Nuclear DNA Analysis Research Articles

Morphological variation, genetic differentiation and phylogeography of the East Asia cicada Hyalessa maculaticollis (Hemiptera: Cicadidae)

AbstractThe cicada Hyalessa maculaticollis is widely distributed in East Asia, and is noted for its great morphological variability. The variation in this species and its allies has been a long‐standing controversy. The population differentiation, genetic structure and phylogeography of this species are explored based on morphological observations, mitochondrial and nuclear DNA analyses, and comparison of the calling song structure of males. Our results reveal that the abundant intraspecific morphological variations are consistent with high levels of genetic divergence in this species, but incongruence between the morphological variations and genetic divergence is found in a few lineages. Phylogenetic and network analyses indicate that H. maculaticollis is composed of two major lineages – China and Japan. The East China Sea (ECS) land bridge acted as a dispersal corridor for H. maculaticollis during the glacial period. The climatic oscillations in the Pleistocene and the terrain structure of East Asia influenced population differentiation. The divergence time between the two sides of the East China Sea is estimated to be ∼1.05 (95% CI = 0.80–1.30) Ma, which was about the same period during which the sea level increased rapidly during the ‘Ryukyu Coral Sea Stage’ (0.2–1.3 Ma). Populations of H. maculaticollis are structured phylogeographically, with the China populations differentiated into a greater number of highly structured haplogroups. Qinling Mountains and the mountainous regions around the Sichuan Basin are presumed to have been major refugia for H. maculaticollis in glacial periods, and a recent population expansion has been detected for populations distributed in the area to the north of Qinling Mountains. The high degree of haplotype and nucleotide diversity shown in East China populations suggests that the flat terrain with low‐altitude hills are suitable for the survival of H. maculaticollis. The species H. fuscata, treated as an independent species from H. maculaticollis by some researchers based on acoustic analyses of the calling song structure, is confirmed to be a junior synonym of H. maculaticollis based on the results of our analyses of morphological variation, calling song structure and acoustic playback experiments.

Call types of Bigg’s killer whales (Orcinus orca) in western Alaska: using vocal dialects to assess population structure

Bigg’s killer whales (Orcinus orca; i.e. ‘transient’ ecotype), as apex predators, are important to the dynamics of marine ecosystems, but little is known about their population structure in western Alaska. Currently, all Bigg’s killer whales in western Alaska are ascribed to a single broad stock for management under the US Marine Mammal Protection Act. However, recent nuclear microsatellite and mitochondrial DNA analyses indicate that this stock is likely comprised of genetically distinct sub-populations. In accordance with what is known about killer whale vocal dialects in other locations, we used the spatial distribution of group-specific call types to investigate the population structure of Bigg’s killer whales in this part of Alaska. Digital audio recordings were collected from 33 Bigg’s killer whale encounters throughout the Aleutian and Pribilof Islands in the summers of 2001–2007 and 2009–2010. Recorded calls were qualitatively classified into discrete types and then quantitatively described using 12 structural and time-frequency measures. Resulting call categories were validated using a random forest approach. A total of 36 call types and subtypes were identified across the entire study area, and regional patterns of call type use revealed three distinct dialects which correspond to proposed genetic delineations. Our results suggest that there are at least three acoustically and genetically distinct sub-populations in western Alaska, and we present an initial catalogue for this area describing the regional vocal repertoires of Bigg’s killer whale call types.

The Lactate Dehydrogenase Gene LDH-C1, a New Molecular Marker for Phylogenetic Analysis of Salmonid Fishes (Salmoniformes: Salmonidae)

We tested the locus of the nuclear lactate dehydrogenase gene (LDH-C1) as a phylogenetic marker in specimens of 11 salmonid genera (Thymallus, Coregonus, Hucho, Brachymystax, Salmo, Salmothymus, Acantholingua, Parahucho, Salvelinus, Parasalmo, and Oncorhynchus). All the sequences were veraciously clustered according to their taxonomic affiliation at the species and genus levels. It is shown that used complex of characters contains a phylogenetic signal that represents specific information about the phylogenesis process. This allows us to recommend the LDH-C1 locus to specify the phylogeny of salmonids in the combined analysis of several independent nuclear genes and mitochondrial DNA.

179 - Dietary Selenomethionine Supplementation Limits Extracellular Trap Formation and Reduces Lesion Burden in a Mouse Model of Atherosclerosis

Atherosclerosis is a chronic inflammatory disease of the vasculature characterised by the infiltration of activated neutrophils and macrophages at sites of damage within the vessel wall, which contributes to lesion formation and plaque progression. This is in part due to the formation of extracellular traps (ETs) by these invading immune cells, with inhibition of ET formation previously shown to reduce lesion formation. Selenomethionine (SeMet) is the organic form of selenium (Se), an essential trace element that functions in the regulation of the immune response. The aim of this study was to evaluate the effect of dietary SeMet supplementation within normal chow and high fat diet (HFD) fed apolipoprotein E deficient (apoE-/-) mice, as a model of atherosclerosis. Dietary supplementation with 2 mg/kg SeMet (w/w) over 12 weeks resulted in the significant reduction of atherosclerotic plaque formation in aortas from mice fed SeMet compared to controls in both the normal (0.40% vs 1.26%) and HFD (4.70% vs 6.21%) fed cohorts, as assessed by Oil Red O staining of lipid content. Isolation of bone marrow derived cells from the mice and subsequent ex vivo induction of ET formation through stimulation with phorbol 12-myristate 13-acetate (PMA) resulted in markedly less ET formation within the SeMet supplementated cohorts compared to controls as assessed through SYTOX Green staining of extracellular DNA and qPCR analysis of mitochondrial and nuclear genomic DNA within culture supernatants. Together, these data support a novel role for SeMet to mitigate ET formation and reduce atherosclerotic lesion development and highlight the potential beneficial effect of SeMet supplementation as a therapeutic strategy for atherosclerosis.

Isolation of Mitochondrial DNA from Single, Short Hairs without Roots Using Pressure Cycling Technology.

Hairs are commonly submitted as evidence to forensic laboratories, but standard nuclear DNA analysis is not always possible. Mitochondria (mt) provide another source of genetic material; however, manual isolation is laborious. In a proof-of-concept study, we assessed pressure cycling technology (PCT; an automated approach that subjects samples to varying cycles of high and low pressure) for extracting mtDNA from single, short hairs without roots. Using three microscopically similar donors, we determined the ideal PCT conditions and compared those yields to those obtained using the traditional manual micro-tissue grinder method. Higher yields were recovered from grinder extracts, but yields from PCT extracts exceeded the requirements for forensic analysis, with the DNA quality confirmed through sequencing. Automated extraction of mtDNA from hairs without roots using PCT could be useful for forensic laboratories processing numerous samples.

Molecular taxonomy of the Sympetrum vulgatum (Odonata: Libellulidae) complex in the West Palaearctic

The Sympetrum vulgatum (Linnaeus, 1758) complex is composed of the subspecies S. vulgatum vulgatum, S. vulgatum decoloratum (Selys, 1884) and S. vulgatum ibericum Ocharan, 1985 in the West Palaearctic. These taxa have parapatric distributions and noticeable morphological differences in colour and body size, and their taxonomic status is debated. Here we revise the systematics of this group using molecular taxonomy, including molecular analyses of mitochondrial (cytochrome c oxidase subunit I, COI) and nuclear (internal transcribed spacer, ITS1) DNA taking into account known morphological differences. Each subspecies has a unique and differentiated COI haplotype, although divergences among them are low (0.4% maximum uncorrected p-distance). The subspecies are not differentiated by the nuclear marker ITS1. The genetic results for these taxa contrast with the deep divergence of the sister species S. striolatum (Charpentier, 1840). Given current evidence, we propose to maintain the subspecific status of the S. vulgatum complex and hypothesize their biogeographical history. It is likely that the three subspecies became isolated during one of the latest glacial periods, each in a different refugium: S. vulgatum ibericum possibly occupied the Iberian Peninsula, S. vulgatum vulgatum the Balkan Peninsula or territories further east and S. vulgatum decoloratum Anatolia.

Comparison of three comercial kits to the establishment of str genetic profiles on critical samples

Nuclear DNA analysis provides great and crucial information to personal identifications and to kinship analysis. Nevertheless, nuclear DNA from human skeletal remains usually shows several problems related with its scarcity (LTD or Low Template DNA) and its modifications. Such problems compromise its analysis and results interpretation. For this reason, there have been developed different technics to face up to these problems, many of them based on the improvement of STR amplifying kits, for example the amplifying of miniSTRs instead of classical STRs.In the last few years, there have been developments of many of these kits, mainly those ones that concern autosomal STR amplification on critical samples. Through the present study, we would like to evaluate the efficiency of three of them (AmpFℓSTR® MiniFiler™ PCR Amplification Kit, AmpFLSTR® NGM™ PCR Amplification Kit, and PowerPlex® ESX 17 System). For this purpose, there was carried out a STR analysis on 84 skeletal and/or dental samples from 42 skeletal human remains (two samples from each individual) dated back between 2000 and 5000 years of antiquity. Here we discuss the obtained results evaluating the performance of each kit.

Molecular and morphological analyses revealed a cryptic species of dojo loach Misgurnus anguillicaudatus (Cypriniformes: Cobitidae) in Japan.

Although it has been reported that populations of the Japanese dojo loach Misgurnus anguillicaudatus (Cypriniformes: Cobitidae) belong to two distinct mitochondrial (mt)DNA (Type I and Type II), the taxonomic status of the species remains unresolved. To address this question, nuclear DNA and morphological analyses were performed on M. anguillicaudatus population in the Nakaikemi Wetland, where Type I and Type II lineages are sympatric. Results suggest the existence of a cryptic species (Type I) within the Japanese dojo loach.

The systematics and biogeography of African tailorbirds (Cisticolidae: Artisornis) with comment on the choice of Bayesian branch-length prior when analyzing heterogeneous data

The Long-billed Tailorbird (Artisornis moreaui), one of Africa’s rarest birds, has a strikingly disjunct distribution, the origin of which has long puzzled biogeographers. One small population (subspecies moreaui) occurs in sub-montane forest in the East Usambara Mountains, a sky island near the coast of northern Tanzania, and another (subspecies sousae) on Serra Jeci in northwestern Mozambique, 950km away. The African Tailorbird, the putative sister-species of Long-billed Tailorbird, also occurs in the East Usambara Mountains and on Serra Jeci, but in addition occupies all the Eastern Arc Mountain forests between these disjunct sites. Stuart (1981) hypothesized that the two tailorbird distributions could be explained by strong ecological competition, with African Tailorbird populations having eliminated Long-billed Tailorbird populations via competitive exclusion in montane forests between the East Usambara and Serra Jeci. If such competitive exclusion explains these geographic distributions, the co-occurrence of the two species in the East Usambara and at Serra Jeci may be ephemeral, with the status of Long-billed Tailorbird especially in doubt. We sought to (1) determine whether the two species of African tailorbirds are indeed sister-species, and (2) test predictions from Stuart’s (1981) competitive exclusion hypothesis using genetic data. Phylogenetic analyses of our seven gene dataset (3 mtDNA, 4 introns; 4784bp) indeed place these two species together in the genus Artisornis. Instead of finding shallow divergence among African Tailorbird populations and deep divergence between Long-billed Tailorbird populations as expected from Stuart’s hypothesis, we recover deep genetic divergence and geographic structure among populations of both tailorbird species. This result is consistent with long-term co-existence of the two species at East Usambara and Serra Jeci. Observational data from both the East Usambara and Serra Jeci suggest that the two species have diverged in use of forest canopy strata. From a conservation standpoint, our results suggest that extinction of the Long-billed Tailorbird as a function of competition with African Tailorbird is highly unlikely, and should not be viewed as imminent. Threats to its survival are instead anthropogenic, and conservation measures should take this into account. Finally, our empirical results suggest that mis-specification of the branch-length prior in Bayesian analyses of mitochondrial DNA data can have a profound effect on the overall tree-length (sum of branch-lengths), whereas the topology and support values tend to remain more stable. In contrast, mis-specification of the branch-length prior had a lesser impact on all aspects of the nuclear-only DNA analyses. This problem may be exacerbated when mitochondrial and nuclear DNA analyses are combined in a total evidence approach.

Evaluation of the Systematic Status of Geographical Variations in Arcuphantes hibanus (Arachnida: Araneae: Linyphiidae), with Descriptions of Two New Species.

The systematic status of geographical variants of Arcuphantes hibanus Saito, 1992 belonging to the A. longiscapus species group, indigenous to western Honshu and Shikoku, Japan, was evaluated using morphological and molecular data. Two species, A. enmusubi Ihara, Nakano and Tomikawa, sp. nov. and A. occidentalis Ihara, Nakano and Tomikawa, sp. nov., are described, and A. hibanus is redescribed with redefinition of its taxonomic status. These three species are diagnosed by the characteristics of paracymbium, pseudolamella, and epigynal basal part. Phylogenetic trees obtained with mitochondrial cytochrome c oxidase subunit I and 16S rRNA markers showed that the variants are mutually genetically highly diverged. However, the mtDNA phylogenies failed to recover the monophyly of A. hibanus redefined herein. Contrary to the mtDNA phylogenetic analyses, a neighbor-network analysis of nuclear internal transcribed spacer 1 sequences of A. hibanus, A. enmusubi and A. occidentalis spiders showed that each of them forms a cluster. The results of mitochondrial and nuclear DNA analyses in each of the three species are briefly discussed, along with their taxonomic identities.

DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation.

Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array “waves”, and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance.

Molecular Phylogenies indicate a Paleo-Tibetan Origin of Himalayan Lazy Toads (Scutiger)

The Himalaya presents an outstanding geologically active orogen and biodiversity hotspot. However, our understanding of the historical biogeography of its fauna is far from comprehensive. Many taxa are commonly assumed to have originated from China-Indochina and dispersed westward along the Himalayan chain. Alternatively, the “Tibetan-origin hypothesis” suggests primary diversification of lineages in Paleo-Tibet, and secondary diversification along the slopes of the later uplifted Greater Himalaya. We test these hypotheses in high-mountain megophryid anurans (Scutiger). Extensive sampling from High Asia, and analyses of mitochondrial (2839 bp) and nuclear DNA (2208 bp), using Bayesian and Maximum likelihood phylogenetics, suggest that the Himalayan species form a distinct clade, possibly older than those from the eastern Himalaya-Tibet orogen. While immigration from China-Indochina cannot be excluded, our data may indicate that Himalayan Scutiger originated to the north of the Himalaya by colonization from Paleo-Tibet and then date back to the Oligocene. High intraspecific diversity of Scutiger implies limited migration across mountains and drainages along the Himalaya. While our study strengthens support for a “Tibetan-origin hypothesis”, current sampling (10/22 species; 1 revalidated: S. occidentalis) remains insufficient to draw final conclusions on Scutiger but urges comparative phylogeographers to test alternative, geologically supported hypotheses for a true future understanding of Himalayan biogeography.

Taxonomic study on Japanese Salvia (Lamiaceae): Phylogenetic position of S. akiensis, and polyphyletic nature of S. lutescens var. intermedia.

Both Salvia akiensis and S. lutescens (Lamiaceae) are endemic to Japan. Salvia akiensis was recently described in 2014 in the Chugoku (= SW Honshu) region, and each four varieties of S. lutescens distributed allopatrically. Among varieties in S. lutescens, var. intermedia show a disjunctive distribution in the Kanto (=E Honshu) and Kinki (= W Honshu) regions. Recent field studies of S. lutescens var. intermedia revealed several morphological differences between the Kanto and Kinki populations. Here, I evaluated these differences among Salvia lutescens var. intermedia and its allies with morphological analysis and molecular phylogenetic analyses of nuclear ribosomal DNA (internal and external transcribed spacer regions) and plastid DNA (ycf1-rps15 spacer, rbcL, and trnL-F) sequences. Both morphological analysis and molecular phylogenetic analyses showed that S. lutescens var. intermedia from the Kinki region and var. lutescens were closely related to each other. However, var. intermedia from the Kanto region exhibited an association with S. lutescens var. crenata and var. stolonifera, which also grew in eastern Japan, rather than var. intermedia in the Kinki region. These results indicated that S. lutescens var. intermedia is not a taxon with a disjunctive distribution, but a combination of two or more allopatric taxa. Present study also suggested that S. akiensis was most closely related to S. omerocalyx.

Evaluating differential nuclear DNA yield rates and osteocyte numbers among human bone tissue types: A synchrotron radiation micro-CT approach

Molecular human identification has conventionally focused on DNA sampling from dense, weight-bearing cortical bone tissue, typically from femora or tibiae. A comparison of skeletal elements from three contemporary individuals demonstrated that elements with high quantities of cancellous bone yielded nuclear DNA at the highest rates, suggesting that preferentially sampling cortical bone may be suboptimal (Mundorff & Davoren, 2014). Despite these findings, the reason for the differential DNA yields between cortical and cancellous bone tissues remains unknown. The primary goal of this work is to ascertain whether differences in bone microstructure can be used to explain differential nuclear DNA yield among bone tissue types observed by Mundorff and Davoren (2014), with a focus on osteocytes and the three-dimensional (3D) quantification of their associated lacunae. Osteocytes and other bone cells are recognized to house DNA in bone tissue, thus examining the density of their lacunae may explain why nuclear DNA yield rates differ among bone tissue types. Lacunae were visualized and quantified using synchrotron radiation-based micro-Computed Tomographic imaging (SR micro-CT). Volumes of interest (VOIs) from cortical and cancellous bone tissues (n=129) were comparatively analyzed from the three skeletons sampled for Mundorff and Davoren’s (2014) study. Analyses tested the primary hypothesis that the abundance and density of osteocytes (inferred from their lacunar spaces) vary between cortical and cancellous bone tissue types. Results demonstrated that osteocyte lacunar abundance and density vary between cortical and cancellous bone tissue types, with cortical bone VOIs containing a higher lacunar abundance and density. We found that the osteocyte lacunar density values are independent of nuclear DNA yield, suggesting an alternative explanation for the higher nuclear DNA yields from bones with greater quantities of cancellous bone tissue. The use of SR micro-CT allowed for a scale of analysis that revealed a high range of variation in lacunar abundance in both tissue types. Moreover, high-resolution SR micro-CT imaging revealed potential soft tissue remnants within marrow spaces not visible macroscopically. It is hypothesized that soft tissue remnants observed among the trabeculae of skeletal elements with high quantities of cancellous bone tissue are responsible for the high nuclear DNA yields. These findings have significant implications for bone-sample selection for nuclear DNA analysis in a forensic context when skeletal remains are recovered from the ground surface.

Genetic variability and health of Norway spruce stands in the Regional Directorate of the State Forests in Krosno

Abstract The study was conducted in 2015 in six spruce stands situated in different forest districts administratively belonging to the Regional Directorate of State Forests in Krosno. Each spruce population was represented by 30 trees and assessed in terms of their current health status. Genetic analyses were performed on shoot samples from each tree using nine nuclear DNA markers and one mitochondrial DNA marker (nad1). The health status of the trees was described according to the classification developed by Szczepkowski and Tarasiuk (2005) and the correlation between health classes and the level of genetic variability was computed with STATISTICA (α = 0.05). Nuclear DNA analyses revealed a low level of genetic variability among spruce populations (only 3% of the total genetic variation (F ST = 0.028) and a high variability within populations (97%). The total heterozygosity in all stands (H T) was calculated as 0.646. Based on UPGMA analysis, the most genetically similar populations are spruce stands in the Bieszczady National Park and the Ustrzyki Dolne Forest District, which have the smallest genetic divergence of all populations (D N = 0.0165). Our analysis of the mitochondrial gene nad1 revealed the presence of six different haplotypes ‘a’, ‘a1’, ‘b’, ‘c’, ‘d’ and ‘d1’. Comprising 56% of all haplotypes, ‘a’ was the most common showing a predominant impact on spruce migration from the Carpathian area. The analysis based on mitochondrial markers (by Nei) revealed a heterozygosity of 0.525. Based on the observations of disease symptoms, 29% of the trees belong to health class 1, 30% to class 2, 28% to class 3 and class 4 contains 13% of trees. The comparison between health status and the level of genetic variation in the analysed stands showed a positive correlation. Spruce stands with better health were also characterised by a greater degree of genetic variability. Since most of the investigated spruce populations shared the mitochondrial haplotype ‘a’, we have ascertained their Hercynian–Carpathian origin. Only one stand (Cisna) had a high frequency (43.3%) of the Nordic haplotype ‘c’ suggesting that this provenance is derived from the Baltic post-glacial refugium of P. abies in europe.

Population Structure of Alligator Gar in a Gulf Coast River: Insights from Otolith Microchemistry and Genetic Analyses

AbstractGrowing interest in the Alligator Gar Atractosteus spatula among anglers and fishery managers has inspired efforts to better manage populations. Successful management requires identifying population structure and understanding the distribution of stocks and associated differences in life history. This is particularly important in river systems along the coast of the Gulf of Mexico, where transitions from freshwater rivers to saltwater bays provide the potential for life history diversification. We used otolith microchemistry and genetics to assess population structure of Alligator Gars in the Guadalupe River–San Antonio Bay system, Texas. Lifetime Sr:Ca revealed three, distinct life histories that differed in prevalence across the system. River‐resident fish (i.e., fish exclusive to freshwater) were present throughout the river but were most common in the uppermost river reach (74% of upper river fish). Transient fish that used both river and bay habitats were also found throughout the river but were most prevalent in the lowermost river reach (66% of lower river fish) and bay (91% of bay fish). Bay residents (i.e., fish exclusive to salt water) were detected but comprised only 9% of bay fish. Haplotype diversity based on mitochondrial DNA was lowest in the upper river, indicating limited gene flow compared with the lower river and bay. Similarly, nuclear DNA analyses indicated nonrandom mating between fish from the upper river, lower river, and bay. The differences in Alligator Gar movement and genetics along the river–bay continuum suggest the presence of a river resident stock that predominates the upper river, and a transient stock that predominates the lower river and bay. Therefore, a local‐scale management approach, consistent with the spatial partitioning between stocks, would conserve life history and genetic diversity within the system and provide opportunities to meet the needs of a diverse angling constituency. Understanding how population dynamics differ between stocks is needed to develop appropriate fishery management objectives and corresponding regulations for Alligator Gar.Received May 20, 2016; accepted December 2, 2016 Published online February 27, 2017

Unpacking boxes: Integration of molecular, morphological and ecological approaches reveals extensive patterns of reticulate evolution in box eucalypts

Reticulate evolution by hybridization is considered a common process shaping the evolution of many plant species, however, reticulation could also be due to incomplete lineage sorting in biodiverse systems. For our study we selected a group of closely related plant taxa with contrasting yet partially overlapping geographic distributions and different population sizes, to distinguish between reticulated patterns due to hybridization and incomplete lineage sorting. We predicted that sympatric or proximal populations of different species are more likely to have gene flow than geographically distant populations of the same widespread species. Furthermore, for species with restricted distributions, and therefore, small effective population sizes, we predicted complete lineage sorting. Eastern grey box eucalypt species (Eucalyptus supraspecies Moluccanae) provide an ideal system to explore patterns of reticulate evolution. They form a diverse, recently evolved and phylogenetically undefined group within Eucalyptus, with overlapping morphological features and hybridization in nature. We used a multi-faceted approach, combining analyses of chloroplast and nuclear DNA, as well as seedling morphology, flowering time and ecological spatial differentiation in order to test for species delimitation and reticulate evolution in this group. The multiple layers of results were consistent and suggested a lack of monophyly at different hierarchical levels due to multidirectional gene flow among several species, challenging species delimitation. Chloroplast and nuclear haplotypes were shared among different species in geographic proximity, consistent with hybridization zones. Furthermore, species with restricted distributions appeared better resolved due to lineage sorting in the absence of hybridization. We conclude that a combination of molecular, morphological and ecological approaches is required to disentangle patterns of reticulate evolution in the box eucalypts.

Population genetic structure of the endangered Sierra Nevada yellow-legged frog (Rana sierrae) in Yosemite National Park based on multi-locus nuclear data from swab samples

The mountain yellow-legged species complex (Rana sierrae and Rana muscosa) has declined precipitously in distribution and abundance during the last century. The two primary threats are chytrid epidemic-associated population collapses and predation from the introduction of non-native trout. Widespread declines have occurred throughout the ranges of these species, including populations of R. sierrae in Yosemite National Park. A clear picture of genetic structure of remaining Yosemite R. sierrae populations is critical to short-term management and conservation. We conducted a population genetics study that included samples from 23 geographic sites distributed throughout the range of R. sierrae in Yosemite NP. We used minimally-invasive swab samples to collect genetic data from mitochondrial and nuclear DNA via sequencing (43 transcriptome-derived markers) and analyzed the distribution of genetic variation in a geographic context. Our mtDNA analysis partially confirmed previous results suggesting that two haplotype groups occur in Yosemite: one haplotype group contained high bootstrap support for monophyly while the other did not. However, increased geographic sampling demonstrated that the two haplotypes are not completely geographically partitioned into the two main drainages (Merced drainage and Tuolumne drainage) as previously postulated. Our nuclear DNA analysis revealed a general pattern of genetic isolation by distance, where genetic differentiation was correlated with geographic distance between sites. In addition, our analyses suggested that three clusters of genetically cohesive sites occur in the study area. Understanding population genetic patterns of variability will inform management strategies such as translocations, reintroductions, and monitoring for this endangered frog. Lastly, our next generation sequencing enabled approach allowed us to obtain multi-locus data from minimally-invasive swab samples. Thus researchers can now leverage extensive archives of swab samples (initially collected for pathogen testing) to study host genetics in previously surveyed amphibian populations.

Analysis to Estimate Genetic Variations in the Idarubicin-Resistant Derivative MOLT-3.

Gene alterations are a well-established mechanism leading to drug resistance in acute leukemia cells. A full understanding of the mechanisms of drug resistance in these cells will facilitate more effective chemotherapy. In this study, we investigated the mechanism(s) of drug resistance in the human acute leukemia cell line MOLT-3 and its idarubicin-resistant derivative MOLT-3/IDR through complete mitochondrial and nuclear DNA analyses. We identified genetic differences between these two cell lines. The ND3 mutation site (p.Thr61Ile) in the mitochondrial DNA sequence was unique to MOLT-3/IDR cells. Moreover, we identified five candidate genes harboring genetic alterations, including GALNT2, via CGH array analysis. Sequencing of the GALNT2 exon revealed a G1716K mutation present within the stop codon in MOLT-3/IDR cells but absent from MOLT-3 cells. This mutation led to an additional 18 amino acids in the protein encoded by GALNT2. Using real-time PCR, we determined an expression value for this gene of 0.35. Protein structure predictions confirmed a structural change in GALNT2 in MOLT-3/IDR cells that corresponded to the site of the mutation. We speculate that this mutation may be related to idarubicin resistance.

Interspecific Pair-Bonding and Hybridization In Warbling (Vireo gilvus) and Red-Eyed (Vireo olivaceus) Vireos

ABSTRACT We present field observations and genetic evidence of interspecific mate-selection and nesting resulting in successful hybridization between the Warbling Vireo (Vireo gilvus) and the Red-eyed Vireo (V. olivaceus). Two interspecific nesting pairs were found in western North America, each of which produced two fledglings. We examined the DNA of a salvaged fledgling from the nest of a mixed-species pair in Reno, Nevada, using mitochondrial and nuclear DNA analyses. The mitochondrial DNA of this individual indicated a Red-eyed Vireo mother, while its nuclear DNA was heterozygous and consistent with a hybrid origin. Phenotypic traits of the fledglings from the nest of a second mixed-species pair near Juneau, Alaska supported heterospecific parents for those offspring as well. We discuss these findings in the context of reproductive barriers that appear to result in little or no admixture across broad areas of sympatry in these two species.