Mycologia ISSN: 0027-5514 (Print) 1557-2536 (Online) Journal homepage: http://www.tandfonline.com/loi/umyc20 New Meredithiella species from mycangia of Corthylus ambrosia beetles suggest genus-level coadaptation but not species-level coevolution Chase G. Mayers, Craig C. Bateman & Thomas C. Harrington To cite this article: Chase G. Mayers, Craig C. Bateman & Thomas C. Harrington (2018) New Meredithiella species from mycangia of Corthylus ambrosia beetles suggest genus-level coadaptation but not species-level coevolution, Mycologia, 110:1, 63-78 To link to this article: https://doi.org/10.1080/00275514.2017.1400353 Published online: 04 Jun 2018. Submit your article to this journal View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=umyc20 MYCOLOGIA 2018, VOL. 110, NO. 1, 63–78 https://doi.org/10.1080/00275514.2017.1400353 New Meredithiella species from mycangia of Corthylus ambrosia beetles suggest genus-level coadaptation but not species-level coevolution Chase G. Mayers a, Craig C. Bateman b, and Thomas C. Harringtona aDepartment of Plant Pathology and Microbiology, 351 Bessey Hall, Iowa State University, Ames, Iowa 50011; bDepartment of Entomology and Nematology, Institute of Food and Agricultural Sciences, University of Florida, PO Box 110410, Gainesville, Florida 32611 ABSTRACT ARTICLE HISTORY Meredithiella norrisii (Microascales, Ceratocystidaceae) is an ambrosia fungus carried in mycangia Received 25 April 2017 of the North American ambrosia beetle, Corthylus punctatissimus. Reports on the identity of the Accepted 31 October 2017 fungal symbionts of other species of Corthylus have been inconsistent. This study tested the KEYWORDS hypothesis that Meredithiella spp. are the primary symbionts of Corthylus spp. Cultures and/or Ambrosia symbiosis; internal transcribed spacer (ITS) rDNA barcode sequences of Meredithiella spp. were obtained Microascales; mutualism; consistently from beetles and galleries of nine Corthylus spp. The ITS sequences of three putative Scolytinae; 2 new taxa species of Meredithiella were associated with C. consimilis and C. flagellifer in Mexico and C. calamarius in Costa Rica. The symbiont of C. columbianus in the USA was identified as M. norrisii. Two new Meredithiella spp. are described: M. fracta from C. papulans in Florida and Honduras, and M. guianensis associated with C. crassus and two unidentified Corthylus spp. in French Guyana. The Meredithiella spp. propagate in the mycangia of adult females by thallic-arthric growth, and the ambrosia growth in larval cradles comprises bead-like hyphal swellings or conidiophores, with or without terminal aleurioconidia. Bayesian phylogenetic analysis of a combined 18S and 28S nuc rDNA and translation elongation factor 1-α (TEF1-α) data set demonstrated that Meredithiella is a distinct monophyletic clade within the Ceratocystidaceae, but its phylogenetic placement with regard to the other ambrosial genera in the family remains ambiguous. The mycangia of C. punctatissimus and C. papulans are also compared using light microscopy and micro-computed tomography (micro-CT) imaging, revealing that they differ in both size and shape, but these differences may not correlate with different lineages of Meredithiella. INTRODUCTION Corthylus species are only found in the Americas, ranging from Canada to Argentina (Wood 1982; Like other ambrosia beetles (Kirkendall et al. 2015; Wood and Bright 1992), but many of the species Hulcr and Stelinski 2017), species of Corthylus are uncommon and understudied (Wood 2007). (Coleoptera, Curculionidae, Scolytinae, Corthylini, Some species construct galleries in living hosts and subtribe Corthylina) farm and feed upon fungal are important pests. For example, C. columbianus gardens along the walls of their galleries and larval causes a discoloration known as flagworm in hard- cradles in wood. One or both sexes of adult ambro- woods (Nord 1972), and C. zulmae may facilitate sia beetles use organs called mycangia to store and disease in Alnus acuminata in Colombia (Gil et al. transport actively growing propagules of their fungal 2004; Jaramillo et al. 2011). Of the four Corthylus symbionts to establish fungal gardens in new trees species in the USA, C. punctatissimus and C. colum- (Francke-Grosmann 1967;Six2003). Usually the bianus are widespread in the eastern USA, C. petilus adult female has the mycangia, but in Corthylus is native to Arizona and Mexico, and C. papulans males have the mycangia, which consist of a pair (=C. spinifer) is an introduced species in Florida of coiled, reticulated tubes in the prothorax that (Wood 1977, 1982; Wood and Bright 1992). open into the procoxal cavities (Schedl 1962; Ambrosia beetles are associated with a variety of Finnegan 1963;Giese1967;Nord1972). fungi, including mutualistic primary ambrosia fungi Meredithiella norrisii was described as the mycangial that serve as their main food source and have coa- symbiont of C. punctatissimus (Mayers et al. 2015), dapted to be transmitted in mycangia (Batra 1985). but the fungal species associated with mycangia of Auxiliary fungi may be transmitted on the cuticle or other Corthylus have not been clearly identified. in the gut (Batra 1985; Biedermann et al. 2013), or CONTACT Chase G. Mayers [email protected] © 2018 The Mycological Society of America Published online 04 Jun 2018 64 MAYERS ET AL.: NEW MEREDITHIELLA SPP. potentially in mycangia (Bateman et al. 2016), but Michoacán, Ario de Rosales, six females in single such auxiliary fungi may not be important sym- gallery in Persea americana, 23 Sep 2014, S. Ochoa- bionts (Harrington 2005). Many primary ambrosia Ascencio; and C. calamarius: Costa Rica, Cerro de la fungi are species of the genus Raffaelea Muerte, single male in Chusquea subtessellata, 26 Jun (Ophiostomatales) (Harrington et al. 2010; 2010, S. Smith. Dreaden et al. 2014; Simmons et al. 2016), but Two herbarium specimens deposited in KANU some ambrosia beetles with relatively large and spe- (University of Kansas Herbarium) as C. columbianus cialized mycangia host ambrosia fungi in one of gallery material were studied: KANU 376401 and three genera in the Microascales: Ambrosiella, KANU 376400, cited by Batra (1967) as 1893-LRB Phialophoropsis, and Meredithiella (Harrington and 2032-LRB, respectively. Handwritten notes (pre- et al. 2010;Mayersetal.2015). sumably Batra’s) on the single packet in KANU A variety of fungi have been suggested to be the 376401, which contained galleries in wood, identified primary ambrosia fungi of Corthylus. A yeast species in the contents as 1893-LRB but gave collection informa- Pichia was isolated from galleries, observed in mycangia, tion (Oct. 1963, beech, Deer, Arkansas) that was not and interpreted as a primary nutritional symbiont of C. consistent with Batra’s(1967) published notes for 1893- columbianus (Wilson 1959; Kabir and Giese 1966;Giese LRB (May 1965, Ulmus sp., Deer, Arkansas), so the 1967; Gil et al. 2004). Batra (1967) identified the primary material inside could not be verified. The other speci- symbiont of C. columbianus as Ambrosiella xylebori, men (KANU 376400) contained three packets, collected although illustrations suggest that the symbiont was by J. C. Nord in Jun 1966 from Acer rubrum wood with similar to M. norrisii, the recently described symbiont C. columbianus galleries, as well as a dried-down cul- of C. punctatissimus (Mayers et al. 2015). Mayers et al. ture isolated from the gallery tunnels; the written notes (2015) also detected DNA of another putative species of matched Batra’s(1967) published notes for 2032-LRB. Meredithiella in mycangia of C. consimilis. Males of C. consimilis and males and galleries of Several species of Corthylus were encountered in C. punctatissimus, as well as M. norrisii cultures, were ongoing studies by the authors and collaborators, present- available from a previous study (Mayers et al. 2015). ing opportunities to clarify the primary ambrosia fungi of Corthylus. The goal of this study was to characterize Corthylus mycangia, identify their fungal contents, isolate Mycangium dissection and observation.—Mycangia symbionts from the beetles, and characterize ambrosia were dissected and removed from freshly killed males growth in their galleries and larval cradles. or ethanol-preserved specimens. For light microscopy, the prothorax was separated and bisected longitudinally and excess body tissue was trimmed carefully with a MATERIALS AND METHODS scalpel until only the front leg, procoxa, and Sample collection. —The following beetle and gallery mycangium remained. The leg/coxa/mycangium was specimens were studied: Corthylus papulans: USA: then mounted in cotton blue for imaging with Florida, Gainesville, Austin Cary Forest (29.732161, Normarski interference contrast microscopy (BH-2 −82.219386), ethanol-baited light trap, two males kept compound microscope; Olympus, Melville, New alive on moistened tissue paper until dissection, Spring York). Images were captured using a Leica DFC295 2016, C. Bateman; Honduras, Francisco Morazán, camera and Leica Application Suite 3.6 (Leica campus of Zamerano Pan-American Agricultural Camera, Allendale, New Jersey). Some images taken at School, ethanol-baited light trap, one male different focus levels and combined into a single focus- immediately preserved in 95% ethanol, Summer 2013, stacked composite image using CombineZP (Alan C. Storer; C. crassus: French Guiana, near Kaw Hadley). Mountain (4.55892, −52.19662),