Entomopathogens of Amazonian stick insects and locusts are members of the Beauveria species complex (Cordyceps sensu stricto) Sanjuan, T., Tabima, J., Restrepo, S., Læssøe, T., Spatafora, J. W., & Franco-Molano, A. E. (2014). Entomopathogens of Amazonian stick insects and locusts are members of the Beauveria species complex (Cordyceps sensu stricto). Mycologia, 106(2), 260-275. doi:10.3852/106.2.260 10.3852/106.2.260 Allen Press Inc. Version of Record http://cdss.library.oregonstate.edu/sa-termsofuse Mycologia, 106(2), 2014, pp. 260–275. DOI: 10.3852/106.2.260 # 2014 by The Mycological Society of America, Lawrence, KS 66044-8897 Entomopathogens of Amazonian stick insects and locusts are members of the Beauveria species complex (Cordyceps sensu stricto) Tatiana Sanjuan1 clade, suggest that the holomorphs of these species may include Beauveria or Beauveria-like anamorphs. The varying host specificity of the beauverioid Cordyceps species suggest the potential importance of identifying the natural host taxon before future consideration of strains for use in biological control of pest locusts. Key words: Anamorph-teleomorph connection, entomopathogenic fungi, host specificity, Neotropics, Orthoptera, phylogeny Laboratorio de Taxonomı́a y Ecologı́a de Hongos, Universidad de Antioquia, calle 67 No. 53 – 108, A.A. 1226, Medellin, Colombia Javier Tabima Silvia Restrepo Laboratorio de Micologia y Fitopatologia, Universidad de Los Andes, Cra 1 No. 18A- 12, Bogotá, Colombia Thomas Læssøe Department of Biology, Universitetsparken 15 DK-2100, Copenhagen Ø, Denmark INTRODUCTION Joseph W. Spatafora Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331 Entomopathogenic fungi of the genus Cordyceps sensu Kobayasi et Mains (Mains 1958) have a complex taxonomy in spite of being extensively studied since the 19th century. A recent phylogenetic classification based on molecular data separated the species into four genera (Cordyceps s. str., Elaphocordyceps, Metacordyceps and Ophiocordyceps) across three families, Cordycipitaceae, Clavicipitaceae and Ophiocordycipitaceae, with numerous species of undetermined phylogenetic affinity retained in a residual Cordyceps sensu lato (Sung et al. 2007b). In the study of Sung et al. (2007b) the species of cordycipitoid fungi recorded on Orthoptera were classified in Cordycipitaceae for C. locustiphila Henn. and Ophiocordycipitaceae for Ophiocordyceps amazonica (Henn.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora. Species such as C. uleana Henn. from Peru, C. lilacina Moreau from Congo and C. neogrillotalpae Kobayashi, C. stiphrodes Syd. and C. ctenocephala Syd. from New Guinea were not assigned to any family and retained in Cordyceps s.l. due to the lack of conclusive information. Cordyceps locustiphila and C. uleana were described by Hennings (1904) from collections sent by Ernesto Ule from Perú. He described C. locustiphila as a species with gregarious or solitary, claviform and yellow stromata, but surprisingly no information regarding partspores was given, although this character is commonly used in Cordyceps taxonomy. The original illustration shows the fungus emerging from the abdomen and the coxa (the basal segment of the insect leg attached to the thorax) of an adult locust (Orthoptera: Acrididae). While C. locustiphila is frequently collected in the Amazon, there have been few taxonomic studies of this species (Evans 1982). The type specimen of C. locustiphila was studied only by Petch, who made a revision of Cordyceps species Ana Esperanza Franco-Molano Laboratorio de Taxonomı́a y Ecologı́a de Hongos, Universidad de Antioquia, calle 67 No. 53 – 108, A.A. 1226, Medellin, Colombia Abstract: In the Amazon the only described species of Cordyceps sensu stricto (Hypocreales, Cordycipitaceae) that parasitize insects of Orthopterida (orders Orthoptera and Phasmida) are Cordyceps locustiphila and C. uleana. However, the type specimens for both taxa have been lost and the concepts of these species are uncertain. To achieve a more comprehensive understanding of the systematics of these species, collections of Cordyceps from the Amazon regions of Colombia, Ecuador and Guyana were subjected to morphological, ecological and molecular phylogenetic studies. Phylogenetic analyses were conducted on partial sequences of SSU, LSU, TEF, RPB1 and RPB2 nuclear loci. Two new species are proposed including C. diapheromeriphila, a parasite of Phasmida, and C. acridophila, a parasite of the superfamily Acridomorpha (Orthoptera), which is broadly distributed across the Amazon. For C. locustiphila a lectotypification and an epitypification are made. Cordyceps locustiphila is host specific with Colpolopha (Acridomorpha: Romaleidae), and its distribution coincides with that of its host. The phylogenetic placement of these three species was resolved with strong support in the Beauveria clade of Cordyceps s. str. (Cordycipitaceae). This relationship and the morphological similarity of their yellow stromata with known teleomorphs of the Submitted 14 Jan 2013; accepted for publication 5 Mar 2013. 1 Corresponding author. E-mail: email@example.com 260 SANJUAN ET AL.: AMAZONIAN CORDYCEPS parasitizing Orthoptera (Petch 1934). He considered O. amazonica, collected in Brazil, and O. striphodes, collected in New Guinea, as synonyms of C. locustiphila because all have clavate stromata with a short stalk. Concerning the C. uleana type, Petch said ‘‘C. uleana has clavae with a short stout stalk and a globose head with strongly projecting perithecia.’’ Petch did not report any information about partspores or the host. In addition, Petch (1933) compared in detail the type of C. uleana, preserved in alcohol, with two specimens collected from stick insects (Phasmida) in Madagascar. He identified them as C. uleana due to their similarity in the globose stromata head and projecting perithecia. The partspores of the Madagascar material is 3–9 mm long (TABLE I). Finally Moureau (1949) identified two specimens from Congo on Mantodea ootheca as C. uleana because they had yellow, globose stromata with an echinulate surface. He described the partspores as 5–10 mm long and filiform (TABLE I). As we have summarized above, there is no consensus on the taxonomy, phylogenetic position or ecological relationship of Cordyceps on Orthoptera and Phasmida, particularly for C. locustiphila and C. uleana sensu Hennings. Unfortunately, during the Second World War (WWII), Hennings’s types deposited in Berlin were destroyed. The only original material supporting these species is a copy of an original illustration deposited in Kew Botanical Garden and the original paper (Hennings 1904). Thus, it is necessary to define the position of these species based on morphological and molecular phylogenetic analyses of freshly collected specimens. We clarify species and type concepts of Amazonian Cordyceps s. str. on the superorder Orthopterida (Orthoptera and Phasmida; Grimaldi and Engel 2005) through the examination of morphological characters, host association, geographic distribution and phylogenetic analyses of molecular data obtained from specimens of C. locustiphila-like and C. uleanalike material from Colombia, Ecuador and Guyana, MATERIALS AND METHODS Field collections.— Most specimens in this study were collected in Colombia, Department of Amazonas, Municipality of La Chorrera, which is a Uitoto indigenous territory and Municipality of Leticia Town in El Zafire reserve (4u09210S, 69u539550W). Two kinds of forests were sampled: (i) ‘‘chagra’’ which is characterized by low canopy and mixed forest surrounded by the typical indigenous agriculture crops, and (ii) high, intact canopy forest with relatively low human disturbance that is limited to use by indigenous peoples for hunting and timber. Initial collections were made in Mar and then May–Jul 2010 and 2011, which typically is the rainy season in this region. Additional field 261 trips were made to a lowland tropical forest of the interAndes valley at the municipal forest, Mariquita, Tolima Department, in Jan 2011, and the second at the El Amargal Biological Station Nuqui, Chocó Department, in Mar 2012. All areas are 20–200 m, average temperatures is 28 C, and relative humidity is 90%. Specimen collection at each site occurred over a 2 h interval and avoided human trails. Collecting involved careful examination of leaf litter, downed wood and elevated plant structures (e.g. leaves, twigs) to detect the emergence of stromata from insect cadavers. Additional data on forest characteristics, including habitat and live, unparasitized host specimens also were collected. Dried specimens were placed in plastic bags with silica gel and stored at the Antioquia University Herbarium (HUA). Morphological observations.— Collected material as well as material provided by the National Herbarium of Colombia (COL), the National Herbarium of Ecuador (QCNE) and Dr M. Catherine Aime’s personal collection (MCA) were rehydrated in sterilized water and stained with Congo red or lactophenol cotton blue. Perithecia, asci, ascospores and partspores were examined in a Leica Dm 1000 compound microscope and fluorescent Olympus B60 microscope. Methuen Handbook of Color (Kornerup and Wanscher 1984) was used for color descriptions of stromata. DNA extraction, PCR and sequencing.—In the field small pieces of fresh tissue from stromata were placed in 50 mL CTAB extraction buffer (1.4 M NaCl; 100 mM Tris-HCl pH 8.0; 20 mM EDTA pH 8.0; 2% CTAB w/v), and DNA was extracted following the method in Kepler et al. (2011). Five nuclear loci were amplified: small subunit ribosomal RNA (SSU) and large subunit ribosomal RNA (LSU), elongation factor-1a (TEF), and the largest and second largest subunits of RNA polymerase II (RPB1 and RPB2). Nuclear ribosomal internal transcribed spacer region (ITS) also was amplified and sequenced for all samples. PCR amplification was performed in 25 mL MasterAmp 23 PCR premix E (Epicenter, Madison, Wisconsin), 0.2 mM of each dNTP’s, 0.5 mM amplification primers, 0.1–0.2 mg template DNA and 1.25 U Taq DNA polymerase (Fermentas, Glen Burnie, Maryland). Amplification of SSU and LSU were performed respectively with NS1/NS4 (White et al. 1990) and LROR/ LR5 primers (Vilgalys and Sun 1994). ITS was amplified with primers ITS1f and ITS4 (White et al. 1990). Amplification of TEF was performed with the primers 983F and 2218R (Rehner and Buckley 2005). Amplification of RPB1 was performed with primers cRPB1-1aF and cRPB1-CaR (Castlebury et al. 2004), and amplification of RPB2 was performed with primers fRPB2-5f2 and fRPB2-7cR (Liu et al. 1999). The PCR reactions were performed in a thermocycler 1000 (BIORAD, Hercules, California) programmed as follows: 94 C for 3 min; 10 cycles of 94 C for 30 s, 55 C for 1 min, and 72 C for 2 min; 35 cycles of 94 C for 30 s, 50 C for 1 min, and 72 C for 2 min; one cycle of 72 C for 3 min and 4 C indefinitely (Kepler et al. 2011). Sequencing was performed with the amplification primers at Macrogen (Seoul, South Korea) sequencing service. New DNA sequences of genes generated in this study were submitted to GenBank (TABLE II). nymph and adult: Acridomorpha (Or) Locustidae adult (Or) Oothec (Ma) Diapheromeridae adult (Ph) Staphylinidae larvae (Co), (He) Scarabeidae adult (Co) Gryllotalpa (Or) Colombia, Ecuador, Guyana Peru Congo Ecuador Guyana Japan Korea New Guinea New Guinea Nepal Claviform Colpolopha sp. (Or) Colombia, Ecuador Semi-immersed 7–20 3 5–7 Cylindrical, simple Claviform, * 150–200 3 3–3.5 Semi-immersed, wide wall Subimmersed 25 3 2.5 620–680 3 320–380 400 3 4 275–350 3 200–250 135–150 3 4–4.5 6–8 3 1 10 3 1 3–4 3 1 300 3 100–110 Semi-immersed 30–40 3 2–4 (6–)8–12 3 0.8–1 320–600(–700) 200–400 3 3.5–4 3 280–350(–400) Semi-immersed aggregates 150–180 3 3 5–10 3 1 440–650 3 250–350 200–350 3 2.4–4 Semi-immersed 2–3 3 0.6–0.8 (3)4–7(10) 3 1 3–5(8) 3 1 Size (mm) Size (mm) 500–600 3 250–300 270–300 3 4 mm 150 ca. Size (mm) Partspores Asci Immersed to 450–730 3 200–440 260–450 3 3–4 semi-immersed wide wall Subimmersed 300–350 3 200–250 250–300 3 4.5–5 aggregates Immersed, aggregates Arrangment 10–15 Size (mm) Claviform, 7–27 3 3–4 echinulate dry Globular, 6–8 scattered. caespitose Globosus, 30 3 2 gregarious (3–)7–16 Globose, gregarious, equinulate Claviform, 55 3 15 solitary Claviform Colpolopha sp. (Or) Features Peru Host Perithecium * Information not provived in the original description; Or: Orthoptera, Ph: Phasmida, Ma: Mantodea, Co: Coleoptera, He: Hemiptera. C. uleana sensu Moureau (1949) C. diapheromeriphila sp. nov. Sanjuan & Restrepo C. staphylinidicola Kobayasi & Shimizu (1982) C. scarabeicola Kobayasi and Shimizu (1976) C. neogryllotalpae Kobayasi and Shimizu (1976) C. uleana Henning (1904) C. locustiphila Henning (1904) C. locustiphila sensu Sanjuan et al. C. acridophila sp. nov. Sanjuan &Franco Distribution Stromata A morphological comparison of beauverioid Cordyceps species Species with yellow stromata TABLE I. 262 MYCOLOGIA HUA ATCC BCC AEG ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF G.J.S. CBS GAM NHJ HUA HUA HUA HUA MCA QCNE QCNE OSC OSC CBS QCNE QCNE MCA OSC EFCC HUA HUA OSC 772 62321 8105 96-15a 1969 4622 1478 6215 2251 2567 7032 7760 1855 1681 2694 2922 71-328 114056 12885 11343 179220 179222 179219 179221 1181 186720 186726 110988 93609 101247 186272 186714 1557 76404 5886 179218 179217 93623 Akanthomyces aculeatus Aphysiostroma stercorarium Aschersonia badia Balansia epichloë Beauveria amorpha Beauveria australis Beauveria bassianna Beauveria brongniartii Beauveria caledonica Beauveria caledonica Beauveria kipukae Beauveria malawesi Beauveria pseudobassiana Beauveria sungii Beauveria varroae Beauveria vermiconia Bionectria cf. aureofulva Bionectria ochroleuca Claviceps purpurea Conoideocrella luteorostrata Cordyceps acridophila Cordyceps acridophila Cordyceps acridophila Cordyceps acridophila Cordyceps acridophila Cordyceps acridophila Cordyceps acridophila Cordyceps bifusispora Cordyceps cardinalis Cordyceps confragosa Cordyceps diapheromeriphila Cordyceps diapheromeriphila Cordyceps diapheromeriphila Cordyceps gunni Cordyceps kyusyuënsis Cordyceps locustiphila Cordyceps locustiphila Cordyceps militaris Taxon Acrididae: Ommatolampis sp. Proscopidae: Apioscelis columbica Acrididae: Ommatolampis sp. Acrididae: Ommatolampis sp. Romaleidae:Tropidacris cristata Acrididae: Ommatolampis sp. Romaleidae:Prionacris sp. Lepidoptera, pupa Lepidoptera, larvae Coccus viridis (Hemiptera) Phasmida: Diapheromeridae Phasmida: Diapheromeridae Phasmida: Diapheromeridae Lepidoptera, pupa Lepidoptera Romaleidae: Colpolopha sinuata Romaleidae: Colpolopha sp. Lepidoptera, pupa Bark Poaceae Lepidoptera: Sphingidae Cow dung Hemiptera: Coccidae Poaceae Coleoptera: Curculionidae Orthoptera: Acridiidae Hemiptera: Pentatomidae Coleoptera: Scarabaeidae Coleoptera Soil Homoptera: Delphacidae Coleoptera: Cerambycidae Coleoptera: Scotlitydae Coleoptera: Scarabaeidae Coleoptera: Curculionidae Soil Host/substratum nLSU KC519368 KC519370 AF543769 AF543792 DQ522537 DQ518752 EF468949 nSSU TEF GenBank no. KC519366 AF543782 DQ522317 EF468743 AY531907 HQ880996 AY531981 AY531890 HQ880990 AY531912 AF339570 AF339520 EF469057 HQ881005 DQ376246 HQ880999 AY531899 HQ881004 AY531920 DQ862044 DQ862027 DQ862029 DQ862013 AY489684 AY489716 AY489611 AF543765 AF543789 AF543778 EF468995 EF468850 EF468801 JQ958600 JQ895527 JQ895536 JQ958614 JQ958602 JQ895528 JQ895538 JQ958616 JQ895541 JQ958613 JQ958601 JQ895526 JQ895537 JQ958615 JQ958607 JQ895542 JQ958604 JQ895531 JQ895539 JQ958617 JQ958605 JQ895532 JQ895540 JQ958618 EF468953 EF468807 EF468747 AY184973 AY184962 DQ522325 AF339604 AF339555 DQ522359 JQ958599 JQ895530 JQ895534 JQ958610 JQ958603 JQ895529 JQ895533 JQ958611 JQ958608 JQ958612 AF339572 AF339522 AY489616 EF468960 EF468813 EF468754 JQ958606 JQ895525 JQ895535 JQ958619 JQ958609 JQ958598 JQ958597 AY184977 AY184966 DQ522332 KC519371 ITS Taxon, specimen voucher and sequence information for specimens used in this study Voucher no. TABLE II. AY489633 DQ522363 EF468851 HQ880879 HQ880862 HQ880836 HQ880853 HQ880891 EF469086 HQ880875 HQ880897 HQ880868 HQ80881 HQ880874 HQ880894 EF469135 DQ842031 AY489648 EF468906 JX003852 JX003849 JX003857 JX003853 JX003856 JX003854 JX003855 EF468855 DQ522370 DQ522407 JX003848 JX003850 JX003851 AY489650 EF468863 JX003846 JX003847 DQ522377 RPB1 AY545732 DQ522426 EF468917 JX003845 JX003844 EF468910 DQ522422 DQ522466 JX003841 JX003843 JX003842 HQ880947 HQ880969 HQ880940 HQ880953 HQ880946 HQ880966 EF469001 DQ522415 DQ522417 EF469103 DQ522411 EF468908 HQ880950 HQ880934 HQ880908 HQ880925 HQ880963 RPB2 SANJUAN ET AL.: AMAZONIAN CORDYCEPS 263 38.165 5691 5693 10684 5718 12623 111002 114050 71233 110990 110991 106405 71235 309.85 56429 513 567.95 12525 36093 208838 89-104 76479 12525 6279 111007 902 350.85 726.73a 402.78 164.70 101270 478.75 5413 101244 5714 3145 2037 EGS EFCC EFCC NHJ ARSEF NHJ OSC CBS OSC OSC OSC OSC OSC CBS ATCC FAU CBS NHJ ATCC ATCC GJS ATCC OSC EFCC OSC HUA CBS CBS CBS CBS CBS CBS ARSEF CBS ARSEF ARSEF ARSEF Voucher no. Taxon Cordyceps ninchukispora Cordyceps ochraceostromata Cordyceps pruinosa Cordyceps pruinosa Cordyceps scarabeicola Cordyceps staphylinidicola Cordyceps takeomontana Cordyceps takeomontana Cordyceps tuberculata Cosmospora coccinea Elaphocordyceps capitata Elaphocordyceps fracta Elaphocordyceps japonica Elaphocordyceps ophioglossoides Elaphocordyceps subsessilis Engyodontium aranearum Epichloë typhina Glomerella cingulata Haptocillium sinense Hirsutella sp. Hydropisphaera erubescens Hypocrea lutea Hypocrella nectrioides Hypomyces polyporinus Isaria farinosa Isaria farinosa Isaria tenuipes Isaria tenuipes Lecanicillium antillanum Lecanicillium aranearum Lecanicillium attenuatum Lecanicillium fusisporum Lecanicillium psalliote Leuconectria clusiae Mariannaea pruinosa Metacordyceps chlamydosporia Metacordyceps taii Metarhizium anisoplae Metarhizium flavoridae TABLE II. Continued Lepidoptera Lepidoptera, limacodid pupa Lepidoptera, limacodid pupa Coleoptera: Scarabeidae Coleoptera, Staphylinid pupa Lepidoptera Lepidoptera Lepidoptera: Noctudidae Hymenomycetes: Inonotus Euascomycetes: Elaphomyces sp. Euascomycetes: Elaphomyces sp. Euascomycetes: Elaphomyces sp. Euascomycetes: Elaphomyces sp. Coleoptera: Scarabaeidae Arachnida Poaceae: Festuca rubra Rosaceae: Fragaria sp. Nematode Hemiptera adult Laxmanniaceae: wood Hemiptera: Coccidae Hymenomycetes: Trametes versicolor Lepidoptera pupa Lepidoptera pupa Lepidoptera: Noctudidae Lepidoptera: Noctudidae Hymenomycete: Agaric Arachnida Hemiptera: Coccidae Hymenomycetes: Coltricia perennis soil soil Lepidoptera: Iragoides fasciata Diplopoda Lepidoptera Coleoptera Hemiptera Host/substratum ITS nSSU EF468991 EF468964 EF468966 EF468968 AF339574 EF468981 EF468984 AB044631 DQ522553 AY489702 AY489689 DQ522545 DQ522547 AY489691 EF469124 AF339576 U32405 U48427 AF339594 EF469125 AY545722 AF543768 U32409 AF543771 DQ522558 EF469127 DQ522559 KC519367 AF339585 AF339586 AF339614 AF339598 EF469128 AY489700 AY184979 DQ522544 AF543763 AF339579 AF339580 EF468846 EF468819 EF468821 EF468823 AF339524 EF468836 EF468838 AB044637 DQ518767 AY489734 AY489721 DQ518759 DQ518761 AY489723 EF469077 AF339526 U17396 U48428 AF339545 EF469078 AY545726 AF543791 U47832 AF543793 DQ518772 EF469080 DQ518773 KC519369 AF339536 AF339537 AF339565 AF339549 EF469081 AY489732 AY184968 DQ518758 AF543787 AF339530 AF339531 nLSU DQ522338 AY489629 AY489615 DQ522328 DQ522330 AY489618 EF469061 DQ522341 AF543777 AF543772 DQ522343 EF469063 DQ522344 AF543781 DQ522347 AF543784 DQ522348 EF469065 DQ522349 KC519365 DQ522350 EF468781 EF468782 EF468783 EF469066 AY489627 DQ522351 DQ522327 AF543775 AF543774 DQ522353 EF468795 EF468759 EF468762 EF468761 DQ522335 EF468776 EF468778 TEF GenBank no. RPB1 RPB2 DQ522396 EF468887 EF468888 EF468889 EF469095 AY489664 DQ522397 DQ522372 DQ522383 DQ522399 DQ522400 DQ522384 AY489667 AY489649 DQ522373 DQ522375 AY489652 EF469090 DQ522387 AY489653 DQ858454 DQ522389 EF469092 DQ522390 AY489662 DQ522393 AY489663 DQ522394 EF469094 DQ522395 EF469113 EF469114 DQ522451 DQ522424 DQ522434 DQ522453 DQ522454 DQ522450 EF468934 EF468935 DQ522449 DQ522435 DQ522438 DQ522421 DQ522425 DQ522428 DQ522429 EF469108 DQ522439 DQ522440 DQ858455 DQ522443 EF469111 AY545731 DQ522446 DQ522448 EF468900 EF468867 EF468921 EF468869 EF468871 DQ522380 DQ522431 EF468881 EF468884 EF468932 264 MYCOLOGIA 128574 111003 109876 2181 431.87 284.36 38.166 145.70 91-164 6564 116.25 101267 102308 1915 101237 384.81 460.88 OSC OSC CBS ARSEF CBS CBS EGS CBS GJS EFCC CBS CBS CBS ARSEF CBS CBS CBS Nomuraea atypicola Ophiocordyceps acicularis Ophiocordyceps agriotidis Ophiocordyceps brunneipunctata Ophiocordyceps entomorrhiza Ophiocordyceps gracilis Ophiocordyceps heteropoda Ophiocordyceps melolonthae Ophiocordyceps nigrella Ophiocordyceps nutans Ophiocordyceps sinensis Ophiocordyceps sobolifera Ophiocordyceps sphecocephala Ophiocordyceps tricentri Ophiocordyceps unilateralis Ophiocordyceps variabilis Ophionectria trichospora Purpureocillium lilacinum Purpureocillium lilacinum Purpureocillium lilacinum Phytocordyceps ninchukispora Pochonia bulbillosa Roumegueriella rufula Shimizuomyces paradoxus Simplicillium lamellicola Simplicillium lanosoniveum Sphaerostilbella berkeleyana Torrubiella ratticaudata Torrubiella wallacei Verticillium epiphytum Verticillium incurvum Taxon Arachnida Coleoptera Coleoptera Coleoptera Coleoptera larvae Lepidoptera larvae Hemiptera: Cicadae nymph Coleoptera: Scarabeidae Lepidoptera larvae Hemiptera: Pentatomidae Lepidoptera pupae Hemiptera: Cicadae nymph Hymenoptera: Vespidae Hemiptera: Cicadae adult Hymenoptera: Formicidae Diptera larvae on liana soil nematode soil Beilschmiedia erythrophloia root of Picea abies Globodera rostochiensis (Nematoda) Smilacaceae: Smilax sieboldii Hymenomycete: Agaricus bisporus Uredinales: Hemileia vastatrix Hymenomycete: Polypore Arachnida Lepidoptera Uredinales Hymenomycete: Ganoderma lipsiense Host/substratum ITS EF468987 EF468950 DQ522540 DQ522542 EF468954 EF468955 EF468957 DQ522548 EF468963 DQ522549 EF468971 EF468972 DQ522551 AB027330 DQ522554 EF468985 AF543766 AF339583 AY624188 AY624189 EF468992 AF339591 EF469129 EF469130 AF339601 AF339603 AF543770 DQ522562 AY184978 DQ522361 AF339600 nSSU EF468841 EF468805 DQ518754 DQ518756 EF468809 EF468810 EF468812 DQ518762 EF468818 DQ518763 EF468827 EF468828 DQ518765 AB027376 DQ518768 EF468839 AF543790 AF339534 EF468844 AY624227 EF468847 AF339542 EF469082 EF469083 AF339552 AF339554 U00756 DQ518777 AY184967 DQ522409 AF339551 nLSU RPB1 DQ522339 EF468779 AF543779 EF468790 EF468791 EF468792 EF468794 EF468796 EF469070 EF469072 DQ522356 DQ522357 AF543783 DQ522360 EF469073 DQ522469 DQ522362 DQ522385 EF468885 AY489669 EF468896 EF468897 EF468898 EF468901 EF468902 EF469099 EF469101 DQ522404 DQ522405 AY489671 DQ522408 EF469102 DQ522532 DQ522410 EF468892 EF468852 DQ522368 DQ522369 EF468857 EF468858 EF468860 DQ522376 EF468866 DQ522378 EF468874 EF468875 DQ522336 DQ522381 EF468786 EF468744 DQ522322 DQ522324 EF468749 EF468750 EF468752 DQ522331 EF468758 DQ522333 EF468767 TEF GenBank no. EF468943 EF469116 EF469118 DQ522462 DQ522463 DQ522465 DQ522467 EF469119 EF469053 DQ522470 EF468940 EF468941 DQ522436 EF468933 DQ522457 EF468924 EF468925 DQ522432 EF468920 DQ522418 DQ522420 EF468911 EF468913 EF468914 RPB2 AEG, Anthony E. Glenn personal collection; ARSEF, USDA-ARS Collection of Entomopathogenic, Ithaca, New York, USA; ATCC, American Type Culture Collections, Manassas, Virginia, USA; BCC, BIOTEC Culture Collection, Klong Luang, Thailand; CBS, Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands; EFCC, Entomopathogenic Fungal Culture Collection, Chuncheon, Korea; FAU., F.A. Uecker personal collection; EGS, E, G. Simmons personal collection; GAM, Julian H. Miller Mycological Herbarium Athens, Georgia; GJS, G. J. Samuels personal collection; HUA, Herbarium Antioquia University, Medellin, COL; KEW, mycology collection of Royal Botanical Garden, KEW, Surrey, UK; MCA, Marie Catherine Aime personal collection; NHJ, Nigel Hywel-Jones personal collection; OSC Oregon State University Herbarium, Corvallis, Oregon, USA; QCNE, National Herbarium of Ecuador, Quito, ECU. 744.73 110987 5692 128576 53484 3101 10125 110993 9247 110994 7287 78842 110998 CBS OSC ARSEF OSC KEW EFCC EFCC OSC EFCC OSC EFCC KEW OSC Voucher no. TABLE II. Continued SANJUAN ET AL.: AMAZONIAN CORDYCEPS 265 266 MYCOLOGIA Sequence alignment and phylogenetic analyses.—Sequenceswere edited with Geneious Pro 4.8.5 (Drummond et al. 2009) and combined with sequences from Cordycipitaceae, Clavicipitaceae and Ophiocordycipitaceae used by Sung et al. (2007b) and sequences of Beauveria from Rehner et al. (2011). (All sequences in this study are in TABLE II.) Glomerella cingulata and the various species of the Bionectriaceae, Nectriaceae and Hypocreaceae were used as the outgroup taxa based on phylogenetic analyses of Castlebury et al. (2004), Sung et al. (2007a) and Kepler et al. (2011). A preliminary alignment of the sequences was performed with Clustal W (Thompson et al. 1994) as part of BioEdit 7.0. (Hall 1999). A re-alignment of SSU and LSU was performed with Muscle (Edgar 2004) with the default settings from Geneious Pro 4.8.5 (Drummond et al. 2009); re-alignment of protein coding genes TEF, RPB1and RPB2 was performed with MAFFT (Katoh and Toh 2010) as part of the CIPRES gateway (Miller et al. 2010). Finally, each alignment was refined manually in BioEdit. Maximum likelihood (ML) analyses were performed with RAxML-VI-HPC 2.0 using a GTR-GAMMA model of evolution (Stamatakis 2006) with 1000 bootstrap replicates. All five genes were concatenated into a single dataset and 11 data partitions were defined: one each for SSU and LSU, plus nine for each of the three codon positions for the protein coding genes TEF, RPB1 and RPB2 (Kepler et al. 2012). Bayesian Inference was performed with MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001) and partitions were specified as in RAxML analyses. 10 000 000 MCMCMC generations were performed, using a sample frequency of 500 generations and a burn-in of 25% of the total run. Two runs using four chains each (one cold and three heated chains) were performed and each run was examined with Tracer 1.5 (Drummond and Rambaut 2007) to verify burnin parameters and convergence of individual chains. For the analysis of combined ITS and TEF we performed RAxML and Bayesian analyses with the same parameter mentioned before but with partitions per gene. For ITS the GTR + G + I model was used, while the HKY + G model was applied to TEF. Each of these models were obtained by using the findings of Rehner et al. (2011) and Kepler et al. (2012) and corroborated by model reconstruction with JModelTest 2.1.3 (Posada 2008). Nodes were considered supported by bootstrap values greater than 70% and posterior probability equal to or greater than 0.95. RESULTS Fifty-eight sequences were obtained from the 12 specimens analyzed (TABLE II). The concatenated alignment was 4770 bases long, with 1094 bases from the SSU, 926 bases from LSU, 1007 bases from TEF, 1049 bases from RPB2 and 690 bases from RPB1. The alignment is available from TreeBASE (S13829). Phylogenetic analyses resolved three families of entomopathogenic fungi, Cordycipitaceae, Ophiocordycipitaceae and Clavicipitaceae, with strong statistical support (bootstrap, MLBS 5 100, posterior probability, PP 5 1.00) in the ML and Bayesian analyses, respectively (FIG. 1, SUPPLEMENTARY FIG. 1). The Orthoptera and Phasmida pathogens—hereafter, named the Orthopterida clade—are nested within the Beauveria clade (MLBS 5 67, PP 5 0.95) and comprise three clades with strong support (MLBS 5 100, PP 5 1.00). These three clades also are supported in the separate analysis of each of the individual five genes (SUPPLEMENTARY TABLE 1). The concordance of the five independent gene genealogies (Taylor et al. 1999) provides additional support for recognition of two different phylogenetic species that parasitize locusts and one that is a pathogen of stick insects (FIG. 1). To analyze the relationships and host affiliation of the Orthopterida clade further, phylogenetic analyses were conducted on the combined dataset of ITS (13 sequences of 586 bp) and TEF (11 sequences of 998 bp) with C. staphylinidicola as the outgroup, available from TreeBASE (S13829). C. locustiphila, C. diapheromeriphila sp. nov. and C. acridophila sp. nov. each received support that was consistent with the previous five-gene phylogenetic tree (MLBS 5 100, PP 5 1.00) (FIG. 4). C. locustiphila was restricted to the genus Colpolopha (Romaleidae: Orthoptera) and C. diapheromeriphila was restricted to the Diapheromeridae family (Phasmida). C. acridophila had three subclades weakly to moderately supported (MLBS 5 45, 71, 80; PP 5 0.59, 0.67, 0.92 respectively) that do not correspond with a particular family of Acridomorpha (FIG. 2). However, these three groups correspond to three geographic zones: (i) Napo area (Ecuador) close to the Andes foothills; (ii) western Amazon basin in Colombia, Perú and Brazil; and (iii) Amazon region of Guyana. Within each locality the hosts of the specimens collected belong to different families of Acridomorpha that normally are distributed throughout the Amazon region (FIG. 2). TAXONOMY Cordyceps locustiphila Henn., Fungi amazonici II. a cl. Ernesto Ule collecti. Hedwigia 43:246 (1904). Lectotype (selected here): Hennings, P. Fungi amazonici II. a cl. Ernesto Ule collecti. Tab IV f. 3. (1904) FIG. 3a–d Stromata gregarious, claviform, simple, bright yellow (4B4), 12–20 mm long. Fertile head clavate, slightly echinulate from protruding perithecial ostioles, bright yellow (4A4), 5–7 3 2–5 mm. Stipe fleshy, terete, sometimes caespitose, grayish yellow (4B6), 3–5 3 1–2 mm. Perithecia semi-immersed, perpendicular orientation, ovoid, 550–600(–650) 3 250–320 mm (n 5 20), wall less than 50 mm wide. Asci cylindrical, 270–300 3 4 mm (n 5 40), apical apparatus 3 3 4 mm. Ascospores filiform, hyaline, 225 3 0.8 mm (n 5 10); SANJUAN ET AL.: AMAZONIAN CORDYCEPS 267 FIG. 1. Bayesian 50% majority rule consensus phylogenetic tree based on combined dataset of SSU rRNA, LSU rRNA, TEF, RPB1 and RPB2 of Cordyceps species that parasitize Orthopterida. Bayesian posterior probabilities and ML bootstrap support are given respectively at first and second position, above or below the branches. Bionectriaceae, Nectriaceae, Hypocreaceae, Clavicipitaceae, Ophiocordycipitaceae are collapsed for emphasis of Cordyipitaceae, but a fully expanded tree is available (SUPPLEMENTARY FIG. 1). 268 MYCOLOGIA FIG. 2. Host associations of C. acridophila complex species mapped on phylogeny inferred from Bayesian inference and maximum likelihood analyses of combined nuclear rDNA ITS and elongation factor TEF with C. staphylinidicola as outgroup. Host identity codes are indicated at branch tips. The map of the Amazon region indicates sampling localities and their relation to the centers of distribution for the following acridid taxa: 1. Napo, 2. Amazonian west, 3. Guyana. Host identification symbols include Diapheromeridae (Phasmida) Apioscelis columbica (Proscopidae) Ommatolampis spp. (Acrididae). Romaleidae: &Tropidacris cristata X Prionacris compressa cColpolopha spp. N breaking into truncate partspores, 3–5(–8) 3 1 mm (n 5 50). Host: On imago of Colpolopha cf. sinuata Stål (1873), Orthoptera: Romaleidae. Known distribution: Colombia and Ecuador. Specimens examined: COLOMBIA. TOLIMA: Mariquita Municipal Forest, 5u119290N, 74u549400W, 560 m. Jan 2011, T. Sanjuan 881 (EPITYPE, HUA 179218). CHOCO: Nuquı́, Biological Station El Amargal, 5u349390N, 77u309490S, 40 m. 26 Jan 2000, T. Sanjuan 208 (PARATYPE, HUA 179217). ECUADOR. ORELLANA: Tiputini Research Station, 00u409S, 76u099040W, 235 m. 16 Jun 2004, T. Læssøe 11514 (PARATYPE, QCNE 186267). NAPO: Jatunsacha Reserve, 1u049S, 77u369W, 450 m. 18 Jul 2004, C. Padilla 1423. Notes: The host of the lectotype is likely a species of Colpolopha because it has a thin and conspicuous prothoracic crest typical of this genus (FIG. 3a). Colpolopha has wide distribution in the Amazon, with the center in the Amazonian foothills of Peru, Colombia and Ecuador but is also known from Costa Rica (Carbonell 2004). The host of the epitype was identified as Co. sinuata Stål (determination by C. Carbonell), as well as another collection examined from Colombia (HUA 179217). The host species of the epitype has been reported only from lowlands in Colombia (Eades 2012). The host of the Ecuadorian specimen (QCNE 186267, FIG. 3C) was identified as Co. latipennis Stål, which is distributed in Ecuador and Peru (Eades 2012). Specimen QCNE 186267 was collected in the Amazon foothills of Ecuador, the same region in which specimen C. Padilla (1423) also was collected. Cordyceps acridophila T. Sanjuan et A.E. MolanoFranco, sp. nov. FIG. 3e–g MycoBank MB801975 Etymology: Referring to the host from superfamily group Acridomorpha. Stromata capitate-stipitate, gregarious, or solitary, 7–27 mm long. Fertile part clavate, papillate when fresh and echinulate when dry, pale yellow (4A2/3), bright yellow, 6–12 3 (2.5–) 4–5 mm. Stipe fleshy, terete, cylindrical, grayish yellow (4B5), 5–14 3 2– 3 mm. Perithecia immersed when young and semiimmersed when mature, perpendicular in orientation. Ostioles pale yellow (4A/B5), ovoid to ellipsoid, 450– 730 3 200–440 mm (n 5 20), wall 50–100 mm wide. Asci hyaline, cylindrical 260–450 3 3–4 mm, apical apparatus 2.4–3 3 3–4 mm (n 5 40). Ascospores parallel, smooth, filiform, hyaline, breaking regularly into truncate part spores, (3–)4–7(–10) 3 1 mm (n 5 50). SANJUAN ET AL.: AMAZONIAN CORDYCEPS 269 FIG. 3. Cordyceps locustiphila (a–d) and Cordyceps acridophila (e–g). a. Lectotype, original ilustration by Hennings. b. Cross section of semi-immersed perithecia in the epitype (HUA 179118). c. Stromata of C. locustiphila on Colpolopha laetipenis (QCNE 186267). d. Ascus tip and part-spores (in cotton blue/lactic acid). e. Type on Ommatolapis spp. (Orthoptera: Acrididae) (HUA 179219). f. Part spores inside ascus stained with Congo red (QCNE 186720). g. Cross section of semiimmersed perithecia (in cotton blue/lactic acid) of holotype (HUA 179219). Bars: d, f 5 10 mm; g 5 100 mm. 270 MYCOLOGIA Host: On imago of superfamily group Acridomorpha, Orthoptera. Known distribution: Colombia, Ecuador and Guyana. Specimens examined (molecular and morphological data): COLOMBIA. AMAZONAS: La Chorrera, San Francisco Uitoto community, 1u269580S, 72u479380W, 150 m, on Ommatolampis sp. (Acrididae) 1 Jul 2010, T. Sanjuan 916 (HOLOTYPE, HUA 179219). Tarapaca, El Zafire Reserve, 4u09210S, 69u539550W, on Ommatolampis sp., 15–24 Mar 2011, A. Vasco 1815 & 1845 (PARATYPE, HUA 179220, 179221); ibid., on Apioscelis sp. (Proscopidae) A. Vasco 1872 (HUA 179222). ECUADOR. SUCUMBIOS: Jatunsacha Reserve, 1u049S, 77u369W, 450 m, on Ommatolampis sp., 18 Jul 2004, M. Villegas 2498 (PARATYPE, QCNE 186720), (FIG. 3F); ibid., on Prionacris compressa Stål. (Romelidae), M. Villegas 2509 (QCNE 186726). GUYANA. REGION 8 POTARO SIPARUNI: Pakaraima Mountains, Upper Potaro River Basin, on Tropidacris cristata L., 10 Jun 2010, M.C. Aime 1181. Specimens examined morphologically: COLOMBIA. AMAZONAS: Puerto Santander, Peña Roja, on Ommatolampis sp., 24 Jun 1986, G. Galeano 1228 (COL 329175). META. Acacias, Penal de Oriente, on Agriacris plagiata Walker (Romaleidae), 15 Aug 1981, C. Schulet 290 (COL 214201). PUTUMAYO: Villagarzon, Cabildo Chalguayaco, on Omura congrua Walker (Pyrgomorphidae), 15 Jan 1997, T. Sanjuan 126, ECUADOR. NAPO: Jatunsacha Reserve. 1u049S, 77u369W, 450 m, on T. cristata, 18 Jul 2004, C. Padilla 1428. Notes: Insect species in six acridid genera, all belonging to families of the superfamily Acridomorpha with centers of distribution in the Amazon, so far have been identified as hosts of C. acridophila (Carbonell 2004). Cordyceps diapheromeriphila T. Sanjuan et S. Restrepo, sp. nov. FIG. 4a–f MycoBank MB801976 Etymology: Referring to its affinity with the host family Diapheromeridae. Stromata gregarious, capitate-stipitate, simple, (3–) 7–16 mm high. Fertile head globose, echinulate from protruding ostioles, light yellow (4A4), 2–4 mm diam. Stipe fleshly, terete, light yellow (4A5), 4–12 3 2 mm. Perithecia semi-immersed, aggregated, ovoid to ellipsoid 320–600 3 280–350 mm. Asci cylindrical, 200–400 3 3.5–4 mm (n 5 10), apical apparatus 4–5 mm. Ascospores parallel within asci, filiform, hyaline, breaking into 30 to 40 truncate part spores, (6–)8– 12 3 0.8–1 mm (n 5 40). Conidia hyaline, cylindric to ellipsoid, 5–6 3 1.5–3 mm (n 5 20). Host: Adult of Diapheromeridae, Phasmida. Known distribution: Amazonian region of Ecuador and Guyana. Specimens examined: ECUADOR. ORELLANA: Yasuni National Park, Tiputini Research Station, 00u409S, 76u09904 0W, 235 m, 15 Jul 2004, T. Læssøe 11390 (HOLOTYPE, QCNE 186272). NAPO: Jatunsacha Reserve, 1u049S, 77u369W, 450 m, 17 Sep 2004, M. Villegas 2492 (PARATYPE, QCNE 186714). GUYANA. REGION 8 POTARO SIPURUNI: Pakaraima Mountains, Upper Potaro River Basin, 21 May 2001, M.C. Aime 1557. Notes: Because the original reports of C. uleana are from locust, we propose this new taxon for material collected from Phasmida, family Diapheromeridae. All specimens have globose, pale yellow stromata and aggregated perithecia that are similar in appearance to C. uleana as depicted in the original publication. The measurements of the part spores as well as the order of the host, however, are inconsistent with the original description (TABLE I). It is possible that the original description differs due to the conservation of the specimen (in alcohol), but we have no means of verification because the type material no longer exists to support Hennings’ description. DISCUSSION Distribution and typification of C. locustiphila.— Phylogenetic analyses presented here support the recognition of three species of Cordyceps that parasitize species of Orthopterida in the Neotropics: C. locustiphila, epitypified above and the two new proposed species, C. acridomorpha and C. diapheromeriphila. Collectively these species are referred here as the ‘‘Orthopterida clade’’. The continued use of the name C. locustiphila is adopted because the original illustration in Hennings (1904) is diagnostic both regarding the fungus and the host specimen (FIG. 3a). According to Carbonell (pers comm) the host illustrated is Colopolopha (Romaleidae: Acridomorpha: Orthoptera), the same genus identified as the host for specimens examined herein. In contrast, there is a difference in the size of the perithecia and asci of the examined material compared to the protolog of C. locustiphila. This could be explained by the preservation procedure of the original material, which was stored in alcohol, and might have resulted in dehydration of the cells. Also, Hennings (1904) did not mention part spores, perhaps because the material was immature, which is consistent with his description of the immersed position of perithecia in his specimen. The description of C. locustiphila is also very similar to that of C. neogryllotalpae from New Guinea (Kobayasi and Shimizu 1976) (TABLE I). The measurements and shape of C. neogryllotalpae are consistent with the description of C. locustiphila species, but host families differ between the two taxa; C. neogryllotalpae infects Gryllotalpa, a grasshopper with Austral-Asiatic distribution, whereas C. locustiphila parasitizes Colpolopha, a locustid with Neotropical distribution (Eades 2012). It remains important SANJUAN ET AL.: AMAZONIAN CORDYCEPS 271 FIG. 4. Cordyceps diapheromeriphila, sp. nov. a. Type, stromata on stick imago (Phasmida: Diapheromeridae) (QCNE 186272). b. Longitudinal section of stroma showing ellipsoid perithecia and the central core. c. Asci with partspores inside and details of apex stained with cotton blue with lactic acid. d. Original illustration of C. uleana by Hennings. e. Stromata and conidial cushion on body of stick insect (QCNE186716). f. Conidia. Bars: b 5 200 mm, c 5 10 mm. f 5 5 mm. 272 MYCOLOGIA to make new collections of C. neogryllotalpae to confirm its taxonomic and phylogenetic status and its relationship to C. locustiphila, which is suggested by their morphology similarity. Host range of orthopteroid species of Cordyceps.—The morphology of claviform, light yellow pigmentation, papillate and fleshy stromata; semi-immersed, ovoid perithecia; cylindrical asci with filiform ascospores that disarticulate into truncate part spores is not unique to C. locustiphila. Phylogenetic analyses resolve the cryptic species C. acridophila among specimens that are morphologically consistent with older descriptions of C. locustiphila. These two species share similar morphology (TABLE I) but differ in their host affiliation. While most species of entomopathogenic fungi have been recorded from a limited host range, definitive assessments of the specificity of host affiliation have been documented for only a few species. Examples include that of the Ophiocordyceps unilateralis species complex (Ophiocordycipitaceae), which infect ants primarily in the genus Camponotus (Sanjuan et al. 2001, Mongkolsamrit et al. 2011, Kobmoo et al. 2012), and O. kniphofioides var. kniphofioides, which is a pathogen of the ant species Cephalotes atratus (Evans and Samson 1984, Sanjuan et al. 2001). If we define host specificity as the extent to which a parasite is restricted to the number of host species used at a given stage in the life cycle (Poulin 2007), this work demonstrates differing levels of host specificity between phylogenetic species of Cordycipitaceae that are pathogens of Orthoptera. Cordyceps locustiphila is apparently limited to the genus Colpolopha of the family Romaleidae, a common Amazonian family of locust (Amegdanato and Descamps 1982). Colpolopha inhabits lowland, tropical rainforest in the arboreal stratum with five species reported from the Neotropics (Eades et al. 2012). The host of the epitype is C. sinuata, which is recorded to date only from Colombia, while C. laetipennis is the host of the paratype, and is more broadly distributed from Colombia to Peru (FIG. 2). Since Hennings original description of C. locustiphila, most of the records have originated from western Amazon (Hennings 1904, Petch 1933, Evans 1982), the center of the distribution of Romaleidae (Amegdanato and Descamps 1982). By contrast, hosts of C. acridophila include species in at least six families of the superfamily Acridomorpha (TABLE I). Based on the phylogenetic tree inferred from combined dataset of ITS and TEF, the fungus does not segregate by Acridomorpha families (FIG. 2). Instead collections are grouped in a manner consistent with the three biogeographical areas reported by Amedegnato and Descamps (1982), who studied the dispersal centers of Acridids in the Amazon (FIG. 2). Region 1 is located in the Napo (circle) and is a biogeographic zone in Ecuador and Colombia known for its endemic assemblages of birds, insect and plants. It is hypothesized that its high endemism could be the product of ancient age and proximity to the Andean foothills (Morrone 2000). Region 2, located in Western Amazon, which is defined as west of the Madeira River in Brazil, east of the Andes and south of the Guyana Shield, is an area significantly influenced by the Amazon floodplain (Amedegnato and Descamps 1982). Finally, region 3 is the Guyana Shield, which dates to the Cretaceous and is considered the most ancient terrestrial landscape of South America (Morrone 2000). These three biogeographic regions also are consistent with the Neotropical areas of distribution of Amazonian biota proposed by Cracraft (1985) and corroborated by the distributions of primates (Da Silva and Oren 1996) and the woodcreeper Glyphyorynchus spirurus (Marks et al. 2002). Although additional sampling is necessary, C. acridophila might provide a taxonomic example from fungi that correlates with biogeographic patterns seen in other taxa. C. diapheromeriphila is supported as sister to the C. locustiphila/C. acridophila clade (MLBS 5 99/100; PP 5 1.00; FIGS. 1, 2) and represents a host shift among more distantly related orthopterida taxa that occupy a common forest habitat. The morphology of C. diapheromeriphila is similar to that of C. locustiphila s.l., and the name C. uleana, also a pathogen of locust, was applied to collections on Phasmida by Petch based on morphological similarities (e.g. yellow, papillate stromata), which are likely symplesiomorphies of the Orthopterida clade of Cordyceps s. str. Moureau (1949) also described specimens of C. uleana with the same morphology, but the host remains were identified as oothecae of Mantodea (TABLE I). If host affiliation is diagnostic of species boundaries, as supported by the phylogenetic analyses presented here (FIGS. 1, 2), then Moureau’s description may be based on a misidentified host. The ootheca of Phasmida, Orthoptera and Mantodea are very similar in morphology and in many cases are accurately identified only by molecular techniques (Hunter 2002). Thus, we suggest here that Moureau’s specimen may represent the C. diapheromeriphila lineage, in agreement with the pantropical distribution of the family Diapheromeridae (Grimaldi and Engel 2005). Based on our sampling and the morphological similarities of our specimens with those reported in the literature, the host range of C. diapheromeriphila is probably limited to the order Phasmida and more accurately to stick insects belonging to Diapheromeridae. This group includes the largest known species of SANJUAN ET AL.: AMAZONIAN CORDYCEPS stick insects, Diapherodes gigantea Gmélin, which as in the case of the host of the type specimen of C. diapheromeriphila, was 20 cm long. Host affiliations in Orthopterida’s clade and the host habitat/host relatedness hypotheses.— Phylogenetic analyses support the recognition of three species, C. locustiphila, C. acridophila and C. diapheromeriphila, that share a most recent common ancestor within the Beauveria clade of Cordyceps. This phylogeny poses interesting questions with respect to the phylogenetic distribution of their hosts and possible patterns of host switching. The majority of Beauveria and Cordyceps s.s. are pathogens of Coleoptera and Lepidoptera in soil and leaf litter. Thus, the host affiliations of Orthoptera and Phasmida are likely derived host character states. Of note, all three Cordyceps species of the Orthopterida clade attack hosts in arboreal habitats and are consistent with different elements of both the ‘‘host habitat hypothesis’’ and the ‘‘host relatedness hypothesis’’ (Brooks and McLennan 1991), which have been demonstrated in other cordycipitoid fungi. Nikoh and Fukatsu (2000) showed that closely related species of Elaphocordyceps attack subterranean hosts from two kingdoms: Cicadae (Insecta, Animalia) and Elaphomyces (Ascomycota, Fungi). This was further argued likely to be the result of a single shift to Elaphomyces followed by phylogenetic diversification of the pathogens of Elaphomyces (host relatedness) with independent shifts onto subterranean stages of insects (host habitat) (Spatafora et al. 2007, Sung et al. 2007b). As in Elaphocordyceps, these two mechanisms appear to play a role in diversification of the three species of the Orthopterida clade. The host habitat hypothesis explains how the common ancestors shift among more distantly related taxa (e.g. Orthoptera vs. Phasmida) that occupy a common arboreal habitat. The host relatedness hypothesis is consistent with diversification of C. locustiphila and C. acridophila among the more closely related taxa of the Acridomorpha. The presence of cryptic phylogenetic species with different host ranges also is illustrative of the challenges of characterizing host ranges of cordycipitoid fungi and cautions against broad extrapolation of host ranges among closely related taxa. While C. locustiphila and C. acridophila are closely related and both parasitize Orthoptera, phylogeny and host affiliations suggest that the former is more specialized and the latter a generalist, albeit occupying a common habitat. These differential host ranges, combined with the apparent specificity of C. diapheromeriphila to Phasmida, provide additional evidence for host specificity being a potential isolation mechanism in speciation of cordycipitoid fungi. 273 Yellow, claviform stroma is a synapomorphy of the Beauveria clade.—The entomopathogens of Orthopterida sampled here are supported as members of the Beauveria clade of Cordyceps s. str. (FIG. 1). Beauveria is linked to species of Cordyceps with yellow stromata as C. staphylinidicola and C. scarabaeicola (Rehner 2001), and teleomorph-anamorph connections have been established for Cordyceps bassiana-B. bassiana (Li et al. 2001). In this study this connection is demonstrated for C. scarabaeicola-B. sungii and C. staphylinidicola-B. bassiana (FIG.1). The results presented here provide additional evidence that the yellow pigmentation and claviform stromata of these species are morphological synapomorphies for teleomorphs of the clade, and we extend this concept to species of C. locustiphila, C. acridophila and C. diapheromeriphila. Numerous species of Beauveria have been described from South America including B. amorpha, B. velata and B. vermiconia (Samson and Evans 1982). Because Beauveria has been demonstrated to be monophyletic (Rehner and Buckley 2005) and because the orthopterida species of Cordyceps are nested within the Beauveria clade, we predict that future preparation of cultures from freshly collected material, which was not possible as part of this work, will reveal Beauveria or Beauveria-like anamorphs for the three orthopterida species of Cordyceps. The species of Cordyceps within the Beauveria clade are known mostly from Asia and collectively possess a wide host range that includes Coleoptera and Lepidoptera in (TABLE I). Here we extend the collective host range of beauverioid Cordyceps species to include Orthoptera and Phasmida. Further examination and determination of teleomorph-anamorphs links from additional Neotropical species in this clade are important to fully understand the phylogenetic and ecological diversity of the clade with respect to host range and specificity and the role that host switching, habitat preference and biogeography play in speciation and phylogenetic diversification of this clade. ACKNOWLEDGMENTS We thank Oscar Cadena and Carlos Carbonell for their support in the identification of the Orthoptera host, Ryan Kepler for his advice on phylogenetic analyses and Aida Vasco, M. Catherine Aime and Margarita Villegas for making collections available for this study. The photographs of C. acridophila and C. diapheromeriphila were supplied by Thomas Læssøe and Jens H. Petersen from the Fungi of Ecuador Project (http://www.mycokey.com/Ecuador.html) financially supported by Danish foreign aid organization (RUF). 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