Mitochondrial COI and morphological specificity of the mealy aphids (Hyalopterus ssp.) collected from different hosts in Europe (Hemiptera, Aphididae)

Abstract Forty three European population samples of mealy aphids from various winter and summer host plants were attributed to respective species of Hyalopterus by means of their partial sequences of mitochondrial COI gene. Used Hyalopterus samples emerged as monophyletic relative to outgroup and formed three major clades representing three host specific mealy aphid species in the Neighbor joining, Maximum parsimony, Maximum likelihood and Bayesian inference trees. Hyalopterus pruni and Hyalopterus persikonus emerged as a sister species, whilst Hyalopterus amygdali was located basally. Samples representing different clades in the molecular trees were used for canonical discrimination analysis based on twenty two morphological characters. Length of the median dorsal head hair enabled a 97.3 % separation of Hyalopterus amygdali from the remaining two species. No single character enabled satisfactory discrimination between apterous viviparous females of Hyalopterus pruni and Hyalopterus persikonus. A modified key for the morphological identification of Hyalopterus species is suggested and their taxonomic status discussed.


Introduction
Mealy aphids of the genus Hyalopterus Koch are reported to be serious pests of stone fruits all over the World , Blackman and Eastop 2000, Lozier et al. 2009). Therefore, their morphology, biology, systematics, evolution, invasion history and potential harmfulness have been substantially studied (Smolarz 1970, Tscharntke 1989, Mosco et al. 1997, Poulios et al. 2007, Lozier et al. 2008, Tewksbury et al. 2002, Penvern et al. 2010, Symmes et al. 2012, for more and earlier references see Blackman and Eastop 2000). Nonetheless, the species level classification of mealy aphids remains unclear despite the long lasting debate. Since the very beginning, mealy aphids inhabiting various prunoideous plants have been described as a single species, Hyalopterus pruni (Geoffroy, 1762). Later on, almond inhabiting aphids were separated as Hyalopterus amygdali Blanchard, 1840. Such a viewpoint has been subjected for a long lasting controversy (e.g. Börner 1952, Shaposhnikov 1972, Eastop and Hille Ris Lambers 1976, Stroyan 1984, Heie 1986, Remaudiere and Remaudiere 1997. Recently, in addition to the two above mentioned species, Hyalopterus persikonus Miller, Lozier and Foottit, 2008 has been separated from H. amygdali by Lozier et al. (2008). For the present, three host plant associated Hyalopterus species are recognized. All three might inhabit reeds (Phragmites) as a summer hosts, but are different in their winter host specificity: H. amygdali is associated with almonds, whilst H. pruni and H. persikonus with plums and peaches, respectively. Nonetheless, apricot has been reported as a shared resource among the three Hyalopterus species supporting the possibility of interspecific hybridization (Lozier et al. 2007, Poulios et al. 2007, Lozier et al. 2008. Hyalopterus species, although well-defined on molecular level (Lozier et al. 2008), still remain difficult to separate by their morphological characters (Basky and Szalay-Marszό 1987, Blackman and Eastop 1994, 2000, including the most recent identification key (Lozier et al. 2008). For example, mealy aphids, collected on apricots in Lithuania, run to H. amygdali in the key of Blackman and Eastop (2000), but appeared difficult to identify by means of the key suggested by Lozier et al. (2008) (Kudirkaitė-Akulienė andRakauskas 2009). Moreover, the above keys do not concern mealy aphid populations on summer hosts, reeds. Host plant mediated developmental pathways might influence morphological characters, therefore, samples from reeds must be included in the analysis, together with those from stone fruit crops.
The aim of this study was to elaborate morphological identification key of the genus Hyalopterus based on the material from Europe that was identified by means of partial CO-I sequences.

Material studied
Forty three population samples of mealy aphids from five European countries were collected from various winter and summer host plants (Table 1). The entire data set has been subdivided: 21 samples (bolded in Table 1) were used for canonical discrimination procedures and subsequent evaluation of the received discrimination functions was performed on remaining 22 samples.

DNA extraction, PCR amplification and sequencing
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 were PCR-amplified using previously published primers (Turčinavičienė et al. 2006). PCR amplification was carried out in a thermal cycler (Eppendorf ) in 50 µl volumes containing 1-2 µl genomic DNA, 5 µl of each primer (10 µM), 5 µl of PCR-reaction buffer, 5 µl of dNTP mix (2mM each), 4-8 µl of 25mM MgCl 2 and 1.25 U of AmpliTaq Gold 360 polymerase (5U/µl) and ddH 2 O to 50 µl. The cycling parameters were as follows: denaturizing at 95°C for 10 min (1 cycle), denaturizing at 95°C for 30", annealing at 49°C for 30" and extension at 72°C for 30" (32-37 cycles in total), and a final extension for 5 min (1 cycle). PCR products were subjected to electrophoresis on 2% TopVision agarose (Fermentas, Lithuania), stained with ethidium bromide and sized against a MassRuler Low Range DNA ladder (Fermentas, Lithuania) under UV light. PCR products were purified and sequenced at Macrogen Europe (Amsterdam, the Netherlands). The amplification primers were also used as sequencing primers. DNA sequences for each specimen were confirmed with both sense and anti-sense strands and aligned in the BioEdit Sequence Alignment Editor (Hall 1999). Partial sequences of COI gene were tested for stop codons and none were found. The sequence data have been submitted to the GenBank, Accession numbers JX943517-JX943559.

Analysis of DNA sequences
Forty three sequences of three Hyalopterus species were analyzed. Sequences of Aphis gossypii Glover, 1877 (Aphidini) and Nasonovia ribisnigri (Mosley, 1841) (Macrosiphini) were selected as outgroups for the phylogenetic analyses, which included Neighbor joining (NJ), Maximum parsimony (MP), Maximum likelihood (ML) and Bayesian inference in phylogeny (BI). NJ, MP and ML analyses were performed using MEGA  (Ronquist and Huelsenbeck 2003) using General Time Reversible model with Gamma distribution (GTR+G), which was selected by jModeltest (Posada 2008). Four simultaneous chains, 3 heated and 1 "cold", were run for 3 000 000 generations with tree sampling every 1000 generations. The topologies obtained by NJ, MP, ML and BI were similar, so only ML tree is shown with values of NJ/MP and ML/BI bootstrap support and posterior probabilities indicated above and below branches respectively.

Morphological study and discrimination analysis
Samples representing different clades in the molecular trees were used for canonical discrimination analysis: 2 samples from almond (H. amygdali clade), 10 samples from cultivated plums (H. pruni clade), and 9 samples from peaches (H. persikonus clade) ( Table 1). Based on the earlier references (Poulios et al. 2007, Lozier et al. 2008, twenty two metric (in mm) characters were studied: A2L -length of antennal segment 2; A2W -width of antennal segment 2; A3BW -basal width of antennal segment 3; A3L -length of antennal segment 3; A3SLlength of the longest hair on antennal segment 3; A4L -length of antennal segment 4; A5L -length of antennal segment 5; A6BL -length of basal part of antennal segment 6; A6TPL -length of terminal process of antennal segment 6; AT8SL -length of submedian hair on abdominal tergite 8; BL -body length (excluding cauda); CL -length of cauda; DT3L -length of the second segment of hind tarsus; F3L -length of hind femur; FSL -length of the frons hair; HW -width of the head across eyes; MDHSL -length of median dorsal head hair; MDHSW -distance between the bases of median dorsal head hairs. SL -length of siphunculus; T3L -length of hind tibia; URL -length of ultimate rostral segment; URW -basal width of ultimate rostral segment.
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 (Statsoft 2007) was exploited for data analysis. Pearson's correlation coefficients were calculated to evaluate the correlation of morphometric characters with body length. Characters with strong (| r | ≥ 0.50) statistically significant (p<0.05) correlation with body length were removed from the further analysis: BL (r=1.00), F3L (r=0.58), T3L (r=0.59), A2L (r=0.57), HW (r=0.51). Remaining seventeen characters were used for forward stepwise discriminant analysis with host plant species as grouping variable followed by canonical analysis. Discriminant analysis was conducted in three steps. The first step was performed to discriminate between the all three mealy aphid species emerged in the COI dendrogram (H. Canonical scores were visualized as scatter plots. The morphological interrelationships among different samples were examined using hierarchical cluster analysis based on squared Mahalanobis distances (linkage method -UPGA).
Characters that contributed most in canonical discrimination functions were evaluated as having potential for species separation. The eventual species identification key based on these morphological characters and host plant information was constructed. Afterwards, it was applied on mealy aphid samples that were not used for the construction of the identification key (Table 1). Lozier et al. (2008) reported partial COI sequences being the most variable in Hyalopterus aphids and suggested them as a possible tool for the identification of the mealy aphid species complex. Forty three partial COI sequences of 3 Hyalopterus species from 5 countries were included in analysis. The alignment contained 564 bases in final set with 79 variable sites, 35 of which appeared parsimony informative. The sequences were heavily biased towards A and T nucleotides. The average base composition was A = 34.3 %, C = 14.1 %, G = 12.0 % and T = 39.7 %. The overall transition/transversion ratio R = 2.805 for all sites.

Partial sequences of mitochondrial (COI)
The maximum parsimony (MP) analysis of partial COI sequences resulted in 425 equally parsimonious trees (length = 152, CI=0.76, RI=0.95). ML tree (T92+G model) showed similar topology, the same as NJ analysis (Kimura 2-parameter distances) and BI (GTR+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 Fig. 1. One can ensure that used Hyalopterus samples emerge as monophyletic relative to outgroup and form three major clades representing three host specific mealy aphid species. H. pruni and H. persikonus are placed as a sister species, whilst H. amygdali is located basally.

Morphology
The scatter plot of the first two canonical variates for samples from 18 different geographical localities representing three mealy aphid species (apterous viviparous females) is shown in Fig. 2. All individuals were reclassified correctly into their a priori specified groups. The following characters proved to be important predictors when separating between three Hyalopterus species: MDHSL, URW, T3L/CL ( Table 2). The post hoc classification of samples gave 96.7 % correct identification of H. persikonus (n=46), 100 % of H. amygdali (n=10) and 99% of H. pruni (n=94) specimens.  To discriminate between apterous viviparous females of H. amygdali and H. pruni samples the following canonical function (for character acronyms see above) was obtained: -2.2645*SL-18.6609*MDHSL+1. The values of canonical scores were >0 for H. amygdali and <0 for H. pruni. This combination of canonical variables separated 94.5 % of H. amygdali (n=18) and 100% of H. pruni (n=67) specimens involved in the analysis with a priori specified group membership. The post hoc classification gave 100 % correct identification of H. amygdali (n=10) and 94.7% of H. pruni (n=94) specimens.
Out of eleven morphological characters included in the canonical function discriminating between sampled apterous viviparous females of mealy aphid species complex, the length of median dorsal head hair (MDHSL) enabled separation of 97.3 % H. amygdali specimens. Namely, the lengths of median dorsal head hair from 0.026 to 0.039 mm were characteristic of H. amygdali, whilst 0.036 -0.067 mm -for other two species. Yet we failed to find any single character or ratio enabling satisfactory discrimination between apterous viviparous females of H. pruni and H. persikonus. For the present, the following morphological identification key might be suggested to identify apterous viviparous females of the mealy aphid species complex.

Discussion and conclusions
Our analysis shows the morphological separation of mealy aphid species complex being a really difficult task which is in accordance with the earlier references (Poulios et al. 2007, Lozier et al. 2008. Nonetheless, it appeared that certain morphological char- When performing discriminant analyses, the body length should be eliminated from the data set together with characters that have strong and statistically significant correlation with the body length. In our case, when the entire data set of morphological characters was used for discriminant analysis, samples from reeds appeared the most different  Table 1. ar samples from Prunus armeniaca, d P. domestica, du P. dulcis, p P. persica, ph Phragmites communis. (not shown). Contrary, after the body length and correlated characters were removed from analysis, samples from reeds scattered amongst samples from plum and peach.
The results of cluster analysis based on morphological data (Fig. 3) show H. persikonus being more distantly related with H. pruni and H. amygdali. This contradicts the results of morphological analysis by Poulios et al. (2007) and supports the opinion of Mosco et al. (1997) on the early separation of H. persikonus from H. pruni/amygdali stem, which was also supported by the subsequent molecular analyses (Lozier et al. 2007(Lozier et al. , 2008. Such long lasting controversy might be explained by the fact that all three species share apricot as a winter host (see Lozier et al. (2008) for broader discussion), enabling interspecific gene flow. To clear the matter, precise studies of the host specificity and life cycles of the three taxa (including experimental transfers from plums to reeds and vice versa), together with hybridization trials, are needed. For the present, phylogenetic relationships of the three Hyalopterus species remain uncertain.