The cotton aphid, Aphis gossypii, is one of the most biologically diverse species of aphids; a polyphagous species in a family where most are host specialists. It is economically important and belongs to a group of closely related species that has challenged aphid taxonomy. The research presented here seeks to clarify the taxonomic relationships and status of species within the A. gossypii group in the North American Midwest. Sequences of the mitochondrial cytochrome oxidase 1 (COI), nuclear elongation factor 1-α (EF1-α), and nuclear sodium channel para-type (SCP) genes were used to differentiate between A. gossypii and related species. Aphis monardae, previously synonymised with A. gossypii, is re-established as a valid species. Phylogenetic analyses support the close relationship of members of the A. gossypii group native to North America (A. forbesi, A. monardae, A. oestlundi, A. rubifolii, and A. rubicola), Europe (A. nasturtii, A. urticata and A. sedi), and Asia (A. agrimoniae, A. clerodendri, A. glycines, A. gossypii, A. hypericiphaga, A. ichigicola, A. ichigo, A. sanguisorbicola, A. sumire and A. taraxicicola). The North American species most closely related to A. gossypii are A. monardae and A. oestlundi. The cosmopolitan A. gossypii and A. sedi identified in the USA are genetically very similar using COI and EF1-α sequences, but the SCP gene shows greater genetic distance between them. We present a discussion of the biological and morphological differentiation of these species.

Aphid, host plant, morphology, phylogeny, sequence divergence, status novus

Host plant association is often one of the main characters used to distinguish between closely related aphid species. However, host association can also be one of the main sources of misidentification of host-alternating aphids. These aphids migrate between taxonomically distant hosts, usually between woody and herbaceous plants. Taxonomic problems have been created when aphid morphs from primary (woody) host plants have been treated as separate species from those found living on secondary (herbaceous) or summer host plants. Host alternation provides an opportunity for aphids to acquire new hosts and may be a key to the rapid diversification of some groups of aphids (Eastop 1971, Dixon 1973, von Dohlen and Moran 2000), thereby leading to hard-to-distinguish species complexes. The evolution of Aphis, the largest aphid genus by a margin, is associated with the rapid diversification of herbaceous angiosperms (Heie 1996).

The Aphis gossypii group contains economically important and taxonomically problematic species, with A. gossypii Glover itself being the most biologically diverse and hence taxonomically challenging (Blackman and Eastop 2007). It has many different primary and secondary host plants and exhibits both holocyclic and anholocyclic life cycles (Kring 1955, Blackman and Eastop 2006, Margaritopoulus et al. 2006). Its taxonomic complexity is attested to by its 42 available synonyms, including the native North American species, Aphis monardae Oestlund (Favret 2014). Eastop and Hille Ris Lambers (1976) established this synonymy without comment. Lagos (2007) found that A. monardae is distinct morphologically from A. gossypii and treated it as a valid species. No type specimen of A. monardae could be found at the time, and no molecular or biological evidence was available to support this decision. The research presented here contains both molecular and biological evidence as well as an examination of material collected by Oestlund from Monarda spp.

In Europe, there are approximately 20 aphid species morphologically similar to A. gossypii (Stroyan 1984, Heie 1986). Several studies using mitochondrial, nuclear, and intron length polymorphism in the sodium channel para-type (SCP) genes have achieved some resolution discriminating A. gossypii and other Aphis species (Coeur d’acier et al. 2007, Foottit et al. 2008, Coccuzza et al. 2009, Carletto et al. 2009b, Kim et al. 2010a, Komazaki et al. 2010, Favret and Miller 2011). In North America, morphological studies show that species of the A. gossypii group can be misidentified easily (Voegtlin et al. 2004, Lagos 2007). The discrimination of species closely related to A. gossypii is of particular importance due to the recent introduction into North America of the soybean aphid, A. glycines Matsumura (Voegtlin et al. 2004). This species is obligately holocyclic and heteroecious, feeding on soybean, Glycine max (L.) Merr., as secondary host, and on Rhamnus spp. as primary host. Aphis gossypii has also been reported to colonize soybean in North America (Blackman and Eastop 2007), and while its colonization on soybeans is uncommon in the north central United States, some soybean-collected insect samples from Alabama, Georgia, Kansas, Louisiana, and Mississippi contained only A. gossypii or a mixture of both species (personal observation, Illinois Natural History Survey (INHS) insect collection records). These collections suggest that A. gossypii may be more common on soybeans in southern regions. There are no records of the exotic A. nasturtii Kaltenbach feeding on soybeans, and attempts to culture A. nasturtii on soybeans were not successful (David Ragsdale, personal communication); however, this species shares the primary host, Rhamnus spp., with both A. glycines and A. gossypii.

We here elucidate the phylogenetic relationship of species morphologically close to A. gossypii and the taxonomic status of A. monardae in the North American Midwest.

Aphid collections: Aphids were collected from their primary and/or secondary host plants from different sites in China, France, Italy, Japan, Spain and the USA, with the majority of the material originating from the Midwest of the USA. When possible, aphids were collected alive and reared on the host plant for the maturation of late instar nymphs. Adults were preserved in 95% ethanol and stored at -20°C until DNA extraction and microscope slide preparation. Collection data with INHS Insect Collection specimen voucher numbers are presented in Suppl. material 1.

Morphology: Archival microscope slides were prepared using the technique described by Pike et al. (1991). Individuals were selected from the same colonies as those selected for DNA extraction. Photographs of mounted specimens and measurements were taken using a Leica DM 2000 digital camera and SPOT Software 4.6 (Diagnostic Instruments, Inc). Analyses of variance of diagnostic characters, such as the distance from the base of the third antennal segment to the first secondary sensorium, the ratio of the lengths of the processus terminalis and the base of the sixth antennal segment, and the ratio of the lengths of the siphunculus and the cauda, were tested using JMP, Version 7 (SAS Institute Inc., Cary, NC, 1989–2007). Species identification of slide-mounted material was done by the first author, using published keys (Oestlund 1887, Gillette 1927, Hottes and Frison 1931, Palmer 1952, Kring 1955, Cook 1984, Voegtlin et al. 2004) and authoritatively identified specimens in the insect collections of the INHS and the University of Minnesota. Identifications of slide-mounted specimens were referenced to the aphid colony-mates used in the molecular analyses.

DNA extraction, PCR amplification, and sequencing: Two or three specimens per colony were sequenced individually. Individual specimens were crushed in a 1.5 ml microcentrifuge tube and DNA was extracted and purified using the QIAamp DNAmicrokit (QIAGEN Inc., Valencia, CA). The mitochondrial gene Cytochrome Oxidase I (COI) was amplified in two overlapping fragments: 5’ fragment with forward primer C1-J-1718 (Simon et al. 1994) and internal reverse primer C1-J-2411 (Lagos et al. 2012); 3’ fragment with internal forward primer C1-N-2509 (Lagos et al. 2012) and reverse primer TL2-N-3014 (Simon et al. 1994). The nuclear gene Elongation Factor-1-α (EF1-α) the following primers were used: EF3F (Lagos et al. 2012) and EF2 (Palumbi 1996). The length polymorphism of an intron in SCP was sequenced using the primers Aph13 and Aph15 (Carletto et al. 2009a). All primers were synthesized by Invitrogen™ Corporation (Carlsbad, CA). PCR used PuReTaq™ Ready-To-Go™ PCR 0.2 ml beads (GE Healthcare UK) mixed with 20 µl of PCR-grade water, 1 µl of F and R primers at 10 µM, and 3 µl of genomic DNA solution. The thermal cycler protocol used to amplify COI and EF1-α was: 95°C 2 min (95°C 30s; 53°C 30s; 72°C 120s) 40x. For SCP, it was: 95°C 3 min (94°C 60s, 55°C 45s, 72°C 60s) 40x. PCR products were run on a 1% agarose gel for 40 min at 90 v, and visualized with GelGreen nucleic acid stain (Biotium Inc, California, USA). Most PCR products were purified using QIAquick^® (QIAGEN Inc.) kit. PCR products that included the co-amplification of non-specific bands were gel purified using Zymoclean ™ gel DNA recovery kit (Zymo Research, USA). The concentration of PCR products was measured using a NanoDrop^® ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). PCR products were sequenced in both directions using 3 µl of a mixture of BigDye^® Terminator v3.1, dGTP BigDye Terminator v3.0, and buffer in a ratio of 2:1:1 respectively, 1.6 µl of 2 µM primer primers, differing amounts of DNA, and 1 µl of dimethyl sulfoxide (DMSO) (SIGMA-ALDRICH^®, St Louis, MO). Sequencing reactions were run using the following protocol: 96°C 2 min (95°C 20s; 50°C 5s; 60°C 240s) 25x. Sequencing reactions were cleaned using Performa^® DTR Ultra 96-Well Plates (EdgeBioSystems, Gaithersburg, MD) and run on ABI 3730 at the Keck Center (University of Illinois at Urbana-Champaign). Raw sequence data were examined and assembled using Sequencher 4.7 (Gene Codes Corporation, Ann Arbor, MI). Sequences were then aligned with Clustal X (version 2.0, 2007; Larkin et al. 2007). Three introns in EF1-α were identified and used in this study. Nucleotide sequences were deposited in GenBank (Suppl. material 1). Pairwise distances were obtained using PAUP 4.0b10 based on the Kimura two-parameter model (Swofford 2001).

The COI sequence of the A. gossypii neotype specimen (GU591547) and 25 EF1-α sequences of Aphis spp. (especially those of species closely related to A. gossypii) were retrieved from GenBank: EU019867, EU019869, EU019871, EU019872, EU019873, EU019874, EU019875, EU019876, EU019878, EU019879, EU358904, EU358907, EU358911, EU358915, EU358916, EU358917, EU358924, EU358926, EU358927, GU205375 and GU205376.

Phylogenetic analysis: Modeltest 3.7 (Posada and Crandall 1998) was used to select the best-fit nucleotide substitution model. Single sets of gene sequences were analyzed using MrBayes 3.1.2 (Huelsenbeck and Ronquist 2003) to execute Bayesian analyses. For single analysis, four chains were run. The number of generations was 5,000,000 with a burn-in of 250 trees and frequency sampling of 100 generations with rates equal to variable gamma as a model of substitution of nucleotides. Rhopalosiphum maidis (Fitch) (Aphidinae: Aphidini), and Hyadaphis tataricae Aizenberg and Uroleucon helianthicola (Olive) (Aphidinae: Macrosiphini) were selected as outgroups.

Aphid biology: Two growth chambers were used to examine various aspects of the biology of A. monardae, A. gossypii, and A. sedi Kaltenbach, in order to discern differences in their life cycle. Experimental plants were grown in a greenhouse in 12.7 cm diameter pots and isolated in 13.5 by 13.5 by 22.5 inches cages. Chamber A was set at 12°C and short photoperiod (8L:16D), conditions that will trigger the development of sexual morphs. Colonies of A. monardae on Monarda fistulosa L., A. sedi on Hylotelephium telephium (L.) H.Ohba, and A. gossypii on Cucurbita pepo L. and Rhamnus cathartica L. were exposed to these conditions for extended lengths of time. Samples of A. monardae and A. sedi were collected on a weekly basis from the host plants listed above and examined for the presence of sexual morphs. In the cages of A. gossypii weekly samples were taken from R. cathartica.

The B chamber was set at 24°C with constant illumination (24 hours) to keep colonies and test host plant specificity of the three species mentioned above. The following experiments were done in chamber B: a Monarda fistulosa plant infested with A. monardae was placed into a cage with an aphid-free C. pepo plant and left for a several weeks. Biweekly examination of the C. pepo plants was made to determine if A. monardae had colonized them. A Cucurbita pepo plant infested with A. gossypii was placed into a cage with aphid-free M. fistulosa and H. telephium and left for several weeks. Biweekly examination of M. fistulosa and H. telephium was made to see if A. gossypii had colonized them. A Hylotelephium telephium plant infested with A. sedi was placed into a cage with aphid-free C. pepo and left for several weeks. Biweekly examination of C. pepo was made to see if A. sedi had transferred to them. An entire tree of R. cathartica infested with A. gossypii was isolated in a 2 by 2 by 2-m walk-in cage in May of 2011 on the grounds of the South Farms of the University of Illinois (Suppl. material 1). The temperature ranged between 10 and 22 °C, http://www.isws.illinois.edu/atmos/statecli/cuweather/. Aphid-free C. pepo, H. telephium and Glycine max were placed into the cage to document the potential infestation of these secondary hosts under natural environmental conditions.

The analysis of different species included in this study largely corroborates the results obtained by Coeur d’acier et al. (2007), Kim and Lee (2008), Kim et al. (2010a), Kim et al. (2010b), Kim et al. (2011) and Lagos et al. (2014b). The gossypii complex in the North American Midwest contains the following native species, A. oestlundi and A. monardae, and the invasive species A. gossypii and A. sedi. Collection host records for A. gossypii show that it has been collected on Oenothera and Monarda, the host plants of the native Aphis species listed above (Blackman and Eastop 2006). Collection records for the native species suggest a very limited host range, in contrast with the highly polyphagous A. gossypii. Our results indicate that these species can be differentiated by morphological characters as well as host association. Data from this study confirms the finding of Lagos (2007) that A. monardae is a valid species and not a synonym of A. gossypii (Eastop and Hille Ris Lambers 1976). The novel character (distance from the base of antennal segment III to its first secondary sensorium, DBIII) is useful to differentiate alate viviparae of A. monardae and A. gossypii when they are collected together in traps. The sexual morphs collected on Monarda under laboratory and field conditions indicate that A. monardae has a monoecious holocyclic life cycle. A neotype of A. monardae has to be designated according to the Article 75.3 of the International Code of Zoological Nomenclature (International Commission on Zoological Nomenclature 1999). Concomitant with the redescription of the species, we here designate a neotype of A. monardae from the state of Minnesota on Monarda fistulosa. Slides deposited by O.W. Oestlund in the Insect Collection of the University of Minnesota show collection data no earlier than 1896. However, the first description of Aphis monardae was published in 1887 and the original description specified neither a type nor the type locality (Oestlund 1887). The comparison of apterae, alatae and oviparae of Oestlund’s collections match the morphological characters of those collected recently (Suppl. material 1). Some slides made by Oestlund were remounted in 1968 so it was possible to better see the characters. For a neotype we chose a more recently collected specimen taken in Minnesota as it more clearly shows color pattern and other characters used in the redescription.

The discrimination of A. gossypii and A. sedi is clear when the aphids are alive (Figure 8). The identification problem arises when we examine samples that have lost their color by being stored in ethanol. Molecular data also are helpful. The pair-wise sequences divergences between these species using SCP are higher than for COI and EF1-α sequences (Tables 1 and 2). This marker also successfully differentiated the cryptic species A. gossypii and A. frangulae (Carletto et al. 2009a). Results obtained in this study corroborate the biological and morphological findings of Kring (1955), who found that A. sedi is holocyclic monoecious on Hylotelephium. In this study, only apterous oviparae were collected under laboratory conditions conducive to the production of sexuales (Figure 4C). Kring’s morphological observations showed that the ratio of the processus terminalis to the base of the last antennal segment (PT/B), and the ratio of the length of the siphunculus to the length of the cauda (SIPH/CA), are both greater in A. gossypii than A. sedi for all morphs (Suppl. material 2). Although the above characters are useful to differentiate these species, their identification (especially the alate viviparae) is still problematic because of their similar morphology and because these ratios overlap (Figures 5B–C).

The inclusion of A. glycines, A. gossypii and A. nasturtii in strongly supported clades (Clade A, Figures 1 and 2) is consistent with the findings of Foottit et al. (2008) but in disagreement with those of Kim et al. (2010a). Interestingly, these three invasive species share a winter host plant, Rhamnus spp., but this is not the only known overwintering host for A. gossypii. This indiscriminate behavior, in addition to multiple species sharing winter hosts, raises the possibility of interspecies hybridization (Müller 1986, Rakauskas 2003). Hybridization may or may not be successful but should be detectable in studies of gene flow and phenotypic characterization of putative hybrids.

The species regarded here as members of the A. gossypii complex, A. gossypii, A. sedi, A. oestlundi and A. monardae (Clade D), exhibit interesting biological, morphological and molecular patterns. Aphis gossypii has been shown to colonize numerous secondary host plants including those of closely related taxa (Stroyan 1984, Heie 1986, Blackman and Eastop 2006). Moreover, it is one of the few Aphis species with multiple primary host plants (Blackman and Eastop 2006). By contrast, the native taxa related to it and found in the Midwest have or are presumed to have monoecious holocyclic life cycles (see Suppl. material 1 for host plant information). Aphis oestlundi, A. monardae and A. sedi have wingless males, a characteristic that would contribute to the genetic isolation of these species. These sibling species possess morphological characters useful for diagnostic purposes (Suppl. material 2) and the values that support interspecific sequences divergences (Table 2) are similar to those found by Foottit et al. (2008) and Favret and Miller (2011). The identification of species related to A. gossypii is made more difficult because they feed on host plants that can also serve as host to A. gossypii. Interestingly, however, their colors in life differ and can be useful for identification. For example, A. gossypii is dark green or light brownish and its siphunculi are dark throughout (Figures 8A, E), although this can vary in summer dwarf specimens. Aphis gossypii is mostly darker than A. monardae, which is light yellow or green (Figures 8B, C), and A. oestlundi is light yellow (Figure 8F). The color of A. sedi is dark green (Figure 8G), like A. gossypii, although it has more white wax on its body (García Prieto et al. 2005).

The COI sequence divergence values obtained in this study are similar to those obtained in other studies (Cocuzza et al. 2009, Cognato 2006, Coeur d’acier et al. 2007, Foottit et al. 2008, Favret and Miller 2011, Wang and Qiao 2009). Moreover, the low pair-wise sequence divergences found between some species such as A. gossypii and A. sedi (Table 1) are consistent with those obtained by other workers such as Piffaretti et al. (2012). While COI data have been found useful to discern the phylogenetic relationships of many taxa, the use of COI sequence divergences to set cut-off points that can differentiate Aphis species should be used with caution, since it may lead to the misidentification of new species, a conclusion drawn by other studies for several orders of insects (Blaxter 2004, Hebert et al. 2004, Nadler 2002, Will and Rubinoff 2004, Smith et al. 2008).

Our work suggests the possible existence of three undescribed Midwestern species (Aphis spp. 1, 2, and 3) within the gossypii complex. Further studies need to be done to validate their status. It is likely that additional new species will be found within this group as material is gathered from a larger geographical area and combined molecular, morphological and biological data are used to analyze the new taxa. The use of multiple primary hosts is unusual for any species, thus lineages within the gossypii complex that select and limit themselves to specific hosts may be driving the speciation process within this group (Peccoud et al. 2010, Kim et al. 2011).

Support was provided by funds from the North Central Soybean Research Program, Illinois Soy Board to D. Voegtlin and HATCH funds to R. Giordano. We are very grateful to Armelle Coeur d’acier, Thelma Heidel, Wayne Ohnesorg, Benjamin Puttler and Andrew Williams for supplying with specimens used in this study. Many thanks to the associate curator of the University of Minnesota Insect Collection, Robin Thomson, who kindly sent Oestlund’s slides and provided quick correspondence. We gratefully acknowledge the comments and examination of specimens by Drs. Juan Nieto Nafría, Shun’ichiro Sugimoto and Giuseppe E. Cocuzza. The manuscript benefited greatly from the comments provided by Susan Halbert, two anonymous reviewers, and the subject editor, Roger Blackman.