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Home , Alycaulini, Astereae, Asteromyia, Brintonia, Chrysoma, Chrysothamnus

Southern Illinois University Carbondale OpenSIUC

Honors Theses University Honors Program

December 2017 Phylogenetics of (: ) and euthamiae (: ) Marisa Szubryt [email protected]

Follow this and additional works at: http://opensiuc.lib.siu.edu/uhp_theses

Recommended Citation Szubryt, Marisa, "Phylogenetics of Euthamia (Asteraceae: Astereae) and Asteromyia euthamiae (Cecidomyiidae: Alycaulini)" (2017). Honors Theses. 436. http://opensiuc.lib.siu.edu/uhp_theses/436

This Dissertation/Thesis is brought to you for free and open access by the University Honors Program at OpenSIUC. It has been accepted for inclusion in Honors Theses by an authorized administrator of OpenSIUC. For more information, please contact [email protected]. Phylogenetics of Euthamia (Asteraceae: Astereae) and Asteromyia euthamiae (Cecidomyiidae: Alycaulini) Marisa Blake Szubryt

A thesis submitted to the University Honors Program in partial fulfillment of the requirements for the Honors Diploma

Southern Illinois University 31 October 2017

Acknowledgments

I would like foremost to thank my primary advisor, Dr. Kurt Neubig, for his guidance in this project and aiding with my intellectual development these past two years. Our collaborator,

Dr. Lowell Urbatsch, at State University, has been instrumental in this project's success and has spent countless hours examining Euthamia specimens. Others who have indirectly assisted in this project, including Drs. Pamela Weisenhorn and Nancy Garwood, also deserve considerable thanks. The many botanical collectors, multiple cooperative herbarium curators (Louisiana State University Shirley Tucker Herbarium, Southern Illinois University

Carbondale Herbarium, New York Botanical Garden Herbarium, University of

Herbarium, Michigan State University Herbarium), and funding sources (Southern Illinois

University, Botanical Society of America, National Science Foundation) across the country have made this study possible. I lastly owe thanks to my departmental colleagues, my academic siblings, and some genuinely wonderful friends for sharing their perspectives and providing support over this past year in particular.

Introduction

The field of systematics encompasses evolution, biodiversity, and studies

(Bremer and Wanntorp 1978; Hodkinson and Pamell 2006; Stuessy 2009) and provides a means of identifying species and broader taxonomic groups. Because evolution is the fundamental process of change through time, phylogenetic relationships are instrumental to explaining many biological phenomena (Resh and Unzicker 1975; Tautz et al. 2003).

Taxonomic organization (i.e., classification) of organisms may convey descriptive information about important features of their morphology (Heiser et al. 1963), ecological niche partitioning

(Grace and Wetzel 1981), or biochemical characteristics (Giannasi 1978) which relate evolutionary relationships to discernable phenotypes and ecological relationships (Ferris and

Ferris 1989; Munstermann and Conn 1997).

Understanding organismal relationships can further explain symbiotic associations with pathogens or susceptibility to specific toxins, which may have agronomic and economic utility, for example, regarding herbicide susceptibility and disease transmission (Farrell and Mitter

1994; Pemberton 2000; Rausher 2001; Westerdahl and Getsinger 1988). Studies on genera or more inclusive taxa provide insight into evolution with biogeographic changes and symbionts, which have been economically important in industries such as timber production (Hadley and

Veblen 1993; Orwig et al. 2002; Richardson and Rajmanek 2004). Many taxa have specialized symbiotic associations (Kennedy et al. 2011; Pawson et al. 2010; Pelser et al. 2016; Thorogood et al. 2009) and specific niches (Gabriel and Bates 2005); this information helps evaluate ecological implications and possible conservation concerns of particular organisms (Behling and

Pillar 2007; Griffiths et al. 2005; Swarts and Dixon 2009)

Although closely related organisms appear similar and frequently share symbiont relationships, these groups do not evolve together. Rather, species are the only unit subjected to natural selection (Simpson 1951; Wiley 1978) although similar, specific adaptations may occur across different species and appear coordinated. Closely related species frequently occupy ecological niches and ranges which favor slightly different morphological and biochemical features (Harper et al. 1961; Losos 2008; Wiens 2004) which may be utilized for identification purposes (Liu et al. 2011; Okuyama and Kato 2009; Reznicek 1990).

Many taxonomic groups based on particular morphological or anatomical characters may not represent phylogenetic history, however. Since taxonomists may begin without knowing which characters are evolutionarily significant, taxonomic groups may be poorly circumscribed initially (Nesom 2007; Olmstead et al. 2001). Different features carry importance for different groups, and what is systematically informative in one circumstance may be uninformative for others. Furthermore, variable environmental conditions may further confound species delimitation because of the accompanying complexity of morphological information (Slova´k et al. 2012; Zouhar 2009). Groups with structurally unambiguous morphological synapomorphies can be circumscribed with certainty, but many lineages lack such clear and easily observed characters. When morphological features do not provide clear resolution, numerous forms of molecular data can aid in determining evolutionary relationships and taxonomic classifications are based on those relationships (Despres et al. 2002; Giannasi 1978; Vaezi and Vrouillet

2009). Morphologically ambiguous groups that have been clarified using DNA data include many genera in the sunflower family Asteraceae (Markos and Baldwin 2001; Roberts 2002;

Roberts and Urbatsch 2003; Roberts and Urbatsch 2004; Suh and Simpson 1990; Urbatsch et al. 2000; Urbatsch et al. 2003). Such research provides crucial information regarding biogeographic ranges of these taxa and ecosystem interactions including the potential for uncovering symbiont specificity (Stireman III et al. 2010). Many genera relevant to this study, such as , , and , have had infrageneric relationships established using molecular DNA data, but relationships within other genera are still unresolved.

The Euthamia represents one such group without adequate phylogenetic study.

Although it superficially resembles and species of both may co-occur in mesic fields (Abrahamson et al. 2005), Euthamia was uncertainly placed as a subgenus in Solidago (Nuttall

1818) before being recognized as a distinct genus (Cassini 1825; Creech 1973; Urbatsch et al.

2003). This separation is supported by phylogenetic analysis of DNA data because Euthamia is not closely related to Solidago (Urbatsch et al. 2003). While numerous floristic treatments are available and taxonomic revisions exist for Euthamia, there are considerable disagreements regarding which taxa are biologically real species and what the ranges of these entities are

(Ebringer et al. 2010; Friesner 1933; Nuttall 1818; Sieren 1981). Widespread species such as

Euthamia graminifolia or E. caroliniana have numerous synonyms and have been inconsistently classified (Johnson 1995); DNA data, which is largely absent in Euthamia, may provide clarity to taxonomic treatments (Urbatsch et al. 2003). The first purpose of this study was to determine the monophyly of Euthamia, to circumscribe species in Euthamia using phylogenetic data, and to determine the native ranges of these taxa.

The second purpose of this study was to assess host (Euthamia) to parasite

(Asteromyia) specificity using phylogenetic tools. Asteromyia euthamiae, commonly known as

“gall midges”, live specifically on Euthamia . All Asteromyia utilize a fungal host,

Botryosphaeria dothidea, upon which they feed and live during the larval stage; the fungus gains sustenance through consuming living tissues and comprises the “gall” structure

(Heath and Stireman III 2010; Janson et al. 2010). Other North American Astereae, closely related to Euthamia, including the genera Chrysothamnus, , , ,

Gutierrezia, and Solidago, host other species of gall midges (Stireman III et al. 2010). Most midge species have a high level of host-specificity at the generic level of the host.

Because closely related midges occupy specific niches on closely related plant hosts, we considered that the level of host-parasite specificity may exist at even finer levels (Stireman III et al. 2010).

Materials and Methods

Sampling - Representative Euthamia species described by Sieren in 1981, primarily in the Great Lakes and southeast coast regions, were selected for analyses. Samples were either field collected and dried in silica (Chase and Hills 1991) or removed from herbarium specimens with permission (Table 1). All Asteromyia samples were taken directly from galls on herbarium specimens, although some data included in this study were taken from GenBank (Table 2).

Table 1. Voucher information for sampled Euthamia. “Collection ID” refers to voucher information; “Euthamia Taxon” refers to the identified species. “Accession number” refers to the herbarium accession number, and “Locality” refers to the state and county/parish the specimen was collected. Two specimens have not yet been accessed and placed in a herbarium. Collection ID Euthamia Taxon Accession number Locality Gilmore 3457 E. gymnospermoides LSU00014452 LA, Calcasieu Heineke 3801 E. graminifolia SIU000105117 IL, Jackson Abbott 26919 E. caroliniana NA NA Urbatsch 10098 E. scabra LSU00132419 MS, Hancock Urbatsch 10364 E. graminifolia LSU00131848 , Quebec Urbatsch 10827 E. caroliniana LSU00134814 FL, Walton Urbatsch 10473-1 E. gymnospermoides LSU00131989 TX, Urbatsch 10757 E. scabra LSU00183284 LA, Tangipahoa Urbatsch 10790 E. scabra LSU00183771 LA, Livingston Urbatsch 10809 E. gymnospermoides LSU00188025 LA, Acadia Urbatsch 10818 E. gymnospermoides LSU00134804 VA, Sussex Urbatsch 11019 E. graminifolia LSU00135092 VA, Prince George Urbatsch 11040 E. gymnospermoides LSU00135115 AL, Mobile Urbatsch 11123 E. gymnospermoides LSU00137575 WI, Sauk Urbatsch 11135 E. graminifolia LSU00137587 IN, Pulaski Urbatsch 11212 E. leptocephala LSU00137674 TX, Jasper Urbatsch 11219 E. gymnospermoides LSU00137681 TX, Newton Urbatsch 11220 E. graminifolia LSU00137682 NH, Grafton Urbatsch 11228 E. scabra LSU00176921 MS, Harrison Urbatsch 11231 E. scabra LSU00190444 AL, Baldwin Urbatsch 11232 E. hirtipes LSU00176946 FL, Wakulla Urbatsch 11234 E. hirtipes LSU00190447 FL, Wakulla Urbatsch 11235 E. scabra LSU00176961 FL, Wakulla Urbatsch 11236 E. scabra LSU00090451 FL, Wakulla Urbatsch 11253 E. caroliniana LSU00176938 FL, Taylor Urbatsch 11255 E. scabra LSU00176948 FL, Taylor Urbatsch 11257 E. hirtipes LSU00190472 FL, Taylor Urbatsch 11258 E. hirtipes LSU00190473 FL, Taylor Urbatsch 11278 E. scabra FLA00176960 FL, Bay Urbatsch 11283 E. scabra LSU00177102 LA, St. Tammany Urbatsch 11285-08 E. caroliniana LSU00177261 LA, St. Tammany Urbatsch 11285-09 E. caroliniana LSU00177262 LA, St. Tammany Urbatsch 11285-17 E. caroliniana LSU00177263 LA, St. Tammany Urbatsch 11287-14 E. scabra LSU00177266 LA, St. Tammany Urbatsch 11305 E. hirtipes LSU00176664 FL, Charlotte Urbatsch 11306 E. hirtipes LSU00177665 FL, Charlotte Urbatsch 11309 E. hirtipes LSU00176680 FL, Charlotte Urbatsch 11326 E. scabra LSU00177309 LA, Urbatsch 7724 E. occidentalis LSU00061862 CA, Inyo Urbatschdna951 E. leptocephala LSU00131815 LA, Calcasieu Reid 9000 E. gymnospermoides LSU00139903 TX, Calhoun Reid 9045 E. gymnospermoides LSU00139902 TX, Calhoun Reid 9058 E. gymnospermoides LSU00139901 TX, Calhoun Reznicek 9214 E. gymnospermoides MICH1217652 Canada, Windsor Utech 80-153 E. graminifolia SIU00094076 PN, Westmoreland Urbatsch 11561 E. leptocephala LSU00179139 LA, Tangipahoa Urbatsch 11570 E. scabra LSU00179161 LA, Tangipahoa Urbatsch 10799 E. scabra LSU00132516 LA, Livingston Urbatsch 11223 E. scabra LSU00137715 LA, St. Tammany Urbatsch 11030 E. caroliniana LSU00135103 SC, Horry Urbatsch 10744 E. caroliniana LSU00132437 MS, Hancock Urbatsch 11035 E. caroliniana LSU00135110 GA, Ware Urbatsch 10825 E. caroliniana LSU00134812 FL, Bay Urbatsch 10772 E. caroliniana LSU00132473 MS, Hancock Urbatsch 10761 E. leptocephala LSU00132454 LA, Tangipahoa Szubryt 6 E. graminifolia LSU00179184 IL, Will Urbatsch 10974 E. caroliniana LSU00179332 LA, Washington Urbatsch 10766 E. caroliniana LSU00132467 LA, St. Tammany Urbatsch 10771 E. caroliniana LSU00132472 MS, Hancock Urbatsch 10737 E. caroliniana LSU00132430 MS, Hancock Urbatsch 10824 E. caroliniana LSU00134811 LA, St. Tammany Urbatsch 10766 E. caroliniana LSU00132467 LA, St. Tammany Urbatsch 10781 E. scabra LSU00132488 LA, Washington Urbatsch 10758 E. scabra LSU00132451 LA, Tangipahoa Urbatsch 10761 E. leptocephala LSU00132454 LA, Tangipahoa Urbatsch 11041 E. scabra LSU00135116 AL, Mobile SH6 s.n. E. hirtipes NA MS Urbatsch 10819 E. leptocephala LSU00134805 LA, Acadia Urbatsch 7896 Gutierrezia sarothrae LSU00191714 NM, Socorro

Table 2. Asteromyia cytochrome oxidase I DNA data from GenBank. “GenBank Accession”, “Identified Taxon”, “Host Taxon”, and “State” are listed and refer to their source, Asteromyia species, host species, and collection locality when applicable, respectively. GenBank Accession Identified Taxon Host Taxon State EU439828.1 A. carbonifera Solidago altissima MA FJ803322.1 A. modesta Solidago altissima OH FJ803284.1 A. laeviana Symphyotricum urophyllum NY FJ803338.1 A. chrysothamni Chrysothamnus sp. NM FJ803289.1 A. gutierreziae Gutierrezia sarothrae NM FJ803290.1 A. gutierreziae Gutierrezia sarothrae NM FJ803307.1 A. gutierreziae Gutierrezia sarothrae TX FJ803316.1 A. gutierreziae Gutierrezia sp. NM FJ803319.1 A. gutierreziae Gutierrezia sp. NM FJ803320.1 A. gutierreziae Gutierrezia sp. NM FJ803331.1 A. gutierreziae Gutierrezia sarothrae TX FJ803334.1 A. gutierreziae Gutierrezia sarothrae TX FJ803339.1 A. gutierreziae Gutierrezia sarothrae TX FJ803352.1 A. gutierreziae Gutierrezia sarothrae UT FJ803353.1 A. gutierreziae Gutierrezia sarothrae UT FJ803354.1 A. gutierreziae Gutierrezia sp. UT DQ241865.1 A. euthamiae Euthamia sp. NA EU439782.1 A. euthamiae NY EU439810.1 A. euthamiae Euthamia graminifolia MA FJ803279.1 A. euthamiae Euthamia graminifolia ME FJ803296.1 A. euthamiae Euthamia graminifolia Canada FJ803310.1 A. euthamiae Euthamia graminifolia OH FJ803323.1 A. euthamiae Euthamia graminifolia IN FJ803324.1 A. euthamiae Euthamia graminifolia IN FJ803325.1 A. euthamiae Euthamia graminifolia IN KC166219.1 willistoni None, outgroup NA

DNA extraction, PCR, and sequencing - Dried tissues were ground to a fine powder using a bead mill and had DNA extracted using a modified CTAB method (Doyle and Doyle

1987) where the aqueous supernatant from the chloroform/isoamyl alcohol purification step was further purified using a silica column to remove any impurities (Neubig et al. 2014); the resulting

DNA was stored in 100 μl of Tris-EDTA buffer. Euthamia DNA samples were amplified for the nuclear ribosomal regions ITS and ETS (internal and external transcribed spacers, respectively). The primers Y5 and Y4 were utilized for ITS amplification; ETS-B and 16S-IGS primers were used to amplify ETS (Table 3). The GoTaq reagent kit (Promega) used included 14.5 μl water, 5.0 μl 5x GoTaq Buffer, 2.0 μl 25mM Mg, 0.5 μl dNTPs, 0.5 μl forward primer, 0.5

μl reverse primer, 0.15 μl Taq polymerase, and 1.0 μl of template DNA. Both utilized a program for 120 seconds at 98°C followed by 35 cycles of 95°C for 15 seconds, 55°C for 15 seconds, and 72°C for 60 seconds. The program terminated in two minutes of 72°C and then a holding temperature at 8°C. Asteromyia DNA samples were amplified for the mitochondrial cytochrome oxidase I (COI) subunit using the primers HCO2198 and LCO1498 (Table 3) and the same concentrations of aforementioned reagents. The same program was used for Asteromyia amplification but with an annealing temperature at 40°C, in accordance with previous work

(Geller et al. 2014). PCR productions were run electorphoretically on 1% agarose gels to confirm amplification.

The PCR products were diluted to approximately equal levels of DNA concentration by visual approximations and Sanger sequenced at the Eurofins Genomics sequencing facility in

Louisville, KY on and ABI3730 capillary sequencer. Ab1 files of forward and reverse reads were edited in Geneious version R10 and trimmed based on quality (Kearse et al. 2012).

Polymorphisms and inaccurately designated nucleotide bases were manually detected and corrected.

Table 3. Primers used for Euthamia ITS, Euthamia ETS, and Asteromyia COI. “Primer Name” includes the original name of the primer used from another paper with the region it is amplifying in parenthetical adjacent to it. “Sequence” refers to the actual nucleotide composition of the primers; “Source” indicates where the original primer was described. Primer Name Sequence Source Y5 (ITS) 5’- TAG AGG AAG GAG AAG TCG TAA (Hoshi et al. 2008) CAA -3’ Y4 (ITS) 5’- CCC GCC TGA CCT GGG GTC GC -3’ (Hoshi et al. 2008) 18S-IGS (ETS) 5’- GAG ACA AGC ATA TGA CTA CTG (Baldwin and Markos GCA GGA T -3’ 1988) ETS-B (ETS) 5’- ATA GAG CGC GTG AGT GGT G -3’ (Beardsley and Olmstead 2002) HCO2198 (COI) 5’- TAA ACT TCA GGG TGA CCA AAA (Geller et al. 2014) AAT CA -3’ LCO1490 (COI) 5’- GGT CAA CAA ATC ATA AAG ATA (Geller et al. 2014) TTG G -3’

Data alignment and phylogenetic tree construction - Sequences were exported as fasta files and aligned using Muscle (Edgar 2004; Galtier et al. 1996) Parsimony searches with the tree bisection reconnection (TBR) model were made using the heuristic search method utilizing simple stepwise additions with the closely related Gutierrezia sarothrae as an outgroup

(Urbatsch et al. 2003) in PAUP* (Swofford 2003). Bootstrap support values (Felsenstein 1985) were estimated from heuristic searches using the TBR model where up to one tree with a score of ten or more was saved, for 100 random-addition replicate trees with a TBR model.

Results

Euthamia taxa were partially resolved by ITS, ETS, and combined phylogenetic analysis

(Figs. 1-3). Nuclear ribosomal ITS alignments consisted of 662 base pairs while ETS alignments were 535 base pairs in length. ITS and ETS analysis resulted in trees of 62 and 52 steps, respectively; the concatenated phylogram had 84 steps. Analysis of each dataset consistently indicated that the following form clades: Euthamia hirtipes, E. leptocephala, and E. graminifolia

+ caroliniana. The monophyly of E. occidentalis could not be assessed because only one specimen was sampled and a + scabra clade was only supported by bootstrap analyses in the ITS (68%) and concatenated dataset (84%).

The Euthamia graminifolia + caroliniana complex forms a well-supported clade (87%,

83%, 100% for ITS, ETS, and concatenated trees respectively); only one poorly supported group of E. graminifolia (68% in ITS, 82% in combined) emerged but did not include all E. graminifolia specimens. The combined ITS and ETS dataset provided the best overall support of relationships. Concatenated trees produced better support for some Euthamia graminifolia

(82%) and some (67%) groups, but this reciprocal monophyly did not include each specimen. Bootstrap support for the Euthamia gymnospermoides + scabra complex was 68% in the

ITS analysis, below 50% in the ETS analysis, and 84% in the concatenated bootstrap analysis.

Conspicuous subclades were not present in either ITS or ETS trees, and the two poorly supported clades in the concatenated trees emerged without including all taxa (62% for a predominantly Euthamia scabra group and under 50% for a Euthamia gymnospermoides group).

Euthamia leptocephala, essentially endemic to eastern Texas, Louisiana, and southern

Arkansas, was genetically distinct and strongly supported by bootstrap values in all trees.

Euthamia occidentalis, was similarly distinctive from other species in all trees and is most likely sister to the strictly eastern Euthamia graminifolia + caroliniana complex. Euthamia hirtipes, found along the eastern coast, was monophyletic in every tree with 59%, 96%, and 98% bootstrap support in ITS, ETS, and concatenated trees, respectively, and was consistently sister to all other Euthamia taxa.

The Asteromyia COI cladogram resulted in trees of 113 steps (Fig. 4). The topology showed a paraphyletic grade of Asteromyia euthamiae with respect to monophyletic A. gutierreziae. However, the bootstrap analysis supported neither the paraphyly nor monophyly of

A. euthamiae. The Asteromyia gutierreziae clade was well supported (96% bootstrap), and one unique midge from in Louisiana was sister to the A. gutierreziae clade, albeit with poor bootstrap support (<50%). Multiple A. euthamiae clades were strongly supported: one specifically associated with Euthamia graminifolia (99%) while another (97%) contained Euthamia caroliniana and E. scabra subclades, both well-supported (78% and 97%, respectively). This Euthamia caroliniana + scabra host clade was separate from the remaining groups in parsimony analyses. Another, albeit poorly supported, clade associated with Euthamia caroliniana emerged as genetically distinct. A remaining clade emerged, consisting of one subclade associated with Euthamia graminifolia and another with E. gymnospermoides, E. scabra, E. hirtipes, and E. leptocephala, which collectively bore 64% bootstrap support.

Figure 1. Parsimony phylogram based on ITS data for Euthamia, with branch lengths and parsimony bootstrap support percentages above and below the branches, respectively. Note the poor resolution within the Euthamia graminifolia + caroliniana and E. gymnospermoides + scabra clades.

Figure 2. Parsimony phylogram based on ETS data for Euthamia, with branch lengths and parsimony bootstrap support percentages above and below the branches, respectively. Reciprocal monophyly is unsupported in the Euthamia graminifolia + caroliniana and E. gymnospermoides + scabra clades.

Figure 3. Euthamia concatenated ITS and ETS parsimony phylogram with branch lengths and parsimony bootstrap support percentages above and below the branches, respectively. Reciprocal monophyly is unsupported in the Euthamia graminifolia + caroliniana and E. gymnospermoides + scabra clades.

Figure 4. Asteromyia COI parsimony phylogram with branch lengths and parsimony bootstrap percentages above and below the branches, respectively. Midge (Asteromyia) name, identification number, collector name, collector number, host Euthamia, state locality, and county locality are listed for each specimen, if applicable. Outgroups and additional Asteromyia sampling from GenBank are listed in Table 2 (see Stireman III et al. 2010). Discussion

Euthamia phylogenetics - The currently recognized and accepted Euthamia species, according to the U.S. Department of Agriculture, are Euthamia graminifolia, E. caroliniana, E. gymnospermoides, E. leptocephala, E. galetorum, and E. occidentalis (USDA 2017). Our phylogenetic analyses do not perfectly match that taxonomic scheme. In particular, our data highlight two difficult to resolve lineages within the genus: E. gymnospermoides + scabra and E. graminifolia + caroliniana. These are both well-supported clades, but the species within are poorly differentiated by parsimony and bootstrap searches using nuclear ribosomal internal and external transcribed spacer data. Additional sampling of E. gymnospermoides, E. scabra, E. graminifolia, and E. caroliniana may distinguish each species more effectively, but their lack of reciprocal monophyly is either biologically real, or alternatively caused by low sequence divergence.

Euthamia graminifolia and E. caroliniana are easily distinguished morphologically and overlap minimally in their native ranges. Conspicuous differences in leaf size, pubescence, resin pit content, and capitula characteristics have led to their separation as distinct species by most authors (Johnson and M. 1995; Semple et al. 1984; Sieren 1981). The same has not been true for Euthamia scabra, which was mostly neglected after its description (Greene 1902).

Subsequently, its potentially emerging monophyly (although poorly supported) and morphological distinctness, characterized principally by pubescent abaxial veins and young stems, was surprising. We favor the recognition of E. scabra, despite the poor phylogenetic resolution, because of its morphological distinctiveness compared to its closest relative, E. gymnospermoides.

One additional taxon, E. hirtipes, which has largely been ignored in taxonomic literature, is monophyletic. When E. hirtipes was originally described, it was suggested to be a hybrid between E. graminifolia and E. microcephala (synonym of E. tenuifolia sensu Sieren 1981, and a synonym of E. caroliniana, herein). However, our data of E. hirtipes are not consistent with hybridization. Few authors have recognized its morphological distinctness and accurate geographic distribution (Barger et al. 2013). Our data indicate that E. hirtipes occurs along the east coast, from to New Jersey; both E. graminifolia and E. caroliniana can occur within the same states along the east coast (chiefly Virginia and North Carolina). However, E. hirtipes contains small and sparse translucent dots on its leaves, a feature only shared with E. leptocephala which has more conspicuous dots.

Other putative Euthamia species, chiefly E. remota and E. galetorum, have not been addressed in this thesis and remain evolutionarily ambiguous despite morphological distinctions.

Wide ranging taxa like Euthamia graminifolia, E. caroliniana, and E. gymnospermoides have been variously split up previously into different species, but our morphological assessments of such herbarium specimens could not find sufficient justification for many of these. Specimens of

Euthamia microcephala, E. minor, and E. tenuifolia fall within the standard morphological range of E. caroliniana. Euthamia pulverulenta appear to be thinner-leaved variants of E. gymnospermoides in the western part of its range, but the variability of leaf size within likely indicates that they are regional acclimations to drier habitats rather than biologically significant characteristics. Euthamia remota (Bush 1918; Friesner 1933; Heimlich 1921; Hill 2003) and E. galetorum (McJannet et al. 1995) are morphologically distinct and geographically specific, but their monophyly and relationships to other species has not been evaluated phylogenetically yet.

Nuclear ribosomal internal and external transcribed spacer (ITS and ETS) regions may provide sufficient resolution for differentiating some Euthamia species. However, low sequence divergence inherently indicates that these data alone will likely be inadequate to resolve all relationships; other DNA data may disentangle relationships more successfully. Chloroplast genes or entire plastomes for Euthamia may provide sufficient data to resolve these relationships. The low substitution and evolution rate of plant mitochondrial genes (Muse 2000) indicate that mitochondrial DNA is unlikely to have phylogenetic utility in Euthamia. Unfortunately, poor resolution makes it unlikely that any additional and cryptic species will be discovered for Euthamia.

Asteromyia phylogenetics – We used the mitochondrial gene cytochrome oxidase I

(COI) in order to elucidate the relationships of Asteromyia euthamiae, a parasite of Euthamia.

Our data indicate that the monophyly of A. euthamiae is unclear. Two major clades of

Asteromyia euthamiae emerged in parsimony heuristic searches: one on Euthamia caroliniana and E. scabra with two reciprocally monophyletic subgroups and another with midges associated with E. graminifolia, E. caroliniana, E. leptocephala, E. hirtipes, E. gymnospermoides, and E. scabra. Many of these A. euthamiae specimens exhibit host clade specificity. For example, one clade of A. euthamiae appear to specifically parasitize most

Euthamia scabra sampled. The presence of two distinct A. euthamiae clades on E. graminifolia and two distinct groups associated with E. caroliniana may indicate frequent host swapping or the midges identifying two distinct E. caroliniana forms. One E. caroliniana specimen harbored midges from both clades, though, indicating that the latter is not the case. Additionally, a lone specimen from Euthamia leptocephala in Texas sometimes formed a clade with the Asteromyia gutierreziae group, indicating that host genus swapping may have occurred more than once to

Euthamia. However, this was unsupported in the bootstrap analysis, which yielded four genetically distinct lineages of A. euthamiae, all distinct from A. gutierreziae.

Conclusions – Increased sampling of Euthamia graminifolia, E. caroliniana, E. gymnospermoides, and E. scabra may better support the monophyly of each species and uncover subclades corresponding to varieties or other species. Improved geographic sampling of taxa like may uncover previously unseen variation or geographic trends. Sampling hypothetical species, such as Euthamia remota and E. galetorum, may support the biological reality of such taxa. Additional sampling of Euthamia from heterogeneous

“species” may uncover additional taxa as well, but support for such remains to be seen and may be unlikely given the low sequence divergence of this genus. Unfortunately, certain species boundaries remain partially ambiguous given the nature of low divergence of DNA data here and certain morphologically heterogeneous specimens described as E. graminifolia, E. caroliniana, or E. gymnospermoides. Further investigation into hypothetical taxa like E. pulverulenta or E. tenuifolia may support the existence of these taxa or their correct placement in E. gymnospermoides or E. caroliniana, respectively.

Increased sampling of more Asteromyia from different Euthamia species in additional geographic areas, particularly in the western, central, and northeastern , may also elucidate relationships further. Inclusion of additional E. hirtipes, E. leptocephala, E. gymnospermoides, and E. scabra associated Asteromyia may clarify the “mixed” clade of A. euthamiae as a heterogeneous group or one legitimately possessing greater host specificity than estimated from the data seen to date. Because we did not sample galls from taxa such as

Euthamia occidentalis, E. remota and E. galetorum, other undocumented Asteromyia lineages may exist. The presence of clades of Asteromyia euthamiae specializing on taxa such as

Euthamia scabra and E. caroliniana (Fig. 4) indicates the ability of midge parasites to identify and distinguish lineages of Euthamia.

The addition of other gene regions will likely provide greater support to phylogenetic relationships of Asteromyia. The data presented here appear to display high host-specificity.

The appearance of multiple clades, often specific to Euthamia host species, may indicate that

Asteromyia euthamiae actually comprises one or more unnamed species. This may also indicate that parasite speciation is more frequent than previously believed on fungivorous

Asteromyia specializing on North American Astereae.

References

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