Corresponding author: Frederico F. Salles (
Academic editor: L. Pereira-da-Conceicoa
The imago stages of three species of the
Salles FF, Domínguez E, Mariano R, Paresque R (2016) The imagos of some enigmatic members of the
Since the delimitation of the
While expedient on one hand, the description of new leptophlebiid taxa based on nymphs alone has, on the other hand, generally added more uncertainty to our understanding of the delimitations and relationships of taxa within this incredibly diverse mayfly family. As part of ongoing taxonomic and phylogenetic studies of the
Habitus images of preserved specimens were taken using a Leica M165C stereomicroscope with a DFC420 digital camera or a Zeiss STEMI 2000-C stereomicroscope with a ERC5 digital camera. In order to produce final images with enhanced depth of field, a series of stacked images were processed with the program Leica Application Suite version 3.4.1 or Helicon Focus®. Living specimens were photographed in the field, in a small acrylic aquarium, with a Nikon D800, a 105 mm objective and a Nikon macro flash. Line drawings based on photographs were made with Adobe Illustrator CC® and were prepared according to
DNA was extracted using a Wizard SV Genomic DNA Purification System Kit (Promega®) based on the protocol for animal tissue. For imago specimens, the abdomen and wing were removed, and all remaining portions were placed in extraction buffer; for nymphs, three legs were used, and the rest of each specimen was retained as voucher material. A 658 base pair portion of COI was amplified for for each specimen, and PCR was performed in a 25-µL mixture containing: approximately 20 ng/µL DNA template, 1X PCR buffer, a 2.0 mM concentration of MgCl2, and a 30µM concentration of each primer (LCO 1490 and HCO 2198) (Folmer et al. 1994), a 100µM concentration (each) of dATP, dCTP, dGTP, and dTTP), 1U Taq Platinum DNA Polimerase Invitrogen® and ultrapure water to complete 25µL. Initial PCR consisted of a preheating at 94°C for 5 min; 40 cycles of 94°C for 45 s, 47°C of annealing temperature for 45 s and 72°C for 45 s, and incubation at 72°C for 5 min. Negative controls were used that contained all elements of the reaction mixture except DNA. Successful bands were detected on 1.5% agarose gel in 1X TAE buffer. Products were purified using ExoSAP-IT® for PCR Product Cleanup (GE Heathcare). All samples were sequenced by Macrogen®. The alignment of sequences was relatively unambiguous as all specimens were length invariable. Sequences were aligned and trimmed to length using Geneious R8, resulting in 658 characters. The basic sequence statistics including nucleotide frequencies and transition/transversion (Ts/Tv) ratio; variabilities in different regions of sequences were analyzed using Jmodeltest V0.1 (Posada 2008), DAMBE (Xia and Xie 2001) and DnaSP v5.0 (Librado and Rozas 2009). Pairwise numbers of nucleotide differences were calculated with MEGA, version 6.06 (Tamura et al. 2013), using the ‘Calculate distances’ option and the Kimura 2-parameter model of evolution (Kimura 1980).
Voucher material is deposited in the following institutions:
Instituto Nacional de Pesquisa da Amazônia Manaus, Brazil
Florida A&M University, Tallahassee, Florida, USA
Instituto de Biodiversidad Neotropical, Tucumán, Argentina
Coleção Zoológica Norte Capixaba, São Mateus, Brazil
The male imago of
(in alcohol).
Four ♂ imagos: Brazil, Mato Grosso State, Ribeirão Cascalheira, Gleba Maria Tereza, córrego “corgão”,
The wide projections of the styliger plate readily distinguish
Variation in body lengths and colouration were encountered among specimens, with some individuals clearly darker than others. The overall shape of genitalia, however, was the same, and thus we are concluding for now that all of this material belongs to a single species. Unfortunately, since it could help in the identification of potential cryptic species, we were unable to extract and/or amplify DNA from all localities (see COI divergence section below).
With the description of this species, the diagnoses of the adults of the genus must be expanded in the following way: 1) Forks of veins MA and MP of fore wings asymmetrical; 2) cross vein close to MA fork slanted or not; 3) vein Sc of hind wings ending in transverse vein near base of costal projection; 3) vein MP of hind wings unforked; 4) costal projection of hind wings acute or rounded at apex; 5) tarsal claws of a pair dissimilar, one apically hooked, other blunt; 6) penis divided in apical 1/2 to totally divided, each lobe with median spine-like projection; 7) styliger plate with spines close to base of forceps or with two wide projections; 8) prosternum with short to long median carina; and 9) female sternum IX apically cleft.
The male imago of
(in alcohol).
This is the only species of the genus known from a male imago. Therefore, it is impossible to ascertain at this time the characteristics that will distinguish it from its congeners.
(in alcohol).
One reared ♂ imago: Brazil, Roraima, Boa Vista, Rio Cauamé,
Imagos of
In Roraima, nymphs were predominantly captured on a small stream leading to Rio Branco, at the Bem Querer falls, and in Boa Vista, at the Cauamé River (Fig.
The male imago of
(in alcohol).
(in alcohol).
This is the only species of the genus. Therefore, it is impossible to ascertain at this time the characteristics that will distinguish it from its congeners.
(in alcohol).
General coloration: grayish-brown.
(Fig.
Similar to male imago, except as follows: head yellowish-orange, except central longitudinal line on posterior part of dorsum of head; anterior margin of head, line connecting ocelli and area behind lateral ocelli washed with black. Eyes black. Ninth sternite yellowish-white.
Three ♂ imagos: Brazil, Mato Grosso State, Nova Xavantina, córrego Benedito Ferreira, 06.xii.2006, light trap, Mariano R., Calor A.R. & Mateus S. (
This species appears to be unique, in particular reference to the development of the labial palpi in the nymph (
Nymphs (Fig.
Living specimens:
General aspect of the Cauamé River, Roraima, Brazil.
GenBank Accession numbers are given in Table
Collection information for specimens analysed in this study. Specimen information includes: species name, voucher number, locality ( , State of Espírito Santo , State of Roraima , Brazil
Species | Voucher | Locality | GenBank |
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4014 a | Serra, |
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4014 b | Serra, |
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4015 a | Bom Jesus do Norte, |
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6100 a | Iúna, |
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5607 e | Boa Vista, |
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5607 h | Boa Vista, |
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5607 i | Boa Vista, |
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5607 k | Boa Vista, |
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5607 l | Boa Vista, |
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5607 n | Boa Vista, |
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5761 a | Bonfim, |
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6086 a | Bonfim, |
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Kimura-2-Parameter , , ,
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0.176 | 0.184 | ||||||||||
0.176 | 0.184 |
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0.173 | 0.180 |
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0.172 | 0.179 |
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0.164 | 0.171 | 0.218 | 0.218 | 0.215 | 0.211 | ||||||
0.158 | 0.164 | 0.215 | 0.215 | 0.211 | 0.207 |
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0.158 | 0.164 | 0.218 | 0.218 | 0.215 | 0.211 |
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0.168 | 0.175 | 0.215 | 0.215 | 0.211 | 0.207 |
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0.158 | 0.164 | 0.218 | 0.218 | 0.215 | 0.211 |
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0.164 | 0.171 | 0.226 | 0.226 | 0.222 | 0.218 |
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Genetic species delimitations were highly congruent with our morphological species identifications and showed a high level of confidence. Sequence differences smaller than 3% are frequently observed in intraspecific distances of DNA barcodes (
Since the description of
The imago of
The male imago of
Despite the similarities between
The species in the
1 | Styliger plate without projections (Fig. 151d of |
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– | Styliger plate with sublateral (Figs 144j, 144l, 150e of |
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2 | Styliger plate with single medial projection (Fig. 4d, e and fig. 174e of |
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– | Styliger plate with paired sublateral projections (Figs 2d, 6d and figs 144j, l, 150e of |
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3 | Medial projection of styliger plate of various shapes, but never curved toward penis lobes (Fig. 174e of |
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– | Medial projection of styliger plate robust, curved towards penis lobes (Fig. |
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4 | Length of body ca. 5 mm; costal area of fore wing hyaline |
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– | Length of body ca. 10 mm; costal area of fore wing brown |
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5 | Paired projections wide, partially or almost completely covering the penis lobes (Figs |
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– | Paired projections subtriangular, not covering the penis lobes (figs 144j, l, 150e of |
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6 | Paired projections fused (Fig. |
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– | Paired projections separated (Fig. |
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7 | Abdominal coloration contrasting, with segments II–VI translucent and segments VII–X reddish-brown (fig. 13a, b of |
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– | Abdominal coloration not contrasting, segments II–X all similarly washed with black (Fig. |
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8 | Paired projections with small distal spines; penis lobes each with a strong spine-like projection, which is medially bowed and ventrally directed (fig. 35 of |
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– | Paired projections without small distal spines; penis lobes each with a strong spine-like projection posteriorly directed (Fig. |
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9 | Eyes meeting on meson of head | |
– | Eyes not meeting on meson of head (separated by a distance equal to 1.5 times width of lateral ocellus) |
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10 | Projections of penis lobes broad and parallel (figs 144k, 144l of |
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– | Projections of penis lobes spine-like and convergent (figs 144j, m, 150e, 169e of |
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11 | Spine-like projection of penis lobes straight (sometimes slightly curved at apex) (fig. 150e of |
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– | Spine-like projection of penis lobes strongly curved (Figs 144j, m, 169e of |
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12 | Projections of styliger plate short and blunt (fig. 150e of |
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– | Projections of styliger plate long and pointed (fig. 24 of |
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13 | Apex of penis lobes pointed; projections of styliger plate relatively short (fig. 24 of |
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– | Apex of penis lobes somwehat truncate; projections of styliger plate relatively long (fig. 6 of |
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14 | Projections of styliger plate long; distolateral corner of penis lobe less developed than inner corner (fig. 169e of |
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– | Projections of styliger plate short; distolateral corner of penis lobe more developed than inner corner (as in figs 144j, m of |
Partial view of South America, with emphasis on Brazil (yellow), showing the distribution of the species treated herein. Dashed lines, Brazilian states limits.
We would like to express our gratitude to the CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for financial support (projects 479967/2013-0, 474789/2011-0) and for a productivity grant to FFS. We thank FAPES (Fundação de Amparo à Pesquisa do Espírito Santo) for financial support of the project “Diversidade de Taxonomia de Insetos Aquáticos na Porção Capixaba da Bacia do Rio Doce” (Process number 61938408/2013) and for a “taxa de pesquisa” grant to FFS (process number 600166004/2012), as well as FAPESB (DCR 6576/2009) and UESC (PROPP/UESC 00220.1100.1265). We thank the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) and Instituto Brasileiro do Meio Ambiente e Recursos Naturais Renováveis (IBAMA) (Process number 12777-1) for collection permissions that allowed the completion of this work. We are extremely grateful to Luke M. Jacobus and Jean-Luc Gattolliat for reviewing the manuscript and to Neusa Hamada, Helena Cabette, Rafael Boldrini, Jeane Nascimento, Jesine Falcão, and Jaime Gama Neto for field assistance and/or loan of the material.