Western North American Naturalist

Volume 77 Number 3 Article 9

10-27-2017

Reproductive strategies matter for rare conservation: pollination, phenology, and mating in an endemic plant of the Sky Islands of Arizona, lemmonii A. Gray (Lemmon's fleabane) ()

Pamela Bailey United States Army Engineer Research and Development Center, Vicksburg, MS, [email protected]

Peter Kevan University of Guelph, [email protected]

Follow this and additional works at: https://scholarsarchive.byu.edu/wnan

Recommended Citation Bailey, Pamela and Kevan, Peter (2017) "Reproductive strategies matter for rare plant conservation: pollination, phenology, and mating in an endemic plant of the Sky Islands of Arizona, Erigeron lemmonii A. Gray (Lemmon's fleabane) (Asteraceae)," Western North American Naturalist: Vol. 77 : No. 3 , Article 9. Available at: https://scholarsarchive.byu.edu/wnan/vol77/iss3/9

This Article is brought to you for free and open access by the Western North American Naturalist Publications at BYU ScholarsArchive. It has been accepted for inclusion in Western North American Naturalist by an authorized editor of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Western North American Naturalist 77(3), © 2017, pp. 385–397

REPRODUCTIVE STRATEGIES MATTER FOR RARE PLANT CONSERVATION: POLLINATION, PHENOLOGY, AND MATING IN AN ENDEMIC PLANT OF THE SKY ISLANDS OF ARIZONA, ERIGERON LEMMONII A. GRAY (LEMMON’S FLEABANE) (ASTERACEAE)

Pamela Bailey1 and Peter G. Kevan2

ABSTRACT.—Erigeron lemmonii A. Gray is extremely rare, but not endangered. There are fewer than 1000 individu- als, but they compose an apparently stable, heterozygotic, diploid (2n = 18) population. The are perennial and endemic to one small location (about 0.5 km2) in Scheelite Canyon, Huachuca Mountains (Cochise County), Fort Huachuca Army Base, part of the Sky Island region, Arizona. Erigeron lemmonii grows from crevices in cliffs, ledges, and rock faces where it can form mats by spreading vegetatively from the roots as they proliferate, especially in microsites where soil accumulates. Our goals were to (1) investigate floral anatomy for reproductive structures (inflores- cences, florets, and pollen), (2) record flowering phenology, and (3) determine the ’ breeding and mating system. anatomy and development are typical for Erigeron with ray (pistillate) and disc (bisexu al) florets. Stigmas of the disc florets do not fully exsert and reflex, though they do present and expose the small (17-m-diameter), spiny, and mostly viable (85%) pollen by pushing it from the anthers below to the florets’ entrances during anthesis. Such charac- teristics are appropriate for entomophily. Erigeron lemmonii has an extended (5–6 month) bimodal (May and August–September) sexual reproduct ive period. Each capitulum lives about 3 weeks (bud to cypsela-set) and each floret lasts about 5 days. Despite the small population, E. lemmonii is obligately xenogamous and genetically diverse, reliant on insect pollinators to produce wind-dispersed cypselae. It also reproduces clonally. We compare our results to the findings of others on congeners with similar growth habits. Our findings relate to management and conservation of this plant because, to persist, its habitat must support the diversity of pollinating insects on which it is reliant for sexual reproduction.

RESUMEN.—Aunque extremadamente rara, la especie Erigeron lemmonii, A. Gray, no se encuentra en peligro de extinción. Existen menos de 1000 ejemplares, pero con una población aparentemente estable, heteróciga, diploide (2n = 18). Las plantas son perennes y endémicas de un área pequeña (aprox. 0.5 km2), compuesta por el Cañón de Scheelite, las montañas de Huachuca (condado de Cochise), la Base Militar de Fort Huachuca y parte de la región de Sky Island, Arizona. Esta especie crece en las grietas de los acantilados, en las cornisas y en las rocas donde puede formar esteras al propagarse vegetativamente desde las raíces a medida que éstas proliferan, especialmente en micrositios donde se acu- mula tierra. Nuestros objetivos fueron: (1) investigar la anatomía floral de las estructuras reproductivas (inflorescencias, flósculos y polen), (2) registrar la fenología en época de floración, y (3) determinar su sistema reproductivo y de aparea- miento. La anatomía y el desarrollo de la inflorescencia son típicos de la especie Erigeron, con flósculos de rayos (pisti- lada) y de disco (bisexuales). Los estigmas de los flósculos del disco no sobresalen ni se reflejan completamente, aunque sí presentan y exponen al polen pequeño (17 m de diámetro), espinoso y sobre todo viable (85%) empujándolo de las anteras por debajo de las entradas de los flósculos durante la antesis. Tales características son típicas de la entomofilia. La especie Erigeron lemmonii tiene un período reproductivo prolongado (5–6 meses) y bimodal (mayo y de agosto a septiembre). Cada inflorescencia vive alrededor de 3 semanas (brotes de conjuntos de cipselas) y cada flósculo dura alrededor de 5 días. A pesar de su pequeña población, es obligadamente xenógama, genéticamente diversa y depende de insectos polinizadores para producir cipselas dispersadas por el vie nto. También se reproduce clonalmente. Compara- mos nuestros resultados con los hallazgos de otros sobre congéneres con hábitos de crecimiento similares. Nuestros hallazgos se relacionan con el manejo y la conservación de esta planta porque, para persistir, su hábitat debe mantener la diversidad de insectos polinizadores de los cuales depende para su reproducción sexual.

Populations of rare organisms must be amy] and resulting hybrid vigor). Many plants reproductively active if they are to persist. If do not rely on a single mating system. Mixed those organisms are sexually reproductive, mating systems confer benefits of heterozy- they must have mates (even if themselves: gosity with assured (Richards 1997, Kar- autogamy and geitonogamy in plants) and the ron et al. 2012). Some plants, including rare potential to thrive and adapt vigorously species, eschew, in full or in part, sexual through exchange of genetic information be - recombination and can reproduce asexually tween individuals (cross-fertilization [xenog- (Richards 1997).

1United States Army Engineer Research and Development Center, 3909 Halls Ferry Road, Vicksburg, MS 39180. E-mail: [email protected] 2Canadian Pollination Initiative (CANPOLIN), School of Environmental Sciences, University of Guelph, Guelph, ON NIG 2W1, Canada.

385 386 WESTERN NORTH AMERICAN NATURALIST [Volume 77

B

A C

Fig. 1. (A) Typical habitat for Erigeron lemmonii in the slot canyon Scheelite Canyon, Cochise County, Arizona (see Fig. 2 for map). (B) E. lemmoni habitus showing matted growth form and capitula in various stages of development. (C) Mat of E. lemmonii at peak flowering in May 2011 showing numerous white ligulate florets and a compact array of yellow disc florets on capitula.

For plant conservation, emphasis is mostly 1999, Noyes 2000b), E. lemmonii is recognized placed on habitat, with little emphasis on as distinct. reproductive biology despite acknowledgment Erigeron lemmonii is adapted to the natu- of the consequences of inbreeding, loss of ge - rally fragmented canyon landscape (Fig. 1a) netic diversity (adaptability), and loss of vigour where it grows along the slot canyon bottom (Falk and Holsinger 1991; but see Kevan 1975, (Nesom 2006, Malusa 2006); on shady south-, Ambrose and Kevan 1990, Karron 1991, Spira north-, and west-facing cliff walls; and on 2001). Since about 2000 there has been an large boulders of Escabrosa limestone (Warren upsurge in studies linking the importance of et al. 1991). It is typically found in crevices pollination (and other mutualisms) to plant and on ledges where its taproots penetrate the conservation (Kearns et al. 1998, Spira 2001, highly organic, accumulated trapped soil Gascoigne et al. 2009, Barber and Soper Gor- (Bailey 2013). Field observations and root scan don 2015). The comprehensive study by analysis (Bailey 2013) show that E. lemmonii Tepedino et al. (2014) on humilis can also reproduce vegetatively from its tangled Coville (), a rare plant of the system of lateral roots to produce flowering extreme northeastern Mojave desert in Utah is mats. That sort of vegetative propagation an excellent example. occurs in alpine western North American Erigeron lemmonii was described by Asa congeners E. vagus and E. lanatus (Spongberg Gray (1883, p. 2) from specimens collected 1971) and in E. karvinskianus, a widely known by John Gill Lemmon in 1882. Cronquist’s horticultural plant which grows well on rocky (1947) monograph included E. lemmonii with sites (Lisci and Pacini 1993). High genetic what is now recognized as E. piscaticus diversity (Edwards et al. 2014) indicates that Nesom, leading to its putative broader range E. lemmonii also reproduces sexually. from the Mexican border to the Salt River Aboveground, E. lemmonii is a prostrate (Kearney and Peebles 1960). Since the taxo- perennial growing in mats spreading 10–25 cm nomic revision of Erigeron by morphological (occasionally up to 60 cm; Fig. 1b). From its and molecular analyses (Nesom 1989, 2000, taproots grow prostrate to decumbent and Nesom and Noyes 1999, Noyes and Rieseberg ascending stems that are greenish, minutely 2017] POLLINATION AND CONSERVATION FOR LEMMON’S FLEABANE 387 glandular and hirsute to piloso-hispid (Nesom but only if the plant is self-compatible can 2006). The basal leaves are ephemeral, not geitonogamy result in set. Some species forming rosettes. The cauline leaves are green - are agamospermous or have agamospermous ish with proximal blades oblanceolate to ovate, biotypes, notably those with high ploidy, 5–12 mm by 1–3 mm, margins with 1–2 pairs such as the widespread E. compositus (Noyes of teeth or shallow lobes, or entire, with faces et al. 1995). that are piloso-hirsute and minutely glandular. The ancestral chromosome number in It is reported to (Fig. 1c) from August Astereae is x = 9 (Semple and Watanabe 2009), through October, and possibly into December, and most Erigeron species are diploid (2n = depending on weather (Nesom 2006). 18) with normal chromosomal associations and Although little is known about the mecha- divisions in meiosis (Spongberg 1971). That is nisms of pollination and sexual reproduction the case for E. lemmonii (Noyes and Bailey in E. lemonnii, inferences from the genus can 2014). The latter study suggests that self- be made. In Erigeron, capitula are generally incompatibility and obligate xenogamy apply heteromorphic; the peripheral ray (ligulate) and that agamospermy is unlikely. zygomorphic florets are pistillate and surround The most recent, comprehensive popula- fertile, bisexual (perfect), tubular (stereomor- tion survey of E. lemmonii by Malusa (2006), phic) disc florets (Mani and Saravanan 1999). following that of Gori et al. (1990), reports Erigeron is reported as protandrous (Spongberg approximately 954 individuals. The species’ 1971), but because the pistillate ray florets open native habitat covers some 50 ha (124 acres; first and because subsequent anthesis pro- Fig. 2) with no more than 50 m (164 ft) gresses centripetally and stylar elongation between any 2 plants (Malusa 2006). Malusa pushes the pollen from the corolla tubes of the defined “one plant” in the same crack in a rock disc florets (see below), this designation may as an individual if it was at least 10 cm from be functionally irrelevant from the viewpoint another. Both censuses state that the plants of pollination. The mechanism of pollen pre- flowered and set fruit, indicating that the sentation and stylar growth in Asteraceae has population was, at both times, healthy and been understood for about 250 years (Müller reproducing. Other plant surveys taken in 1883, pp. 315–364; Knuth 1908, volume 2, pp. adjacent canyons indicated no other popula- 568–703; Small 1915, 1917, Percival 1950, tions (Gori et al. 1990, Warren et al. 1991). Thiele 1988, Yeo 1993, Lane 1994, Mani and The Army Resource Manager at Fort Saravanan 1999, Torres and Galetto 2007, Huachuca has monitored E. lemmonii since Hipólito et al. 2013). Style elongation in bisex- 2008 by maintaining permanently identified ual florets through the column of 5 anthers photo-monitoring plots. That monitoring indi- passively carries pollen to the opening of the cates the population remains stable (S. Stone, floret where the stigmatic lobes reflex with personal communication, 2010). A cooperative the pollen on the abaxial, nonreceptive sur- agreement with the U.S. Fish and Wildlife face. In some species, irritable contraction of Service (1) ensures continued monitoring, (2) the filaments after being touched by visiting promotes adaptive management, (3) restricts insects causes the anthers to retract over the recreational activities, and (4) encourages stylar piston (Small 1917). Variations on the research into the life history and population general plan of inflorescence (capitulum) and biology. The species is listed as a critically floret development and anatomy occur so that imperiled G1 plant (NatureServe 2012), which gynomonoecy, protogyny and a diversity of means the species has fewer than 1000 indi- pollination relations (syndromes) eventuate viduals and is defined as being at very high (Lane 1994, Mani and Saravanan 1999). Autog - risk of extinction by extreme rarity (5 or amy reportedly occurs in some species, espe- fewer populations). Nevertheless, in October cially during later floral stages, by contraction 2012, the U.S. Fish and Wildlife Service of the stylar branches so that the stigmatic (USFWS) dropped E. lemmonii from the list surfaces are brought into contact with pollen of candidates proposed under the U.S. of the same flower (see also Müller 1883, Endangered Species Act because of changes Knuth 1908). At least some self-pollination is to previous rules (USFWS 1996a, 1996b) in almost inevitable because of the proximity the Federal Register and because the main and small size of the densely packed florets, threat, wildfire, is considered unlikely. The 388 WESTERN NORTH AMERICAN NATURALIST [Volume 77

Fig. 2. Locations of study plants of Erigeron lemmonii in Scheelite Canyon, Huachuca Range, Cochise County, Arizona, within the plant’s tiny range of approximately 50 ha (31.45854N, 110.35275W). Stars indicate our 10 collection and 11 study sites. Main map derived from www.topozone.com/arizona/cochise-az/valley/scheelite-canyon/ (accessed 5 June 2017) with general location in western USA as indicated.

Desert Botanical Garden, Phoenix, Arizona, with steep, cliffy topography (Fig. 2), which stores of E. lemmonii in its seed bank provides shade, cool-air drainage, higher in case of catastrophe. humidity, and lower temperatures than the surrounding landscape. METHODS Scheelite Canyon is within the Madrean Evergreen Woodland Vegetation Association Habitat Description (Kearney and Peebles 1960, Epple and Epple Erigeron lemmonii is a rare, highly local- 1995, Malusa 2006). Erigeron neomexicanus is ized endemic plant growing only in Scheelite found growing in the sandy ground directly Canyon (31.45854N, 110.35275W) on Fort below the cliffs. Huachuca Army Base in the Huachuca Moun- tains (Cochise County) of the Sky Islands Staining and Microscopy region of Arizona between elevations of 1900 Flowering heads of Erigeron lemmonii and 2200 m (6300 and 7300 feet; Fig. 2). The were collected from 26 individual plants from Huachuca Mountains support a comparatively Scheelite Canyon (May 2011). Florets were rich flora within the geographical context of removed, dissected, examined, and photo - the Arizona locale, with 29%–39% more graphed at the Monsanto Center, Missouri species than expected compared with other Botanical Garden (June and December 2011 localities of similar size (Warshall 1994). The and April 2012) (Missouri Botanical Garden region is semiarid, with an average annual 2011). To photographically record floral devel- maximum temperature of 23.8 °C, average opment, especially pistil development, pollen annual minimum temperature of 9.4 °C, and presentation, and pollen grain structure, sam- average precipitation of 39.7 cm over 2 pluvial ple preparations were made for examination by periods during the spring and midsummer light microscope (using Calberla’s pollen stain; monsoon rains (WRCC 2015). Elevation, sub- Dafni et al. 2005) at 4×, 10×, and 40× magni- strate complexity, and spring-fed, well-watered fication and by scanning electron microscope canyon habitats contribute to the high species (SEM; Nikon–JEOL Neoscope JCM-500 scan- diversity (Bowers and McLaughlin 1994). ning electron microscope). SEM samples were Scheelite Canyon is a narrow slot canyon prepared using 2,2-dimeth oxy propane (DMP). 2017] POLLINATION AND CONSERVATION FOR LEMMON’S FLEABANE 389

The field-collected dry samples were rehy- were labeled. The 5 treatments randomly drated in deionized water. Anthers were placed assigned to each of the 15 (3 per subsite) study into acidified DMP for 20–30 min, then trans- plants were (1) control, 3 plants bagged with ferred to pure acetone for about 3 min before no further manipulations (but one treatment being critical-point dried in CO2 (using ace- was destroyed, presumably by a small mam- tone; Erdtman 1969). The dried pollen was mal); (2) outcrossed, 3 plants bagged and the mounted on stubs with double-stick tape, manually cross-pollinated by placed in a Denton-Desk V Sputter Coater, applying a blooming capitulum from a differ- and sputter coated with gold for viewing. ent plant to the study capitulum; (3) self- To test for vi ability, pollen grains were also within-same-flower (autogamy), 3 plants bagged mounted on microscope slides and treated and florets dusted with their own pollen with lactophenol cotton blue for 4 days, then (success of this treatment was hard to discern evaluated with Bright field microscopy at at the time the experiment was set up because 400× magnification (Stanley and Linskens of the small size of individual florets and later 1974); stained pollen was deemed viable because of possible geitonogamous pollination (Stanley and Linskens 1974, Dafni et al. 2005). within the study capitula); (4) self-within- same-plant (geitonogamy), 3 plants bagged Blooming Phenology and pollen transfers made as for treatment 2 Observations were made on single, marked but with capitula from the same plant; and capitula from 55 fully visible or physically (5) open-pollination, 3 plants that remained and safely accessible individual plants grow- unbagged and untreated manually. All bagged ing on 10 different cliff locations within the and treated plants were rebagged immediately site (approximately 5% of the population) after treatment. After the experiment was set once per month over the flowering period up and running, the study plants were revis- (April–September 2012). The terrain pre- ited during every 24 h until the flowering vented full, and often any, access to most of heads became dry and/or developed seeds the plants in the population. Numbers of with their pappi fully expanded (spent flower formed buds, actively blooming , and and fruiting stage). At that time, the flowering spent flowers (peduncles with and without heads were picked and each one placed in a cypselae) were recorded. At the same times, separate labeled envelope. Each harvested numbers of individual plants were recorded head was later examined in the laboratory, and each plant was mapped. then placed individually on microscope slides stained with Aniline Blue (Dafni et al. 2005) Mating System for microscopic examination at 40× magni - Our methods were designed to examine fication (Swift dissecting microscope). The the relative importance of outcrossing (polli- achenes from each flowering head were scored nator dependent), selfing (possibly pollinator as full or shriveled (aborted) and counted, as mediated, at least in part), or agamospermy were the floret scars on the capitula (floret (probably pollinator independent) (Dafni et scar counts combine disc and ray florets, al. 2005). Our studies were made in May 2012 which are indistinguishable by scars), and the on 3 separate safely accessible cliff areas cypsela : floret ratio was recorded. within the site (referred to as subsites) to the A larger experiment using more subsites extent that our study permit allowed. At each and more plants per subsite was not possible subsite, 5 treatments were set up within a because of accessibility and safety issues and group of 5 or more fully accessible plants study permit restrictions, especially for re- (additional to those used for phenological rec - moval of material from the site. Our experi- ords), as per Dafni et al. (2005). First, all open ment was made in the only site available, capitula on the study plants were removed. with our experimental units being the study One or 2 days after excising open capitula plants in the site as a whole and treatments and bagging the plants as necessary, the new as described. capitula opened and treatments were made. tatistical Analysis Then, each plant, except those identified for S open-pollination, was bagged in fine mesh (1 To test that the capitula on each plant were mm) to exclude pollinators, and all study plants similar, regardless of the treatments to which 390 WESTERN NORTH AMERICAN NATURALIST [Volume 77 the plats were assigned, we used one-way filament is inserted in the upper third of the ANOVA (SAS 2008) to compare the numbers corolla tube and tightly appressed to it. The of capitula per plant and numbers of florets anthers ranged in length from 0.08 mm to 0.1 per capitulum on plants assigned to the differ- mm. The ovary is on average 1 mm in length ent treatments, regardless of the subsite from with 2 distinct whorls of pappi attached to the which they were taken. Because the data top, one whorl of 7 pappus bristles (3.0–3.2 were Poisson distributed, log transformation mm long) and a second whorl with numerous log(y + 1) was applied. The results from stud- tiny bristles (2.6–2.8 mm long) seen as the ies on mating system and pollination were dark band above the ovary (Fig. 3). Style analyzed by contingency test (2 for more than length in the disc floret was 3 mm (average). 2 proportions; Zar 2009) under the null The ray florets lack stamens and the stigmatic hypothesis (H0) that the total numbers of lobes reflex at floral maturity. cypselae produced per capitulum and per From comparative observations of disc flo- plant would be the same regardless of treat- rets at different stages of development from ment. Too few plants were available for rig- unopen to postanthesis, floret phenology does orous truly replicated experimental ANOVA not follows the standard pattern for Asteraceae design even though the statistical software (see Introduction). As the style emerges from (SAS 2008) generated results (pseudoreplica- the corolla tube, the 2 stigmatic lobes do sepa- tion). Graphical analyses of the floral phenol- rate and so cannot reflex. After the stigma des- ogy for the 31 (of 55 marked) plants that iccates, the style shrivels, receding into the flowered were made by Origin8.6® (Origin corolla tube. The unisexual, pistillate ray flo- Lab Corporation 2012). rets are, on average, 8 mm long and 1 mm wide (at the widest point). The ovary, style, RESULTS and pappus is the same as in the disc florets, but the stigmatic lobes do open and reflex. Over the period of our 6-month field study in 2011, thirty-one of our 55 marked plants Pollen Presentation and Pollen Morphology flowered (not counting the 15 that were used It was not possible to determine when the for experimentation). The number of buds on anthers dehisced (as generally noted by Yeo a plant ranged from a minimum of 3 to a maxi- 1993), but pollen presentation in E. lemmonii mum of 122. Not all buds opened during the appeared to be standard for Asteraceae (i.e., season. The total number of capitula available the pollen is carried on the elongating pistil to for study was 828 on the 55 plants marked beyond the opening of the floret where it is and ranged from 0 (i.e., some plants did not exposed during anthesis). bloom) to 90 inflorescences per plant with a Pollen of E. lemmonii is typical for Astera - mean of 15. ceae as tricolporate, spinescent, and spherical (Fig. 4). We measured the average diameter at Floral Morphology 16.5 m with 2 m spines. Pollen viability by Capitula (Fig. 1c) from 26 plants of the 55 staining in Cotton Blue in lactophenol was plants used for phenological observations were 85.8% (Noyes and Bailey 2014). examined (ranging from 1 to 3 per plant on small plants to 1 to 47 per plant on larger Phenology and Flowering plants). Capitula ranged from 2.5 to 5.1 mm Over winter, plants of E. lemmonii are dor- in diameter as also noted by Nesom (2006). mant with much dead leaf material visible on On average there were 56 yellow disc florets the top of the mat. As weather warms, the (28 minimum and 73 maximum) and 45 white brittle, dead leaves break off, blow away, and ray florets (35 minimum and 60 maximum ray are replaced with new growth from the florets) per capitulum. The mean value of flo- crown and live stems. Once the plants become rets per capitulum ranged from 84 to 109 over green and vibrant, small green tight buds the 5 mating system treatments (Table 1). appear and grow over about 6–7 d. Figure 3 shows a typical mature disc floret We recorded 2 blooming periods (Fig. 5) in anthesis and its parts. The corolla tube of with a peak in early May and another in mid- the d isc florets was 3.2–3.4 mm in length. Fila- August. The peak of cypsela release was in ment length was approximately 1 mm, and the mid-July (Fig. 5). 2017] POLLINATION AND CONSERVATION FOR LEMMON’S FLEABANE 391 < 0.0001). The ANOVA was preceded f 0 j P ratio (%)* per capitulum* = 41.43,

Cypselae : floret: Mean cypselae Cypselae a 12.1 (3.1) e 11.6 (3.0) h F = 0.05 (SAS 2008) or by chi-square analysis followed by  Fig. 3. Disc floret with inferior ovary. The style is in the center of the corolla tube, and the 2-lobed stigma at the top of the style is in the process of retracting (4×). = 0.35) and followed by Tukey’s test at P = 1.16, F 0 2 0 108 (24.0) a 0 1 6 1 87.1(13.6) a 1.8 (0.6) f 1.6 (0.6) j

§ Fig. 4. Pollen grain of Erigeron lemmonii showing spinescent exine with 2-m spines. iven column are not significantly different from each other (ANOVA: overall

< 0.0001. Enlarged buds (visible in Fig. 1b), with white ray florets just visible above the involu- > 44, P

2 cre, take about 24–30 h to fully open with the

in Scheelite Canyon, Arizona, in 2012: Uniformity of florets per capitulum (boldface column header) in the various treatments used for mating experi- ray florets fully extended (Fig. 1c). At this time, the ray florets open and the stigma is

 = 0.05 (Zar 2009). presented at the opening of the floret, but reflexing and exposure of the stigmatic lobes is hardly evident (Fig. 3). The disc florets open centripetally, starting about 6–12 h la ter, with Erigeron lemmonii the stigmas extending to the apex of the 1. corolla tube and then exposing pollen they

ABLE have carried. It can take several days for all One bag on a control plant was removed by bird or mammal. Plants with Capitula Capitula with Mean florets multiple comparisons at T Treatment Plants fruit-set sampled cypselae per capitulum Bagged and untreated (control) 2 Open pollination 3 3 7 7 109.2 (12.8) a 18.7 (4.4) d 20.4 (18.8)Hand-cross-pollinated (outcrossed) 3 g 3 7 7 95.8 (14.3) *Proportions are statistically highly different;  § Artificial autogamy 3 1 6 1 84.1 (15.0) a 1.1 (0.3) f 0.9 (0.3)Artificial geitonogamy j 3 From phenological observationsFrom 31 of 55 bloomed 31 828 100% All were observed to set cypselae in parentheses) followed by the same letter in any g by Levene’s test to establish uniformity of variance (Levene’s ments and cypsela development for the 5 pollination and mating experiments, and general observations on plants used for phenological observations. Means (with standard deviation the disc florets to open. The florets remain 392 WESTERN NORTH AMERICAN NATURALIST [Volume 77

Fig. 5. Total production of buds, flowers, and capitulae with withered or fallen ligulate florets (spent flowers) for the 55 study plants of Erigeron lemonnii at a subset of 5 sites in Scheelite Canyon, Arizona, over 6 months in 2012. Observations numbered 330 for buds, 275 for blooming heads (flowers), and 275 for spent flowers. Overall the periods are significantly different in bud (F = 15.09, P < 0.0001), flowering head (F = 43.6, P < 0.0001), and spent head (F = 43.6, P < 0.0001) production. open day and night and the capitulum, con- lum used for the various treatments (Table 1). tinuing to open its disc florets, remains The proportions of cypsela-set and the showy for another 6–9 d, before becoming cypsela : floret ratios differed statistically (2 > spent. At the end of flowering, the rays turn 44, P < 0.0001). The cypsela-set per capitu- from white to light pink to lavender with age, lum and per plant were about half when out- and the disc florets often turn brown and crossing was done by hand versus by natural some appear not to open fully. The entire pollination, and those differences were statis- process seems to depend on weather, primar- tically significant. Both values are even more ily temperature and moisture. The develop- significantly different from the equivalent val- mental progression from tight bud to being ues resulting from experimental treatments of spent for a single capitulum is about 3 d in autogamy (about 1 or 2 cypselae per capitu- natural conditions. The cypselae remain on lum or per plant), geitonogamy (about 1 or 3 the capitulum until they are dispersed by cypselae per capitulum or per plant) and wind, gravity, or animals, after the 3-week automatic self-fertilization (control; 0 cypse- flowering period. lae) (Table 1). In short, the results (Table 1) are definitive, Pollination and Mating System indicating that automatic self-fertilization and Table 1 presents the results of open polli- human-assisted self-pollination are ineffective nation, artificial cross pollinations of bagged and that cross-pollination is required for plants, autogamous and geitonogamous arti- cypsela-set, despite the small sample sizes ficial self-pollinations, and no pollination (numbers of plants) used. When the results (plants bagged and not treated further) for all were analyzed by captitulum (technically subsites combined. No statistical differences pseudoreplication) with ANOVA, the same were found for numbers of florets per capitu- differences were found (Table 1). 2017] POLLINATION AND CONSERVATION FOR LEMMON’S FLEABANE 393

DISCUSSION Allphin et al. 2002). Erigeron karvinskianus DC (E. mucronatus DC) lives naturally in Erigeron lemmonii is a highly localized habitats similar to those of E. lemmonii (e.g., endemic on cliffs and rock faces in the Sky rock gardens and walls ; Lisci and Pacini 1993), Islands area of Arizona (Fig. 2). Its total popu- but where it has been studied in India, it is a lation is estimated to be fewer than 1000 indi- tetraploid apomict which is vigorously vege- viduals in a range of <0.5 km2. Therefore, tative (Mehra and Gill 1972, Bala et al. 2010). there is concern for its continued survival. Xenogamy can create new genetic combi- Understanding the reproductive biology of nations and reduce the potentially negative rare plants is important if management is consequences of inbreeding. An initial genetic required to maintain populations with suffi- investigation of E. lemmonii was made to cient genetic diversity to avoid extinction obtain microsatellite markers (Lindsay et al. (Ambrose and Kevan 1990, Karron 1991, 2012). Eight loci that exhibit a range of het- Kearns and Inouye 1993, Fritz and Nelson erozygosity and genetic diversity within the 1994, Taki and Kevan 2007). Our study was population were identified. Edwards et al. aimed at elucidating the importance of sexual (2014) provide genetic evidence that E. lem- reproduction in the species, even though it moni is highly outcrossed. Allphin and Wind- can reproduce easily and clonally by vegeta- ham (2002) found a positive correlation between tively rooting from nodes on its stems and heterozygosity and longevity in E. kachinen - roots (Bailey 2013). sis, highlighting the evolutionary value of Our results (Table 1) show that E. lemmonii longevity for cliff-growing perennials. Many has sexually functional flowers, sets seed cliff-growing species are long-lived perenni- (cypselae) by obligate outcrossing, and so is als, and once established, populations can not genetically constrained (as is also indicated remain relatively stable (Larson et al. 2000). by its genetic diversity; Edwards et al. 2014) by For E. lemmonii, although individuals grow at genetic drift, exclusive reliance on vegetative different rates depending on microhabitat, the reproduction, apomixis (agamospermy which population appears to be stable (S. Stone, per- our experimental results indicate does not sonal communication, 2012) as determined by occur), or self-pollination (autogamy or geito - the U.S. Fish and Wildlife Service (USFWS nogamy, again which our experimental results 1996a, 1996b). indicate does not occur, or possibly occurs at The inflorescence and floral forms in E. very low rates; the few inflorescences and lemmonii are typical of many Asteraceae with minimal number of cypselae probably resulted ray and disc florets, as in the genus Erigeron from contamination during manipulations). (see Introduction). The white ray florets are Over our study period, the plants that flowered unisexually female but the ray florets are bi - were highly successful in producing cypselae. sexual. Both types of florets are typical for Our findings are consistent with the Erigeron (and many other Asteraceae), but the hypothesis that E. lemmonii possesses sporo- ray florets’ stigmas do not exsert from the phytic incompatibility (Noyes and Bailey corolla and reflex to become exposed even 2014), as has been described for other Aster- though they do push the pollen from the aceae (Gerstel 1950, de Nettancourt 1977, anthers lower in the corolla to be exposed and Noyes and Rieseberg 2000, Noyes 2000a). dispersed. The stigmas of the ray florets do Erigeron kachinensis is another endemic species become exserted and reflexed. The anatomy of with a similar life history found growing on the capitulum, its florets, and the presentation cliffs in the arid southwestern USA (Allphin of the infloresences suggest that self-pollination and Harper 1994, 1997). It is an obligate out- would be a common occurrence within florets crosser, with a putative homomorphic sporo- (autogamy), within and between capitula of the phytic type of self-incompatibility (Allphin same plant (genet) (geitonogamy), as presum- and Windham 2002) . In small, isolated popula- ably occurs in many Asteraceae. Nevertheless, tions, self-incompatibility could reduce sexual such pollinations are ineffective in fertilizing reproductive success by severely limiting ovules and do not result in seed set in E. mating and mate choice (Byers and Meagher lemmonii (Table 1). Self-incompatibility is 1992), reducing seed set, and allowing expres- widespread in Asteraceae, and our evidence sion of lethal genes (Wiens et al. 1987, 1989, indicates it is operational in E. lemmonii. 394 WESTERN NORTH AMERICAN NATURALIST [Volume 77

The small size of the pollen grains (16.5 m reported (Bailey 2013) and has 2 peaks (Fig. in diameter), with their spherical shape and 5). The duration that individual inflorescences spinescent exine with 2-m spines (Fig. 4), is on the plant remain apparently fresh (6–9 d) characteristic of Erigeron species (Noyes and suggests that E. lemmonii has evolved to Bailey 2014). As Hesse’s review (Hesse 2000) favour the occurrence of cross-pollination by a points out, it is often difficult to make corre- wide range of insects, including flies, native lations between pollen exine features and and wasps, beetles, butterflies, and noc- pollination mechanisms, but spines are often turnal moths. This cross pollination results in associated with entomophily as a feature xenogamy (Bailey 2013). Pleasants (1983) sug- facilitating trapping amongst insects’ setae gests that temporal division of blooming in and transport during pollination. The pollen plant communities suppo rts a greater number produced shows high viability (85%; Noyes and diversity of insect visitors over the entire and Bailey 2014). In general, plants that pro- blooming period of a given plant species. He duce pollen of uniform size and high viability reported that Erigeron has diurnal separation are noted for reproducing sexually (Noyes et of pollinators and visitation-based midday al. 2006) (cf. E. karvinskianus, a tetraploid dehiscence (which we did not try to deter- apomict with high pollen sterility; Bala et al. mine), but that idea seems not to apply to 2010). In some genera of Asteraceae, the flo- E. lemmonii. rets can protect the pollen from moisture Seed (cypsela) set from florets that opened (Müller 1883, Knuth 1908) as the stamens are in the second (August–September) bloom was enclosed in the corolla tube and the pollen is high, though we did not assess that quantita- further enclosed in an anther tube. We did tively. The prolonged production of cypselae not detect staminal irritability and retraction from June until October (Fig. 5) indicates that in E. lemmonii, in which the anthers are sexual reproduction is an important compo- attached to the inner wall of the corolla tube nent of this plant’s bionomics. at the upper third (Fig. 4); because of this Recently, E. lemmonii has been removed attachment, retraction cannot occur. It was from the Candidate List for protection under the U.S. Endangered Species Act (USFWS difficult to tell when the anthers dehisced 2012) because human disturbance and wild- (see also Yeo 1993). According to Thiele fire do not appear to be significant threats to (1988), the anthers of many Asteraceae dehisce this cliff-adapted species. The plant is well at the beginning of anthesis which is often adapted to its environment and has many around noon. We did not gather information physical and reproductive traits that allow the for calculation of the pollen: ovule ratio, which population to maintain itself. However, further if high would be an indicator of xenogamous genetic studies of the population, building on reproduction (Cruden 1977). Edwards et al. (2014), would be useful to We were unable to observe how pollen from determine more about the genetic structure the disc florets of E. lemmonii is transferred and possible adaptive constraints that can onto the bodies of pollinating insects (several afflict small populations in isolation (e.g., small flies [Diptera], wasps and bees [Hymen - inbreeding depression, genetic drift, Allee optera], and beetles [Coleoptera]; Bailey 2013) effect). Phylogenetic analysis would also be or if the form and size of the pappus has any interesting in determining lineage, evolution, influence (Horsburgh et al. 2011). Likewise, we and evolutionary potential of E. lemmonii. were unable to determine how pollen could be dosed onto the nonreflexing adaxial stigmatic ACKNOWLEDGMENTS lobes, the presumed receptive side for pollen germination, of the disc florets. Our attempts to The authors express thanks to Mr. Sheri- check for stigmatic receptivity by H2O2 or by dan Stone, Natural Resource Manager at Fort staining with neutral red (Dafni et al. 2005) were Huachuca for Army base assistance, Ms. Kim not successful and would be worth repeating. Whitley for her assistance with fieldwork Flowering is associated with 2 pluvial peri- throughout the project, Jacob Higgins for ods, spring and the late-summer monsoon producing t he map (Fig. 2), Mr. Dale Magoun rains. The prolonged duration of flowering for assistance and review of statistics, Dr. (about 3 weeks) is longer than previously Richard Noyes for advice, and Dr. David 2017] POLLINATION AND CONSERVATION FOR LEMMON’S FLEABANE 395

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