A DNA investigation into the mysterious disappearance of the Rocky Mountain grasshopper, mega-pest of the 1800s.

The mysterious extinction of the Rocky Mountain Grasshopper, Melanoplus spretus, a major pest species on the prairies and plains of the 1800s, is truly a remarkable event in the history of agriculture. Recently, we obtained specimens of M. spretus from museums and from 400-year-old glacial deposits in Wyoming. We report success in obtaining mitochondrial DNA sequences from both sources. This permitted us to examine two issues surrounding the species' disappearance. First, a long-standing view that M. spretus and the extant species Melanoplus sanguinipes are (if not phase transforms of one another) sister taxa, is disputed, but cannot be rejected with certainty. Interestingly, there is some evidence that suggests there may be a closer affinity with another member of the Mexicanus species-group, Melanoplus bruneri. Second, because M. spretus still possesses considerable nucleotide diversity (1.15+/-0.19%), a depletion of variation cannot be considered a factor contributing to its demise.

Out of South America? Additional evidence for a southern origin of melanopline grasshoppers.

Molecular phylogenetic methods provide a useful tool for critically evaluating competing biogeographic hypotheses. This paper focuses on conjectures at the intercontinental level. An earlier phylogeographic study of the grasshopper subfamily Melanoplinae examined relationships among taxa occupying the Americas and Eurasia. The objective was to test which of three scenarios best described the subfamily's origins and patterns of intercontinental movement. It was tentatively proposed that the melanopline grasshoppers evolved somewhere in the Americas and spread to the Old World. The present article, by including additional Neotropical species, not only upholds that conjecture, but is able to identify South America as the site of origin. Phylogenetic analysis indicates a direction of dispersal: South America-->North America-->Eurasia, that is opposite to what was previously believed. This study also provides, for the first time, a preliminary molecular phylogeny of selected South American melanopline genera.

A molecular phylogenetic study of two relict species of melanopline grasshoppers.

Two species belonging to the large grasshopper genus Melanoplus, Melanoplus gaspesiensis and Melanoplus madeleineae, are thought to have survived in refugia in eastern Canada during the Wisconsin glaciation period and perhaps during earlier glacial episodes as well. It has been proposed that either Melanoplus borealis, which is widely distributed today, or its antecedent gave rise to the two relict species. Our research, based on standard phylogenetic analyses of mitochondrial sequences, examines this claim by establishing the positions of M. gaspesiensis and M. madeleineae within Melanoplus. There is a very close and strongly supported connection between M. madeleineae and M. borealis (d = 1.36%), whereas M. gaspesiensis is more distant from the latter (d = 2.08%) and appears closely aligned with two other species. Molecular clock estimates of the times of separation of M. madeleineae and M. gaspesiensis from M. borealis are 0.68 and 1.04 million years ago, respectively. Within-species diversities of 1.01 and 0.70% for M. madeleineae and M. gaspesiensis, respectively, are of comparable magnitudes to that of the continentally distributed Melanoplus sanguinipes (0.86%), clearly indicating that neither species endured bottleneck effects despite isolation.

A molecular phylogeographic perspective on a fifty-year-old taxonomic issue in grasshopper systematics.

This paper addresses a decades-old taxonomic controversy surrounding a species in the grasshopper subfamily Melanoplinae. Melanoploid grasshoppers fall into two tribes, the Nearctic-restricted Melanoplini and the Holarctically distributed Podismini. The current view regarding one member, Bohemanella frigida, is that it belongs to the latter tribe and that North American populations were established by dispersal from Eurasia via the Bering Land Bridge. Over the past 50 years, this opinion has changed a few times; this species was once regarded as part of the tribe Melanoplini and, as such, deemed to be the only Holarctically distributed Orthopteran insect with New World antecedents. A molecular phylogenetic study of this species was thus performed to verify its phylogenetic position and to establish a probable direction of dispersal. Portions of three mitochondrial genes (cyt b, COII, and ND2) were sequenced and phylogenetically analysed using weighted and unweighted parsimony, neighbour-joining, and maximum likelihood methods. Support for the inclusion of B. frigida within the tribe Melanoplini and the use of its original name, Melanoplus frigidus, was strong using all methods. Placement in the tribe Melanoplini leads to an acceptance of an earlier hypothesis regarding direction of dispersal across the Bering Land Bridge, making this grasshopper a unique case in orthopteran insects in this respect.

A molecular biogeographic analysis of the relationship between North American melanoploid grasshoppers and their Eurasian and South American relatives.

The Melanoplinae constitute one of the two largest subfamilies of Acrididae. Distributed mainly throughout the New World and parts of Eurasia, this group of grasshoppers includes over 100 genera and 800 species. Over the past five decades there has been considerable speculation on the origins of North and South American taxa. The most favored hypothesis proposes an ancient division of Laurasian taxa accompanying the separation of North America and Eurasia, with subsequent radiations within those continents, followed by a recent incursion of Nearctic melanoplines into the southern hemisphere with the establishment of the Isthmus of Panama. This research tests that scenario by phylogenetic analysis using as characters portions of five mitochondrial gene sequences, totaling 2285 bp. Three tree-building methods, maximum-parsimony, neighbor-joining, and maximum-likelihood, strongly support the different view that melanopline grasshoppers originated somewhere in the Americas and spread to the Old World. The feasibility of these findings is discussed within a geological context.

Mitochondrial DNA variation in North American oedipodinae.

Restriction fragment analysis of mitochondrial DNA (mtDNA) was used to examine genetic variation and population structure in 13 species of banded-winged grasshoppers (subfamily Oedipodinae). Total DNA of 246 individuals was digested with 11 restriction enzymes and probed with three cloned EcoRI fragments representing the entire mitochondrial genome of Melanoplus sanguinipes. On average, members of this subfamily were five times more variable than those in another subfamily, Melanoplinae, previously examined. This would appear to lend support to Vickery's claim that the time of origin of Nearctic oedipodines is more ancient than that of melanoplines. With respect to population structure, a few different patterns were exhibited. Species such as Camnula pellucida had populations containing a mixture of haplotypes, some widespread and some geographically restricted. In contrast, Chortophaga viridifasciata populations were characterized by unique assemblages of diverse haplotypes. Phylogeographic hypotheses are advanced to account for these observations.

A feasibility study of the use of random amplified polymorphic DNA in the population genetics and systematics of grasshoppers.

Single, short primers of arbitrary nucleotide sequence were used in polymerase chain reactions to amplify regions of DNA isolated from several melanopline and oedipodine grasshoppers collected from local Saskatchewan populations. This represents one of the first applications of the method, called randomly amplified polymorphic DNA (or RAPD), to natural populations. Twenty-four different oligonucleotide primers, nine nucleotides in length, yielded clear and reproducible bands corresponding to amplified products and separable by agarose gel electrophoresis. On average, about 8.1 bands (range 0-17) were obtained per primer per individual. The mean percent similarity between band profiles of conspecific individuals was 51.2%, whereas the mean value for individuals representing different species or genera was 35.0%. Clearly, greater numbers of insects and primers will be required to achieve a satisfactory level of phylogenetic resolution. Given RAPDs technical advantages and ease of execution, however, this should not be problematic to the molecular systematist.

Characterization of a reduced-eye mutant of the grasshopper, Melanoplus sanguinipes.

A reduced-eye (re) mutant grasshopper of Melanoplus sanguinipes has been characterized by small flat compound eyes lacking facets, no lateral ocelli and only a remnant of the median ocellus. The re grasshoppers walk, jump, fly and feed in a normal manner, but do not respond to visual and auditory stimuli, suggesting they may be blind and deaf. Extracellular recordings from the ventral nerve cord of re mutants verified the lack of neural activity in response to visual and auditory inputs, yet the mutants detected mechanical and tactile stimuli. Electroretinograms implied that a visual deficit may be within the photoreceptors of the compound eye. Histological examination of the compound eyes and ocelli indicated that the cells of the mutant compound eye incompletely differentiate. The optic lamina underlying the retina is missing, as is the outer optic chiasma. The medulla and lobula of the mutant optic lobe are present, however, the neuropil of the medulla lacks the characteristic axonal projection patterns of wild-type grasshoppers. The re grasshopper also lacks all ocellar nerves. Ocellar nerves are normally formed from processes of second order ocellar neurons (SONs), suggesting that if the mutant SONs are present within the protocerebrum, their morphology is drastically altered. Comparison of embryos and juvenile nymphs supports the suggestion that the alterations in the re visual system are the result of abnormal differentiation during development. Even though there is clear evidence of morphological alterations in second and third order optic lobe interneurons, one higher order visual interneuron of the midbrain, the descending contralateral movement detector (DCMD), has the same morphology as the DCMD in a wild-type brain. In this instance, the complete deprivation of the primary sensory input does not appear to alter cellular development.

Correlations between chromosome segments and fitness in Drosophila melanogaster. IV. Fecundity, viability and the third chromosome.

Unmarked segments within the third chromosome of three different Drosophila melanogaster lines were assessed for their effects on egg production and egg viability. By making a series of crosses among original and derived recombinant lines, it was possible to estimate parameters representing additive, dominance (for egg production), and interaction effects of the segments. Each segment influences both traits, but to extend which are dependent on the genetic background provided by an adjacent segment. There is no clear pattern, however, with respect to the segments' joint effects on the two characters. Unlike in the previous study involving the X chromosome, the majority of the derived recombinant lines were superior in fitness to their original lines. The agricultural implications of this result with respect to the manipulation of chromosomal segments in order to achieve higher yields are discussed.

Correlations between Chromosome Segments and Fitness in DROSOPHILA MELANOGASTER. II. the X Chromosome and Egg Viability.

Unmarked segments within the X chromosomes of four different Drosophila melanogaster isogenic lines were assessed with respect to egg-to-adult viability. The results were compared with those of an earlier study involving egg production. All segments influence both traits, but to extents that are dependent upon the strains being compared. Segmental effects are also a function of the genetical background, which, in this case, constitutes material within the same chromosome. With respect to both traits, the segments are not necessarily parallel in their effects. A segment that increases fecundity, for example, may or may not augment viability. The possibility of manipulating chromosomal segments to improve "yield" in organisms is explored.

Correlations between chromosome segments and fitness in Drosophila melanogaster III. Differential genetic responses to zinc sulfate and selenocystine.

Genetic X environmental interactions are examined at an intrachromosomal level in Drosophila melanogaster. With respect to two fitness components, egg production and egg-to-adult viability, evidence is provided that different segments of the X chromosome are affected differently by each of the chemical substances, zinc sulfate and selenocystine. The extent of a segment's effect on a trait is not always parallelled by the extent of its association with that trait's sensitivity to chemical treatment. Both attributes are functions of the genetical background. The degree of dominance of each segment is not always greater in the chemical environments, a finding inconsistent with Parson's concept of "extreme-environment heterosis".

Correlations between chromosome segments and fitness in Drosophila melanogaster. I. The X chromosome and egg production.

Unmarked segments within the X chromosomes of four different Drosophila melanogaster isogenic lines were assessed with respect to egg production. By making a series of crosses among original and derived recombinant lines, it was possible to estimate parameters representing additive, dominance and interaction effects of the segments. It was shown that whereas most of the segments were additive for egg production when homozygous, they all displayed dominance in the heterozygous condition. Two of the strains were characterized by intersegmental interaction. A possible position effect was detected for these same two strains, with flies in the coupling phase laying more eggs than those in the repulsion configuration. There was no apparent relationship between the number of eggs laid and the amount of heterozygosity within the X chromosome.

An exact test of the Hardy-Weinberg law.

An exact distribution of a finite sample drawn from an infinite population in Hardy-Weinberg Equilibrium is described for k-alleles. Accordingly, an exact test of the law is presented and compared with two x2-tests for two and three alleles. For two alleles, it is shown that the "classical" c2-test is very adequate for sample sizes as small as ten. For three alleles, it is shown that a simpler formulation based on Leven's distribution approximates the exact test of this paper rather closely. However, it is recommended that researchers continue to employ the standard x2-test for all sample sizes and abide by it if the corresponding probability value is not "too close" to the critical level; otherwise, an exact test should be used.

The Location of Viability Genes within a Testcross Framework.

The maximum likelihood method is applied to the problem of estimating the positions and effects of viability genes. Whenever testcross linkage data indicate the presence of differential viability, it is hypothesized that there exists one viability gene between each marker. Estimation is possible only for two-point data since the number of independent expectation expressions is less than the number of parameters for three or more markers. It is pointed out that within the two-point testcross system, it is impossible to distinguish between pleiotropic effects of the marker genes and the effect of a middle viability gene, if existent. The methods outlined will be useful in their application to experiments specifically designed to locate induced viability genes.