Aphis intybi Koch is recorded for the first time in North and South America. Measurements for the identification of this species in comparison with Aphis craccivora Koch are given. Aphis cytisorum Hartig is recorded for the third time in Argentina. Data on morphological variation, geographical distribution, and host plants of these species in Argentina are provided. Their morphological identification is supported by comparison of DNA sequences of the mitochondrial cytochrome oxidase subunit 1 (DNA barcode) and subunit 2.
Seventy percent of the aphid species recorded so far in South America (233 of 335) are not native to the subcontinent, and this figure is increasing continually (Mier Durante et al. 2011). A study of Aphis Linnaeus, 1758 samples from Argentina, deposited in the Zoology Collection of the University of Leon (CZULE), enabled Aphis intybi Koch, 1855 to be recorded for the first time in South America, and increased our knowledge of the presence of A. cytisorum Hartig, 1841 and Aphis craccivora Koch, 1854 in Argentina. Both of the latter species are also adventive in South America and are phylogenetically related to Aphis intybi Koch, 1855. Although recorded as a frequent interception at ports of entry in the USA (Stoetzel & Russell 1991), specimens (Canadian National Collection of Insects, Ottawa, Canada) of A. intybi from the Montréal region, Québec, Canada provide the first indication of its presence in North America.
MATERIALS and METHODS
Specimens of viviparous females in all cases and alate viviparous females in most of the 118 samples were studied (Table 1). They were collected by M. P. Mier Durante, J. M. Nieto Nafría and J. Ortego during 4 expeditions carried out in Jan 2000, Nov 2002, Nov 2006, and Dec 2009, and by J. Ortego at different times from 1994 onwards.
Sample colonies were split, with a part preserved in 70% ethanol for morphological study and a part in 95% ethanol for molecular work. Methods for slide mounting of specimens and morphometric measurements were those normally used in the taxonomic study of aphids.
LIST OF THE STUDIED ARGENTINEAN SAMPLES. GENBANK ACCESSION NUMBERS ARE GIVEN IN PARENTHESES BELOW VOUCHER IDENTITIES (ARG- PLUS NUMBER) IN THE UNIVERSITY OF LEON ZOOLOGICAL COLLECTION.
DNA sequences for the 5′ end of the cytochrome oxidase subunit 1 (COI) gene (DNA barcode region) were obtained through the facilities of the Biodiversity Institute of Ontario using techniques described in deWaard et al. (2008). Sequence data are available on GenBank under the accession numbers given in Table 1 and in Fig. 1. Reference sequences (GenBank accession numbers given in Fig. 1) for samples from various geographic regions are taken from Foottit et al. (2008), Komazaki et al. (2010), Valenzuela et al. (2007), and additional sequences released with this study (GenBank accession numbers JX438172 to JX438176). Between-sequence distances were calculated using the Kimura two-parameter model and the resultant matrix summarized in a neighbor-joining tree (Fig. 1). DNA sequences for the tRNAleucine/cytochrome oxidase subunit II genes (COII) were obtained using methods of DNA extraction, gene amplification, and sequencing as described in von Dohlen & Teulon (2003). In addition to Argentinean samples, 2 A. craccivora samples from USA and Spain were sequenced in this study: 99EM0214, from Medicago sativa L. (Fabaceae), Colorado, 1999, and LE2904, from Cytisus scoparius (L.) Link (Fabaceae), Spain, León, San Martín del Agostedo, 11 Sep 2004 (GenBank accessions JX624107 and JX624108). Reference sequences from other geographic regions were downloaded from GenBank (accession numbers in Fig. 2.). Between-sequence distances were calculated using the Tamura-Nei model of evolution, as the best fit model determined by ModelTest (Posada & Crandall 1998); the resultant matrix was summarized in a neighbor-joining tree with Geneious Pro software (Drummond et al. 2011) (Fig. 2).
RESULTS and DISCUSSION
Aphis intybi Koch, 1855
This species closely resembles Aphis craccivora Koch, but can be differentiated by: (1) when alive the specimens are covered with a whitish waxy powder, (2) the shape of the cauda, which is more triangular than digitiform, (3) some small differences in measurements (see below), and (4) the males are apterous.
According to Blackman & Eastop (2006), 2 metric characteristics differentiate the apterous viviparae in A. intybi from A. craccivora: (first) “terminal filament/base of antennal segment VI”, 2.4–3.8 in A. intybi and 1.7–2.7 in A. craccivora, and (second) “apical rostral segment / base of antennal segment VI” 1.25–1.75 times in A. intybi and 0.85–1.20 in A. craccivora.
However, based on data (including photographs) provided by García Prieto & Nieto Nafría (2005) for both species, and on the measurements of specimens from Argentina, the values for Aphis craccivora are (first) 1.2–3.4 times and (second) 0.8–1.3 (exceptionally 1.5) times. Thus, the first of the 2 characters is of little use.
According to our data, identification can also be based on “ultimate rostral segment/second segment of hind tarsus”, 1.1–1.5 times in A. intybi and 0.7–1.3 times in Aphis craccivora.
Analysis of COI barcodes demonstrates that the Argentinean sample is similar to material identified as A. intybi from Lithuania and Canada. The COI sequence clusters corresponding to A. intybi and A. craccivora are defined by 4 fixed differences but the minimum between-species K2P distance between the groups is small (0.77%) compared to the maximum within-species divergence of 0.61%, (Fig. 1).Despite the small magnitude of the interspecific distance relative to intraspecific variation, the concordance of these clusters with the morphological differences indicated above supports the recognition of A. intybi as a distinct entity.
While COII sequences were collected primarily to quantify genetic variation within A. craccivora, these data also demonstrate that A. intybi is well differentiated from all samples of A. craccivora, which form a distinct cluster. The minimum distance of A. intybi to A. craccivora is 1.43%, while the maximum distance within A. craccivora is 0.81%. Although our COII data do not measure within-species variation for A. intybi, the genetic distance of the single A. intybi representative to A. craccivora samples supports the distinction of the 2 species, consistent with COI data and morphology.
The specimens were collected in 5 localities in the provinces of Rio Negro, Neuquen and Mendoza, on the usual host plant, Cichorium intybus L. (Asteraceae), which is adventive in Argentina (see Table 1).
Aphis cytisorum Hartig, 1841
The first record of A. cytisorum in Argentina (and also in South America), and the only one so far, on Tipuana tipu Lillo (Fabaceae) in the province of Buenos Aires, was made by Nieto Nafría et al. (1994), with a correction by Ortego (1998). This species was also recorded from Santa Fe province on Spartium junceum L. (Fabaceae) by Ortego et al. (2002). The presence of the aphid species in Argentina is confirmed on 2 of its usual host plants in Europe: Cytisus scoparius (L.) Link (Fabaceae) and Spartium junceum, which are adventive in Argentina.
MINIMUM and MAXIMUM LIMITS OF THE QUANTITATIVE CHARACTERS IN APHIS CRACCIVORA.
The collection localities are situated in the provinces of Mendoza, Río Negro, Neuquen and Santa Cruz, which would indicate that the species occurs throughout the country.
The qualitative and quantitative characteristics of the collected specimens are within the limits known for the species (Blackman & Eastop 2006, and Garcia Prieto & Nieto Nafría 2005). The COI sequence for the Argentinean sample is identical to sequences obtained from samples from Canada.
Aphis craccivora Koch, 1854
Aphis craccivora is subcosmopolitan and one of the polyphagous species in the genus, and its most important characteristics vary considerably, as Mehrparvar et al. (2012) have also shown.
Some of the measurements corresponding to some of the studied specimens are not within the known limits for the species (Blackman & Eastop 2006; García Prieto & Nieto Nafría 2005) (Table 2). However, the identification of clone-mates of these specimens as A. craccivora was confirmed by molecular analysis (Figs. 1 and 2). Our results address certain comments by Mehrparvar et al. (2012), because they analyzed samples from 4 plant genera (Astragalus, Atriplex, Chenopodium and Robinia) of a total of 6, which are also present in our study: “The results presented here for the morphological structure of A. craccivora provide the framework to investigate the genetic and biological differences among these entities so that we could be able to exactly evaluate their taxonomic situation”.
Aphis craccivora is here mentioned for first time, according to Blackman & Eastop (2006), on species of Baccharis (Asteraceae), Bulnesia (Zygophyllaceae), Condalia (Rhamnaceae), Gazania (Asteraceae), Hoffmanseggia (Fabaceae), Larrea (Zygophyllaceae), Prosopidastrum (Fabaceae), Proustia (Asteraceae) and Tricomaria (Malpighiaceae), and on species of another 21 genera of several plant families in Argentina (Table 1). While the specimens recorded on Condalia microphylla were identified with some uncertainty by Nieto Nafría et al. (2008) as Aphis conflicta Nieto Nafría, Ortego & Mier Durante, 2008, COII sequences here confirm these specimens as A. craccivora (Fig. 2). COII sequences further illustrate the high genetic similarity among Argentinean populations, which nevertheless exploit a broad taxonomic range of host plants.
Aphis craccivora is recorded for the first time in the provinces of Chubut, San Juan, Neuquen, Rio Negro and Salta, and is now known in 17 of the 23 provinces forming the Republic of Argentina.
The authors acknowledge Prof. R. Rakauskas, Vilnius University, for the Lithuanian sample of Aphis intybi. Dr. Lee Humble (Canadian Forest Service, Victoria, British Columbia, Canada) provided specimens of Aphis cytisorum and Ms. Claude Pilon (Repentigny, Quebec, Canada) provided specimens of A. intybi. We thank Mr. E. Maw for the preparation of Fig. 1. C. D. von Dohlen's research was supported by the Utah Agricultural Experiment Station, approved as journal paper #8461. M. P. Mier Durante and J. Ortego's research was supported by grants (LE45/02, LE034A05 and LE036A08) of the Regional Government of Castilla y Leon, Spain.