A review of Diphuia ( Diptera : Ephydridae ) with description of two new species from southern Brazil

The species of Diphuia Cresson, 1944 are reviewed with an emphasis on the fauna from southern Brazil, where two new species have been discovered and herein are described: Diphuia antonina sp. nov. and Diphuia grandis sp. nov. All known species are placed into two species groups (the anomala and nitida groups), which are characterized, and a key to these species is included. To facilitate identification of species of this uncommon genus, we have included diagnoses of the genus and tribe Hecamedini and have also provided an annotated key to New World genera in the tribe and to the known species of Diphuia. We have also provided illustrations of structures of the male terminalia of all included species.The species from southern Brazil, including the new ones, are illustrated.

Specimens are usually collected in habitats associated with brackish water but we have also found specimens in freshwater environments.Nothing is known about the immature states.
To facilitate identification of species of this uncommonly collected genus, we have included a diagnosis of the genus and of the tribe Hecamedini and have also provided an annotated key to New World genera in the tribe and to the known species of Diphuia.We have also provided illustrations of structures of the male terminalia for all included species.

MATERIAL AND METHODS
The descriptive terminology, with the exceptions noted in MATHIS (1986) and MATHIS & ZATWARNICKI (1990a), follows that published in the Manual of Nearctic Diptera (MCALPINE 1981).Because specimens of Diphuia are small, usually less than 2.0 mm in length, study and illustration of the male terminalia required use of a compound microscope.We have followed the terminology for most structures of the male terminalia that other workers in Ephydridae have used (see references in MATHIS 1986, MATHIS & ZATWARNICKI 1990a, b), such as surstylus.ZATWARNICKI (1996) suggested that the pre-and postsurstylus correspond with the pre-and postgonostylus and that the subepandrial plate is the same as the medandrium.The terminology for structures of the male terminalia is provided in the legends.
A review of Diphuia (Diptera: Ephydridae) with description of two new species from southern Brazil Wayne N. Mathis 1 & Luciane Marinoni 2 ZOOLOGIA 27 (5): 803-812, October, 2010 Dissections of male and female genitalia and descriptions were performed using the method of CLAUSEN & COOK (1971) and GRIMALDI (1987).Microforceps were used to remove abdomens, which were macerated in a potassium hydroxide solution.Cleared genitalia were rinsed in a weak solution of acetic acid and then transferred to glycerin for observation and illustration.If necessary for proper orientation, the specimen was transferred from glycerin to glycerin jelly.The glycerin jelly was heated, and the specimen appropriately oriented.After cooling, the embedded specimen in glycerin jelly became immobilized.The abdomen was placed in a plastic microvial filled with glycerin and attached to the pin supporting the remainder of the insect from which it was removed.
The new species descriptions are composite and not based solely on holotypes.Two head and two venational ratios used in the descriptions are based on three specimens (largest, smallest, and one other): Eye ratio: maximum width/maximum height; gena-to-eye ratio: genal height (immediately below maximum eye height)/eye height; costal vein ratio: the straight line distance between the apices of R 2+3 and R 4+5 /distance between the apices of R 1 and R 2+3 ; M vein ratio: the straight line distance along vein M between crossveins dm-cu and r-m/distance apicad of dm-cu.
Specimens for this study are in the Universidade Federal do Paraná (DZUP: Coleção Entomológica Padre Jesus Santiago Moure, Departamento de Zoologia, Curitiba) and the National Museum of Natural History, Smithsonian Institution, Washington, D. C. (USNM).
Diagnosis.Head: arista with 3-5 dorsally branching rays, longer two or three rays subequal, inserted toward aristal base; compound eye bare of microsetulae or the latter very sparse.Thorax: usually with a gray to silvery stripe on thorax from postpronotum through ventral portion of notopleuron; anterior supra-alar seta lacking; posterior notopleural seta inserted at distinctly elevated position, especially as compared to anterior seta; anepisternum usually two toned, dorsal portion concolorous with mesonotum, ventral portion gray; anepisternum with two subequal setae inserted along posterior margin.Wing: venation of wing generally pale colored; vein R 2+3 elongate, section III much shorter than section II; apical section of vein M longer than section between crossveins r-m and dm-cu; alula wide, width subequal to that of costal cell.Abdomen: male terminalia: pregonite either lacking or fused indistinguishably with postgonite; subepandrial sclerite lacking; postgonite generally elongate and bearing few setulae, usually only two are conspicuous.Diagnosis.Small shore flies, body length 1.35-1.80mm; mostly black, subshiny to shiny, microtomentum usually sparse.Head: wider than high; face width-to-head width ratio 0.28; frons black, mostly unicolorous, lacking distinctively colored ocellar triangle; frons wider than long, frontal length-to-width ratio 0.58; frontal vestiture variable; ocellar seta well-developed, inserted slightly in front of alignment of anterior ocellus and at about the same distance apart as between posterior ocelli; pseudopostocellar setae usually well-developed, length subequal to ocellar setae, proclinate, slightly divergent; one reclinate and one proclinate fronto-orbital seta present, reclinate seta inserted slightly anteromediad of proclinate seta; both medial and lateral vertical setae present; ocelli in isosceles triangle, with distance between posterior ocelli larger than between anterior ocellus and either posterior ocellus.Antenna exerted; pedicel with well-developed, proclinate, dorsal seta; aristal length subequal to antennal length and bearing 4-5 dorsal rays, with basal three rays longer than apical 1-2, latter subequal.Eye apparently bare.Face black in both sexes and with silvery white, microtomentose antennal grooves and with two lines, sometimes irregular, paralleling parafacials, these and similarly invested and colored ventral margin (microtomentum sometimes interrupted at middle) form a facial triangle that has a small microtomentose area below facial prominence; face not carinate between antennal bases but slightly, conically protruding at middle (best seen in lateral view); ventral facial margin shallowly emarginate; face bearing two facial setae, the dorsal seta very slightly larger, both inserted near parafacials; parafacials densely microtomentose, silvery white; clypeus very sparsely microtomentose, black; palpus blackish brown to black; mouthparts not geniculate, labella shorter than mediproboscis.Thorax: generally black, vestiture of microtomentum variable with species, although generally sparse; pleural areas lacking stripes of distinctly colored microtomentum.Chaetotaxy with mesonotal setae poorly developed except for those at posterior margin; mesonotal setulae numerous and not in well-defined setal tracks; prescutellar acrostichal setae much larger than other acrostichal setulae and more widely set apart; only one dorsocentral seta, inserted posteriorly; intra-alar setulae irregularly seriated; presutural seta well-developed, length subequal to notopleural setae; two scutellar setae and scutellar disc with sparse, scattered setulae; postpronotal seta 1; postalar seta 1; notopleural setae 2, insertion of posterior seta elevated dorsally above anterior one; anepisternal setae 2, inserted along posterior margin; katepisternal seta well-developed, conspicuous.Wing: membrane mostly hyaline to very slightly milky white; veins behind costa pale, usually yellowish to yellowish brown; vein R 2+3 extended well beyond level of crossvein dmcu, costal section II at least 1.5X longer than section III; alular marginal setulae short, less than 1/2 alular height.Legs: femora black; tibiae dark basally, concolorous with femora, apices yellowish.Abdomen: fifth segment of male well-sclerotized, elongate, not normally visible from a dorsal view, usually retracted within 4 th segment; 5 th tergite and sternite of male united anteriorly to form a complete annulus.Male terminalia as follows: cercus rod shaped, bearing 2-3 conspicuously longer setae at ventral margin; surstylus well-developed, well-sclerotized, and conspicuous, length as long as cercus.
Although Diphuia is clearly a monophyletic group, its sister-group relationships are unclear.The most recent cladistic analysis of the genera of Hecamedini (MATHIS 1991) placed Diphuia at the base of a lineage also giving rise to Allotrichoma and its included subgenera (Pseudohecamede Hendel, 1936 and an undescribed subgenus).The evidence for this relationship is not totally convincing, however, and Diphuia could be more closely related to Hecamede (MATHIS 1997).
We recognize two species groups in Diphuia that are based primarily on characters of the abdomen (see species group diagnosis below).The anomala group

Key to species of Diphuia
Diagnosis.This species group is distinguished by the following combination of characters: Thorax: mesonotum moderately densely microtomentose; pleural region almost entirely but thinly invested with whitish gray microtomentum.Abdomen: 5 th tergite of male wider than long and with anterior margin deeply emarginate, broadly V-shaped.Male terminalia: epandrium bulbous, shiny, usually evident from a dorsal view; ZOOLOGIA 27 (5): 803-812, October, 2010 aedeagus robust, length in lateral view about twice height, apex broadly rounded and ventrally produced as an acute point.
Discussion.In addition to D. anomala, D. zatwarnickii also belongs to this species group.Diagnosis.This species is distinguished from congeners by the following combination of characters: Small shore-fly species, body length 1.60-1.80mm.Head: frons moderately invested with brownish microtomentum, microtomentum sparse or lacking on two small areas laterad of posterior ocelli and on two spots along anterior margin.Thorax: mesonotum densely invested with brownish to golden brown microtomentum, especially medially, along posterior portion of scutum and scutellum; anepisternum with fine investment of whitish microtomentum.Wing with costal vein ratio 0.47-0.51;M vein ratio 0.40-0.42.Abdomen: 5 th tergite (Figs 1 and 2) almost as high as long, anterior margin in dorsal view with deep, broadly V-shaped emargination, posterior margin with sparse setae; 5 th sternite (Fig. 1) clearly divided into 2 broad sternites that are connected only anteroventrally.Male terminalia (Figs 3-7): epandrium bulbous, shiny, in lateral view (Fig. 3) almost as wide as high; surstylus long (Fig. 3), narrow, parallel sided, width and length subequal to that of cercus, apex angulate, pointed anteriorly, and bearing a few setulae; cercus bearing two elongate, prominent setae at ventral margin; gonite (Fig. 5) broad basally, with posteriorly extended process sheathing aedeagus, posterior apex of gonite curved anteroventrally; phallapodeme triangular in lateral view, narrowly produced dorsally; aedeagus in lateral view broad, thumb-like, produced posteroventrally to a ventral point, in dorsal view becoming wider apically, apex broadly rounded; hypandrium in ventral view longer than wide, anterior margin with a small, anterior process.
Remarks.This is the type species of Diphuia.It is very similar externally to D. zatwarnickii Mathis and can be distinguished only by reference to structures of the male terminalia.From D. nitida it may be distinguished by the following characters: Frons and mesonotum invested moderately densely with brownish to golden brown microtomentum; anepisternum invested with fine, grayish to whitish microtomentum, anteroventral portion not bare, shiny; second costal section long, costal vein ratio 0.50; and several characters of the male terminalia (see description).

The nitida group
Diagnosis.This species group is distinguished by the following combination of characters: Thorax: mesonotum very thinly microtomentose, subshiny to shiny; pleural region with anteroventral portion bare or microtomentum, shiny or very thinly whitish gray microtomentose.Abdomen: 5 th tergite of male longer than wide and with anterior margin truncate or very shallowly emarginate.Male terminalia: epandrium narrow, not conspicuously evident from dorsal view, thinly microtomentose; aedeagus elongate, 3-4 times longer than wide, tapered to thinly rounded or sharp apex that is not ventrally produced as an acute point.
Discussion.Three species, including both newly described species (D. antonina sp.nov., D. grandis sp.nov., and D. nitida), belong to this species group.
Etymology.The species epithet, antonina, is the name of a colorful, colonial port town on the Paranaguá Bay of the Brazilian state of Paraná.
Remarks.This species is similar and closely related to D. nitida.Structures of the male terminalia are also very similar, especially the bifurcate aedeagal apex.These characters demonstrate that these two species form a monophyletic lineage.
Etymology.The species epithet, grandis, is of Latin derivation, meaning large, and refers to the large size of this species relative to congeners.