A new species of Cordyligaster Macquart, reared from caterpillars in Area de Conservacion Guanacaste, northwestern Costa Rica

Abstract We describe a new species of Cordyligaster Macquart (Diptera: Tachinidae) from Area de Conservacion Guanacaste (ACG) in northwestern Costa Rica. Cordyligaster capellii sp. n., is described and photographed. All specimens of C. capellii were reared from Syngamia florella (Stoll, 1781) (Lepidoptera, Crambidae, Spilomelinae), a leaf-rolling caterpillar collected in ACG rain forest. By coupling morphology, photographic documentation, life history and molecular data, we provide a clear and concise description of this new species. In addition the authors provide new distribution and host records for C. fuscipennis (Macquart) reared in ACG.


Introduction Geographic area of the study and rearing intensity
All flies and rearing information described here were found by the 35+ year-old ongoing inventory of the caterpillars, their food plants and their parasitoids of the dry forest, rain forest, cloud forest, and intergrades, in the 125,000+ ha terrestrial portion of Area de Conservación Guanacaste (ACG) in northwestern Costa Rica (Rodriguez et al. 2012, Janzen and Hallwachs 2011, Smith et al. 2006, Smith et al. 2008, Fernández-Triana et al. 2014). The tachinid rearing methods are described at http://janzen.bio.upenn.edu/caterpillars/ methodology/how/parasitoid_husbandry.htm. In brief, caterpillars (and sometimes pupae) are found in the wild at all instars by a wide variety of search methods, and reared in captivity on the food plant species on which they were found, until they produced an adult, a parasitoid, or died of other causes. Each caterpillar is documented as an individual, the emerging parasitoid flies are first treated as a rearing event and later as individuals once they are barcoded.
In the near 4 decades since its inception in 1978, this inventory has reared approximately 600,000 wild-caught caterpillars. All frequencies of parasitization reported here must be considered against the background of this inventory. Equally, it is patently obvious that although the inventory is carried out throughout the year, there is a bias towards certain environments, types of vegetation and distance off the ground. Comparison of reared species of parasitoids with those collected by net or Malaise traps demonstrates that to date, the caterpillar inventory has so far encountered well less than half the species of caterpillar parasitoids present in ACG. The largest unsampled void is the upper foliage of the canopy above about 3-4 m above the ground.
The treatment reported here is focused on placing names on the species reared, thereby preparing them for later detailed ecological and behavioral accounts and studies that will normally extend across ACG ecological groups, whole ecosystems, and taxonomic assemblages much larger than a genus. DNA barcodes (standardised 5' region of the mitochondrial cytochrome c oxidase I (COI) gene) for all ACG inventory specimens were obtained using DNA extracts prepared from single legs using a glass fibre protocol (Ivanova et al. 2006). Total genomic DNA was resuspended in 30 μl of dH O, and a 658-bp region near the 5' terminus of the CO1 gene was amplified using standard primers (LepF1-LepR1) following established protocols , Smith et al. 2006, Smith et al. 2008. All information for the sequences associated with each individual specimen (including GenBank and BOLD accession) can be retrieved from the Barcode

Imaging and Dissections
Our descriptions of new species are deliberately brief and only include some differentiating descriptions of body parts and colors that are commonly used in Tachinid identification. These brief descriptions are complemented with an extensive series of color photos of every species to illustrate the readily observed differences among these species.
Habitus photographs were taken using a Canon T3i digital SLR, using a 65mm Macro Photo Lens 1:2.8 (MP-E 65mm), mounted on a microscope track stand (AmScope, Model: TS200) modified to accept a Manfrotto QR 200PL-14 quick release plate. Images were shot in aperture priority, allowing the camera to control shutter speed at f/4.5, over 40 images at equal distance increments. Illumination was provided with a homemade reflective dome (instruction for dome creation can be found at: http://www.cdfa.ca.gov/ plant/ppd/entomology/Dome/kd-200.html) placed over a 144 LED ringlight (AmScope, Model: LED-144-YK).
Adult fly dissections followed standard practice (O'Hara 1983). Photographs of male terminalia were taken using a Canon S110 digital camera adaptor mounted to the eyepiece of a Leitz-Wetzlar dissecting scope. Preparations were mounted on a depression slide in a small quantity of Rexall brand hand sanitizer gel (NPN# 80007138). This allowed the specimen to remain steady in a given position, and therefore provided a better environment for photography. After mounting and photographing, the terminalia were rinsed in a small quantity of pure distilled water, before being replaced in the glycerine-filled microvial.
The photographic series were created using Photoshop CS6, and Zerene Stacker Software v1.04. So as to maximize image quality and depth of field, photo series were digitally stacked to produce a final composite image.
The terminology used for genitalia (which refers here only to the sclerotized parts of the genitalia, and not to the soft internal structures) and other body parts follows Cumming and Wood (2009).
All specimens listed as examined are considered paratypes, except for the holotype, which is noted separately.

Voucher specimen management
All caterpillars reared from ACG efforts receive a unique voucher code in the format of yy-SRNP-xxxxx. Any parasitoid emerging from this caterpillar receives the same voucher code, and then if/when later the parasitoid is dealt with individually, it receives a second voucher code unique to it, in the format of DHJPARxxxxxxx. The voucher codes and collateral data assigned to both host and emergent parasitoids are available at http:// janzen.bio.upenn.edu/caterpillars/database.lasso. To date, all DHJPARxxxxxxx coded tachinids have had one leg removed for attempted DNA barcoding at the Biodiversity Institute of Ontario (BIO) in the University of Guelph, with all collateral data and all successful barcodes permanently and publically deposited in the Barcode of Life Data System (BOLD, www.boldsystems.org) (Suppl. material 2), and later migrated to GenBank as well. A neighbor-joining (NJ) tree (Saitou and Nei 1987) for all Cordyligaster reared and DNA barcoded by this inventory through 2013 is included as Suppl. material 1. The inventory grows continually and new specimens can be found by searching the genus Cordyligaster in BOLD. Each barcoded specimen also has an accession code in the Barcode of Life Data System (BOLD) and GenBank.
Inventoried Tachinidae were collected under Costa Rican government research permits issued to DHJ since 1978, and likewise exported under permit by DHJ from Costa Rica to Philadelphia, and then to the final depository in the Canadian National Insect collection in Ottawa, Canada. Tachinid identifications for the inventory were done by DHJ in coordination with a) visual inspection by AJF and DMW, b) DNA barcoding by BIO, MAS, and BOLD, and c) correlation with host caterpillar identifications by DHJ and WH through the inventory itself. Dates of capture of each reared fly in the inventory are the dates of eclosion of the fly, and not the date of capture of the caterpillar. This is because the fly eclosion date is much more representative of the time when that fly species is on the wing than is the time of capture of the caterpillar or (rarely) finding a parasitized pupa. However, the collector listed is the parataxonomist who found the caterpillar, rather than the person who retrieved the newly eclosed fly from its rearing bag or bottle, and processed it by freezing, pinning, labeling and oven-drying. Fly biology and degrees of parasitization by these flies will be the detailed subject of later papers.  (Sabrosky 1973: 221 Description Male (Fig. 1); Head: fronto orbital plate with silver tomentosity; parafacial silver; pedicel black; antenna black with plumose arista; trichiae at base, 6 times as long as base of arista is wide tapering to half that length before apex; eye bare; ocellar bristles parallel and proclinate approximately twice the length of the ocellar triangle; fronto-orbital plate narrowing at apex enclosing only the ocellar triangle; proclinate orbital bristles absent in male; palpus black. Thorax: at first glance appears glabrous black, but under certain angles of light a very light tomentum is often apparent however no vittae are visible. Three post-sutural supra alar bristles, (two strong anterior, and third one weak; second bristle strongest, 1.5X thickness of first post sutural supra alar bristles) (Fig. 3), apical scutellar bristles long, up to 3/4 length of subapical scutellars; subapical scutellar bristles parallel or divergent (forming a wide V); katepisternum bearing 2 bristles, very lightly tomentose (same as dorsum), lacking the tomentose bands apparent in C.  . 2); Head: fronto orbital plate with silver tomentosity except along facial ridge which appears red; pedicel black; antenna black with plumose arista; trichiae at base, 6 times as long as base of arista is wide tapering to half that length before apex; eye bare; ocellar bristles parallel and proclinate approximately twice the length of the ocellar triangle; fronto-orbital plate narrowing at apex enclosing only the ocellar triangle; 2 proclinate orbital bristles present; palpus black, with distinctive gold tomentosity on tip. Thorax: at first glance appears glabrous black, however under certain angles of light a very light tomentum is apparent. Three post-sutural supra alar bristles, (two strong anterior, and third one weak); second bristle strongest, 1.5X thickness of first post-sutural supra alar; apical scutellar bristles long, equal in length of subapical scutellars; scutellar bristles divergent (forming a wide V); katepisternum a b c Figure 2.  Detailed dorsolateral view of Cordyligaster capellii; square indicates the magnified inset; circles indicate 3 post-sutural supra-alar bristles.

Diagnosis
Cordyligaster capellii posesses exceptionally large calypteres, a trait also shared by C. nyomala, and C. minuscula. While C. minuscula (Wulp) can also sometimes have the variable character of 3 post-sutural supra alars, C. capellii is distinguished by the presence of 3 postsutural supra-alars (Fig. 3b), smoky yellow wings (Fig. 4), and a black antennal pedicel and black palpi. C. minuscula possesses a orange brown pedicel, and reddish brown palpi. A very light tomentum covers much of the thorax as well as T5, but this trait is visible only under certain angles of light. C. minuscula also possesses only the posterior katepisternal bristle, while C. capellii has two. The holotype specimen has the DNA barcode given below: Genetic comparison to the type specimens of previously know species was outside the scope of this paper, however the authors have selected to give the barcode data here as a diagnostic character such that it is readily available for future works which may undertake the barcoding of those previously described types.

Etymology
This species is named to honor Sr. Luciano Capelli of San Jose, Costa Rica in recognition and appreciation of his enthusiastic and superb photography of all aspects of ACG specifically, and Costa Rica's conserved wildlands more broadly, and for allowing ACG and Costa Rican conservation in general to freely use these photographs to explain conserved wildlands to the public.

Ecology
Hosts: Crambidae, Syngamia florella (Stoll, 1781). While more than 500 species of Crambidae have been reared from more than 65,000 leaf-rolling crambid caterpillars in ACG dry forest, rain forest and cloud forest (and intergrades among them), generating 3,000+ tachinid rearings, Cordyligaster capellii has been reared just 10 times and always from the leaf roller Syngamia florella feeding on Spermacoce exilis (L.O. Williams) (Rubiaceae) herbs in the dry-rain forest ecotone on the northern intermediate elevation slopes of Volcan Orosi and Cerro Orosilito, Sector Del Oro and Sector Pitilla, of ACG. These ten rearings were spread among 144 S. florella wild-caught caterpillars. It is likely to be the only species of host for this fly in ACG.   (Fig. 6); Head: fronto orbital plate with silver tomentosity; parafacial silver; antenna black with plumose arista; eye bare; ocellar bristles parallel and proclinate approximately twice the length of the ocellar triangle; fronto-orbital plate slightly narrowing at apex to almost the width of the ocellar triangle; frontal vitta 2 times as wide as face; proclinate orbital bristles present; palpus black. Thorax: at first glance appears glabrous black, however under certain angles of light a very light tomentum becomes apparent. Three post-sutural supra alar bristles, (two strong anterior, and third one weak; second bristle strongest, 1.5X thickness of first pssa; apical scutellar bristles long, equal in length of subapical scutellars; scutellar bristles divergent (forming a wide V); katepisternum bearing 2 bristles, tomentose bands as in male, these bands apparent when viewed laterally. Wings: smoky black in colour, dark amber towards a b c Figure 6.

Diagnosis
This species is easily recognized by its relatively large size, black palpus, black antenna, and all black abdomen. It is distinguished from C. petiolata (Wiedemann 1830), by the lack of yellow spots on T3 (Sabrosky 1973). Species has the DNA barcode recorded below: AACTTTATACTTTATTTTCGGTGCTTGATCAGGAATACTAGGAACATCTTTAAGAATTT TAATTCGAACAGAATTAGGACATCCAGGTTCACTAATTGGAGATGATCAAATTTATAA  CGTAATTGTAACAGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCAATTATAATT  GGAGGATTTGGAAATTGATTAGTTCCTTTAATATTAGGAGCTCCAGATATAGCTTTTC  CTCGAATAAATAATATAAGATTTTGACTACTTCCCCCTTCTTTATTACTTCTCCTAATT  GGTAGAATAGTTGAAAATGGAGCTGGAACAGGATGAACAGTTTACCCTCCTTTATCT  TCTAATATTGCACATAGAGGATCTTCTGTTGACTTAACTATTTTTTCACTACATTTAGC  AGGTATTTCTTCTATTATAGGAGCTGTAAATTTTATTACAACAGTAATTAATATACGATC  AACAGGAATTACATTTGATCGAATACCTTTATTTGTTTGATCTGTAGCAATTACAGCA  TTATTATTACTTTTATCTTTACCTGTATTAGCAGGAGCTATTACCATATTATTAACTGAT  CGAAATATAAATACTTCTTTTTTTGACCCAGCAGGAGGAGGAGANCCTATTTTATACC  AACATTTATTT Genetic comparison to the type specimens of previously know species was outside the scope of this paper, however the authors have selected to give the barcode data here as a diagnostic character such that it is readily available for future works which may undertake the barcoding of those previously described types.

Distribution
Costa Rica, ACG, Prov. Guanacaste, rain forest, 153 -640m elevation. Originally described from "South America", this species has been found to be very widely distributed, from Brazil, west to Bolivia and Peru, and North to Guatemala.

Ecology
Hosts: Three species of leaf-rolling spilomeline Crambidae feeding on leaves of rain forest Urticaceae.

Revised key to the species of Cordyligaster Macquart
Key adapted from Sabrosky 1973 to include all species presently occuring within the genus.

Analysis
Mitochondrial DNA barcodes from the two species of Cordyligaster displayed no heteroplasmy or double banding that might suggest the inadvertent amplification and sequencing of a nuclear pseudogene. Sequences displayed the characteristic AT bias of insect mitochondrial DNA (70%) and showed very little intraspecific variation within their DNA barcode region (min 0.05%, min 0.31% respectively) with greater inter-specific variation evident between them (6.83%).