Research Article |
Corresponding author: Nicolás Pérez Hidalgo ( nicoperh@gmail.com ) Academic editor: Roger Blackman
© 2019 Adonay Zúñiga-Centeno, Izayana Sandoval-Carvajal, Mauricio Montero-Astúa, William Villalobos-Muller, Nguyễn Bảo Quốc, Nicolás Pérez Hidalgo.
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:
Zúñiga-Centeno A, Sandoval-Carvajal I, Montero-Astúa M, Villalobos-Muller W, Quốc NB, Pérez Hidalgo N (2019) A molecular study of Neophyllaphis varicolor (Hemiptera, Aphididae) in Costa Rica. ZooKeys 865: 123-135. https://doi.org/10.3897/zookeys.865.35213
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The genus Neophyllaphis (Takahashi) (Aphididae: Neophyllaphidinae) is composed of 18 species; however, in the Americas only nine species have been reported previously. A new species, Neophyllaphis varicolor Miller & Halbert, was described in 2014 in USA. Colonies resembling those of this new species have been observed in Costa Rica on Podocarpus spp. In order to determine if N. varicolor is also present in Costa Rica, we sampled Neophyllaphis colonies from Podocarpus falcatus and P. chinensis. Additionally, we sampled individuals from Podocarpus sp. in Spain and Vietnam. DNA of each sample was extracted and used to amplify and sequence the cytochrome c oxidase subunit I (COI) and elongation factor I (EF-1α) partial regions. According to morphological characteristics, sequences comparisons done in GenBank and BOLD, and phylogenetic analyses, the colonies collected from Podocarpus spp. in Costa Rica and the colony from Vietnam corresponded to the species N. varicolor. To the best of our knowledge this is the first report of the presence of N. varicolor in Central America and Vietnam.
Aphids, cytochrome c oxidase subunit I, DNA barcoding, elongation factor I, integrative taxonomy, phylogenetic analysis, Podocarpus
Neophyllaphis Takahashi (Aphididae: Neophyllaphidinae) is a genus occurring predominantly in the southern hemisphere and composed of 18 species characterized by a body covered with pulverulence and waxy secretions, antenna with a short terminal process, siphuncular pores on small cones, cauda with a constriction in the middle and with a caudal knob, and annular secondary rhinaria that are only present in alatae (
In the Americas there are nine Neophyllaphis species (
The newly recorded species N. varicolor, described by
Thirteen aphid colonies were sampled for this study. Nine samples from colonies on Podocarpus spp. L’Hér.ex Pers. were collected from different localities in San José province and samples from two colonies of N. araucariae were collected on Araucaria spp. in San José and Cartago provinces (Costa Rica). Additionally, two samples, one of N. podocarpi from Gerona (Spain) and one Neophyllaphis sp. multicolored colony from a Podocarpus sp. shrub in Vietnam were collected for comparison (Table
Samples of Neophyllaphis spp. colonies (individual aphids per colony indicated by a, b, and c) and the corresponding accession numbers of COI and EF-1α sequences deposited at GenBank.
Species | Colony code | Collection place | Host plant | Accession numbers | ||
COI | EF-1α | |||||
N. varicolor | CR14-002 | Montes de Oca, San José (9.935764°N, 84.057778°W) | Podocarpus falcatus (Thunb.) Mirb. | a | MK174294 | ND |
b | MK174295 | MK174326 | ||||
c | MK174296 | MK174327 | ||||
N. varicolor | CR14-004 | Montes de Oca, San José (9.934636°N, 84.058056°W) | Podocarpus chinensis Wall. ex J.Forbes | a | MK174297 | ND |
b | MK174298 | ND | ||||
c | MK174299 | MK174328 | ||||
N. varicolor | CR14-005 | Montes de Oca, San José (9.934567°N, 84.059167°W) | Podocarpus falcatus (Thunb.) Mirb. | a | MK174300 | ND |
b | MK174301 | MK174329 | ||||
c | MK174302 | ND | ||||
N. varicolor | CR14-012 | Goicoechea, San José (9.946283°N, 84.053056°W) | Podocarpus falcatus (Thunb.) Mirb. | a | MK174303 | MK174330 |
b | MK174304 | ND | ||||
c | MK174305 | ND | ||||
N. varicolor | CR14-013 | Curridabat, San José (9.923417°N, 84.033056°W) | Podocarpus falcatus (Thunb.) Mirb. | a | MK174306 | MK174331 |
b | MK174307 | ND | ||||
c | MK174308 | ND | ||||
N. varicolor | CR14-033 | Montes de Oca, San José (9.943450°N, 84.018889°W) | Podocarpus chinensis Wall. ex J.Forbes | b | MK174309 | ND |
c | MK174310 | MK174332 | ||||
N. varicolor | CR14-034 | Montes de Oca, San José (9.939783°N, 84.009444°W) | Podocarpus falcatus (Thunb.) Mirb. | a | MK174311 | ND |
b | MK174312 | MK174333 | ||||
c | MK174313 | ND | ||||
N. varicolor | CR14-127 | San José, San José (9.929947°N, 84.070278°W) | Podocarpus falcatus (Thunb.) Mirb. | a | MK174314 | ND |
b | MK174315 | ND | ||||
c | MK174316 | MK174334 | ||||
N. varicolor | CR14-295 | Vázquez de Coronado, San José (9.969086°N, 84.016944°W) | Podocarpus falcatus (Thunb.) Mirb. | a | MK174317 | MK174335 |
b | MK174318 | ND | ||||
c | MK174319 | ND | ||||
N. araucariae | CR14-364 | Paraíso, Cartago (9.852750°N, 83.807500°W) | Araucaria araucana (Molina) K. Koch | b | MK174320 | MK174336 |
c | MK174321 | MK174337 | ||||
N. podocarpi | CR14-398 | Gerona, Spain (41.676944°N, 2.801944°W) | Podocarpus neriifolius D.Don | c | MK174325 | MK174341 |
N. varicolor | CR14-397 | Long Dinh, Vietnam (10.384510°N, 106.166800°W) | Podocarpus sp. L’Hér.ex Pers. | a | MK463550 | MK463553 |
b | MK463551 | MK463554 | ||||
c | MK463552 | MK463555 | ||||
N. araucariae | CR14-423 | Vázquez de Coronado, San José (9.970153°N, 84.030833°W) | Araucaria heterophylla (Salisb.) Franco | a | MK174322 | MK174338 |
b | MK174323 | MK174339 | ||||
c | MK174324 | MK174340 |
Individuals preserved in 70% ethanol were cleared using KOH and acetic acid and mounted in slides with Canada balsam. The morphological identification of the specimens was done using a Leica Z16 microscope. We measured structures with taxonomic value and used the keys from
DNA was extracted from three individual aphid specimens per colony (preserved in 95% ethanol) using the animal tissue protocol of NucleoSpin Tissue extraction kit (Macherey-Nagel, Germany) following the manufacturer instructions with a modification at the elution step; it was made by duplicate, using 50 µL of elution buffer each time.
For the molecular identification and phylogenetic analysis of the Neophyllaphis spp. samples, we amplified the cytochrome c oxidase subunit I (COI) and the elongation factor 1α (EF-1α) genes. To amplify COI we used the primer pair C1-J-1490 (= LepF) and C1-N-2198 (= LepR) to obtain an amplicon of 658 bp (
Amplicons of COI and EF-1α were directly sequenced after purification in reverse and forward directions by the Sanger method (Macrogen, Korea). The final contigs were obtained using BIOEDIT 7.0 (
Phylogenetic analyses of Neophyllaphis spp. samples were done using partial sequences of the COI gene. Additionally, partial sequences of COI (nucleotide positions from 94 to 570 according to the reference sequence KF199852) and EF-1α (nucleotide positions from 81 to 546 according to the reference sequence KF199851) were concatenated using BIOEDIT tool (
Sequences obtained from GenBank (www.ncbi.nlm.nih.gov/Genbank) of the species N. varicolor (COI: KF199852; EF-1α: KF199851, USA), N. podocarpi (COI: EU701821, Japan and JQ920926, China), Neophyllaphis brimblecombei Carver (COI: JF883870, Australia) and from BOLD Systems (www.barcodinglife.org) for Neophyllaphis totarae Cottier (COI: RFBAD211_08, New Zealand) were included for comparison in the phylogenetic analyses. Sequences of Greenidea anonae (Pergande) (COI: JQ926000; EF-1α: KF856808, China) and Greenidea psidii van der Goot (COI: JQ925937 and EF-1α: KF856814, China, and COI: EU701673, USA) were used as an outgroup because the genus Greenidea clustered relatively close to Neophyllaphis in a COI phylogeny (
The metric and meristic characters (including color when alive) of the approximately 70 apterous specimens (Figure
A total of 39 individual aphids was analyzed by molecular means; COI sequence data were obtained for 35 individuals and EF-1α sequences for 19 individuals. We did not obtain final sequences for all three aphid individuals processed per colony because amplifications failed for some individuals or the sequencing reaction rendered low quality reads. All COI and EF-1α sequences obtained from samples morphologically identified as N. varicolor corresponded to this species according to the identification made in BOLD systems with 99.5% (KF199852.1) and 99.7% (KF199851.1) of similarity, respectively. It was not possible to corroborate the identification of the species N. araucariae by sequence identity comparison because data for this species is not available at GenBank or BOLD.
All COI sequences of N. varicolor from Costa Rica were identical, while, EF-1α sequences showed 0.6% difference. Sequences of COI and EF-1α of N. araucariae did not show intraspecific variation. Interspecific genetic variation between N. varicolor and N. araucariae was of 11.4% in COI sequences and 13.4% in EF-1α sequences.
All 26 partial sequences of COI from individuals morphologically identified as N. varicolor were grouped within the same clade, together with the Neophyllaphis sp. sample from Vietnam and the reference sequence of N. varicolor (GenBank Acc. No. KF199852) from Florida. This clade clustered independently from available sequences for N. araucariae, N. brimblecombei, N. podocarpi, and N. totarae. Similarly, all COI sequences of N. araucariae were grouped in the same clade, supporting identifications by morphological characters for both species. The N. araucariae cluster showed more relatedness to N. totarae than to the clade comprising N. podocarpi, N. brimblecombei, and N. varicolor.
The phylogenetic analysis made with a concatenated sequence composed of partial COI and EF-1α sequences showed a clade grouping all the sequences of N. varicolor and another clade with the sequences of N. araucariae, in accordance with the COI phylogenetic tree (Figure
Phylogenetic analysis of Neophyllaphis araucariae Takahashi, Neophyllaphis brimblecombei Carver, Neophyllaphis podocarpi Takahashi, Neophyllaphis totarae Cottier, and Neophyllaphis varicolor Miller & Halbert, using sequences of cytochrome c oxidase subunit I, made with Bayesian inference and using as outgroup Greenidea psidii van der Goot. and Greenidea anonae (Pergande). Sequence codes: species name - colony code - (a, b or c represent the specimen sampled) - country code. Key: AUS: Australia, CHN: China, CRI: Costa Rica, ESP: Spain, JPN: Japan, NZL: New Zealand, USA: United States, VNM: Vietnam. Scale bar represents 0.03 changes per site.
Phylogenetic analysis of Neophyllaphis araucariae Takahashi, Neophyllaphis podocarpi Takahashi and Neophyllaphis varicolor Miller & Halbert, using concatenated sequences of cytochrome c oxidase subunit I and elongation factor 1α made by Bayesian inference and using as outgroup Greenidea psidii van der Goot. and Greenidea anonae (Pergande). Sample names: species name - colony code - (a, b or c represent the specimen sampled) - country code. Key: CHN: China, CRI: Costa Rica, ESP: Spain, USA: United States, VNM: Vietnam. Scale bar represents 0.03 changes per site.
According to our results, the recently described aphid species N. varicolor also is present in Costa Rica and Vietnam. In addition to N. varicolor sequence information for Costa Rica and Vietnam, we also generated and submitted partial COI and EF-1α sequences for the species N. araucariae to GenBank for the first time. Indeed, we found few sequences available for the genus Neophyllaphis in public databases. Publicly available sequence information for all the describe species is important for comprehensive studies of the genus.
The morphological studies and molecular comparisons of COI and EF-1α sequences, supported the description of the new, distinct species, N. varicolor on Podocarpus spp. (
The COI gene was characterized by a high interspecific variation (
According to our phylogenetic analyses made with COI region, the species N. podocarpi and N. brimblecombei are most related to N. varicolor, similar to findings by
Our discoveries of N. varicolor in Costa Rica and Vietnam represent the first time that N. varicolor is reported outside of the USA. Currently, the genus Neophyllaphis is thus represented by two species in Costa Rica: N. varicolor and N. araucariae.
The presence in Vietnam of N. varicolor suggests that it is a species native to Southeast Asia. However, the genus Neophyllaphis presents taxonomic problems (
This research, and the scientific visit of NPH to the CIBCM in 2014, were funded by University of Costa Rica (grants 801-B7-169 and 801-A1-801) and the investigation in Spain was conducted in the context of Project CGL2015-68188-P, funded by “Ministerio de Economía, Industria y Competitividad” (MIMECO) of Spain. The authors appreciate the comments and corrections made by Susan Halbert and Roger Blackman.