The aim of this study was to investigate the level of evolutionary divergence among five isofemale lineages of Metopolophium dirhodum Walker (Hemiptera: Aphididae) using sequences of the Internal Transcribed Spacer (ITS) of ribosomal DNA (rDNA). These isofemalelineages were derived from parthenogenetic females, previously genetically characterized by having strong association of RAPD (Random Amplified Polymorphic DNA) molecular markers and host plant preference. Out of 25 sequences, only 17 containing ITS2 (the second spacer) were used for analysis due to their quality. Within 250 nucleotide sites originated by multiple alignments, 10.4% were variable, with the predominance of indels over substitutions. The average of nucleotide diversity (π) within isofemale lineages was similar to the diversity found among all isofemale lineages. The topology inferred by the Neighbor-Joining method showed that no evolutionary divergence inferred by ITS2 sequences among isofemale lineages typified by RAPD could be associated with host preference. The ITS2 sequence differences found within lineages and their causes are discussed.
Metopolophium dirhodum (Walker, 1849) is an important pest of cereals, not only because of the direct damage that this aphid causes by sap suction but also indirectly by transmission of plant virus and losses in cereal production are reported between 7% - 17 % [
There are no records of the occurrence of the sexual phase in Brazil [
Insect species that cyclically alternate sexual and asexual phases, as aphids, provide an excellent opportunity to study changes in multi-gene families, which may have arisen after reproductive isolation periods [
In parthenogenetic organisms, rDNA may be homogeneous due to recombination [
Despite variation of ITS, which is a limitation for phylogenetic studies of closely related species, ITS1 and ITS2 sequences are useful to detect cryptic species or even species before their formal description [
The primary interest of this investigation was to verify the existence of intragenomic variability in the internal transcribed spacer sequence of rDNA within and among isofemales lineages (clonal lineages derived from a single female clone) collected in cereal producing regions in southern Brazil [
The aphid lineages used in this work were selected based on their differences in the RAPD profiles, which DNA polymorphisms showed a strong relationship to the host-plant field preference of individual clones [
DNA extraction was performed according to Carvalho and Vieira’s protocol [
Clonal lineage ID | Field and sequencing data | ||
---|---|---|---|
Sequence codes | Collection site | Genbank accession | |
O6 | O6-1, O6-2, O6-3, O6-4 | Panambi, RS, Brazil | EU685264, EU685265, EU685266, EU685267 |
O14 | O14-1, O14-2, O14-3 | Selbach, RS, Brazil | EU685268, EU685269, EU685270 |
W7 | W7-1, W7-2 | Ijuí, RS, Brazil | EU685254, EU685255 |
W9 | W9-1, W9-2, W9-3 | Cruz Alta,RS, Brazil | EU685256, EU685257, EU685258 |
W10 | W10-1, W10-2, W10-3, W10-4, W10-5 | El Batan, Mexico | EU685259, EU685260, EU685261, EU685262, EU685263 |
DNA extraction with reasonable quality for analysis. Five clones of each lineage from a single parthenogenetic female were macerated in 1.5 mL microtubes with a 200 mL extraction buffer (200 mM Tris-HCl, pH 8.0; 2 M NaCl; 0.5 M EDTA) and 50 mL sarcosyl. The resultant suspension was heated on a warming plate (65˚C) for 30 min and centrifuged at 10,000 rpm for 15 min. The supernatant was poured into a new microtube, and 110 mL ammonium acetate (10 M) with 250 mL cold isopropanol was added and both are necessary for DNA precipitation at −20˚C for 8h. After a freezing process, the microtubes were centrifuged at 10,000 rpm during 15 min. The DNA pellets were washed with 70% ethanol, re-suspended by addition of a 25 µL TE buffer (Tris-EDTA, pH 8.0 with RNAse) and then stored at −20˚C. The PCR reactions were carried out in 0.6mL microcentrifuge tubes containing 400 mM of dNTP, 2.5 mM of primers ITS-4: 5’-TCCTCCGCTTTATTGATATGC- 3’ and ITS-5: 5’AAGTAAAAGTCGTAACAAGG-3’ [
After gel staining with ethidium bromide, the band was excised by the “freezing-thaw” method, and recovered inside a 1.5 mL microtube and DNA was precipitated using sodium acetate 3 M (pH 5.2, 10% v/v) and 2× the amount of absolute ethanol at 0˚C. After centrifugation (10,000 rpm) for 5 min, the precipitate was washed with 200 mL ethanol 70% and dried at room temperature. DNA was re-suspended in 10 mL TE buffer +RNAse. The fragment of the ITS2 region was cloned using the vector PCR 2.1-TOPOâ cloning kit (Invitrogen™), according with manufacturer’s specifications and inserted in the Escherichia coli strain DH5-α by electroporation. Five transformed clones per Petri dish were selected each one representing one copy of ITS2 of each isofemale lineage of M. dirhodum. Plamidial DNA was extracted by alkaline lise [
DNA sequence alignments were performed using the Clustal W package with a gap-opening penalty of 5 and a gap-extension penalty of 2. Manual adjustments were made with the BioEdit software [
Nucleotide diversity (p) was calculated as: p = P/L, where P = average nucleotide differences between two sequences randomly taken from the population and L = length of sequence (number of nucleotides) [
The phylogenetic tree was drawn using the MEGA 2.1 software (Molecular Evolution Genetic Analysis) [
Acyrthosiphon pisum Harris (Hemiptera: Aphididae) was chosen as an out-group because it is the species which is phylogenetically most closely related to M. dirhodum [
From 25 sequences obtained, only 17 were utilized for ITS2 analysis due to their sequence quality. Although sequence information of over 900 bp from the internal transcribed spacer region (ITS1, 5.8S and ITS2) was obtained, only sequences from the ITS2 region were considered in this study. These sequences ranged from 236 to 243 bp, resulting in 250 positions suitable for analysis in multiple alignments.
The sequences differed by six substitutions (three transversions and three transitions) and they presented 21 indels. In the position 221 of multiple alignment was observed a substitution and a deletion. Differently from human lice (P. humanus) where the occurrence of deletions of 10 - 25 bp has been reported [
The nucleotide composition of the M. dirhodum ITS2 partial fragment reveals a predominance of C:G (approximately 70%). The predominance of C:G over A:T in the ITS region is common, with the exception of Drosophila, where the percentage of G:C content of the ITS2 ranges from 18% to 26% [
Overall, the most divergent distance between lineages was the pair W9-W10 (they shared the same host). The less divergent was W7-014 (isofemales from different host species). The Mexican line (W3) had greatest genetic distances among their sequences and also from sequences of other isofemale lines (
The average ITS2 nucleotide diversity within an isofemale lineage was similar to the diversity found among the five lineages. Within the clonal lineages previously characterized by RAPD, the lowest nucleotide diversity was within lineage W9 (pi = 1.3%), and the highest within clonal lineage O14 (pI = 3.3%). Between clonal lineages, the highest p was found within pairs that contained O14 lineage, especially the sequence pair O14-1 and O6-2 (p = 5.0%).
These results showed that the nucleotide diversity within the ITS2 region of M. dirhodum is distributed homogeneously within (pi) and among lineages (pe) (
The amount of variable sites found within ITS2 sequences of M. dirhodum clonal lineages analyzed in this work (10.4%) was less than that found in Pediculus humanus (34.4 %) [
Clonal Lineage ID | D |
---|---|
O6 | 0.00425 |
O14 | 0.00281 |
W7 | 0.00768 |
W9 | 0.00212 |
W10 | 0.00564 |
O6 | O14 | W7 | W9 | W10 | |
---|---|---|---|---|---|
O6 | 0.00000 | ||||
O14 | 0.00282 | 0.00000 | |||
W7 | 0.00318 | 0.00211 | 0.00000 | ||
W9 | 0.00495 | 0.00423 | 0.00390 | 0.00000 | |
W10 | 0.00725 | 0.00596 | 0.00620 | 0.00797 | 0.00000 |
Index | Value (%) |
---|---|
1.98 | |
1.83 | |
1.74 |
the existence of six-point mutations within ITS1 of M. persicae [
Some organisms have highly conserved ITS2 sequences, such as Drosophila (Diptera) species [
Variation is not found only in non-coding regions as ITS, IGS and External Transcribed Spacers and a highly divergent rDNA groups was found within single individuals of Podisma pedestris (Orthoptera: Acrididae) [
The consensus bootstrap neighbor-joining tree obtained after 1000 bootstraps replicates indicated that the hypothesis of monophyly associated to the host plant should be rejected (
Host-race evolution is described in grass aphids like Schizaphis graminum (Rondani) which divergence among sequences of cytochrome oxidase I (mitochondrial gene) among biotypes were consistently observed [
The particularities of aphid reproduction systems have important evolutionary consequences. The genetic variation that appears within obligatory parthenogenetic clones is not caused only by mutations. Exclusively asexual lineages can produce males, which only mate with females of sexual lineages, giving rise to new asexual and sexual lineages [
chromosomes) [
In this present work, it was showed for the first time that intragenomic ITS2 sequence variation occurs within M. dirhodum. This finding lends support to the hypothesis of rapid changes in clonal lineages of aphids [
The variation in ITS2 sequences of the ITS2 region in M. dirhodum populations collected in southern Brazil suggests that concerted evolution has not homogenized all rDNA copies within individuals. Despite the level of intragenomic variation found, based on information obtained from ITS2 sequences, the previously RAPD typified isofemales lineages are not evolutionary divergent by host use or preference.
High diversity in ITS regions of rDNA is a clear indication of occurrence of sexual phase in M. dirhodum at certain level. In the case of aphids, this can be a source of new clonal variants and high probability of emergence of new-host races. This aspect of biology of M. dirhodum in South America is important especially for plant breeding programs to incorporate resistance to this pest.
We are indebted to Hugo Bruno Molinari and Elizabeth Keiko Takahashi for their assistance in the work of cloning and sequencing and, Rick Harder for his review of this article.