Taxonomy and Phylogeny of the Asphondylia

Bucknell University

From the SelectedWorks of Warren G. Abrahamson, II

2015

Taxonomy and phylogeny of the Asphondylia species (Diptera: Cecidomyiidae) of North American goldenrods: challenging morphology, complex host associations, and cryptic speciation Netta Dorchin, Tel Aviv University Jeffrey B. Joy, Simon Fraser University Lukas K. Hilke, University of Bonn Michael J. Wise, Roanoke College Warren G. Abrahamson, II, Bucknell University

Available at: https://works.bepress.com/warren_abrahamson/154/ bs_bs_banner

Zoological Journal of the Linnean Society, 2015, 174, 265–304. With 105 ﬁgures

Taxonomy and phylogeny of the Asphondylia species (Diptera: Cecidomyiidae) of North American goldenrods: challenging morphology, complex host associations, and cryptic speciation

NETTA DORCHIN1*, JEFFREY B. JOY2, LUKAS K. HILKE3, MICHAEL J. WISE4 and WARREN G. ABRAHAMSON5

1Department of Zoology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel 2Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC, Canada V5A 1S6 3University of Bonn, Regina-Pacis-Weg 3, D-53113 Bonn, Germany 4Department of Biology, Roanoke College, Salem VA 24153, USA 5Department of Biology, Bucknell University, Lewisburg, PA 17837, USA

Received 12 October 2014; revised 29 November 2014; accepted for publication 15 December 2014

Reproductive isolation and speciation in herbivorous insects may be accomplished via shifts between host-plant resources: either plant species or plant organs. The intimate association between gall-inducing insects and their host plants makes them particularly useful models in the study of speciation. North American goldenrods (Asteraceae: Solidago and Euthamia) support a rich fauna of gall-inducing insects. Although several of these insects have been the subject of studies focusing on speciation and tritrophic interactions, others remain unstudied and undescribed. Among the latter are at least seven species of the large, cosmopolitan gall midge genus Asphondylia Loew (Diptera: Cecidomyiidae), the taxonomy and biology of which are elucidated here for the ﬁrst time using morphological, molecular, and life-history data. We describe Asphondylia pseudorosa sp. nov., Asphondylia rosulata sp. nov., and Asphondylia silva sp. nov., and redescribe Asphondylia monacha Osten Sacken, 1869 and Asphondylia solidaginis Beutenmüller, 1907, using morphological characters of adults, immature stages, and galls, as well as sequence data from both nuclear and mitochondrial genes. A neotype is designated for A. solidaginis, the type series of which is considered lost. We also provide information on the life history of all species, including a description of two inquilinous cecidomyiids commonly found in the galls, Clinodiplosis comitis sp. nov. and Youngomyia podophyllae (Felt, 1907), and on parasitoid wasps associated with the gall midges. Asphondylia johnsoni Felt, 1908, which was described from an unknown gall on an unknown Solidago host, is assigned to nomina dubia. Our phylogenetic analyses show that some of the Asphondylia species associated with goldenrods induce two different types of galls during their life cycle, some exhibit host alterations, and some do both. In the absence of reliable morphological differences, recognising species boundaries and deciphering host associations of species must rely heavily on molecular data. Our analysis suggests that radiation in this group has been recent and occurred through shifts among host plants.

ADDITIONAL KEYWORDS: cryptic speciation – gall midges – host association – inquilines – Solidago.

*Corresponding author. E-mail: [email protected]

INTRODUCTION 2008; Adair et al., 2009), this has been demonstrated only in the genus Asteromyia Felt, 1910 (Heath & Phytophagous insects are well-known models for study- Stireman, 2010; Janson et al., 2010). In general, gall ing diversification and speciation (Futuyma & Agrawal, midge genera with such known associations with fungi 2009; Nosil, 2012). The evolution of host associations have a broader host range and are more diverse than among phytophagous insects has been shown to be gall midge genera that do not exhibit fungal associa- caused by both spatial and temporal shifts between tions, suggesting that the fungus is important in me- host-plant species and within host-plant species (to dif- diating relationships with the host plant (Joy, 2013). ferent plant organs or different time periods of plant The hypothesis that females actively collect conidia in growth; Joy & Crespi, 2012); however, the relative im- specialized structures on their ovipositors (Borkent & portance and the context of between- versus within- Bisset, 1985; Rohfritsch, 1997, 2008) remains untest- host shifts remains unknown. Gall midges (Diptera: ed, and the mechanistic role of the fungus in Cecidomyiidae) are excellent models for studying the Asphondylia galls is still unknown. importance and context of such shifts because of the The life histories of Asphondylia species vary con- intimate nature of their relationships with their host siderably, depending largely on zoogeographical region plants, and because closely related groups of gall midges and host-plant phenology (Yukawa, 1987; Uechi & are known to diversify through shifts between hosts Yukawa, 2006a, b; Tokuda, 2012). Many species are and through shifts between organs within a host- univoltine and enter extended diapause, whereas others plant species (e.g. Jones, Gagné & Barr, 1983; Hawkins, are bi- or multivoltine and present complex host as- Goeden & Gagné, 1986; Joy & Crespi, 2007; Stireman, sociations (reviewed in Tokuda, 2012). The great ma- Devlin & Abbot, 2012). jority of gall-inducing cecidomyiids are considered Asphondylia Loew, 1850 belongs to the tribe monophagous or narrowly oligophagous, but system- Asphondyliini, currently with 505 species, and within atic and molecular studies have revealed some Asphondyliini to the subtribe Asphondyliina, which is Asphondylia species to be polyphagous, as they develop most diverse in the Neotropics (Gagné & Jaschhof, on several host plants from two or more unrelated plant 2014). The subtribe contains 19 genera that share clear families. Documented examples of polyphagous species morphological apomorphies, but no cladistic or other include Asphondylia gennadii Marchal, 1904 (Gagné phylogenetic analysis has been conducted on it to date. & Orphanides, 1992), Asphondylia yushimai Yukawa All genera included in this group other than Asphondylia & Uechi, 2003 (Yukawa et al., 2003), and Asphondylia are small (with between one and 11 species), and most websteri Felt, 1917 (Gagné & Jaschhof, 2014). Some are restricted to South America. By contrast, of these species are multivoltine and alternate between Asphondylia is one of the largest genera in the family hosts that are available at different times of the year Cecidomyiidae, with 320 described species that feed (Tokuda, 2012), whereas others alternate between dif- on a great diversity of plant families worldwide (Gagné ferent organs on the same host plant (Parnell, 1964; & Jaschhof, 2014), and the number of undescribed Plakidas, 1988). On the other end of the scale are groups species in this genus is probably far greater. Despite of strictly monophagous species that are plant-organ descriptions of numerous new Asphondylia species in specific and have apparently radiated, or are in the recent years from South America and Australia process of radiating, in situ on a single plant genus (Veenstra-Quah, Milne & Kolesik, 2007; Kolesik & (Hawkins et al., 1986; Gagné & Waring, 1990; Joy & Veenstra-Quah, 2008; Maia et al., 2008; Maia, Santos Crespi, 2007; Stokes et al., 2012). & Fernandes, 2009; Kolesik, Adair & Eick, 2010), knowl- Asphondylia taxonomy is challenging because adult edge about the fauna of the southern hemisphere is morphology differs little among species. All species share greatly lacking, in particular that of the Afrotropical the neckless, cylindrical antennal flagellomeres in both region, and there is little doubt that it includes hun- sexes, the needle-like ovipositor and conspicuously en- dreds of undescribed species (ND, unpublished data). larged seventh sternite in the female, and the compact The North American fauna of plant-feeding gall midges male genitalia with the spherical gonostyli posi- is relatively well known (Gagné, 1989), but undescribed tioned dorsally rather than apically (Gagné, 1994). The species are still continuously discovered, and many of third-instar larvae possess a well-developed, usually these belong to Asphondylia. four-toothed spatula on the first thoracic segment. All Asphondylia species are gall inducers, and the Because pupation in all Asphondylia species takes place galls are almost always associated with a fungus that inside the gall rather than in the soil, the larvae do lines the inside walls of the gall. Similar associations not need the spatula for digging; thus the function of are known in other cecidomyiid genera, and al- the spatula in this genus is unknown. The pupae are though several studies suggested that the larvae of such characterized by well-developed horn-like antennal bases, ‘ambrosia’ gall midges feed on the fungus rather than a varying number of facial horns, and transverse dorsal on the plant tissue (Bisset & Borkent, 1988; Rohfritsch, rows of spines on the abdominal segments (Gagné, 1989,

1994) that assist in breaking out of the galls just before utes of the goldenrod- and Aster-associated species adult eclosion. While larvae and pupae sometimes offer suggest that they are a monophyletic group. useful taxonomic characters – for example in the species groups associated with Larrea Cavanilles, 1800 (Zygophyllaceae) and Atriplex Linnaeus, 1753 MATERIAL AND METHODS (Chenopodiaceae) in North America (Hawkins et al., COLLECTING AND REARING OF INSECTS 1986; Gagné & Waring, 1990) – often the only way to In this work, the term ‘goldenrods’ refers to Solidago verify species identity is by conducting molecular analy- and Euthamia, which were historically treated as a ses (Yukawa et al., 2003; Uechi, Yukawa & Yamaguchi, single genus (Solidago) but are currently recognized 2004; Kolesik et al., 2010). as distinct genera (Semple & Cook, 2006). Golden- Here, the Asphondylia fauna of North American gold- rods were extensively surveyed for galls in numerous enrods was studied as part of a general review of gall- localities in central Pennsylvania in 2005–2007, and inducing cecidomyiids on these plants. Goldenrods [the occasionally thereafter. Other occasional collecting was genera Solidago Linnaeus, 1753 and Euthamia Nuttal performed in New York (Lake District) and Virginia ex Cassini, 1825; (Asteraceae)], which include many (Roanoke area), and material has also been received common herbs throughout eastern USA, support a re- from Maine (Perry and Sipp Bay) and Massachusetts markably diverse community of insects (McEvoy, 1988; (Nantucket). Goldenrod species that were surveyed on Gagné, 1989; Maddox & Root, 1990; Root & Cappuccino, a regular basis were Solidago altissima Linnaeus, 1753, 1992; Fontes, Habeck & Slansky, 1994). Because they Solidago gigantea Aiton, 1789, Solidago rugosa Miller, are easy to manipulate in field and greenhouse ex- 1768, Solidago juncea Aiton, 1789, Solidago caesia periments, goldenrods have served as model systems Linnaeus, 1753, and Euthamia graminifolia (Linnaeus) for numerous studies on evolutionary and ecological Nuttall, 1840. Species that were sampled intermit- aspects of speciation and tritrophic interactions (e.g. tently included Solidago bicolor Linnaeus, 1767, Soli- Abrahamson & Weis, 1997; Stireman et al., 2005, 2006, dago canadensis Linnaeus, 1753, Solidago erecta Banks 2012; Crutsinger, Cadotte & Sanders, 2009; Dixon, Craig ex Pursh, 1813, Solidago flexicaulis Linnaeus, 1753, & Itami, 2009; Wise, Cole & Carr, 2010; Craig et al., Solidago nemoralis Aiton, 1789, Solidago sempervirens 2011; Hakes & Cronin, 2012). Of the 50 or so insect Linnaeus, 1753, and Solidago ulmifolia Muhlenberg species known to induce galls on goldenrods, about 30 ex Willdenow, 1803. Additionally, Asphondylia species species are cecidomyiids. Of these, 16 belong to the from several other Asteraceae were reared and studied genus Rhopalomyia Rübsaamen, 1892 (Dorchin et al., for comparison, including Asphondylia recondita Osten 2009), three belong to the genus Asteromyia (Stireman Sacken, 1875 (on Aster novae-angliae Linnaeus, 1753), et al., 2010), and two species belong to the genus Asphondylia rudbeckiaeconspicua Osten Sacken, 1870 Dasineura Rondani, 1840 (Dorchin et al., 2007). To date, (on Rudbeckia laciniata Linnaeus, 1753), and only two Asphondylia species have been described from Asphondylia eupatorii Felt, 1911 [on Ageratina altissima goldenrods: one in the 19th and the other in the early (L.) King & Robinson, 1970]. 20th century (Gagné, 1989); however, in the course of Galls were either bagged in the field or collected and our work on goldenrod-galling cecidomyiids, we regu- brought to the laboratory, where they were kept at room larly encountered numerous types of Asphondylia galls temperature (20–25°C) in ventilated rearing cages until that have not been previously recorded or attributed adult emergence. Some of the galls were dissected under to species. a stereomicroscope to obtain the immature stages of In the present study, we provide a systematic review the gall midges as well as those of parasitoids and of Asphondylia species from goldenrods using a com- inquilines. bination of morphological, ecological, and molecular data. We describe three new species, re-describe the two known species, add numerous host records, and provide TAXONOMY detailed information about species life histories. We Immature stages and emerging adults of the gall midges, also describe two common inquilinous cecidomyiids and their parasitoids, and inquilines were preserved in 70% provide information on parasitic Hymenoptera that are ethyl alcohol for morphological study, and the gall found regularly in the galls. Furthermore, we con- midges were later mounted on permanent micro- ducted a molecular phylogenetic analysis based on both scope slides in euparal, according to the method out- mitochondrial and nuclear genes, which assisted with lined in Gagné (1989). Illustrations were made with taxonomic decisions and provided insight into the role the aid of a drawing tube mounted on a Leica DM1000 of host use in the radiation of species in this group. LED compound microscope. Pupae were also studied A revision of Asphondylia via extensive sampling across under a scanning electron microscope. Some adults the genus is a major task that is beyond the scope of and immature stages were preserved in 96% ethanol this paper; however, the distinct morphological attrib- for molecular study, which limited the number of

© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015, 174, 265–304 268 N. DORCHIN ET AL. specimens that were available for the morphological Sequences were aligned using the local alignment study in some of the species. Relevant material de- tool MAFFT (Katoh, Asimenos & Toh, 2009), and then posited in the National Museum of Natural History inspected and adapted by eye using Se-Al (Rambaut, (USNM), Smithsonian Institution, Washington, DC, 1996). Models of molecular evolution for each gene were USA, was also examined. The terminology for adult assessed using jModeltest (Posada, 2008). The codon morphology follows McAlpine et al. (1981), and the ter- position (CP) model (Shapiro, Rambaut & Drummond, minology for immature morphology follows Gagné (1989). 2006) was employed for each gene. Bayesian Taxonomy and nomenclature of the host plants follow phylogenetic analyses were performed on the result- Semple & Cook (2006); thus the name S. canadensis ing data partitions under an uncorrelated lognormal used by Felt is referred to here as S. altissima. All taxo- relaxed molecular clock model (Drummond et al., 2006), nomic decisions were made by the first author. The with a birth–death speciation tree prior (Gernhard, material examined in this work is deposited in the Na- 2008), as implemented in BEAST 1.7.1 (Drummond tional Collection of Insects, Zoological Museum, Tel Aviv et al., 2012). Four independent chains were run for University (TAUI), unless otherwise indicated. All types 50 million generations. Convergence was assessed are mounted on permanent microscope slides. Holotypes graphically using TRACER 1.5 (Rambaut & Drummond, of newly described species are deposited in TAUI. Other 2007), and through evaluation of the effective sample types are deposited as indicated in the relevant section size (ESS) values (Drummond et al., 2006). Following of the species descriptions. burn-in and a combination of the four runs, ESS values of greater than 200 for all parameters were taken as evidence of parameter stability and an adequate run MOLECULAR METHODS time. The maximum clade credibility (MCC) phylogenetic Genomic DNA was extracted from whole adult or im- tree was produced from the resulting distribution of mature midges using a BioSprint96 magnetic bead ex- trees using TreeAnnotator 1.7.1 (Drummond et al., 2012). tractor and corresponding extraction kits by Qiagen We reconstructed the evolution of host use as an un- Inc. (Valencia, CA, USA and Hilden, Germany). We PCR- ordered multistate character on our phylogenetic tree amplified one mitochondrial and one nuclear gene using using the maximum parsimony approach, as imple- the following procedures: a fragment of ∼680 bp of the mented in MESQUITE (Maddison & Maddison, 2011). mitochondrial cytochrome oxidase c subunit I (COI) was amplified using the primers LCO1490 (F) and HCO2198 (R) (Folmer et al., 1994); and a fragment of ∼570 bp RESULTS of the elongation factor 1 alpha gene (EF-1α) was amplified using the primers EF1aF and EF1aR (Joy & MORPHOLOGY Crespi, 2007). As the Asphondylia species associated with golden- Following enzymatic clean up (Exo/SAP), double- rods are very similar, we could not differentiate among stranded sequencing was conducted on an automated them based on morphology alone. Although larval ABI 3730XL sequencer (Applied Biosystems) at the and pupal characters often offer better morphological Pennsylvania State University nucleic acid facility or characters for this purpose in Asphondylia than adult at the Macrogen facility, Amsterdam, the Nether- characters, this is generally not the case in the lands. Sequences were initially inspected for sequenc- goldenrod-associated species. Larvae are robust, light ing errors using CodonCode Aligner 1.5.2 (CodonCode to dark orange, and have a greatly reduced terminal Corporation, Dedham, MA, USA). segment that is typical of the genus. The most conspicuous larval character is the strong, four-toothed sternal spatula, which is accompanied by five setose PHYLOGENETIC ANALYSES lateral papillae on each side (Fig. 31); however, in the The phylogenetic analysis included 54 individuals species associated with goldenrods, the intraspecific vari- from goldenrods (Solidago and Euthamia), one from ation in the shape of the spatula is often so great that Aster, two from other Asteraceae (Chrysothamnus it is of no taxonomic value. Nuttall, 1840 sp. and Gutierrezia Lagasca, 1807 sp.), Pupae of all species in this group have well-developed and one from Larrea (Zygophyllaceae). The last three antennal bases that form the ‘antennal horns’, two upper were used as out-group taxa. Two additional species, and one lower facial horns, long and pointed prothoracic A. rudbeckiaeconspicua and A. conglomerata De Stefani, spiracles, and minute cephalic setae (e.g., Fig. 65). On 1900, yielded problematic sequences that could not be each side of the lower facial horn there are two pa- aligned with the rest of the data set, and were there- pillae, one of which has a short seta. This arrange- fore eliminated from the analysis. Data on all speci- ment may not be unique to the species from goldenrods, mens that were included in the analysis are provided but it differs at least from some Asphondylia species in Table 1. that develop on other North American Asteraceae. For

© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015, 174, 265–304 05TeLnenSceyo London, of Society Linnean The 2015 © Table 1. Samples used for DNA analysis with GenBank accession numbers

Name of sequence Host plant Collecting site Collection date Collector COI accession no. EF-1α accession no.

Asphondylia bigeloviabrassicoides Chrysothamnus sp. Canada, BC, Oliver 2005 J.B. Joy EF189995 EF190018 Asphondylia clavata Larrea tridentata USA, AZ, Tucson 2005 J.B. Joy EF189979 EF190010 Asphondylia sp. Gut Gutierrezia sp. USA, CA, Jacumba 2005 J.B. Joy EF189996 EF190019 Asphondylia recondita Aster novae-angliae USA, PA, Mauses Creek 19 September 2005 N. Dorchin KP208470 – Asphondylia monacha ul Solidago uliginosa USA, ME, Winter Harbor 7 September 2007 R.J. Gagné KP208469 – Asphondylia monacha er1 Solidago erecta USA, VA, Roanoke 5 September 2012 M.J. Wise KP208519 – Asphondylia monacha er2 Solidago erecta USA, VA, Roanoke 5 September 2012 M.J. Wise KP208518 – Asphondylia monacha jun1 Solidago juncea USA, PA, Rt 642 24 August 2007 N. Dorchin KP208492 KP208551 Asphondylia monacha jun2 Solidago juncea USA, PA, Mauses Creek 16 September 2005 N. Dorchin KP208491 KP208550 Asphondylia monacha jun3 Solidago juncea USA, PA, Lewisburg, Stein Ln. 22 August 2005 N. Dorchin KP208493 KP208552 Asphondylia monacha alt1 Solidago altissima USA, NY, Canton 1 May 2005 N. Dorchin KP208494 – Asphondylia monacha jun4 Solidgo juncea USA, PA, Lewisburg, Stein Ln. 22 August 2005 N. Dorchin KP208495 – Asphondylia monacha alt2 Solidago altissima USA, PA, Lewisburg, Furnace Rd. 28 April 2005 N. Dorchin KP208496 KP208541 Asphondylia monacha jun5 Solidago juncea USA, PA, Rt 642 2 September 2005 N. Dorchin KP208497 –

olgclJunlo h ina Society Linnean the of Journal Zoological Asphondylia monacha er3 Solidago erecta USA, VA, Roanoke 28 August 2010 MJ Wise & N. Dorchin KP208467 – Asphondylia monacha er4 Solidago erecta USA, VA, Roanoke 28 August 2010 MJ Wise & N. Dorchin KP208468 – Asphondylia rosulata sp. nov. bud1 Solidago rugosa USA, PA, Black Moshannon State Park 14 July 2007 N. Dorchin & M.J. Wise KP208486 KP208546 Asphondylia rosulata sp. nov. bud2 Solidago rugosa USA, PA, Lairdsville 2 July 2007 N. Dorchin KP208485 KP208540 Asphondylia rosulata sp. nov. gig1 Solidago gigantea USA, PA, Lewisburg, Furnace Rd. 20 June 2006 N. Dorchin KP208479 KP208544 Asphondylia rosulata sp. nov. sn4 Solidago rugosa USA, PA, Montour Preserve 30 June 2006 N. Dorchin KP208480 KP208543 Asphondylia rosulata sp. nov. sn1 Solidago rugosa USA, PA, Millersburg 30 June 2007 N. Dorchin & M.J. Wise KP208483 KP208539 Asphondylia rosulata sp. nov. sn2 Solidago rugosa USA, PA, Montour Preserve 30 June 2006 N. Dorchin KP208484 KP208525 Asphondylia rosulata sp. nov. sn3 Solidago rugosa USA, PA, BU Natural Area 26 July 2006 N. Dorchin KP208482 KP208542

Asphondylia rosulata sp. nov. gig2 Solidago gigantea USA, PA, Mauses Creek 4 July 2007 N. Dorchin & D. Ryan KP208481 KP208526 GOLDENROD OF SYSTEMATICS Asphondylia rosulata sp. nov. bud4 Solidago rugosa USA, PA, Lairdsville 12 August 2005 N. Dorchin KP208478 KP208545 Asphondylia silva sp. nov. 1 Solidago caesia USA, PA, Dale’s Ridge 19 June 2006 N. Dorchin KP208487 – Asphondylia silva sp. nov. 2 Solidago caesia USA, PA, Dale’s Ridge 19 June 2006 N. Dorchin KP208488 – Asphondylia silva sp. nov. 3 Solidago caesia USA, PA, Shikellamy State Park 30 June 2007 N. Dorchin & M.J. Wise KP208490 KP208537 Asphondylia silva sp. nov. 4 Solidago caesia USA, PA, Dale’s Ridge 4 September 2005 N. Dorchin KP208489 KP208538 Asphondylia pseudorosa sp. nov. b1 Euthamia graminifolia USA, PA, Millersburg 30 June 2007 N. Dorchin & MJ Wise KP208513 KP208521 Asphondylia pseudorosa sp. nov. f1 Euthamia graminifolia USA, PA, BU Natural Area 7 August 2007 N. Dorchin KP208511 KP208532 Asphondylia pseudorosa sp. nov. f2 Euthamia graminifolia USA, PA, Hughesville 2 August 2007 N. Dorchin KP208512 KP208520 Asphondylia pseudorosa sp. nov. b2 Euthamia graminifolia USA, PA, BU Natural Area 7 August 2007 N. Dorchin KP208508 KP208536 Asphondylia pseudorosa sp. nov. b3 Euthamia graminifolia USA, PA, Lewisburg, Stein Ln. 4 July 2007 N. Dorchin & D. Ryan KP208516 KP208535 Asphondylia pseudorosa sp. nov. b4 Euthamia graminifolia USA, PA, Hughesville 2 August 2007 N. Dorchin KP208517 KP208534 2015, , Asphondylia pseudorosa sp. nov. f3 Euthamia graminifolia USA, PA, BU Natural Area 5 August 2006 N. Dorchin KP208507 KP208524 Asphondylia pseudorosa sp. nov. b5 Euthamia graminifolia USA, PA, RB Winter State Park 22 July 2007 N. Dorchin & M.J. Wise KP208510 KP208531 Asphondylia pseudorosa sp. nov. f4 Euthamia graminifolia USA, PA, Mauses Creek 8 August 2005 N. Dorchin KP208515 KP208523

174 Asphondylia pseudorosa sp. nov. f5 Euthamia graminifolia USA, PA, Rt 642 18 August 2006 N. Dorchin KP208514 KP208533 Asphondylia pseudorosa sp. nov. f6 Euthamia graminifolia USA, PA, BU Natural Area 5 August 2006 N. Dorchin KP208509 KP208522

265–304 , Asphondylia solidaginis bud1 Solidago altissima USA, PA, Rt. 642 20 July 2007 N. Dorchin KP208506 – Asphondylia solidaginis bud2 Solidago altissima USA, PA, Rt. 642 20 July 2007 N. Dorchin KP208505 – Asphondylia solidaginis sn1 Solidago altissima USA, PA, BU Natural Area 16 June 2005 N. Dorchin KP208502 KP208547

Asphondylia solidaginis sn2 Solidago altissima USA, PA, Mauses Creek 19 July 2005 N. Dorchin KP208501 KP208529 ASPHONDYLIA Asphondylia solidaginis sn3 Solidago altissima USA, PA, Furnace Rd. 20 June 2006 N. Dorchin KP208500 KP208530 Asphondylia solidaginis sn4 Solidago altissima USA, Lewisburg, Dale’s Ridge 19 June 2006 N. Dorchin KP208503 KP208549 Asphondylia solidaginis gig Solidago gigantea USA, PA, BU Natural Area 26 June 2005 N. Dorchin KP208504 KP208548 Asphondylia solidaginis sn5 Solidago altissima USA, PA, BU Natural Area 28 June 2007 N. Dorchin KP208498 KP208528 Asphondylia solidaginis bud3 Solidago altissima USA, PA, Lairdsville 19 July 2007 N. Dorchin KP208499 KP208527 Asphondylia sp. ul Solidago uliginosa USA, ME, Winter Harbor 7 September 2007 R.J. Gagné KP208471 – Asphondylia sp. bic1 Solidago bicolor USA, VA, Havens 9 October 2011 M.J. Wise KP208477 – Asphondylia sp. bic2 Solidago bicolor USA, VA, Havens 9 October 2011 M.J. Wise KP208476 – Asphondylia sp. bic3 Solidago bicolor USA, VA, Sharp Top, Bedford 15 October 2011 M.J. Wise KP208474 – Asphondylia sp. bic4 Solidago bicolor USA, VA, Havens 9 October 2011 M.J. Wise KP208475 –

Asphondylia sp. semp1 Solidago sempervirens USA, ME, Nantucket 9 September 2011 C. Eiseman KP208472 – 269 Asphondylia sp. semp2 Solidago sempervirens USA, ME, Nantucket 9 September 2011 C. Eiseman KP208473 – 270 N. DORCHIN ET AL. example, the pupae of A. rudbeckiaeconspicua and tractable ovipositor (Fig. 58). The male terminalia are A. eupatorii have two upper and three lower facial horns. round and compact, with gonocoxites that are short Each of the abdominal terga bears one straight row and wide, extending into a short mediodistal lobe and a couple of less ordered rows of pointed spines (Fig. 29). The gonostyli are round–ovoid, situated dor- (Fig. 76), except for the first abdominal segment, which sally on the gonocoxites, and bear a crescent-shaped is bare. The only useful taxonomic character we found tooth. Male genitalia are very similar in all species for distinguishing among pupae of the goldenrod- from goldenrods, and the only variation we found was associated species is the shape of the antennal horns. in the shape of the hypoproct, which is notched to a The species fall into one of two groups in this respect: varying degree in different species (in A. silva sp. nov. those with relatively short and wide-based horns it is virtually entire). Given the general lack of reli- (Asphondylia monacha Osten Sacken, 1869), and those able morphological differences, identification of species with longer and more slender horns (Asphondylia in this group must rely heavily on their galls and on solidaginis Beutenmüller, 1907, Asphondylia molecular markers. In the species descriptions that rosulata sp. nov., Asphondylia silva sp. nov., and follow, the two previously described species, A. monacha Asphondylia pseudorosa sp. nov.). An exception is the and A. solidaginis, are re-described first, followed by morphology of A. pseudorosa sp. nov. pupae that develop descriptions of the new species. Characters that are in inflorescences, which have very short and blunt horns common to all species are not mentioned again. and thus differ markedly from the pupae of the same species that develop in buds. We consider this to be an intraspecific variation as a result of adaptation to LIFE HISTORY the galled organ. The Asphondylia galls on goldenrods can be found Adults of all species from goldenrods (as well as from early spring to late autumn (April–October). species from Aster) are basically black, with black- They develop either in buds or leaves, and and-white banded legs due to a thick covering of black A. pseudorosa sp. nov. also galls inflorescences. Al- scales and patches of white scales on the tibiae and though bud galls are diverse in size and shape, they first tarsomeres (Fig. 18). Wings are dark grey and are similar in basic structure: they contain a rigid densely covered by black hairs. Tarsal claws are central chamber made of shortened leaves and sur- untoothed on all legs. As in all members of the genus rounded by one or more whorls of short leaves at shoot and tribe, there are 12 antennal flagellomeres in both tips. When such a gall develops singly and is accom- sexes, which are all cylindrical except for the succes- panied by a small number of leaves, it is hardly no- sively shorter and rounder flagellomeres 10–12 in the ticeable (as in A. silva sp. nov., Fig. 19), but when it female. Male flagellomeres bear twisting circumfila, is surrounded by numerous leaves, the gall is much whereas circumfila on female flagellomeres consist of more conspicuous, as in the summer-generation galls only two horizontal bands connected by two longitu- of A. solidaginis and A. rosulata sp. nov. (Figs 13, 14). dinal strands (Figs 23, 24). The first antennal When several individual galls develop in aggrega- flagellomere in females of all species from Solidago is tion, they form complex, spherical structures, as in the much longer than the more distal flagellomeres, whereas summer generation of A. monacha (Fig. 3). In all of these in the species from Euthamia these flagellomeres are cases, the larval chamber is rigid to the touch and con- equal or subequal in length (compare Figs 46 and 56). tains white fungal mycelium that sometimes spills out To compare this character among species, the length of the chamber. Although the leaf galls on goldenrods of flagellomere 1 relative to the length of flagellomere 5 are simple blisters, they are unusual because they join is given for each species in the following descriptions together two or more leaves in a snap, in which one (referred to as ‘flagellomere 1/flagellomere 5 ratio’). The leaf usually forms the bottom part of the gall and the palpal segments of all species from Solidago are suc- other forms the upper part. These ‘snap galls’ also cessively longer, whereas in the species from Euthamia, contain a thick layer of white mycelium. the third segment is not appreciably longer than the Host associations in goldenrod Asphondylia species second segment (compare Figs 45 and 46 with Figs 55 are complex in that several species use more than one and 56). Species may otherwise differ in overall size host and/or induce different types of galls during the (e.g. A. monacha is a big species, whereas A. silva sp. nov. season. In such cases, we were able to associate the and A. pseudorosa sp. nov. are relatively small species), different generations of an individual species only with and in the length of the ovipositor relative to overall the aid of molecular data. For example, A. monacha is size (larger species have relatively longer oviposi- bivoltine and induces two types of galls on different tors). As in all members of the genus, females have a host plants in spring and late summer. The solitary, pair of cerci-like appendages at the end of the eighth inconspicuous spring gall of this species (Figs 1, 2) de- abdominal segment, a sternite 7 that is much longer velops quickly in young sprouts of Solidago altissima, than the preceding sternites, and a needle-like, re- whereas the late-summer generation takes several

Figures 1–6. Asphondylia bud galls: 1, 2, Asphondylia monacha spring galls on Solidago altissima sprouts; 3, Asphondylia monacha summer-generation gall on Solidago juncea;4,Asphondylia monacha summer-generation gall on Solidago erecta; 5, Asphondylia sp. gall on Solidago sempervirens (photo: Charley Eiseman); 6, Asphondylia sp. gall on Solidago bicolor.

© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015, 174, 265–304 272 N. DORCHIN ET AL. months to develop in the aggregated galls on S. juncea, gall is opened, in contrast to the much more docile S. erecta, and S. uliginosa (Figs 3, 4). Females of the Asphondylia larvae. Although the larva of the gall inducer late-summer generation probably lay their eggs in plant was not observed in galls inhabited by Y. podophyllae, tissue close to the ground, and the first-instar larvae it is not entirely clear if the latter are predators or that will develop in S. altissima sprouts in the follow- inquilines because direct predation has not been ob- ing spring must overwinter in dormant buds on rhi- served. In the present study, Y. podophyllae larvae were zomes. The life history of the undescribed species from often found in the galls of all the surveyed Asphondylia S. bicolor and S. sempervirens is probably similar. The species, but they were especially common in remaining species on goldenrods are multivoltine, and A. pseudorosa sp. nov. and A. monacha galls. In the tiny some of them induce two types of galls at different times gall of A. silva sp. nov., a single inquiline larva per gall during the season. Interestingly, the snap galls that was found. A second inquilinous gall midge species found are all structurally similar on S. altissima, S. rugosa, in the galls is Clinodiplosis comitis sp. nov., which is and Euthamia graminifolia belong to three different described in the present paper. The tiny larvae of this species, each of which also induces bud galls on the gall midge were found among leaves that surround the same host. Two of these species use S. gigantea as a main chamber of the rosette galls of A. monacha and supplementary host. Larvae of the last generation of A. pseudorosa sp. nov., and did not appear to cause any all multivoltine species must overwinter in dormant damage to the gall or to the Asphondylia larva. plant tissues underground and complete their devel- More than ten species of parasitic Hymenoptera were opment in inconspicuous galls in early spring, but this reared from Asphondylia galls on goldenrods, the most has not been confirmed in the present study. common of which by far is Galeopsomyia haemon. This Several other gall midges and the tephritid fly species forms ‘a gall within a gall’ during its devel- Procecidochares atra (Loew, 1862) develop in bud galls opment. Larvae of G. haemon induce the develop- on goldenrods, but their galls can be easily distin- ment of numerous dark, spherical structures inside the guished from those of the Asphondylia species. The very gall that are clearly made of plant tissues (Fig. 22). common rosette galls of Rhopalomyia solidaginis Loew, Each of these ball-like structures contains a single larval 1862 on S. altissima contain white, soft-walled larval chamber with a single wasp larva. As the balls grow chambers that are hidden among the rosette leaves. larger, they appear to physically crush the larva of the By contrast, the rosette gall of A. solidaginis on the gall inducer, similar to what has been described for same host is smaller and flatter, and the single, rigid Asphondylia atriplicis (Townsend, 1893) galls on saltbush larval chamber is visible in the middle of the gall in Southern California (Hawkins & Goeden, 1982). (Fig. 13). The compact, artichoke-like galls of P. atra Because they do not feed on the midge larvae, the wasps contain a large, semi-open larval chamber, and are can be considered inquilines rather than parasitoids. almost always found in aggregations. The very common By contrast, in galls of Asphondylia borrichiae Rossi bud galls of A. pseudorosa sp. nov. on E. graminifolia & Strong, 1990 on Borrichia frutescens (Linnaeus) de (Fig. 17) cannot be mistaken for the only other bud Candolle, 1836 (Asteraceae), G. haemon seems to behave galler on this host, Rhopalomyia lobata Felt, 1908, the like a gregarious parasitoid (Stiling et al., 1992). In the galls of which are usually much bigger and contain a galls of Asphondylia floccosa Gagné, 1986 on Atriplex large mass of spongy tissue in which the larval cham- polycarpa (Torrey) Watson, 1874 in Arizona, it has not bers are embedded. been determined whether larvae of G. haemon feed on the midge larvae or on the gall tissue (Dixon et al., 1998). When we took the wasp ‘internal galls’ out of INQUILINES AND PARASITOIDS the cecidomyiid gall on goldenrods and isolated them Asphondylia galls on goldenrods suffer remarkable para- in the lab, the wasps did not complete their develop- sitism and inquilinism rates, which often make it ex- ment; however, when we left them inside the cecidomyiid tremely difficult to find a single gall with an intact larva gall, we were able to rear them in great numbers. This or pupa of the original gall inducer, let alone rear its wasp was very common in all snap galls and to a lesser adults. The two most common natural enemies in the extent in the rosette galls of A. solidaginis, galls are the inquilinous gall midge Youngomyia A. rosulata sp. nov., and A. pseudorosa sp. nov. podophyllae Felt, 1907 and the parasitoid Galeopsomyia Additional hymenopteran parasitoids that were reared haemon (Walker, 1847) (Hymenoptera: Eulophidae: from the goldenrod galls in much smaller numbers in- Tetrastichinae). cluded the following: Rileya insularis Ashmead, 1894; Youngomyia podophyllae is a big and very distinct Tenuipetiolus teredon (Walker, 1843) and Tenuipetiolus cecidomyiid that is very common in galls of different Bugbee, 1951 sp. (Eurytomidae); Torymus advenus Asphondyliini (Gagné, 1989, 1994). Several of its long (Osten Sacken, 1870) and Torymus Dalman, 1820 sp. and slender larvae are usually found together inside (Torymidae); Aprostocetus Westwood, 1833 sp.; the gall. The larvae begin wriggling violently when the Neochrysocharis Kurdjumov, 1912 sp. (Eulophidae); and

KEY TO ASPHONDYLIA GALLS ON NORTH AMERICAN GOLDENRODS 1. Bud gall...... 2 – Leaf or inflorescence gall...... 9 2. Gall in young Solidago altissima sprouts (in April–May; Figs 1, 2)...... Asphondylia monacha Osten Sacken, 1869, spring generation – Gall in apical (usually) or lateral bud of mature goldenrod spp...... 3 3. Inconspicuous gall, up to 1 cm long, composed of two or three leaves that form a single chamber in shoot tips (Fig. 19) ...... 4 – Gall composed of many leaves that form a rosette, at least 4 cm wide ...... 5 4. Gall on Solidago caesia ...... Asphondylia silva sp. nov. – Gall on Solidago nemoralis ...... Asphondylia sp. 5. Gall forms spherical structure, 5–8 cm in diameter, composed of many individual units, each with a central larval chamber (Figs 3–6) ...... 6 – Gall not spherical, containing single, central chamber...... 7 6. On Solidago juncea, Solidago erecta, and Solidago uliginosa...... Asphondylia monacha Osten Sacken, 1869, summer generation –OnSolidago sempervirens and Solidago bicolor...... Asphondylia spp. 7. Gall dorsoventrally flat, up to 5 cm wide; on Solidago spp. (Figs 13, 14)...... 8 – Gall narrower, resembling, a rosebud when young; on Euthamia graminifolia (Fig. 17)...... Asphondylia pseudorosa sp. nov. 8. Gall on Solidago altissima...... Asphondylia solidaginis Beutenmüller, 1907, summer generation – Gall on Solidago rugosa...... Asphondylia rosulata sp. nov. 9. Leaf gall, joining two or more adjacent leaves on various goldenrods...... 10 – Gall in inflorescence of Euthamia graminifolia...... Asphondylia pseudorosa sp. nov. 10. Gall on Solidago spp...... 11 – Gall on Euthamia graminifolia...... Asphondylia pseudorosa sp. nov. 11. Gall on Solidago altissima...... Asphondylia solidaginis Beutenmüller, 1907 – Gall on Solidago rugosa...... Asphondylia rosulata sp. nov.

Lyrcus Walker, 1842 sp. (Pteromalidae). Some of these early-spring generation was found only on S. altissima parasitoids have been recorded from other Asphondylia in April–May. Galls were discovered accidentally in early galls on Asteraceae (e.g. Rossi et al., 2006), and appear April while digging out rhizomes, as they developed to have specialized on this cecidomyiid group. in buds that grew from the rhizomes and were barely visible above ground (Fig. 1). Galled buds were wider and felt harder to the touch than normal buds, were SPECIES DESCRIPTIONS 5 cm long and 2 cm wide, and contained a single In the following descriptions, the identity of the host chamber, the internal walls of which were lined by a plants and the description of the galls appear first, because thick layer of white mycelium. Each gall contained a these are the clearest and most obvious manifesta- single larva or pupa. In May, some galls were found tions of the species. One is more likely to recognize in much longer sprouts (∼15 cm long) that still ap- species in this group (as in many other cecidomyiids) peared stunted and somewhat thicker than normal by the appearance and structure of their galls than by sprouts (Fig. 2). The larval chamber in these galls was the morphology of adults or immature stages. situated at the very tip of the shoot. Adults of the spring generation emerged in May. The much more conspicuous summer-generation gall of this species on S. juncea ASPHONDYLIA MONACHA OSTEN SACKEN, 1869 (the host was incorrectly identified as S. canadensis Asphondylia monacha Osten Sacken, 1869: 299. in the original description) is a rosette bud gall that is found in great numbers (Fig. 3). The galls become Hosts plants apparent in mid-June and reach their final size while Solidago juncea, S. erecta, S. uliginosa (summer gen- the larvae inside them are still tiny first instars. They eration), and S. altissima (spring generation). are usually composed of 15–30 individual units, each with a single larval chamber that is surrounded by Gall and biology shortened leaves and lined internally by white myce- This species has two generations a year that induce lium. These units form a spherical structure on shoot distinct bud galls on different Solidago species. The tips that is 4–7 cm in diameter and can be spotted from

Figures 7–14. Galls: 7, 8, Asphondylia solidaginis snap galls on Solidago altissima;9,Asphondylia solidaginis snap galls on Solidago gigantea; 10, Asphondylia solidaginis pupa in snap gall on Solidago altissima – gall was cut open to show pupa in fungus-lined chamber; 11, 12, Asphondylia rosulata sp. nov. snap galls on Solidago rugosa; 13, Asphondylia solidaginis rosette gall on Solidago altissima; 14, Asphondylia rosulata sp. nov. rosette gall on Solidago rugosa.

Figures 15–22. Galls: 15, Asphondylia pseudorosa sp. nov. inﬂorescence gall on Euthamia graminifolia; 16, Asphondylia pseudorosa sp. nov. snap gall on Euthamia graminifolia; 17, Asphondylia pseudorosa sp. nov. rosette galls on Euthamia graminifolia; 18, Asphondylia sp. from Solidago sp. (photo: Tom Murray); 19, 20, Asphondylia silva sp. nov. galls on Solidago caesia; 21, Clinodiplosis comitis sp. nov. larvae around Asphondylia pseudorosa sp. nov. bud gall; 22, Galeopsomia haemon ‘internal galls’ inside Asphondylia pseudorosa sp. nov. bud gall.

© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015, 174, 265–304 276 N. DORCHIN ET AL. a distance. Adults of the summer generation emerged summer generation, 2.91–3.11 mm in males (N = 2) and in late August to mid-September. Although it was not 3.29–3.59 mm in females (N = 5) of spring genera- observed, we assume that adults of the autumn gen- tion; R1 joins C proximal to mid-length of wing, R5 eration lay their eggs in plant tissue close to the ground joins C posterior to wing apex, M weak, CuA forked and the hatching first-instar larvae overwinter next into CuA1 and CuA2. to dormant buds in the rhizomes. Old galls turn black and may remain on dry shoots of S. juncea through- Female abdomen (Fig. 26): Dorsum covered by black out winter and into the next spring. Galls of similar scales, pleuron and venter with silvery hair-like scales. structure were found on S. erecta (Fig. 4) and Tergites 1–7 rectangular, with posterior one or two rows S. uliginosa, and our molecular analysis indicates that of strong setae and otherwise evenly covered by scales; they are all induced by A. monacha. No morphologi- tergite 8 narrower than preceding tergite, saddle- cal differences were found among adults from these like, without setae. Sternites 2–6 with posterior row three host plants. Asphondylia monacha galls super- of setae and several setae on mid part; sternite 7 much ficially resemble those of Rhopalomyia solidaginis on longer than preceding sternite, narrowed posteriorly, S. altissima, but they are not as wide and flat as the with group of strong setae on posterior half. Oviposi- Rhopalomyia galls, are never found on S. altissima, tor relatively long: sclerotized part 1.96–3.04 times as and their structure is different, as indicated by Osten long as sternite 7 (N = 55) in summer-generation females, Sacken (1869) in his original description of A. monacha. 2.62–3.25 times as long in spring-generation females (N = 6). Adult General colour black. Male abdomen (Fig. 27): Colour pattern as in female. Tergite 1 narrow, band-like, without setae; tergites 2–7 Head: Eye facets round. Palpus three-segmented, seg- rectangular, with posterior row of strong setae, few setae ments successively longer, with several strong setae on basal area, and evenly scattered scales; tergite 7 and otherwise covered by microtrichia. Labella slight- more setose than preceding tergite; tergite 8 narrow, ly pointed, with numerous strong setae on lateral surface. band-like, without setae. Sternites 2–6 rectangular, with posterior row of strong setae and several strong setae Antenna: Scape and pedicel with long, dark setae. Male medially, otherwise evenly covered by scales. Sternite 7 flagellomeres cylindrical, flagellomere 1 slightly longer more setose than preceding sternite. Sternite 8 with than succeeding flagellomere, apical flagellomere small but strongly setose sclerotized area. slightly shorter than preceding flagellomere, all covered by anastomosing loops of circumfila, numerous strong setae, and microtrichia (Fig. 23); flagellomere 1/ Male terminalia (Figs 28–30): Gonocoxite compact, wide, flagellomere 5 ratio = 1.13–1.34 (N = 23). Female and short, with short apical projection extending me- flagellomeres 1–9 cylindrical with well-developed dially; bearing numerous strong setae and evenly circumfila, with two transverse connections, numer- setulose. Gonocoxal apodeme extending on both sides ous strong setae, and otherwise covered by microtrichia of aedeagus to form complex, strongly sclerotized struc- (Fig. 24); flagellomere 1 conspicuously longer than suc- ture (Figs 28, 30). Gonostylus round–ovoid, with nu- ceeding flagellomere, flagellomere 1/flagellomere 5 merous strong setae and otherwise evenly setulose, ratio = 1.41–1.62 (N = 38); flagellomeres 7 and onwards bearing crescent-shaped apical tooth. Aedeagus wide successively shorter; flagellomeres 10–12 with two whorls at base, tapered towards rounded apex, curved ante- of circumfila and several longitudinal connections, nu- riorly in lateral view (Fig. 30). Hypoproct wide at base, merous strong setae, and otherwise covered by deeply divided into two lobes apically, setose and microtrichia (Fig. 25); flagellomere 10 slightly longer setulose, with two longer setae apically on each lobe. than wide; flagellomere 11 slightly wider than long; Cerci completely or almost completely separated, flagellomere 12 rudimentary. bulbous, strongly setose and setulose throughout.

Thorax: Legs: densely covered by black scales other Larva (third instar) (Fig. 31) than a patch of white scales from apical part of femur Orange; integument covered by spicules. Length 2.06– to base of tibia, and from base of tarsomere 1 to ﬁrst 2.84 mm (N = 6). Antennae about 1.5 times as long as third of tarsomere 2; ventral part with silvery hair- wide; cephalic apodeme as long as head capsule. Spatula like scales, coxae with long black setae. Tarsal claws shape variable (Figs 32–37): lateral teeth slightly or thick, evenly curved; empodia longer than bend in claw. conspicuously longer and more pointed than median Wing: dark grey, densely covered by dark hair-like teeth, gap between median teeth slightly or clearly microtrichia (Fig. 18); length 2.00–2.80 mm in males deeper than gaps between lateral and median teeth, (N = 41) and 2.01–3.30 mm in females (N = 57) of shaft thick and well-sclerotized in summer-generation

Figures 23–27. Asphondylia monacha: 23, male flagellomere 5; 24, female flagellomere 5; 25, female flagellomeres 10– 12; 26, female abdomen; 27, male abdomen. Scale bars: 0.1 mm. larvae (Figs 35–37), thinner and less sclerotized in slender (Fig. 63), and are tapered at apex in frontal spring-generation larvae (Figs 32–34). view (Fig. 62). Other attributes are similar in pupae of both generations, as follows. Cephalic seta minute. Pupa (Figs 62–65) Upper facial horn divided into two apices separated The pupae of the summer and spring generations differ by shallow, curved notch. Lower facial horn curved dor- from each other in the shape of the antennal horns. sally at apex, on each side with two papillae, one bearing In summer-generation pupae antennal horns are robust, a relatively long seta. Frons on each side with three wide at base, slightly arched (Fig. 65), with apices flat lateral papillae: one setose and two asetose. Prothoracic and finely serrated in frontal view (Fig. 64); in spring- spiracle long and slender, with widened base; trachea generation pupae antennal horns are longer, more ends at apex. Abdominal segments, except for first, each

Figures 28–30. Asphondylia monacha, male terminalia: 28, dorsal; 29, ventral; 30, lateral. Scale bars: 0.1 mm. with posterior straight row and two or three anterior generation of A. monacha induces galls on other Soli- less ordered rows of spikes. dago species in areas where S. altissima is uncom- mon, but such galls have not been found in the present Notes study. Aggregated bud galls that are very similar to We could not find any substantial morphological dif- those of A. monacha are also found on S. sempervirens ferences among populations from S. juncea, S. erecta, (Fig. 5) and S. bicolor (Fig. 6), and although we could and S. uliginosa, and our molecular analysis indi- not find morphological differences between A. monacha cates that all belong to A. monacha. The galls on and individuals from these populations, our molecu- S. uliginosa are somewhat smaller than those on the lar analysis indicates that the latter belong to one or other two host plants, and were also found in lateral more undescribed species. There are additional Soli- buds, whereas galls on S. juncea and S. erecta almost dago species on which similar composite galls have been always develop in apical buds. Adults reared from galls observed, and further molecular study will probably on S. uliginosa were likewise smaller than those from be necessary to determine whether they belong to the two other hosts. Additional molecular work on the A. monacha or to undescribed species. Felt (1908, 1916) S. uliginosa population may show that it represents attributed rosette and inflorescence galls on Euthamia a separate species. ‘lanceolata’, as well as leaf galls on S. gigantea or Adults of the spring generation that develop on S. Canadensis,toA. monacha, but these galls belong S. altissima are clearly bigger than those of the summer to different species discussed in the present paper. generation. Based on the collection date, one individual from the Felt collection represents the spring gen- Material examined eration of A. monacha, but the host from which it was Spring generation (from S. altissima bud galls): 4Ǩ, reared is not indicated. It is possible that the spring 1 exuviae, 3 larvae, USA, PA, Route 642 (40°59.114′N

Figures 31–37. Asphondylia monacha, larva: 31, head and prothorax; 32–34, spatula of spring-generation larvae; 35– 37, spatula of summer-generation larvae. Scale bars: 0.1 mm.

76°38.567′W), 28 April 2005, N. Dorchin; 3Ǩ, 2 exuviae, N. Dorchin; 1ǩ, USA, PA, Mauses Creek, 16 Septem- USA, PA, Montour Environmental Preserve, ber 2005, N. Dorchin; 3ǩ,12Ǩ, 7 exuviae, USA, PA, 25 May 2007, N. Dorchin; 1 larva, USA, PA, Bucknell Lewisburg, 23 August 2007, N. Dorchin; 1ǩ,USA,PA, University Chillisquaque Creek Natural Area, Lewisburg, 30 August 2007, N. Dorchin; 12ǩ,11Ǩ, USA, 1 May 2005, N. Dorchin; 1ǩ, 2 exuviae, USA, PA, VA, Bedford, Sharp-top, 15 Spetember 2012, M.J. Wise; Bucknell University Chillisquaque Creek Natural Area, 9 pupae, USA, PA, multiple localities, August– 13 May 2005, N. Dorchin; 5 pupae, USA, PA, multi- September 2005–2007, N. Dorchin (on SEM stubs). ple localities, May 2005–2007, N. Dorchin (on SEM stubs). From S. erecta: 4ǩ,3Ǩ, 2 exuviae, 1 larva, USA, VA, Roanoke, 28 August 2010, N. Dorchin and M.J. Wise; Material from Felt collection with no indication of host: 3ǩ,5Ǩ, USA, VA, Roanoke, Spetember 2010, M.J. Wise; 1Ǩ, USA, NJ, Orange Mountain, May 1907? (Felt 3ǩ,9Ǩ, USA, VA, Roanoke, Havens, 5 Septem- no. 813; USNM). ber 2012, M.J. Wise; 3ǩ,3Ǩ, USA, VA, Roanoke, Forest Acre Trail, 9 September 2012, M.J. Wise; 12 pupae, USA, From S. juncea: Gall (syntype), USA, NY, near Brook- VA, Roanoke, August–September 2010, 2012, M.J. Wise lyn, 1867 (USNM); 2 larvae, USA, MD, Wheaton Park, (on SEM stubs). 22 August 1976, R.J. Gagné (USNM); 1 larva, USA, PA, Pittsburgh, 13 August 1991, J. Plakidas (USNM); 1ǩ, From S. uliginosa: 7ǩ,6Ǩ, USA, ME, Winter Harbor, USA, PA, Warrendale, 30 August 1991, J. Plakidas 7 September 2007, R.J. Gagné (USNM). (USNM); 1ǩ,1Ǩ, 3 larvae, USA, PA, Lewisburg, 22 August 2005, N. Dorchin; 1ǩ,2Ǩ, USA, PA, Route Material from Felt collection with no indication of host 642 (40°59.114′N 76°38.567′W), 16 Spetember 2005, and collector (recognized by Felt as A. monacha; all in

USNM): 1Ǩ, USA, NY, Albany, 11 June 1906 (Felt grow. The later-developing rosette galls are probably no. 208); 1Ǩ, USA, NY, Albany, 20 July 1906 (Felt induced in more mature buds that develop more slowly. no. 650a); 1Ǩ,1ǩ, USA, NY, Karner, 5 August 1906 (Felt no. 1583); 1 exuviae, USA, NY, Albany, 4 Sep- Adult tember 1906 (Felt no. 1200); 1Ǩ, USA, NY, Albany, Characters as described in A. monacha except for the 21 August 1906 (Felt no. 761); 1ǩ, USA, NY, Nassau, following. 17 September 1906 (Felt no. 1336); 1Ǩ, USA NY, Albany, 20 July 1907 (Felt no. 1568a); 1ǩ, USA, NY, Bath, Head: Flagellomere 1/ﬂagellomere 5 ratio = 1.19–1.35 24 July 1907 (Felt no. 1568a); 1 larva, USA, NY, Albany, in male (N = 7), 1.53–1.72 in female (N = 5). 24 July 1907 (Felt no. 1583); 1Ǩ, USA, NY, Bath, 16 July 1907 (Felt no. 1568a); 1ǩ, USA, NY, Nassau, Thorax: Wing length 2.15–2.62 mm in males (N = 7), 7 August 1907 (Felt no. 1583a); 1Ǩ, 1 exuviae, USA NY, 2.26–2.80 mm in females (N = 5). Bath, 18 July 1907 (Felt no. 1268); 1ǩ, USA, MA, Mag- nolia, 11 August 1908 (Felt no. 1879). Female abdomen: Sclerotized part of ovipositor 2.44– 3.20 times as long as sternite 7 (N = 6).

ASPHONDYLIA SOLIDAGINIS BEUTENMÜLLER, 1907 Male terminalia: Aedeagus cylindrical, same width Asphondylia solidaginis Beutenmüller, 1907: 305. throughout length, tapered at apex.

Larva (third instar) Host plants Orange; integument covered by round, flat bumps. Solidago altissima and S. gigantea. Length 2.66–3.59 mm (N = 7). Antennae about 1.5 times as long as wide; cephalic apodeme as long as head Gall and biology capsule. Spatula shape relatively uniform among larvae This species has several generations a year and it forms from S. altissima (Figs 38, 39), with lateral teeth the two very different types of galls. The galls that are same length or slightly longer than median teeth, and formed in spring and early summer are blister leaf galls equal gaps between all teeth. In larvae from S. gigantea, that join two (and sometimes three or four) leaves to- median teeth considerably smaller than lateral teeth, gether like a snap (Figs 7–10). When made of two leaves, and gap between median teeth much deeper than gap one leaf contributes the bottom part of the gall and between lateral and median teeth (Figs 42–44). Shaft the other contributes the upper part to form a single- well sclerotized in larvae from both host plants. chambered gall that is thickly lined by white mycelium on the inside. An individual leaf can participate Pupa (Figs 66, 67) in multiple galls, forming the upper part of some and Characters as described in A. monacha, except for the the bottom part of others. These ‘bifoliate’ galls (termed following: antennal horns long and slender, almost snap galls in this paper) are very abundant on straight (Fig. 67), apices splayed, pointed and finely S. altissima and can sometimes also be found on serrated in frontal view (Fig. 66); cephalic seta minute; S. gigantea. Adults emerge from the galls in June and upper facial horn divided into two apices separated by July. From June to August, a rosette gall also appears deep notch. in the apical or axillary buds of S. altissima. These rosette galls are small (3–5 cm in diameter), com- Notes posed of shortened leaves, and contain a single chamber We did not find morphological differences among adults at their centre (Fig. 13). The chamber is formed by or immature stages from the two types of galls induced several very short leaves that are attached together by this species, and our molecular analysis suggests to form a small, rigid cone, and is lined internally by that individuals from these galls belong to the same white mycelium. Adults emerge from these rosette galls species, as the respective haplotypes are intermingled. from late June to late August, when the leaf snap galls Although adult morphology is virtually identical between become less abundant. Although the snap galls domi- individuals from A. solidaginis and A. monacha, their nate in early summer and the rosette galls dominate pupae differ in the shape of the antennal horns, which in late summer, the two types overlap in June–July, are shorter and wider in A. monacha than in A. solidaginis and the type of gall that will result from an oviposition (as well as than those of all other Asphondylia species event is apparently determined by the location in the from Solidago described in this paper). Our molecular plant where the egg is laid. As is nicely described by analysis shows that A. solidaginis uses S. gigantea as Beutenmüller (1907), the snap galls are formed in im- a complementary host on which it induces snap galls mature leaves in young, rapidly growing buds, so that (but never bud galls), whereas snap galls on S. rugosa the leaves that form the gall remain attached as they and E. graminifolia belong to different species.

Figures 38–44. Asphondylia larval spatulae: 38, 39, Asphondylia solidaginis; 40, 41, Asphondylia rosulata sp. nov.; 42–44, spatulae of larvae taken from Solidago gigantea snap galls. Scale bars: 0.1 mm.

Asphondylia solidaginis snap galls on S. altissima cannot (USNM); 1 larva, USA, PA, Dale’s Ridge, 19 June 2006, be mistaken for any other gall on this host, but the N. Dorchin; 1 larva, USA, PA, Bucknell University bud galls induced by this species later in the season Chillisquaque Creek Natural Area, 21 June 2006; 1 may superﬁcially resemble bud galls of other Diptera larva, USA, PA, Montour Environmental Preserve, on this plant. The differences among these galls were 30 June 2006, N. Dorchin; 1 larva, USA, PA, Bucknell summarized above. University Chillisquaque Creek Natural Area, 7 June 2007, N. Dorchin; 1ǩ,2Ǩ, USA, PA Montour Material examined Environmental Preserve, 12 June 2007, N. Dorchin; 1ǩ, The type series of A. solidaginis, consisting of several 3Ǩ, USA, PA, Mifflinburg, 14 June 2007, N. Dorchin; male and female specimens from New York, New Jersey, 1 larva, USA, PA, Dale’s Ridge, 17 June 2007, and North Carolina, could not be located in the N. Dorchin; 3ǩ,1Ǩ, USA, PA, Bucknell University American Museum of Natural History in New York, Chillisquaque Creek Natural Area, 24 June 2007, where it was originally deposited, and is presumed to N. Dorchin; 8 pupae, USA, PA, multiple localities, June– be lost (D. Grimaldi, pers. comm.). We therefore des- July 2006, 2007, N. Dorchin (on SEM stubs). ignate a neotype for this species as follows.

Neotype: Pupal exuviae, USA, PA, Dale’s Ridge, From S. altissima rosette galls: 2 larvae, USA, PA, ǩ 18 June 2005, N. Dorchin; taken from leaf snap gall Lairdsville, 19 July 2007, N. Dorchin; 2 exuviae, 1 , Ǩ ′ ′ on S. altissima. The neotype is mounted on a perma- 1 , USA, PA, Route 642 (40°59.114 N 76°38.567 W), ǩ Ǩ nent microscope slide in Euparal and is deposited in 20 July 2007, N. Dorchin and D. Ryan; 1 ,1 , USA, the USNM. PA, Lewisburg, Furnace Road, 26 July 2007, D. Ryan; Ǩ Other material examined in this study includes the 1 , USA, PA, Lewisburg, Stein Lane, 15 August 2007, following. N. Dorchin; 2 pupae, USA, PA, Lairdsville, July 2007, N. Dorchin (on SEM stubs). From S. altissima snap galls: 4 exuviae, USA, MD, Beltsville, July 1979, R.J. Gagné; 1 larva, USA, PA, From S. gigantea snap galls: 1 larva, USA, PA, Montour Pittsburgh, Weible Rd., 22 June 1991, J. Plakidas Environmental Preserve, 20 June 2006, N. Dorchin; 1

© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015, 174, 265–304 282 N. DORCHIN ET AL. larva, 1 exuviae, USA, PA, Dale’s Ridge, 19 June 2006, inflorescence galls were also found to contain inquilinous N. Dorchin and C. Clarkin; 1ǩ, USA, PA, Bucknell Uni- larvae, usually with one found per gall. The larvae of versity Chillisquaque Creek Natural Area, 24 June 2007, the gall inducer and the inquiline are both orange, but N. Dorchin; 1 larva, 1 exuviae, 1ǩ, USA, PA, Dale’s the latter are much bigger, longer, and more active, Ridge, 5 July 2007, N. Dorchin and D. Ryan; 4 pupae, and the two species are easily distinguished by their USA, PA, multiple localities, June–July 2006, 2007, spatula shape. Larvae of a second species of gall midge N. Dorchin (on SEM stubs). inquiline, Clinodiplosis comitis sp. nov., were often found among the outer leaves of the bud gall. These are much smaller than both the Asphondylia and the Youngomyia ASPHONDYLIA PSEUDOROSA DORCHIN SP. NOV. larvae, and are pale yellow. Other natural enemies found Host plants in the galls are two species of parasitic wasps: one that Euthamia graminifolia. induces dark, spherical endo-galls, such as those found in Asphondylia snap galls, and the other that pupates Gall and biology freely inside the gall. Both are usually found in groups This species has several generations between late June of between four and eight individuals. Parasitism rates and mid-September, during which it forms two types in galls may reach 90%. of galls in buds and inflorescences. The bud galls are single-chambered and contain only one larva; they are Adult composed of a cluster of leaves that are wider and Colour pattern as in A. monacha. Individuals from bud shorter in the outer part of the gall, and thinner and galls are notably larger than those from inflores- longer in its inner part, resembling a rosebud (Fig. 17). cence galls. Some of the wider leaves have a very wide central longitudinal vein that is lighter in colour than the re- Head (Figs 45, 46): Eye facets round. Frons with two mainder of the leaf. Galls are 40–200 mm wide at the groups of seven to ten setae. Palpus three-segmented, base, and most often found in the apical bud but some- segment 3 about as long as segment 2, all segments times also in lateral buds. The base of the galled bud with several strong setae and otherwise covered by is wide and somewhat rigid. The innermost leaves are microtrichia. Labella slightly pointed, with numer- joined to form a conical, apically tapered chamber, the ous strong setae on lateral surface. internal walls of which are lined by white mycelium. As the gall matures, the innermost leaves turn black. The galls are very common from late June to mid- Antenna: Scape and pedicel with long, dark setae. Male August, and become scarcer thereafter. Adults were flagellomere 1 about as long as succeeding flagellomere, reared from them from late June to early September. apical flagellomere slightly shorter than preceding Beginning from August, when inflorescences start de- flagellomere, all flagellomeres covered by anastomosing veloping on the host plants, these are also galled by loops of circumfila, numerous strong setae, and A. pseudorosa sp. nov. Infested inflorescence buds do not microtrichia; flagellomere 1/flagellomere 5 ratio = 1.04– usually show any external signs of infestation, al- 1.27 (N = 14). Female flagellomeres 1–9 cylindrical, with though they may appear somewhat wider than normal two circumfila whorls, numerous strong setae, and other- buds (Fig. 15). The larva feeds on the developing in- wise covered by microtrichia; flagellomere 1 only slight- florescence within the closed bud, and the gall is ac- ly longer than succeeding flagellomere (flagellomere 1/ companied by a white fungal mycelium that lines its flagellomere 5 ratio = 1.31–1.42, N = 7); flagellomeres 7– internal walls. Larvae and pupae are found in the in- 12 successively shorter; flagellomeres 10 and 11 with florescences during August, and adults emerge from two whorls of circumfila and one or two longitudinal mid-August to mid-September. The bud galls of this connections, numerous strong setae, and otherwise species are heavily attacked by the predator/inquiline covered by microtrichia; flagellomere 12 spherical, with Youngomyia podophyllae, the large orange larvae of a single loop of circumfila (Fig. 47). which usually occupy the central chamber of the gall, but are occasionally found among its outer leaves. Thorax: Legs: densely covered by black scales other Between two and ten inquiline larvae can be found than a patch of white scales from base of tarsomere 1 in a single gall, usually with no signs of the larva of to first third of tarsomere 2; ventral part with silvery the gall inducer, but it is not clear whether they ac- hair-like scales, coxae with long black setae. Tarsal claws tually prey on it before taking over the gall. The in- thick, evenly curved; empodia longer than bend in claw. quiline larvae are very active and squirm out of the Wing: dark grey, densely covered by dark hair-like gall when disturbed. When they are present in the gall, microtrichia; wing length 2.38–2.56 mm in males (N =7), the fungus lining the central chamber is not as ap- 2.33–2.98 mm in females (N = 3) from bud galls; 1.57– parent as when the Asphondylia larva is present. Some 1.92 mm in males (N = 9), 1.62–1.75 mm in females

Figures 45–49. Asphondylia pseudorosa sp. nov.: 45, male head; 46, female head; 47, female ﬂagellomeres 10–12; 48, male terminalia, dorsal; 49, male hypoprocts showing intraspeciﬁc morphological diversity. Scale bars: 0.1 mm.

(N = 5) from inﬂorescence galls; R1 joins C before mid- individuals from bud galls; 2.23–2.53 times as long length of wing, R5 joins C behind wing apex, M very as sternite 7 (N = 5) in individuals from inﬂorescence weak, CuA forked. galls.

Male abdomen: Colour pattern as in female. Tergite 1 Female abdomen: Dorsum covered by black scales, narrow, band-like without setae; tergites 2–7 rectan- pleuron and venter with silvery hair-like scales. gular, with posterior row of setae and evenly scat- Tergites 1–7 rectangular, with posterior row of strong tered scales; tergite 7 more setose than preceding tergite; setae and otherwise evenly covered by scales; tergite 8 tergite 8 narrow, band-like dorsally, widened ven- narrower than preceding tergite, without setae. trally, sometimes completely unsclerotized, without setae. Sternites 2–6 with posterior row of setae and several Sternites 2–6 rectangular, with posterior row of setae setae on mid-dorsal part; sternite 7 much longer than and several setae medially, otherwise evenly covered preceding sternite, narrowed posteriorly, with group by scales. Sternite 7 more setose than preceding sternite. of strong setae on posterior half. Sclerotized part of Sternite 8 with small but strongly setose sclerotized ovipositor 2.10–2.61 as long as sternite 7 (N =2)in area.

Figures 50–54. Asphondylia pseudorosa sp. nov. larval spatulae: 50, 51, from inﬂorescence galls; 52–54, from bud galls. Scale bars: 0.1 mm.

Male terminalia (Fig. 48): Gonocoxite compact, wide, Pupa (Figs 70–73) and short, with short apical projection extending me- Antennal horns clearly different between pupae from dially; bearing numerous strong setae and evenly buds and pupae from inflorescences. In pupae from buds, setulose. Gonocoxal apodeme extending on both sides antennal horns long, almost straight, apices pointed of aedeagus to form strongly sclerotized structure. and serrated in frontal view; in pupae from inflores- Gonostylus ovoid, with numerous strong setae and other- cences, antennal horns very short, wide and blunt in wise evenly setulose, bearing crescent-shaped apical frontal view. Cephalic setae minute. Upper facial horn tooth. Aedeagus cylindrical, same width throughout divided into two apices separated by shallow, curved length, tapered at apex. Hypoproct wide at base, divided notch. Lower facial horn curved dorsally at apex, on into two lobes apically by notch of variable size: deep each side with two papillae, one bearing relatively long in some individuals, very shallow in others (Fig. 49), seta. Frons on each side with three lateral papillae, setose and setulose, with one long seta apically on each one setose, two asetose. Prothoracic spiracle long and lobe. Cerci separated to or almost to base, bulbous, slender, with widened base; trachea ending at apex. strongly setose and setulose throughout. Abdominal segments, except for first, each with posterior straight row and two or three anterior, less ordered, rows of spines. Larva (third instar) Orange; integument covered by tiny, shallow bumps. Length 1.44–3.26 mm (N = 18). Spatula (Figs 50–54): Diagnosis variable in shape; median teeth shorter than lateral Asphondylia pseudorosa sp. nov. can be easily distin- teeth, sometimes rudimentary (Fig. 52); gap between guished from other Asphondylia spp. on goldenrods by median teeth clearly deeper than gaps between lateral association with its host plant and galls: the bud gall and median teeth, shaft well-sclerotized, especially along is single-chambered, resembling a small rosebud, and mid-section. is not as flat as the galls of A. rosulata sp. nov. and

A. solidaginis on Solidago rugosa and S. altissima, re- Winter State Park, 22 July 2007, N. Dorchin. Ex in- spectively. Pupae developing in buds are very similar ﬂorescence galls: 2ǩ, USA, PA, Bucknell University morphologically to those of A. rosulata sp. nov., Chillisquaque Creek Natural Area, 19 August 2005, A. solidaginis, and A. silva sp. nov., which all differ from N. Dorchin; 1ǩ, USA, PA, Lewisburg, 29 July 2005, pupae of A. monacha in having more slender, tapered N. Dorchin; 1ǩ, USA, PA, Mauses Creek, 16 Septem- antennal horns. Individuals of A. pseudorosa sp. nov. ber 2005, N. Dorchin; 1ǩ, USA, PA, Hughesville, that develop in inﬂorescences are the smallest of the 2 August 2006, N. Dorchin; 1ǩ, USA, PA, Hughesville, Asphondylia species on goldenrods, and are the only 9 August 2006, N. Dorchin & Michael Wise; 3ǩ,5Ǩ, ones known to use this plant organ. Their pupa differs USA, PA, Route 642 (40°59.114′N 76°38.567′W), from that of all other goldenrod-associated Asphondylia 18 August 2006, N. Dorchin (1Ǩ USNM, others TAUI); in having short antennal horns that are distally trun- 11 larvae, USA, PA, Hughesville, 27 August 2007, cate rather than tapered. N. Dorchin.

Etymology Other material examined The name pseudorosa refers to the typical appear- Ex bud galls: 1ǩ, USA, PA, RB Winter State Park, ance of the galls, which resemble rosebuds. 22 July 2007, N. Dorchin; 1ǩ, USA, PA, Pine Creek, Hampton, 8 August 2010, J. Plakidas (Plakidas, Notes private collection); 1Ǩ, USA, PA, Dorseyville Rd. Smaller bud galls of similar structure to those de- Fox Chapel, 8 August 2010, J. Plakidas (Plakidas, private scribed above commonly develop in lateral buds on this collection); 7 pupae, USA, PA, various localities, host, and at this point it is unclear whether they rep- June–August 2006–2007, N. Dorchin (on SEM stubs). resent a separate species. The adults and immature Ex inflorescence galls: 1ǩ, USA, PA, Route 642 stages described here were taken from the larger bud (40°59.114′N 76°38.567′W), 18 August 2006, N. Dorchin. galls. Some pupae taken from small bud galls have 2 pupae, USA, PA, Mauses Creek, 8 August 2006, shorter, blunter antennal horns than those of pupae N. Dorchin (on SEM stub). from large galls, representing a transitional condi- tion between pupae from large buds and from inflorescences. More detailed morphological and molecular ASPHONDYLIA ROSULATA DORCHIN SP. NOV. work will be needed in order to determine whether the Host plants different types of bud galls on Euthamia graminifolia Solidago rugosa, S. gigantea. represent different species. Our molecular analysis failed to separate between individuals from bud and inflo- Gall and biology rescence galls, hence they are described here as be- This species has several generations a year and forms longing to the same species. snap and bud galls (Figs 11, 12, 14), similar to the situa- Leaf snap galls are uncommonly found on tion in A. solidaginis. Snap galls join two or more leaves E. graminifolia (Fig. 16), and we were unable to rear together, are formed in spring and early summer, and adults from them. We attribute these galls to are usually found on leaves very close to the apical A. pesudorosa based on the pupal morphology and on bud, rather than on more mature leaves farther down the similar situation observed in A. solidaginis and the shoot. In this respect, the galls of A. rosulata sp. nov. A. rosulata sp. nov., where the same species induces both constitute intermediate steps between snap and rosette snap and bud galls on its host plant. galls, and the distinction between these two types is not as clear as in A. solidaginis (Figs 7, 8, 13). As in Type material A. solidaginis, this species appears to use S. gigantea Holotype: Ǩ, USA, PA, RB Winter State Park, 10 Sep- as a complementary host, as indicated by our molecu- tember 2006, N. Dorchin, ex. bud gall on Euthamia lar analysis, but the bud galls are found only on the graminifolia (TAUI). primary host: S. rugosa. The small rosette galls develop only in apical buds and can be locally very common. Paratypes: Ex bud galls: 1 larva, USA, PA, Lewisburg, These galls are composed of several shortened leaves 31 July 2005, N. Dorchin; 1 exuviae, USA, PA, Bucknell that surround a single central, rigid chamber made University, Chillisquaque Creek Natural Area, of closely attached leaves (Fig. 14), and are lined by 26 July 2006, N. Dorchin; 5 larvae, USA, PA, Lewisburg, white mycelium on the inside. Adults emerge from these 4 July 2007, N. Dorchin; 1 larva, USA, PA, White Deer galls from late June to late August. Creek, 4 July 2007, N. Dorchin; 1ǩ,1Ǩ, USA, PA, Lewisburg, 13 July 2007, N. Dorchin; 1ǩ, USA, PA, Bucknell University Chillisquaque Creek Natural Area, Adult 16 July 2007, N. Dorchin; 2 exuviae, 3ǩ, USA, PA, RB Characters as in A. monacha, except for the following.

Head: Flagellomere 1/ﬂagellomere 5 ratio = 1.11–1.18 Paratypes: 1 exuviae, USA, MD, Silver Spring, un- in male (N = 5), 1.38–1.48 in female (N = 5). speciﬁed date, R.J. Gagné (USNM); 1 larva, USA, PA, Pittsburgh, Weible Road, 15 August 1991, J. Plakidas Thorax: Wing length 2.62–2.85 mm in males (N = 6), (USNM); 1Ǩ, USA, PA, Lairdsville, 9 August 2006, 2.61–2.78 mm in females (N = 6). N. Dorchin; 4 exuviae, 4ǩ,4Ǩ, USA, PA, Black Moshannon State Forest, 8 July 2007, N. Dorchin and Female abdomen: Sclerotized part of ovipositor 2.31– M.J. Wise (1ǩ,1Ǩ USNM, others TAUI), 2 larvae, USA, 2.56 as long as sternite 7 (N = 5). PA, Lairdsville, 19 July 2007. N. Dorchin & D. Ryan.

Male terminalia: Aedeagus cylindrical, same width Other material examined throughout length, tapered at apex. 1ǩ,1Ǩ, 1 exuviae, USA, PA, Fox Chapel, Squaw Run Road, 25 June 2010, J. Plakidas (Plakidas, private Larva (third instar) collection). Orange; integument covered by round, flat bumps. Length 2.18–3.46 mm (N = 3). Antennae about as long ASPHONDYLIA SILVA DORCHIN SP. NOV. as wide; cephalic apodeme slightly longer than head Host plants capsule. Spatula (Figs 40, 41) with lateral teeth longer Solidago caesia. than median teeth, and gap between median teeth much deeper than between lateral and median teeth. Gall and biology Pupa (Figs 68, 69) This species induces very small, single-chambered Characters as in A. monacha, except for the follow- galls in apical shoot tips (Figs 19, 20). The gall is ing: antennal horns long and slender, almost straight, composed of several very short leaves that are pressed apices tapered and finely serrated along median margins together to form a conical chamber, the internal walls in frontal view. of which are lined by a white layer of mycelium. Each gall contains a single larva. Galls are 4.5–7.5 mm long and 1.5–3.0 mm wide, and are barely noticeable. They Diagnosis may be very common in some localities, but absent in Asphondylia rosulata sp. nov. can be distinguished from others. The species has at least two generations between other Asphondylia spp. on goldenrods by its host plant June and September; galls were first apparent in early and gall structure. The bud gall resembles that of June and adults emerged from them in early July. In A. solidaginis on S. altissima, and is flatter than that early September, galls were found among flower buds of A. pseudorosa sp. nov. Pupae are morphologically on the shoot tips, and all were already empty, some similar to those of A. solidaginis, A. pseudorosa sp. nov., with pupal skins stuck in them (Fig. 20). This species and A. silva sp. nov., all having antennal horns that is heavily parasitized, and thus very few galls yielded are more slender than those of A. monacha. adult midges in the laboratory. Out of six regularly surveyed localities in central PA, galls were found in Etymology only two (Shikellamy State Park and Dale’s Ridge), The species is named after its gall, which forms a small where they were consistently abundant. rosette in apical buds. Adult Notes Characters as in A. monacha, except for the following. We did not find morphological differences among adults from the two types of galls induced by this species, and our molecular analysis indicated that they belong Head: Flagellomere 1/flagellomere 5 ratio = 1.09–1.23 to the same species. The small rosette galls of this in male (N = 3), 1.41–1.56 in female (N = 4). species may be mistaken for the superficially similar galls of Rhopalomyia solidaginis on S. rugosa but differ Thorax: Wing length 2.37–2.63 mm in males (N = 3), from them in being flatter rather than spherical, com- 2.15–2.51 in females (N = 4). posed of a smaller number of leaves, and containing a central, rigid larval chamber that is lined by white Female abdomen (Fig. 58): Sclerotized part of oviposi- mycelium, similar to the rosette galls of A. solidaginis. tor 2.15–2.24 times as long as sternite 7 (N = 4).

Type material Male terminalia (Fig. 59): Aedeagus about same width Holotype: ǩ, USA, PA, Lewisburg, 26 June 2007, G. Lee throughout length, slightly tapered towards rounded and D. Ryan, from Solidago rugosa leaf snap gall (TAUI). apex. Hypoproct with very shallow notch apically.

Figures 55–61. Asphondylia silva sp. nov.: 55, male head; 56, female head; 57, female ﬂagellomere 5; 58, female abdomen; 59, male terminalia, dorsal; 60, 61, larval spatulae. Scale bars: 0.1 mm.

Larva (third instar) though we did not ﬁnd morphological differences Orange; integument covered by small bumps. Length between them and A. monacha, our molecular analy- 2.02–3.26 mm (N = 6). Antennae about 1.0–1.5 times sis suggests that they belong to at least two undescribed as long as wide; cephalic apodeme as long as head species. These species are not described here because capsule. Spatula shape variable (Figs 60, 61), lateral additional molecular data are needed in order to de- teeth longer than median teeth, gap between median termine their boundaries and host associations. Their teeth as deep as, or clearly deeper than, gaps between galls are locally common and appear similar in struc- lateral and median teeth, shaft thick and well ture to those of A. monacha, although some galls on sclerotized. S. sempervirens develop in lateral rather than apical buds (C. Eiseman, pers. comm.). Galls are composed Pupa (Figs 74, 75) of numerous individual units, each with a single central Characters as in A. monacha, except for the follow- chamber surrounded by several short leaves that are ing. Antennal horns long and slender, only slightly aggregated together to form a spherical structure on curved, apices pointed and ﬁnely serrated in frontal apical buds. Adult midges emerged from S. sempervirens view. from mid-August to mid-September, and from S. bicolor from early September to mid-October. Like other Diagnosis Asphondylia galls from goldenrods, these are also com- This is the second smallest Asphondylia species from monly attacked by the inquiline Youngomyia podophyllae goldenrods and the only one that is found on S. caesia, and by several parasitoid Hymenoptera species. One hence it can be easily recognized from its host and tiny individual from a rosette gall on S. uliginosa is also bud gall. Other than their size and the slightly dif- included in the sempervirens/bicolor clade, whereas ferent shape of the male hypoproct, adults of this species another individual from this host grouped with do not differ morphologically from those of A. solidaginis, A. monacha, suggesting that S. uliginosa is used by two A. rosulata sp. nov., and A. pseudorosa sp. nov., but mo- different species. lecular data consistently indicate that this is a dis- Leaf snap galls were observed once during this study tinct species most closely related to A. rosulata sp. nov. on S. nemoralis, but we were unable to rear adults from them or to characterize them with molecular tools. Etymology Similar galls may be found on other Solidago hosts The name silva (Latin for forest) refers to the typical but the identity of the species that induce them re- habitat in which this species is found. quires further study. Similarly, small rosette galls that resemble those of A. rosulata sp. nov. were observed by Type material R.J. Gagné (pers. comm.) on Solidago tortifolia Elliott, Holotype: ǩ, USA, PA, Shikellamy State Park, 1823 in Maryland in late October, but the identity of 30 July 2007, N. Dorchin and M.J. Wise, from S. caesia the gall inducer is currently unknown. bud gall (TAUI). Asphondylia johnsoni Felt (1908) has been described from an unknown species of Solidago and an Paratypes: 1 larva, USA, PA, Dale’s Ridge, 18 June 2006, unknown gall collected in Lansdowne, PA, USA, and N. Dorchin; 6 larvae, USA, PA, Dale’s Ridge, is separated from A. monacha based on differences in 19 June 2006, N. Dorchin; 4 exuviae, USA, PA, adult colour, which is hardly a reliable character. The Shikellamy State Park, 30 June 2007, N. Dorchin; 1 type we examined and Felt’s more detailed descrip- larva, 1ǩ, USA, PA, Dale’s Ridge, 5 July 2007, tion of the male and pupa do not offer conclusive taxo- N. Dorchin and D. Ryan; 1 exuviae, 1ǩ,4Ǩ, USA, PA, nomic information (Felt, 1916). Without a known host Shikellamy State Park, 30 July 2007, N. Dorchin and and gall, it is impossible to determine whether M.J. Wise (exuviae and 1Ǩ USNM, others TAUI). A. johnsoni is a distinct species or to verify its identity through future collections. We therefore assign this species to nomina dubia. OTHER ASPHONDYLIA SPECIES ON GOLDENRODS Aggregate bud galls that resemble those of A. monacha INQUILINES IN ASPHONDYLIA GALLS ON GOLDENRODS on S. juncea and S. erecta are known from several other goldenrod species, including S. patula, S. odora, and CLINODIPLOSIS COMITIS DORCHIN SP. NOV. an undetermined Solidago species from Florida, based Hosts and biology on material in the National Museum of Natural History, This species lives as an inquiline in Asphondylia Washington, D.C. (USNM). Populations that are as- monacha and A. pseudorosa sp. nov. galls. Dozens of sociated with S. sempervirens in Maine and Massa- tiny, yellowish larvae were found frequently among the chusetts (Fig. 5), and with S. bicolor in Virginia (Fig. 6), rosette leaves (Fig. 21), but we never observed them were studied in detail in the present work, and al- actually interacting with larvae of the gall inducer. They

Figures 62–69. Pupal heads: 62, 63, Asphondylia monacha, spring generation; 64, 65, Asphondylia monacha, summer generation; 66, 67, Asphondylia solidaginis; 68, 69, Asphondylia rosulata sp. nov. Scale bars: 200 μm.

Figures 70–77. Pupal heads: 70, 71, Asphondylia pseudorosa sp. nov. pupae from inﬂorescence galls; 72, 73, Asphondylia pseudorosa sp. nov. pupae from bud galls; 74, 75, Asphondylia silva sp. nov.; 76, Asphondylia silva sp. nov., dorsal spines on pupal abdominal segments; 77, Clinodiplosis comitis, female cerci. Scale bars: 200 μm, except 80 μm in Fig. 76.

Figures 78–83. Clinodiplosis comitis sp. nov.: 78, head; 79, male flagellomere 3; 80, female flagellomere 9; 81, female flagellomere 12; 82, wing; 83, acropod. Scale bars: 0.1 mm. leave the gall to pupate in the ground and adults emerge segment 1, segment 4 about 4.0 times as long as 10–14 days later. This is an extremely delicate segment 1; all segments with fine, long setae and other- cecidomyiid whose dark eyes stand out on the back- wise covered by microtrichia. Face with four or five ground of an otherwise whitish body. Several consecu- short setae on each side. Labrum and labella pointed, tive generations appear to develop during spring and strongly setose. Antenna: 12 flagellomeres in both sexes; summer, and larvae of the last generation most prob- first two flagellomeres fused. Male flagellomeres 1– ably overwinter in the ground. 11 trinodal (Fig. 79): first node setulose, with basal whorl of strong setae, distal whorl of long-looped circumfila, Adult followed by long, bare neck; second node setulose, with Tiny whitish-hyaline gall midge with black eyes. one median circumfilar whorl, followed by third setulose node, with basal whorl of strong setae and whorl of Head (Fig. 78): Small dorsal projection at top of vertex long-looped circumfila, followed by long, mostly bare present just behind eyes, bearing two setae. Eye facets neck. Flagellomere 12 with third node followed by long round. Palpus four-segmented; segment 1 about as long and thin vestigial, setulose appendage, 0.2–0.3 times as wide, segments 2 and 3 about 2.5 times as long as as long as flagellomere. Both proximal and distal necks

Figures 84–87. Clinodiplosis comitis sp. nov.: 84, female post-abdomen; 85, larva head and prothorax; 86, male terminalia, dorsal; 87, larva terminal segment with associated papillae. Scale bars: 0.1 mm. longer in distal flagellomeres than in proximal of setae; sternites with uniformly scattered setae; no flagellomeres: neck 1 to node 1 ratio 0.83–1.37 for discernible trichoid sensilla. Ovipositor protractible, flagellomere 3 (N = 8), 1.10–1.47 for flagellomere 7 about 3.5 as long as sternite 7. Cerci large, setose and (N = 7); neck 2 to nodes2+3 ratio 0.66–0.90 for setulose, ventral and apical areas with numerous thicker, flagellomere 3 (N = 8), 0.89–1.14 for flagellomere 7 blunt sensory setae ending in apical pore (Fig. 77). (N = 7). Female flagellomeres cylindrical, setose and setulose, with simple circumfila and long bare necks of same relative length throughout antenna (Fig. 80); Male abdomen: Segments and setation as described in neck to node ratio for flagellomere 7, 0.61–0.80 (N = 12). female. Flagellomere 12 with long vestigial appendage, about 0.3 times as long as flagellomere, setose and setulose, Male terminalia (Fig. 86): Gonocoxites slender, with rounded apically (Fig. 81). strong setae on mid-distal half. Gonostylus long and slender, only slightly arched, about same width Thorax: Legs: tarsal claws bent beyond mid-length, throughout length, with numerous evenly spread untoothed (Fig. 83); empodia almost reaching bend in short setae, bearing small apical tooth. Aedeagus wide, claw. Wing (Fig. 82): completely transparent, with sparse, conical, extending far beyond hypoproct, with two pairs delicate hairs; length 1.60–2.00 mm in male (N = 11), of pits on apical third; slightly constricted at level of 1.40–2.20 mm in female (N = 10). R1 joins C at third apical pits towards rounded apex. Hypoproct widest of wing length, R5 joins C far beyond wing apex, Rs distally, with wide, deeply concave notch apically, evenly incomplete, situated around midlength of R1; M weak, setulose dorsally except for bare, widened sections along CuA forked, with CuA2 strongly curved posteriad. apicolateral margins. Cerci trapezoid, separated almost to base by a deep notch, narrowing abruptly at Female abdomen (Fig. 84): Sclerites rectangular, vir- half-length to form triangular lobes; evenly setulose, tually undifferentiated from surrounding membrane; with several long setae apically and one strong median tergites with posterior row and one or two median rows seta at base of triangular lobe.

Pupa USA, PA, Rt. 487 (41°21.2′N, 76°17.8′W), 31 July 2006, Unknown. N. Dorchin, ex Asphondylia monacha bud galls on Soli- dago juncea;3ǩ,6Ǩ, same data as holotype (1ǩ & Larva (third instar) (Figs 85, 87) 1Ǩ USNM, others TAUI); 4ǩ,3Ǩ, USA, PA, White Pale yellow, slender. Length: 1.36–2.19 mm (N = 12). Deer Creek, 4 July 2007, N. Dorchin and D. Ryan, ex Integument covered by shallow, acute bumps. Anten- Asphondylia pseudorosa sp. nov. bud galls on Euthamia nae about twice as long as wide; cephalic apodeme as graminifolia (TAUI). long as head capsule. Spatula with two triangular teeth and long, slender shaft, on each side with two groups Other material examined of three tiny lateral papillae with no perceptible setae 2ǩ,2Ǩ, USA, PA, Millersburg, 29 June 2007, N. Dorchin (Fig. 85). Terminal segment with one large and two & M.J. Wise; 4ǩ,3Ǩ, USA, PA, White Deer Creek, smaller pairs of coniform papillae, and fourth pair 4 July 2007, N. Dorchin & D. Ryan. bearing long setae (Fig. 87).

YOUNGOMYIA PODOPHYLLAE FELT, 1907 Diagnosis This species is unique among all 45 described species Dicrodiplosis podophyllae Felt, 1907: 30. of Clinodiplosis in North America for the shape and setation of the male hypoproct and the large female Hosts and biology cerci with their ventral group of blunt sensory setae. The six species in this genus are inquilines, or pos- sibly predators, in galls of various cecidomyiids in the Etymology Nearctic, Neotropical, and Oriental regions (Gagné, 1989; The species name is derived from the Latin word for Gagné & Jaschhof, 2014). Two species are currently ‘companion’, with reference to the fact that it accom- known from North America, Youngomyia quercina Felt, panies Asphondylia galls without causing apparent 1911 from Quercus in California and Y. podophyllae, damage to the gall inducer. which is particularly associated with Asphondyliini galls on Asteraceae host plants in the north-eastern US. In Notes the present study, the large and very active larvae Clinodiplosis is a large cosmopolitan genus represent- of Y. podophyllae were regularly found in groups of ed in North America by 45 described species and many between three and five in galls of all the surveyed undescribed species (Gagné, 1994; Gagné & Jaschhof, Asphondylia species, but were especially common in 2014). Whereas most European species appear to be A. pseudorosa sp. nov. and A. monacha galls. It is unclear mycophagous, many New World species are inquilines, whether they actually prey on the gall inducer’s larva, gall inducers, or even predators (Gagné & Jaschhof, or kill it indirectly by feeding on gall tissue, because 2014), but the life history of many is unknown because feeding behaviour has not been observed directly. they were caught in flight. Morphological attributes Youngomyia podophyllae larvae were abundant in galls of the male genitalia, and in particular of the cerci, from June to September, and the species probably com- are the best taxonomic characters in the genus, al- pletes at least two generations during this time. The though in some cases there may be considerable larvae leave the gall to pupate in the ground, and those intraspecific variability that renders these characters of the last (fall) generation most probably overwinter unusable (Skuhravá, 1973). The shape and setation of in the soil as third instars. the male hypoproct and the large female cerci, with their blunt sensory setae, in C. comitis make this species Adult unique among all described North American species General colour brownish orange. (R. Gagné, pers. comm.). At present we do not know how specific its association with Asphondylia galls on Head (Fig. 88):Eye facets round–hexagonal. Palpus four- goldenrods is, but we never reared it from galls of other segmented, with distinct palpiger; segment 1 slightly cecidomyiids on these plants. longer than wide, segment 2 about 3.5 times as long as segment 1, segments 3–4 about 0.75 times as long Type material as segment 2; all segments with several strong setae Holotype: ǩ, USA, PA, Millersburg, 29 June 2007, and otherwise covered by microtrichia. Face with two N. Dorchin and M.J. Wise, ex Asphondylia pseudorosa or three short setae on each side. Labrum and labella sp. nov. bud gall on Euthamia graminifolia (TAUI). elongate, pointed, and strongly setose. Antenna: 12 flagellomeres in both sexes; first two flagellomeres fused. Paratypes: 8 larvae, USA, PA, Lewisburg, 8 August 2005, Male flagellomeres 1–11 trinodal (Figs 93–95): first node N. Dorchin, ex Asphondylia pseudorosa sp. nov. bud galls setulose, with basal whorl of strong setae and distal on Euthamia graminifolia (4 USNM, 4 TAUI); 7 larvae, whorl of long-looped circumfila, followed by long, bare

Figures 88–95. Youngomyia podophyllae: 88, head; 89, female flagellomere 3; 90, female flagellomere 12; 91, acropod (second tiny tooth on claw not shown); 92, wing; 93, male flagellomere 3; 94, male flagellomere 8; 95, male flagellomere 12. Scale bars: 0.1 mm, except 1 mm for wing. neck; second node setulose, with one median circumfilar flagellomeres than in proximal ones (Figs 93, 94): neck 1 whorl, followed by short setulose neck; third node to node 1 ratio 0.67–1.34 for flagellomere 3 (N = 17), setulose, with basal whorl of strong setae and whorl 1.13–1.98 for flagellomere 10 (N = 10); neck 2 to of long-looped circumfila, followed by long, bare neck. nodes2+3ratio 0.44–0.54 for flagellomere 3 (N = 17), Flagellomere 12 (Fig. 95) with third node followed by 0.54–0.79 for flagellomere 10 (N = 10). Female smaller, vestigial setulose appendage bearing few setae, flagellomeres cylindrical, setose and setulose, with simple ending in short, apically rounded neck; both proxi- circumfila and short, setulose necks of same relative mal and distal flagellomere necks longer in distal length throughout antenna (Fig. 89): neck to node ratio

Figures 96–101. Youngomyia podophyllae: 96, male terminalia, dorsal; 97, male terminalia, inset, ventral; 98, female post-abdomen; 99, proximal part of larval spatula with associated papillae; 100, larval head and prothorax; 101, pupal exuviae, head. Scale bars: 0.1 mm. for ﬂagellomere 5, 0.24–0.28 (N = 5). Flagellomere 12 ent, covered by hair-like microtrichia; length 2.47– with short vestigial appendage, setose and setulose, 3.70 mm in male (N = 19), 2.49–4.22 in female (N = 17). rounded apically (Fig. 90). R1 joins C at third of wing length, R5 joins C far beyond wing apex, Rs incomplete, situated slightly beyond Thorax: Legs: very long and slender. Tarsal claws curved midlength of R1; M weak, CuA forked. close to base, with long, thin tooth and a tiny, barely visible proximal second tooth (Fig. 91); empodia much Female abdomen (Fig. 98): Sclerites rectangular, weakly shorter than bend in claw. Wing (Fig. 92): transpar- sclerotized, with posterior row of setae, several scat-

© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015, 174, 265–304 296 N. DORCHIN ET AL. tered setae on mid-proximal part, and pair of proxi- Material examined mal trichoid sensilla. Abdomen not protractible. Cerci 1 larva, USA, NY, Albany, 26 August 1907, EP. Felt, large and bulbous, bearing evenly scattered setae and ex gall on Solidago patula (USNM); 1 larva, USA, densely covered by short, peg-like setulae. NY, Albany, 26 August 1907, E.P. Felt, ex gall on Solidago odora (USNM); 1 larva, USA, MA, Magno- Male abdomen: Tergites 1–7 rectangular, with posteri- lia, 26 August 1908, EP. Felt, ex Asphondylia gall on or row of setae, a group of setae at mid-proximal part, Aster sp. (Felt no. 1891) (USNM); 2 larvae, USA, MD, and pair of proximal trichoid sensilla; tergite 8 com- Wheaton Pk. 15 July 1968, R.J Gagné, ex. gall on Soli- pletely undifferentiated from surrounding mem- dago altissima (USNM); 2 larvae, USA, MD, Wheaton brane. Sternites 2–7 rectangular, with posterior row Pk., 22 August 1970, R.J. Gagné, ex Asphondylia of setae, numerous setae on proximal half, and a pair monacha gall on Solidago juncea (USNM); 12 larvae, of closely adjacent trichoid sensilla. Sternite 8 smaller USA, PA, Lewisburg, 8 August 2005, N. Dorchin, ex and more setose than preceding sternite. Asphondylia monacha galls on S. juncea;10ǩ,4Ǩ, USA, PA, White Deer Creek, 4 July 2007, N. Dorchin and Male terminalia (Figs 96, 97): Gonocoxites slender, widely D. Ryan, ex Asphondylia pseudorosa sp. nov. galls on ǩ Ǩ splayed, with numerous very strong setae on distal half, Euthamia graminifolia;5 ,7 , USA, PA, Mauses a prominent angular lobe basoventrally, and conspicu- Creek, 4 July 2007, N. Dorchin and D. Ryan, ex ous setose lobe basodorsally. Gonostylus long, slender, Asphondylia pseudorosa sp. nov. galls on Euthamia ǩ Ǩ evenly arched, with numerous setae on distal two- graminifolia;3 ,2 , USA, PA, Millersburg, 4 July thirds, bearing small apical tooth. Aedeagus wide, almost 2007, N. Dorchin and D. Ryan, ex Asphondylia heart-shaped at apex, extending far beyond hypoproct. pseudorosa sp. nov. galls on Euthamia graminifolia;5 ǩ Ǩ Hypoproct conspicuously and densely covered by exuviae, 1 ,5 , USA, PA, Bucknell University short, blunt setae, rounded at apex. Cerci thin, deeply Chillisquaque Creek Natural Area, 15 July 2012, separated and splayed, with several strong setae along N. Dorchin, ex Asphondylia pseudorosa sp. nov. galls posterior margin, setulose throughout. on Euthamia graminifolia.

Larva (third instar) (Fig. 100) PHYLOGENETIC ANALYSIS Bright orange, very long and slender. Length 2.19– The complete molecular data set of cytochrome c oxidase 5.14 mm (N = 19). Integument covered by tiny spic- subunit I (COI, 53 sequences) and elongation factor 1 ules, spiracles conspicuously protruding above body alpha (EF-1α, 33 sequences) consisted of 1047 posi- surface. Antennae about 1.5 times as long as wide; ce- tions (690 COI and 357 EF-1α). All sequences are de- phalic apodeme much longer than head capsule. Spatula posited in GenBank (http://www.ncbi.nlm.nih.gov/) and (Figs 99, 100) long and robust, with two large trian- accession numbers are provided in Table 1. Tree to- gular teeth and long shaft, on each side with two groups pologies inferred from the mitochondrial COI and from of three tiny lateral papillae; two setose, one asetose the nuclear EF-1α genes are largely compatible in each group. Terminal segment on each side with four (Figs 102, 103). The combined phylogenetic analysis setiform papillae on slightly elevated bases. yielded five well-supported clades representing at least six species of Asphondylia on goldenords (Fig. 104). Pupa (Fig. 101) The previously described species A. monacha Small pointed antennal horns; no facial horns. Face and A. solidaginis, and the newly described species on each side with pair of papillae medially, one setose, A. pseudorosa sp. nov., A. rosulata sp. nov., and one asetose, and a group of three asetose papillae A. silva sp. nov., are well supported in this analysis, laterally. Prothoracic spiracle conspicuously long, corroborating inferences drawn from morphological and pointed and strongly sclerotized; trachea ends at apex. life-history data. A fifth, early branching clade in- Abdominal segments, except for first and last, each with cludes individuals from three different Solidago one proximal row of long and slender barbed spikes, hosts as well as Asphondylia recondita from Aster novae- and otherwise covered by tiny spicules. angliae. This clade requires further morphological and molecular sampling before systematic conclusions can Notes be reached; the taxa represented by this clade are there- Several larvae of this species were found in Felt’s slide- fore not described in the present paper. mounted collection, in which they were attributed to Analysis of EF-1α (Fig. 102) provides enhanced reso- Asphondylia because they were taken from Asphondylia lution and support at deeper nodes in the tree, but galls; however, larvae of the two genera can be easily does not differentiate between A. silva sp. nov. and distinguished from each other and the labels on the A. rosulata sp. nov., which form distinct species in relevant specimens were therefore amended. the COI tree (Fig. 103), where shallower nodes are

A. clavata 0.9926 A. sp. Gut A. bigeloviabrassicoides A. monacha jun1 A. monacha alt2 0.7178 A. monacha jun2 A. monacha jun3 A. Silva 3 0.9105 0.3187 A. rosulata gig2 A. silva 4 A. rosulata sn2 1 0.2233 A. rosulata gig1 A. rosulata bud4 A. rosulata sn3 0.2726 A. rosulata sn1 0.9392 A. rosulata bud1 A. rosulata bud2 A. rosulata sn4 A. solidaginis bud3 A. solidaginis sn3 0.3981 A. solidaginis sn4 0.9325 A. solidaginis sn1 1 A. solidaginis gig A. solidaginis sn2 A. solidaginis sn5 A. pseudorosa f2 A. pseudorosa b1 0.1705 A. pseudorosa f3 A. pseudorosa f4 A. pseudorosa b2 0.9999 A. pseudorosa f6 A. pseudorosa f5 A. pseudorosa f5 0.5251 A. pseudorosa f1 A. pseudorosa b4 A. pseudorosa b3

3.0E-4

Figure 102. Phylogenetic tree of Asphondylia species associated with goldenrods based on Bayesian analysis of partial sequence of the elongation factor 1α (EF-1α) gene. Support values are shown next to nodes.

strongly supported. Other discrepancies between the early and retained this host, inducing galls in both two gene trees include the division in the EF-1α tree buds and inﬂorescences. The history of host use to Euthamia-associated and Solidago-associated clades within the A. monacha clade is uncertain, with (Fig. 102), whereas in the COI tree the Euthamia clade four different hosts used by the same gall midge is nested within the Solidago + Aster clade (Fig. 103). species. In both trees, A. monacha is most closely related to Our phylogenetic analyses yielded novel insights into A. rosulata sp. nov. and A. silva sp. nov., and it is clearly contexts of host shifts and the evolution of Asphondylia separated from A. solidaginis. host–plant relationships in the goldenrod species Reconstruction of the evolution of host-plant pref- complex. Further, ﬁner scale patterns of host-plant use erences among Asphondylia spp. on goldenrods (Fig. 105) (plant-part and gall-type associations) within individ- reveals a complex history of host-plant use. Maximum- ual species are revealed with greater granularity parsimony analyses suggest substantial uncertainty (Fig. 104). Asphondylia solidaginis is shown to include in host-plant usage at deeper nodes in the phylog- individuals from two very different types of galls – a eny. Our analyses suggest that S. rugosa is the an- rosette bud gall and a leaf snap – that represent the cestral host for the A. rosulata sp. nov. clade with overlapping spring and summer generations of this subsequent shifts to S. gigantea, and that A. silva sp. nov. species on S. altissima. This species appears to use shifted onto and retained S. caesia as its host. The S. gigantea occasionally as a supplementary host for Euthamia-feeding clade shifted onto E. graminifolia the leaf snap galls. Asphondylia rosulata sp. nov.

0.9764 A. bigeloviabrassicoides A. clavata A. sp. Gut A. sp. semp1 A. sp. bic3 0.7018 A. sp. bic4 0.7813 A. sp. bic2 1 A. sp. ul 0.999 A. sp. bic1 A. sp. semp2 A. recondita A. solidaginis bud1 1 A. solidaginis bud2 A. solidaginis gig A. solidaginis sn4 1 A. solidaginis sn5 A. solidaginis bud3 A. solidaginis sn1 A. solidaginis sn2 A. solidaginis sn3 A. pseudorosa b2 A. pseudorosa b3 1 A. pseudorosa b4 0.7334 A. pseudorosa f6 A. pseudorosa f5 A. pseudorosa b5 A. pseudorosa f4 A. pseudorosa f3 A. pseudorosa f1 A. pseudorosa f2 A. pseudorosa b1 A. silva 2 1 A. silva 1 1 A. silva 3 1 A. silva 4 A. rosulata bud1 A. rosulata bud2 0.9986 A. rosulata sn3 A. rosulata sn4 A. rosulata sn1 A. rosulata gig2 A. rosulata gig1 A. rosulata sn2 0.9725 A. rosulata bud4 A. monacha jun3 A. monacha jun5 A. monacha jun4 A. monacha alt2 A. monacha alt1 A. monacha ul A. monacha jun1 A. monacha jun2 1 A. monacha er3 A. monacha er1 A. monacha er2 A. monacha er4

0.002

Figure 103. Phylogenetic tree of Asphondylia species associated with goldenrods based on Bayesian analysis of partial sequence of the cytochrome c oxidase subunit I (COI) mitochondrial gene. Support values are shown next to nodes. exhibits a very similar pattern but uses S. rugosa rather dago caesia, on which it completes multiple genera- than S. altissima as its primary host. Despite the mor- tions in tiny bud galls throughout the season. The A. phological and biological similarity between these two monacha clade comprises individuals from complex Asphondylia species, they are not closely related. rosette galls on S. juncea, S. erecta, and S. uliginosa Instead, the sister species to A. rosulata sp. nov. is that represent the summer generation of this species. A. silva sp. nov., which is found exclusively on Soli- Also included in this clade are individuals from simple

Figure 104. Phylogenetic tree of Asphondylia species associated with goldenrods based on combined Bayesian analysis of the cytochrome c oxidase subunit I (COI) mitochondrial and elongation factor 1α (EF-1α) genes. Support values are shown next to nodes. Colours refer to host-plant species; icons on branches refer to galled plant organ. Rectangular brack- ets on right indicate species boundaries.

Figure 105. Reconstruction of the evolution of host-plant use by the Asphondylia species associated with goldenrods under maximum parsimony. Branch colours correspond to host species. bud galls on S. altissima that represent the previous- than in other genera in the family (Tokuda, 2012). This ly unknown spring generation of A. monacha. situation makes it very difficult to circumscribe host ranges and associate populations of the same species that develop in different plant species and/or gall types DISCUSSION at different times of the year (e.g. Plakidas, 1988; The systematics and taxonomy of Asphondylia is chal- Yukawa et al., 2003; Uechi et al., 2004). In such cases, lenging because of the general morphological uniform- recognizing species and deciphering their host asso- ity among species and the complexities of their host ciations must rely on the morphology of immature stages and gall-type associations. The ability of certain species and on molecular data. in this genus to exploit numerous unrelated host plants In the present study, we observed only minor dif- is unusual in the Cecidomyiidae, and it is not known ferences between the results of the analyses based on why this phenomenon occurs in Asphondylia more often the mitochondrial and nuclear genes (Figs 102, 103),

© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015, 174, 265–304 SYSTEMATICS OF GOLDENROD ASPHONDYLIA 301 which lends strong support to our taxonomic deci- goldenrod species in the north-eastern USA: S. altissima, sions and conclusions about evolutionary trends in the S. gigantea, S. rugosa, S. juncea, S. caesia, and studied group. Discordance between mitochondrial and E. graminifolia. Although different habitat prefer- nuclear markers may result from limited phylogenetic ences and growth forms among these plant species signal or from introgression resulting from continued result in differences in small-scale distribution pat- gene flow in a recently radiating group (Fontenot, terns (Abrahamson et al., 2005), they are largely Makowsky & Chippindale, 2011). Alternatively, dis- sympatric in most places where we sampled them. cordance could arise from the retention of ancestral Therefore, the pattern of host use by gall midges that polymorphism (Petit & Excoffier, 2009) or incomplete we revealed here cannot be attributed to the avail- lineage sorting among nuclear genes, which typically ability or absence of certain goldenrod species in a have longer coalescent times relative to mitochondrial given area. genes (Stelkens & Seehausen, 2009). The topology of It is noteworthy that some of the Asphondylia our combined tree is influenced to a greater extent by species use several host plants that are not closely the mitochondrial gene, in accordance with the mor- related: for example, A. monacha develops in four Soli- phological and life-history data. Consequently, we cur- dago species, each belonging to a different subsection rently recognize five species of Asphondylia on (of Zhang 1996 and Semple & Cook, 2006). Similarly, goldenrods, and another one or two species that induce A. rosulata sp. nov. uses S. rugosa (subsection Venosae) complex bud galls on S. bicolor and S. sempervirens, and S. gigantea (subsection Triplinerviae). It there- which require further work. Unlike other species com- fore appears that the dominant factor for gall plexes in the genus (e.g. Hawkins et al., 1986; Gagné midges colonizing new host plants in this system is & Waring, 1990; Kolesik et al., 2010), immature stages not the taxonomic relatedness of the plants but local in this group do not offer reliable morphological char- abundance and physical proximity. This could also acters that can be used to distinguish among them, account for the position of the Euthamia-associated and many of the taxonomic decisions in this paper rely clade, which is more closely related to species that are on molecular evidence. Any work that is done on this associated with abundant goldenrod hosts than to those group as additional morpho-species are found will like- from plants with more restricted distributions. Had taxo- wise warrant the use of molecular markers to verify nomic relatedness of the host plants played a more im- species identities and host associations. portant role in the speciation of the gall midges in this group, the Euthamia-associated clade could be expect- ed to form a sister group to the Solidago-associated HOST ASSOCIATIONS clade, rather than being nested within it. The radiation of the Asphondylia species associated with goldenrods apparently occurred through shifts to new host species, which are likely to have driven the evo- INTRASPECIFIC MORPHOLOGICAL DIFFERENCES IN lution of reproductive isolation. This may be a con- RELATION TO GALLED ORGAN tinuing process that will eventually lead to the Superimposing the type of gall on the phylogenetic tree establishment of distinct species of the host-associated (Fig. 104) suggests that bud galling is the ancestral populations within A. monacha. Assuming that the clade habit among goldenrod Asphondylia, with one as a whole is monophyletic, ancestral state reconstruc- switch to inflorescence galls (by A. pseudorosa sp. nov.) tion reveals a complex history of host associations in and three switches to leaf galls (by A. solidaginis, this group; however, such analysis is complicated in A. rosulata sp. nov., and A. pseudorosa sp. nov.). Each the present case by the large number of states (11 hosts) of the leaf-galling species also induces bud galls, and and small number of tips (51), yielding low statisti- the relative proportions of the two types in the popu- cal power to make inferences about deeper nodes in lation depend on the season. The ability to cause these the phylogeny. two types of galls is less surprising once the struc- Although the systematic relations within Solidago ture of the galls is examined closely. Some snap galls have not been sufficiently resolved, the currently avail- – most often those of A. rosulata sp. nov. – are found able phylogeny of the genus (Zhang, 1996) allows us in small apical leaves near the shoot apex, and appear to make some assumptions about the evolution of as- to constitute an intermediate stage between a ‘true’ sociations between the gall midges and their golden- snap gall and an apical bud gall. In all cases, snap rod hosts. An emerging pattern from the present study galls are more common early in the season, whereas and previous ones (e.g. McEvoy, 1988; Gagné, 1989; rosette galls of the same species dominate later on. A Dorchin et al., 2007, 2009) is that the gall midges possible explanation for this phenomenon may be that are most numerous and diverse on the most common the plant’s growth rate is much faster early in the goldenrods. The five Asphondylia species discussed season, so that fully expanded leaves remain at- in this paper develop on some of the most common tached around the larva after an oviposition event to

© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015, 174, 265–304 302 N. DORCHIN ET AL. form a snap gall far below the shoot tip. Later in the al Museum of Natural History, Washington DC) for season, oviposition on slower growing leaves around access to material in the USNM and comments on the apical bud stunts their growth and causes a rosette the manuscript, to Klaus Klass (Museum of Zoology, gall with a central larval chamber. Senckenberg Natural History Collections, Dresden, It is remarkable that the snap-like structure is so Germany) for valuable advice and comments on an similar on all host plants on which these galls are found, earlier version of this manuscript, and to Michael W. and yet they unequivocally belong to three different Gates (USDA, National Museum of Natural History, species. The same is true for the bud galls induced by Washington DC) for the identification of the parasitoids. these species. Together, the similarity in these snap- This study was supported by Bucknell University’s and bud-gall structures induced by different species David Burpee Plant Genetics Chair endowment and suggests convergent evolution of these structures as by the Zoologisches Foschungsmuseum Alexander a result of the same selection pressures on different Koenig, Bonn, Germany. plant species (probably from a combination of natural enemies and host plant-mediated selection). Alterna- REFERENCES tively, if this galling strategy evolved only once among Abrahamson WG, Ball Dobley K, Houseknecht HR, Pecone goldenrod Asphondylia, then A. silva sp. nov. and CA. 2005. Ecological divergence among five co-occurring species A. monacha must have lost the ability to induce leaf of old-field goldenrods. Plant Ecology 177: 43–56. galls and thus develop only in bud galls throughout the season; however, A. monacha does cause a differ- Abrahamson WG, McCrea KD. 1985. Seasonal nutrient dy- ent type of bud gall in early spring in young shoots namics of Solidago altissima (Compositae). Bulletin of the of S. altissima, which is the only Solidago species for Torrey Botanical Club 112: 414–420. which sprouts are available for galling in the field at Abrahamson WG, Weis AE. 1997. Evolutionary ecology across three trophic levels: goldenrods, gallmakers, and natural that time. The structure of this solitary bud gall gen- enemies. Princeton, NJ: Princeton University Press, 456. erally resembles that of the individual units that Adair RJ, Burgess T, Serdani M, Barber P. 2009. Fungal compose the large spherical gall of this species much associations in Asphondylia (Diptera: Cecidomyiidae) galls later in the season on other hosts. Asphondylia from Australia and South Africa: implications for biological pseudorosa sp. nov. is the only species in this complex control of invasive acacias. Fungal Ecology 2: 121–134. that develops also in inflorescences, and the differ- Beutenmüller W. 1907. Descriptions of new species of ences in size and pupal morphology between individ- Cecidomyiidae. The Canadian Entomologist 39: 305–307. uals from buds and inflorescences in this species Bisset J, Borkent A. 1988. Ambrosia galls: the significance probably arise from the type of plant tissue they use, of fungal nutrition in the evolution of the Cecidomyiidae as they do not appear to have a genetic basis. The shift (Diptera). In: Pirozynski KA, Hawksworth DL, eds. Coevo- to inflorescence buds occurs in summer, once these lution of fungi with plants and animals. New York: Aca- become available, and probably offers superior nutri- demic Press, 203–225. tional conditions for the larvae than the now slower- Borkent A, Bisset J. 1985. Gall midges (Diptera: developing vegetative buds (Abrahamson & McCrea, Cecidomyiidae) are vectors for their fungal symbionts. 1985). One wonders why the ability to gall inflores- Symbiosis 1: 185–194. cences did not develop in Asphondylia species on other Craig TP, Itami JK, Ohgushi T, Ando Y, Utsumi S. 2011. goldenrods that flower around the same time. The ability Bridges and barriers to host shifts resulting from host plant of many Asphondylia species to use different host plants genotypic variation. Journal of Plant Interactions 6: 141– and host tissues according to their availability at dif- 145. ferent times is probably a key factor in the success- Crutsinger GM, Cadotte MW, Sanders NJ. 2009. Plant ge- ful radiation of this genus worldwide. netics shapes inquiline community structure across spatial scales. Ecology Letters 12: 285–292. Dixon KA, Lerma RR, Craig TP, Hughes KA. 1998. Gall ACKNOWLEDGEMENTS morphology and community composition in Asphondylia floccosa (Cecidomyiidae) galls on Atriplex polycarpa We are grateful to Gina Lee and Dan Ryan (Bucknell (Chenopodiaceae). Environmental Entomology 27: 592– University) for their help with fieldwork, to Claudia 599. Etzbauer (Museum Koenig) for her invaluable help Dixon MD, Craig TP, Itami JK. 2009. The geographic mosaic with processing samples for the molecular analysis, of co-evolution and the natural enemies of Eurosta solidaginis. to Charley Eiseman for material from and informa- Evolutionary Ecology Research 11: 871–887. tion about the midges from S. sempervirens, to John Dorchin N, Clarkin CE, Scott ER, Luongo MP, Plakidas for his kind permission to examine material Abrahamson WG. 2007. Taxonomy, life history, and popu- from his collection and access to unpublished infor- lation sex ratios of North American Dasineura (Diptera: mation, to Tom Murray for permission to use his photo Cecidomyiidae) on goldenrods (Asteraceae). Annals of the for Figure 18, to Raymond J. Gagné (USDA, Nation- Entomological Society of America 100: 539–548.

Dorchin N, McEvoy MV, Dowling TA, Abrahamson WG, Hawkins BA, Goeden RD. 1982. Biology of a gall-forming Moore JG. 2009. Revision of the goldenrod-feeding Tetrastichus (Hymenoptera: Eulophidae) associated with gall Rhopalomyia (Diptera: Cecidomyiidae) in North America. midges on saltbush in southern California. Annals of the En- Zootaxa 2152: 1–35. tomological Society of America 75: 444–447. Drummond AJ, Ho SYW, Phillips MJ, Rambaut A. 2006. Hawkins BA, Goeden RD, Gagné RJ. 1986. Ecology and Relaxed Phylogenetics and Dating with Confidence. PLoS taxonomy of the Asphondylia spp. (Diptera: Cecidomyiidae) Biology 4: e88. forming galls on Atriplex spp. (Chenopodiaceae) in south- Drummond AJ, Suchard MA, Xie D, Rambaut A. 2012. ern California. Entomography 4: 55–107. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Heath JJ, Stireman JO. 2010. Dissecting the association Molecular Biology and Evolution 29: 1969–1973. between a gall midge, Asteromyia carbonifera, and its sym- Felt EP. 1907. New species of Cecidomyiidae. Albany, NY: New biotic fungus, Botryosphaeria dothidea. Entomologia York State Education Department, Albany. Experimentalis et Applicata 137: 36–49. Felt EP. 1908. Appendix D. Pp. 286–422, 489–510, pls. 33– Janson EM, Peeden ER, Stireman JO, Abbot P. 2010. 34. In his 23d report of the State Entomologist on injurious Symbiont-mediated phenotypic variation without co-evolution and other insects of the State of New York 1907. New York in an insect-fungus association. Journal of Evolutionary Biology State Museum Bulletin 124: 5–541. 23: 2212–2228. Felt EP. 1916. Appendix: a study of gall midges IV. Pp. 101– Jones RG, Gagné RJ, Barr WF. 1983. Biology and taxono- 172, 199–203, pls. 14–18. In his 31st Report of the State my of the Rhopalomyia gall midges (Diptera: Cecidomyiidae) Entomologist on injurious and other insects of the State of Artemisia tridentata Nuttall (Compositae) in Idaho. of New York 1915. New York State Museum Bulletin 186: Contributions of the American Entomological Institute 21: 7–215. 1–79. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. 1994. Joy JB. 2013. Symbiosis catalyses niche expansion and di- DNA primers for amplification of mitochondrial cytochrome versification. Proceedings of the Royal Society of London Series c oxidase subunit I from diverse metazoan invertebrates. Mo- B 280: 2212–2228. lecular Marine Biology and Biotechnology 3: 294–299. Joy JB, Crespi BJ. 2007. Adaptive radiation of gall- Fontenot BE, Makowsky R, Chippindale PT. 2011. inducing insects within a single host-plant species. Evolu- Nuclear-mitochondrial discordance and gene flow in a recent tion 61: 784–795. radiation of toads. Molecular Phylogenetics and Evolution 59: Joy JB, Crespi BJ. 2012. Island phytophagy: explaining the 66–80. remarkable diversity of plant-feeding insects. Proceedings of Fontes EMG, Habeck DH, Slansky F Jr. 1994. Phytophagous the Royal Society B 279: 3250–3255. insects associated with goldenrods (Solidago spp.) in Gaines- Katoh K, Asimenos G, Toh H. 2009. Multiple alignment of ville, Florida. Florida Entomologist 77: 210–221. DNA sequences with MAFFT. Methods in Molecular Biology Futuyma DJ, Agrawal AA. 2009. Macroevolution and the bio- (Clifton, N.J.) 537: 39–64. logical diversity of plants and herbivores. Proceedings of the Kolesik P, Adair RJ, Eick G. 2010. Six new species of National Academy of Sciences of the United States of America Asphondylia (Diptera: Cecidomyiidae) damaging flower buds 106: 18054–18061. and fruit of Australian Acacia (Mimosaceae). Systematic Ento- Gagné RJ. 1989. The plant feeding gall midges of North mology 35: 250–267. America. Ithaca, NY: Cornell University Press. Kolesik P, Veenstra-Quah A. 2008. New gall midge taxa Gagné RJ. 1994. The gall midges of the neotropical region. (Diptera: Cecidomyiidae) from Australian Chenopodiaceae. Ithaca, NY: Cornell University Press. Australian Journal of Entomology 47: 213–224. Gagné RJ, Jaschhof M. 2014. Update for a Catalog of the Maddison WP, Maddison DR. 2011. Mesquite: a modular Cecidomyiidae (Diptera) of the World. 3rd edition, digital system for evolutionary analysis. Version 2.75. Available at: version 2. Available at: http://www.ars.usda.gov/SP2UserFiles/ http://mesquiteproject.org (accessed 3 Oct 2014). Place/12454900/Gagne_2014_World_Cecidomyiidae_Catalog Maddox GD, Root RB. 1990. Structure of the encounter _3rd_Edition.pdf (accessed 11 May 2014). between goldenrod (Solidago altissima) and its diverse insect Gagné RJ, Orphanides GM. 1992. The pupa and larva of fauna. Ecology 71: 2115–2124. Asphondylia gennadii (Diptera: Cecidomyiidae) and taxo- Maia VC, Santos JC, Fernandes GW. 2009. Asphondylia nomic implications. Bulletin of Entomological Research 82: fructicola, a new species of Cecidomyiidae (Diptera) associ- 313–316. ated with Solanum sp. (Solanaceae) from Brazil. Revista Gagné RJ, Waring GL. 1990. The Asphondylia (Cecidomyiidae: Brasileira de Entomologia 53: 166–170. Diptera) of creosote bush (Larrea tridentata) in North America. Maia VC, Silveira FAO, Oliveira LA, Xavier MF. 2008. Proceedings of the Entomological Society of Washington 92: Asphondylia gochnatiae, a new species of gall midge (Diptera: 649–671. Cecidomyiidae) associated with Gochnatia polymorpha (Less.) Gernhard T. 2008. The conditioned reconstructed process. Cabrera (Asteraceae). Zootaxa 1740: 53–58. Journal of Theoretical Biology 253: 769–778. McAlpine JF, Peterson BV, Shewell GE, Teskey HJ, Hakes AS, Cronin JT. 2012. Successional changes in plant Vockeroth JR, Wood DM, eds. 1981. Manual of nearctic resistance and tolerance to herbivory. Ecology 93: 1059– diptera, Vol. 1. Ottawa: Research Branch, Agriculture Canada, 1070. Monograph No. 27.

McEvoy MV. 1988. The Gall Insects of Goldenrods (Compositae: Cecidomyiidae), a gall maker on Borrichia frutescens. Florida Solidago) with a Revision of the Species of Rhopalomyia Entomologist 75: 130–137. (Diptera: Cecidomyiidae). M.S. Thesis, Cornell University, Stireman JO III, Delvin H, Carr TG, Abbot P. 2010. Evo- Ithaca, New York. lutionary diversification of the gall midge genus Asteromyia Nosil P. 2012. Ecological speciation. Oxford: Oxford Univer- (Cecidomyiidae) in a multitrophic ecological context. Molecu- sity Press. lar Phylogenetic and Evolution 54: 194–210. Osten Sacken CR. 1869. Biological notes on Diptera. (Galls Stireman JO III, Devlin H, Abbot P. 2012. Rampant on Solidago). Transactions of the American Entomological host- and defensive phenotype-associated diversification in Society 2: 299–303. a goldenrod gall midge. Journal of Evolutionary Biology 25: Parnell JR. 1964. Investigation on the biology and larval mor- 1991–2004. phology of the insects associated with the galls of Asphondylia Stireman JO III, Nason JD, Heard SB. 2005. Host- sarothamni H. Loew (Diptera: Cecidomyiidae) on broom associated genetic differentiation in phytophagous insects: (Sarothamnus scoparius (L.) Wimmer). Transactions of the general phenomenon or isolated exceptions? Evidence Royal Entomological Society of London 116: 255–273. from a goldenrod-insect community. Evolution 59: 2573– Petit RJ, Excoffier L. 2009. Gene flow and species delimi- 2587. tation. Trends in Ecology and Evolution 24: 386–393. Stireman JO III, Nason JD, Heard SB, Seehawer JM. 2006. Plakidas JD. 1988. The newly discovered spring crown gall Cascading host-associated genetic differentiation in parasitoids of Asphondylia rudbeckiaeconspicua (Diptera: Cecidomyiidae) of phytophagous insects. Proceedings of the Royal Society, B on Rudbeckia laciniata (Asteraceae) in Pennsylvania. 273: 523–530. Proceedings of the Entomological Society of Washington 90: Stokes K, Stiling P, Gilg MR, Rossi AM. 2012. The gall 393. midge Asphondylia borrichiae (Diptera: Cecidomyiidae): an Posada D. 2008. jModelTest: phylogenetic model averaging. indigenous example of host-associated genetic divergence in Molecular Biology and Evolution 25: 1253–1256. sympatry. Environmental Entomology 41: 1246–1254. Rambaut A. 1996. Se-Al: sequence alignment editor. Avail- Tokuda M. 2012. Biology of Asphondyliini (Diptera: able at: http://tree.bio.ed.ac.uk/software/seal (Accessed 3 Oct Cecidomyiidae). Entomological Science 15: 361–383. 2014). Uechi N, Yukawa J. 2006a. Host range and life history of Rambaut A, Drummond A. 2007. Tracer v1. 4. Available at: Asphondylia sphaera (Diptera: Cecidomyiidae): use of short- http://beast.bio.ed.ac.uk/Tracer term alternate hosts. Annals of the Entomological Society of Rohfritsch O. 1997. Morphological and behavioral adapta- America 99: 1165–1171. tions of the gall midge Lasioptera arundinis (Schiner) (Diptera, Uechi N, Yukawa J. 2006b. Life history patterns and host Cecidomyiidae) to collect and transport conidia of its fungal ranges of the genus Asphondylia (Diptera: Cecidomyiidae). symbiont. Tijdschrift voor Entomologie 140: 59–66. In: Ozaki K, Yukawa J, Ohgushi T, Price PW, eds. Galling Rohfritsch O. 2008. Plants, gall midges, and fungi: a three arthropods and their associates: ecology and evolution. Tokyo: component system. Entomologia Experimentalis et Applicata Springer-Verlag, 275–285. 128: 208–216. Uechi N, Yukawa J, Yamaguchi D. 2004. Host alternation Root RB, Cappuccino N. 1992. Patterns in population change by gall midges of the genus Asphondylia (Diptera: and the organization of the insect community associated with Cecidomyiidae). Bishop Museum Bulletin in Entomology 12: goldenrod. Ecological Monographs 62: 393–420. 53–66. Rossi AM, Murray M, Hughes K, Kotowski M, Moon DC, Veenstra-Quah A, Milne J, Kolesik P. 2007. Taxonomy Stiling P. 2006. Non-random distribution among a guild of and biology of two new species of gall midge (Diptera: parasitoids: implication for community structure and host Cecidomyiidae) infesting Sarcoronia quinqueflora survival. Ecological Entomology 31: 557–563. (Chenopodiaceae) in Australian salt marshes. Australian Semple JC, Cook RE. 2006. Solidago. In: Flora of North Journal of Entomology 46: 198–206. America Editorial Committee, ed. Flora of North America Wise MJ, Cole JA, Carr DE. 2010. A field study of poten- North of Mexico, Vol. 20. New York and Oxford: Oxford Uni- tial ecological costs of resistance by ‘stem ducking’ in tall gold- versity Press, 107–166. enrod, Solidago altissima. Entomologia Experimentalis et Shapiro B, Rambaut A, Drummond AJ. 2006. Choosing ap- Applicata 136: 271–280. propriate substitution models for the phylogenetic analysis Yukawa J. 1987. Life history strategies of univoltine gall making of protein-coding sequences. Molecular Biology and Evolu- Cecidomyiidae (Diptera) in Japan. Phytophaga 1: 121–139. tion 23: 7–9. Yukawa J, Uechi N, Horikiri M, Tokuda M. 2003. Descrip- Skuhravá M. 1973. Monographie der Gallmückengattung tion of the soybean pod gall midges, Asphondylia yushimai Clinodiplosis Kieffer, 1894 (Cecidomyiidae, Diptera). Studie sp.n. (Diptera: Cecidomyiidae), a major pest of soybean and Cˇ eskoslovenská Akademie cˇ 17: 1–80. findings of host alternation. Bulletin of Entomological Re- Stelkens R, Seehausen O. 2009. Genetic distance between search 93: 73–86. species predicts novel trait expression in their hybrids. Evo- Zhang JJ. 1996. A molecular biosystematic study on North lution 63: 884–897. American Solidago and related genera (Asteraceae: Astereae) Stiling P, Rossi AM, Strong DR, Johnson DM. 1992. Life based on chloroplast DNA RFLP analysis. PhD thesis, Uni- history and parasites of Asphondylia borrichiae (Diptera: versity of Waterloo, Waterloo.