Corresponding author: Keith R. Hopper ( email@example.com )
Academic editor: María Bressa
© 2017 Vladimir E. Gokhman, Kristen L. Kuhn, James B. Woolley, Keith R. Hopper.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Gokhman VE, Kuhn KL, Woolley JB, Hopper KR (2017) Variation in genome size and karyotype among closely related aphid parasitoids (Hymenoptera, Aphelinidae). Comparative Cytogenetics 11(1): 97-117. https://doi.org/10.3897/CcompCytogen.v11i1.10872
Genome sizes were measured and determined for the karyotypes of nine species of aphid parasitoids in the genus Aphelinus Dalman,1820. Large differences in genome size and karyotype were found between Aphelinus species, which is surprising given the similarity in their morphology and life history. Genome sizes estimated from flow cytometry were larger for species in the A. mali (Haldeman, 1851) complex than those for the species in the A. daucicola Kurdjumov, 1913 and A. varipes (Förster,1841) complexes. Haploid karyotypes of the A. daucicola and A. mali complexes comprised five metacentric chromosomes of similar size, whereas those of the A. varipes complex had four chromosomes, including a larger and a smaller metacentric chromosome and two small acrocentric chromosomes or a large metacentric and three smaller acrocentric chromosomes. Total lengths of female haploid chromosome sets correlated with genome sizes estimated from flow cytometry. Phylogenetic analysis of karyotypic variation revealed a chromosomal fusion together with pericentric inversions in the common ancestor of the A. varipes complex and further pericentric inversions in the clade comprising Aphelinus kurdjumovi Mercet, 1930 and Aphelinus hordei Kurdjumov, 1913. Fluorescence in situ hybridization with a 28S ribosomal DNA probe revealed a single site on chromosomes of the haploid karyotype of Aphelinus coreae Hopper & Woolley, 2012. The differences in genome size and total chromosome length between species complexes matched the phylogenetic divergence between them.
Aphelinidae, Aphelinus, parasitoid, genome size, flow cytometry, karyotype
Genome size estimates and karyotypic studies provide data for comparative research at various taxonomic levels and allow evaluation of phylogenetic associations (
Genome size estimates have been published for more than 13,000 species of animals and plants (Animal Genome Size Database, http://www.genomesize.com; Plant DNA C-values Database, http://data.kew.org/cvalues; accessed 29 August 2014). There are currently 930 estimates of insect genome size in the Animal Genome Size Database, 152 of which are for species of Hymenoptera, and these genome sizes range from 98 to 1115 Mb. Genome size is usually considered constant within species, and limited intraspecific variation is a standard assumption in measurement and comparison of genome sizes. However, genome size can vary widely between closely related species (
Here we report genome size estimates and karyotypes for males and females in nine species of Aphelinus Dalman, 1820 (Hymenoptera: Chalcidoidea: Aphelinidae) all of which are parasitoids of aphids. Parasitoids are free-living as adults, but are parasitic as larvae, and represent one of the most species-rich groups of insects, constituting more than 10% of all described insect species (
The parasitoid species studied, the sources of the colonies, and the permit and voucher numbers are listed in Table
The nine Aphelinus species studied, the year and country of their collection, permit and voucher numbers.
|Species complex||Species||Authority||Year||Country||Permit and voucher|
|A. varipes||A. atriplicis||Kurdjumov, 1913||2000||Georgia||P526P-15-04274, VGg00_Dn|
|A. varipes||(Förster, 1841)||2009||France||P526P-13-02503, VFr09_Rp|
|A. certus||Yasnosh, 1963||2001||Japan||P526P-01-53096, VJp01_TU|
|A. kurdjumovi||Mercet, 1930||2000||Georgia||P526P-13-02503, VGg00_Rp|
|A. hordei||Kurdjumov, 1913||2011||France||P526P-15-04274, VFr11_Dn|
|A. daucicola||A. daucicola||Kurdjumov, 1913||2013||USA||P526P-15-04274, DUSA12_UD|
|A. mali||A. glycinis||Hopper et Woolley, 2012||2007||China||P526P-08-02142, MKor09_M|
|A. coreae||Hopper et Woolley, 2012||2009||Korea||P526P-01-72318, MCh04_Bj|
|A. rhamni||Hopper et Woolley, 2012||2005||China||P526P-01-53096, MCh05_Bj|
Live Aphelinus were sexed, flash frozen in liquid nitrogen, and stored at −80°C. To estimate genome sizes, we used the flow cytometry protocol described by
The haploid content of DNA in megabases (Mb) was calculated for each Aphelinus sample from the ratio of mean fluorescence of the sample to mean fluorescence of the standard times the genome size of the standard. We report genome size estimates in megabases, but also give estimates in picograms (pg) calculated by dividing the amount of DNA in Mb by the standard 1C value of 978 Mb.
Chromosome preparations were made from cerebral ganglia of prepupae using a modified version of the technique in
A custom biotinylated fragment from the 28S rDNA gene was used to probe A. coreae chromosomes with fluorescence in situ hybridization (FISH). To prepare the probe, we extracted DNA from ~50 adult parasitoids using a Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA). From this DNA, we amplified a ~650 nt fragment of the 28S rDNA gene using the following primers and PCR protocol: reaction mix - 5 µl NEB PCR buffer and 0.5 µl Taq polymerase (New England Biolabs, Ipswich, MA, USA), 4 µl each of 2.5 mM dATP, dCTP, dGTP, 4 µl 0.25 mM dTTP plus 1 µl 1mM biotinylated-11-dUTP, 1 µl 10 µM forward primer (5’-cgt gtt gct tga tag tgc agc) and 1 µl 10 µM reverse primer (5’-tca aga cgg gtc ctg aaa gt), 4 µl genomic DNA (50 ng/µl), 21.5 µl ultrapure H2O; cycling - 3 min at 95 °C, then 35 cycles 95 °C for 30 sec, 55°C for 30 sec, and 72°C for 60 sec, and a final extension at 72 °C for 3 min. Unincorporated dNTPs, primers, and other unwanted components were removed from the PCR product using precipitation with sodium acetate and ethanol, and the resulting pellet was resuspended in 50 µl ultrapure H2O, yielding a solution of probe at 300 ng/µl.
Chromosomes were prepared for probing using the protocol described above for karyotyping. To probe the chromosomes, a protocol modified from
Genome sizes and total lengths of chromosome sets were compared among species and between sexes in generalized linear models with species and sex as fixed main-effects and Poisson error distributions using the glm function in R (R Core Team 2014). The set of relative lengths among species in a multivariate analysis of variance was compared with the Pillai–Bartlett statistic and the manova function in R. Centromeric indexes among species in generalized linear models were compared for each chromosome with species as a fixed effect and Poisson error distributions using the glm function in R. Because chromosomal formulae were different for the A. varipes complex versus the A. mali and A. daucicola complexes, we analyzed the effects of species on relative lengths and centromeric indexes separately within these groups. For genome size, the experimental unit was either 15 heads of female parasitoids and one D. melanogaster head pooled or 15 heads of male parasitoids and 15 heads of female parasitoids pooled. For total lengths of chromosome sets and relative lengths and centromeric indexes of chromosomes, the experimental unit was an individual mitotic cell. Post-hoc comparisons of means were done using the glht and cld functions in the multcomp package in R. We tested the relationships between genome sizes from flow cytometry and total lengths of chromosome sets with linear regression using the lm function in R. Data are archived on the Ag Data Commons website (data.nal.usda.gov; DOI 10.15482/USDA.ADC/1329930).
Haploid genome sizes of Aphelinus differed among species (model deviance = 444.0; residual deviance = 5.4; df = 6, 60; P < 0.0001). Female genome sizes ranged from 330 to 483 Mb so the largest was 1.5 times the smallest (Table
Haploid genome sizes of nine Aphelinus species estimated from flow cytometry. Shared letters after means indicates that they do not differ significantly.
|Species complex||Species||Sex||n replicates||Genome size||95% CI|
|A. varipes||A. atriplicis||female||6||0.361||353a||338–368|
|A. daucicola||A. daucicola||female||4||0.337||330a||313–348|
|A. mali||A. glycinis||female||6||0.442||432c||416–449|
Species in the A. varipes complex had four chromosomes in haploid males and thus eight chromosomes in diploid females, whereas species in the A. mali and A. daucicola complexes had five chromosomes in haploids and thus ten chromosomes in diploids (Table
Karyotypic features of nine Aphelinus species. Shared letters after means indicate that they do not differ significantly within each sex.
|species complex||species||number nuclei measured||number chromosomes||chromosomal formula||total length of chromosome set (µm)|
|mean||95% confidence interval|
|female||A. varipes||A. atriplicis||14||8||4M + 4A||15.6ac||13.7–17.9|
|A. varipes||16||8||4M + 4A||15.1ab||13.3–17.1|
|A. certus||19||8||4M + 4A||14.0a||12.4–15.8|
|A. kurdjumovi||13||8||2M + 6A||16.8ac||14.8–19.2|
|A. hordei||8||8||4M + 4A||14.3ab||11.9–17.1|
|A. daucicola||A. daucicola||15||10||10M||16.8ac||14.8–19.0|
|A. mali||A. glycinis||5||10||10M||19.6ac||16.1–23.9|
|male||A. varipes||A. atriplicis||3||4||2M + 2A||16.3ab||12.3–21.6|
|A. certus||3||4||2M + 2A||25.0bc||19.9–31.3|
|A. hordei||21||4||2M + 2A||17.8b||16.1–19.7|
|A. daucicola||A. daucicola||7||5||5M||23.7ac||20.4–27.6|
|A. mali||A. coreae||6||5||5M||29.8c||25.8–34.5|
Relative lengths of chromosomes in Aphelinus species. Means with 95% confidence intervals in parentheses.
|A. varipes||A. atriplicis||40||26||18||16|
|A. daucicola||A. daucicola||24||22||19||18||18|
|A. mali||A. glycinis||24||22||21||18||18|
Centromeric indexes of chromosomes in Aphelinus species. Means (95% confidence intervals); shared letters within a species complex and chromosome indicate means that are not significantly different.
|A. varipes||A. atriplicis||46a||47b||0||0|
|A. mali and A. daucicola||A. daucicola||45a||45a||41a||39a||38a|
Haploid mitotic karyograms of six Aphelinus species. a A. atriplicis b A. certus c A. hordei d A. coreae e A. rhamni f A. daucicola. Species in the A. varipes complex have n = 4 versus n = 5 in the A. mali and A. daucicola complexes. Scale bar: 10 µm.
Diploid mitotic karyograms of nine Aphelinus species. a A. atriplicis b A. certus c A. hordei d A. kurdjumovi e A. varipes f A. coreae g A. glycinis h A. rhamni, i A. daucicola. Species in the A. varipes complex have 2n = 8 versus 2n = 10 in the A. mali and A. daucicola complexes. Scale bar: 10 µm.
Centromeric indexes for chromosome 1 did not differ among species, and centromeric indexes for chromosome 2 did not differ among species in the A. mali and A. daucicola complexes. However, in the A. varipes complex, the centromeric index of chromosome 2 in A. hordei was significantly lower than in other members of the A. varipes complex. Centromeric indexes for chromosomes 3 and 4 in A. daucicola were significantly lower than those for A. rhamni, and the centromeric index for chromosome 5 in A. daucicola was significantly lower than those for A. coreae and A. rhamni (Table
Analysis of deviance for differences in centromeric indexes among species of Aphelinus; acrocentric chromosomes were not included in these analyses.
|A. mali and A. daucicola||1||3||1.8||86||20.7||0.62|
Total length of chromosome set (µm) versus flow cytometry genome size (Mb) for nine Aphelinus species. Error bars are 95% confidence intervals.
Hybridization with a 28S rDNA probe revealed a single rDNA cluster on chromosomes of the haploid set and two rDNA clusters in the diploid set (Fig.
Fluorescence in situ hybridization with 28S rDNA probe. a metaphase chromosomes of the haploid karyotype and b prometaphase chromosomes of the diploid karyotype of A. coreae. Red = hybridization signal (a single rDNA cluster in the haploid set and paired clusters in the diploid set), blue = counterstaining of chromosomes with DAPI.
The large genome size differences between the A. varipes complex versus the A. mali complex matched the phylogenetic divergence between these complexes (
Genome size has been hypothesized to depend on several factors, including eusociality, parasitism, and developmental biology in insects (
Mapping karyotypic data on a molecular phylogeny of Aphelinus and two outgroup species allowed reconstruction of karyotype evolution in the species we studied (Fig.
Chromosomal formulae and genome sizes on phylogeny of Aphelinus species and several outgroups. Blue = 5 metacentric chromosomes; aqua = 1 metacentric and 4 acrocentric chromosomes; green = 2 metacentric and 2 acrocentric chromosomes; dark green = 1 metacentric and 3 acrocentric chromosomes; yellow = 2 metacentric, 1 subtelocentric, and 1 acrocentric chromosome; grey = unknown. Numbers after species names are genome sizes estimated from flow cytometry; values for Aphelinus abdominalis and E. formosa are from (
Chromosomes in the A. varipes complex differ from those in the A. mali complex in relative length and centromeric indices. The longest metacentric chromosome in species in the A. varipes complex is much longer than the other chromosomes. We suggest that this metacentric chromosome resulted from a fusion of two smaller chromosomes from an ancestral karyotype with five chromosomes. Species in the A. varipes complex have two smaller acrocentrics that, in turn, could originate from metacentric chromosomes of the ancestral karyotype via pericentric inversions. Moreover, the position of the centromere of the second largest chromosome underwent further changes in two sister species in the A. varipes complex, A. kurdjumovi and A. hordei. The centromere is significantly shifted in A. hordei (CI= 41 versus 46 in A. certus, and 47 in A. atriplicis and A. varipes), and is further moved to a terminal position in A. kurdjumovi (CI= 0). We propose that consecutive pericentric inversions in A. hordei and A. kurdjumovi would be the most parsimonious explanation. These chromosomal rearrangements in the A. varipes complex are an example of a general trend in karyotype evolution in parasitic Hymenoptera, namely, karyotypic dissymmetrization, which involves an increase in size differentiation between chromosomes and an increase in the proportion of acrocentric chromosomes (
A recent review of the distribution of rDNA sites on chromosomes of parasitic Hymenoptera showed that the number of these sites correlates with chromosome number (
Total chromosomal length was correlated with genome size in Aphelinus, but this was because of the difference in chromosome length between the A. mali and A. varipes complexes. Although a similar correlation was found for species in the family Figitidae (
Differences as large as 44% were found in genome size between Aphelinus species, which is surprising given the similarity in their morphology and life history. Mean total chromosome length correlated with mean genome size. The differences in genome size and total chromosome length between species complexes matched the phylogenetic divergence between species complexes. Chromosomal rearrangements in the A. varipes complex are an example of karyotypic dissymmetrization, which involves an increase in size differentiation between chromosomes and an increase in the proportion of acrocentric chromosomes, which is a general trend in karyotype evolution in parasitic Hymenoptera.
We thank Kathryn Lanier and Joshua Rhoades, USDA-ARS, Newark, Delaware, for rearing the Aphelinus cultures. We thank Deni Galileo in the CTCR Core Facility, University of Delaware, for assistance with the flow cytometer analyses and Jeffrey Caplan and Jean Ross in the Bio-Imaging Center, Delaware Biotechnology Institute, for providing facilities and assistance with imaging of fluorescence in situ hybridizations. These core facilities are supported by the Delaware INBRE program, with a grant from the National Institute of General Medical Sciences grant (P20 GM103446) from the National Institutes of Health and the state of Delaware and by a National Science Foundation EPSCoR grant (IIA-1301765). This research was funded by the United States Department of Agriculture, Agriculture Research Service and by Award DEB 1257601 from the National Science Foundation to JBW and KRH and Award 15-04-07709 from the Russian Foundation for Basic Research to VEG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.