Research Article |
Corresponding author: Doris Lagos-Kutz ( dlagos@illinois.edu ) Academic editor: Roger Blackman
© 2014 Doris Lagos-Kutz, Colin Favret, Rosanna Giordano, David Voegtlin.
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:
Lagos-Kutz D, Favret C, Giordano R, Voegtlin D (2014) Molecular and morphological differentiation between Aphis gossypii Glover (Hemiptera, Aphididae) and related species, with particular reference to the North American Midwest. ZooKeys 459: 49-72. https://doi.org/10.3897/zookeys.459.7850
|
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 (
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 (
In Europe, there are approximately 20 aphid species morphologically similar to 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
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 (
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 (
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.
A total of 160 COI sequences from 28 species, 133 EF1-α sequences from 36 species, and 13 SCP sequences from 6 species were used in this study. After alignment and excluding the primer sites, 1,290, 1,078 and 703 bp for COI, EF1-α (including gaps and introns) and SCP were used in the analysis, respectively. COI sequence divergence between species of the A. gossypii species group ranged from 0.08% (between A. gossypii and A. sedi) to 3.04% (between A. gossypii and A. monardae). The sequence divergence of A. glycines and A. nasturtii (sharing a winter host plant with A. gossypii), as compared with the species of the gossypii group, ranged from 5.25% (between A. gossypii and A. glycines) to 6.97% (between A. nasturtii and A. sedi) (Table
The cladograms using COI (Figure
The dendrogram inferred by MrBayes using EF1-α (Figure
The SCP gene was difficult to amplify and thus we only acquired sequences for six taxa. The Bayesian cladogram using SCP (Figure
Range of Kimura 2 Parameter pair-wise inter- and intraspecific sequence divergence (%) for COI sequences.
A. forbesi | A. glycines | A. gossypii | A. monardae | A. nasturtii | A. oestlundi | A. sedi | |
---|---|---|---|---|---|---|---|
A. forbesi | 0.00 | ||||||
A. glycines | 5.49–5.73 | 0.00 | |||||
A. gosyypii | 6.27- 6.35 | 5.25–5.92 | 0.00–0.54 | ||||
A. monardae | 6.27 | 5.75–5.85 | 2.70–3.04 | 0.00–0.08 | |||
A. nasturtii | 5.73–5.77 | 7.03–7.15 | 6.66–6.89 | 6.50–6.73 | 0.00–0.08 | ||
A. oestlundi | 6.35 | 5.67–5.85 | 2.37–2.57 | 1.57–1.81 | 6.57–6.68 | 0–0.16 | |
A. sedi | 6.2–6.35 | 5.51–5.76 | 0.08–0.70 | 2.62–3.02 | 6.54–6.97 | 2.37–2.77 | 0.00–0.54 |
Range of Kimura 2 Parameter pair-wise inter- and intraspecific sequence divergence (%) for EF1-α and SCP sequences.
A. gossypii | A. monardae | A. oestlundi | A. sedi | |||||
EF1-α | SCP | EF1-α | SCP | EF1-α | SCP | EF1-α | SCP | |
A. gossypii | 0.40–0.87 | 0.14–0.84 | ||||||
A. monardae | 0.54–0.97 | 1.12–1.98 | 0.00–0.11 | 0.14–0.28 | ||||
A. oestlundi | 0.76–1.20 | 1.12–1.83 | 0.87–0.98 | 0.42–0.64 | 0.00 | 0.00 | ||
A. sedi | 0.11–0.76 | 0.84–1.84 | 0.65–0.76 | 1.26 | 0.87 | 1.26 | 0.00–0.22 | 0.00 |
Cladogram inferred based on analysis of COI with MrBayes. Support values (Posterior Probabilities) are below branches. Values under 0.95 are not presented. Species names are followed by collection locality (USA: AL (Alabama), CO (Colorado), IA (Iowa), IL (Illinois), IN (Indiana), KS (Kansas), LA (Louisiana), MO (Missouri), MN (Minnesota), OH (Ohio), SD (South Dakota), WI (Wisconsin)), and number of haplotypes.
After four weeks under conditions of reduced temperature and photoperiod, colonies of A. monardae reared on M. fistulosa produced oviparae and apterous males (Figure
The outdoor experiments located at the South farms of the University of Illinois were evaluated after 25 days. Alate viviparae of A. gossypii were seen on H. telephium and G. max but they did not produce offspring, however, alates that moved to C. pepo did produce apterous and alate viviparae. Voucher slides are deposited in the INHS insect collection numbers: 512851-512857. The colonies of A. gossypii reared on C. pepo were set in a growth chamber B where they grew rapidly. Potted M. fistulosa were placed in this chamber and were colonized by A. gossypii. Clean plants of C. pepo that were later exposed in the same chamber to a colony of A. monardae were not colonized. A colony of A. sedi begun with fundatrices from H. telephium was exposed to C. pepo in growth chamber B for several weeks, but the aphids did not transfer to and establish on this plant.
Analysis of variance of morphological characters useful to discriminate A. gossypii, A. monardae, and A. sedi. The gray line represents the median. The gray diamond represents the means and standard deviation. A 95% level indicates a significant difference. A distance from the base of antennal segment III to the first secondary sensorium (DBIII) between A. gossypii and A. monardae B ratio of length of processus terminalis (PT) to the base of last antennal segment B between A. gossypii and A. sedi C ratio of length of siphunculi (SIPH) to the length of cauda (CA) between A. gossypii and A. sedi.
In both the COI and EF1-α analyses, A. monardae was readily distinguished from A. gossypii (Figure
Diagnosis: Siphunculi of apterous morph pale, dark distally. When alive, light yellow to light green, body covered with white wax (Figure
Neotype: Apterous viviparous female. USA: Minnesota; Douglas County; on Monarda fistulosa L.; 45.8160°N, 95.7472°W; 19.viii.2010; D. Lagos. Neotype apterous viviparous female (INHS Insect Collection 513070). Body1.4, URS 0.09, accessory setae 2, antennal segments: III 0.16, IV 0.08, V 0.09, B 0.08, Pt 0.18, LHIII 0.010, hind tibiae 0.50, HT2 0.08, width of tubercle on abdominal tergite I 0.020, width of tubercle on abdominal tergite VII 0.018, siphunculus 0.19, cauda 0.12, with 5 setae, abdominal tergite VIII with 2 setae, sub-genital fig with 3 setae on anterior part.
See Suppl. material 2 for morphological measurements of the four morphs of A. monardae. Additional images of A. monardae can be found in
Apterous viviparae (n= 40). Color in life (Figure
Alate viviparae (n= 59). Color in life (Figure
Oviparae (n= 26). Color in life (Figure
Alate male (n=17). Color in life (Figure
Aphis species of the A. gossypii complex. A Apterous vivipara of A. gossypii on Rhamnus cathartica B Nymphs, apterous and alate viviparae of A. monardae on Monarda fistulosa C Apterous ovipara of A. monardae D Nymphs and apterous male (brownish in the center of the image) of A. monardae E Nymphs and alate vivipara of A. gossypii on Cucurbita pepo F Nymphs and apterous vivipara of and A. oestlundi on Oenothera biennis G Apterous vivipara (top) and apterous ovipara (bottom) of A. sedi on Hylotelephium telephium.
The distinction of A. sedi from A. gossypii is supported by phenotypic characters of specimens in collections included in Tables S1 and S2. In addition, morphological characters such as the ratio of the lengths of the processus terminalis and the base of the sixth antennal segment (Suppl. material 2, Figure
These species are sometimes misidentified because they share some morphological characters on either apterous or alate morphs. Moreover, the pair-wise sequence divergences using COI sequences between A. gossypii and A. forbesi Weed, A. glycines and A. nasturtii are up to 5% (Table
Many dichotomous keys to subsets of Aphis have been written (
1 | Cauda pale, most often with constriction at midpoint, with 4–7 setae. Antennae five or six segmented. Siphunculi pale, distally dusky. Summer morphs. Polyphagous (Figure |
A. gossypii |
– | Cauda dusky or dark | 3 |
3 | Siphunculi dark all throughout | 4 |
– | Siphunculi dusky or lighter at the base | 7 |
4 | Cauda constricted | 5 |
– | Cauda not constricted | 7 |
5 | Cauda spoon-shaped, distinctly constricted, with 4–7 setae. Ratios PT/B 2.6–4.1, SIPH/CA 1.3–2.5. Polyphagous (Figure |
A. gossypii |
– | Cauda slightly constricted | 6 |
6 | Cauda slightly constricted at midpoint, with 4–5 setae. Ratios PT/B 2.0–2.7, SIPH/CA 1.5–2.2. On Oenothera spp. (Figure |
A. oestlundi |
– | Cauda elongate, parallel-sided, with acute tip and slight constriction at the base, and with 4–8 setae. Ratios PT/B 1.8–2.5, SIPH/CA 0.9–1.6. On Hylotelephium spp. (and elsewhere recorded from Sedum spp. and some other Crassulaceae) (Figure |
A. sedi |
7 | Siphunculi lighter at the base, dusky distally. Cauda tongue-shaped, with 6–9 setae. Ratios PT/B 1.7–2.9, SIPH/CA 1.3–1.7. On Monarda spp. (Figure |
A. monardae |
– | Siphunculi dusky. Cauda tongue-shaped, with 4–7 setae. Ratios PT/B 2.6–4.1, SIPH/CA 1.3–2.5. Polyphagous (Figure |
A. gossypii |
1 | Cauda tongue-shaped, with 3–9 setae, without sclerites on dorsum abdominal segments I, II, and III. Secondary sensoria on antennal segment III (4–9), IV (0–3). DBIII 0.07–0.12 (Figure |
A. monardae |
– | Cauda constricted, sometimes with sclerites on dorsum of abdominal segments I, II, and III | 2 |
2 | Antenna VI PT/B 2.1–3.6. Secondary sensoria on antennal segment III (4–10) DBIII 0.04–0.07 (Figure |
A. gossypii |
– | Antenna VI PT/B 1.9–2.3. Secondary sensoria on antennal segment III (7–10) and IV (0–2) (Figure |
A. sedi |
– | Antenna VI PT/B 2.2–2.9. Secondary sensoria on antennal segment III (2–8) (Figure |
A. oestlundi |
The analysis of different species included in this study largely corroborates the results obtained by
The discrimination of A. gossypii and A. sedi is clear when the aphids are alive (Figure
The inclusion of A. glycines, A. gossypii and A. nasturtii in strongly supported clades (Clade A, Figures
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 (
The COI sequence divergence values obtained in this study are similar to those obtained in other studies (
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 (
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.
Table S1. Collection information.
Data type: species data
Explanation note: Collection information for specimens included in this study. INHS voucher and GenBank accession numbers are for specimens originating from a specific collection.
Table S2. Morphological characters useful to discriminate A. gossypii, A. monardae, A. oestlundi and A. sedi.
Data type: measurement data
Explanation note: Morphological characters useful to discriminate A. gossypii, A. monardae, A. oestlundi and A. sedi. For all measurements and counts the range is given and the mean is in parentheses. All measurements in mm. Abbreviations: B base of last antennal segment, CA cauda, DBIII: Distance from the base of antennal segment III to the first secondary sensorium, HT2 second hind tarsus, LHIII longest Hair on ant. segm. III, PT: Processus terminalis, SIPH siphunculi, URS ultimate rostral segment. Data of oviparae of A. gossypii from