Research Article |
Corresponding author: Giuseppe Massimino Cocuzza ( giuseppe.cocuzza@gmail.com ) Academic editor: Roger Blackman
© 2015 Giuseppe Massimino Cocuzza, Silvia Di Silvestro, Rosanna Giordano, Carmelo Rapisarda.
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:
Cocuzza GEM, Di Silvestro S, Giordano R, Rapisarda C (2015) Congruence between cytochrome oxidase I (COI) and morphological data in Anuraphis spp. (Hemiptera, Aphididae) with a comparison between the utility of the 5’ barcode and 3’ COI regions. ZooKeys 529: 123-144. https://doi.org/10.3897/zookeys.529.6081
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The discrimination of species in the genus Anuraphis is particularly difficult due to the overlap of morphological characters. In this study, we used the 5’ (barcode) and 3’ regions of cytochrome oxidase I (COI) to test their utility in the identification of species in this genus as well as closely related species. Both regions were useful to discriminate all the species tested. However the non-barcode 3’ region resulted in higher resolution and support for species relationships when the data were analyzed using both Maximum Likelihood and MrBayes. We propose the development of an integrated database that encompasses morphological, molecular, life-cycle, host plant and bibliographic information to facilitate and increase the accuracy of aphid identification.
Insects, aphids, taxonomy, species identification
Aphids are sap-sucking insects. Currently there are 5012 valid species (
A. farfarae (pear-colt’s foot aphid) and A. subterranea (pear-hogweed aphid) have been reported in the literature as pests of pear, where they cause direct damage to young foliage in spring (
All species belonging to the genus Anuraphis are morphologically similar to each other but easily discriminated from other genera. The main morphological features of the genus are an almost flat frontal profile, as a result of the minimally developed antennal tubercles, and a short cauda. Moreover, Anuraphis shares with a few other genera of Macrosiphini a typical spinulose ornamentation of siphunculi and a well-developed, often almost complete set of dorsal tubercles (both marginal and spinal). However, as already reported for other groups of aphids, the morphometric similarity among Anuraphis species leads to an overlap that renders their discrimination to species level difficult (
In some genus (e.g. Aphis), a recurrent and difficult problem in using only morphological characters to identify aphids is that for many species there are insufficient diagnostic characters, resulting in their identification being partially based on host plant association and life cycle characteristics (
Ideally the description of a species should result from a synthesis of information that encompasses morphological, molecular, biological, biogeographical, physiological, ecological and bibliographical data (
This study was undertaken to improve the current taxonomic knowledge of the various taxa belonging to the genus Anuraphis by testing the utility of the COI gene, specifically comparing the widely used barcode 5’ region with the much less studied 3’ region, as a molecular tool for their identification. A further goal is to compare the results obtained with the COI gene to those previously published using only morphological characters (
This study was conducted with seven species (Table
Voucher code | Species | Host plant | Location | Sampling date | GeneBank accession N° |
---|---|---|---|---|---|
S03189 | Anuraphis farfarae Koch | Tussilago farfara |
40,0970N/15,8131E Lauria (Potenza, Basilicata) |
25 Jun. 03 | KT878791 |
S03190 | A. farfarae | Tussilago farfara |
39,8762N/16,0050E Mormanno (Cosenza, Calabria) |
25 Jun. 03 | KT878792 |
S13572 | A. farfarae | Tussilago farfara |
46,5606N/12,1285E Cortina d’Ampezzo (Bolzano, Trentino Alto Adige) |
18 Sep. 13 | KT878793 |
S03157 | A. pyrilaseri Shaposhnikov | Magydaris pastinacea |
37,9795N/12,7637E Buseto Palizzolo (Trapani, Sicily) |
6 Jun. 03 | KT878794 |
S03171 | A. pyrilaseri | Thapsia garganica |
37,9258N/15,7602E Rognudi (Reggio Calabria, Calabria) |
9 Jun. 03 | KT878795 |
S03141 | A. pyrilaseri | Ferula communis |
37,6345N/15,0744E Trecastagni (Catania, Sicily) |
15 May 03 | KT878797 |
S03146 | A. pyrilaseri | Ferula communis |
38,0229N/15,3890E Fiumedinisi (Messina, Sicily) |
17 May 03 | KT878799 |
S03152 | A. pyrilaseri | Thapsia garganica |
37,8152N/15,1869E Piedimonte Etneo (Catania, Sicily) |
28 May 03 | KT878796 |
S03147 CBGP#ACOE2024 GBMIN37806 CBGP#ACOE2050 CBGP#ACOE1998 |
A.
pyrilaseri
A. pyrilaseri A. pyrilaseri A. pyrilaseri A. pyrilaseri |
Ferula communis Not reported Not reported Not reported Not reported |
38,0440N/15,4309E Itala (Messina, Sicily) 37,7863N/15,2337E Fiumefreddo (Catania, Sicily) Not reported 37,7826N/15,1325E Sant’Alfio (Catania, Sicily) 37,7827N/15,1418E Linguaglossa (Catania, Sicily) |
17 May 03 27 May 06 Not reported 30 May 06 23 May 06 |
KP714117
ACEA860 GU568501 ACEA880 ACEA839 |
S03144 | A. catonii HRL | Pimpinella major |
38,0505N/15,4343E Itala (Messina, Sicily) |
17 May 03 | KT878815 |
S03173 | A. catonii | Pimpinella peregrina |
37,9937N/15,9250E Bova (Reggio Calabria, Calabria) |
9 Jun. 03 | KT878816 |
S12477 | A. catonii | Pimpinella peregrina |
37,1334N/15,0165E Sortino (Siracusa, Sicily) |
25 May 12 | KT878817 |
S03179 | A. cachryos Barb. & Str. | Cachrys sicula |
37,3619N/15,0219E Scordia (Catania, Sicily) |
15 Jun. 12 | KT878818 |
S03180 | A. cachryos | Cachrys sicula |
36,7765N/14,5989E Donnalucata (Ragusa, Sicily) |
15 Jun. 12 | KT878819 |
S12423 | A. cachryos | Cachrys sicula |
36,7766N/14,5990E Donnalucata (Ragusa, Sicily) |
2 May 12 | KT878820 |
S14599 CPGP#ACOE1057 |
A.
cachryos
A. cachryos |
Cachrys libanotis Not reported |
37,3080N/14,8587E Lentini (Siracusa, Sicily) 42,7869N/3,0361 Languedoc-Roussillon (France) |
13 Jun. 13 30 Oct. 00 |
KT878821
ACEA353 |
S03181 | A. subterranea (Walker) | Heracleum pyrenaicum |
37,9756N/14,9516E Floresta (Messina, Sicily) |
22 Jun. 03 | KT878800 |
S03182 | A. subterranea | Heracleum pyrenaicum |
37,9808N/15,1435E Novara di Sicilia (Messina, Sicily) |
22 Jun. 03 | KT878801 |
S12517 | A. subterranea | Heracleum sphondylium |
37,9020N/13,9999E Isnello (Palermo, Sicily) |
3 Jul. 12 | KT878804 |
S03191 | A. subterranea | Pastinaca sativa |
39,8761N/16,0038E Mormanno (Cosenza, Sicily) |
25 Jun. 03 | KT878805 |
S03163 | A. subterranea | Heracleum pyrenaicum |
37,8801N/14,0283E Petralia Sottana (Palermo, Sicily) |
6 Jun. 03 | KT878802 |
S03184 CBGP#ACOE2053 CBGP#ACOE2060 CBGP#ACOE645 CBGP#ACOE1068 |
A.
subterranea
A. subterranea A. subterranea A. subterranea A. subterranea |
Heracleum pyrenaicum Not reported Not reported Not reported Not reported |
37,9756N/14,9516E Floresta (Messina, Sicily) 37,9216N/14,957E Randazzo (Catania, Sicily) 37,9921N/14,9306E Floresta (Messina, Sicily) 44,8893N/1,4062E Peryllac-et-Millac (France) 42,8742N/2,1829E Quillan (France) |
22 Jun. 03 30 May 06 30 May 06 2 Jun. 99 21 May 01 |
KT878803
ACEA883 ACEA890 ACEA164 ACEA367 |
S03160 | A. shaposhnikovi Barb. & Coc. | Magydaris pastinacea |
37,9795N/12,7637E Buseto Palizzolo (Trapani, Sicily) |
6 Jun. 03 | KT878808 |
S03143 | A. shaposhnikovi | Opopanax chironium |
37,9075N/15,1211E Francavilla di Sicilia (Messina, Sicily) |
16 May 03 | KT878809 |
S03166 | A. shaposhnikovi | Opopanax chironium |
37,9917N/15,9309E Bova Sup. (Reggio Cal., Calabria) |
9 Jun. 03 | KT878810 |
S14589 CBGP#ACOE438 CBGP#ACOE2052 |
A.
shaposhnikovi
A. shaposhnikovi A. shaposhnikovi |
Opopanax chironium Not reported Not reported |
37,6324N/14,9859E Belpasso (Catania, Sicily) 44,1891N/6,7477E Entraunes (France) 37,9216N/14,957E Randazzo (Catania, Sicily) |
21 Apr. 14 24 Jul. 98 30 May 06 |
KT878811
ACEA035 ACEA882 |
S12413 | Nearctaphis bakeri (Cowen) | Trifolium pratense |
45,0877N/7,6387E Torino (Piemonte) |
16 Apr. 12 | KT878807 |
S13562 CBGP#ACOE824 CBGP#ACOE1020 |
N.
bakeri
N. bakeri N. bakeri |
Trifolium pratense Not reported Not reported |
41,2367N/13,9319E Sessa Aurunca (Caserta, Campania) 43,7337N/3,5500 Saint-Guillerme-le-Desert (France) 47,9862N/-4,4642E Plouhinec (France) |
12 Jun. 13 8 Apr. 00 30 Jul.00 |
KT878806
ACEA242 ACEA331 |
S06340 CBGP#ACOE460 |
Aphis fabae Scopoli A. fabae |
Vicia faba Not reported |
36,9251N/14,7423E Ragusa (Sicily) 44,0105N/3,6058E Levignan (France) |
20 Apr. 06 1 Jul. 98 |
KT878822
ACEA050 |
S04230 | Roepkea marchali HRL | Prunus mahaleb |
43,2235N/13,1518E S. Severino (Macerata, Marche) |
20 May 04 | KT878812 |
S14613 | R. marchali | Prunus mahaleb |
50,0810N/14,4029E Prague (Czech Rep.) |
31 May 14 | KT878813 |
S14623 CBGP#ACOE1674 |
R.
marchali
R. marchali |
Prunus mahaleb Not reported |
50,0871N/14,4172E Prague (Czech Rep.) 43,6833N/3,9262E Teyran (France) |
1 Jun. 14 26 Jun. 0 |
KT878814
ACEA723 |
S03145 GBMIN10086 |
Brachycaudus jacobi Stroyan B. jacobi |
Myosotis
sylvatica
Myosotis sylvatica |
38,0505N/15,4343E Itala (Messina, Sicily) 38,0505N/15,4343E Itala (Messina, Sicily) |
15 May 03 15 May 03 |
EU189690
EU196598 |
Whole aphid specimens for DNA sequencing were stored in 95% ethanol at -20 °C, those used for morphological observations were stored in 70% ethanol and at room temperature.
Total genomic DNA was extracted by macerating entire single individuals using the DNeasy Blood & Tissue kit (Qiagen®, Hilden, Germany) in 50 µl of extraction buffer and stored at -20 °C. To compare the utility of the 5’, barcode region, and the 3’ region of COI we amplified the following regions: for the 5’ end, a 600 bp region using primers LCO1490 and HCO2198 (
The COI sequences were edited manually using BioEdit (
COI was easily amplified for all specimens analysed using the primers indicated above. No frame shift or premature stop codons were detected.
The five prime end (5’) constituted a 601 base pair (bp) fragment. With total bp frequencies of 75.3% for A/T and 24.7% for G/C. These latter results concur with those found for other aphid species (
The three prime end (3’) sequences analysed consisted of 648 bp with frequencies of 74.9% A/T and 25.1% G/C. The 3’ end showed that there were 521 constant and 127 variable sites of which 111 were parsimony informative. The percentage of variable sites was slightly higher for the 3’ (19.6%) than the 5’ end (18.99%).
Considering the 5’ region, the mean genetic distance of Anuraphis species from N. bakeri, R. marchali, B. jacobi and A. fabae were 6.5%, 6.7%, 8.0% and 9.2%, respectively, whereas slightly higher distance values were observed for most comparisons of the 3’ region (7.5%, 7.9, 8.1 and 8.6%, respectively). The genetic differences recorded in the 5’ barcode region among Anuraphis species (Table
p-distance and nucleotide divergences (expressed as percentage) of Anuraphis spp. and species used as outgroup.
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
5’ | 3’ | 5’ | 3’ | 5’ | 3’ | 5’ | 3’ | 5’ | 3’ | 5’ | 3’ | 5’ | 3’ | 5’ | 3’ | 5’ | 3’ | ||
1 | Anuraphis farfarae | ||||||||||||||||||
2 | Anuraphis pyrilaseri | 1.7 | 3.2 | ||||||||||||||||
3 | Anuraphis subterranea | 5.7 | 7.2 | 5.8 | 7.4 | ||||||||||||||
4 | Anuraphis shaposhnikovi | 5.3 | 6.9 | 5.0 | 6.6 | 3.7 | 4.7 | ||||||||||||
5 | Anurahis catonii | 5.5 | 6.9 | 5.2 | 6.6 | 3.9 | 4.8 | 0.2 | 0.8 | ||||||||||
6 | Anuraphis cachryos | 6.6 | 7.0 | 6.7 | 6.6 | 4.3 | 5.9 | 5.6 | 3.3 | 5.6 | 3.8 | ||||||||
7 | Nearctaphis bakeri | 6.8 | 8.3 | 6.9 | 7.9 | 5.6 | 7.6 | 6.6 | 6.9 | 6.6 | 7.0 | 6.7 | 7.1 | ||||||
8 | Roepkea marchali | 7.3 | 7.9 | 6.7 | 8.2 | 6.2 | 8.0 | 6.7 | 7.6 | 6.7 | 7.7 | 6.8 | 8.1 | 5.5 | 7.4 | ||||
9 | Brachycaudus jacobi | 8.5 | 8.4 | 8.9 | 8.5 | 7.1 | 8.5 | 7.8 | 7.5 | 7.8 | 7.6 | 7.8 | 8.2 | 7.5 | 8.0 | 6.9 | 6.8 | ||
10 | Aphis fabae | 10.0 | 9.9 | 9.1 | 8.1 | 9.1 | 9.1 | 9.0 | 7.9 | 9.0 | 8.0 | 8.9 | 8.4 | 8.1 | 8.1 | 8.5 | 10.0 | 10.0 | 9.5 |
Our results indicate that there is high genetic homogeneity within Anuraphis species, despite differences in geographic origin and host plant. Anuraphis farfarae is the only member of the genus that uses Asteraceae, nevertheless its position in Anuraphis is well supported (Fig.
Little to no intraspecific differences were found among the various geographic samples of each Anuraphis species (0.3% only for some populations of A. catonii, A. cachryos and A. pyrilaseri). Phylogenetic analysis with Neighbour Joining (NJ), Maximum Likelyhood (ML) and Bayesian (MrBayes) using the 5’ and 3’ end of the COI gene showed two discreet clades: one comprising A. farfarae and A. pyrilaseri; the other including A. cachryos, A. subterranea, A. catonii, and A. shaposhnikovi respectively (Figs
The clade including A. farfarae and A. pyrilaseri shows a genetic distance between the two species of 3.2% when using the 3’end and 1.7% when using the 5’ end of COI. The various samples of A. farfarae were highly similar, regardless of host plant, locality and COI region examined. Similarly, the populations of A. pyrilaseri showed low genetic variability (0.3%). Differences in body colour, possibly due to host plant effects, as well as differences in dorsal abdominal sclerotisation, do not correlate with the low genetic diversity observed with the COI gene. The various samples of A. subterranea showed no genetic differences, regardless of their geographic origin, host plant or COI region used for the analysis. Genetic difference (3.7% with 3’ and 4.7% with 5’ region) between A. subterranea and A. shaposhnikovi clearly distinguishes the two species, despite the small morphological differences observed (length of ultimate rostral segment and number and distribution of abdominal spinal tubercles). A. shaposhnikovi and A. catonii showed the lowest genetic divergence (<1%) regardless of the COI region considered. However, while with 5’ COI barcode showed a pairwise distance of 0.2%, the 3’ region showed a difference of 0.8%.
A result similar to the one based on COI was found using a multivariate discriminant analysis with 16 morphometric characters (
The molecular analysis based on the 3’ and 5’ COI gene regions indicates that the genus Anuraphis is a homogeneous taxonomic group. However, COI also provides information to distinguish the taxa at the species level as evidenced by the level of support, 89% bootstrap or more, on the likelihood tree (Fig.
We observed a difference in genetic distances when using the 5’ barcode or the 3’ regions of COI. Most “barcode” studies on aphids are carried out using the 5’ region of COI that has produced some ambiguous results (
The genetic results observed here in Anuraphis spp. closely mirror previous morphometric findings. The lack of appreciable differences in morphological characters is a phenomenon well known in various groups of aphids (
We are grateful to Prof. Sebastiano Barbagallo and Prof. Felipe Soto, for a critical review as well as suggestions provided during the research. Many thanks to Dr. Vincenzo Cavalieri for technical support in molecular analysis and Dr. Giuseppe Scuderi for valuable suggestions. Finally, we are particularly grateful to the anonymous reviewer and to Dr. R.L. Blackman for their valuable comments.