Corresponding author: Rimantas Rakauskas (
Academic editor: R. Blackman
Partial sequences of the mitochondrial COI gene of forty eight European and two Turkish population samples of
Rakauskas R, Havelka J, Zaremba A, Bernotienė R (2014) Mitochondrial COI and morphological evidence for host specificity of the black cherry aphids
Black cherry aphid
A similar species complex is found in the mealy plum aphid,
The
Fifty population samples of apterous viviparae of black cherry aphids from nine European countries and Turkey were collected in 2004–2013, mostly from
Aphid material used in the present study. Samples used for morphology-based discriminant analysis are given in bold.
Place, date, collection number; (number of individual apterae per sample) | GenBank Accession No |
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Alytus, Lithuania, 2012.05.30, 12-30; (8) |
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Molėtai, Lithuania, 2012.06.19, 12-72; (8) |
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Kraujaliai, Molėtai distr., Lithuania, 2012.07.10, 12-120; (8) |
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Žičkai, Molėtai distr., Lithuania, 2012.07.13, 12-132; (8) |
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Bagnolo Mella, Brescia prov., Italy, 2013.05.01, 13-27; (8) |
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Poncarale, Brescia prov., Italy, 2013.05.02, 13-33; (8) |
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Suginčiai, Molėtai distr., Lithuania, 2013.06.15, 13-83; (8) |
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Akmeniai, Lazdijai distr., Lithuania, 2013.05.30, 13-57; (8) |
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Karsava, Ludza distr., Latvia, 2013.07.17, 13-133; (8) |
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Gorodok, Vitebsk distr., Belarus, 2008.06.17, 08-6; (6) |
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Zadrachje, Vitebsk distr., Belarus, 2008.06.18, 08-18; (8) |
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Riga, Latvia, 2008.07.03, 08-73; (8) |
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Cluj Gilau, Romania, 2012.06.19, Z12-116; (8) |
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Poncarale, Brescia prov., Italy, 2013.05.02, 13-30; (8) |
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Mezöpeterd, Hajdu-Bihar distr., Hungary, 2012.06.20, Z12-122; (8) |
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Skirgiškės, Vilnius distr., Lithuania, 2012.06.05, 12-39; (7) |
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Frankfurt/Main, Germany, 2012.06.30, 12-104; (8) |
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Preila, Neringa, Lithuania, 2012.08.13, 12-199; (7) |
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Rondo, Katowice, Poland, 2011.05.13, 11-10; (8) |
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Tekir, Karamanmarash distr., Turkey, 2011.05.21, 11-25; (5) |
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Göksun, Karamanmarash distr., Turkey, 2011.05.21, 11-27; (8) |
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Zafferana, Catania, Italy, 2004.06.28, 04-49; (5) |
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Costinesti, Romania, 2012.06.15, Z12-90; (8) |
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Cluj Gilau, Romania, 2012.06.19, Z12-117; (7) |
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Carpendolo, Brescia prov., Italy, 2013.04.27, 13-12; (7) |
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Akmeniai, Lazdijai distr., Lithuania, 2013.05.30, 13-60; (8) |
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Wojslawice, Lower Silesia, Poland, 2013.06.20, 13-98; (8) |
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Wojslawice, Lower Silesia, Poland, 2013.06.20, 13-97; (8) |
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Dobele, Latvia, 2013.07.03, 13-119; (8) |
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Medias, Sibiu distr., Romania, 2012.06.19, Z12-113; (8) |
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Zmejinyje ostrova, Kanev distr., Cherkasy reg., Ukraine, 2006.06.16, 06-74 |
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For molecular analysis, a single aphid individual from one sampled plant was considered as a unique sample. Total genomic DNA was extracted from a single aphid using the DNeasy Blood & Tissue kit (Qiagen), which involved at least a 2 h digestion of tissue with proteinase K. Partial sequences of mitochondrial COI gene were PCR-amplified using earlier published primers (
In addition to the sequences from 50 samples of
Samples representing different clades in the molecular tree and haplotype network were used for stepwise discriminant analysis followed by canonical analysis: 10 samples from sour cherry,
Based on earlier taxonomic work (
Measurements of the slide-mounted apterous viviparous females were performed by means of interactive measurement system Micro-Image (Olympus Optical Co. GmbH). STATISTICA 8 version software (
Morphological characters that contributed most to canonical discrimination functions were evaluated as having potential for separation of taxa. An identification key was constructed based on these discrimination functions. The key was then tested on the 30 aphid samples that had not been used in its construction (listed in normal font in
Fifty partial COI sequences of
Five COI haplotypes were detected (
Haplotype network (TCS 1.21 software:
COI haplotypes of three
Haplotype number | Number of sequences | Sequence length (bp) | Sample numbers |
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1 | 2 | 616 | 13-33; 13-27. |
2 | 23 | 616 | 08-6;12-32; 12-176; 12-30; 08-18; z12-122; 12-120; z12-112; 12-132; 12-25; 12-43; 12-191; 12-203; 12-70; 12-72; 08-73; 13-83; 13-133; 11-46; z12-116; 12-37; 13-30; 13-57 |
3 | 23 | 616 | 11-10; 12-39; z12-110; 12-182; 12-104; 12-56; 12-83; 12-111; 12-199; z12-90; z12-102; 12-48; 12-188; z12-113 ( |
4 | 2 | 616 | 11-27; 11-25 |
5 | 1 | 616 | 06-74 |
Range of pairwise interspecific sample divergences of mitochondrial COI gene fragment (K2P model) for three species of
Species 1 | Species 2 | Range of divergence, % |
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0.2 – 0.5 | ||
6.6 – 6.8 | ||
6.8 |
The maximum parsimony (MP) analysis of partial COI sequences resulted in 930 equally parsimonious trees (length = 43, CI = 1.00, RI = 1.00). The ML tree (T92 model) showed similar topology, as did NJ (K2P distances) and BI (HKY+I+G model) analyses. NJ, MP and ML bootstrap values over 50% together with BI posterior probabilities over 0.50 are given at respective nodes of the same tree in
Maximum likelihood (ML) tree showing phylogenetic relationships among
When morphometric data of apterous viviparous females from 20 different geographical localities were subjected to discriminant analysis with sample collection number as the grouping variable, the first two canonical variates (
Plot of the mean scores of the first two canonical variates for 20 samples of
Contributions of 11 morphological characters to the canonical function discriminating 20 samples of
Wilks’ Lambda | Partial Wilks’ Lambda | F-remove | p-level | Toler. | 1-Toler. (R-Sqr.) | |
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0.34 | 0.65 | 72.00 | 0.00 | 0.46 | 0.54 |
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0.30 | 0.73 | 49.26 | 0.00 | 0.31 | 0.69 |
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0.27 | 0.83 | 28.31 | 0.00 | 0.79 | 0.21 |
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0.24 | 0.91 | 12.99 | 0.00 | 0.71 | 0.29 |
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0.23 | 0.93 | 9.51 | 0.00 | 0.65 | 0.35 |
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0.23 | 0.94 | 8.69 | 0.00 | 0.47 | 0.53 |
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0.23 | 0.97 | 4.62 | 0.01 | 0.49 | 0.51 |
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0.23 | 0.97 | 4.23 | 0.02 | 0.86 | 0.14 |
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0.23 | 0.97 | 3.70 | 0.03 | 0.85 | 0.15 |
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0.22 | 0.98 | 2.38 | 0.10 | 0.57 | 0.43 |
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0.22 | 0.99 | 1.75 | 0.18 | 0.56 | 0.44 |
To discriminate between apterous viviparous females of host-specific black cherry aphid samples representing different clades in the haplotype network and the phylogenetic tree (
Plot of the mean scores of the individual LDF values (number of specimens per sample is given in
1 | Value of LDF [3.92× (length of siphunculus) - 5. 67×(length of terminal process of antennal segment 6) - 32.55×(maximal length of the ventral body hairs) + 1] greater than zero. On |
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– | Value of LDF less than zero. On |
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The combination of genetic distance evaluation with phylogenetic tree-building methods and multivariate analyses of morphometric data has been successfully applied to solve taxonomic problems in aphids, particularly in the genera
Pairwise sample divergences of 1145 bp mitochondrial COI gene fragment (K2P model) between three subspecies of
Subspecies 1 | Subspecies 2 | Mean and range of divergence, % |
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1.2 (0.97–1.42) | ||
0.15 (0.00–0.26) | ||
1.05 (0.97–1.15) |
This research was funded by a grant (No LEK-04/2012) from the Research Council of Lithuania. Kind assistance of S. Barbagallo and G. Cocuzza (Catania, Sicily), V. Zhuravlev (Kyiv, Ukraine), S. Buga (Minsk, Belarus), V. Spuņģis (Riga, Latvia), A. Stalažs (Dobele, Latvia), M. Badini (Bagnolo Mella, Italy), A. A. Işıkber and M. M. Aslan, (Kahramanmaraş, Turkey) during field sampling in respective countries cannot be overestimated.