Paul T. Smith

PCR primers for the amplification of four insect mitochondrial gene fragments.

Insect mitochondrial genome (mtDNA) analysis is a powerful tool for the study of population genetics and phylogenetics. In the past few years primer sequences for the PCR amplification of various insect mtDNA genes have been published. The objectives of this study were (1) present new primer sequences for six insect mitochondrial genes and (2) test primers designed in our laboratory and some previously published primers on a wide range of insects to determine if amplification of the target fragment could be obtained. The primers for the amplification of the two ribosomal RNA gene (16S and 12S rRNA) fragments are universal for insects and related groups; the primers for NADH5 and NADH4 dehydrogenase gene fragments and cytochrome c oxidase I gene fragment are applicable broadly.

Phylogenetic relationships among Bactrocera species (Diptera: Tephritidae) inferred from mitochondrial DNA sequences

Several members of the dipteran family Tephritdae are serious pests because females lay eggs in ripening fruit. The genus Bactrocera is one of the largest within the family with over 500 described species arranged in 28 subgenera. The phylogenetic relationships among the various species and subgenera, and the monophyly of specific groups have not been examined using a rigorous phylogenetic analysis. Therefore, phylogenetic relationships among 24 Bactrocera species belonging to 9 subgenera were inferred from DNA sequence of portions of the mitochondrial 16S rRNA, cytochrome oxidase II, tRNA(Lys), and tRNA(Asp) genes. Two morphological characters that traditionally have been used to define the four groups within the subgenus Bactrocera were evaluated in a phylogenetic context by mapping the character states onto the parsimony tree. In addition, the evolutionary trend in male-lure response was evaluated in a phylogenetic context. Maximum parsimony analyses suggested the following relationships: (1) the genus Bactrocera is monophyletic, (2) the subgenus B. (Zeugodacus) is paraphyletic, (3) the subgenus B. (Daculus) is a sister group to subgenus B. (Bactrocera), and (4) the subgenus B. (Bactrocera) is monophyletic. The mapping analyses suggested that the morphological characters exhibit a simple evolutionary transition from one character state to another. Male-lure response was identified as being a labile behavior that has been lost on multiple occasions. Cue-lure response was plesiomorphic to methyl-eugenol response, and the latter has evolved independently within the Bactrocera and Zeugodacus groups of subgenera. The implications of our results for devising a coherent, consolidated classification for Bactrocera is discussed.

Phylogenetic Analysis of Mitochondrial DNA Supports the Monophyly of Dacini Fruit Flies (Diptera: Tephritidae)

Abstract The tribe Dacini (Diptera: Tephritidae: Trypetinae) currently consists of ≈40 genera and 1,000 described species. Among others, it includes the genera Bactrocera, Ceratitis, and Dacus, all of which are pests of agricultural importance because they oviposit in ripening fruit. Although previous studies, based on morphological and molecular data, suggested that the members of Dacini are monophyletic, no extensive molecular phylogenetic studies have been published on Bactrocera, Ceratitis, or Dacus. Therefore, we undertook a phylogenetic analysis of 34 tephritid taxa, including 16 species of the tribe Dacini, utilizing 1,391 bp from the mitochondrial 16S rRNA, 12S rRNA, and cytochrome oxidase II + tRNALys + tRNAAsp genes. Combined maximum parsimony analysis suggested the following with strong quantitative support: (1) the subfamilies Tephritinae and Trypetinae are monophyletic, (2) Dacini is monophyletic, and (3) members of the subtribe Dacina are monophyletic. In addition, Ceratitidina + Gastrozonina are basal to Dacina. Within Dacina, Dacus and Bactrocera were each monophyletic. The results obtained here are generally congruent with those based on morphology.

Mitochondrial DNA variation among host races of Eurosta solidaginis Fitch (Diptera: Tephritidae)

Eurosta solidaginis Fitch (Diptera: Tephritidae) induces galls on two species of goldenrod, Solidago (Compositae), in the northern regions of the United States. Recent studies have demonstrated that E. solidaginis is comprised of two host races that differ in adult emergence times, mate preference, and host preference. However, it is not known how much genetic variation, if any, exists among E. solidaginis host-associated populations west of Minnesota where the two host races occur in sympatry. Sequencing analysis was used to characterize two mitochondrial gene fragments: (1) NADH1 dehydrogenase (ND1: 539 bp) and (2) cytochrome oxidase II + tRNA(Lys) + tRNA(Asp) (CO2KD: 396 bp) from sympatric, host-associated populations of E. solidaginis in North Dakota. Our results indicated that two genetically distinct lineages exist among E. solidaginis in North Dakota that correspond with host-plant association.

The bee-killing flies, genus Melaloncha Brues (Diptera: Phoridae): a combined molecular and morphological phylogeny

The phylogeny of the bee‐killing flies, genus Melaloncha Brues (Diptera: Phoridae) is analysed using six genes –cytochrome oxidase I, 16S ribosomal DNA, 12S ribosomal DNA, NADH1 dehydrogenase, 28S ribosomal DNA and CAD– plus 47 morphological characters. A total of 91 specimens, including eight out‐groups and 83 Melaloncha (representing 70 species) were included in the analyses. Parsimony analysis of the combined data set produced a single most parsimonious tree with varied Bremer and bootstrap support of interior nodes. Bayesian analysis of molecules only and of morphology + molecules produced trees largely in agreement with parsimony results, although with a few differences. Supported groups included subfamily Metopininae, genus Melaloncha, and subgenera Melaloncha s.s. and Melaloncha (Udamochiras) Enderlein. Within the subgenera, the previously recognized Melaloncha furcata, Melaloncha cingulata, Melaloncha ungulata and Melaloncha stylata groups were recovered, as well as some new groupings. The M. furcata group was placed as the sister group of other Melaloncha s.s., which is consistent with known host‐attacking behaviour.

Molecular Evidence for Parthenogenesis in the Sierra Garter Snake, Thamnophis couchii (Colubridae)

Abstract We used microsatellite analysis to investigate possible reproduction by parthenogenesis in the bisexual Sierra garter snake, Thamnophis couchii. The genotypes of four microsatellite loci were determined for each of four individuals, including three T. couchii, two of which were a female and her offspring, and T. ordinoides. The female T. couchii and her offspring were homozygous and identical for all four microsatellite loci; however, Ts3 and Ts4 are the only loci that supported reproduction by parthenogenesis as Ts1 and Ts2 were uninformative. These data along with absence of a unique paternal allele and known absence of an opportunity to mate prior to and during captivity is consistent with reproduction by parthenogenesis.

Utility of DNA Sequences for Inferring Phylogenetic Relationships and Associating Morphologically Dissimilar Males and Females of the Bee-Killing Flies, Genus Melaloncha (Diptera: Phoridae)

Abstract The bee-killing flies, genus Melaloncha, are parasitoids of bees of the family Apidae, including stingless bees, bumble bees, and the western honey bee, Apis mellifera L. With >160 described species, Melaloncha is among the largest genera in the family Phoridae. Most species are known only from females as the males typically show too few differences to be useful in characterizing species. The monophyly of Melaloncha and its two subgenera (Udamochiras and Melaloncha s.s.) is strongly supported by morphological characters, but the phylogenetic relationships among the various species and species groups are not well understood. Here, we report on a preliminary molecular phylogenetic study of 30 exemplar Melaloncha species representing both subgenera and seven species groups by using a combination of nuclear (28S and CAD) and mitochondrial (12S, 16S, ND1, and CO1) genes for a total of 3,306 bp. Maximum parsimony analysis suggested the following relationships: 1) Melaloncha is monophyletic, 2) the subgenus M. (Udamochiras) is monophyletic and is a sister-group to M. (Melaloncha), and 3) each of the species groups for which we had multiple species were monophyletic with the M. furcata-group being the sister-group to all other Group II taxa. These results support hypotheses of relationships based on morphological characters. The utility of the molecular data for associating morphologically dissimilar males and females is discussed.

A Molecular Phylogenetic Analysis of the Genus Dohrniphora (Diptera: Phoridae)

ABSTRACT The genus Dohrniphora Dahl 1898 (Diptera: Phoridae) is an extremely diverse assemblage of species that exhibit fascinating larval lifestyles, including, scavenging, fungivory, kleptoparasitism, predation, and parasitism. Moreover, some species are known to exhibit interesting associations with social insects. Here we report on the first in-depth molecular phylogenetic study of the genus Dohrniphora based on 2,327 bp of DNA sequence data from portions of the nuclear CAD (rudimentary) and mitochondrial ND1, CO1, and 16S rDNA genes. Maximum parsimony and Bayesian analysis each recovered Dohrniphora as a monophyletic group. Within Dohrniphora, the New World species were paraphyletic with respect to the Old World clade. We also provide evidence that members of the termite-parasitizing genus Dicranopteron Schmitz 1931 are simply highly specialized members of Dohrniphora and revive the species status of Dohrniphora obscuriventris Borgmeier 1925. Finally, biogeography and hind femoral morphology are examined in a phylogenetic context.

FOOD HABITS OF THE BLUNT-NOSED LEOPARD LIZARD (GAMBELIA SILA)

Abstract We quantified the food habits of the endangered blunt-nosed leopard lizard (Gambelia sila), an endemic species of the San Joaquin Valley, California. Based on analyses of stomach contents and prey remains in scats, we determined that this species is an opportunistic predator that eats large numbers of orthopterans, coleopterans, and hymenopterans, with dipterans, hemipterans, and lizards taken in lesser numbers. These data are similar to the qualitative or unpublished data of previous studies, and also are similar to the food habits of the congeneric Gambelia wislizenii.

Genetic Variation Among Populations of the Endangered Giant Kangaroo Rat, Dipodomys ingens, in the Southern San Joaquin Valley

Abstract Populations of the endangered giant kangaroo rat (Dipodomys ingens) have decreased because of habitat fragmentation and isolation over the past 100 y. Changes in population structure due to habitat fragmentation can significantly affect the population size and the dispersal of these animals compromising long-term sustainability of each fragmented population. We collected small ear clippings from male and female giant kangaroo rats from six sites in the southern San Joaquin Valley to determine the genetic population structure of this species in this part of their range. Multilocus F-statistics indicated that the six populations of giant kangaroo rat are not composed of randomly mating populations and that genetic drift and inbreeding are major determinants of population structure. Furthermore, F-statistics confirmed a significant decrease in observed heterozygosity. Genetic distance analyses did not support the hypothesis that geographically distant populations would exhibit greater genetic differentiation. We also compared our data to published estimates of genetic diversity of giant kangaroo rats in populations to the west and north, the other large population centers of this species.