Models of development for blowfly sister species Chrysomya chloropyga and Chrysomya putoria

Abstract Developmental curves for the sister species Chrysomya chloropyga (Wiedemann, 1818) and Chrysomya putoria (Wiedemann, 1830) (Diptera: Calliphoridae) were established at eight and 10 different constant temperatures, respectively, using developmental landmarks and body length as measures of age. The thermal summation constants (K) and developmental threshold (D₀) were calculated for five developmental landmarks using a previously described method. Isomorphen and isomegalen diagrams were also constructed for the purpose of estimating postmortem intervals (PMIs). Chrysomya chloropyga had an average developmental threshold value (D₀) of 10.91 °C (standard error [SE] = 0.94 °C, n = 5), significantly lower than that of C. putoria (13.42 °C, SE = 0.45 °C, n = 5) (paired t‐test: t = ? 4.63, d.f. = 8, P < 0.00). Similarly, K values for C. chloropyga were larger than those for C. putoria for all developmental events except onset of the wandering phase. These are the first data that can be used to calculate minimum PMIs and predict population growth of C. chloropyga and C. putoria in Africa; the data indicate that developmental data for one of these species cannot be used as surrogate data for the sister species.     

The tropical African latrine blowfly, Chrysomya putoria (Wiedemann)

The status of the tropical African latrine blowfly Chrysomya putoria (Wiedemann, 1830) is recognized as distinct from the sub-tropical African blowfly C.chloropyga (Wiedemann, 1818) (Diptera: Calliphoridae). The biology of C.putoria in laboratory culture is described briefly with emphasis on the differences in development found in three strains of this species from Tanzania, Liberia and Brazil. All three strains were reproductively compatible and there was no evidence of F1 hybrid sterility or hybrid breakdown in subsequent generations. Larval development was significantly faster in the Brazilian strain than in the strain from Tanzania. Sexual maturity was significantly faster in adults of the Liberian and Brazilian strains compared to that found in the strain from Tanzania. This latter strain was affected by the source of protein available as adult food. These differences between strains did not appear to be due to selection in laboratory culture, but rather evidence of the evolution of physiological divergence within a species occupying different geographical regions.     

The larvae of some blowflies of medical and veterinary importance

Diagnostic features are described as a series of couplets that enable separation of the third instar larvae of the following pairs of closely related forms of blowflies of medical and veterinary importance: Chrysomya chloropyga (Wiedemann) and Ch.putoria (Wiedemann), Chrysomya albiceps (Wiedemann) and Ch.rufifacies (Macquart), Cochliomyia hominivorax (Coquerel) and Co.macellaria (Fabricius), Lucilia sericata (Mergen) and L. cuprina (Wiedemann), Calliphora augur (Fabricius) and C. stygia (Fabricius).     

Methods for the recovery of mitochondrial DNA sequences from museum specimens of myiasis-causing flies

Mitochondrial DNA (mtDNA) sequences from eight species of myiasis-causing flies, stored for up to 50 years, were amplified successfully. Universal primers were used to amplify six specific regions from total genomic DNA, including five mtDNA genes. The comparison of phenol/chloroform, DNAzol and Chelex techniques for DNA extraction showed that the DNAzol reagent was the most efficient in retrieving DNA from museum specimens, although the Chelex extraction procedure is currently the most frequently reported method. Comparison of the universal primer sequences with the homologous sequences of Cochliomyia hominivorax Coquerel and Chrysomya putoria Wiedemann (Diptera: Calliphoridae) revealed mismatches that could contribute to the low recovery of a short sequence from subunit II of cytochrome oxidase. The ability to characterize mtDNA markers from museum specimens should be useful in comparative studies of contemporary samples and should help in elucidating species introduction, colonization and dispersal.     

Amplified fragment length polymorphism confirms reciprocal monophyly in Chrysomya putoria and Chrysomya chloropyga: a correction of reported shared mtDNA haplotypes

Reinvestigation of mitochondrial haplotypes previously reported to be shared between the Afrotropical blowflies Chrysomya putoria Weidemann and Chrysomya chloropyga Weidemann (Diptera: Calliphoridae) revealed an error resulting from the misidentification of specimens. Preliminary amplified fragment length polymorphism (AFLP) analysis of the original and additional individuals again failed to find reciprocal monophyly, leading to a re-examination of the specimens for diagnostic male genitalic characters that were first described following the earlier study. Four of the original study specimens were found to have been misidentified, and definitive analysis of both mtDNA and AFLP genotypes using phylogenetic analysis and genetic assignment showed that each species was indeed reciprocally monophyletic. In addition to correcting the earlier error, this study illustrates how AFLP analysis can be used for efficient and effective specimen identification through both phylogenetic analysis and genetic assignment, and suggests that the latter method has special advantages for identification when no conspecific specimens are represented in the reference database.     

mtDNA中COⅠ分子标记在常见食尸性蝇类鉴定中的应用

死后不同时间,在尸体上出现不同种类食尸性蝇类的演替规律,可用于准确推断死亡时间。传统上仅依据蝇类形态学特征来判断种属,但由于蝇类的形态结构复杂和种间形态差异微小等特点,对蝇类尤其是对蝇类幼虫的种属鉴别很难。因此应用分子生物学方法对食尸性蝇类及其幼虫进行种属鉴定非常重要。本研究主要是利用此方法对我国西部部分地区常见双翅目食尸性蝇类包括：开普黑蝇、大头金蝇、丝光绿蝇及部分卵,铜绿蝇、棕尾别麻蝇及部分幼虫和蛹的线粒体DNA(mtDNA)上细胞色素氧化酶辅酶Ⅰ(COⅠ)中278 bp的基因序列进行鉴别。除个别蝇类如丝光绿蝇与铜绿蝇外,该方法均能有效地将上述食尸性蝇类鉴定到种属水平。在我国,它将成为法医鉴别食尸性蝇类种属的可靠依据。     

C-banding and FISH in chromosomes of the blow flies Chrysomya megacephala and Chrysomya putoria (Diptera, Calliphoridae)

The blow flies Chrysomya putoria and C. megacephala have 2n=12 chromosomes, five metacentric pairs of autosomes and an XX/XY sex chromosome pair. There are no substantial differences in the karyotype morphology of these two species, except for the X chromosome which is subtelocentric in C. megacephala and metacentric in C. putoria and is about 1.4 times longer in C. putoria. All autosomes were characterized by the presence of a C band in the pericentromeric region; C. putoria also has an interstitial band in pair III. The sex chromosomes of both species were heterochromatic, except for a small region at the end of the long arm of the X chromosome. Ribosomal genes were detected in meiotic chromosomes by FISH and in both species the NOR was located on the sex chromosomes. These results confirm that C. putoria was the species introduced into Brazil in 1970s, and not C. chloropyga as formerly described.     

Selective attractiveness of rodent-baited traps for female blowflies

Five species of Calliphoridae, mostly females, were collected using fly-traps baited with freshly killed rodent carcasses. Female blowflies were examined to determine the stages of ovarian development. The traps were selective for certain ovarian stages which varied according to the species. Phaenicia eximia (Wiedemann) was attracted to the traps mainly to oviposit, whereas females of Chrysomya albiceps (Wiedemann), C. putoria (Wiedemann) and C. megacephala (Fabricius) were captured in intermediate stages of oogenesis but seldom with eggs mature for oviposition. It is concluded that this kind of baited trap is unsuitable for the collection of male blowflies and that samples of female blowflies are specifically biased in favour of certain ovarian conditions.     

Concordant divergence in proteins and mitochondrial DNA between two vole species in the genusClethrionomys

The bank vole (Clethrionomys glareolus) and the northern red-backed vole (C. rutilus) are two closely related species where interspecific crosses result in fertile female but sterile male offspring. Mitochondrial DNA (mtDNA) fromC. rutilus has passed the species barrier and is found inC. glareolus from northern Fennoscandia. The present report shows that the genetic distance between the two species, calculated from enzyme data (Nei'sD), is 0.64. Isoelectric focusing of muscle proteins resolved around 55 bands, of which each species had 6 or 7 bands not present in the other species. Sequence divergence of mtDNA from the two species is 13.9%. A comparison between protein and mtDNA distances in other species pairs reveals a high correlation between the two measures, indicating that differences in mtDNA between taxa are not random when compared to divergence in protein-coding nuclear genes. The relationship between genetic divergence in proteins and that in mtDNA betweenClethrionomys glareolus andC. rutilus is similar to that found in other species pairs. It is also shown that despite large differences on the protein level it is still, in some cases, possible for species pairs to produce fertile hybrid females.This study was sponsored by the Swedish Natural Science Research Council, the Erik Philip-Sörensen Foundation, and the Nilsson-Ehle Foundation.     

Malignant tumor affects the developmental pattern of feeding larvae of Chrysomya albiceps (Wiedemann) and Chrysomya putoria (Wiedemann) (Diptera: Calliphoridae)

Larvae of Chrysomya albiceps (Wiedemann) and Chrysomya putoria (Wiedemann) were reared on liver tissues from a rabbit that had a malignant tumor in the thoracic cavity. Larval rearing of both blowfly species was conducted at ambient temperature. Larvae that fed on tissues from the rabbit with the tumor developed at significantly faster rates than those feeding on tissues from the control animal.     

Ovarian development in the blowflies Chrysomyaputoria and C. megacephala on natural diets

Fresh human, chicken and cattle faeces were compared as diets for adult female blowflies Chrysomya megacephala (Fabricius) and C.putoria (Wiedemann) (Diptera: Calliphoridae). As controls, sugar and raw beef liver were used. For females of both species, fresh beef liver was the best of these diets: oogenesis was completed in 10-13 days. Human or chicken faeces facilitated slower oogenesis in 27-48 days. On a diet of cattle faeces, C.putoria females began vitellogenesis only 17 days after emergence, whereas females of C.megacephala remained previtellogenic until the end of the experiment (56 days).     

Phylogenetic relationships among four members of Stizostedion (Percidae) determined by mitochondrial DNA and allozyme analyses

Phylogenetic relationships among four Stizostedion species were examined using mitochondrial DNA (mtDNA) and allozyme analyses. Twenty-six allozyme loci were scored, and mtDNA variation was examined using 24 restriction endonucleases, yielding 48–57 restriction sites among the species. Genetic distance analyses show that the two North American species ( S. canadense and S. vitreum ) cluster in one group, while the two European species ( S. hciopercu and S. vogense ) form a second group. Nei's genetic distance between these two groups was 0.7 ± 0.2 for allozymes, while the corresponding mtDNA sequence divergence was 14.8 ± 2.0%, suggesting that these two groups diverged approximately 10 million years ago. Thus, these data are consistent with the hypothesis that Stizostedion colonized North America during the Pliocene.     

Matching loci surveyed to questions asked in phylogeography

Although mitochondrial DNA (mtDNA) has long been used for assessing genetic variation within and between populations, its workhorse role in phylogeography has been criticized owing to its single-locus nature. The only choice for testing mtDNA results is to survey nuclear loci, which brings into contrast the difference in locus effective size and coalescence times. Thus, it remains unclear how erroneous mtDNA-based estimates of species history might be, especially for evolutionary events in the recent past. To test the robustness of mtDNA and nuclear sequences in phylogeography, we provide one of the largest paired comparisons of summary statistics and demographic parameters estimated from mitochondrial, five Z-linked and 10 autosomal genes of 30 avian species co-distributed in the Caucasus and Europe. The results suggest that mtDNA is robust in estimating inter-population divergence but not in intra-population diversity, which is sensitive to population size change. Here, we provide empirical evidence showing that mtDNA was more likely to detect population divergence than any other single locus owing to its smaller N_e and thus faster coalescent time. Therefore, at least in birds, numerous studies that have based their inferences of phylogeographic patterns solely on mtDNA should not be readily dismissed.     

GENETIC STRUCTURE OF THE BLUE RIDGE DUSKY SALAMANDER (DESMOGNATHUS ORESTES): INFERENCES FROM ALLOZYMES, MITOCHONDRIAL DNA, AND BEHAVIOR

Abstract The plethodontid salamander Desmognathus orestes, a member of the D. ochrophaeus species complex, is distributed in southwestern Virginia, eastern Tennessee, and western North Carolina. Previous allozyme analyses indicate that D. orestes consists of two distinct groups of populations (D. orestes‘B’ and D. orestes‘C’) with extensive intergradation and probable gene flow between these two groups. Spatially varying allele frequencies can reflect historical associations, current gene flow, or a combination of population‐level processes. To differentiate among these processes, we use multiple markers to further characterize divergence among populations of D. orestes and assess the degree of intergradation between D. orestes‘B’ and D. orestes‘C’, specifically investigating variation in allozymes, mitochondrial DNA (mtDNA), and reproductive behavior among populations. On a broad scale, the mtDNA genealogies reconstruct haplotype clades that correspond to the species identified from previous allozyme analyses. However, at a finer geographic scale, the distributions of the allozyme and mtDNA markers for D. orestes‘B’ and D. orestes‘C’ are discordant. MtDNA haplotypes corresponding to D. orestes‘B’ are more broadly distributed across western North Carolina than predicted by allozyme data, and the region of intergradation with D. orestes‘C’ indicates asymmetric gene flow of these markers. Asymmetric mating may contribute to observed discordance in nuclear versus cytoplasmic markers. Results support describing D. orestes as a single species and emphasize the importance of using multiple markers to examine fine‐scale patterns and elucidate evolutionary processes affecting gene flow when making species‐level taxonomic decisions.     

Evolutionary and taxonomic relationships among Far-Eastern salmonid fishes inferred from mitochondrial DNA divergence

Mitochondrial DNA (mtDNA) restriction analysis was used to examine the evolutionary and taxonomic relationships among 11 taxa of the subfamily Salmoninae. The genera Brachymystax and Hucho were closely related, diverging by sequence divergence estimates of 3.1%. Because the mtDNA sequence divergence between blunt- and sharp-snouted forms of Brachymystax (2.24%) was similar to divergence level of Brachymystax and Hucho , then taking into account the distinct morphological, ecological and allozyme differences between them, it is possible to recognize these forms as two separate species. The subgenus Parahucho formed a very distinct group differing by 6.35–7.08% (sequence divergence estimate) from both Brachymystax and Hucho and must be considered as a valid genus. The UPGMA and neighbour-joined phenograms showed that the five genera studied are divided into two main groupings: (1) Hucho, Brachymystax and Salvelinus ; and (2) Oncorhynchus and Parahucho species. The mtDNA sequence divergence estimates between these groupings were about 8.1%. However, the subsequent bootstrap analysis of mtDNA RFLP data did not support the monophyly of the latter grouping. The concordance of morphological and mtDNA phylogenetic patterns is discussed.     

Chromosomenverhältnisse,konstitutives Heterochromatin und Geschlechtsbestimmung bei einigen Arten der GattungChrysomya (Calliphoridae,Diptera)

Compared with the strongly monogenic blowfliesChrysomya albiceps andC. rufifacies investigated previously, the closely related speciesC. chloropyga, C. putoria andC. varipes turned out to be amphogenic, i.e. each female of these species produces both sexes in nearly equal frequencies.C. chloropyga, C. putoria andC. varipes possess similar karyotypes consisting of five pairs of long identifiable autosomes and one pair of smaller sex chromosomes, which are homomorphic (XX) in the female and heteromorphic (XY) in the male. In all species the Y is shorter than the X. Among the cytologically known amphogenicChrysomya speciesC. varipes has the smallest heterochromosomes. Male meiosis inC. chloropyga, C. putoria andC. varipes is achiasmatic. During mitosis the autosomes show a high degree of somatic pairing but heterochromosomes, which form a heteropyknotic mass in the interphase nuclei, usually separate in the course of early mitotic prophase. A comparative study of C-banding patterns in mitotic and meiotic chromosomes ofC. chloropyga, C. putoria, C. varipes andC. rufifacies revealed that constitutive heterochromatin is present in almost all chromosomes. In the five pairs of large chromosomes it is confined to short regions adjacent to the kinetochores. An interstitial C-band has been observed only in the shorter arm of autosome no. 3 inC. putoria. With the exception of the tiny Y chromosome ofC. varipes, which seems to be completely euchromatic, all the other sex chromosomes of the amphogenicChrysomya species as well as the small chromosome no. 6 ofC. rufifacies possess longer or shorter C-segments. The karyotype ofC. varipes resembles closely that of the monogenic speciesC. albiceps andC. rufifacies thus indicating that the monogenic species could have evolved from an ancestral species closely related toC. varipes. The occurrence of two exceptional XO females and one exceptional XXY male among normal XX and XY animals in a wild stock ofC. chloropyga demonstrates that the Y chromosome of C. chloropyga and most probably of all amphogenicChrysomya species bears an epistatic male determining factor.

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Herrn Professor Dr. G. Krause zum 70. Geburtstag gewidmet     

Evolutionary basis of mitonuclear discordance between sister species of mole salamanders (Ambystoma sp.)

Distinct genetic markers should show similar patterns of differentiation between species reflecting their common evolutionary histories, yet there are increasing examples of differences in the biogeographic distribution of species‐specific nuclear (nuDNA) and mitochondrial DNA (mtDNA) variants within and between species. Identifying the evolutionary processes that underlie these anomalous patterns of genetic differentiation is an important goal. Here, we analyse the putative mitonuclear discordance observed between sister species of mole salamanders (Ambystoma barbouri and A. texanum) in which A. barbouri‐specific mtDNA is found in animals located within the range of A. texanum. We test three hypotheses for this discordance (undetected range expansion, mtDNA introgression, and hybridization) using nuDNA and mtDNA data analysed with methods that varied in the parameters estimated and the timescales measured. Results from a Bayesian clustering technique (structure ), bidirectional estimates of gene flow (migrate ‐n and IMa2) and phylogeny‐based methods (*beast , buck y) all support the conclusion that the discordance is due to geographically restricted mtDNA introgression from A. barbouri into A. texanum. Limited data on species‐specific tooth morphology match this conclusion. Significant differences in environmental conditions exist between sites where A. texanum with and without A. barbouri‐like mtDNA occur, suggesting a possible role for selection in the process of introgression. Overall, our study provides a general example of the value of using complimentary analyses to make inferences of the directionality, timescale, and source of mtDNA introgression in animals.     

Genetic relationships among the shads (Alosa) revealed by mitochondrial DNA analysis

We surveyed restriction site differences in mitochondrial DNA. (mtDNA) among five species of shad ( Alosa ) from North America and Europe. Allis shad, Alosa alosa and twaite shad, Alosa fallax shared two divergent genotype groups, suggesting that the two forms are either a single species, or are distinct species that have hybridized. Phenetic and cladistic analyses of the relationships among the mitochondrial genotypes defined two groups of shad, corresponding to the subgenera, Alosa and Pomolobus . The mean estimated sequence divergence between the mtDNAs of these two groups of shad was 6.5%. Taken in conjunction with fossil data, this divergence estimate suggests that the rate of mtDNA divergence between the two subgenera has been almost 10-fold lower than the 'conventional' clock calibration for mtDNA.     

The incomplete natural history of mitochondria

Mitochondrial DNA (mtDNA) has been used to study molecular ecology and phylogeography for 25 years. Much important information has been gained in this way, but it is time to reflect on the biology of the mitochondrion itself and consider opportunities for evolutionary studies of the organelle itself and its ecology, biochemistry and physiology. This review has four sections. First, we review aspects of the natural history of mitochondria and their DNA to show that it is a unique molecule with specific characteristics that differ from nuclear DNA. We do not attempt to cover the plethora of differences between mitochondrial and nuclear DNA; rather we spotlight differences that can cause significant bias when inferring demographic properties of populations and/or the evolutionary history of species. We focus on recombination, effective population size and mutation rate. Second, we explore some of the difficulties in interpreting phylogeographical data from mtDNA data alone and suggest a broader use of multiple nuclear markers. We argue that mtDNA is not a sufficient marker for phylogeographical studies if the focus of the investigation is the species and not the organelle. We focus on the potential bias caused by introgression. Third, we show that it is not safe to assume a priori that mtDNA evolves as a strictly neutral marker because both direct and indirect selection influence mitochondria. We outline some of the statistical tests of neutrality that can, and should, be applied to mtDNA sequence data prior to making any global statements concerning the history of the organism. We conclude with a critical examination of the neglected biology of mitochondria and point out several surprising gaps in the state of our knowledge about this important organelle. Here we limelight mitochondrial ecology, sexually antagonistic selection, life-history evolution including ageing and disease, and the evolution of mitochondrial inheritance.     

Retrospective Estimation of Genetic Diversity of an Extinct Oriental White Stork (Ciconia boyciana) Population in Japan Using Mounted Specimens and Implications for Reintroduction Programs

The Japanese regional population of the Oriental white stork (Ciconia boyciana) became extinct in 1986. Mitochondrial DNA (mtDNA) D-loop region from 20 mounted specimens preserved at public facilities in Toyooka City, Hyogo Prefecture, Japan and its vicinity (n = 17), the area inhabited by the last of the Japanese population, and samples originating from China (n = 3) which were kept at a zoo was analyzed. After extracting DNA from small pieces of skin from mounted specimens, a 1210-bp region of the mtDNA D-loop region was analyzed. The haplotypes among 11 specimens of storks captured or found dead at Toyooka City just before the population became extinct were completely identical. Four haplotypes observed among the mounted specimens preserved in the vicinity of Toyooka City were differentiated from those of captive storks originating from China and Russia in a previous study. Therefore, the last Japanese population might have been a genetically unique group. However, phylogenetic analysis using the maximum likelihood method showed that haplotypes found in the Japanese regional population were closely related to the Chinese and Russian lineages (sequence difference = 2.1%). One mounted specimen collected in 1935 at Izushi village, in the vicinity of Toyooka City, showed the same haplotype as the captive storks from China, suggesting that genetic flow may have historically occurred between the populations of Japan and the continent. Recently, reintroduction for the Oriental white stork has been planned in Toyooka City. The planning for the recovery of extinct populations should not only involve translocation of species to the range from which it disappeared, but also reconstruction of regional populations while considering the genetic lineage between the extinct and introduced populations.