Research
Molecular Ecology & Phylogenetics of silkmoths
Molecular phylogeny of silkmoths
Characterizing geographic patterns of genetic variation within and among populations is a necessary precursor to understanding mechanisms of population differentiation and speciation. The mitochondrial sequences are useful for describing such patterns since mitochondrial DNA is maternally inherited, does not recombine, and houses selectively neutral variation. Mitochondrial sequence variation provides a record of maternal lineages that allows inferences to be made about evolutionary processes such as demographic changes and dispersal. The mitochondrial genome of insects is circular, ~16kb in size and encodes for about 37 genes. More is known about the arrangement of mt genes for the class Insecta than for any other invertebrates. All the insects studied so far have the same arrangement of protein-coding genes, rRNA genes, and most tRNA genes. Mitochondrial genome is highly useful in understanding evolutionary relationships between species, because of its relatively simple genetic structure, and a rapid rate of sequence change. Very few studies describe nucleotide variation in the mitochondrial sequences at the interspecific level in silkmoth species and our objective was to investigate plausible progenitor of domesticated silkworm Bombyx mori and to understand the phylogenetic relationship between different saturniid and bombycoid silkmoth species based on mitochondrial sequences.
The present study suggests that the mitochondrial 12S, COI, and the CR are suitable for resolving the phylogenetic relationships of the species of the superfamily Bombycidae. The analysis based on chromosome number, transition bias ratio and tandem triplication of a 126-bp repeat element in mitochondrial DNA only in Japanese mandarina, suggest that B. mori must have diverged from its wild chinese relative. Another wild species of Bombycidae family, Theophila religiosa, appears to have shared a common ancestor along with B. mori and B. mandarina.
Figure 1: Maximum Likelihood tree inferred from all informative sites of COI for six silkmoth species and comparison of 126 bp repeat element in Japanese mandarina, Chinese mandarina, and B. mori. Arrow marks represent inverted repeats and their direction. The 64 bp repeat elements are represented in red and 62 bp elements in green.
Our studies on repeat structure of Antheraea roylei, A. pernyi, and A. proylei support the paternal inheritance of mitochondria. Interestingly, this diagnostic repeat structure is absent in other saturniids, A. assama, A. mylitta, and S. c. ricini. This repeat region is probably inserted after the divergence of A. pernyi and A. roylei from other saturniids. The phylogenetic analysis using three mitochondrial genes and CR shows that A. pernyi and A. roylei appear to have evolved recently. Previous reports performed cytogenetic assessment of A. pernyi, A. roylei, and A. proylei, and concluded that, in spite of allopatry and karyotypic divergence, a high degree of homology exists between the chromosomal complements of A. pernyi and A. roylei and A. pernyi possibly evolved from A. roylei through chromosomal fission. The results on mitochondrial sequence based phylogenetic analysis also suggest close relationship between the two species.
Conservation of microsatellites in heterologous silkmoth species
To test the conservation of B. mori microsatellite loci in heterologous species, we used 30 B. mori microsatellite loci. All species except H. armigera were from the Bombycoidea superfamily. Twenty-two (73.3%) B. mori microsatellite loci were found to be conserved in B. mandarina, the ancestral species of B. mori, and 16 (53.3%) were conserved in A. assama, an Indian muga silkmoth, endemic to northeastern India. The species of S. c. ricini, A. mylitta, A. yamamai, and H. armigera shared four (13.3%); A. pernyi shared three (10%); whereas A. roylei and A. proylei shared only two (6.6%) conserved microsatellite loci with B. mori.
Figure 2: Repeat regions of the four microsatellite loci are mapped onto phylogeny of the silkmoths. The number of repeat units is indicated as subscripts. The allele sizes (in base pairs) are given in parentheses. NA indicates no PCR amplification and ND represents alleles for which sequence could not be determined.
The cross-species amplification of silkworm microsatellite loci shows that mutation patterns of microsatellites are often complex: some loci tend to gain repeats, while others tend to lose repeats, and size variations at microsatellite loci are caused by indels in the flanking sequences as well. Such studies in lepidopteran microsatellites can be particularly useful for population studies and for increasing understanding of the origin, mutational processes, and structure of microsatellites, in addition to allowing more informed interpretation of genotyping data.
The successful amplification of silkworm SSR markers across related wild species particularly in B. mandarina and A. assama provides "connectivity" for future consolidation of genetic and physical maps and provides the foundation for association of these maps with particular traits of interest. Also, it provides an opportunity to use SSR markers for investigating the wide range of genetic diversity that exists in wild species outside the gene pool of domesticated silkworm, B. mori.
Projects being pursued
- Molecular ecology and phylogenetics of silkmoths and other insects
- Population genetic analysis of insects with special importance on silkmoths
Publications
- Johny S, Chakraborty S, Gadagkar R, Nagaraju J (2009) Polymorphic microsatellite loci for primitively eusocial wasp Ropalidia marginata. Molecular Ecology Resources 9: 2203-2205.
- Kanginakudru S, Metta M, Jakati RD, Nagaraju J (2008) Genetic evidence from Indian red jungle fowl corroborates multiple domestication of modern day chicken. BMC Evolutionary Biology 8:174.
- Biju SD, Bocxlaer IV, Giri VB, Roelants K, Nagaraju J, Bossuyt F (2007) A new rightfrog, Nyctibatrachus minimus sp. nov. (Anura: Nyctibatrachidae): The smallest frog from India. Current Science 93: 854-858.
- Arunkumar KP, Nagaraju J (2006) Unusually long palindromes are abundant in mitochondrial control regions of insects and nematodes. PLoS ONE 1: e110.
- Bocxlaer IV, Roelants K, Biju SD, Nagaraju J, Bossuyt F (2006) Late cretaceous vicariance in gondwanan amphibians. PLoS ONE 1: e74.
- Arunkumar KP, Metta M, Nagaraju J (2006) Molecular phylogeny of silkmoths reveals the origin of domesticated silkmoth, Bombyx mori from Chinese Bombyx mandarina and paternal inheritance of Antheraea proylei mitochondrial DNA. Molecular Phylogenetics and Evolution 40: 419-427.
- Nagaraja, Nagaraju J, Ranganath HA (2004) Molecular phylogeny of the nasuta subgroup of Drosophila based on 12S rRNA, 16S rRNA and CoI mitochondrial genes, RAPD and ISSR polymorphisms. Genes and Genetic Systems 79: 293-299.
- Nageswara Rao S, Muthulakshmi M, Kanginakudru S, Nagaraju J (2004) Phylogenetic relationships of three new microsporidian isolates from the silkworm, Bombyx mori. Journal of Invertebrate Pathology 86: 87-95.
- Nagaraju J (2004) Spider silks: A possible key to evolution of spiders. Heredity 93: 520-521.
- Prasad MD, Han SJ, Nagaraju J, Lee WJ, Brey PT (2003) Cloning and characterization of an eukaryotic initiation factor-2alpha kinase from the silkworm, Bombyx mori. Biochimica et Biophysica Acta 1628: 56-63.
- Prasad MD, Nagaraju J (2003) A comparative phylogenetic analysis of full-length mariner elements isolated from the Indian tasar silkmoth, Antheraea mylitta (Lepidoptera: saturniidae). Journal of Biosciences 28: 443-453.
- Prasad MD, Nurminsky DL, Nagaraju J (2002) Characterization and molecular phylogenetic analysis of mariner elements from wild and domesticated species of silkmoths. Molecular Phylogenetics and Evolution 25: 210-217.