Research Article |
Corresponding author: Frederico F. Salles ( ffsalles@gmail.com ) Academic editor: Lyndall Pereira-da-Conceicoa
© 2016 Frederico F. Salles, Eduardo Dominguez, Rodolfo Mariano, Roberta Paresque.
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:
Salles FF, Domínguez E, Mariano R, Paresque R (2016) The imagos of some enigmatic members of the Hermanella complex (Ephemeroptera, Leptophlebiidae). ZooKeys 625: 45-66. https://doi.org/10.3897/zookeys.625.9874
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The imago stages of three species of the Hermanella complex are described mostly based on material from Roraima, northern Brazil: Hydrosmilodon gilliesae, Hydromastodon sallesi and Leentvaaria palpalis. Male imagos of Hydrosmilodon gilliesae and Leentvaaria palpalis both have a pair of large, broad projections at the posterior margin of the styliger plate, nearly covering the penis lobes; in L. palpalis, however, these projections are fused. The male imago of Hydromastodon sallesi resembles Hydrosmilodon plagatus in that both species have a styliger plate with a robust projection that is curved towards the penis lobes. DNA barcoding is likely to be a powerful investigative tool for identifying and understanding species limits among these Ephemeroptera taxa, especially if it is used within an integrative taxonomic context. An updated identification key to the genera of the Hermanella complex is proposed.
Taxonomy, diversity, Atalophlebiinae , mayflies, Neotropical Region, key, barcoding
Since the delimitation of the Hermanella generic complex (Ephemeroptera: Leptophlebiidae: Atalophlebiinae) by
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 Hermanella complex, an important group of Neotropical Leptophlebiidae is examined here. The male imagos of Hydrosmilodon gilliesae and Hydromastodon sallesi, as well as the male and female imagos of Leentvaaria palpalis are described for the first time. Additionally, the first DNA barcode sequences is reported for these species, and their use for stage associations is assessed as part of a combined morphological and molecular approach. Based on the discovery of these metamorphic stages, an updated identification key is provided to the genera of the Hermanella complex.
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:
FAMU Florida A&M University, Tallahassee, Florida, USA
IBN Instituto de Biodiversidad Neotropical, Tucumán, Argentina
CZNC Coleção Zoológica Norte Capixaba, São Mateus, Brazil
The male imago of Hydrosmilodon gilliesae can be distinguished from the other species of the genus by the following combination of characters: 1) Eyes separated on meson of head by a short distance — less than 0.5 times width of median ocellus (Fig.
(in alcohol). Lengths: body, 4.1–5.5 mm; fore wings: 5.4-5.8 mm; hind wings: 0.7–0.9 mm.
Head (Fig.
Thorax (Fig.
Abdomen (Fig.
Four ♂ imagos: Brazil, Mato Grosso State, Ribeirão Cascalheira, Gleba Maria Tereza, córrego “corgão”, S12°43.040, W52°03.345, 09.x.2007, light trap, Pinho L.C., Mateus S., Torali L. & Silva F.R. (
The wide projections of the styliger plate readily distinguish H. gilliesae from all other members of the complex except for Leentvaaria palpalis, but this latter species has the projections fused (see “Discussion” below).
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).
Hydrosmilodon gilliesae was found to occur in several localities in Brazil, ranging from relatively close to its type-locale in French Guiana (state of Roraima), to central (Mato Grosso and Mato Grosso do Sul), Northeast (Pernambuco and Bahia) and southeast parts of the country (Espírito Santo and São Paulo) (Fig.
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 Hydromastodon can be distinguished from the other genera of the Hermanella complex by the following combination of characters: 1) Eyes meeting on meson of head (Fig.
(in alcohol). Head (Fig.
Thorax: Prosternum with rather wide, X-shaped median carina, with similar anterior and posterior arms; similar to Needhamella, as shown by
Wings (Fig.
Legs. Ratio of segments of male forelegs, 0.6:1.0 (0.62 mm): 0.03:0.31:0.28:0.15: 0.08. Claws on each leg dissimilar, with one apically hooked and one blunt, pad-like.
Abdomen: Genitalia (Fig.
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). Lengths: body, 4.6–5.6 mm; fore wings: 4.8–5.6 mm; hind wings: 0.8–0.9 mm. General coloration: light brown.
Head (Fig.
Thorax (Fig.
Abdomen (Fig.
One reared ♂ imago: Brazil, Roraima, Boa Vista, Rio Cauamé, 2°52'5.30"N / 60°44'25.40"W, 76 m asl, 21.v.2014, R. Boldrini col. (CZNC); one ♂ imago (partially molted) and two ♂ subimagos, same data as previous, except 03.ii.2007, J. Falcão col. (CZNC); 16 nymphs, same data as previous, except for 20.iii.2014, F.F. Salles, E. Domínguez, R. Boldrini, J. Gama-Neto col. (11 nymphs CZNC and 5 nymphs IBN); ten ♂ imagos: Brazil, Rondônia, Nova Londrina, Rio Urupá, 11°02'05"N / 62°08'34"W, 182 m asl, 02.ix.2012, N. Hamada leg. (5
Imagos of Hydromastodon sallesi are readily distinguished from all members of the complex, except for Hydrosmilodon plagatus, by the shape of the forceps and by the presence of a strong and dorsally curved, medial projection at the styliger plate. Body color pattern (compare Fig.
Hydromastodon sallesi was described based on a few nymphs from Mato Grosso (Rio Pindaíba, Nova Xavantina) and Roraima (Bem Querer falls, Rio Branco, Caracaraí). The material examined in the present paper was collected from the states of Roraima and Rondônia, the latter of which extends the known distribution of the genus and species to the east.
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 Leentvaaria can be distinguished from other genera of the Hermanella complex by the following combination of characters: 1) Eyes separated on meson of head by a short distance—less than 0.5 times the width of the median ocellus (Fig.
(in alcohol). Head (Fig.
Thorax: Prosternum with narrow, straight median carina, similar to Hermanella and Hylister, but with longer anterior arms, as in Fig.
Wings (Fig.
Legs. Ratio of segments in male forelegs, 0.6:1.0 (1 mm): 0.03:0.35:0.30:0.15:0.06. Claws of each pair dissimilar, one apically hooked and one blunt, pad-like.
Abdomen. Genitalia (Fig.
(in alcohol). Lengths: body, 4.7–4.9 mm; fore wings, 4.9–5.2 mm; hind wings, 0.8–0.9 mm.
Head: Eyes (Fig.
Abdomen: Ninth sternum deeply cleft apically.
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). Lengths: body, 4.7–4.9 mm; fore wings, 4.6–4.8 mm; hind wings, 0.8–0.9 mm.
General coloration: grayish-brown.
Head (Fig.
Thorax (Fig.
Abdomen (Fig.
(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 (
Leentvaaria palpalis was originally described from Surinam, but it seems to be a widespread species. Recently
Nymphs (Fig.
GenBank Accession numbers are given in Table
Collection information for specimens analysed in this study. Specimen information includes: species name, voucher number, locality (ES, State of Espírito Santo; RR, State of Roraima; BR, Brazil) and GenBank Accession Number.
Species | Voucher | Locality | GenBank |
---|---|---|---|
Hydrosmilodon gilliesae | 4014 a | Serra, 20°3'33"S/ W40°22'42’, ES - BR | KX831900 |
Hydrosmilodon gilliesae | 4014 b | Serra, 20°3'33"S/ W40°22'42’, ES - BR | KX831901 |
Hydrosmilodon gilliesae | 4015 a | Bom Jesus do Norte, 21°6'53"S/41°41'31"W, ES - BR | KX831902 |
Hydrosmilodon gilliesae | 6100 a | Iúna, 20°21'06"S/41°31'58"W, ES, BR | KX831903 |
Hydromastodon sallesi | 5607 e | Boa Vista, 2°52'5"N/60°44'25"W, RR - BR | KX831904 |
Hydromastodon sallesi | 5607 h | Boa Vista, 2°52'5"N/60°44'25"W, RR - BR | KX831905 |
Hydromastodon sallesi | 5607 i | Boa Vista, 2°52'5"N/60°44'25"W, RR - BR | KX831906 |
Hydromastodon sallesi | 5607 k | Boa Vista, 2°52'5"N/60°44'25"W, RR - BR | KX831907 |
Hydromastodon sallesi | 5607 l | Boa Vista, 2°52'5"N/60°44'25"W, RR - BR | KX831908 |
Hydromastodon sallesi | 5607 n | Boa Vista, 2°52'5"N/60°44'25"W, RR - BR | KX831909 |
Leentvaaria palpalis | 5761 a | Bonfim, 3°21'4"N/59°545'13"W, RR - BR | KX831910 |
Leentvaaria palpalis | 6086 a | Bonfim, 3°21'4"N/59°545'13"W, RR - BR | KX831911 |
Kimura-2-Parameter (K2P) genetic distances for COI barcodes between Ephemeroptera specimens; specimens represented by voucher numbers (see Table
Lp 5761a | Lp 6086a | Hg 4014a | Hg 4014b | Hg 4015a | Hg 6100a | Hs 5607e | Hs 5607h | Hs 5607i | Hs 5607k | Hs 5607n | |
---|---|---|---|---|---|---|---|---|---|---|---|
Lp 6086a | 0.011 | ||||||||||
Hg 4014a | 0.176 | 0.184 | |||||||||
Hg 4014b | 0.176 | 0.184 | 0.000 | ||||||||
Hg 4015a | 0.173 | 0.180 | 0.003 | 0.003 | |||||||
Hg 6100a | 0.172 | 0.179 | 0.019 | 0.019 | 0.017 | ||||||
Hs 5607e | 0.164 | 0.171 | 0.218 | 0.218 | 0.215 | 0.211 | |||||
Hs 5607h | 0.158 | 0.164 | 0.215 | 0.215 | 0.211 | 0.207 | 0.031 | ||||
Hs 5607i | 0.158 | 0.164 | 0.218 | 0.218 | 0.215 | 0.211 | 0.028 | 0.003 | |||
Hs 5607k | 0.168 | 0.175 | 0.215 | 0.215 | 0.211 | 0.207 | 0.008 | 0.028 | 0.025 | ||
Hs 5607l | 0.158 | 0.164 | 0.218 | 0.218 | 0.215 | 0.211 | 0.028 | 0.003 | 0.000 | 0.025 | |
Hs 5607n | 0.164 | 0.171 | 0.226 | 0.226 | 0.222 | 0.218 | 0.017 | 0.025 | 0.022 | 0.014 | 0.022 |
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 Hs. gilliesae and Hs. mikei the diagnosis and consequently the monophyly of the genus Hydrosmilodon have been questioned (
The imago of Hs. gilliesae described here does not conform with the diagnosis of the male imago of Hydrosmilodon given by
The male imago of Hydromastodon sallesi, in turn, shares some important characteristics with the male imago of another recently described species of Hydrosmilodon, Hs. plagatus. Besides the shape and morphology of forceps segment I, which is more elongate than in other members of the complex (Fig.
Despite the similarities between Hs. gilliesae and L. palpalis, and between Hs. plagatus and Hm. sallesi, we will follow the classification scheme of
The species in the Hermanella complex group present a tendency to bear some kind of projections on the styliger plate. These projections can be paired, submedial and of different width, from narrow and pointed (as in Needhamella and some species of Hermanella) to broad (Hydrosmilodon gilliesae), or single and medial as in Paramaka convexa(Spieth), Hydromastodon sallesi and Hydrosmilodon plagatus. With the imagos described here, interesting questions could be raised: is the plate that completely covers the penis found in Leentvaaria palpalis (and also in Traverella insolita Nascimento & Salles) a single projection resulting from the medial fusion of the mentioned paired projections, of which Hs. gilliesae is an intermediate development (from narrow, to wide projections to totally fused)? Is the origin of the expansion of a medial projection similar to that of Paramaka convexa, or is there a different explanation for this character? We hope that these questions will be answered with the new evidence we are gathering from several new taxa recently collected and with the ongoing phylogenetic analysis of the group.
1 | Styliger plate without projections (Fig. 151d of |
Hylister (in part, plaumanni) |
– | Styliger plate with sublateral (Figs 144j, 144l, 150e of |
2 |
2 | Styliger plate with single medial projection (Fig. 4d, e and fig. 174e of |
3 |
– | Styliger plate with paired sublateral projections (Figs 2d, 6d and figs 144j, l, 150e of |
5 |
3 | Medial projection of styliger plate of various shapes, but never curved toward penis lobes (Fig. 174e of |
Paramaka (convexa, pearljam, incognita) |
– | Medial projection of styliger plate robust, curved towards penis lobes (Fig. |
4 |
4 | Length of body ca. 5 mm; costal area of fore wing hyaline | Hydromastodon (sallesi) |
– | Length of body ca. 10 mm; costal area of fore wing brown | Hydrosmilodon (plagatus) |
5 | Paired projections wide, partially or almost completely covering the penis lobes (Figs |
6 |
– | Paired projections subtriangular, not covering the penis lobes (figs 144j, l, 150e of |
9 |
6 | Paired projections fused (Fig. |
7 |
– | Paired projections separated (Fig. |
8 |
7 | Abdominal coloration contrasting, with segments II–VI translucent and segments VII–X reddish-brown (fig. 13a, b of |
Traverella (in part, insolita) |
– | Abdominal coloration not contrasting, segments II–X all similarly washed with black (Fig. |
Leentvaaria (palpalis) |
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 |
Hylister (in part, chimaera) |
– | Paired projections without small distal spines; penis lobes each with a strong spine-like projection posteriorly directed (Fig. |
Hydrosmilodon (in part, gilliesae) |
9 | Eyes meeting on meson of head | Traverella (in part, bradley, calingastensis, longifrons, montium, valdemari) |
– | Eyes not meeting on meson of head (separated by a distance equal to 1.5 times width of lateral ocellus) | 10 |
10 | Projections of penis lobes broad and parallel (figs 144k, 144l of |
Hermanella (in part, amere, guttata, thelma) |
– | Projections of penis lobes spine-like and convergent (figs 144j, m, 150e, 169e of |
11 |
11 | Spine-like projection of penis lobes straight (sometimes slightly curved at apex) (fig. 150e of |
12 |
– | Spine-like projection of penis lobes strongly curved (Figs 144j, m, 169e of |
14 |
12 | Projections of styliger plate short and blunt (fig. 150e of |
Hydrosmilodon (in part, primanus, saltensis) |
– | Projections of styliger plate long and pointed (fig. 24 of |
13 |
13 | Apex of penis lobes pointed; projections of styliger plate relatively short (fig. 24 of |
Hermanella (in part, mazama) |
– | Apex of penis lobes somwehat truncate; projections of styliger plate relatively long (fig. 6 of |
Traverella (in part, albertana) |
14 | Projections of styliger plate long; distolateral corner of penis lobe less developed than inner corner (fig. 169e of |
Needhamella (ehrhardti) |
– | Projections of styliger plate short; distolateral corner of penis lobe more developed than inner corner (as in figs 144j, m of |
Hermanella (in part, froehlichi, maculipennis, nigra) / Hylister (in part, obliquus) |
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.