The imagos of some enigmatic members of the Hermanella complex (Ephemeroptera, Leptophlebiidae)

Abstract 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 Leentvaaria 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.


Introduction
Since the delimitation of the Hermanella generic complex (Ephemeroptera: Leptophlebiidae: Atalophlebiinae) by Domínguez and Flowers (1989), significant new data have come to light, including the descriptions of several new taxa. The genus Hydrosmilodon Flowers & Domínguez (1992) was established for the species Thraulus primanus Eaton and the new species H. saltensis Domínguez & Flowers. Domínguez et al. (2001) redescribed the nymphs of the monotypic genus Leentvaaria Demoulin, which is known only from nymphs, and studied the phylogenetic relationships of the genera of the Hermanella complex as it was understood at the time. Thomas et al. (2004) described two new species of Hydrosmilodon, H. gilliesae Thomas & Péru and H. mikei Thomas & Boutonnet, based on nymphs from French Guiana. Later, Polegatto and Batista (2007) erected the new genus Hydrosmastodon for H. mikei and described a new species Hydrosmastodon sallesi Polegatto & Batista, also based solely on nymphs. More recently, Kluge (2007) considered Hydrosmilodon and Paramaka Savage & Domínguez, 1992 as junior synonyms of Needhamella Domínguez & Flowers, 1989 and placed all remaining genera as subgenera of Hermanella Needham & Murphy, 1924, a vision not followed by Nascimento and Salles (2013) nor in the present paper. Currently, therefore, the Hermanella complex is composed by the following taxa: Hermanella; Hydromastodon; Hydrosmilodon; Hylister Domínguez & Flowers, 1989;Leentvaaria;Needhamella;Paramaka;and Traverella Edmunds, 1948. 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.

Methods
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 Coleman (2003Coleman ( , 2006.

Sequence data
DNA was extracted using a Wizard SV Genomic DNA Purification System Kit (Pro-mega®) 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 MgCl 2 , and a 30µM concentration of each primer (LCO 1490and HCO 2198) (Folmer et al. 1994), a 100µM concentration (each) of dATP, dCTP, dGTP, and dTTP), 1U Taq Platinum DNA Polimerase Invit-rogen® 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. Diagnosis. 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. 1a); 2) Fore wings hyaline, slightly tinged with brown at base (Fig. 2a); 3) Coloration of abdominal segments II -IX with blackish anterior and posterior stripes, and variable submedial marks as in Fig. 1a, b; 4) Styliger plate with two wide projections that nearly cover the penis (Fig. 2d); 5) Penis lobes totally divided with distomedial spines converging medially (Fig. 2d). Description of male imago (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. 1a, b): brown, upper portions of eyes light orange-brown, lower portions blackish. Eyes separated on meson of head by short distance -less than 0.5 times width of median ocellus. Ocelli white surrounded with black. Antennae: light brown. Thorax (Fig. 1a, b): brownish with lighter sutures, mesoscutellum darker, and white spot on each anterolateral corner of posterior scutellar protuberance. Prosternum ( Fig. 1c) similar to Hydrosmilodon primanus and Hs. saltensis, but with carina longer and slightly wider. Pleurae yellowish and heavily washed with black. Wings ( Fig. 2a, b, c): membrane of fore and hind wings hyaline, slightly tinged with brown at bases, longitudinal veins yellowish-brown, cross veins yellowish. Fork of MA asymmetrical and fork of MP slightly asymmetrical (MP2 connected to MP1 by crossvein); crossvein above MA not slanted; vein ICu 2 attached at base to ICu 1 by crossvein. Legs: fore leg yellowish-brown, with apex of femur and base of tibia darker; mid and hind legs generally lighter.
Abdomen (Fig. 1a, d): terga light yellowish-brown, translucent on segments I-VII, segment I completely washed with black, segments II-IX with blackish anterior and posterior stripes, and variable submedial mark as in Fig. 1a Comments. 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. 9).
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.

Hydromastodon Polegatto & Batista, 2007 Figures 3, 4, 7
Diagnosis. 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. 3a); 2) Cross vein above fork of MA slanted (Fig. 4a); 3) Fork of MA asymmetrical and fork of MP slightly asymmetrical (MP2 connected to MP1 by a crossvein); 4) Styliger plate with a strong dorsally curved median projection (Fig. 4d, e); 5) Penis divided, each lobe with a long spine ventromedially directed (Fig. 4d, e).
Description of male imago (in alcohol). Head (Fig. 3a, b): Eyes meeting on meson of head, lower portion of eyes slightly < ½ length of upper portion.
Thorax: Prosternum with rather wide, X-shaped median carina, with similar anterior and posterior arms; similar to Needhamella, as shown by Domínguez and Flowers (1989: fig. 18).
Wings (Fig. 4a, b, c): Maximum width of fore wings 1/3 their maximum length; maximum width of hind wings about ½ their maximum length; maximum length of hind wings 1/6 maximum length of fore wings. Fore wings (Fig. 4a): vein Rs forked slightly > 1/4 distance from base of vein to margin, fork of vein MA asymmetrical and forked at ½ distance from base of vein to margin, cross vein above fork of MA slanted; fork of vein MP slightly asymmetrical and forked 1/3 distance from base of vein to margin; vein ICu 1 attached at base to vein CuA by crossvein; vein ICu 2 free basally.
Hind wings (Fig. 4b, c): costal projection well-developed, acute and located ½ distance from base to apex; vein MP unforked; apex of wings rounded; vein Sc ½ distance from base to wing margin, ending in crossvein; 5 cross veins present.
Abdomen: Genitalia (Fig. 4d, e) with segment II of forceps subequal to segment III; segment II of forceps 1/5 length of segment I; styliger plate with strong, dorsally curved, median projection. Penis divided, each lobe with long spine ventromedially directed. Caudal filaments broken off and lost. Figures 3, 4, 7a, b Diagnosis. 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.
Head (Fig. 3a, b): yellowish-white, tinged with orange between ocelli; upper portion of eyes orangeish, lower portion black; ocelli white, surrounded with black and orange. Antennae light yellow-brown.
Material examined. One reared ♂ imago: Brazil, Roraima, Boa Vista, Rio Cauamé, 2°52'5. Comments. 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. 3a herein to figs 2-4 of Lima et al. 2012), body length (around 5 mm in Hm. sallesi, but around 10 mm in Hs. plagatus) and details of penis morphology are enough to separate these two taxa. Geographic distribution may also prove helpful with identification, as Hs. plagatus is a typical Atlantic Forest species that seems to be restricted to the Brazilian coast, while Hm. sal- lesi is found in western and northern Brazil in transitional areas between the Amazon forest and Brazilian savannah.
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. 8). In the Cauamé River, nymphs (Fig. 7a, b) of this species were found under rocks, close to the river margins, and they were much less abundant than the nymphs of Leentvaaria palpalis (see immediately below).

Leentvaaria Demoulin, 1966
Figures 5, 6, 7c, d Diagnosis. 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. 5a); 2) Fork of MA asymmetrical and fork of MP slightly asymmetrical (Fig. 6a); 3) Crossvein above fork of MA not slanted (Fig. 6a); 4) Styliger plate enlarged posteriorly, completely covering penis lobes in ventral view (Fig. 6d); 5) Penis divided, each lobe with a long spine apically curved (Fig. 6e).
Description of male imago (in alcohol). Head (Fig. 5a, b, c, e): Eyes separated on meson of head by short distance-less than 0.5 times width of median ocellus (Fig. 5a, b, c), lower portion of eyes slightly < ¼ length of upper portion.
Thorax: Prosternum with narrow, straight median carina, similar to Hermanella and Hylister, but with longer anterior arms, as in Fig. 5d.
Wings (Fig. 6a, b, c): Maximum width of fore wings 1/3 their maximum length; maximum width of hind wings about ½ their maximum length; maximum length of hind wings 1/5 maximum length of fore wings. Fore wings: vein Rs forked slightly > 1/6 distance from base of vein to margin, fork of vein MA asymmetrical and forked at ½ distance from base of vein to margin, cross vein above fork of MA not slanted; fork of vein MP slightly asymmetrical and forked 1/3 distance from base of vein to margin; vein ICu 1 attached at base to vein CuA by crossvein; vein ICu 2 attached at base to vein ICu 1 by crossvein. Hind wings: costal projection well-developed, acute; located ½ distance from base to apex; vein MP unforked; apex of wings rounded; vein Sc ½ distance from base to wing margin, ending in crossvein; 4-6 crossveins present.
Abdomen. Genitalia (Fig. 6d, e): Styliger plate: length of segment II of forceps subequal to length of segment III; segment II of forceps 1/9 length of segment I; styl-iger plate enlarged posteriorly, completely covering penis lobes in ventral view. Penis divided, each lobe with long spine apically curved. Caudal filaments: terminal filament longer than cerci.
Head: Eyes (Fig. 5e) separated on meson of head by 6 times width of lateral ocellus. Abdomen: Ninth sternum deeply cleft apically. Demoulin, 1966 Figures 5, 6, 7c Diagnosis. 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. Description of male imago (in alcohol). Lengths: body, 4.7-4.9 mm; fore wings, 4.6-4.8 mm; hind wings, 0.8-0.9 mm.
Thorax (Fig. 5a, b, c): brown, washed with black (faded in figures) with lighter sutures. Wings (Fig. 6a, b, c): membranes of fore and hind wings hyaline with base tinged with yellow. Base of C of both wings tinged with black basally. Longitudinal veins yellowishbrown, cross veins yellowish. Legs: fore leg yellowish, with base of coxa washed with black. Femur washed with grey. Remainder of fore leg and mid & hind legs yellowish.
Description of female imago (Fig. 5e)     Comments. This species appears to be unique, in particular reference to the development of the labial palpi in the nymph (Domínguez et al. 2001) and the subgenital plate in the male imago. The wide projections of the styliger plate are fused into a single structure (Fig. 6d), as mentioned in the discussion of Hs. gilliesae (see above), which readily distinguishes L. palpalis from all the other members of the complex.
Leentvaaria palpalis was originally described from Surinam, but it seems to be a widespread species. Recently Lima et al. (2012) reported its presence from the states of Espírito Santo and Pernambuco, representing the Brazilian coast and Atlantic Forest. In the present paper we report material from Mato Grosso and Roraima, western and northern Brazil, which represents the Amazon and Cerrado transition zones. Nymphs (Fig. 7c) were found under rocks. In the case of the Cauamé River (where all the species treated herein were found, Fig. 8), L. palpalis is one of the most abundant species of mayflies.

COI intra-and interspecific divergence
Genbank Accession numbers are given in Table 1. Identifications of the three morphologically defined species treated herein, their metamorphic stages, and the average sequence distance (K2P) among haplotypes are given in Table 2. Intraspecific distances ranged from 1.10-1.86% (values in bold in Table 2) with an average of 1.32%. Distances between species ranged from 16.50-21.50% with an average of 18.60%.
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 (Ferguson 2002;  Hebert et al. 2003;Hebert et al. 2004;Ball et al. 2005;Cardoni et al. 2015;Gattolliat et al. 2015;Angeli et al. 2016). Whereas the examined specimens of Hm. sallesi for the barcode analysis were from the type-locality and those of L. palpalis were from an area relatively close to its type-locality, the specimens of Hs. gilliesae were from southeastern Brazil. Genetic distance of these specimens, when compared to those found in French Guyana (type-locality of the species), could be high due to geographic distance (e.g. Webb et al. 2012).

Discussion
Since the description of Hs. gilliesae and Hs. mikei the diagnosis and consequently the monophyly of the genus Hydrosmilodon have been questioned (Sartori 2005). This idea was later confirmed when Polegatto and Batista (2007) transferred Hs. mikei to the new genus Hydromastodon. Lima et al. (2012) described Hydrosmilodon plagatus, which presented some conflicting characters with the diagnosis of the genus (see below), as also happened with the adult of Hs. gilliesae described for the first time in this paper.
The imago of Hs. gilliesae described here does not conform with the diagnosis of the male imago of Hydrosmilodon given by Flowers and Domínguez (1992) in the following features: 1) Eyes of male separated on meson of head by a short distance-less than 0.5 times width of median ocellus; 2) Crossvein above MA not slanted; 3) Two wide projections almost covering the penis; and 4) Distomedial spines of penis converging medially. Most of these characteristics, in fact, are also present in Leentvaaria palpalis, indicating that these two species are probably closely related. The only difference is that the styliger projections are divided in Hs. gilliesae, while they are fused in L. palpalis.
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. 4d), there is a medial projection at the posterior margin of the styliger plate, which is curved and directed towards the penis lobes (Fig. 4d, e).
Despite the similarities between Hs. gilliesae and L. palpalis, and between Hs. plagatus and Hm. sallesi, we will follow the classification scheme of Nascimento and Salles (2013). When describing species and commenting on the status of the generic arrangement in the Hermanella complex, Nascimento and Salles (2013) argued that no further classification changes should be made without a formally constructed phylogeny for the group. As there is a cladistic analysis in progress, we will wait to make any necessary changes until after formal hypotheses of relationships are presented. Also for this reason, no emendations to the generic diagnosis of Hydrosmilodon are presented here.
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.
Updated Key to the male imagos of the Hermanella complex 1 Styliger plate without projections (Fig. 151d of Domínguez et al. 2006 Medial projection of styliger plate of various shapes, but never curved toward penis lobes (Fig. 174e  Abdominal coloration not contrasting, segments II-X all similarly washed with black (Fig. 5a); paired projections forming two small plates (Fig. 6d)