During expeditions to Costa Rica in 2008, 3 of the authors (M.D., P.H., N.N.) and W. Villalobos (Villalobos Muller et al. 2010) collected apterous and alate viviparous female macrosiphines (Aphididae, Aphidinae, Macrosiphini) on Bidens pilosa (Asteraceae, Asteroideae) in the University of Costa Rica main campus. Additional individuals collected on Bidens, Schkuhria, Senecio and Stevia in several Mexican localities, and kept in the collection génerale d'aphides of the Muséum national d'Histoire naturelle (Paris, France), were studied; these specimens had been provisionally identified by the Prof. G. Remaudière as Hyperomyzus sp. and marked as possible new species.

Morphological, statistical (PCA) and molecular (genes COI and EF 1α) studies have been carried out and have shown that (i) the studied specimens belong plausibly to a single species independently from their geographical origin or host plant, and (ii) it is a new species, which can not be included in any known Macrosiphini genus, and so, a new genus and its type species are established.

MATERIAL AND METHODS

Ten samples collected in 1 Costa Rican and 6 Mexican localities have been studied (see “Types” section), considering a sample as the group of specimens collected on the same species of plant in a locality on a specific date. Specimens were preserved in microscopic slides with a water-soluble mounting medium (Nieto Nafría & Mier Durante 1998). Aphids for molecular analyses were preserved in 96% ethanol until processing; these individuals were caught together with the holotype on the same plant specimen.

Morphological measurements were made according to Nieto Nafría & Mier Durante (1998). In the description, measurements are lengths except when indicated otherwise as width or diameter.

The comparative morphological study was conducted on species (i) whose apterae possess swollen siphunculi and have been recorded in North America or (ii) are known to feed on Asteroideae species over the World. Specimens belonging to diverse genera presumably related with the new one and also specimens of the most part of American species currently classified Neonasonovia Hille Ris Lambers, 1949 (subgenus of Hyperomyzus Börner 1933), in the collections of the Muséum national d'Histoire naturelle (Paris, France) and the Universidad de León (Leon, Spain) were examined. Several taxonomic works, mainly Blackman & Eastop (2006), Blackman (2010), Foottit et al. (1993), Heie (1992, 1994, 1995), Miyazaki (1971), and Nieto Nafría et al. (in press), were consulted.

A Leica DC digital camera with IM 1000 version 1.10 software was used for the photomicrographs.

The variables used in the PCA (principal components analysis) are the metric and meristic characters mentioned in the description of 39 apterous viviparous females; all samples are represented. Variables were standardized to zero mean and unit standard deviation prior to the analysis to give the same weight to all of them.

Molecular phylogenetic analyses were done (i) on a fragment of the mitochondrial DNA containing the 5′ region of the cytochrome c oxidase 1 (COI) and (ii) on a fragment of the nuclear gene coding for elongation factor-1 alpha (EF1α) of 3 specimens of the Costa Rican sample.

DNA extraction was done following the HotSHOT (Hot Sodium HidrOxide and Tris) method (Truett et al. 2000).

PCR amplifications of each mentioned fragment were done on 3 µL of the extracted DNA. A 710 bp fragment of the 5′ region of the COI gene was amplified using primers LCO1490 and HCO2198 described by Folmer et al. (1994). PCR conditions for COI amplification were as follows: 94 °C for 1 min, 35 cycles of 94 °C for 30 s, 48 °C for 1 min, and 68 °C for 1 min; a final extension step of 7 min at 68 °C was included after cycling. Amplification of the EF1α fragment was done using 2 consecutive PCR reactions with primers Efs175 (Moran et al. 1999) and Efr1 (5′GTGTGGCAATSCAANACNGGAGT3′) in the first reaction and then primers Efs175 and Efr2 (5′TTGGAAATTTGACCNGGGTGRTT3′) in the second semi-nested reaction. PCR conditions used in the first reaction were 94 °C for 1 min; 40 cycles of 94 °C for 30 s, 50 °C for 1 min and 68 °C for 1.5 min; a final extension step of 7 min at 68 °C was included after cycling. The semi-nested PCR was done similarly but using 52 °C for the annealing step and 1 µL of the first PCR product.

PCR products were purified by ammonium precipitation and reconstituted in 10 mL of LTE buffer (10 mM Tris, 0.1 mM EDTA). Direct sequencing of the amplified fragments was done on both directions using PCR primers (Efr2 was used as reverse primer for sequencing the EF1α fragment). Sequencing was conducted using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) following manufacturer's instructions, and samples were loaded into an ABI 3700 automated sequencer.

Chromatograms were revised and sequences assembled using the Staden package v1.6.0 (Bonfield et al. 1995). Multiple alignments were done with Clustal X v1.81 (Larkin et al. 2007) with gap opening and gap extension penalties of 10.0 and 0.2, respectively.

Molecular phylogenetic analyses were conducted using MEGA 5 (Tamura et al. 2011).

RESULTS AND DISCUSSION

Apterous Viviparous Females. Fig. 1. Based on 66 Specimens.

Color in life dull pale green, with yellowish appendages and siphunculi. Body 1.65–2.45 mm including cauda.

Head. Pale-brown; cuticular ornamentation reduced to fine and scattered striae. Frontal margin undulated and frontal tubercles rounded; lateral tubercles divergent and low, but bigger than the medial. Dorsal setae with rounded apex, 15–17 µm and 0.42–0.70 times the basal diameter of the antennal segment III [henceforth D]; ventral setae pointed and similar in size. Ventral margin of the antennal socket with a striate protuberance. Antennae are 1.75–2.62 mm and 0.93–1.28 times the body length. Antennal segments I and II pale brown and delicately rugous on the inner margin. Antennal segment III 0.41–0.70 mm and 1.1–2.1 times IV, pale brown (on a proximal portion) to dark brown, with scales (ventrally on paler portion) and striae, blunt setae 7–15 µm and 0.2–0.6 times D, and 1–20 secondary sensoria with double-lined and non-ciliate margin placed in a ventral line of the basal third of the segment. Antennal segments IV to VI brown to dark brown and imbricated; IV, 0.25–0.50 mm; V, 0.23–0.37 mm; processus terminalis of VI 0.55–0.82 mm, 0.9–1.7 times antennal segment III and 5.3–8.2 times base, which is 0.09–0.12 mm. Rostrum as pigmented as head, smooth and reaching the hind coxae. Ultimate rostral segment triangular with straight margins, 0.12–0.14 mm, 1.8–2.9 times its basal diameter, 1.1–1.4 times base of the antennal segment VI and 1.1–1.3 times second tarsal segment of the hind legs; with 3–6 accessory setae, which are 12–20 µm and 0.50–0.66 times D.

Thorax. Membranous, very pale and with pale spiracular and inconspicuous intersegmental sclerites. Prothorax with 2 spinal and 2 pairs of marginal setae, similar to other dorsal setae in shape and size; spiracular apertures rounded and only slightly wider than those on the abdomen. Coxae, trochanters pale brown, femora basad pale brown and darkening to the apex, tibiae brown to dark brown, tarsi dark brown. Femur and tibia of the hind legs 0.58–0.81 and 0.97–1.50 mm, respectively. Second tarsal segment of hind legs 0.10–0.12 mm. Setae on femora scattered, blunt and short, those on hind legs are 12–18 µm and 0.41–0.60 times D. Setae on basal part of tibiae like the femoral ones, others are pointed and longer than those (20–25 µm and 0.61–1.0 times D). First tarsal segments with 3 setae.

Abdomen. Membranous, with small and unpigmented intersegmental and spiracular sclerites, pale but rugous postsiphuncular sclerites, and sometimes marginal sclerites on segments VII and VIII also pale and rugous or with spinules. Spiracular apertures subcircular to reniform. Papillae absent. Dorsal setae on anterior segments blunt, 8–10 per segment, 7–12 µm and 0.2–0.4 times D. Ventral setae pointed, more numerous and 12–30 µm. Abdominal segment VIII with 4–6 setae, 8–20 µm and 0.2–0.8 times D. Siphunculi asymmetrically swollen (outer edge is almost straight), with broad base and well-defined preapical incision and flange; 0.37–0.50 mm and 0.18–0.25 times body length; 4.7–5.3 times diameter at base (0.075–0.100 mm), 6.8–11.5 times diameter of peduncle at middle (0.03–0.07 mm) and 5.0–8.3 times greatest diameter of swollen part (0.06–0.09 mm); pigmented like femora and darkening apically; mostly smooth, with striae on incision and few striae or wrinkles on peduncle. Genital plate pale, with 2 discal and 6–12 posterior setae, all of them with pointed apex. Anal plate and cauda pale brown, paler than siphunculi. Cauda lanceolate, 0.21–0.34 mm, 1.6–2.6 times its basal width, 0.60–0.74 times the siphunculus, and carrying 5–7 fine, curved and pointed setae.

Fig. 1.

Apterous viviparous female: A, habitus; B, head; C, III antennal segment; D, dorsal seta on III abdominal segment; E, setae on hind tibiae.

Alate Viviparous Females. Fig. 2. Based on 42 Specimens.

Identical to apterae in qualitative, metric and meristic features, including the ranges of these last two, except the following ones: those being properly alatae (e.g. pterothorax), slightly more pigmented legs and sometimes antennae, pale brown marginal and sometimes spiracular abdominal sclerites, and 25–43 scattered secondary sensoria on antennal segment IV, 5–19 aligned ones on segment IV and 0–2 on segment V. The wing venation is not noticeably.

Bionomics. Aphids of this species form dense colonies on the stems of plants of genera Bidens, Schkuhria, Senecio and Stevia, and perhaps of other species belonging to subfamily Asteroideae (Asteraceae). Nothing is known regarding host alternation or lack thereof.

Fig. 2.

Alate viviparous female: A, habitus; B, III antennal segment; C, IV and V antennal segments.

Taxonomic Discussion

Principal Components Analysis. PCA summarizes multivariate data in a reduced number of dimensions which are linear combinations of the original variables.

The first 2 axes of the PCA represent 42.6% of the total variance (Fig. 3). Individuals from different Costa Rican and Mexican localities and caught on diverse host plants (belonging to genera Bidens, Schkuhria, Senecio and Stevia) are shown intermixed with each other. This suggests that all individuals belong to a single species no matter their procendence or host plant. Remarkably, some individuals from Costa Rica, which were bigger than the others, scored high on the first axis, which was dominated by antennae, leg, siphunculus and cauda variables.

Fig. 3.

Plot of Principal Components Analysis (PCA) of apterous viviparous females of Ucrymyzus villalobosi sp. n. from Costa Rica () and Mexico () recorded on different host-plant species.

MOLECULAR RESULTS AND DISCUSSION

A 710 bp DNA fragment containing a portion of the mitochondrial COI gene was amplified through PCR from the 3 individual aphids analyzed. After removing primer sequences, useful sequences consisted of 658 nucleotides. Sequences for the 3 sampled aphids were identical so a single sequence was finally deposited in Genbank (accession number KC110893). The online identification engine available at the Barcode of Life Data Systems (BOLD) (Ratnasingham & Hebert 2007) using the COI species database, failed to find any record corresponding to any identified species that matched our sequence. After a BLASTN search against the non-redundant nucleotide database at the NCBI, sequences from different aphid genera of the tribe Macrosiphini, such as Aulacorthum, Acyrthosiphon, Hyperomyzus, Macrosiphum, Nasonovia and Uroleucon were most similar to our sequence (94–96% identical). Since the COI sequence from U. villalobosi sp. n. did not match any sequence available at the DNA barcode reference library, we must conclude that it corresponds to a species not represented in the database, which currently hosts sequences from near 300 species from 70 genera within the subfamily Aphidinae. Moreover, we were also unable to assign the new species to any previously described genus based on the COI sequence as both strict tree-based and best close match assignment criteria (Wilson et al. 2011) were not fulfilled. Phylogenetic reconstructions using a set of available sequences from different Macrosiphini representatives were not informative about the phylogenetic affiliation of U. villalobosi sp. n., which grouped with Delphiniobium hanla, although with very low support, since they differed at 3.8% of the aligned positions (results not shown). This lack of resolution of COI for phylogeny inference is not surprising and it may be a weak phylogenetic signal compared with other markers as previously reported (Wilson 2010).

For the elongation factor-1 alpha gene fragment (EF1α) we obtained an identical sequence of 910 bp from the 3 analyzed individuals that was deposited in Genbank with accession number KC110894. Using sequences available for EF1α at NCBI for different Macrosiphini species, a Maximum Likelihood tree, which included the sequence obtained for our species, was built (Fig. 4). Contrary to COI, phylogenetic analysis using EF1α did not support the grouping of U. villalobosi sp. n. with the only Delphiniobium sequence available in GenBank (excluded from the final analysis because of its shorter length), but rather it grouped within a highly supported clade formed by 3 groups of sequences. One group contained sequences from species of genera such as Acyrthosiphon, Aulacorthum, Macrosiphum, Metopolophium and Sitobion. A second group included sequences from Nasonovia, Hyperomyzus and Obtusicauda. A third group contained sequences from Uroleucon and Macrosiphoniella (see Fig. 4). U. villalobosi sp. n. occupied a somewhat intermediate position between the first 2 groups of this clade, grouping, albeit with low support, with either of them depending on the precise parameters or the algorithm used in phylogenetic inference. Interestingly, another new species sampled from the same region (Nieto Nafría et al. in press), also grouped within this clade but closer to the third group described above.

Fig. 4.

Maximum Likelihood tree obtained for EF1α sequences from different Macrosophini species including Ucrimyzus villalobosi sp. n. (highlighted). Sequences from aphid species other than U. villalobosi were obtained from the NCBI database and their accession numbers are indicated. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.4096)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 47.0010% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated leaving a total of 816 positions in the final dataset. Percentage bootstrap support values obtained after 200 replicates are indicated above branches when higher than 50%.