Monotropa</I> and <I>Monotropsis</I>: Two Mycohet
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American Journal of Botany 96(7): 1337–1347. 2009. C OMPARATIVE ANALYSIS OF THE REPRODUCTIVE ECOLOGY OF MONOTROPA AND MONOTROPSIS : TWO MYCOHETEROTROPHIC 1 GENERA IN THE MONOTROPOIDEAE (ERICACEAE) M ATTHEW R. KLOOSTER 2,3 AND THERESA M. CULLEY 2 2 Department of Biological Sciences University of Cincinnati Cincinnati, Ohio 45221-0006 Studies of mycoheterotrophs, defi ned as plants that obtain carbon resources from associated mycorrhizal fungi, have fundamen- tally contributed to our understanding of the importance and complexity of symbiotic ecological interactions. However, to date, the reproductive ecology of these organisms remains empirically understudied, with existing literature presenting hypotheses about traits including a generalist pollination syndrome and autogamous self-pollination. To address this gap in our knowledge of the reproductive ecology of mycoheterotrophic plants, we comparatively analyzed three species of two monotropoid genera, Monotropa and Monotropsis . During three consecutive years of fi eld observations and manipulations of four populations of Monotropa unifl ora , seven of M. hypopitys (both red and yellow color forms), and two of Monotropsis odorata , we investigated fl owering phenology, pollination ecology, breeding system, fl oral herbivory, and reproductive effort and output. Contrary to previ- ous predictions, our results revealed that taxa are largely outcross-pollinated and specialized toward Bombus pollinators. Addition- ally, species differ in breeding system, timing and duration of reproductive development, fl uctuations in reproductive effort and output, and fi tness impacts of herbivory. This study is the fi rst thorough investigation of the reproductive ecology of myco- heterotrophic species and provides insight into possible limitations in reproductive traits imposed by a mycoheterotrophic life history. Key words: breeding system; Ericaceae; Monotropa ; Monotropsis; mycoheterotroph; phenology; pollination; reproductive ecology. Mutualisms and symbioses are some of the most important vegetative structures and expensive photosynthetic pigmenta- ecological interactions driving ecosystem function, but these tion [Leake, 1994]), this evolutionary shift in life history to my- historically have been understudied. Mycoheterotrophy is an coheterotrophy has not come without costs. Consequences of important system of symbiotic interactions that has played a mycoheterotrophy include an obligatory reliance upon mycor- key role in our fundamental understanding of highly special- rhizal associations for carbon infl ux ( Bj ö rkman, 1960 ) and spe- ized ecological interactions and the evolutionary ecology of cialized habitat requirements for survival and propagation nonphotosynthetic plant biology ( Leake, 1994 ; Bidartondo, ( Luoma, 1987; Taylor and Bruns, 1999 ; Leake et al., 2004 ; 2005 ). Defi ned as plant species that obtain carbon resources Moola and Vasseur, 2004 ) that have contributed to rarity, isola- from obligate mycorrhizal associates, nonphotosynthetic myco- tion, and divergence among closely related taxa ( Kruckeberg heterotrophs are a phylogenetically, morphologically, and eco- and Rabinowitz, 1985 ; Bidartondo and Bruns, 2001 ; Taylor et logically diverse group of organisms that evolved from al., 2003 ). Although much is known about mycorrhizal symbio- photosynthetic ancestors ( Wallace, 1975 ; Leake, 1994 ; Cull- ses (e.g., Bidartondo and Bruns, 2001 , 2002 ) and the many con- ings et al., 1996; Bidartondo, 2005). While they are now free vergent life history traits shared by mycoheterotrophic taxa from some of the limitations of “ green ” plants (e.g., complex (e.g., reduced seed size and endosperm, loss of vegetative struc- tures, primary existence as a subterranean root mass [ Wallace, 1975 ; Olson, 1993 ; Leake, 1994 ]), the reproductive ecology of 1 Manuscript received 21 September 2008; revision accepted 21 February these scientifi cally valuable organisms remains empirically 2009. understudied. The authors extend special thanks to M. Becus, D. Boone, E. Buschbeck, In a recent review, Bidartondo (2005) highlighted this gap in D. Conover, B. and G. Culbertson, J. Decker, M. Dodson, S. Dunford, J. E. our understanding of mycoheterotroph reproduction and sug- Klooster, S. Lockwood, E. Maurer, R. Olson, M. Pistrang, V. Soukup, and gested that the limitations of the highly specialized life history D. Taylor for providing support and resources necessary to locate populations and conduct these analyses. They are grateful to M. Bidartondo of mycoheterotrophs may provide direct insight into possible and two anonymous reviewers for critical advice on this manuscript. They reproductive traits of these plants. Bidartondo suggested that it also thank the staff of Cherokee National Forest, Daniel Boone National would be an evolutionarily unstable strategy for a myco- Forest, Germantown MetroPark, The Nature Conservancy, Ohio Department heterotroph already engaged in an obligate symbiotic interac- of Natural Resources; the Culberson family; and the Lockwood family for tion to assume additional compulsory associations, including permission to study these species and for access to populations. Funding those related to reproduction. Subsequently, such taxa would be for this research was made possible through the University Research expected to possess a reproductive ecology free from further Council Fellowship and the Weiman, Wendel, Benedict Award offered by specialization, as evident by traits such as a generalist pollina- the University of Cincinnati to M.R.K. tion syndrome and/or autogamous self-pollination. To date, we 3 Author for correspondence (e-mail: [email protected]); present address: Department of Organismic and Evolutionary Biology, are not aware of any other study that has experimentally ad- Harvard University Herbaria, 22 Divinity Ave., Cambridge, Massachusetts dressed Bidartondo ’ s ideas; existing information consists 02138 USA largely of anecdotal reports of “pollinator ” visitations, informal assessments of breeding systems, and hypothetical commentary doi:10.3732/ajb.0800319 on possible reproductive traits (e.g., Wallace, 1977; Leake, 1337 1338 AMERICAN JOURNAL OF BOTANY [Vol. 96 1994 ; Imamura and Ushimaru, 2007; Ushimaru and Imamura, nodding position and, upon fertilization, assume an erect orientation ( Wallace, 2002 ). To empirically address this fundamental gap in our un- 1975 ). Fruits are dry capsules that contain seeds with wing-like integuments for derstanding of mycoheterotroph reproductive traits, we targeted wind dispersal ( Olson, 1980 ). Within the species M. hypopitys are two distinct color forms, yellow and red, causing much confusion among taxonomists. Once the Monotropoideae (Ericaceae) as an ideal study system. classifi ed as separate species, these color forms are currently thought to simply The Monotropoideae is an entirely nonphotosynthetic sub- represent natural color variation within the species without any distinct ecologi- family with many species having narrow geographic ranges and cal function ( Wallace, 1975 , 1977 ). Because the red and yellow forms have endemism ( Kruckeberg and Rabinowitz, 1985 ) that likely cor- unique fl owering phenologies ( Neyland, 2004 ), rarely occur in sympatry, and relate with the limited distribution of their specialized mycor- are ecologically understudied, we treated each form as a separate experimental rhizal associates ( Bidartondo and Bruns, 2001 ). Most genera unit and separate taxa in this study. Another related taxon with unique reproductive traits is the monotypic genus are morphologically distinct in traits such as color and size of Monotropsis. Monotropsis odorata (sweet pine sap or pygme pipes) is a rare reproductive stems, fl ower number per stem, fl oral fragrance, endemic of the Appalachian Mountains and southeastern United States and is pollen morphology, seed shape, and fruit type (e.g., Copeland, presently listed as threatened or endangered by four states of its limited range 1935 , 1937 , 1938 , 1939 ; Bakshi, 1959 ; Wallace, 1975 , 1977 ; (USDA NRCS, 2009). Found almost exclusively growing in upland, mixed Leake, 1994 ). Additionally, the Monotropoideae has been used oak – pine forests ( Jones, 2005 ), these plants possess coralline roots, which pro- as a model system in pioneering studies of plant – fungal sym- duce one to many reproductive stems that stand 5 –10 cm tall ( Wolf, 1922 ; Cope- land, 1939). Known for their highly fragrant fl owers (similar to baking cloves), bioses (Bj ö rkman, 1960) and for reconciling other myco- these plants are notoriously diffi cult to visually locate due to their small stature heterotrophic life history traits including the complexity of and the seemingly cryptic, brownish bracts and sepals covering the reproductive plant – mycorrhizal associations ( Bidartondo and Bruns, 2001 , stems and encircling each fl ower (M. Klooster, D. Clark [Alma College], and 2002 ; Yang and Pfi ster, 2006 ), developmental biology ( Olson, T. Culley, unpublished manuscript). Flowers are deep pink to lavender, typically 1990 , 1991 , 1993 ), and life history chronology ( Leake et al., with a white tip, and after fertilization, form indehiscent, fl eshy fruits with seeds 2004 ; Bidartondo and Bruns, 2005 ). that are animal dispersed ( Wallace, 1975 ; Leake, 1994 ). The goal of this study was to thoroughly investigate and Site description and sampling— Populations of each taxon were identifi ed compare the reproductive strategies of related mycoheterotrophic in Ohio, Indiana, Kentucky,