Predispersal seed predation by a coleophorid on the threatened Gulf ...

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Predispersal seed predation by a coleophorid on the threatened Gulf of St. Lawrence aster Royce Steeves1 Floristic Diversity Research Group, Biodiversity Institute of Ontario, Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1

Vazrick Nazari Biodiversity Institute of Ontario, Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1

Jean-François Landry Agriculture and Agri-Food Canada, Neatby Building, Central Experimental Farm, Ottawa, Ontario, Canada K1A 0C6

Christian R. Lacroix Department of Biology, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island, Canada C1A 4P3

Abstract—The Gulf of St. Lawrence aster, Symphyotrichum laurentianum (Fernald) G.L. Nesom (Asteraceae), a small annual halophyte endemic to disturbed and highly transient habitats in the Gulf of St. Lawrence, is classified as “threatened” by the Committee on the Status of Endangered Wildlife in Canada. Lepidopteran larvae that are predispersal seed predators of the Gulf of St. Lawrence aster are reported for the first time from populations in Prince Edward Island National Park. DNA barcoding was used to identify the seed predators tentatively as larvae of the casebearing moth Coleophora triplicis McDunnough (Lepidoptera: Coleophoridae), which is typically associated with a related halophyte, Solidago sempervirens L. (Asteraceae). These larvae were found to consume a large proportion of seeds from one of two aster populations in Prince Edward Island National Park and may be yet another risk to the survival of this threatened species.

Résumé—L’aster du golfe du Saint-Laurent, Symphyotrichum laurentianum (Fernald) Nesom (Asteraceae), une petite plante halophyte endémique des habitats perturbés et très éphémères du golfe du Saint-Laurent, est considéré comme “menacé” par le Comité sur le statut des espèces menacées de disparition au Canada. Nous signalons ici pour la première fois l’existence de chenilles prédatrices des graines de l’aster du golfe du Saint-Laurent avant leur dispersion dans des populations du parc national de l’Île-du-Prince-Édouard (PEINP). Nous avons identifié provisoirement ces prédateurs des graines à l’aide des codes-barres de l’ADN comme étant les larves du porte-case Coleophora triplicis McDunnough (Lepidoptera: Coleophoridae) typiquement associé à une plante halophyte apparentée, Solidago sempervirens L. (Asteraceae). Ces chenilles consomment une forte proportion des graines dans l’une des deux populations d’asters du PEINP et elles constituent un danger supplémentaire qui pourrait mettre en péril la survie de cette espèce menacée. [Traduit par la Rédaction] Steeves et al. 305

Introduction Predispersal seed predation is the consumption of part or all of a seed prior to dispersal (Janzen 1971). The vast majority of seed predators are

birds, mammals, or insects. Larger seed predators may also act as dispersers if seeds become attached to their body or are passed through their digestive system intact (Janzen 1971), but insects often act solely as seed predators. Insects in many

Received 27 May 2007. Accepted 23 February 2008. 1

Corresponding author (e-mail: [email protected]).

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orders are predispersal seed predators but this behaviour is most commonly observed in flies (Diptera), beetles (Coleoptera), and moths (Lepidoptera) (Fenner et al. 2002). Casebearing moths (Lepidoptera: Coleophoridae: Coleophorinae) in the genus Coleophora Hubner are represented in North America by 147 species of relatively small moths (wingspan 5.7–26 mm) (Baldizzone et al. 2006). The larvae of most species build and live within silken cases that may be camouflaged with plant material, frass, or sand (Landry 1998; Bucheli et al. 2002). First-instar larvae are obligatory leaf or seed miners (Bucheli et al. 2002). Accurate identification of most Coleophora species relies primarily on genitalic characters of adults and the larvae lack sufficient morphological characters for positive identification to the species level. Constraints such as cost and time associated with preparation of genitalia slides as well as the similarity of larval stages have prompted the use of DNA barcoding (Hebert et al. 2003) for identification of species of Coleophora (J.-F. Landry, unpublished data). The Barcode of Life Database (www.barcodinglife.org) currently contains barcode sequences for nearly 160 species of Coleophora, or about 12% of the world fauna, including about 75% of described (and a number of undescribed) Nearctic species. The Gulf of St. Lawrence aster, Symphyotrichum laurentianum (Fernald) G.L. Nesom (Asteraceae), a rare annual halophyte listed as “threatened” by the Committee on the Status of Endangered Wildlife in Canada (COSEWIC 2004) is endemic to New Brunswick, Prince Edward Island, and Quebec’s Magdalen Islands. It inhabits brackish ponds, dune slacks, sandy beaches, and other habitats in protected estuarine areas along the Gulf of St. Lawrence (Houle and Haber 1990; Stewart and Lacroix 2001). Inflorescences appear in mid-July and the florets, which are believed to be self-fertilized (Houle and Haber 1990), develop into achenes by late August to early September. The objectives of this study were to (i) identify the insect responsible for observed predispersal seed predation on the Gulf of St. Lawrence aster on Prince Edward Island, and (ii) obtain some observational data on the effects of the predator.

Methods Seed-predation analyses The Gulf of St. Lawrence aster has been reported at five locations in Prince Edward Island

National Park, but was found at only three sites at Blooming Point (Tracadie Bay, 46°23′N, 62°59′W) in Prince Edward Island National Park during this study: Dune Slack, East Marsh A (EMA), and East Marsh B (EMB) (EMA and EMB are separated by 400 m). In October 2004, six mature plants with mature infructescences were collected from both EMA and EMB and stored at 4 °C. Only six individuals were collected from each population because of the rarity of these asters. Mature seed heads (n = 47) were dissected under a stereomicroscope within 48 h of collection and all achenes (n = 2247) were counted and classified as unfilled (no embryo), filled (containing an intact embryo), or filled and consumed (all or part of the embryo eaten). Population estimates were obtained for Dune Slack, EMA, and EMB by multiplying the surface area (calculated by marking population edges with stakes and measuring the resulting polygon) they covered on the marsh (m2) by their density (plants/m2). Density was determined by tossing a 100 cm2 wire square at random locations within the population (n = 20 at EMA, n = 100 at EMB) and counting individual plants within the square. Additionally, observations of the behavior of larval seed predators and of case structure were recorded. Molecular identification analyses Attempts were made to rear eight lepidopteran larvae that were found consuming achenes of Gulf of St. Lawrence asters at Blooming Point by placing them in test tubes with aster seeds, but the larvae died within days. However, four of these dead and desiccated larvae were visibly free of mold and were retained for DNA analyses. The seed predators were identified as Coleophora sp. by the presence of a silken larval case, which is unique to this group of moths (Heinrich 1923; Hering 1951; Emmet et al. 1996). Larvae in this genus cannot re Z Zellere assigned to species (J.-F. Landry, unpublished data). Because we had very few larvae (which were in poor condition and could not be reared to adulthood) and were unable to reliably associate any Coleophora adults from these localities with observed damage to Gulf of St. Lawrence asters, we employed DNA barcoding techniques to see if the larvae could be identified to species. In addition to the 4 dead larvae associated with achenes of the Gulf of St. Lawrence aster, 35 adult museum specimens from 18 species of North American seed-boring © 2008 Entomological Society of Canada

Locality

Coleophora acuminatoides McDunnough Coleophora acutipennella Walsingham C. acutipennella

CNCLEP00010408

Coleophora bidens Braun

CNCLEP00010374

C. bidens

CNCLEP00010378

Coleophora borea Braun

CNCLEP00024970

CNCLEP00024954

Quebec: Gatineau, Aylmer Colorado: Grand, St. Louis Creek Road Colorado: Grand, St. Louis Creek Road Ontario: Carleton, Mer Bleue Quebec: Terrebonne, lac Brule Michigan: Van Buren State Park Quebec: Pontiac, Eardley Alberta: Dunvegan Provincial Park and historic site Alberta: Dunvegan Provincial Park and historic site Ontario: Wellington County, Puslinch Township Quebec: Pontiac, Eardley Alberta: Lac La Biche

CNCLEP00024955

Alberta: Lac La Biche

CNCLEP00024964 CNCLEP00024965

C. borea

jflandry1281

Coleophora brunneipennis Braun

jflandry1646

C. brunneipennis

jflandry1647

Coleophora detractella McDunnough C. detractella Coleophora dextrella Braun C. dextrella

2005-ONT-957

jflandry0943

Collector

Deposited in:

22 July 1991

J.-F. Landry

EU409074

1 July 1992

T.S. Dickel

14 June 1992

T.S. Dickel

21 July 1992

J.-F. Landry

25 July 1994

J.-F. Landry

24 Aug. 1996

G. Balogh

21 July 2004

J.-F. Landry

Canadian National Collection Research collection of T.S. Dickel Research collection of T.S. Dickel Canadian National Collection Canadian National Collection Research collection of G. Balogh Canadian National Collection Canadian National Collection Canadian National Collection

EU409082

19 July 2003

19 July 2003

EU409075 EU409076 EU409077 EU409078 EU409079 EU409080 EU409081

29 July 2005

P.D.N. Hebert

Biodiversity Institute of Ontario

EU409083

21 July 2004

J.-F. Landry

EU409084

26 July 1994

G.R. Pohl

26 July 1994

G.R. Pohl

Canadian National Collection Canadian National Collection Canadian National Collection

EU409085 EU409086

299


Voucher ID

GenBank accession No.

Collection date

Steeves et al.

Table 1. Coleophorid specimens used in this study.

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Table 1 (continued).

Coleophora duplicis Braun C. duplicis

Voucher ID CNCLEP00010269 CNCLEP00010465

Coleophora intermediella McDunnough C. intermediella

CNCLEP00010151

Coleophora lineapulvella Chambers

2005-ONT-1000

C. lineapulvella

CNCLEP00010326

jflandry1588 CNCLEP00027952

Coleophora salinoidella McDunnough C. salinoidella

CNCLEP00009894

CNCLEP00027956

CNCLEP00009895

Coleophora simulans McDunnough C. simulans

jflandry1718

Coleophora sp.

HLC-16003

jflandry1719

Ontario: Carleton, Blackburn Anderson Rd Ontario: Wellington County, Puslinch Township Quebec: Verchres, St-Amable Massachusetts: Bristol, Massasoit State Park New Jersey: Middlesex, 3 mi. S Dunnellen Nova Scotia: Victoria, Barrier Beach Nova Scotia: Victoria, Barrier Beach Quebec: Gatineau, Gatineau Park Quebec: Gatineau, Gatineau Park Prince Edward Island: Queens, Blooming Point

Collector J.-F. Landry

19 Aug. 1993

J.-F. Landry

19 July 1992

J.-F. Landry

6 July 1992

J.-F. Landry

31 July 2005

P.D.N. Hebert

4 Aug. 2002 21 Aug. 1973

J.B. Heppner

5 Sept. 1973

J.B. Heppner

15 July 1992

B. Wright

23 June 1992

B. Wright

19 May 1999 12 May 1999 19 Oct. 2004

R. Steeves and K. Bujold

Deposited in: Canadian National Collection Canadian National Collection Canadian National Collection Canadian National Collection

GenBank accession No. EU409087 EU409088 EU409089 EU409090

Biodiversity Institute of Ontario

EU409091

Canadian National Collection Florida State Collection of Arthropods Florida State Collection of Arthropods

EU409092

Canadian National Collection Canadian National Collection Canadian National Collection Canadian National Collection Biodiversity Institute of Ontario

EU409095

EU409093 EU409094

EU409096 EU409097 EU409098 EU409099



Coleophora puberuloides McDunnough C. puberuloides

Locality Ontario: Carleton, Mer Bleue Quebec: Pontiac, Gatineau Park Quebec: Vaudreuil

Collection date 27 Aug. 1993

Collector G. Balogh

17 Aug. 1991

G. Balogh

29 July 1988

Coleophora subapicis Braun C. subapicis

CNCLEP00024895

Locality Michigan: Oceana, Little Point Sable Michigan: Oceana, Little Point Sable Alberta: Jasper

CNCLEP00024896

Alberta: Jasper

29 July 1988

Coleophora therinella Tengström C. therinella

jflandry0669

Quebec: Terrebonne, Ste-Agathe Quebec: Terrebonne, Ste-Agathe Maine: Hancock, Winter Harbor New Brunswick: Albert, Shepody Marsh Quebec: Ste-Agathe, Lac Brule Quebec: Ste-Agathe, Lac Brule

30 June 2004

E.J. van Nieukerken and S. Richter E.J. van Nieukerken and S. Richter J.-F. Landry

7 July 2005

J.-F. Landry

23 Aug. 1997

J.-F. Landry

15 Aug. 2005

R. Steeves

3 July 2002

J.-F. Landry

6 July 2002

J.-F. Landry

Coleophora sparsipulvella Chambers C. sparsipulvella

Coleophora triplicis McDunnough C. triplicis

CNCLEP00024972

jflandry2665 CNCLEP00003260 HLC-16001

jflandry2003 jflandry2009

Deposited in: Research collection of G. Balogh Research collection of G. Balogh Naturalis, Leiden, the Netherlands Naturalis, Leiden, the Netherlands Canadian National Collection Canadian National Collection Canadian National Collection Biodiversity Institute of Ontario Canadian National Collection Canadian National Collection

GenBank accession No. EU409100 EU409101 EU409102 EU409103 EU409104 EU409105 EU409106 EU409107

EU409108 EU409109

301


Coleophora versurella Zeller C. versurella

Voucher ID CNCLEP00024971

Collection date 17 Aug. 1991

Steeves et al.

Table 1 (concluded).

302


Fig. 1. From right to left: dorsal view of a Coleophora triplicis larva, a larval case with pappus bristles attached, and a hermaphroditic floret of a Gulf of St. Lawrence aster, Symphyotrichum laurentianum.

Coleophora spp. (Table 1) were selected for molecular analysis. Total genomic DNA was extracted using a protocol previously described by Hajibabaei et al. (2005). Leg tissue from dried adult voucher specimens was used, except for an adult Coleophora triplicis McDunnough (HLC16001, in 70% ethanol) and the dried Blooming Point larvae. All primer pairs (LepF, LepR, MLepF1, and MLepR1), as well as polymerase chain reaction (PCR) and sequencing programs used to amplify the barcode region (658 base pairs (bp) of cytochrome oxidase subunit 1 of the mitochondrial genome), are described in Hajibabaei et al. (2005). Amplified DNA from each specimen was sequenced in both directions, and final sequencing products were run on an ABI 3730 DNA analyzer (Applied Biosystems, Foster City, California). Complementary strands were assembled into contigs and manually edited, and primers were removed using Sequencher 4.5 (Gene Codes Corporation, Michigan). Sequences and associated voucher information were submitted to the Barcode of Life Database (BOLD; www.barcodinglife.com) and GenBank (Table 1). Kimura’s two-parameter model (K2P) of base substitution (Kimura 1980) was used to calculate genetic distances; results were further contrasted with pairwise distances obtained through the neighbour-joining method (Saitou and Nei 1987) using the QUICKTREE algorithm (Howe et al. 2002).

Results Seed predation and population estimates Dissection of seed heads from EMB revealed that 53.7% (n = 468) of sampled achenes containing an embryo were destroyed, presumably by the coleophorid larvae. No seed predation was observed in the sampled seed heads from EMA. The pale-brown tubular larval cases (Fig. 1) of the coleophorid larvae were constructed of silk with pappus bristles from achenes attached and a trilobed valve (superficially resembling the four- to five-lobed corolla tube of florets) at the posterior end; the bristles and valve appeared to provide some measure of camouflage to each case. The larvae were observed to feed with their heads positioned towards the base of the inflorescence and ate mostly the bottom half of the embryo closest to the receptacle, leaving pappus bristles and a hollowed-out pericarp. The Gulf of St. Lawrence aster populations at EMA, EMB, and Dune Slack covered approximately 100, 1200, and 400 m2 and were estimated to consist of 400–500, 115 000, and 25 000 individuals, respectively. Molecular identification analyses Full-length cytochrome c oxidase I (COI) DNA barcodes were obtained for all samples of identified museum specimens of adult Coleophora spp. © 2008 Entomological Society of Canada

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Fig. 2. A neighbour-joining tree of COI barcode sequence distances (K2P) among 18 North American seed-boring Coleophora species and a Symphyotrichum laurentianum seed predator. Known host plants are indicated on the right side of the tree.

(except C. acuminatoides McDunnough, 311 bp) and one of the four larvae collected from Gulf of St. Lawrence asters. All 18 species of Coleophora were organized into distinct clusters in a K2P neighbor-joining distance tree of COI sequences (Fig. 2). The minimum interspecific K2P distance was 1.2% and the maximum distance between nearest neighbors was found to be 5.7%. The single unidentified larva had a COI sequence that was identical with that of adult C. triplicis specimens collected from Maine and New Brunswick. A total of 12 single nucleotide polymorphisms unambiguously identify the C. triplicis cluster from C. dextrella Braun, the next most similar COI sequence from the taxa included in this study.

Discussion Seed predation and population estimates The Gulf of St. Lawrence aster is an annual plant that is adapted to disturbed habitats and depends solely on its seeds to survive. Previous studies have revealed that populations of the Gulf of St. Lawrence aster have small seed banks of low viability (Kemp and Lacroix 2004) and poor dispersal potential because of interference from surrounding vegetation and the influence of flooding (Lacroix et al. 2007), and that the foliage is consumed by coleopteran and lepidopteran larvae (COSEWIC 2004). Our study provides the first evidence of seed predation on this aster. © 2008 Entomological Society of Canada

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Although no seed predation was detected at EMA, a high seed-predation rate (53%) was noted in the seed-head sample from the largest population (EMB; 115 000 plants). This suggests a substantial potential loss of achenes, considering their low viability and poor dispersal potential. Molecular identification analyses The highly endemic nature and relatively small population size of the Gulf of St. Lawrence aster necessitates rapid identification of any predator of this threatened plant. The larvae were easily identified as Coleophora sp. by the presence of their silken larval case, but morphological features are insufficient to identify Coleophora larvae to the species level, especially considering the desiccated state of the larvae used in this study. The perfectly matching COI barcode sequences (with no base-pair difference) of the unknown larva and two adult specimens of C. triplicis, a granivore of Solidago sempervirens L. (Asteraceae) (McDunnough 1940), lead us to identify this particular larva tentatively as C. triplicis. Coleophora triplicis has been previously recorded from Maine, New Brunswick, and many localities in Nova Scotia (McDunnough 1940, 1946; J.-F. Landry, unpublished data). This is the first apparent record of the species from Prince Edward Island, as well as the first apparent association of it with the Gulf of St. Lawrence aster. All other records of C. triplicis are in association with S. sempervirens (McDunnough 1940; Bucheli et al. 2002), a very common species in our study area. The most notable findings of this study are the tentative identification of the granivorous larva as C. triplicis and the large proportion of seeds consumed at EMB (53.7%) compared with EMA (0%), which is only 400 m away. The reasons for this large difference in seed predation over such a small spatial scale may be the small number of plants (six) sampled from each population and (or) biological factors such as population size and proximity to a species that is a host plant of a polyphagous seed predator. Seed predation tends to be directly related to population size (Johannesen and Loeschke 1996; Kruess and Tscharntke 2000; Colling and Matthies 2004). Munzbergova (2006) found no evidence of seed predation by C. obscenella Herrich-Schäffer in populations of Aster amellus L. (Asteraceae) with fewer than 40 ramets, suggesting that some predators require


a minimum plant population size prior to oviposition. It is also interesting to note that S. sempervirens was found growing near all Gulf of St. Lawrence aster populations, especially the EMB population (50 m separates populations). Thus, possible explanations for the high seed-predation rate observed in the EMB sample are that the difference is a sampling artifact, C. triplicis females attracted to S. sempervirens were also laying eggs on nearby Gulf of St. Lawrence asters, and (or) the aster populations at EMA may be too small to attract C. triplicis adults.

Conclusions This study demonstrates that DNA barcoding is an additional tool for species identification; with the aid of barcoding we were able to identify tentatively the otherwise unidentifiable larval coleophorid feeding on seeds of the threatened Gulf of St. Lawrence aster. Further studies are needed to confirm the identity of the coleophorid. Various aspects of its predation upon Gulf of St. Lawrence aster seeds (especially the hypothesis that proximity of this threatened plant to S. sempervirens and (or) population size has an effect on predispersal predation rates) should be investigated. Surveys of seed-predation rates across the aster’s range and through time to accurately quantify seed predation and monitor which species consume its seeds should also be conducted. This may be difficult, as only about 500 Gulf of Saint Lawrence asters were found in Prince Edward Island National Park during population surveys in 2007 (E. Jenkins, University of Prince Edward Island, personal communication). Predispersal seed predation by this coleophorid may pose a substantial risk to the sustainability of the remaining populations of this threatened aster in Prince Edward Island National Park.

Acknowledgements This research was conducted with the generous aid of a Geoff Hogan Research Grant (University of Prince Edward Island), Genome Canada, and scholarships from the Natural Sciences and Engineering Research Council of Canada (NSERC). The molecular component of this study was supported through funding to the © 2008 Entomological Society of Canada

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Canadian Barcode of Life Network from Genome Canada (through the Ontario Genomics Institute), NSERC, and other sponsors listed at www.bolnet.ca. We also thank Keely Bujold for her help with fieldwork, Stephen Darbyshire (Canadian National Collection, Ottawa, Ontario) for his help with scientific names of plants, Jeremy deWaard (University of British Columbia, Vancouver, British Columbia) for his contribution of Coleophora specimens, and Robb Bennett and several anonymous reviewers for their help in improving the manuscript.

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