HORTSCIENCE 55(12):1980–1986. 2020. https://doi.org/10.21273/HORTSCI15394-20 in Madison County; other populations were most likely extirpated due to habitat loss (Ellis et al., 2006; Matthews et al., 2002). Floral Visitors to Helianthus Helianthus verticillatus is a diploid (2n = 2x = 34) perennial species (Ellis et al., 2006). verticillatus, a Rare Sunflower Species Plants can be propagated clonally via rhi- zomes either in the field or in containers in the Southern United States (Edwards et al., 2017), or more efficiently by rooted cuttings until mid-to-late May or early Nicolas C. Strange, John K. Moulton, and Ernest C. Bernard June (Trigiano et al., 2018). Helianthus Department of Entomology and Plant Pathology, University of Tennessee, verticillatus are 2- to 4-m-tall plants that Knoxville, TN 37996-4560 produce flowers from late August or early September to mid-October (Matthews et al., William E. Klingeman, III 2002) or until a killing frost. This sunflower is Department of Plant Sciences, University of Tennessee, Knoxville, TN 37996- a self-incompatible and largely - pollinated species (R. Trigiano, unpublished 4561 data) that does not lend itself to wind polli- Blair J. Sampson nation (Mandel, 2010). Pollinators of H. verticillatus and other self-incompatible U.S. Department of Agriculture-Agricultural Research Service, Thad plants are likely restricted to the flight range Cochran Southern Horticultural Laboratory, 810 Highway 26 West, of locally available insect visitors (Ackerman, Poplarville, MS 39470 2000; Faegri and Leendert, 1966). Plants and have co-evolved with tightening asso- Robert N. Trigiano ciations between energy expenditures in plants Department of Entomology and Plant Pathology, University of Tennessee, to produce pollen and with the demands of Knoxville, TN 37996-4560 pollinators to obtain nectar and pollen for food (Kevan and Baker, 1983). Because in- Additional index words. Agapostemon species, Bombus species, endangered plant, sect pollination is of paramount importance species, /Dialictus species, pollinators to H. verticillatus propagation and crucial to understanding the biology and conservation Abstract. Helianthus verticillatus Small (whorled sunflower) is a federally endangered of this rare plant, it is imperative to identify plant species found only in the southeastern United States that has potential horticultural potentially important pollinator species (Kevan value. Evidence suggests that H. verticillatus is self-incompatible and reliant on insect and Baker, 1983). pollination for seed production. However, the identity of probable pollinators is Flowers of species in the Asteraceae may unknown. Floral visitors were collected and identified during Sept. 2017 and Sept. be visited by only a single species or numer- 2018. Thirty-six species of visitors, including 25 hymenopterans, 7 dipterans, 2 lepidop- ous species of insects (DeGrandi-Hoffman terans, and 2 other insect species, were captured during 7 collection days at a site in and Watkins, 2000; Horsburgh et al., 2011; Georgia (1 day) and 2 locations in Tennessee (6 days). Within a collection day (0745–1815 Robertson, 1922). Species within the bee HR), there were either five or six discrete half-hour collection periods when insects were genera Apis, Bombus, Halictus, and Meliss- captured. Insect visitor activity peaked during the 1145–1215 and 1345–1415 HR periods, odes are among the common hymenopteran and activity was least during the 0745–0845 and 0945–1015 HR periods at all three visitors to sunflowers (DeGrandi-Hoffman locations. Visitors were identified by genus and/or species with morphological keys and and Watkins, 2000; Robertson, 1922). Some sequences of the cox-1 mitochondrial gene. The most frequent visitors at all sites were common families of pollinators in Diptera, Bombus spp. (bumblebees); Ceratina calcarata (a small carpenter bee species) and including Syrphidae and Bombyliidae, also members of the halictid bee tribe were the second and third most common have a significant role in the pollination of visitors at the two Tennessee locations. Helianthus pollen on visitors was identified by some Helianthus spp. (Robertson, 1922). microscopic observations and via direct polymerase chain reaction of DNA using Native bees are the most efficient pollina- Helianthus-specific microsatellites primers. Pollen grains were collected from the most tors of self-incompatible flowers (Free, 1970; frequent visitors and Apis mellifera (honeybee) and counted using a hemocytometer. Greenleaf and Kremen, 2006), and they have Based on the frequency of the insects collected across the three sites and on the mean co-evolved with sunflowers, which are native number of pollen grains carried on the body of the insects, Bombus spp., Halictus ligatus to North America (Hurd, 1980). Despite this (sweat bee), Agapostemon spp., and Lasioglossum/Dialictus spp., collectively, are the most co-evolution, honeybees, which are not na- probable primary pollinators of H. verticillatus. tive to North America, have been reported to be the most efficient pollinators of commer- The whorled sunflower, Helianthus verti- cial sunflowers (McGregor, 1976). In con- cillatus Small, is a rare and federally endan- trast, Parker (1981) claimed that two oligolectic native bees, Andrena helianthi Received for publication 26 Aug. 2020. Accepted gered plant (U.S. Fish and Wildlife Service, and Melissodes agilis, were much more effi- for publication 28 Sept. 2020. 2014) found in only a few locations in the cient pollinators. A combination of both na- Published online 2 November 2020. southern United States. This plant was de- tive and domesticated bees provided efficient We thank David Paulsen of University of Tennes- scribed in 1892 by Samuel Bain (Ellis et al., see for assistance identifying bee and fly speci- pollination of hybrid commercial sunflowers mens, Kevin Hoyt and his staff at Utah Arboretum 2006; Mandel, 2010; Matthews et al., 2002; (DeGrandi-Hoffman and Watkins, 2000; for plot development and maintenance, and Nature Small, 1898) from collections obtained in Greenleaf and Kremen, 2006). Conservancy for access/permission to work at the Chester County, TN. The species was not There has not been a study of floral Cave Spring, GA site. This research was supported collected again until 1993, when it was dis- visitors of H. verticillatus; therefore, an inte- by funding from USDA Agreement 58-6062-6 covered in Floyd County, GA (Matthews gral part of its reproductive biology is un- Grant and USDA NIFA, Hatch project TEN00494. et al., 2002). A regional census was conduct- known. The goals of this study were as R.N.T. is the corresponding author. E-mail: rtrigian@ ed several years later, and an additional utk.edu. follows: to identify and catalog the diversity This is an open access article distributed under the population was discovered in Cherokee of potential pollinators visiting whorled sun- CC BY-NC-ND license (https://creativecommons. County, AL (Matthews et al., 2002). In flower inflorescences at diverse locations org/licenses/by-nc-nd/4.0/). Tennessee, wild plants are now located only using morphological keys and sequences of

1980 HORTSCIENCE VOL. 55(12) DECEMBER 2020 the cox-1 mitochondrial gene; to verify the insects concurrently in sweep nets. When of Entomology and Plant Pathology Insect pollen composition carried by captured in- sweep netting, specimens could become Museum. sects via DNA amplification with H. annuus cross-contaminated with pollen from other Polymerase chain reactions (PCRs) for primers; and to determine loads of pollen plants, and pollen could potentially be col- the cox-1 mitochondrial gene contained 36 grains carried on captured insects. lected from disturbed flower heads. Second, mL of sterile distilled water, 5 mLof10· sweeping a net over the tops of the inflores- TaKaRa Taq buffer, 2.3 mL MgCl2 (50 mM), Materials and Methods cences would likely capture many flying 3.5 mL dNTPs (10 mM), 0.2 mL TaKaRa hot insects, some of which that may not have start Ex Taq, 3 mLof10mM aliquots of Study sites. Three locations with popula- visited flowers, thus possibly confusing them forward and reverse primers (Table 1), and 1 tions of H. verticillatus were used to assess with potential pollinators. Third, H. verticil- mL of DNA template (various concentra- potential insect pollinators: a rural location latus can grow to a height of 4 m, which is too tions). A touchdown amplification program where the sunflower naturally occurs (native) tall for the practical use of standard sweep (Senatore et al., 2014) was used with the in Cave Spring, GA; plantings at a suburban nets unless the operation is performed using a following modifications: 95 C for 1 min, 10 garden site (suburban) in Maryville, TN; and ladder. Fourth, moving a net over and around cycles of 96 C for 15 s, 58 C for 20 s, 72 C a controlled field trial location at the Univer- these plants could destroy flower heads and for 1 min; 10 cycles of 96 C for 15 s, 50 C sity of Tennessee Forest Resources Research degrade the plants at the location. Potential for 20 s, 72 C for 1 min; and 40 cycles of and Education Center Arboretum (semirural) biases using vials for capture were also 96 C for 15 s, 45 C for 20 s, 72 C for 1 min, in Oak Ridge, TN. introduced, including the following: capture and 72 C for 5 min. PCR products were The rural location is a forested site on a skill among collectors could vary greatly; a separated on a 1% agarose gel (120 V/cm2 for prairie remnant near Cave Spring, GA. Hel- few specimens, such as some large Bombus 30 min), stained with ethidium bromide, and ianthus verticillatus plants at this site were spp. and lepidopterans, were too large to fit visualized with an ultraviolet transillumina- not numerous. Many clusters of a few plants into the lumen of the vials, but these were tor. PCR products were freed from gel pieces were separated from each other either by 1 m rare; some insects were more difficult than and bound to EconoSpin silica columns (Ep- or less or by more than 10 m and distributed others to capture routinely; and visitors to och Life Science, Sugar Land, TX) using 5· among a thick undergrowth of privet, honey- flowers beyond the reach of collectors (more volume of 5 M guanidium thiocyanate (pH = suckle, and grasses. Most plants were grow- than 2 m) were not collected. 5), washed, and eluted in 10 mM Tris (pH = ing in full sunlight, but some individuals were Vials with captured insects were immedi- 8.5). Purified PCR amplicons were cycle- partially shaded under trees. ately placed on ice, transported to the labo- sequenced using 1/16 diluted Big Dye v3.1 The suburban location is in a private ratory, and stored at –20 C until processing terminators (Applied Biosystems, Waltham, garden in Maryville, TN. Plants from natu- for morphological and molecular identifica- MA) and cleaned using Sephadex-50 spin rally occurring populations in West Tennes- tion, enumeration of pollen grains, and mo- columns (Princeton Separations, Adelphia, see and Alabama were collected in 2014 lecular identification of pollen. At all sites, NJ) before being dried and sent to the Uni- before H. verticillatus was declared federally insects were collected during the following versity of Tennessee Genomics Core Se- endangered (United States Fish and Wildlife five 30-min intervals throughout the day: quencing Facility for analysis using an ABI Service, 2014) and transplanted to this site. 0945–1015, 1145–1215, 1345–1415, 1545– 3730 Genetic Analyzer. Both directions were During the study years 2017 and 2018, there 1615, and 1745–1815 HR. In 2018, an addi- sequenced using amplification primers. were 250 stems of sunflower within a 10-m2 tional interval from 0745 to 0815 HR was Resulting chromatograms were reconciled area. Plants grew in filtered sunlight in the added to evaluate potential early morning using Sequencher 4.7 (Gene Codes Corp., morning and direct sunlight in the afternoon. visitors. Ann Arbor, MI) to generate a consensus This site was selected to examine potential Insect morphological and molecular sequence. Resulting sequences were com- pollinators of the sunflower found in a resi- identification. All specimens were examined pared with entries in GenBank (https:// dential garden. with a stereomicroscope and identified to the www.ncbi.nlm.nih.gov/genbank/) and the The Oak Ridge location (semi-rural) at lowest taxonomic level described by avail- Barcode of Life (http://www.barcodingli- the University of Tennessee Arboretum in able resources. For members of the Hyme- fe.org/) databases. Oak Ridge, TN, has 250 acres of exotic and noptera, the work of Mitchell (1960) was Molecular confirmation of pollen identity. native plant species. Clones (Trigiano et al., used; however, the work of McAlpine et al. For some visitors, a hind leg with a pollen- 2018) of 30 H. verticillatus plants collected (1981) was used for Diptera species. bearing scopa was detached for pollen ana- from West Tennessee were established in Following morphological identification, lyses. For visitors without scopae or visitors Oct. 2017 at the arboretum. Plant specimens representatives of each putative morphospe- without visible pollen on their scopae, the were arranged in two sections with three cies were selected for cox-1 gene sequencing. entire body was processed to remove pollen. groups in each section and five plants per Several primers, in addition to the traditional For these individuals, specimens were placed group for uniformity. This location was in- barcoding primers, LepF and Lep R (Hebert in 1.5-mL microcentrifuge tubes with 1 mL termediate for anthropogenic influence be- et al., 2003), were designed using available of Qiagen QX wash buffer (Qiagen, Hilden, tween the other two sites because it mimicked GenBank CoxI sequences from related taxa Germany) added; samples were vigorously the native setting while having suburban to reliably amplify the gene from all captured vortexed for 15 s to dislodge pollen grains areas nearby. taxa (Table 1). DNA was extracted from from the insects. Tubes were centrifuged at Collection and identification of floral insect specimens with the Omega E.Z.N.A. 10,000 gn for 5 min to obtain a pollen pellet. visitors. Floral visitors were collected during Insect DNA Kit (Omega Bio-Tek, Norcross, Insects or body parts were removed, and September and October at the Cave Spring GA). For large specimens (e.g., Bombus spp. pollen samples were stored at –20 C until location and the Maryville location in 2017, and Svastra spp.), one leg was used; for being processed for molecular identification. and only at the Maryville and Oak Ridge smaller taxa (e.g., members of the tribe DNA extraction from the pollen pellet locations in 2018. Floral visitors were caught Augochlorini), the three left legs were used. was completed using the Phire Direct Plant in 27.25- · 70-mm vials (FisherBrand, Wal- For very small insects [e.g., Lasiglossum PCR Kit (ThermoFisher Scientific, Waltham, tham, MA) that were held directly above (Dialictus) spp.], the entire body was used MA) and following the manufacturer’s in- insects on flowers. Contact of the collection with a nondestructive approach (i.e., protein- structions. PCR mixtures contained 4 mL vials with flowers was avoided to prevent ase K–mediated in situ dissolution of tissues). GoTaq (nucleotides included), 0.5 mL di- pollen transfer to the vial. Individual insects As a result, all voucher specimens were methyl sulfoxide (DMSO), 3.5 mL sterile were captured in vials rather than with sweep complete in a taxonomic sense. Extracted distilled water, 1 mL forward primer, 1 mL or aerial nets for several reasons. First, only DNA was stored at –20 C until use. Insect reverse primer, and 1 mL DNA. The DNA pollen collected by individual insects would specimen vouchers were retained at the Uni- concentration was not the same for all sam- be present in comparison with catching many versity of Tennessee-Knoxville Department ples; however, the Phire Direct Plant PCR Kit

HORTSCIENCE VOL. 55(12) DECEMBER 2020 1981 Table 1. Degenerate primers developed for cox-1 sequencing of floral visitors to Helianthus verticillatus flowers. Locus Sequence (5# ! 3#)z Positiony Lep FWD ATAATYGGRGGATTTGGWAAYTG 2000 Beetle FWD ATRGTNATRCCNATYATRATYGG 1985 Hym FWD1 ATRATTTTYTTYATRGTWATRCC 1973 Hym FWD2 CAYGCHTTYMTWATRATTTTYTTYAT 1961 Lep REV GTTARTCCNCCYAGWGTRAA 2841 Beetle/Fly REV ACNACATARTAWGTRTCRTG 2901 Hym REV1 ATNGANARWACRTARTGRAARTG 2928 Hym REV2 ATRATTGMRAAWACWGCYCCYAT 2949 Hym REV3 CCTARRAARTGTTGNGGRAARAA 3075 zDegeneracies are as follows: Y = C or T; R = A or G; W = A or T; N = any base; H = A or C or T; M = A or C. yPositions mapped against the Apis mellifera (honeybee) mitochondrial genome (Crozier and Crozier, 1993; GenBank accession number L06178).

allows for this. A positive control consisted greatly, an attempt to normalize counts was the Maryville garden site. Additionally, of H. verticillatus DNA extracted from completed by dividing the pollen counts by species in the family were cap- leaves; DNA from Cornus florida and dis- the mean length of the insect, which served as tured in much higher numbers at this tilled water were used as negative amplifica- a proxy for the specimen size. Finally, the location than at the other two study sites tion controls. Pollen pellets from the 10 most mean number of pollen grains carried on the (Table 2). frequently captured visitors were tested, and bodies of insects was multiplied by the num- There were some collection biases asso- two amplifications were performed using two ber of collected specimens from all sites to ciated with the Maryville location. Plants at expressed sequence tag–simple sequence re- gain an estimate of which insects were po- this location had many more flower heads peat (EST-SSR) primers from Ellis et al. tentially moving the greatest amount of pol- compared with the other two sites, thus (2006): locus HT1099 (forward 5# GGCTT- len. allowing for more comprehensive and ex- TCGTTTCTCGTTGTC and reverse CAGC- haustive sampling. Insects, especially the TCACTCCTAATTGGTTCC) had an expected Results and Discussion various bee species, remember sources of allele size of 302 bp, and locus HT1123 (forward pollen and nectar (Goulson, 1999; Menzel 5#-3# GGGTTTGTACCAGGCACTTG and re- Collection sites. Fifty-six floral visitors and Erber, 1978; Reinhard et al., 2004) and verse TTCATAGAAATGAGGACCAAAGG) were captured at the location in rural Cave may have been more attracted to the more had an expected allele size of 322 bp. Both Spring, GA (Table 2). The number of visitors abundant H. verticillatus flowers at this site EST-SSRs were developed for H. annuus captured increased throughout the morning compared to other nearby resources. Individ- (Ellis et al., 2006) but cross-amplified and mid-day collection periods (0945–1015, ual stems were growing close to each other, DNA of H. verticillatus (Edwards et al., 1145–1215, and 1345–1415 HR), but activity and sunflowers were not surrounded by un- 2020). The thermocycler protocol com- was lower during the late afternoon period derbrush as they were at the Cave Spring, GA prised the following: 95 Cfor3min;10 (1545–1615 HR). The most commonly cap- site. These biases explain the significantly cycles of 94 C for 30 s, 65 Cfor30s, tured visitors were individuals of the B. greater number of visitors captured in the 72 C for 45 s; 30 cycles of 94 Cfor30s, bimaculatus/impatiens complex. Apis melli- garden setting. 55 C for 30 s, 72 Cfor45s,and72Cfor fera was collected more often at the rural At the University of Tennessee Arbore- 5min;andholdat4C. PCR products were location than at any of the other study sites, tum in Oak Ridge, TN (semirural), 191 floral separated by electrophoresis (100 V/cm2 likely due to hobbyist hives observed close to visitors were collected (Table 2) on two for 1 h) on 2% low-melting-point agarose the site. Megachile spp. were collected more dates. At this site, the lowest number of gels, stained with ethidium bromide, and there than at the sites in Tennessee, and captures occurred during the morning periods visualized on an ultraviolet transillumina- Coelioxys spp. were collected only at the of 0745–0815 and 0945–1015 HR, and in- tor. The detection of amplification prod- Cave Spring site (Table 2). creased during the midday and afternoon ucts, visualized as discrete bands in the gels, Collection limitations were associated collection periods of 1145–1215, 1345– was considered positive identification of Hel- with the rural location. The density and dis- 1415, and 1545–1615 HR. The number of ianthus pollen. tance among the plants affected collection captured insects decreased during the even- Pollen counts. Five specimens of each of efficiency. Additionally, thick underbrush ing period of 1745–1815 HR. The most com- the nine most commonly captured visitors, surrounding individual and clustered plants monly collected visitors at this site belonged plus Apis mellifera, were selected to deter- made travel between them slow and difficult. to the B. bimaculatus/impatiens group. mine the number of pollen grains carried on Overall, collection of floral visitors at this Melissodes spp. and Svastra spp. were also insect bodies. Individuals used for this ex- location was more challenging than that at the commonly caught (Table 2). periment were selected at random from the other two sites, and the lower total number of Insect activity around H. verticillatus was entire collection of that taxon regardless of visitors captured may reflect these limita- sparse during the morning collection period location. The following method was used to tions. of 0745–0815 HR, when the temperature was collect pollen from individual specimens and There were 776 floral visitors captured cooler, and slightly increased during the was slightly modified from the methods re- during 4 d of sampling over 2 years at the 0945–1015 HR period at all three locations. ported by Jones (2012). Then, 1 mL distilled suburban Maryville, TN location (Table 2). Helianthus species secrete nectar early in the water was added to the collection vial, and The number of visitors captured was lowest morning (Neff and Simpson, 1990), and the entire insects were placed in 1.5-mL micro- during the morning collection periods (0745– sparse activity and low diversity of visitors centrifuge tubes, vortexed, and centrifuged at 0815 and 0945–1015 HR); however, the num- present (only those foraging for nectar, such 10,000 gn for 8 min; after which, the insects ber of insects captured at the site increased as some Bombus species and syrphid flies) were removed. Pollen grains were resus- during the midday collection periods (1145– were not unexpected. Neff and Simpson pended with agitation, and the number of 1215, 1345–1415, and 1545–1615 HR) and (1990) also determined that H. annuus an- pollen grains per milliliter was estimated decreased during the late afternoon period thers dehisced in the morning and evening, using a hemocytometer (Trigiano, 2010). (1745–1815 HR). The most commonly cap- and that insect visitation coincided with these Each pollen sample was counted five times, tured visitors at this location were members periods. The data from this study agree with and the mean was calculated from these of the B. bimaculatus/impatiens species this observation because insect visitation in- results. Because insect total surface area group and Ceratina calcarata (a carpenter creased during the 0945–1015 HR period, with and surface composition (e.g., hairs) varied bee), which was found almost exclusively at much greater activity in the late morning and

1982 HORTSCIENCE VOL. 55(12) DECEMBER 2020 Table 2. Insect visitors to Helianthus verticillatus flowers at three collection sites during 2017 and 2018. Taxonomic designation Collection site Orderz Family Species Cave Spring, GA Maryville, TN Oak Ridge, TN Total Apidae Bombus bimaculatus/impatiens 11 210 90 311 Cresson Apidae Ceratina calcarata 0 124 1 125 Robertson Halictidae aurata 081586 Smith Halictidae Halictus ligatus 063467 Say Halictidae Agapostemon virescens 052052 Fabr. Apidae Melissodes agilis 2153350 Laberge Halictidae Agapostemon sericeus 046046 Lepeletier Halictidae Lasioglossum/Dialictus spp.y 040343 Halictidae Augochlora pura 022224 Say Apidae Apis mellifera 82717 L. Megachilidae Megachile spp. 12 2 0 14 Ichneumonidae 0 1 7 8 Apidae Svastra aegis 6017 Laberge Apidae Melissodes dentiventris 0156 Smith Apidae Xylocopa virignica 1506 Lepeletier Apidae Svastra obliqua 0145 Say Andrenidae Andrena helianthi 0055 Robertson Andrenidae Andrena asteroides 4004 Laberge Halictidae Augochloropsis sumptuosa 0101 Smith Apidae Bombus fervidus 0101 Fabr. Braconidae 1 0 0 1 Megachilidae Coelioxys asteris 1001 Crawford Megachilidae Coelioxys sayi 1001 Say Scoliidae Scolia dubia 0101 Say Apidae Xylocopa micans 1001 Lepeletier Diptera Syrphidae Allograpta spp. 3 23 5 31 Syrphidae Eupeodes spp. 2 20 1 23 Bombyliidae Sparnopolius spp. 0 13 5 18 Syrphidae Toxomerus spp. 0 4 4 8 Syrphidae Eristalis spp. 0 3 0 3 Dolichopodidae 0 1 0 1 Syrphidae Lepidophora spp. 1 0 0 1 Lepidoptera Hesperiidae Atalopedes campestris 033235 Boisduval Attevidae Atteva aurea 1023 Cramer Coleoptera Cantharidae Chauliognathus pennsylvanicus 0718 DeGeer Chrysomelidae Diabrotica undecimpunctata 1023 Mannerheim Hemiptera Reduviidae Undetermined 0 3 2 5 Dermaptera Undetermined Undetermined 0 1 0 1 zList of visitors is arranged in order of the most frequently collected and abundance. yLasiogllossum and Dialictus species are difficult to differentiate (Gibbs, 2012); therefore, they were considered one group for this study.

at approximately noon (1145–1215 HR). Fur- activity during the later collection periods, creased throughout the day and decreased thermore, the number of visitors that were the number of visitors captured was some- activity as the temperature decreased during captured peaked during the early afternoon times still greater than those of the morning the evening. sampling time of 1345–1415 HR. Activity was collection periods. The observations of our The B. bimaculatus/impatiens group was steady into the late afternoon (1545–1615 study agree with those of Peat and Goulson the most numerous visitor type captured at HR), and then it decreased into the evening (2005), who reported an increase in bumble- all three sites (Table 2). This species group (1745–1815 HR). Despite this decrease in bee foraging behavior as temperature in- was present during all collection periods.

HORTSCIENCE VOL. 55(12) DECEMBER 2020 1983 Hoverflies (Allograpta spp., Eristalis spp., support the findings of other Helianthus Richards, 2010). The greater availability of Eupeodes spp., and Toxomerus spp.) were not species pollinator studies (Chandler and dead pithy stems could explain the higher captured as frequently as Bombus spp.; how- Heilman, 1982; DeGrandi-Hoffmann and abundance of Ceratina visitors at the longer- ever, they were collected throughout most of Watkins, 2000; Parker, 1981; Posey et al., established suburban sunflower plot. Because the collection periods. The second most 1986; Robertson, 1922). of the prescribed burns that take place at the abundant visitor, C. calcarata, was captured Nonhymenopteran visitors, such as Allog- Cave Spring location, there are fewer dead almost exclusively at the suburban setting, rapta spp. (Diptera: Syrphidae), Atalopedes stems, which explains the lack of Ceratina but only during the 1145–1215 and 1545– campestris (Lepidoptera: Hesperiidae), and visitors at this site. The Oak Ridge location, 1615 HR periods. Augochlorini spp., H. liga- Sparnopolius spp. (Diptera: Bombyliidae), established in late 2017, was planted much tus, Melissodes spp., and others were active carried a relatively low number of pollen more recently than the Maryville location, during the afternoon collection periods at all grains compared with bees (Table 3). Mem- and there were very few closely adjacent locations as well. bers of Apidae (Cresswell, 1999) and Syr- potential nesting sites for Ceratina spp. Visitor abundance does not guarantee phidae seek both nectar and pollen rewards The Cave Spring location had the highest pollination efficiency (Horsburgh et al., (Gilbert, 1981; Kastinger and Weber, 2001). overall diversity of captured visitors but a 2011); however, together with the amount However, syrphid flies primarily use pollen smaller sample size compared with the other of pollen carried by individuals, this may be a as a food source (Horsburgh et al., 2011). In two sites. The Maryville location had the better indicator of pollination potential. Five another study (Gilbert 1981), it was reported lowest diversity of captured visitors; how- specimens of each of the nine most com- that smaller syrphid flies carried less pollen ever, more insects were collected at this site monly collected species or species groups than other pollinators, and that syrphid flies than at the other two collection sites. Despite were selected randomly to estimate the pollen often cleaned pollen from their bodies. Mem- having the highest number of unique individ- load carried, which was expressed as pollen bers of Hesperiidae do consume pollen, but uals captured, a few visitors were collected in grains per millimeter of length for the spec- their primary food is nectar (Gilbert and greater numbers during the collection at imen (Table 3). Including the length to ex- Singer, 1975; Pivnick and McNeil, 1985). Maryville, thus reducing the relative diver- press the pollen load was an attempt to Helianthus pollen was morphologically sity of captured visitors. standardize the data presentation. Addition- identified when counting pollen grains. Sun- At the Maryville location, the second ally, A. mellifera was included because of its flower pollen was overwhelmingly present in collection during late September was more prevalent use as a pollinator of sunflowers in all samples, and some insects contained pol- diverse than the first collection during mid- agricultural settings (Levin, 1983). Dialictus len from other plant species (Echinacea spe- September. This result can be explained by spp. had the greatest mean number of pollen cies and other composite species were often the larger proportion of Agapostemon spp. grains per millimeter of specimen, followed nearby, but they were not identified). Heli- and B. bimaculatus/impatiens group mem- by H. ligatus and members of the B. bima- anthus pollen was also detected using H. bers that were present. Bombus species ex- culatus/impatiens group (Table 3). Of the annuus SSRs, and it is possible that DNA of perience population peaks in the fall, which insects observed visiting H. annuus, female pollen from other local asteraceous plants, could explain their overall abundance in this Melissodes spp. and Bombus spp. were re- such as H. tuberosum and E. purpurea, was study (Neff and Simpson, 1990). Other ported to carry the most pollen grains (Parker weakly amplified by H. annuus primers (R.N. Helianthus pollination studies reported that 1981). Melissodes and Halictus species were Trigiano, unpublished data). Agapostemon spp. were frequent visitors reported as pollinators of other Helianthus Thirty-six insect visitors (25 Hymenop- (Chandler and Heilman, 1982; Posey et al., spp. (DeGrandi-Hoffman and Watkins, 2000; tera, 7 Diptera, 2 Lepidoptera, and 2 other 1986), and this was supported by our study. Robertson, 1922), as was Agapostemon spp. orders) were captured during the two collec- At the Oak Ridge location, the first collection (Chandler and Heilman, 1982; Posey et al., tion seasons across all locations (Table 2). occurred in late September, and that timing 1986). Honeybees carried fewer pollen grains Visitors in the B. bimaculatus/impatiens yielded a much higher floral visitor diversity than most of the native bees recorded by group were present at all locations and during than that observed during the second collec- previously mentioned sunflower studies. In all collection periods (data not shown). Cera- tion period, which occurred in early October. our study, when a measure of pollen-carrying tina calcarata was the second most com- Both collection dates were late in the flower- ability was expressed as insect abundance monly captured visitor in this study, but it ing period for H. verticillatus (Matthews (collected across the three study sites) · mean was found almost entirely at the Maryville or et al., 2002). Furthermore, temperatures on number of pollen grains/length of the insect suburban location. Because this site is in a both collection dates were cool and overcast (Table 3), B. bimaculatus/impatiens, fol- residential garden where the plants have been (data not shown), which were less favorable lowed by H. ligatus, Agapostemon spp., and established since 2014, there are numerous for pollinator activity (Peat and Goulson, Lasioglosum/Dialictus spp., had the potential dead stems, which Ceratina species use for 2005). to disperse more pollen than the other col- nesting; however, they were absent at the Oak Hymenoptera genera (Bombus and Meliss- lected species. Our results are similar to and Ridge and Cave Spring sites (Rehan and odes) and Diptera families (Bombyliidae,

Table 3. Counts of Helianthus verticillatus pollen recovered from the taxa representing the most collected insect visitors and Apis mellifera at the three collection sites. Length of the Mean (range) of Mean pollen count per No. of visitors No. of visitors · mean Insect visitorz visitor (mm) pollen counts ·104y mm insect visitor ·104 collected at all sites (Table 2) pollen count per mm of insect Agapostemon spp. (2) 10–11 4.7 (0.2–11.8) 0.44 98 43.1 Allograpta spp. 7–8 0.3 (0.2–0.4) 0.04 31 1.2 Apis mellifera 12 1.3 (0.6–2) 0.11 17 1.9 Augochlorini spp. (3) 6–8 0.74 (0.4–1.8) 0.11 111 12.2 Bombus bimaculatus/impatiens 9–16 11.0 (1.2–44.6) 0.90 311 279.9 Ceratina calcarata 6.5–8 1.0 (0.4–1.6) 0.14 125 17.5 Halictus ligatus 7–10 15.9 (4.6–25.8) 1.90 67 127.3 Hesperiidae 12–15 0.12 (0–2.0) 0.01 35 0.35 Lasioglossum/Dialictus spp. <5–10 1.4 (0.2–2.4) 0.48 43 20.6 Melissodes spp. (2) 6–9 0.4 (0–1.0) 0.05 56 2.8 zNumber in parentheses indicates the number of species. yFrom five randomly selected specimens.

1984 HORTSCIENCE VOL. 55(12) DECEMBER 2020 Syrphidae) were the most abundant floral States. Front. Genet. 11, doi: 10.3389/fgene. Kevan, P.G. and H.G. Baker. 1983. Insects as visitors to H. verticillatus. Bombus, Hal- 2020. flower vectors and pollinators [Foraging ictus,andLasioglossum species carried Edwards, T.P., R.N. Trigiano, P.A. Wadl, B.H. behavior, nectar secretion, pollination the highest number of Helianthus pollen Ownley, A.S. Windham, and D. Hadziabdic. ecology]. Annu. Rev. Entomol. 28:407– grains and most likely represent important 2017. First report of Alternaria alternata caus- 453, doi: 10.1146/annurev.en.28.010183. ing leaf spot on whorled sunflower (Helianthus 002203. pollinators of this sunflower species. Other verticillatus) in the southeast United States. Levin, M. 1983. Value of bee pollination to US genera in the Halictidae family (Agapos- Plant Dis. 101:632, doi: 10.1094/PDIS-08-16- agriculture. Amer. Entomol. 29:50–51, doi: temon spp., Augochlora spp., and Augo- 1216-PDN. 10.1093/besa/29.4.50. chlorella spp.) were also frequent visitors Ellis,J.R.,C.H.Pashley,J.M.Burke,andD.E. Mandel, J.R. 2010. Clonal diversity, spatial dy- to H. verticillatus inflorescences; how- McCauley. 2006. High genetic diversity in a namics, and small genetic population size in the ever, these visitors carried only limited rare and endangered sunflower as compared rare sunflower, Helianthus verticillatus. Con- numbers of pollen grains. Native bee pol- to a common congener. Molec. Ecol. 15: serv. Genet. 11:2055–2059, doi: 10.1007/ linators (DeGrandi-Hoffman and Watkins, 2345–2355, doi: 10.1111/j.1365-294X.2006. s10592-010-0062-3. 2000; Robertson 1922), rather than the 02937.x. Matthews, J.F., J.R. Allison, R.T. Ware, Sr., and C. exotic A. mellifera, are likely more effi- Faegri, K. and V.D.P. Leendert. 1966. The princi- Nordman. 2002. Helianthus verticillatus Small ples of pollination ecology. Toronto, New (Asteraceae) rediscovered and redescribed. cient pollinators of H. annuus (Parker, York, Toronto, New York, Pergamon Press. Castanea 67:13–24, https://www.jstor.org/sta- 1981), and our findings indicate that this https://books.google.com/books?hl=en&lr=&id= ble/4034312. is probably also true for H. verticillatus. 3zfLBAAAQBAJ&oi=fnd&pg=PP1&ots=3tyLhB- McAlpine, J.F., B.V. Peterson, G.E. Shewell, H.J. Despite some differences in species zGv&sig=42cW1TbKrvm5USiOD5H7OT-itok#v= Teskey, J.R. Vockeroth, and D.M. Wood. 1981. composition, the relative diversity of floral onepage&q&f=false. Manual of nearctic diptera. vol. 1. https://www. visitors from all three locations was simi- Free, J.B. 1970. Insect pollination of crops. Academic cabdirect.org/cabdirect/abstract/19810582668. lar. Helianthus verticillatus attracts a wide Press, London, New York. https://www.cabdirect. McGregor, S.E. 1976. Insect pollination of culti- range of insect visitors regardless of its org/cabdirect/abstract/19710305447. vated crop plants. Agricultural handbook no. location, and species-specific composition Gibbs, J. 2012. Revision of the metallic Lasio- 496, Agricultural Research Service, U.S. De- appears to be dependent on the location. glossum (Dialictus) of eastern North Amer- partment of Agriculture.

HORTSCIENCE VOL. 55(12) DECEMBER 2020 1985 Simuliidae), with three new fast-evolving nu- and orchardgrass leaves, p. 365–371. In: R.N. U.S. Fish and Wildlife Service. 2014. Endangered clear genes for phylogenetic inference. Molec. Trigiano and D.J. Gray (eds.). Plant tissue status for Physaria globosa (Short’s Bladder- Phylogenet. Evol 75:138–148, doi: 10.1016/ culture, development, and biotechnology. CRC pod), Helianthus verticillatus (whorled sun- j.ympev.2014.02.018. Press, Boca Raton, FL. flower), and (Fleshy- Small, J.K. 1898. Studies in the botany of the Trigiano, R.N., S.B. Wilson, and C.N. Steppe. Fruit Gladecress). 79:44712–44718. .

1986 HORTSCIENCE VOL. 55(12) DECEMBER 2020