nDNA Markers Research Articles

Re-establishment of Aechmea laxiflora (Bromeliaceae, Bromelioideae) based on morphological and phylogenetic evidence, with notes on its geographical distribution

Background: Previous phylogenetic analyses suggested that Aechmea bracteata is not a monophyletic species, being each variety an individual lineage within the Aechmea bracteata complex.
 Hypothesis: A phylogenetic analysis based on molecular data and a morphological analysis will provide evidence to support the taxonomic recognition of A. bracteata var. pacifica as a distinct species.
 Studies species: A. bracteata var. bracteata, A. bracteata var. pacifica and related species.
 Study site and dates: Mexico to Northern South America; 17 populations from Southeastern and Western Mexico. Analyses were performed between 2021 and 2023.
 Methods: A linear morphometric analysis was conducted with 85 herbarium specimens testing 28 quantitative and three qualitative variables. Potential diagnostic traits were reviewed in living specimens. A phylogenetic analysis was performed with two nDNA markers (ETS and g3pdh) and one cpDNA marker (trnL-F). A total of 373 records were projected into biogeographical provinces of the Neotropics.
 Results: The morphometric analysis allowed to separate each variety into a group, also, nine of the evaluated traits resulted statistically significant through a univariate analysis. Five additional diagnostic traits from the inflorescence and leaves were recognized. According to nDNA both taxa present reciprocal monophyly, however, cpDNA groups A. bracteata var. pacifica with Central American species, showcasing a potential hybrid origin. Geographically, the Sierra Madre del Sur acts as a barrier between both taxa.
 Conclusions: Due to the gathered evidence is possible to recognize A. bracteata var. pacifica as an endemic species to Mexico, reestablishing the basionym A. laxiflora.

Highly divergent sympatric lineages of Leptotila verreauxi (Aves: Columbidae) suggest a secondary contact area in the Isthmus of Tehuantepec, Mexico

Due to a complex geological and biotic history, the Isthmus of Tehuantepec (IT), has been long recognized as a driver for the evolutionary divergence of numerous lowland and highland taxa. Widely distributed in the lowlands of the American continent, the White-Tipped Dove (Leptotila verreauxi) is a polytypic species with 13 recognized subspecies. Four of these have been recorded in Mexico, and the distribution of three abuts at the IT, suggesting a contact zone. To estimate phylogenetic patterns, divergence times and genetic differentiation, we examined two mtDNA (ND2 and COI) and one nDNA (β-fibint 7) markers. We also used correlative ecological niche models (ENM) to assess whether ecological differences across the IT may have acted as a biogeographical boundary. We estimated paleodistributions during the Middle Holocene, Last Glacial Maximum and Last Interglacial, to evaluate the influence of climate changes on the distribution and demographic changes. Our results showed genetically distinct lineages that diverged approximately 2.5 million years ago. Climatic and ecological factors may have played a dual role in promoting differentiation, but also in the formation of a secondary contact zone in the southern IT. Our ecological niche comparisons indicated that the ecological niche of sympatric lineages at the IT are not identical, suggesting niches divergence; in addition, environmental niche models across the region indicated no abrupt biogeographic barriers, but the presence of regions with low suitability. These results suggest that genetic differentiation originated by a vicariant event probably related to environmental factors, favored the evolution of different ecological niches. Also, the absence of a biogeographic barrier but the presence of less suitable areas in the contact regions, suggest that secondary contact zones may be also maintained by climatic factors for the eastern group, but also by biotic interactions for the western group.

Ecological and physiological analysis of immune reactions of <i>Pelophylax ridibundus</i> and <i>P. lessonae</i> (Amphibia: Ranidae) in anthropogeneously transformed territories

The purpose of the work is to assess the state of Anura populations living in an anthropo-genic territory according to a set of indicators of the body’s immune homeostasis. The objects of the study were Pelophylax ridibundus (Pallas, 1771) and P. lessonae (Camerano, 1882), living in the natural populations of reservoirs in Nizhny Novgorod. Priority chemical pollutants of the water bodies were determined by spectrophotometry. Species were identified using a multiplex PCR test system. The identification results were confirmed by sequencing of the mitochondrial ND2 gene and a fragment of the SAI protein. We counted the numbers of erythrocytes and leukocytes, determined the leukocyte profile and the level of immune complexes in all frogs. An excess of the water quality standard for the content of heavy metals was revealed in all the water bodies. Molecular genetic diagnostics showed the presence of both “pure” P. ridibundus and individuals with introgressive mtDNA of the Anatolian form of the lake frog (P. cf. bedriagae) in the sample of lake frogs. In the sample of pond frogs, all studied individuals had only species-specific mt- and nDNA markers of P. lessonae. P. ridibundus differed from P. lessonae by an increased content of erythrocytes, neutrophils, basophils, small immune complexes, and a reduced content of lymphocytes. The revealed changes in the immunohematological parameters of green frogs were caused by the complex henotoxic effect of pollutants in the water bodies. A decrease in the lymphocytes/eosinophils ratio index was shown with an increase in the concentration of nitrites, an increased activity of humoral immune responses in conditions of sulfate pollution of the aquatic environment, an increase in the proportion of myelocytes in the blood of frogs with an increased concentration of manganese and nitrates in water. Under conditions of environmental stress, the blood regulatory systems of frogs reflected a stress-induced reaction, which was more pronounced in the body of lake frogs compared to pond ones.

Unravelling morphological overlap of the rock-dwelling snails Pyramidula saxatilis (Hartmann, 1842) and P. pusilla (Vallot, 1801)

ABSTRACT Pyramidula saxatilis and P. pusilla are two Central European rock-dwelling snail species that frequently co-occur and show substantial overlap in overall shell morphology. The two species can be separated from each other by differences in mitochondrial and nuclear DNA (mtDNA and nDNA) sequences. Recent studies have not shown consistent differences between these species in shell shape but have suggested possible species-specific features in shell microstructure. We investigated this issue by studying variation in the microsculpture of the upper shell surface (based on five shells per population from a total of nine populations for each species) and by analysing the geometric morphometrics of shell shape (based on 51 individuals and 12 populations of P. saxatilis and 54 individuals and 14 populations of P. pusilla), with species identifications based on mtDNA and nDNA markers. While geometric morphometrics and canonical variance analysis did reveal some statistical differences in overall shell shape, these differences were too subtle to be consistently detected by the eye. However, the density and development of growth ridges on the upper shell surface of the two species were found to be statistically different, with P. saxatilis having denser and very regular ridges. As a final verification of the utility of shell miscrosculpture, we were able to separate these two species even in two mixed populations with highly overlapping shell phenotypes, the shell-based identifications being confirmed by cytochrome c oxidase subunit I sequence data for all of the collected individuals. We therefore recommend using shell microsculpture to distinguish these two species.

Emesis planeca n. comb. (Lepidoptera: Riodinidae): a new combination revealed by molecular evidence with a description of its morphological variation.

We transfer Apodemia planeca R. de la Maza E. J. de la Maza E. 2017 to Emesis as Emesis planeca (R. de la Maza E. J. de la Maza E.) n. comb. based on phylogenetic hypotheses estimated with one mtDNA and two nDNA markers. This is a rare and poorly known metalmark, endemic to the central region of Balsas Basin in Michoacán, Mexico, originally described from material collected 23 years ago. Here we analyze new specimens from the type locality not included in the original description. With this new material, we describe the morphological variation of adults, including male and female genitalia. This variation is then discussed and compared with the original description. Emesis planeca n. comb. is restricted to the Tropical Deciduous Forest and the adults fly only in the dry season. Due to the spatial, temporal, and ecological rareness of Emesis planeca n. comb., considering it has not been collected in more than two decades and that the Tropical Deciduous Forest is one of the most threatened habitats in Mexico; we propose assigning a protection status to this species.

Evidence of strong population bottleneck in genetics of endangered Brazilian Merganser (Mergus octosetaceus)

The Brazilian Merganser is one of the rarest birds of South America with three known remnant populations in Brazil and only 250 individuals existing in the wild. Thus, evaluating the genetic variability within the remaining population is essential to its conservation. This study assessed genetic diversity of remnant populations by mtDNA (Dloop and cytb) and nDNA (MUSK 13 intron, five microsatellites) markers, giving support to management and conservation efforts. Seventy-one samples were collected during eight years from Chapada dos Veadeiros National Park (PNCV), Serra da Canastra National Park (PNSC), and Alto do Paranaiba region (APR). Cytochrome b, Dloop, and MUSK intron showed only one haplotype, indicating a loss of genetic variability for the Brazilian Merganser. The microsatellites confirmed low genetic variability (1–4 alleles). Despite the low variability observed, it was possible to infer minor differentiation between remnant populations, with PNCV locale being the most differentiated subgroup, due to its being isolated by greater distance from the others. Low genetic diversity observed in Brazilian Merganser, is expected to be found in Critically Endangered species, due to small population size, being result of high inbreeding rates, and gene drift effects. Thus, our genetic data have confirmed the extremely critical status of the Brazilian Merganser, making it necessary to create an immediate recovery program for their populations, and develop a management plan to integrate and preserve all remnant populations to avoid the species extinction.

Genetic Relationship between Lake and Marine Forms of Pacific Herring Clupea pallasii

Polymorphism in the variable fragment of the mtDNA control region (D-loop, 373 bp) and four microsatellite loci in 683 specimens of the Pacific herring Clupea pallasii sampled at different locations from the Asian part of range has been studied. It is shown that the Pacific herring lake forms inhabiting the three lakes on Sakhalin Island (Ainskoe) and in the Kamchatka Peninsula (Vilyuy and Nerpiche) have significantly diverged from the marine forms according to the data on the mtDNA and nDNA markers. The genetic variations among the herring forms inhabiting different lakes (FST = 0.034−0.066) and between the lake herring forms and marine herring forms (FST = 0.004−0.055) are revealed. The level of the genetic differentiation among the lake herring forms is much higher than that among the samples of the marine herring forms, which may be explained by the founder effect. Evolution of the lake forms occurred along with the geological processes producing the lakes.

Invasion origin, rapid population expansion, and the lack of genetic structure of cotton bollworm (Helicoverpa armigera) in the Americas.

In 2013, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) was officially declared as present in Brazil and, after two years, the species was detected in the Caribbean and North America. Information on genetic features and accurate distribution of pests is the basis for agricultural protection policies. Furthermore, such knowledge is imperative to develop control strategies, understand the geographical range, and genetic patterns of this species in the Americas. Here, we carried out the widest sampling of H. armigera in the South American continent and Puerto Rico, after we estimated the diversity, demographic parameters, and genetic structure. The Internal Transcribed Spacer 1 (ITS1) nuclear marker was used to investigate the presence of putative hybrids between H. armigera and H. zea, and they were observed at a frequency of 1.5%. An ABC analysis, based in COI gene fragment, suggested Europe as the origin of South America specimens of H. armigeraand following a movement northward through the Caribbean. Three mtDNA genes and three nDNA markers revealed high genetic diversity distributed without the defined population structure of H. armigera in South America. Most of the genetic variation is within populations with a multidirectional expansion of H. armigera among morphoclimatic regions. High genetic diversity, rapid population expansion, and hybridization have implications for pest management since they suggest that adaptive alleles are spread through wide areas in South America that favor rapid local adaptation of H. armigera to new and disturbed environments (e.g., in agricultural areas).

Phylogeny, phylogeography and hybridization of Caucasian barbels of the genus Barbus (Actinopterygii, Cyprinidae)

Phylogenetic relationships and phylogeography of six species of Caucasian barbels, the genus Barbus s. str., were studied based on extended geographic coverage and using mtDNA and nDNA markers. Based on 27 species studied, matrilineal phylogeny of the genus Barbus is composed of two clades – (a) West European clade, (b) Central and East European clade. The latter comprises two subclades: (b1) Balkanian subclade, and (b2) Ponto-Caspian one that includes 11 lineages mainly from Black and Caspian Sea drainages. Caucasian barbels are not monophyletic and subdivided for two groups. The Black Sea group encompasses species from tributaries of Black Sea including re-erected B. rionicus and excluding B. kubanicus. The Caspian group includes B. ciscaucasicus, B. cyri (with B. goktschaicus that might be synonymized with B. cyri), B. lacerta from the Tigris-Euphrates basin and B. kubanicus from the Kuban basin. Genetic structure of Black Sea barbels was influenced by glaciation-deglaciation periods accompanying by freshwater phases, periods of migration and colonization of Black Sea tributaries. Intra- and intergeneric hybridization among Caucasian barbines was revealed. In the present study, we report about finding of B. tauricus in the Kuban basin, where only B. kubanicus was thought to inhabit. Hybrids between these species were detected based on both mtDNA and nDNA markers. Remarkably, Kuban population of B. tauricus is distant to closely located conspecific populations and we consider it as relic. We highlight revealing the intergeneric hybridization between evolutionary tetraploid (2n = 100) B. goktschaicus and evolutionary hexaploid (2n = 150) Capoeta sevangi in Lake Sevan.

Differential Rates of Male Genital Evolution in Sibling Species of Drosophila

Fil: Stefanini, Manuel Ignacio. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Instituto de Ecologia, Genetica y Evolucion de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecologia, Genetica y Evolucion de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Ecologia, Genetica y Evolucion; Argentina

Analysis of sequence divergence in redfin (Cypriniformes, Cyprinidae, Tribolodon) based on mtDNA and nDNA markers with inferences in systematics and genetics of speciation

To clarify relationship of species of the genus Tribolodon in the Russian part of their distribution ranges, two mitochondrial markers (Co-1 and Cyt-b), a nuclear marker (Rho), and a gene marker of rDNA internal transcribed spacer (ITS-1,2) were used. Depending on the marker, different numbers of species groups were detected by the ABGD method, but in combination with the analysis of phylograms, these data generally support the known species clusters and regional intraspecies groups. A complex analysis of sequences from three redfin species within the area of the study, based on four marker genes and using the methods of molecular phylogenetics, ordination of genetic distances, recombinant analysis, and population genetic approaches, has revealed clusters of three commonly recognized species, regional intraspecific groups or individuals of local populations, and few hybrid individuals. DNA barcoding technique proved to be efficient with the use of two mtDNA markers: Co-1 and Cyt-b. It has been found that analysis of insertions and substitutions within the ITS-1,2 gene marker is also suitable for identification of Tribolodon species. Results of the studies of local groups do not confirm a sufficient level of differences for defining any new taxa of a species rank in the genus Tribolodon.

Genetic differentiation of the Schizothorax species complex (Cyprinidae) in the Nujiang River (upper Salween)

Phenotypically diverse species from recently evolved groups always share allele/haplotype due to insufficient differentiation in the early process. In this study, we performed population genetics analyses using sequences from the mitochondrial cytochrome b gene, and two nuclear genes to investigate the genetic differentiation of the closely related Schizothorax species complex, comprising a group of alpine fish living in the Nujiang River. The results from both mtDNA and nDNA markers revealed relatively low but pronounced genetic differentiation among the three Schizothorax species, i.e., Schizothorax gongshanensis, S. lissolabiatus, and S. nukiangensis. However, haplotype sharing was frequently occurred among the three species. Divergence time estimation suggested the last glaciation on the Tibetan Plateau (0.075–0.01 Ma) might drive the divergence of the species complex. Gene flow might contribute to the haplotype sharing between S. gongshanensis and S. lissolabiatus, and between S. gongshanensis and S. nukiangensis, whereas retention of ancestral polymorphisms seemed to be a better explanation of the haplotype sharing between S. lissolabiatus and S. nukiangensis. In addition, S. lissolabiatus populations should obtain more protection in the future because of their low genetic diversity and habitat fragmentation. In summary, our study assesses genetic differentiation among the three closely related Schizothorax species and explores the possible driving forces for their differentiation.

Cytonuclear Discordance, Cryptic Diversity, Complex Histories, and Conservation Needs in Vietnamese Bent-toed Geckos of the Cyrtodactylus irregularis Species Complex

Examples of cytonuclear discordance (CD), i.e. differing branching patterns from mitochondrial and nuclear DNA data, are increasing. This raises concerns about combining data from the two genomes for the purpose of reconstructing phyletic history. Because of this, we explore the efficacy of DNA barcoding based on the gene cytochrome c oxidase subunit I ( COI ; Entrez COX1 ) from the perspective of two nuclear DNA (nDNA) loci: RPL35 and RAG1 . Our analyses use 164 samples of Vietnamese bent-toed geckos of the Cyrtodactylus irregularis species complex (Reptilia: Gekkonidae) collected from 24 localities and 306 new sequences. DNA barcoding infers the presence of ten described species and identifies at least 11 unknown lineages from different areas. K2P genetic distances between sites average 16.6 ± 4.6%. Phylogenetic analyses of nuclear DNA data resolve two strongly supported main clades that correspond with northern and southern groups of geckos and presence or absence of enlarged fermoral scale rows. The matrilineal genealogy and nDNA phylogeny are concordant in resolving major lineages, yet trees derived from the two sets of data, and a combined mtDNA-nDNA dataset, are significantly incompatible (AU test: p < 0.01). The extent of CD precludes combining the two genomes for a single analysis. Analyses of both genomes suggest the presence of at least five undescribed cryptic species. Geckos at one site, Ta Kou Mountain, have a complex history involving the unification of two divergent matrilines (K2P = 10.2%). Analyses of nDNA markers alone does not recover this event because gene flow has erased the historical signal. Finally, the independent and geographically isolated lineages face a battle for survival owing to continued habitat destruction. Urgent conservation planning and implementation is necessary for many sites.

Ecomorphological adaptation in three mudskippers (Teleostei: Gobioidei: Gobiidae) from the Persian Gulf and the Gulf of Oman

We hypothesise that the body shapes of three mudskipper species (Boleophthalmus dussumieri, Periophthalmus waltoni, and Scartelaos tenuis) are ecomorphological adaptations to different epi- and infaunal habitats. We investigated: (i) the association between burrow density and selected ecological variables; (ii) the phylogenetic relationships among these species, based on two mtDNA and one nDNA markers; (iii) their geometric morphometrics and ancestral shape reconstructions, based on two-dimensional landmark configurations; and (iv) their body surface-to-volume ratios (SAV), based on a geometric model. Boleophthalmus dussumieri and S. tenuis are more closely related, and more elongated than P. waltoni; the body of S. tenuis also has a higher SAV, and its elongated shape appears to be a divergent trait. P. waltoni is found in a wide range of conditions, including vegetated habitats; B. dussumieri is associated with low-energy conditions and fine unvegetated deposits; and S. tenuis is associated with high-energy conditions and coarser, non-cohesive sediments. These results suggest that body elongation in Scartelaos species is a divergent locomotory adaptation to marine habitats characterised by semi-liquid and less cohesive sediments. The more compact body shape of Periophthalmus species is hypothesised to have evolved from a gobionelline ancestor, and is proposed to be a preadaptation to subaerial locomotion in semi-terrestrial habitats.

Genetic characterization of Amur sturgeon Acipenser schrenckii and its hybrid caught around Hokkaido

Genetic characterization was performed in five individuals of wild Amur sturgeon Acipenser schrenckii, and/or its presumed hybrid caught around Hokkaido, using a mitochondrial DNA (mtDNA) marker and two markers of nuclear DNA (nDNA). Genetic analyses indicated that two of the five fish had the mtDNA haplotype of Kaluga, Huso dauricus, whereas the nDNA markers indicated signs for both A. schrenckii and H. dauricus genotypes, referring to a hybrid origin. The other three fish were plausibly pure A. schrenckii. The results indicated the importance of combined usage of mtDNA and nDNA markers for correct species identification in sturgeon.

Discomark: nuclear marker discovery from orthologous sequences using draft genome data.

High-throughput sequencing has laid the foundation for fast and cost-effective development of phylogenetic markers. Here we present the program discomark, which streamlines the development of nuclear DNA (nDNA) markers from whole-genome (or whole-transcriptome) sequencing data, combining local alignment, alignment trimming, reference mapping and primer design based on multiple sequence alignments to design primer pairs from input orthologous sequences. To demonstrate the suitability of discomark, we designed markers for two groups of species, one consisting of closely related species and one group of distantly related species. For the closely related members of the species complex of Cloeon dipterum s.l. (Insecta, Ephemeroptera), the program discovered a total of 78 markers. Among these, we selected eight markers for amplification and Sanger sequencing. The exon sequence alignments (2526 base pairs) were used to reconstruct a well-supported phylogeny and to infer clearly structured haplotype networks. For the distantly related species, we designed primers for the insect order Ephemeroptera, using available genomic data from four sequenced species. We developed primer pairs for 23 markers that are designed to amplify across several families. The discomark program will enhance the development of new nDNA markers by providing a streamlined, automated approach to perform genome-scale scans for phylogenetic markers. The program is written in Python, released under a public licence (GNU GPL version 2), and together with a manual and example data set available at: https://github.com/hdetering/discomark.

No more time to stay 'single' in the detection of Anisakis pegreffii, A. simplex (s. s.) and hybridization events between them: a multi-marker nuclear genotyping approach.

A multi-marker nuclear genotyping approach was performed on larval and adult specimens of Anisakis spp. (N = 689) collected from fish and cetaceans in allopatric and sympatric areas of the two species Anisakis pegreffii and Anisakis simplex (s. s.), in order to: (1) identify specimens belonging to the parental taxa by using nuclear markers (allozymes loci) and sequence analysis of a new diagnostic nuclear DNA locus (i.e. partial sequence of the EF1 α-1 nDNA region) and (2) recognize hybrid categories. According to the Bayesian clustering algorithms, based on those markers, most of the individuals (N = 678) were identified as the parental species [i.e. A. pegreffii or A. simplex (s. s.)], whereas a smaller portion (N = 11) were recognized as F1 hybrids. Discordant results were obtained when using the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) of the internal transcribed spacer (ITS) ribosomal DNA (rDNA) on the same specimens, which indicated the occurrence of a large number of 'hybrids' both in sympatry and allopatry. These findings raise the question of possible misidentification of specimens belonging to the two parental Anisakis and their hybrid categories derived from the application of that single marker (i.e. PCR-RFLPs analysis of the ITS of rDNA). Finally, Bayesian clustering, using allozymes and EF1 α-1 nDNA markers, has demonstrated that hybridization between A. pegreffii and A. simplex (s. s.) is a contemporary phenomenon in sympatric areas, while no introgressive hybridization takes place between the two species.

Effect of oceanic straits on gene flow in the recently endangered little brown bat (Myotis lucifugus) in maritime Canada: implications for the spread of white-nose syndrome

White-nose syndrome is rapidly spreading in eastern North America, causing mass mortality of hibernating bats. We characterized levels of genetic diversity and population structure of the little brown bat (Myotis lucifugus (Le Conte, 1831)) in eastern Canada to infer the extent to which oceanic straits may be barriers to movement. To quantify metrics of gene flow and infer movement dynamics, we genotyped 679 M. lucifugus at nine nuclear microsatellites (nDNA) and sequenced a portion of the mitochondrial DNA (mtDNA). We found high levels of genetic diversity and little population structure, with ≈13-fold higher differentiation of mtDNA than nDNA markers, suggesting that structuring patterns largely result from female philopatry. Discriminant analysis of principle components suggested that the subtle underlying structure was not concordant with sampling site. Regional differentiation (FST, Dest, Mantel test residuals) is mostly consistent with genetic isolation by distance. However, samples from Newfoundland showed genetic differentiation over and above the effects of distance, lower levels of genetic diversity, and less genetic connectivity with other sampled regions. Despite this, oceanic straits in the Gulf of Saint Lawrence do not appear to create an impenetrable barrier to movement, therefore it may be possible for white-nose syndrome to spread to Newfoundland.

Identification of the Mislabeled Breast Cancer Samples by Mitochondrial DNA Haplotyping

The task to identify whether an archival malignant tumor specimen had been mislabeled or interchanged is a challenging one for forensic genetics. The nuclear DNA (nDNA) markers were affected by the aberration of tumor cells, so they were not suitable for personal identification when the tumor tissues were tested. In this study, we focused on a new solution - mitochondrial single nucleotide polymorphism (mtSNP) haplotyping by a multiplex SNaPshot assay. To validate our strategy of haplotyping with 25 mtSNPs, we analyzed 15 pairs of cancerous/healthy tissues taken from patients with ductal breast carcinoma. The haplotypes of all the fifteen breast cancer tissues were matched with their paired breast tissues. The heteroplasmy at 2 sites, 14783A/G and 16519C/T was observed in one breast tissue, which indicated a mixture of related mitochondrial haplotypes. However, only one haplotype was retained in the paired breast cancer tissue, which could be considered the result of proliferation of tumor subclone. The allele drop-out and allele drop-in were observed when 39 STRs and 20 tri-allelic SNPs of nDNA were applied. Compared to nDNA markers applied, 25 mtSNPs were more stable without interference from aberrance of breast cancer. Also, two cases were presented where the investigation of haplotype with 25 mtSNPs was used to prove the origin of biopsy specimen with breast cancer. The mislabeling of biopsy specimen with breast cancer could be certified in one case but could not be supported in the other case. We highlight the importance of stability of mtSNP haplotype in breast cancer. It was implied that our multiplex SNaPshot assay with 25 mtSNPs was a useful strategy to identify mislabeled breast cancer specimen.

Systematics, biogeography, and character evolution of Sparganium (Typhaceae): Diversification of a widespread, aquatic lineage

Sparganium (Typhaceae) is a genus of aquatic monocots containing ±14 species, with flowers aggregated in unisexual, spherical heads, and habit ranging from floating to emergent. Sparganium presents an opportunity to investigate diversification, character evolution, and biogeographical relationships in a widespread temperate genus of aquatic monocots. We present a fossil-calibrated, molecular phylogeny of Sparganium based on analysis of two chloroplast and two nuclear markers. Within this framework, we examine character evolution in both habit and stigma number and infer the ancestral area and biogeographic history of the genus. • Sequence data from two cpDNA and two nDNA markers were analyzed using maximum parsimony, maximum likelihood, and Bayesian inference. We used the program BEAST to simultaneously estimate phylogeny and divergence times, S-DIVA and Lagrange for biogeographical reconstruction, and BayesTraits to examine locule number and habit evolution. • Two major clades were recovered with strong support: one composed of S. erectum and S. eurycarpum; and the other containing all remaining Sparganium. We realigned the subgenera to conform to these clades. Divergence time analysis suggests a Miocene crown origin but Pliocene diversification. Importantly, the floating-leaved habit has arisen multiple times in the genus, from emergent ancestors-contrary to past hypotheses. • Cooling trends during the Tertiary are correlated with the isolation of temperate Eurasian and North American taxa. Vicariance, long-distance dispersal, and habitat specialization are proposed as mechanisms for Sparganium diversification.