Is it possible to identify habitat for a rare species? Shortjaw Cisco (<Emphasis Type="Italic">Coregonus zenithicus</Emphasis>) in Lake Huron as a case study

The Government of Canada is considering a recommendation by the Committee on the Status of Endangered Wildlife in Canada to list shortjaw cisco (Coregonus zenithicus) as “Threatened” throughout its range under the Species At Risk Act (SARA). If the listing is approved, shortjaw cisco will receive legal protection, including protection for its ‘critical habitat.’ This study focused on habitat characteristics associated with specimens identified as shortjaw cisco collected with targeted sampling in Lake Huron of the Laurentian Great Lakes. Competing habitat-use models were developed using available data for three physical habitat variables: Water Depth, Substrate Slope, and Cliff Distance, and the models were evaluated using binary logistic regression and ranking of Akaike information criteria. For the habitat factors examined, Water Depth was the most important variable for explaining the observed distribution of identified shortjaw cisco, although this factor alone was not sufficient to adequately represent habitat for this taxonomically uncertain and rare animal. Future habitat discrimination for this hypothesized species at risk must be based on (a) reduction of taxonomic uncertainty, (b) expansion of sampling effort, and (c) consideration of additional physical and ecological habitat factors.     

Phylogenetic relationships among the subfamily Coregoninae as revealed by mitochondrial DNA restriction analysis

Mitochondria1 DNA (mtDNA) restriction analysis was used to assess phylogenetic patterns among 21 taxa of the subfamily Coregoninae. The genus Prosopium formed a very distinct group differing by 10% (sequence divergence estimate) from other species. Coregonus and Stenodus species were closely related, diverging by sequence divergence estimates of less than 5.6%. These species split into two major sister groups. One comprised all 'true whitefish' (subgenus Coregonus ) and four cisco species (subgenus Leucichrhys ). The most distant species within this assemblage was the Acadian whitefish ( C. huntsmani ). The other group included all other cisco species and also the Inconnu ( Stenodus leucichthys ). These results supported a polyphyletic origin of the ciscoes, and did not support Stenodus as a sister taxon of the genus Coregonus . The levels of sequence divergence observed suggested that most extant coregonines radiated during the Pleistocene.     

Identification of palearctic coregonid fish species using mtDNA and allozyme genetic markers

Two types of molecular genetic markers were used for genetic identification of species and local stocks of palearctic coregonids (Coregonidae, Salmoniformes, Teleostei). Seven nominate species of whitefishes and ciscoes Coregonus , spp. of Eurasia Arctic Sea basin and inconnu Stenodus leucichthys nelma , represented by specimens from North America were studied. Using restriction analysis of PCR-amplified products of the ND-1 gene of mitochondrial DNA (mtDNA) and allelic composition at several allozyme loci discrimination was successful between C. lavaretus pidschian , Siberian whitefish, C. nasus , broad whitefish, C. autumnalis , Arctic cisco, C. migratorius , Baikal omul, C. peled , peled, and C. sardinella , least cisco. Muksun C. muksun , was indistinguishable from Siberian whitefish. Creatine kinase (CK) isozyme patterns and Rsa , I restriction patterns of ND-1 were the most effective markers allowing discrimination among species. Intra-specific differentiation in mtDNA was found in all species but was much less pronounced than inter-species variation. In several specimens composite haplotypes typical of another species were found that reflect probable gene introgression by hybridization. A combination of mtDNA and nuclear genetic markers is suggested for reliable identification of both typical species representatives and hybrids.     

Molecular systematics of the western rattlesnake, Crotalus viridis (Viperidae), with comments on the utility of the D-loop in phylogenetic studies of snakes.

Crotalus viridis, the western rattlesnake, ranges throughout western North America and has been divided into at least eight subspecies. However, the validity of and relationships among these subspecies and the monophyly of C. viridis as a whole are questionable. We used mitochondrial DNA sequence data from the D-loop region and ND2 gene to examine the relationships among 26 populations of C. viridis and to test the monophyly of this species. These data were analyzed separately and combined using maximum-likelihood and maximum-parsimony. The C. viridis group was monophyletic in all combined analyses, consisting of two strongly divergent clades. We recommend that these clades be recognized as two distinct evolutionary species: C. viridis and C. oreganus. Crotalus viridis should be restricted to the subspecies viridis and nuntius and the remaining subspecies be assigned to the species C. oreganus. Our data do not allow strong evaluation of the validity of the subspecies. We found that the ND2 gene had greater sequence divergences among closely related individuals than the D-loop region, but this relationship reversed at higher levels of divergence. This pattern is apparently due to: (1) ND2 third positions evolving faster than the D-loop but becoming saturated at higher levels of divergence, and (2) the D-loop evolving faster than ND2 second (and possibly first) positions. Our results suggest that the ND2 gene is preferable for examining intraspecific relationships and the D-loop may better resolve relationships between species of snakes. The latter result is contrary to the common perception of the phylogenetic utility of the D-loop. Another unusual result is that the 145 bp spacer region, adjacent to the 5' end of the light strand of the D-loop, provides greater phylogenetic resolution than the 1030 bp D-loop.     

Mitochondrial D-loop sequence variation among the 16 maternal lines of the Lipizzan horse breed

Mitochondrial DNA from 49 Lipizzan horses representing 16 maternal lines from the original stud at Lipica was used for SSCP analysis and DNA sequencing. The SSCP analysis of the 444 bp long fragment of the D-loop region extending from the tRNA(Pro) gene to the central conserved sequence block revealed three distinct groups of SSCP patterns. Both ends of the D-loop region (378 bp and 310 bp), which are considered as the most variable regions within the mammalian mitochondrial DNA, were sequenced. According to 49 polymorphic sites identified within the both parts of the D-loop region, the 16 maternal lines were grouped into 13 distinct mitochondrial haplotypes. The minimal difference between two different haplotype DNA sequences was one nucleotide and the maximal 24 nucleotides. The inheritance of mitochondrial haplotypes was stable and no sequence variation potentially attributable to mutation within maternal line was observed. Considerable DNA sequence similarity of Lipizzan mitochondrial haplotypes with the haplotypes from other breeds was observed. Phylogenetic analysis of the sequence data revealed a dendrogram with three separated branches, supporting the historical data about the multiple origin of the Lipizzan breed.     

Evolution of the cetacean mitochondrial D-loop region.

We sequenced the mitochondrial DNA D-loop regions from two cetacean species and compared these with the published D-loop sequences of several other mammalian species, including one other cetacean. Nucleotide substitution rates, DNA sequence simplicity, possible open reading frames (ORFs), and potential RNA secondary structure were investigated. The substitution rate is an order of magnitude lower than would be expected on the basis of reports on human sequence variation in this region but are consistent with interspecific primate and rodent D-loop sequence variation and with estimates of substitution rates from whole mitochondrial genomes. Deletions/insertions are less common in the cetacean D-loop than in other vertebrate species. Areas of high sequence simplicity (clusters of short repetitive motifs) across the region correspond to areas of high sequence divergence. Three regions predicted to form secondary structures are homologous to such putative structures in other species; however, the presumptive structures most conserved in cetaceans are different from those reported for other taxa. While all three species have possible long ORFs, only a short sequence of seven amino acids is shared with other mammalian species, and those changes that had occurred within it are all nonsynonymous. We conclude that DNA slippage, in addition to point mutation, contributes to the evolution of the D-loop and that regions of conserved secondary structure in cetaceans and an ORF are unlikely to contribute significantly to the conservation of the central region.     

Differentiation between Diphyllobothrium dendriticum and D. latum using isozymes,restriction profiles and ribosomal gene probes

Plerocercoids of Diphyllobothrium dendriticum from cisco (Coregonus artedii) and whitefish (C. clupeaformis) and of Diphyllobothrium latum from pike (Esox lucius) and walleye (Stizostedion vitreum) were collected at Quigly Lake, Manitoba. Adult tapeworms were obtained by experimental infection of hamsters (Mesocricetus auratus) and were used to verify species identity. Isozyme analysis showed no differences between adult and plerocercoid stages. The enzymes esterase and malate dehydrogenase differed between species. Comparison of DNA restriction fragment profiles showed species specific differences, as did Southern hybridization analysis using nematode ribosomal gene probes.     

High frequency of mitochondrial DNA D-loop mutations in Ewing’s sarcoma

Somatic mutations and polymorphisms in the noncoding displacement (D)-loop of mitochondrial DNA (mtDNA) are present in a variety of human cancers. To investigate whether Ewing’s sarcoma (EWS) harbors genetic alterations within the D-loop region and their potential association with EWS carcinogenesis, we analyzed and compared the complete mtDNA D-loop sequences from 17 pairs of tumor tissues and corresponding peripheral blood samples using the direct DNA sequencing method. Our results revealed that 12 of the 17 EWS tumor specimens (70.6%) carried 19 somatic mutations in the D-loop of mtDNA, including 11 single-base substitutions, 3 insertions and 5 deletions. Among the tested 17 patients, we screened a total of 40 germline polymorphisms including one novel sequence variant in the D-loop fragment. Most of these identified mutations and germline variations were clustered within two hypervariable segments (HVS1 and HVS2) as well as the homopolymeric C stretch between nucleotide position 303 and 309. In addition, there was no significant correlation between mtDNA D-loop mutations and various clinicopathological factors of EWS. In conclusion, our study reports for the first time that mtDNA D-loop mutations occur at a high frequency in EWS. These data provide evidence of mtDNA alterations’ possible involvement in the initiation and/or progression of this rare malignancy.     

Genetic diversity of moose (Alces alces L.) in Eurasia

Polymorphism of nucleotide sequence of D-loop fragment of the mitochondrial DNA was studied in 20 moose from several local populations on the territory of Eurasia. Three main haplotype variants of D-loop were detected by molecular phylogenetic method, which formed three clusters named European, Asian and American. Intraspecies variation in the length of HVSI of D-loop of the mitochondrial DNA of moose was revealed. In the Far Eastern and Yakutian moose, haplotypes with a 75-bp deletion were found, which were most similar with haplotypes (also with the deletion), earlier observed in North American moose [1]. The highest diversity of the haplotypes of mitochondrial DNA is characteristic of Yakutia and the Far East (where three haplotype variants were found), which demonstrates the probable role of the region as the center of the species or as the region of ancient population mixture. The geographic region might be considered as a probable source of ancient moose migrations from Asia to America, basing on the data of distribution of mitochondrial haplotypes of D-loop and alleles of MhcAlal-DRB1. Divergence of nucleotide sequences of haplotypes with the 75-bp deletion (forming the American cluster on the phylogenetic tree) was the lowest (0.4%), which evidences respectively recent origin of the group of haplotypes. In Europe, only haplotypes of mitochondrial DNA referred to European variant were observed. Basing on analysis of variation of nucleotide sequences of D-loop, exon 4 of kappa-Casein and exon 2 of MhcALal-DRB1, we demonstrated that Eurasian moose studied belong to the unique species, which has probably passed through a bottle neck. The time of the origin of modern diversity of D-loop haplotypes of the species was estimated as 0.075-0.15 Myr ago.     

Metazoan parasites of whitefish Coregonus clupeaformis (Mitchill) and cisco C. artedii Lesueur from Southern Indian Lake, Manitoba

Metazoan parasites of whitefish Coregonus clupeaformis and cisco C. artedii from Southern Indian Lake, Manitoba were studied to reveal: species composition, differences with host age, sex, and location and season of capture. Whitefish hosted 19 species, 18 of which were also in cisco with generally lower intensity levels. Parasites exhibited definite patterns of abundance with host age and season, the primary causes being dietary and behavioural. No differences in parasite abundance existed between host sexes. Ranking of cisco parasites was significantly different between two sampling sites while whitefish parasites did not differ. Whitefish and cisco from sites 40 miles (64 km) apart had significantly different abundances of Tetracotyle intermedia but not Triaenophorus crassus . An increase in the abundance of copepod-vectored cestodes with a concomitant decrease in abundance of amphipod-vectored parasites is predicted after flooding and diversion.     

A phylogenetic study of the genus Apodemus by sequencing the mitochondrial DNA control region

The sequences of the mitochondrial DNA control region (D-loop) and flanking tRNA genes (about 1000 bp) of 20 samples of wood mice (genus Apodemus ) were analyzed in order to clarify the relationships between different species belonging to the genus. The phylogenetic trees obtained using different methods showed similar topologies with distinct Karstomys ( Apodemus epimelas and Apodemus mystacinus ) and Sylvaemus ( Apodemus alpicola , Apodemus flavicollis , Apodemus hermonensis , Apodemus sylvaticus and Apodemus uralensis ) subtrees. Within Sylvaemus all species appeared to be closely related to each other, probably as result of a bush-like radiation event. Nevertheless, A. hermonensis seemed to be the first diverging branch followed by A. sylvaticus ; A. alpicola and A. flavicollis appeared to be very closely related. Three individuals of uncertain taxonomical status were included in the analysis: hypotheses as to their status are discussed. Further phylogenetic analysis was carried out combining the D-loop sequences of part of the samples of certain taxonomical status with 12S rRNA and cytochrome b sequences obtained by other researchers. Furthermore, I present a structure analysis of the D-loop in Apodemus as compared other rodent species.     

SSCP analysis of pig mitochondrial DNA D-loop region polymorphism

The sequence polymorphism that occurs in the mitochondrial DNA (mtDNA) displacement (D)-loop region is useful as a cytoplasmic DNA marker. We cloned the mtDNA D-loop regions of five breeds of pig by polymerase chain reaction (PCR) and determined their sequences. The sequence diversities in D-loop regions among five breeds of pig were located in the starting area of heavy-strand replication. From these sequences, we designed primers for PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis that amplified the most polymorphic 227 bp fragment of the D-loop region. The results of PCR-SSCP analysis clearly showed that four types of polymorphism (A to D) are found in Landrace (A), Large White (A, B), Duroc (A), Göttingen miniature pig (B) and Meishan (C, D). The same polymorphisms were also detected from each porcine embryo by this method. Our results show that PCR-SSCP analysis is useful in detecting polymorphisms in the D-loop region of pigs and pig embryos.     

Influence of cisco (Coregonus artedi,Lesueur) on muskellunge (Esox masquinongy,Mitchill) mean length,population size structure,and maximum size in northern Wisconsin lakes

Population size structure and maximum size of managed sportfish populations are dictated by abiotic, biotic, ecosystem, and anthropogenic influences. In their native ranges of northern Wisconsin, muskellunge (Esox masquinongy) and cisco (Coregonus artedi) are co-adapted cool- and cold-water species where cisco presence may influence population size structure and maximum size of muskellunge. We tested whether muskellunge size structure indices (length-frequency distributions, proportional size distribution), mean length, and mean maximum length of muskellunge differed when cisco were present or absent in Ceded Territory of Wisconsin (CTWI) lakes during 2015–2018. Cisco presence had a positive influence on size structure and mean length of individual muskellunge within populations. In contrast, cisco presence had no influence on the mean maximum length of muskellunge observed in CTWI populations suggesting that other factors may be better predictors of this metric than cisco presence. In cisco lakes, mean muskellunge length was negatively correlated with mean cisco length suggesting that gape limitation may be a factor influencing population size structure and individual growth rates. Therefore, cisco populations with primarily large individuals may be unavailable to muskellunge as forage. Our results suggest that cisco are an important forage species for some aspects of muskellunge population ecology; however, other factors may also contribute to muskellunge population size structure and maximum size outcomes. As such, conservation of remaining cisco populations in Wisconsin is critical because they influence muskellunge population ecology in lakes where the species coexist. Future research is needed to better understand the interactions of cisco, abiotic and biotic factors, and anthropogenic influences on muskellunge growth dynamics.     

Identification of a soybean chloroplast DNA replication origin-binding protein

Replication of chloroplast DNA (ctDNA) in several plants and in Chlamydomonas reinhardii has been shown to occur by a double displacement loop (D-loop) mechanism and potentially also by a rolling circle mechanism. D-loop replication origins have been mapped in several species. Minimal replication origin sequences used as probes identified two potential binding proteins by southwestern blot analysis. A 28 kDa (apparent molecular weight by SDS-PAGE analysis) soybean protein has been isolated by origin sequence-specific DNA affinity chromatography from total chloroplast proteins. Mass spectrometry analysis identified this protein as the product of the soybean C6SY33 gene (accession number ACU14156), which is annotated as encoding a putative uncharacterized protein with a molecular weight of 25,897 Da, very near the observed molecular weight of the purified protein based on gel electrophoresis. Western blot analysis using an antibody against a homologous Arabidopsis protein indicates that this soybean protein is localized specifically in chloroplasts. The soybean protein shares some homology within a single-stranded DNA binding (SSB) domain of E. coli SSB and an Arabidopsis thaliana mitochondrial-localized SSB of about 21 kDa (mtSSB). However, the soybean protein induces a specific electrophoretic mobility shift only when incubated with a double-stranded fragment containing the previously mapped ctDNA replication oriA region. This protein has no electrophoretic mobility shift activity when incubated with single-stranded DNA. In contrast, the Arabidopsis mtSSB causes a mobility shift only with single-stranded DNA but not with the oriA fragment or with control dsDNA of unrelated sequence. These results suggest that the 26 kDa soybean protein is a specific origin binding protein that may be involved in initiation of ctDNA replication.     

Characterization of mitochondrial genome of Formosan sambar (Rusa unicolor swinhoei)

The complete mitochondrial genome sequence of the Formosan sambar (Rusa unicolor swinhoei) was obtained by DNA sequencing based on PCR fragments amplified by 26 primer pairs designed by ourselves. The results indicated that the mtDNA is 16,505 bp in size. This is the first report on mitochondrial DNA (mtDNA) sequence analysis of the Formosan sambar and the sequence was deposited in the GenBank database under the accession number DQ989636. The complete mitochondrial sequence included the following gene sequences: 12S and 16S rRNAs, 22 tRNAs and 13 protein-coding genes. The base composition of the sequence was as follows: A, 33.51%; T, 28.97%; C, 24.07%; and G, 13.46%. The mitochondrial D-loop region was also analyzed for comparative purposes in the Formosan sambar and 13 other species within the Cervidae family using neighbour-joining method. The phylogenetic tree demonstrated that there are two separate groups, a European type and an Asian type, within the Cervidae family. The D-loop sequences of mtDNA of 24 Formosan sambar animals were compared, and the results showed that the Formosan sambar can be divided into two clades.     

Mitochondrial DNA coding region sequences support the phylogenetic distinction of two Indian wolf species

Two small endangered populations of Indian wolves were recently shown to be distant from other wolf and dog mtDNA lineages characterized so far. None of the inner branches in the tree of canid species based on partial hypervariable D-loop sequences were, however, statistically supported by the data raising the question whether the two Indian wolf lineages represent two new species, occupying an intermediate position between Canis latrans and C. lupus or have diverged from the sub-species of C. lupus due to isolation and drift. Here we report complete D-loop, cytochrome b, and 16S rRNA sequences data for 23 additional wolves from India analysed in the context of other canid species. Extended analyses of D-loop data and partial sequences of 16S rRNA showed highly reticulated pattern and were unable to resolve unambiguously the phylogenetic relationship of Indian wolves among other canid species. The phylogenetic reconstructions of cytochrome b sequences, however gave significant statistical support for the inner branches supporting genetic distinction of the two Indian wolf lineages within themselves as well as from all other wolves of the world, including individuals belonging to subspecies C. lupus chanco and C. lupus pallipes to which the two Indian wolf populations have been traditionally assigned. Their genetic differentiation relative to worldwide variation of wolves supports the suggestion to treat them as separate wolf species, C. himalayensis and C. indica .     

Genetic Diversity of Moose (Alces alces L.) in Eurasia

Polymorphism of nucleotide sequence of D-loop fragment of the mitochondrial DNA was studied in 20 moose from several local populations on the territory of Eurasia. Three main haplotype variants of D-loop were detected by molecular phylogenetic method, which formed three clusters named European, Asian, and American. Intraspecies variation in the length of HVSI of D-loop of the mitochondrial DNA of moose was revealed. In the Far Eastern and Yakutian moose, haplotypes with a 75-bp deletion were found, which were most similar with haplotypes (also with the deletion), earlier observed in North American moose [1]. The highest diversity of the haplotypes of mitochondrial DNA is characteristic of Yakutia and the Far East (where three haplotype variants were found), which demonstrates the probable role of the region as the center of the species origin or as the region of ancient population mixture. The geographic region might be considered as a probable source of ancient moose migrations from Asia to America, basing on the data of distribution of mitochondrial haplotypes of D-loop and alleles of MhcAlal-DRB1. Divergence of nucleotide sequences of haplotypes with the 75-bp deletion (forming the American cluster on the phylogenetic tree) was the lowest (0.4%), which evidences respectively recent origin of the group of haplotypes. In Europe, only haplotypes of mitochondrial DNA referred to European variant were observed. Basing on analysis of variation of nucleotide sequences of D-loop, exon 4 of -Casein and exon 2 of MhcAlal-DRB1, we demonstrated that Eurasian moose studied belong to the unique species, which has probably passed through a bottle neck. The time of the origin of modern diversity of D-loop haplotypes of the species was estimated as 0.075–0.15 Myr ago.     

以川陕哲罗鲑为目标物种的水样环境DNA分析流程的优化

水样环境DNA分析包括水样采集、DNA提取和分析等流程,已成为监测濒危水生生物种群分布调查的重要手段.为减少在监测目标物种尤其濒危物种中的不确定性,对水环境DNA分析流程的优化至关重要.本研究以川陕哲罗鲑为目标物种,采用滤膜法采集养殖池中的水样,设计了 250 mL、500 mL、1 L和2 L等4种水样采集量,分别采用 PoweWater DNA Isolation kit和DNeasy Tissue and Blood DNA extraction kit 提取水样环境DNA(eDNA),使用物种mtDNA D_loop区特异性引物进行PCR扩增,通过研究滤膜法、水样采集量和水样DNA提取方法对水样eDNA中目标基因检出率的影响,探索适宜的eDNA分析操作方案.结果表明: 使用DNeasy Tissue and Blood DNA extraction kit提取的水样DNA中目的基因的检出率为100%,效果明显优于PoweWater DNA Isolation kit(目标基因的检出率为0);目标基因扩增条带的亮度随水样采样量的增加而增加,其中2 L水样目标基因的扩增效果较理想;序列比对结果显示,本试验从水样DNA中成功扩增得到了川陕哲罗鲑mtDNA Dloop区部分序列.表明DNA提取方法和水样采集量对目标物种的检出率有显著的影响,滤膜法、2 L水样采集量、DNeasy Tissue and Blood DNA extraction kit更适宜进行水样的DNA分析,mtDNA D-loop区可作为川陕哲罗鲑识别的特异性分子标记.