Comp. Parasitol. 77(2), 2010, pp. 125–136

Nubenocephalus nickoli n. sp. and Nubenocephalus xunantunichensis n. sp. (Apicomplexa: Eugregarinida: Actinocephalidae) Parasitizing (: Zygoptera) in Belize, Central America

1,4 2 3 RICHARD E. CLOPTON, TAMARA J. COOK, AND JOANNA J. CIELOCHA 1 Department of Natural Science, Peru State College, Peru, Nebraska 68421, U.S.A. (e-mail: [email protected]), 2 Department of Biological Sciences, Sam Houston State University, Huntsville, Texas 77341-2116, U.S.A. (e-mail: [email protected]), and 3 Department of Ecology and Evolutionary Biology and the Biodiversity Institute, University of Kansas, Lawrence, Kansas 66045, U.S.A. (e-mail: [email protected])

ABSTRACT: Two new species of Nubenocephalus (Apicomplexa: Eugregarinida: Actinocephalidae) are described from adult damselflies sampled in Cayo District, Belize, Central America. Nubenocephalus nickoli n. sp. is described from the American Rubyspot , americana, and the Smoky Rubyspot damselfly, Hetaerina titia (Odonata: Zygoptera: Hetaerinidae), and Nubenocephalus xunantunichensis n. sp. is described from the Comanche Dancer, chelata (Odonata: Zygoptera: ). New, complete morphological data sets are reported for populations of Nubenocephalus nebraskensis and Nubenocephalus secundus, and a complete, synoptic reevaluation of diagnostic morphological characters used to discriminate species within Nubenocephalus is presented for all known species of the genus in the New World. Nubenocephalus nebraskensis is reported from Argia apicalis for the first time. KEY WORDS: Acanthosporinae, Actinocephalidae, Apicomplexa, Argia apicalis, Argia chelata, Belize, Central America, Coenagrionidae, damselfly, Eugregarinida, Gregarine, Hetaerinidae, Hetaerina americana, Hetaerina titia, Nubenocephalus mutabilis, Nubenocephalus nebraskensis, Nubenocephalus nickoli n. sp., Nubenocephalus secundus, Nubenocephalus xunantunichensis n. sp., Odonata, Septatorina, Stylocephaloidea, Zygoptera.

The eugregarine genus Nubenocephalus consists of 1 from adults of the American Rubyspot damselfly, 3 species parasitizing Nearctic and Palearctic dam- Hetaerina americana (Fabricius) (Odonata: Zygop- selflies. Known Nearctic species are reported from tera: Hetaerinidae), and the Smoky Rubyspot dam- hosts in the genus Argia (Odonata: Zygoptera: selfly, Hetaerina titia (Drury) (Odonata: Zygoptera: Coenagrionidae). The type species, Nubenocephalus Hetaerinidae). The gregarines recovered are referable nebraskensis Clopton, Percival and Janovy, 1993, is to the genus Nubenocephalus but are taxonomically described from the Seepage Dancer, Argia bipunctu- distinct from known species of Nubenocephalus. lata (Hagen) in Nebraska, U.S.A. Nubenocephalus Herein, we describe these new taxa and reevaluate secundus Hays, Clopton, T. J. Cook, 2007, is diagnostic morphological characters used to discrim- reported from the Blue-Ringed Dancer, Argia sedula inate species within Nubenocephalus. (Hagen), and the Dusky Dancer, Argia translata (Hagen in Selys), both in Texas, U.S.A. The single MATERIALS AND METHODS Palearctic species assigned to the genus, Nubenoce- Argia apicalis adults were collected with aerial nets along phalus mutabilis (K. Hoshide, 1977) Clopton and T. Long’s Creek, north of Auburn, Nemaha County, Nebraska, J. Cook, 2007 (5Ancyrophora mutabilis K. Hoshide, U.S.A. (40u24911.80N; 95u50981.80W), from July to August, 1977), is described from the Pied Bush Dart, Copera 2007 (n 5 71). Two specimens vouchering Argia apicalis are deposited in the Sam Houston State University annulata (Selys) (Odonata: Zygoptera: Platycnemi- Collection (SHSUIC), Department of Biological Sciences, didae), in Yamaguchi, Yamaguchi Prefecture, Japan. Sam Houston State University, Huntsville, Texas, U.S.A. No species of Nubenocephalus is reported from the Individual accession numbers are not assigned by SHSUIC. Neotropics. These specimens can be distinguished by the following 5- During a survey of damselfly and eugregarine line determination labels: Line 1—‘‘Argia’’; Line 2— ‘‘apicalis (Say, 1839)’’; Line 3—‘‘MALE: REC070372’’ diversity in Belize, Central America, we collected 2 or ‘‘FEMALE: REC070465’’; Line 4—‘‘SYMBIOTYPE- heretofore unknown gregarine species: 1 from adults VOUCHER’’; Line 5—‘‘det. R E Clopton 2007.’’ of the Comanche Dancer, Argia chelata Calvert, and Argia chelata adults were collected in May 2008 with aerial nets near Hammock Bridge on the Mopan River at Branch Mouth, Cayo District, Belize, Central America (17u10965.60N; 89u4981.30W), and along Barton’s Creek at 4 Corresponding author. Caesar’s Place, Imperial Mile Marker 60, Western Highway

125 126 COMPARATIVE PARASITOLOGY, 77(2), JULY 2010 near Unitedville, Cayo District, Belize, Central America Damselflies were eviscerated, and their alimentary canals (17u12933.50N; 88u57920.50W) (n 5 22 and 4, respectively). were dissected in insect muscle saline (Belton and Argia sedula adults were collected with aerial nets along Grundfest, 1962). Permanent parasite preparations were Harmon Creek at the Sam Houston State University Center made following the techniques described by Clopton for Biological Field Studies, Walker County, Texas, U.S.A. (2004a). Observations of trophozoites, sporonts, and (30u44944.70N; 95u28946.20W), June to October 2006 (n 5 gamonts were made using an Olympus B-Max 41 compound 94). These are the original type collections for Nubeno- microscope with 310, 340, and 3100 universal planapo- cephalus secundus, and data provided herein for this species chromatic objectives with either bright field or phase are taken from the original type data set. Hetaerina contrast condensers. Digital photographs were taken using americana adults were collected using an aerial net from an Olympus DP-12 digital camera through the aforemen- the following localities in the U.S.A: Cedar Creek at the tioned microscope. Measurements were taken from digitized Paxton-Sarben Road, Keith County, Nebraska, U.S.A. images of preserved specimens using Image Pro Express v. (41u11902.80N; 101u21961.30W), July 1996 (n 5 47), June 4.5 image analysis software (Media Cybernetics, L.P., Silver 1997 (n 5 36), and September 2003 (n 5 17); Clear Creek Spring, Maryland). Images of preserved specimens used in at Highway 92, Keith County, Nebraska, U.S.A. the photographic plates were taken using the Olympus DP- (41u19920.90N; 102u02924.90W) in June 1998 (n 5 36), 70 digital camera attached to the Olympus B-Max 50 and June 1999 (n 5 28); Long’s Creek, north of Auburn, compound microscope described above. Photographic plates Nemaha County, Nebraska, U.S.A. (40u24911.80N; were processed and assembled using AdobeH PhotoShopH 95u50981.80W), July to August 2007 (n 5 92); Otter Creek 7.0.1 software (Adobe Systems Inc., San Jose, California, at Highway 92, Keith County, Nebraska, U.S.A. U.S.A.). (41u18908.30N; 101u55939.00W), June 1996 (n 5 30); and Individual gregarine gametocysts were preserved for Turkey Creek in Johnson County, Nebraska, U.S.A. future genetic analysis after transfer through 3 changes of (40u15975.30N; 96u21940.40W), July to August 2003 (n 5 TMS and 3 changes of distilled water sterilized by syringe 58). Hetaerina americana adults were collected in May filtration through a 0.2-mm membrane and transferred to 2008 with aerial nets near Hammock Bridge on the Mopan individual microcentrifuge tubes. A hypodermic needle was River at Branch Mouth, Cayo District, Belize, Central used to rupture each gametocyst, and the liberated contents America (17u10965.60N; 89u4981.30W), May 2008, and were dried at ,60uC before capping the microcentrifuge along Barton’s Creek at Caesar’s Place, Imperial Mile tube. All samples were stored at 4uC prior to DNA Marker 60, Western Highway near Unitedville, Cayo extraction. District, Belize, Central America (17u12933.50N; The DNA from each pooled sample of trophozoites and/or 88u57920.50W) (n 5 2 and 6, respectively). Hetaerina titia gamonts was isolated using a PureLink genomic DNA Mini adults were collected in June 2007 and May 2008 with aerial Kit (Invitrogen, Carlsbad, California, U.S.A.). Dried game- nets near Hammock Bridge on the Mopan River at Branch tocyst samples were extracted using the PureLink genomic Mouth, Cayo District, Belize, Central America DNA Mini Kit’s accompanying FTA protocol (Invitrogen, (17u10965.60N; 89u4981.30W) (n 5 29 and 19, respectively), Carlsbad, California, U.S.A.). Isolated DNA samples were and in May 2008 along Barton’s Creek at Caesar’s Place, resuspended in NE buffer (USB Corporation, Cleveland, Imperial Mile Marker 60, Western Highway near United- Ohio, U.S.A.) and stored by aliquot at 4uC for future ville, Cayo District, Belize, Central America (17u12933.50N; analysis. 88u57920.50W) (n 5 15). Adult damselflies were placed in Terminology for developmental stages generally follows 1-liter plastic containers held on ice until processed. that of Levine (1971) for Apicomplexa, with one notable Damselflies were isolated, abdomen-down, in 5-ml exception: Sporont is used herein to indicate a mature plastic test tubes with 1 ml of water. Fecal material in the individual lacking an epimerite that remains attached to the water was examined after 12–24 hr for gregarine host epithelium prior to forming an association (Clopton et gametocysts. Shed gametocysts were freed from feces, al., 1993). Terminology for association and gametocyst life- triple rinsed in Tenebrio molitor muscle saline (TMS) cycle characters follows that of Clopton (2009). Terminol- (Belton and Grundfest, 1962), triple rinsed in deionized ogy for shapes of planes and solids follows Clopton water, and transferred with ,50 ml deionized water to (2004b). General morphometric and epimeritic terminology individual 4 3 12 mm glass microvials (BioQuip Products, follows Hays et al. (2007). The following metric characters Gardena, California, U.S.A.). Vials were sealed with white used herein are consistent with those outlined and illustrated silicon stoppers, and gametocysts were held for maturation for the Acanthosporinae by Clopton (2004a): diamerite and dehiscence. Gametocysts were observed daily to length (DiaL), diamerite width (DiaW), length of deutom- determine time of dehiscence. Fresh preparations of oocysts erite (DL), distance from protomerite–deutomerite septum to were examined as wet mounts or as agar monolayer mounts deutomerite axis of maximum width (DLAM), distance from (Clopton, 2004a). Observations were made using an posterior end of deutomerite to deutomerite axis of Olympus B-Max 50 compound microscope with 310, maximum width (DLPM), length of oocyst dorsal or polar 320, 340, and 360 universal planapochromatic objectives spine (DSL), width of deutomerite at equatorial axis (DWE), with either phase contrast condensers or differential maximum width of deutomerite (DWM), epimerite length interference contrast prisms and an infinity-optics turret (EpiL), epimerite width (EpiW), length of oocyst equatorial image-doubler. Digital photographs were taken with an spine (ESL), distance from nucleus to protomerite–deutom- Olympus DP-70 digital camera through the aforementioned erite septum (NDS), length of nucleus (NL), width of microscope. Oocyst morphometrics were taken from the nucleus (NW), length of oocyst (OL), diameter of oocyst digitized images of oocysts using Image-Pro DiscoveryH residuum (OrD), width of oocyst at equator (OW), width of v. 4.0 image analysis software (Media Cybernetics, L.P., oocyst at polar truncation (OWP), width of protomerite– Silver Spring, Maryland, U.S.A.). deutomerite septum (PDSW), length of protomerite (PL), CLOPTON ET AL.—NUBENOCEPHALUS SPP. FROM BELIZE 127 distance from anterior end of protomerite to protomerite axis tumidus with longitudinal pleats, petals, or lobes not of maximum width (PLAM), distance from protomerite– forming hooks, spines, digitiform or rhizoid process- deutomerite septum to protomerite axis of maximum width (PLPM), width of protomerite at equatorial axis (PWE), es; diamerite present. Anterior margin of sporont maximum width of protomerite (PWM), and total length protomerite expanded to form a broad crateriform (TL). Measurements are presented in mm as mean values adhesive disk. followed parenthetically by range values, standard devia- tions, and sample sizes. Description RESULTS Trophozoite (Fig. 4): Solitary, attached to host ventricular epithelium, with epimerite complex. Two species of Nubenocephalus were collected Epimerite orbicular or spherical, with longitudinal from H. americana, H. titia, and A. chelata pleats. Diamerite oblong, short, less than length of populations sampled in Belize. New samples of N. epimerite. Protomerite broadly ovoid in young nebraskensis were collected from A. apicalis popu- trophozoites, becoming shallowly obpanduriform in lations sampled in Nebraska, U.S.A., and new oocyst mature forms, truncated at protomerite–deutomerite morphometric data were collected for N. secundus septum; protomerite–deutomerite septal constriction using the original type data set. New species are diagnosed and described here. Comparative data and distinct. Deutomerite spatulate in young trophozoites, figures for all 4 species of Nubenocephalus are becoming narrowly obtrullate in mature forms. presented in Figures 1–19 and Tables 1–2. A homo- Nucleus elliptoid. Relative morphometric ratios: EpiL type series representing N. nebraskensis is deposited . DiaL, EpiW . DiaW, EpiL , PL, DiaL , PL, in the Harold W. Manter Laboratory for Parasitology PWM . DWM, PL . PWM. (HWML), Division of Parasitology, University of Sporont (Fig. 8): Solitary, attached to host ven- Nebraska State Museum, Lincoln, Nebraska, U.S.A. tricular epithelium by modified protomerite, epimer- The N. nebraskensis homotype series consists of 32 ite lacking. Protomerite very broadly conical in lateral slides containing trophozoites, gamonts, and associ- aspect, anterior margin expanded to form a broad ations deposited in 1 lot as follows: HWML100023 crateriform adhesive disk that is constricted at (REC07374, REC07375, REC07376, REC07377a–b, protomerite–deutomerite septum; PL 190.5 (127.3– REC07384, REC07385a–c, REC07386a–b, REC 307.9, 636.61, 32); PWE 382.5 (315–482.9, 637.19, 07387a–c, REC07388, REC07394a–b, REC07395, 32); PWM 409.3 (331.5–498.4, 637.04, 32); PLAM REC07448, REC07449, REC07451, REC07452, 137.8 (102.4–181.2, 620.13, 32); PLPM 58.5 (15.2– REC07455, REC07456, REC07458, REC07467, 188.7, 633.54, 32); PDSW 312.3 (243.5–354.5, REC07469, REC07473, REC07484, REC07485, 629.75, 32); PL/PWE 0.5 (0.3–0.7, 60.08, 32); PL/ REC07488, REC07489). The disposition of holotype PWM 0.5 (0.3–0.7, 60.09, 32); PL/PDSW 0.6 (0.4– and paratype specimens for new species described 0.9, 60.13, 32); PLAM/PL 0.7 (0.4–1, 60.13, 32); herein is reported under the individual species PLAM/PLPM 3.1 (0.6–9.8, 61.87, 32); PWM/PWE descriptions that follow. 1.1 (1–1.2, 60.05, 32); PWM/PDSW 1.3 (1.2–1.5, 60.07, 32). Deutomerite very deeply to narrowly Nubenocephalus nickoli n. sp. obtrullate; DL 1017.3 (759.9–1223.2, 687.04, 32); (Figs. 4, 8, 12, 16) DWE 174.4 (118.7–250.4, 626.14, 32); DWM 322.3 (242.4–406.1, 635.46, 32); DLAM 156.9 (93–222.1, Generic diagnosis 623.49, 32); DLPM 864.2 (662–1053.3, 675.58, Eugregarinorida Le´ger, 1892, sensu Clopton 32); DL/DWE 5.9 (3.8–7.2, 60.67, 32); DL/DWM (2002); Septatorina Lankester, 1885, sensu Clopton 3.2 (2.3–3.6, 60.26, 32); DL/PDSW 3.3 (2.7–3.7, (2002); Stylocephaloidea Clopton, 2009, Actinoce- 60.22, 32); DWM/DWE 1.9 (1.5–2.2, 60.14, 32); phalidae Le´ger, 1892, sensu Clopton (2002, 2009); DWM/PDSW 1 (1–1.2, 60.04, 32). Total length Acanthosporinae Le´ger, 1892, sensu Clopton (2002); (TL) 1201.7 (979.7–1461.2, 696.52, 32); TL/PL 6.5 with the characters of the genus Nubenocephalus (4.4–10.2, 61.16, 32); DL/PL 5.5 (3.5–9.2, 61.17, Clopton, Percival, and Janovy, 1993, sensu Hays, 32); DWM/PWM 0.8 (0.7–1, 60.06, 32); TL/DL 1.2 Clopton, Cook, and Cook, 2007. Oocysts dipyramid- (1.1–1.3, 60.04, 32). Nucleus elliptoid, placement ic with polar truncations, hexagonal or septagonal in variable but usually in anterior sixth of deutomerite; equatorial cross section, without equatorial faces, nuclear karyosomes present; NW 68.9 (57.3–87.4, with equatorial and terminal spines. Epimerite a 67.59, 32); NL 117.2 (105.3–132.8, 66.78, 32); 128 COMPARATIVE PARASITOLOGY, 77(2), JULY 2010

Figures 1–16. Comparative morphology of Nubenocephalus nebraskensis, Nubenocephalus secundus, Nubenocephalus xunantunichensis n. sp., and Nubenocephalus nickoli n. sp. 1–4. Young trophozoites, fixed and stained specimens, phase contrast microscopy. 1. N. nebraskensis. 2. N. secundus. 3. N. xunantunichensis. 4. N. nickoli. 5–8. Sporonts, fixed and stained specimens, phase contrast microscopy. 5. N. nebraskensis. 6. N. secundus. 7. N. xunantunichensis. 8. N. nickoli. 9– 12. Oocysts, lateral aspect, sagittal optical section demonstrating residuum and spines, differential interference contrast CLOPTON ET AL.—NUBENOCEPHALUS SPP. FROM BELIZE 129

NDS 136.4 (31.4–432.8, 6104.27, 32); NL/NW 1.7 5-line determination label: Line 1—‘‘Hetaerina’’; (1.4–2.1, 60.18, 32); DL/NL 8.7 (6.8–11.6, 60.85, Line 2—‘‘titia (Drury, 1773)’’; Line 3—‘‘MALE: 32); TL/NL 10.3 (8.8–13.9, 61.03, 32); DL/NDS REC080026’’; Line 4—‘‘SYMBIOTYPE-VOUCH- 11.9 (2.3–31.1, 67.83, 32). ER’’; Line 5—‘‘det. R E Clopton 2008.’’ The second symbiotype vouchers H. americana and can be Association: Association syzygial, frontolateral to distinguished by the following 5-line determination lateral, isogamontic. label: Line 1—‘‘Hetaerina’’; Line 2—‘‘americana Gametocysts: White in color, orbicular with (Fabricius, 1798)’’; Line 3—‘‘MALE: REC080069’’; persistent gelatinous epicyst. Gametocysts stored in Line 4—‘‘SYMBIOTYPE-VOUCHER’’; Line 5— sterile water dehisced by simple rupture in 4 to 6 d; ‘‘det. R E Clopton 2008.’’ oocysts released singly by simple exposure. Other hosts: The American Rubyspot damselfly, Oocysts (Figs. 12, 16): Axially symmetric, dipy- Hetaerina americana (Fabricius) (Odonata: Zygop- ramidic with slight polar truncations OL 7.6 (7.2–8, tera: Hetaerinidae), adults. 60.18, 33); OW 5.1 (4.7–5.6, 60.22, 33); OWP 1.1 (0.8–1.4, 60.16, 33); OL/OW 1.5 (1.4–1.6, 60.07, Other known localities: Barton’s Creek at Caesar’s 33); OL/OWP 6.7 (5.4–9.1, 61.11, 33); OW/OWP Place, Imperial Mile Marker 60, Western Highway 4.6 (3.7–6.3, 60.71, 33); hexagonal in equatorial near Unitedville, Cayo District, Belize, Central cross section, without equatorial faces, bearing 6 America (17u12933.50N; 88u57920.50W). Although equatorial spines, 1 at each equatorial vertex, ESL 4.5 314 adult specimens of H. americana were examined (3.8–5.3, 60.44, 33); terminal spines obliquely postmortem across 5 temperate collection sites in the inserted at each pole, 1 at each vertex created by United States sampled over 13 yr, no infected polar truncations, DSL 3.2 (2.7–3.9, 60.33, 33); OL/ specimen was observed, suggesting that N. nickoli ESL 1.7 (1.4–2, 60.17, 33); OL/DSL 2.4 (1.9–2.9, is restricted to the Neotropics. 60.25, 33); OW/ESL 1.2 (0.9–1.4, 60.11, 33); OW/ Site of infection: Trophozoites, sporonts, and DSL 1.6 (1.2–1.9, 60.18, 33); major oocyst gamonts were collected from the length of the residuum orbicular, smooth, equatorial, abaxial with mesenteron. Gametocysts were collected in host 2 accompanying minor residua, OrD 1.6 (1.3–1.9, hindgut, rectum, and feces. 60.16, 33); OL/OrD 4.9 (3.9–6.2, 60.48, 33); OW/ OrD 3.3 (2.7–4, 60.34, 33). Prevalence: Forty-eight of 48 (100%) and 11 of 15 (73%) individuals of H. titia examined from Taxonomic summary Hammock Bridge and Barton’s Creek, respectively. Two of 2 (100%) and 2 of 6 (33%) individuals of H. Type host: The Smoky Rubyspot damselfly, americana examined from Hammock Bridge and Hetaerina titia (Drury) (Odonata: Zygoptera: Hetaer- Barton’s Creek, respectively. inidae), adults. Specimens deposited: The type series is deposited Type locality: Hammock Bridge on the Mopan in the Harold W. Manter Laboratory for Parasitology River at Branch Mouth, Cayo District, Belize, Central (HWML), Division of Parasitology, University of America (17u10965.60N; 89u4981.30W). Nebraska State Museum, Lincoln, Nebraska, U.S.A. Symbiotype: Two symbiotype specimens are The holotype is a hapantotype slide HWML100029 deposited in the Sam Houston State University Insect (REC080033b). The paratype series consists of 71 Collection (SHSUIC), Department of Biological slides containing trophozoites, gamonts, and associ- Sciences, Sam Houston State University, Huntsville, ations deposited in 4 lots as follows: HWML100026 Texas, U.S.A. Individual accession numbers are not (REC080085, REC080089); HWML100027 (REC assigned by SHSUIC. The first symbiotype vouchers 080090, REC080091, REC080092, REC080093a–b, H. titia and can be distinguished by the following REC080096, REC080098, REC080099, REC

r microscopy. 9. N. nebraskensis. 10. N. secundus. 11. N. xunantunichensis. 12. N. nickoli. 13–16. Oocysts, dorsal aspect, transverse optical section demonstrating residuum and spines, differential interference contrast microscopy. 13. N. nebraskensis. 14. N. secundus. 15. N. xunantunichensis. 16. N. nickoli. 130 COMPARATIVE PARASITOLOGY, 77(2), JULY 2010

080100a–b, REC080101a–b, REC080103); HWML equatorial faces, with equatorial and terminal spines. 100028 (REC07144a–b, REC07145, REC07146, Epimerite a tumidus with longitudinal pleats, petals, REC07147a–b, REC07148, REC07149a–c, REC or lobes not forming hooks, spines, digitiform or 07150a–b, REC07151a–b, REC07152, REC07154, rhizoid processes; diamerite present. Anterior margin REC07155a–b, REC07156, REC07157, REC of sporont protomerite expanded to form a broad 07158a–b, REC07160a–b, REC07161, REC07162, crateriform adhesive disk. REC07163, REC07164, REC07165a–c, REC 07166a–b, REC07168a–b); HWML100030 (REC Description 080027, REC080028a–b, REC080029, REC080030, REC080031a–b, REC080033a, REC080034, REC Trophozoite (Fig. 3): Solitary, attached to host 080035, REC080036, REC080037, REC080038, ventricular epithelium, with epimerite complex. REC080053, REC080054, REC080055, REC Epimerite orbicular or spherical, with longitudinal 080056, REC080057, REC080058, REC080059, pleats. Diamerite oblong, short, length equal to that of REC080072). No specimen from the type series is epimerite. Protomerite elliptoid to dolioform in young retained by the authors. trophozoites, becoming very shallowly dolioform in mature forms, smoothly transitioning at protomerite– Etymology: The specific epithet is an honorific deutomerite septum; protomerite–deutomerite septal and a sign of our gratitude and respect for our early constriction indistinct. Deutomerite spatulate in mentor, Brent B. Nickol. Brent Nickol provided our young trophozoites, becoming narrowly obtrullate (R.E.C. and T.J.C.) initial introduction to systematic in mature forms. Nucleus elliptoid. Relative morpho- theory and philosophy. He is a warm and generous metric ratios: EpiL < DiaL, EpiW . DiaW, EpiL , friend and continues to lend his insightful intellect PL, DiaL , PL, PWM . DWM, PL , PWM. and experience to systematic discussions. His advice in sorting and mitigating the peccadilloes inherent in Sporont (Fig. 7): Solitary, attached to host ven- gregarine nomenclature has been invaluable. In the tricular epithelium by modified protomerite, epimer- mid-1980s, I (R.E.C.) was a student in Brent B. ite lacking. Protomerite very broadly conical in lateral Nickol’s ‘‘Natural History of the Invertebrates’’ field aspect, anterior margin expanded to form a broad course at Cedar Point Biological Station in the western crateriform adhesive disk that is constricted at Nebraska Sandhills. Standing in the cool rush of Clear protomerite–deutomerite septum; PL 164.1 (98.3– Creek and listening to Dr. Nickol’s field lecture on 233.9, 632.49, 32); PWE 255.5 (154.4–477.1, calopterygid damselflies, particularly H. americana, 660.69, 32); PWM 274.3 (156.2–483.1, 664.35, was a pivotal moment in my intellectual development, 32); PLAM 114.8 (38.9–205.2, 637.89, 32); PLPM tutored by a consummate teacher in a captivating 50.7 (10–132, 627.37, 32); PDSW 195.4 (113.2– environment. As a gregarine systematist, I have 296.8, 648.03, 32); PL/PWE 0.7 (0.4–0.9, 60.14, collected and dissected samples of Hetaerina for 32); PL/PWM 0.6 (0.4–0.9, 60.13, 32); PL/PDSW almost 20 yr, searching for a new gregarine species 0.9 (0.5–1.4, 60.21, 32); PLAM/PL 0.7 (0.3–1, to mark that singular experience and teacher. 60.18, 32); PLAM/PLPM 3.5 (0.5–14.4, 63.08, 32); PWM/PWE 1.1 (1–1.3, 60.08, 32); PWM/PDSW 1.4 (1.1–1.9, 60.2, 32). Deutomerite narrowly to very Nubenocephalus xunantunichensis n. sp. narrowly obtrullate; DL 699.6 (446.1–941.3, (Figs. 3, 7, 11, 15) 6136.58, 32); DWE 107.7 (75.1–160.5, 621.82, 32); DWM 199.8 (119.4–291, 648.65, 32); DLAM Generic diagnosis 82.9 (42.5–593, 694.54, 32); DLPM 610.3 (65.8– Eugregarinorida Le´ger, 1892, sensu Clopton 851.5, 6173.36, 32); DL/DWE 6.6 (4.4–9.2, 61.27, (2002); Septatorina Lankester, 1885, sensu Clopton 32); DL/DWM 3.6 (2.7–5.2, 60.57, 32); DL/PDSW (2002); Stylocephaloidea Clopton, 2009, Actino- 3.7 (2.6–5.1, 60.57, 32); DWM/DWE 1.9 (1.4–2.8, cephalidae Le´ger, 1892, sensu Clopton (2002); 60.32, 32); DWM/PDSW 1 (0.9–1.2, 60.05, 32). Acanthosporinae Le´ger, 1892, sensu Clopton Total length (TL) 860.3 (578.8–1149.6, 6157.42, (2002); with the characters of the genus Nubenoce- 32); TL/PL 5.3 (4–7.1, 60.79, 32); DL/PL 4.3 (3– phalus Clopton, Percival, and Janovy, 1993, sensu 6.1, 60.8, 32); DWM/PWM 0.7 (0.5–1, 60.11, 32); Hays, Clopton, Cook, and Cook, 2007. Oocysts TL/DL 1.2 (1.2–1.3, 60.04, 32). Nucleus elliptoid, dipyramidic with polar truncations, hexagonal or placement variable but usually in anterior third of septagonal in equatorial cross section, without deutomerite; nuclear karyosomes present; NW 39 CLOPTON ET AL.—NUBENOCEPHALUS SPP. FROM BELIZE 131

(30.4–50.2, 65.17, 32); NL 59.4 (44.7–73.1, 66.68, near Unitedville, Cayo District, Belize, Central 32); NDS 200.2 (29–739.4, 6204.17, 32); NL/NW America (17u12933.50N; 88u57920.50W). 1.5 (1.1–2.1, 60.25, 32); DL/NL 11.8 (8.5–15, Site of infection: Trophozoites, sporonts, and 61.84, 32); TL/NL 14.5 (10.5–18.7, 62.18, 32); gamonts were collected from the length of the DL/NDS 9.6 (1.2–25.7, 68.24, 32). mesenteron. Gametocysts were collected in host Association: Association syzygial, frontolateral to hindgut, rectum, and feces. lateral, isogamontic. Prevalence: Twenty of 22 (91%) and 2 of 4 (50%) Gametocysts: White in color, orbicular with individuals of A. chelata examined from Hammock persistent gelatinous epicyst. Gametocysts stored in Bridge and Barton’s Creek, respectively. sterile water dehisced by simple rupture in 4 to 6 d; Specimens deposited: The type series is deposited oocysts released singly by simple exposure. in the Harold W. Manter Laboratory for Parasitology Oocysts (Figs. 11, 15): Axially symmetric, dipy- (HWML), Division of Parasitology, University of ramidic with slight polar truncations OL 6.7 (6–7, Nebraska State Museum, Lincoln, Nebraska, U.S.A. 60.22, 25); OW 5.1 (4.8–5.4, 60.15, 25); OWP 0.9 The holotype is a hapantotype slide HWML100025 (0.8–1.2, 60.12, 25); OL/OW 1.3 (1.2–1.4, 60.06, (REC080108). The paratype series consists of 25 25); OL/OWP 7.3 (5.7–8.9, 60.95, 25); OW/OWP slides containing trophozoites, gamonts, and associ- 5.5 (4.4–6.9, 60.69, 25); septagonal in equatorial ations deposited in 1 lot as follows: HWML100024 cross section, without equatorial faces, bearing 7 (REC080019, REC080020, REC080021, REC equatorial spines, 1 at each equatorial vertex, ESL 3.3 080022, REC080023, REC080024, REC080025, (2.6–3.8, 60.34, 25); terminal spines obliquely REC080039, REC080040, REC080041, REC inserted at each pole, 1 at each vertex created by 080043, REC080044, REC080045a–b, REC080060, polar truncations, DSL 2.2 (1.5–2.6, 60.3, 25); OL/ REC080061, REC080062, REC080063, REC ESL 2.1 (1.8–2.7, 60.22, 25); OL/DSL 3.1 (2.5–4.8, 080064, REC080065, REC080066a–c, REC080067, 60.54, 25); OW/ESL 1.6 (1.3–1.9, 60.17, 25); OW/ REC080109). No specimen from the type series is DSL 2.3 (1.9–3.3, 60.36, 25); major oocyst retained by the authors. residuum orbicular, smooth, equatorial, axial with 3 Etymology: The specific epithet is given to 6 subequal minor residua, OrD 1.1 (0.9–1.4, 0.15, recognize the historical significance of the Mayan 6 25); OL/OrD 6.1 (4.8–7.8, 0.88, 25); OW/OrD 4.6 civilization in the type region. The epithet refers to 6 (3.6–6, 0.6, 25). Xunantunich (Mayan, ‘‘Stone Woman’’), a Mayan temple complex overlooking the upper Belize River Taxonomic summary valley from the banks of the Mopan River. Xunantu- Type host: The Comanche Dancer, Argia chelata nich arose around 200 A.D. and thrived during the Calvert (Odonata: Zygoptera: Coenagrionidae), adults. Classical period until the collapse of Mayan civiliza- tion around 900 A.D. The gregarines described herein Type locality: Hammock Bridge on the Mopan are the first described or reported from Belize. River at Branch Mouth, Cayo District, Belize, Central America (17u10965.60N; 89u4981.30W). DISCUSSION Symbiotype: One symbiotype specimen is depos- Hays et al. (2007) asserted that species of ited in the Sam Houston State University Insect Nubenocephalus are differentiated by the form of Collection (SHSUIC), Department of Biological the epimerite. The new species described herein Sciences, Sam Houston State University, Huntsville, demonstrate that while there are notable differences Texas, U.S.A. Individual accession numbers are not in epimerite form within the genus, these differences assigned by SHSUIC. The specimen can be distin- may not be sufficient to diagnose individual species. guished by the following 5-line determination label: The epimerites of N. secundus and N. nebraskensis Line 1—‘‘Argia’’; Line 2—‘‘chelata Calvert, 1902’’; are deltoid with longitudinal pleats and differ largely Line 3—‘‘MALE: REC080105’’; Line 4—‘‘SYM- in that epimerites of N. secundus are deeply deltoid BIOTYPE-VOUCHER’’; Line 5—‘‘det. T. J. Cook while those of N. nebraskensis, although distinctly 2008.’’ deltoid, are more orbicular in nature. Individuals of Other known localities: Barton’s Creek at Caesar’s N. mutabilis, N. nickoli, and N. xunantunichensis all Place, Imperial Mile Marker 60, Western Highway possess distinctly orbicular epimerites that differ in 132 COMPARATIVE PARASITOLOGY, 77(2), JULY 2010

Table 1. Comparative mean and range of morphometric values for sporonts of 4 Nubenocephalus species.*

Nubenocephalus Nubenocephalus Nubenocephalus Nubenocephalus nebraskensis{ secundus{ nickoli§ xunantunichensis§ MetricD Mean Range Mean Range Mean Range Mean Range

PL 434.5 299.4–558.4 198.0 97.9–349.3 190.5 127.3–307.9 164.1 98.3–233.9 PWE 542.2 436.1–675.2 208.4 116.5–422.4 382.5 315.0–482.9 255.5 154.4–477.1 PWM 561.5 446.3–680.3 218.7 127.2–441.7 409.3 331.5–498.4 274.3 156.2–483.1 PLAM 227.1 61.8–414.4 71.9 21.9–171.6 137.8 102.4–181.2 114.8 38.9–205.2 PLPM 208.2 94.3–427.6 122.2 59.8–237.2 58.5 15.2–188.7 50.7 10.0–132.0 PL/PWE 0.8 0.6–1 1.0 0.4–1.9 0.5 0.3–0.7 0.7 0.4–0.9 PL/PWM 0.8 0.5–1 1.0 0.4–1.9 0.5 0.3–0.7 0.6 0.4–0.9 PLAM/PL 0.5 0.1–0.8 0.4 0.2–0.7 0.7 0.4–1.0 0.7 0.3–1.0 PLAM/PLPM 1.4 0.2–3.1 0.6 0.2–2.2 3.1 0.6–9.8 3.5 0.5–14.4 PWM/PWE 1.0 1.0–1.1 1.1 1.0–1.2 1.1 1.0–1.2 1.1 1.0–1.3 DL 1,928.2 1,475.6–2,405 790.2 552.6–1,360.2 1,017.3 759.9–1,223.2 699.6 446.1–941.3 DWE 265.4 166.8–368.3 116.2 62.1–221.2 174.4 118.7–250.4 107.7 75.1–160.5 DWM 483.6 351.5–687.5 172.7 92.7–372.3 322.3 242.4–406.1 199.8 119.4–291.0 DLAM 137.2 79.7–234.7 66.7 26.2–168.7 156.9 93.0–222.1 82.9 42.5–593.0 DLPM 1,790.5 1,338.6–2,322.3 723.6 440.3–1,299.0 864.2 66.02–1,053.3 610.3 65.8–851.5 DL/DWE 7.7 4.4–12.6 7.1 4.8–12.2 5.9 3.8–7.2 6.6 4.4–9.2 DL/DWM 4.1 2.7–5.4 4.9 3.0–8.6 3.2 2.3–3.6 3.6 2.7–5.2 DWM/DWE 1.9 1.4–2.7 1.5 1.1–1.9 1.9 1.5–2.2 1.9 1.4–2.8 TL 2,348.0 1,949.4–2,802.0 987.2 702.2–1,690.6 1,201.7 979.7–1,461.2 860.3 578.8–1,149.6 TL/PL 5.5 3.9–7.8 5.2 3.0–8.0 6.5 4.4–10.2 5.3 4.0–7.1 DL/PL 4.5 2.9–6.8 4.2 2.1–7.1 5.5 3.5–9.2 4.3 3.0–6.1 DWM/PWM 0.9 0.7–1.0 0.8 0.5–1.1 0.8 0.7–1.0 0.7 0.5–1.0 TL/DL 1.2 1.2–1.3 1.3 1.1–1.5 1.2 1.1–1.3 1.2 1.2–1.3 NW 81.2 55.1–110.4 72.5 45.0–108.9 68.9 57.3–87.4 39.0 30.4–50.2 NL 148.7 118.5–199.9 46.4 30.0–80.1 117.2 105.3–132.8 59.4 44.7–73.1 NDS 364.9 40.0–1,389.9 411.5 23.9–1,186.8 136.4 31.4–432.8 200.2 29.0–739.4 NL/NW 1.9 1.3–2.7 0.6 0.3–0.9 1.7 1.4–2.1 1.5 1.1–2.1 DL/NL 13.0 11.1–16.4 17.2 10.8–24.4 8.7 6.8–11.6 11.8 8.5–15.0 TL/NL 15.9 14.0–19.8 21.5 14.7–30.5 10.3 8.8–13.9 14.5 10.5–18.7 DL/NDS 17.0 1.4–43.8 3.9 1.2–34.7 11.9 2.3–31.1 9.6 1.2–25.7

* All measurements are reported as micrometer values. D DL, length of deutomerite; DLAM, distance from protomerite–deutomerite septum to deutomerite axis of maximum width; DLPM, distance from posterior end of deutomerite to deutomerite axis of maximum width; DWE, width of deutomerite at equatorial axis; DWM, maximum width of deutomerite; NDS, distance from nucleus to protomerite– deutomerite septum; NL, length of nucleus; NW, width of nucleus; PL, length of protomerite; PLAM, distance from anterior end of protomerite to protomerite axis of maximum width; PLPM, distance from protomerite–deutomerite septum to protomerite axis of maximum width; PWE, width of protomerite at equatorial axis; PWM, maximum width of protomerite; TL, total length. { N 5 33. { N 5 40. § N 5 32. the shape of the anterior margin (acuminate in N. patterns of variation partitioned by an escalating mutabilis but obtuse in N. nickoli and N. xunantu- diagnostic character set. nichensis) and in the shape and arrangement of Septate gregarine has traditionally incor- longitudinal pleats. Of the known species in the porated characters observed across multiple life-cycle genus, only N. mutabilis is characterized by an stages of the parasite, particularly the morphology and epimerite with an acuminate anterior margin and morphometrics of gamonts or sporonts and oocysts. sepal pleats with sharply acuminate posterior mar- Clopton (2006) used a centroid clustering approach to gins. The similarity and overlap of these characters in demonstrate the efficacy of oocyst morphometrics in large population samples necessitate a more complex diagnosing congeneric species of Xiphocephalus when morphometric solution to species diagnosis within species could not be reliably diagnosed using gamont Nubenocephalus. This is a common taxonomic morphology and morphometrics alone. A similar problem as known diversity within a genus increases: approach is sufficient in the case at hand. Table 1 proliferating diversity reveals increasingly complex and Table 2 present comparative mean and range CLOPTON ET AL.—NUBENOCEPHALUS SPP. FROM BELIZE 133

Table 2. Comparative mean and range of morphometric values for oocysts of 4 Nubenocephalus species.*

Nubenocephalus Nubenocephalus Nubenocephalus Nubenocephalus nebraskensis{ secundus{ nickoli§ xunantunichensis# MetricD Mean Range Mean Range Mean Range Mean Range

OL 7.9 7.6–8.1 6.5 6.1–6.7 7.6 7.2–8.0 6.7 6.0–7.0 OW 4.9 4.5–5.3 4.7 4.4–5.2 5.1 4.7–5.6 5.1 4.8–5.4 OWP 1.5 1.2–1.9 0.9 0.6–1.3 1.1 0.8–1.4 0.9 0.8–1.2 OL/OW 1.6 1.5–1.7 1.4 1.3–1.5 1.5 1.4–1.6 1.3 1.2–1.4 OL/OWP 5.3 4.3–6.5 7.2 5.1–10.1 6.7 5.4–9.1 7.3 5.7–8.9 OW/OWP 3.3 2.6–4.1 5.2 3.6–7.2 4.6 3.7–6.3 5.5 4.4–6.9 ESL 2.9 2.2–3.8 1.1 0.6–1.5 4.5 3.8–5.3 3.3 2.6–3.8 DSL 3.9 3.4–4.7 2.2 1.7–2.5 3.2 2.7–3.9 2.2 1.5–2.6 OL/ESL 2.7 2.0–3.5 6.1 4.1–10.5 1.7 1.4–2.0 2.1 1.8–2.7 OL/DSL 2.0 1.7–2.4 3.0 2.6–3.9 2.4 1.9–2.9 3.1 2.5–4.8 OW/ESL 1.7 1.2–2.3 4.4 3.1–7.4 1.2 0.9–1.4 1.6 1.3–1.9 OW/DSL 1.2 1.0–1.4 2.2 1.8–2.7 1.6 1.2–1.9 2.3 1.9–3.3 OrD 1.2 1.0–1.6 1.0 0.7–1.2 1.6 1.3–1.9 1.1 0.9–1.4 OL/OrD 6.6 5.0–8.4 6.7 5.1–9.4 4.9 3.9–6.2 6.1 4.8–7.8 OW/OrD 4.1 3.1–5.0 4.9 3.8–6.5 3.3 2.7–4.0 4.6 3.6–6.0

* All measurements are reported as micrometer values. D DSL, length of oocyst dorsal or polar spine; ESL, length of oocyst equatorial spine; OL, length of oocyst; OrD, diameter of oocyst residuum; OW, width of oocyst at equator; OWP, width of oocyst at polar truncation. { N 5 41. { N 5 30. § N 5 33. # N 5 25.

morphometric values for sporonts and oocysts, respec- Oocyst length and width have traditionally been tively, across 4 species of Nubenocephalus. used to diagnose differences among congeneric In the case of Nubenocephalus sporonts, the gregarine species. In the case of Nubenocephalus, relative shape of the nucleus as revealed by NL and oocyst length and the lengths of both equatorial and NW values and the total sporont length most clearly dorsal spines clearly separate species (Table 2, separate the 4 species (Fig. 17). Sporonts of Fig. 18). Oocyst length alone partitions species of Nubenocephalus are partially separated among Nubenocephalus into a large-oocyst group (N. species by total sporont length (cf. Table 1, nebraskensis and N. nickoli) and a small-oocyst Fig. 17). Sporonts of N. nebraskensis are markedly group (N. secundus and N. xunantunichensis). There larger than those of any other known species, falling is evident centroid clustering within each group using outside the range values of N. secundus, N. nickoli, traditional oocyst length and width metrics alone and N. xunantunichensis. Although there is sub- (Fig. 19), but clear species diagnosis within each stantial overlap in the range values of these latter group would be difficult. Dorsal and equatorial spine species, sporonts of N. nickoli are generally larger lengths provide clearer centroid separation among than those of N. secundus and N. xunantunichensis. species than oocyst width provides (cf. Figs. 18, 19). Even with large population samples, it is difficult to Thus, N. nebraskensis is characterized by large separate N. secundus and N. xunantunichensis based oocysts with short equatorial spines, and N. nickoli on sporont length alone. However, NW, NL, and is characterized by large oocysts with long equatorial nuclear shape also separate sporonts of Nubeno- spines. Similarly, N. secundus is characterized by cephalus, most clearly the similarly sized sporonts small oocysts with short equatorial spines, and N. of N. secundus and N. xunantunichensis. Nuclear xunantunichensis is characterized by small oocysts shape is partially described by the NL/NW ratio with long equatorial spines. Additionally, N. xunan- (Table 1). Although N. nebraskensis, N. nickoli,and tunichensis can be distinguished from all other N. xunantunichensis all share similarly long, known nubenocephalid species by the number of relatively narrow nuclei, nuclei among N. secundus equatorial spines on the oocyst (7 in N. xunantuni- individuals are typically wider than they are long chensis, 6 in all other known species; cf. Fig. 15 vs. (cf. Table 1, Fig. 17). Figs. 13, 14, 16). 134 COMPARATIVE PARASITOLOGY, 77(2), JULY 2010

Figure 17. Centroid clustering of 4 species of Nubenocephalus based on sporont morphology. Markers represent individual sporonts, and stems signify distance from the taxon centroid. (Circles, Nubenocephalus nebraskensis; diamonds, Nubenocephalus secundus; triangles, Nubenocephalus xunantunichensis n. sp.; squares, Nubenocephalus nickoli n. sp.)

Figure 18. Centroid clustering of 4 species of Nubenocephalus based on oocyst morphology. Markers represent individual sporonts, and stems signify distance from the taxon centroid. (Circles, Nubenocephalus nebraskensis; diamonds, Nubenocephalus secundus; triangles, Nubenocephalus xunantunichensis n. sp.; squares, Nubenocephalus nickoli n. sp.) CLOPTON ET AL.—NUBENOCEPHALUS SPP. FROM BELIZE 135

Figure 19. Centroid clustering of 4 species of Nubenocephalus based on oocyst length and width alone. Markers represent individual sporonts. (Circles, Nubenocephalus nebraskensis; diamonds, Nubenocephalus secundus; triangles, Nubenocephalus xunantunichensis n. sp.; squares, Nubenocephalus nickoli n. sp.)

ACKNOWLEDGMENTS necessarily reflect the views of the National Science We thank Jerry Cook, Debra Clopton, Ellis Foundation. Clopton, Juan Garcia, and Shirley Carrias for expeditionary and collecting assistance in Belize. LITERATURE CITED Collections in Belize were made under permits CD/ Belton, P., and H. Grundfest. 1962. Potassium activation 60/3/06 (28) for 2006–2007 and CD/60/3/08 (31) for and K spikes in muscle fibers of mealworm larva 2008–2009 issued under the Scientific Collection/ (Tenebrio molitor). American Journal of Physiology Research Permit/Wildlife Protection Act No. 4/1981 203:588–594. by the Belize Ministry of Natural Resources and the Clopton, R. E. 2002. Phylum Apicomplexa Levine, 1970: Order Eugregarinorida Le´ger, 1900. Pages 205–288 in Environment, Forest Department, Belmopan, Cayo J. J. Lee, G. Leedale, D. Patterson, and P. C. Bradbury, District, Belize. Julian Sherrard kindly provided eds. Illustrated Guide to the Protozoa, 2nd ed. Society access to Barton’s Creek, Cayo District, Belize. The of Protozoologists, Lawrence, Kansas, U.S.A. Carrias Family, Bullet Tree, Cayo District, Belize, Clopton, R. E. 2004a. Calyxocephalus karyopera g. provided warm hospitality and local collecting nov., sp. nov. (Eugregarinorida: Actinocephalidae: Actinocephalinae) from the Ebony Jewelwing damsel- advice. fly Calopteryx maculata (Zygoptera: ) Dennis J. Richardson served as the acting editor for in southeast Nebraska, U.S.A.: implications for me- this manuscript, arranging reviews and exercising full chanical prey–vector stabilization of exogenous greg- editorial discretion in addition to his continued arine development. Comparative Parasitology 71:141– 153. service on the Editorial Board of Comparative Clopton, R. E. 2004b. Standard nomenclature and metrics Parasitology. The manuscript was markedly im- of plane shapes for use in gregarine taxonomy. proved as a result of his editorial suggestions. We Comparative Parasitology 71:130–140. thank Dr. Richardson for assuming editorial respon- Clopton, R. E. 2006. Two new species of Xiphocephalus sibilities and allowing us to contribute to the in Eleodes tricostata and Eleodes fusiformis (Coleop- tera: Tenebrionidae: Eleodini) from the Sandhills Helminthological Society of Washington through of western Nebraska. Journal of Parasitology 92:569– continued publication in Comparative Parasitology. 577. This material is based in part upon work supported Clopton, R. E. 2009. Phylogenetic relationships, evolution, by the National Science Foundation through grant and systematic revision of the septate gregarines NSF DEB-0340774 to R.E.C. and NSF DEB- (Apicomplexa: Eugregarinorida: Septatorina). Compar- ative Parasitology 76:167–190. 0340782 to T.J.C. Any opinions, findings, and Clopton, R. E., T. J. Percival, and J. Janovy, Jr. 1993. conclusions or recommendations expressed in this Nubenocephalus nebraskensis n. gen., n. sp. (Apicom- material are those of the author(s) and do not plexa: Actinocephalidae) from adults of Argia bipunc- 136 COMPARATIVE PARASITOLOGY, 77(2), JULY 2010

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