Transcript S. altissima

Illuminating species identity with molecular data: The case of the goldenrod gall fly
Carrie F. Olson, Andrew W. Jones & Susan Weller
Dept. of Entomology, University of Minnesota, Dept. of Ecology, Evolution and Behavior
Mil2 gigantea
AH14 gigantea
Fig 5 : Unrooted network of
unique haplotypes. Two
haplotypes were found for
gigantea flies and four haplotypes
were found for altissima flies.
1.00 is the posterior probability
value supporting reciprocal
monophyly of the two host-race
clades.
Introduction
The goldenrod fly (Eurosta solidaginis) is an herbivorous gallmaker found in prairie
and forest habitats in the Upper Midwestern United States (1). The flies attack two species
of goldenrod, Solidago altissima (“altissima flies”) and S. gigantea (“gigantea flies”) (1). A
tumor-like growth (gall) is formed by the host plant after the female fly deposits eggs on the
bud (Fig. 1)(1). Fly galls are subject to predation from birds and other insects, including a
beetle and two parasitic wasps (1). This interaction has been used to demonstrate balancing
selection and to study trophic interactions (1,2,3). An open question remains whether host
plant or habitat specialization is occurring in the goldenrod fly (4). One hypothesis is that
the flies are undergoing speciation driven by the female fly’s selection of either S. altissima
or S. gigantea (2). A competing hypothesis is that the fly is undergoing speciation as the
result of habitat selection (prairie versus forest) (4). In Minnesota, both goldenrod species
co-occur in the two habitats, and these hypotheses can be tested. Two objectives are
addressed here: 1) Test whether E. solidaginis is one or two species using mitochondrial
COI –COII sequences ; 2) Using the haplotype tree, test whether flies are differentiating by
habitat (ecological races) or by host (host races). We also examined whether these fly
populations are stable or expanding, indicating post-glaciation expansion.
1.00
Table2: Specimen codes for goldenrod flies with their collecting sites and host plant
Designation
Collection Location
Host Plant
CA
Carlos Avery Wildlife Management Area
S. altissima
DMR
Des Moines River
S. altissima and S. gigantea
AL
Arnold Lake
S. altissima
CC
Clay County
S. altissima
E
Elko Lake
S. altissima and S. gigantea
FP
Felton Prairie
S. altissima
Mil
Milford
S. altissima and S. gigantea
SL
Shield Lake
S. altissima and S. gigantea
SB
Seven Bridges
S. altissima
NM
New Market
S. altissima
RP
Rahn Park
S. gigantea
AH
Arden Hills
S. altissima and S. gigantea
SP
St. Peter’s
S. gigantea
BN
Bagley Nature Center, Duluth
S. gigantea
CA7 altissima
AL4 altissima
W2-1 altissima DMR1 altissima
Results & Discussion (continued)
Population Analysis – Mismatch distributions show a poisson distribution, reflecting
past exponential population growth (Fig. 6). A significantly negative value for FU’s
Fs (11) confirms this interpretation. This result is not surprising, given pleistocene
glaciation in the northern plains. The time since expansion is approximately twice as
old in gigantea flies as in altissima, as indicated by the mode. This result suggests that
gigantea flies recolonized Minnesota before the altissima flies, post-glaciation.
Fig. 2: Map of Minnesota sampling
locations based on habitat.
Results & Discussion
Table 1. Sampling scheme
Host plant: Gene regions
Solidago altissima
Solidago gigantea
Forest
Prairie
COI
10
9
COII
6
2
Both
23
11
COI
8
9
COII
2
1
Both
13
6
Fig. 1 Gall formed by E.
solidaginis on S. altissima.
Taxa and DNA protocols: Standard insect DNA extractions, PCR amplification and
automated sequencing techniques were used to sample portions of the mitochondrial
gene COI (1491bp) and COII (785bp) for 100 flies from known hosts and habitats
(Table 1, Fig. 3). Sequences were aligned in Sequencher 3.1.1 (Gene Codes Corp., Ann
Arbor) AND exported as NEXUS files for analysis. Flies were reared on either S.
altissima or S. gigantea from either forest or prairie localities by Dr. Itame (U. MN,
Duluth).
Phylogenetic Analysis: To address the question of whether flies were differentiating
by habitat or host, 92 individuals sequenced for COI were analyzed. Neighbor-joining
(NJ) analysis (13) was implemented with PAUP* and bootstrapped (15). Habitat and
host associations were mapped on the resultant consensus tree. These clades were used
to assign individuals for the haplotype mismatch analysis.
To address how many unique haplotypes exist and the structure of their relationships,
56 flies sequenced for both COI and COII (2277 bp) were reduced to unique
haplotypes using Arlequin 2.0 (14). The optimal model of molecular evolution (TrN
+I) was determined using Akaike Index Criterion (AIC) in Modeltest3.6 (10), and then
a phylogenetic hypothesis of relationships generated using MRBAYES 3.2 (12). We
ran the analysis for 2 million generations, with 4 “heated” chains sampled every 100
generations. The first 1000 trees were discarded as the “burn-in”.
Population Analysis: To address whether fly populations were stable or expanding,
we used the large set of 92 flies sequenced for COI. From the NJ analysis, individuals
were assigned to clades. Both clades were analyzed separately in Arlequin 2.0 to
generate mismatch distributions and calculate Fu’s Fs (6).
The 56 flies sequenced for both COI-COII reduced to 6 haplotypes, 4
altissima and 2 gigantea (Fig. 5). The two clades were reciprocally
monophyletic with a posterior probability of 1.00.
Gigantea flies
Altissima flies
180
900
160
800
140
700
120
600
100
Observed
Simulated
80
60
Frequency
Materials and Methods
Fig. 6. Plots of the mismatch distributions for Gigantea and Altissima fly populations.
Plots represent the frequency of pairwise haplotype differences. The mode is indicated
by the vertical black line for each population.
Frequency
Phylogenetic analysis – When the 92 flies sequenced for COI were analyzed,
two distinct clades were recovered. When habitat is mapped on this consensus
tree (Fig. 3), no pattern emerges; however, when host plant is mapped on, the
haplotypes are clearly segregating by host (Fig. 4). Individuals in the two
clades differ by 13 to 27 base pairs. The genetic distance between these
clades is 0.87 to 1.81% over COI, which is consistent with recognition of host
races in flies. These numbers are not corrected.
500
400
300
40
200
20
100
0
0
0
2
4
6
8
Observed
Simulated
0
# Pairwise Differences
Fig 3: Habitat NJ network of 92 flies
sequenced for COI with habitat type for
each individual indicated by color
(Forest habitat = green lines, Prairie
habitat = black lines)
Fig 4: Host plant NJ network of 92 flies
sequenced for COI with natal host plant
for each individual fly indicated by color
(Gigantea reared flies = blue lines,
Altissima reared flies = black lines)
2
4
6
8
# Pairwise Differences
Conclusions
•A 1.2-1.4% genetic difference was found between host-races. Whether these host
races should be recognized as separate species is still unclear. More populations
from across the entire range need to be sampled.
•Genetic differentiation of flies is based on host-plant, not habitat preference
•Limited host switching is occurring. However, one initial case was an artifact of the
amount of sequence (COI vs COI+COII). With more sequence, the fly placed in the
appropriate host clade. Female E. solidaginis are very faithful to their natal hosts.
•Mismatch distribution displays characteristics of expanding populations for both
gigantea and altissima flies.
References:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Abrahamson, W.G. and Weis, A.E. 1997. Evolutionary Ecology across three trophic levels: goldenrods, gallmakers and natural enemies.
Abrahamson, W.G., Eubanks, M.D., Blair, C.P. and Whipple, A.V. 2001. Gall flies, Inquilines, and Goldenrods: A model for Host-race Formation and
Sympatric Speciation. American Zoologist, 41, 928-938. Princeton University Press, Princeton.
Brown, J.M., Abrahamson, W.G. and Way, P.A. 1996. Mitochondrial DNA phylogeography of host races of the goldenrod ball gallmaker, Eurosta
solidagninis (Diptera: Tephritidae). Evolution, 50, 777-786.
Craig et al. 2001. Genetics, experience and host plant preference in Eurosta solidaginis: implications for host shift and speciation. Evolution 55: 773-782.
Felsenstein, J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39:783-791.
Fu, Y.X. (1997) Statistical tests of neutrality of mutations against population growth, hitchhiking and background selection. Genetics, 147, 915-925.
Murmur J. 1961. A procedure for the isolation of deoxyribonucleic acids from microorganisms. Journal of Molecular Biology, 3, 208-218.
Saiki, R.K., Gelfand, D.H, Stoffel, S., Scharf, S.J., Higuchi, R., Horn. G.T., Mullis, K.B. and Erlich. H.A. 1988. Primer-directed enzymatic amplification of
DNA with a thermostable DNA polymerase. Science, 239, 487-491.
Pavlova, A., R. M. Zink, S. V. Drovetski, Y. Red’kin, and S. Rohwer. 2003. Phylogeographic patterns in Motacilla flava and M. citreola: species limits and
population history. Auk 120:744-758.
Posada, D. and Crandall, K.A. 1998. Modeltest: testing the model of DNA substitution. Bioinformatics 14 (9): 817-818.
Rogers A. R., Harpending H. 1992 Population Growth Makes Waves in the Distribution of Pairwise Genetic Differences Molucular Biology and Evolution 9:
552-569
Ronquist, F. and J. P. Huelsenbeck. 2003. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572-1574
Saitou N., Nei M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4: 406-425
Schneider, S., Roessli, D., Excoffier, L. (2000) Arlequin: A software for population genetic data. Genetics and Biometry Laboratory, University of Geneva,
Switzerland.
Swofford, D. L. 2000. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sinauer Associates, Sunderland, Massachusetts.
Acknowledgments: Dr. Susan Weller, Andy Jones, Michelle DaCosta, Dr. Tim Craig, Dr. Joanne Itami, and Crystal Boyd