Jewelled spider flies of North America: a revision and phylogeny of Eulonchus Gerstaecker (Diptera, Acroceridae)

Abstract The spider fly genus Eulonchus Gerstaecker is found throughout the Nearctic Region. Six species are recognized and intraspecific morphological variation is documented in several species. A phylogeny of Eulonchus based on DNA sequence data of three molecular markers (COI, CAD, and 16S) is presented and relationships of species are discussed in the light of biogeography and host usage. All six species of Eulonchus are redescribed using natural language descriptions exported from a character matrix, and a key to species is presented. Lectotypes are designated for Eulonchus sapphirinus Osten Sacken, Eulonchus smaragdinus Gerstaecker, and Eulonchus tristis Loew.


Introduction
Acroceridae are a small group of flies commonly known as spider flies or small-headed flies. The family comprises a morphologically heterogeneous assemblage of taxa, with approximately 550 species in 55 genera (Schlinger et al. 2013) distributed in all major A 1 (as used in Cumming and Wood 2009) was homologized with CuP of Mecoptera. Consequently, the following wing venation terms are used here: CuA 1 (of Cumming and Wood) = M 4 , CuA 2 = CuA, and anal vein (A 1 ) = CuP.
Descriptions were constructed with Lucid Builder 3.5, using a matrix database of character states, which were then exported using the natural language function into a text document (for further editing) and accompanying XML. Specimen images were taken using a digital camera at different focal points and combined into a montage image using Helicon Focus software. Distribution maps were generated using ArcGIS 10.1 software (ESRI 2012). When GPS data were not available on the label, latitude and longitude were obtained by consulting online gazetteers and Google Earth™. Any plants listed as visited by Eulonchus, but without citation, are new records based on label data and collection records from this study.

Material examined
The specimens examined during the course of this study (Table 3) were principally part of the collection amassed by Dr. Evert Schlinger over more than 60 years. This collection now resides at the California Academy of Sciences (CAS). While most belonged to the Schlinger collection, a small minority of these specimens were loaned from other collections, but do not have that information clearly associated with them (either physically or in the incomplete specimen database). Therefore we cannot determine whether the specimen was loaned versus gifted; here we use a conservative approach and list only what is noted in the database or associated labels in the collection.
Annotations of collection label data are included where appropriate in brackets.  Evenhuis (2015).

Phylogenetic analysis
Five species of Eulonchus and two outgroup species (Apsona muscaria Westwood and Lasia corvina Erichson (given as L. carbonanicus in Winterton et al. 2007, a misspelling of L. carbonarius Philippi = L. corvina (Edwards 1930))) were included in the analyses.
Multiple DNA loci, as used previously in the phylogenetic study of spider fly evolution by Winterton et al. (2007), were included here. The dataset comprises sequences of two mitochondrial genes, 16S rDNA and cytochrome oxidase I (COI), as well as a single nuclear gene, the carbamoyl phosphate synthetase (CPSase) active site region of carbamoyl-phosphate synthetase-aspartate transcarbamoylase-dihydroorotase (CAD). Sequences from A. muscaria, L. corvina, E. marialiciae, E. sapphirinus, and E. smaragdinus were downloaded from Genbank, and new sequences for E. marginatus, E. sapphirinus, and E. tristis were added. DNA sequencing for these additional taxa was carried out following the protocol outlined by Winterton et al. (2007). Genbank accession and specimen voucher numbers are presented in Table 1. Sequences were aligned manually, with CAD and COI aligned with reference to translated amino acid sequences using Mesquite 2.75 (Maddison and Maddison 2014). Parsimony analyses were conducted using PAUP* 4.0b10 (Swofford 2003) using a branch and bound search protocol. Bootstrap support values were calculated from 200 heuristic search (TBR) pseudoreplicates of re-sampled data sets, each with 30 random additions (constant characters excluded).  ;Sabrosky 1948 (revision, key);Paramonov 1955: 20 (comments) ;Schlinger 1960Schlinger (revision), 1966Schlinger : 112 (distribution), 1981, 1987 (host records); Coyle and Icenogle 1994 (host records);Poole 1996: 36 (checklist).

Taxonomy
Type species. Eulonchus smaragdinus Gerstaecker, 1856 by monotypy. Common name. North American jewelled spider flies. Diagnosis. Eulonchus can be immediately identified from other Panopinae in the New World by the presence of elongate mouthparts, metallic colouration, and pilose eyes. The only other genus in the New World with metallic colouration is Lasia, which is distinguished from Eulonchus in the eyes being separated below the antennae, and in lacking the palpus and alula. Eulonchus is remarkably similar to the endemic New Zealand genus Apsona and is distinguished from it by the presence of tibial spines (lacking in Apsona), the wing medial veins reaching the wing margin (attenuated in Apsona) and the antennae placed in the middle of the frons (higher in Apsona).
Description. Body length: 7.2-12.8 mm; wing length: 5.2-12.6 mm. Body colouration metallic green, blue or purple, rarely non-metallic (likely due to poor preservation or collection methods). Head hemispherical, width slightly smaller than thorax width; ocellar tubercle slightly to strongly raised, shape variable and either rounded, bifid or trifid, two or three ocelli present; postocular ridge and occiput rounded; eye densely pilose, setae relatively long (2x pedicel length), posterior margin of eye rounded (i.e. not emarginate); antenna located on middle of frons; eye contiguous above and below antennal base; palpus present; proboscis length greater than head length; antennal flagellum elongate, cylindrical to laterally compressed, slightly to strongly tapered apically; in western species male flagellum cylindrical, broader distally, female flagellum more tapered distally; flagellum apex with or without terminal setae; scapes separate (i.e. not fused medially  Gerstaecker, 1856 andE. s. pilosus Schlinger, 1960); Eulonchus tristis Loew, 1872: 60. Distribution (Fig. 20). Eulonchus is distributed throughout the Nearctic, with E. marialiciae found in the eastern state of North Carolina and the remaining species distributed throughout the USA west of the Rocky Mountains, northwards to Canada and south to Baja California, Mexico (Fig. 20). There is significant overlap in the distribution of species of Eulonchus with that of the host (or presumed host) spiders in the families Antrodiaetidae (Aliatypus sp., Antrodiaetus riversi Cambridge, A. unicolor Hentz) and Euctenizidae (Aptostichus stanfordianus Smith) (Fig. 21) (Schlinger 1987), with the range of Eulonchus being clearly inside the range of its hosts. Antrodiaetidae comprise two genera, Aliatypus Smith, and Antrodiaetus Ausserer, with a disjunct distribution in Japan and North America (Coyle 1971;Hendrixson and Bond 2006). Nearctic antrodiaetids inhabit three primary geographic regions: the deciduous forests of the eastern United States (USA), the forested 'sky islands' of the southwestern USA, and various habitats throughout the northwestern USA and adjacent portions of southwestern Canada (Hendrixson and Bond 2006). The euctenizid genus Aptostichus Simon is a genus of trapdoor spiders found predominantly in southern California; A. stanfordianus is commonly called the Stanford Hills Trapdoor Spider, and is distributed throughout California (Bond 2012).
Conservation. Most species of Eulonchus are relatively widely distributed, and locally abundant, except the sole eastern North American species E. marialiciae. This species is known from only a few specimens, all found within a small contiguous area in the Great Smoky Mountains, with collections from Haywood, Jackson, Macon, Transylvania, and Swain counties in North Carolina. Even though the spider host and floral food source of E. marialiciae are distributed over much of western North Carolina, southwestern Virginia, and eastern Tennessee, the species is only found in five mountainous counties of western North Carolina, at elevations of 1250m or higher. Considering the apparent limited and very isolated distribution of this species, the conservation status of E. marialiciae should be further evaluated. Future studies should focus on identifying potential extralimital populations of this species outside of the presently known distribution, as well as understanding potential biotic (e.g. spider host and plant food source range and distribution) and abiotic (e.g. elevation, climate, vegetation and soil type) factors associated with the apparent limited distribution of this species.
Biology. Adult Eulonchus are locally abundant on flowers (Cole 1919;Schlinger 1960) and exhibit behaviours that show constancy to particular plant species (Borkent and Schlinger 2008a, b). Thus, North American jewelled spider flies may form a large and important component of the pollinator fauna for some plant species. Eulonchus visit species from more than 30 plant families (see Table 2), and in multiple habitats (Borkent and Schlinger 2008a, b). However, the ability of Eulonchus species to serve as pollinators needs to be further studied. Coyle (1971) reported a large aggregation of E. marialiciae forming as he was excavating burrows of Antrodiaetus unicolor in North Carolina. A relatively large number of flies (~12 individuals) approached very quickly, hovering close to the ground where the burrows were being excavated, apparently attracted by some chemical released during the process. The author observed multiple adults hovering over and landing near closed burrow entrances. As mentioned above, one of us (C. Borkent) has observed that the newly hatched first instar acrocerid larvae actively search for their hosts, rearing up in search of a spider. Once successful they subsequently penetrate their cuticle and develop as an endoparasitoid (Schlinger 1987). Additionally, Coyle (1971) observed several instances in which the larva, after feeding on the spider in the bottom end of the burrow, climbed up the burrow wall, attached somewhere above the bottom end and completed development within the burrow.
Comments. Species of Eulonchus are very similar to species of Lasia and Apsona, and these three genera, as wells as some species of Panops, are commonly known as jewelled spider flies due to their metallic body colouration. Eulonchus species are commonly called Emeralds or Sapphires, depending on the body colour. Winterton (2012) described the thoracic pile in the Australian genus Panops as being reflective, thus brighter when the individual was viewed anteriorly, a characteristic of many Old World panopine species. This character is absent in Eulonchus and most New World panopine genera, with the thoracic pile being of similar brightness regardless of which angle it is viewed. Phylogenetic relationships among Panopinae genera are still poorly known. Based on DNA sequence data, Winterton et al. (2007) found Eulonchus to be placed between Lasia and more derived genera such as Ocnaea, Archipialea, and Arrhynchus.
Phylogenetic relationships (Fig. 22). The phylogeny performed in this study is based on DNA sequence data and includes five of the six species of Eulonchus. The parsimony analysis resulted in a single most parsimonious tree with length = 904, consistency index (CI) = 0.92, and retention index (RI) = 0.62. The eastern species Eulonchus marialiciae (Fig. 6) and the northwestern E. sapphirinus (Figs 8-9) were recovered as sister taxa in a clade that is sister to the remaining species of the genus. Even though the support for the branch was low (Fig. 22) we are confident in this relationship as E. marialiciae and E. sapphirinus share multiple characters, such as the ovate epandrium (which is thinner at the apex; Fig. 18C-D), the gonocoxite taller than wide , and the aedeagus broad at the apex and not heavily laterally sclerotized (Fig. 17C-D). These two species are typically bright metallic green or blue with yellow legs. This feature is shared with some individuals of the highly variable E. smaragdinus from southern California, USA and Baja California, Mexico. Eulonchus smaragdinus (Figs 10-11) was recovered as an intermediate species that subtends the clade comprising the more northern species, E. tristis and E. marginatus. Eulonchus halli was not included in the phylogenetic analysis due to lack of fresh material for DNA extraction. However, placement of the species in the Eulonchus tree was postulated based on morphology (see dashed lines on Fig. 20). We hypothesize that E. halli is more closely related to E. smaragdinus based on their bifid ocellar tubercles, epandrium somewhat rectangle shaped and wide at the apex, gonocoxite deeply emarginate along anterior margin, fenestrae lacking (Fig. 19A,E) and aedeagus thin at the apex (Fig. 17A, E). The sister-grouping was already suggested by Schlinger (1960). Eulonchus tristis  and E. marginatus (Figs 3-4) have dark brown coloured legs and a thorax and abdomen with dark metallic to brownishblack colouration. Eulonchus tristis is also a highly morphologically variable species with body colour ranging from metallic light blue (Fig. 14) to dark brown (Fig.  13). These two species share multiple genitalia characters such as a somewhat round epandrium, gonocoxite with anterior margin almost straight, with large fenestrae, and aedeagus heavily sclerotized laterally. Margin of calypter dark brown with brown bleeding into calypter membrane; femur completely dark brown, tibia usually dark brown, occasionally with cream yellow on dorsal surface basally (joint between femur and tibia always yellow); pile on abdomen white; aedeagus in lateral view with two rounded points prior to the opening of the aedeagus (Fig. 17B)  Diagnosis. Proboscis curved and extending beyond abdomen apex (but shorter than wing length), ocellar tubercle bifurcate; calypter margin pale; legs dark brownblack; abdomen dark brown with reddish metallic hue; extensive white pile on thorax and as bands on abdomen.

Key to species of Eulonchus
Redescription. Body length: 9.8 -12.0 mm, Wing length 8.0-10.6 mm. Head. Flagellum dark brown, scape and pedicel brown; male flagellum cylindrical, shorter than head height; clypeus elongate, extending beyond oral cavity, shape rounded with raised ridge dorsally, surface glossy black-brown with sparse pubescence; labial palp brown, extending anteriorly beyond proboscis at point of attachment; margin of oral cavity (parafacial) finely pubescent, lacking pile; proboscis length extending to middle of abdomen; ocellar tubercle bifurcate, apices narrowly digitate and subparallel, tubercle height taller than width, median ocellus greatly reduced; occiput metallic blue or metallic purple, occipital pile densely white. Thorax. Metallic green, metallic blue or metallic purple in colour, setal pile white; coxae black with metallic blue sheen; femora dark brown, apices white; tibia brown; tarsi brown; calypter margin yellow or light brown, membrane translucent; haltere entirely dark brown. Abdomen. Metallic green, metallic blue violet or black with metallic green sheen, vestiture white, dominant setae erect, other pile posteriorly directed, marginal band of denser setae on T3-4. Male genitalia (Figs 17A, 18A, 19A). Epandrium rectangular, wide at the apex, with posterior margin concave; gonocoxite deeply emarginate along anterior margin, fenestrae lacking; aedeagus thinned at the apex, only slightly sclerotized.
Ecology. Schlinger (1960) notes that this species has a relatively short adult flight period (ca. six weeks) during spring and has a feeding preference on Cryptantha intermedia flowers (Table 2).
Biology. The larval host for this species is unknown. Comments. Eulonchus halli is a distinctive species that can be distinguished by the dark leg and body colouration, erect white thoracic and abdominal pile, raised bifurcate ocellar tubercle, and a curved proboscis that is shorter than the body length.    Diagnosis. Proboscis straight, approximately reaching apex of abdomen; ocellar tubercle trifurcate; legs dark brown (pale 'knee' joint); calypter margin black or brown.

Eulonchus marginatus Osten Sacken, 1877
Redescription. Body length: 7.2-11.4 mm, wing length: 5.2-9.5 mm. Head. Flagellum dark brown; scape and pedicel brown, male flagellum cylindrical, shorter than head height; clypeus elongate, extending beyond oral cavity, shape rounded with flat area dorsally, clypeus black-brown, glossy with sparse pubescence; labial palp brown, extending anteriorly beyond proboscis at point of attachment; margin of oral cavity (parafacial) pilose, admixed with short pubescence; ocellar tubercle trifurcate, processes narrow (anteromedial process taller), height taller than width; median ocellus greatly reduced or absent; occiput metallic green-blue, metallic blue or metallic purple, pile densely white or yellow. Thorax. Metallic green, blue or purple, setal pile erect, white or yellow; coxae black with metallic blue sheen; femora dark brown, apices white; tibiae brown (whitish basally on dorsal surface); tarsi brown; calypter margin light to dark brown, membrane translucent, with suffused brown marginally; haltere stem dark brown, knob lighter brown. Abdomen. Metallic olive green, green or blue-violet, vestiture white or yellow, dominant setae erect, pile posteriorly directed, marginal band of laterally directed pile on T2-4. Male genitalia. Epandrium round, with posterior margin concave; gonocoxite with anterior margin almost straight, with large fenestrae; aedeagus heavily sclerotized laterally, with a secondary dorsal point just prior to the opening of the aedeagus.
Comments. Eulonchus marginatus is closely related to E. tristis, sharing features such as extensive white thoracic pile and dark colouration on the legs. Eulonchus marginatus is easily distinguished from other species in the genus by the leg colour, trifurcate ocellar tubercle and dark margin of the calypter. This species displays considerable variation in body colour, ranging from metallic green, blue to purple. Brimley, 1925 Figs 6-7, 16E, 17C, 18C, 19C Eulonchus marialiciae Brimley, 1925: 77 References. Brimley 1938: 335 (North Carolina) Adler et al. 1997: 190 (biology, abundance).
Redescription. Body length: 9.9-12.0 mm, Wing length: 9.1-10.2 mm. Head. Flagellum dark brown, male flagellum laterally compressed and variable in amount of distal tapering, longer than head height (pendulous in pinned specimen); scape and pedicel light brown to yellow; clypeus elongate, extending beyond oral cavity, rounded with raised ridge dorsally, surface black-brown, glossy with sparse pubescence; labial palp brown or yellow, length not extending beyond proboscis at point of attachment; margin of oral cavity (parafacial) glabrous or pilose, admixed with pubescence; proboscis straight, shorter than thorax or reaching middle of abdomen; ocellar tubercle trifurcate with processes relatively short and subequal (posterolat-  eral processes often rounded), tubercle height shorter than width; median ocellus present; occiput metallic green-blue or blue, pile densely white or yellow. Thorax. Metallic green, blue or purple, setal pile yellow; coxae black with metallic blue and/ or green sheen; femora yellow; tibiae dark yellow; tarsi dark yellow; calypter margin brown, membrane translucent, with suffused brown marginally; haltere stem dark brown, knob lighter brown. Abdomen. Metallic green or blue-violet, vestiture yellow, dominant setae erect. Male genitalia (Figs 17C,18C,19C). Epandrium ovate, thinned at the apex, with posterior margin straight; gonocoxite taller than wide, with broad fenestrae; aedeagus broad at the apex, bilobate in posterior view, not heavily sclerotized laterally.
Other material examined. Listed in Table 3 (Suppl. material 1). Distribution (Fig. 20) all other species are found in contiguous distributions in the far western part of the continent. This species is the sister species to the north-western E. sapphirinus and both have characteristic bright green metallic colouration, short proboscis, yellow legs and similarities in the male genitalia shape. Eulonchus marialiciae has the shortest proboscis of any species in the genus, as well as a much more elongated and laterally compressed flagellum. Common name. Northern Sapphire or Emerald. Diagnosis. Antennal flagellum relatively short, cylindrical or tapered; proboscis straight, shorter than length of body; ocellar tubercle trifurcate; legs yellow; calypter margin pale; body colour metallic green, blue or purple.
Ecology. Eulonchus sapphirinus has been recorded visiting the flowers of 19 different plant families and 30 different species (Table 2). Eulonchus sapphirinus adults have been observed exhibiting strong fidelity to a single flowering plant species, suggesting their role as important pollinators (Borkent and Schlinger 2008a).

Eulonchus smaragdinus
Common name. Southern Emerald or Sapphire. Diagnosis. Proboscis curved and longer than abdomen apex (as long or longer than wing length); ocellar tubercle nearly flat, weakly bifurcated; legs bright yellow; body colour metallic green, blue or purple; thorax covered in yellowish pile.
Redescription. Body length: 8.3-12.9 mm, Wing length: 6.9-12.6 mm. Head. Flagellum red-brown or dark brown, male flagellum cylindrical, shorter than head height; scape and pedicel brown; clypeus elongate, length equal to oral cavity; rounded with flat area dorsally, black-brown, surface glossy, glabrous; labial palp brown or yellow, extending anteriorly beyond proboscis at point of attachment; margin of oral cavity (parafacial) glabrous, admixed with pubescence; proboscis length extending beyond abdomen; ocellar tubercle bifurcate (processes short and rounded), tubercle height   shorter than width; median ocellus present or greatly reduced; occiput metallic greenblue or blue, pile densely white or yellow. Thorax. Metallic green, blue or purple, pile white or yellow; coxae brown or black with metallic blue (and green) sheen; femora yellow; tibiae dark yellow; tarsi dark yellow (distal tarsomeres often darker); calypter margin yellow or light brown; calypter membrane transparent; haltere entirely light brown to yellow. Abdomen. Metallic green or blue-violet, vestiture white or yellow, dominant setae appressed or erect, pile posteriorly directed, marginal band of laterally directed pile on T2-4. Male genitalia (Figs 17A,18A,19A). Epandrium rectangular, wide at the apex, with posterior margin slightly concave; gonocoxite deeply emarginate along anterior margin, fenestrae lacking; aedeagus thinned at the apex, only slightly sclerotized.
Type  (Schlinger 1987). Comments. Eulonchus smaragdinus is highly variable in size and colour, and is superficially morphologically similar to E. sapphirinus, most notably in the bright yellow legs. However, it can be easily distinguished from the latter in having a proboscis that is curved (rather than straight) that extends beyond the abdomen, and is often longer than body. Male genitalic characters otherwise indicate a closer relationship to E. halli, as suggested by Schlinger (1960) (see Fig. 22). Schlinger (1960) erected the subspecies E. s. pilosus due to the lighter coloured pile of the individuals he collected. In our study we found that these lighter individuals were just one end of the colouration spectrum (golden pile changing progressively to white pile when moving north to south) of E. smaragdinus, and therefore do not recognize it as a distinct subspecies.    Common name. Dusky Sapphire. Diagnosis. Proboscis reaching apex of abdomen; ocellar tubercle trifurcate with three ocelli present (median smaller than laterals); legs mostly dark brown ('knee' pale), calypter margin dark and membrane white or light yellow.

Eulonchus tristis
Redescription. Body length: 7.9-12.8 mm, Wing length: 6.0-11.2 mm. Head. Flagellum dark brown, scape and pedicel brown, male flagellum cylindrical, shorter than head height; clypeus elongate, extending beyond oral cavity, rounded with flat area dorsally, black-brown, surface glossy, glabrous; labial palp brown, length extending anteriorly beyond proboscis at point of attachment; margin of oral cavity (parafacial) pilose admixed with pubescence (faint); proboscis length from middle of abdomen or extending beyond abdomen; ocellar tubercle trifurcate, processes subequal (narrowly digitate), height equal to or shorter than width; median ocellus present, greatly reduced or absent; occiput metallic green-blue, blue or purple, pile densely white or yellow. Thorax. Metallic green, blue or purple, setal pile white or yellow; coxae brown or black with metallic blue sheen; femora dark brown, apicies  white; tibiae dark yellow or brown; tarsi dark yellow; calypter margin dark brown or light brown, membrane translucent; haltere entirely dark brown. Abdomen. Metallic olive green, bright green to blue-violet, vestiture white or yellow, dominant setae appressed or erect, pile posteriorly directed, marginal band of dense thicker setae on T3-4, or posteriorly directed, marginal band of laterally directed pile on T2-4. Male genitalia. Epandrium round, with posterior margin almost straight; gonocoxite as tall as wide, with anterior margin almost straight, with large fenestrae; aedeagus heavily sclerotized laterally. Other material examined. Listed in Table 3 (Suppl. material 1). Distribution (Fig. 20). Nearctic: Northern California, Oregon, Washington, Arizona. Ecology. Pollen loads and diversity from individual E. tristis visiting flowers of Brodiaea elegans (Themidaceae) and Iris douglasiana (Iridaceae) in California has been studied, showing high levels of constancy to a single species (Borkent and Schlinger    (Table 2).
Biology. Host: Aliatypus sp. (Antrodiaetidae) (Schlinger 1987). Comments. Eulonchus tristis is most similar to E. marginatus, with which it shares the ocellar tubercle trifid and the flagellum half as long as the head. Eulonchus tristis can be easily distinguished from E. marginatus by its femur and tibia with yellow markings and the pile on abdomen yellow.  Schlinger (1928Schlinger ( -2014 was a world renowned expert on spider fly taxonomy and biology. This paper is dedicated to him and his legacy.