment-free database searches for unbiased probability estimates is highlighted. Finally, we discuss statistical issues and define minimal standards for mtDNA database searches.