New species of Macunahyphes Dias, Salles & Molineri (Ephemeroptera: Leptohyphidae), with taxonomic notes

Macunahyphes zagaia sp. nov. and M. araca sp. nov. are described based on imagines collected from the Amazon biome in Brazil. Specimens belonging to all species of the genus were examined and a diagnosis for each one is provided. Variation seen in the male imago of M. eduardoi is described, as well as the female and egg for the first time. Together with M. eduardoi, the two new species share the presence of a basal swelling on segment II of the forceps, which raises important questions concerning the evolution of this character in Leptohyphidae. Finally, a re-definition of the genus is proposed.


Introduction
The genus Macunahyphes Dias, Salles & Molineri, 2005 was erected to include Macunahyphes australis (Banks, 1913) originally described by Banks (1913) in the genus Tricorythus Eaton, 1868.This species, described solely on adults, was subsequently transferred to Leptohyphodes Ulmer (Ulmer 1920) and then to Tricorythodes Ulmer (Traver 1958).With the discovery and subsequent description of the nymphs, T. australis was finally transferred to the new genus Macunahyphes (Dias et al. 2005).Among the distinctive characteristics of this species, Traver (1958), as well as Molineri (2002), pointed out the absence of the basal swelling of the second joint of the forceps, absence of the longitudinal vein CuP on male fore wings, and the morphology of the penis, with the presence of a ventral projection.
Based on adults from Venezuela and Brazil, respectively, Molineri et al. (2011) described two additional species: M. pemonensis Molineri et al. and M. incognitus Molineri et al.Despite the absence of forceps in the few available specimens of both species, the presence of a ventral projection on the penis, wing venation and similar color pattern were enough to allocate them to the genus Macunahyphes (Molineri et al. 2011).
Recently, two additional species were found in Brazil: M. pemonensis, recorded from Northern Brazil based on a larger series of male adults, and M. eduardoi Almeida & Mariano, 2015, based on male adults from Northeastern Brazil (Belmont et al. 2015;Almeida & Mariano 2015).Importantly, forceps of both species were unbroken and the basal swelling, absent in M. australis and considered one of the striking characteristics of the genus, was found to be present in M. eduardoi and M. pemonensis.
In the present paper, based on material from the Amazon and Atlantic Forest biomes in Brazil, two new species of Macunahyphes are described.In addition, specimens belonging to all species of the genus are examined, new data are provided for them and a re-definition of the genus is proposed.

Material and methods
Specimens were preserved in 80% ethanol, wings were mounted dry and genitalia were mounted in Euparal ® .Photographs were taken with a digital camera coupled to a Zeiss Axiocam ERc 5s stereo microscope and combined using Helicon Focus 6 ® software.Some of the photographs were used as templates for trace vector graphics in Adobe Illustrator CS6 ® software to produce the illustrations.Material from the following institutions was studied: Coleção Zoológica Norte Capixaba, Universidade Federal do Espírito Santo, São Mateus, Brazil (CZNC) and Coleção Entomológica Professor José Alfredo Dutra (DZRJ), Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.Holotypes and part of the paratypes were deposited in the Coleção de Invertebrados of the Instituto Nacional de Pesquisas da Amazônia, Manaus, Brazil (INPA).The other paratypes were deposited in CZNC and DZRJ.Wings were mounted on dry slides; as all were identical, there was no need to draw them.Eggs were removed from females of Macunahyphes eduardoi Almeida & Mariano, 2015 and Macunahyphes araca sp.nov., then mounted with double sided tape on SEM stubs and sputter coated with gold.They were observed and photographed with a Jeol JSM-6510 scanning electron microscope.The terminology proposed by Koss & Edmunds (1974) was used to describe the eggs.The distribution map was made using the program QGIS 2.12.0-Lyon (QGIS 2015).(Banks, 1913), by monotypy).

Comments
Due to the presence of membranous filaments on the mesoscutellum in females and males in one species, and to the presence of a basal swelling on forceps segment II in four species, we propose an expansion to the diagnosis of the genus provided by Dias et al. (2005) to include characters 5 and 7 above.As M. australis is the only species in which nymphs have been described, the generic diagnostic features of the nymphal stage are maintained.
As M. australis is the only species for which nymphs have been described, the generic diagnostic features of the nymphal stage should also be used for specific diagnosis.

Diagnosis
The male of M. araca sp.nov.can be distinguished from all congeners by the following combination of characteristics: 1) longitudinal vein CuP absent; 2) membranous filaments of mesoscutellum absent;  3) segment II of forceps with a basal swelling; 4) spermatic duct dark purple, visible laterally from outside; 5) styliger plate slightly projected posteriorly as a columnar base for each forceps, with sublateral acute projection on hind margin; 6) penis base subquadrate, narrowing abruptly in median zone; 7) penis with deep apical furrow; 8) ventral structure of penis reaching the deep apical furrow.

Etymology
Aracá is the name of the mountain range where this new species was collected.Legs.Yellowish, inner apex of trochanter tinged with black; fore tibia and fore tarsi slightly washed with dark purple.
abdomen.Terga II -X with medio-longitudinal dark line, terga I -VII translucent, tinged with dark purple, terga VIII -X yellowish brown, washed dark purple; tergum X with medial dark furrow; spermatic duct dark purple, visible from outside.
genitaLia.Forceps translucent, washed with dark purple, except on inner margin of segment I, styliger plate and penes translucent yellowish; styliger plate slightly projected posteriorly as a columnar base for each forceps, with sublateral acute projection on hind margin; forceps segment I weakly attached to styliger plate, basal swelling present on segment II; penes long, with subquadrate base, deep apical furrow and a somewhat protruded ventral structure.Caudal filaments translucent, tinged with dark purple at base, becoming lighter towards apex.
head.Yellowish brown, washed with black, posterior margin darker; scape and pedicel dark brown, flagellum whitish.
thorax.Pronotum tinged with black, lateral corner brown, washed with black; mesonotum brown, washed with black; metanotum yellowish brown, washed with black; membranous filaments of mesoscutellum absent.
Fore wing.Membrane white, longitudinal and cross-veins black; vein CuP absent.
abdomen.Translucent, tinged with black; caudal filaments broken off.4C-D) Yellow; single polar cap, attachment structures with tubercles on surface; three micropylar openings present at opposite pole of polar cap; chorionic surface without sculpture and with six elongated and linear micropylar canals ending at micropylar opening.

Biology
The specimens were collected at light traps during the dry season.The new species was found exclusively at the Serra do Aracá, together with M. eduardoi and M. zagaia sp.nov.Macunahyphes araca sp.nov.was the only species of mayfly found at the base and at the top of the Serra do Aracá.The Serra do Aracá is an elevated area (100 to 1500 m a.s.l.) in the State of Amazonas near the borders of Roraima State and Venezuela.Only five species of mayflies were collected at the top of Serra do Aracá during the same field trip; all of them were new to science at that time.In addition to the Macunahyphes species cited above, the other two species were Askola yanoman Nascimento, Barcelos-Silva & Salles, 2011 and an undescribed species, also found exclusively at the top, suggesting a high level of endemism in this area.

Remarks
Macunahyphes araca sp.nov.and M. pemonensis are the darker species of the genus.Macunahyphes araca sp.nov.can also be distinguished from all other species by the unique spermatic duct, which is visible laterally from outside due to its dark purple coloration, a characteristic also found in M. pemonensis (see Belmont et al. 2015: figs 1, 6).As in M. pemonensis, the styliger plate is slightly projected posteriorly as a columnar base for each forceps, with a sublateral acute projection on the hind margin, unlike the one found in M. incognitus.On the other hand, in the new species the penis base is subquadrate, narrowing abruptly in the median zone, as found in M. incognitus.Despite the very similar genitalia of M. araca sp.nov.and M. incognitus, the ventral structure of the new species reaches the deep apical furrow, while the ventral structure of M. incognitus ends before the apical furrow and the penis lobes are almost completely fused.According to the original description, M. incognitus seems to be similar to M. australis regarding the overall coloration, lighter in comparison to M. araca sp.nov.Distribution (Fig. 1) Brazil (Amazonas).Almeida & Mariano, 2015 Figs 2B, 4E

Diagnosis
According to the original description, the male of M. eduardoi can be distinguished from all congeners by the following combination of characteristics: 1) fore wing translucent, with costal and subcostal area tinged with grey and with longitudinal vein CuP absent; 2) penes opaque yellow, spine elongate with lateral projections.
head.Brown, washed with black; area between lateral ocelli and posterior margin tinged with black.
thorax.Pronotum brown, tinged with black, except for submedial stripe and anterolateral corner; mesonotum brown, washed with black; metanotum yellowish, washed with black; membranous filaments of mesoscutellum present and longer than in males.
Fore wing.Membrane white, longitudinal and cross-veins black; vein CuP absent.
abdomen.Translucent (yellow because of the presence of eggs), washed with black; terga II-V forming a V-shaped unpigmented area; caudal filaments broken off.
Egg (Fig. 4E) Yellow.Shape elongate.One polar cap present.Chorion with longitudinal costae formed by plates, each one with a deep emargination.

Remarks
In the description of the male imago of M. eduardoi there is no reference to the presence of membranous filaments of the mesoscutellum; this character is evident from the fresh material studied here.After examining the holotype of M. eduardoi it was possible to observe the presence of a short filament of about ¼ the length of the mesoscutellum.According to the original description, the hook-shaped lateral expansions present in the penis projection are distributed from its base to the middle region, whereas in the material examined by us the expansions are present throughout the penis projection, decreasing in size towards the apex.This is the first record of M. eduardoi from southeastern (state of Espírito Santo) and northern (state of Amazonas) Brazil.

Diagnosis
According to the original description, the male imago of Macunahyphes incognitus can be distinguished from all congeners by the following combination of characteristics: 1) fore wings with hyaline membrane except around basal half of vein Sc, shaded with gray; 2) styliger with a pair of sublateral acute projections on the hind margin; 3) penes completely fused apically, with subquadrate base, narrowing abruptly in median zone, and with a ventral projection.

Remarks
Macunahyphes incognitus was described based on only one specimen and since then there has been no further record of this species.The forceps are unknown and we only had access to the holotype genitalia.Because of this, the comparisons between species were made based on the genitalia slide and original description.Distribution (Fig. 1) Brazil (Pará).Molineri, Grillet, Nieto, Dominguez & Guerrero, 2011 Macunahyphes pemonensis Molineri, Grillet, Nieto, Dominguez & Guerrero, 2011: 45.

Diagnosis
According to the original description, the male imago of Macunahyphes pemonensis can be distinguished from all congeners by the following combination of characteristics: 1) fore wings hyaline, membrane tinged with gray on basal half of C and Sc regions and basally to vein A; 2) styliger plate very slightly projected posteriorly as a columnar base for each forceps, with a pair of sublateral acute projections on the hind margin; 3) penis long and slender, with apical furrow and a somewhat protruding ventral projection.

Remarks
In the original description of the male imago of M. pemonensis there is no mention of the presence of a dark purple spermatic duct that is laterally visible from outside, a character also found in M. araca sp.nov.and evident from the fresh material of M. pemonensis studied here (see Belmont et al. 2015: figs 2, 6).In addition, a new interpretation is given to the ventral structure present in the penis described by Molineri et al. (2011).The lateral margins of the penis lobe are strongly sclerotized and are ventrally twisted, forming a lateral narrow flap on each side of the penis.The area between these flaps, interpreted as a ventral structure in the original description, is in fact a shallow groove (see Belmont et al. 2015: figs 4-6).

Diagnosis
The male of M. zagaia sp.nov.can be distinguished from all congeners by the following combination of characteristics: 1) longitudinal vein CuP absent; 2) membranous filaments of mesoscutellum absent; 3) segment II of forceps with a basal swelling; 4) penes trident-like, with inward curved lateral projections and acutte medial projection.

Etymology
Zagaia is a spear with three points used in Amazonian artisanal fishery, reminiscent of the characteristic trident-like penis of the new species.

Discussion
According to Dias et al. (2005), unique characters present in imagines of Macunahyphes are: forceps tri-segmented, with first segment distomedially projecting; penis with very wide base, becoming thinner toward a subapical constriction, and then slightly widening again; penis lobes of each side almost completely fused except for apical incision; penis with a ventral projection covered with spines.With the recent discovery of new species, some these features are actually restricted to M. australis.
Concerning the forceps, the presence of a basal swelling in the two new species described here, as well as in adults of M. pemonensis and M. eduardoi, raises important questions concerning the evolution of this character in Leptohyphidae.According to Molineri (2002), the presence of the basal swelling was considered one of the synapomorphies of the genus Tricorythodes.This basal swelling, however, might be a synapomorphy not for Tricorythodes, but for the clade leading to Macunahyphes and Tricorythodes (see Molineri 2002Molineri , 2006)).In that case, its absence in M. australis seems to be an autapomorphic trait.
The ventral projection of the penis covered with spines is clearly an autopomorphy of M. australis, since it is not found in any of the remaining species.In fact, unlike other Leptohyphidae, the morphology of the penis in Macunahyphes is highly variable among species.Any attempt to provide a diagnostic characteristic for that structure, therefore, is unsuccessful.
Wing venation, especially the absence of the vein CuP, was the main characteristic used for the allocation of the new species in Macunahyphes.The shape of the penis, a character somewhat conservative among the genera of Leptohyphidae and historically useful for genus delimitation in mayflies, is surprisingly variable in Macunahyphes.Despite the fact that vein CuP is poorly developed in some species of Tricorythodes (e.g.T. bullus), as well as in some specimens of M. australis, the new species described here should not be allocated to Tricorythodes.In this genus, one of the most diverse and widely distributed of the family, the shape of the penis is similar in all the species described so far (ca 55 species described).The description of the immature stages of most of the species of Macunahyphes will undoubtedly be essential for elucidating the monophyly of the genus and its relationship with Tricorythodes.
To improve our current knowledge of the genus, new efforts must be made in order to find more specimens of some rare species, such as M. incognitus, and, even more important, to find and describe the unknown nymphs of the species described in the last few years.

Fig. 1 .
Fig. 1.Geographical distribution of species of Macunahyphes in South America and Brazil (per state).