Introduction Phytophagous insects make up over one-quarter of the described species on Earth and this incredible diversity seems directly linked to their plant-feeding habits1. Comparative studies of sister groups have shown shifts to herbivory are consistently associated with increased species diversity in insects, but the reasons for this diversification remain unclear2. One hypothesis suggests shifts and subsequent adaptation to new host plants directly promotes herbivorous insect speciation3. Host-shift speciation is a form of ecological speciation, where reproductive isolation evolves between populations as a by-product of adaptation to different hosts4. If host-shift speciation is prevalent, there should be evidence of host-driven divergence within insect species occurring on a wide range of host plants.

Figure 1. Early (A) and late (B) instar larvae of the redheaded pine sawfly, N. lecontei.

A.! B.!

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Methods •  Sawfly populations were collected from the

Arboretum in Lexington, KY from one of three different host trees: Shortleaf pine (SL), Pitch pine (P), and Virginia pine (VA).

•  Pre-mating reproductive isolation was measured using no choice assays in petri dishes (1 male and 1 female per dish, Figure 3A); videos of each assay were then analyzed for mating occurrences.

•  Ecological divergence in female oviposition preference was analyzed using no-choice assays. Mated females were placed singly in sleeve cages containing one of the three hosts.

•  Cages were checked daily for eggs. If oviposition occurred, the number of eggs laid was recorded.

•  All eggs laid were allowed to hatch, and reared on the host to cocoon stage. The cocoons were sexed (Figure 3B), and the mass of female cocoons was recorded as a proxy of fitness.

Results

Jeremy Frederick*, Jordan Wolfe*, Robin K. Bagley, & Catherine R. Linnen University of Kentucky, Department of Biology, Lexington, Kentucky

*These authors contributed equally.

Acknowledgments We would like to thank members of the Linnen lab for assistance at various stages of this project. Alonna Ballinger-Wright assisted in weighing the cocoons. RKB was funded by a University of Kentucky Multi-Year Fellowship and Daniel R. Reedy Award. This work was supported by the University of Kentucky and the National Science Foundation. (DEB-1257739)

Conclusions •  There is no evidence of sexual isolation between the

three sympatric populations of Neodiprion lecontei. •  There is some evidence of host preference/avoidance

among the three populations of N. lecontei found in the Arboretum. P females prefer to oviposit in their natal host, while VA pine females avoided pitch. SL females demonstrate no significant host preference.

•  There is no significant difference in the number of eggs laid once a host has been accepted.

•  While not in the predicted direction, there does appear to be variation in fitness on the three host plants. Sawflies reared on pitch and Virginia pines have significantly higher cocoon masses than those reared on shortleaf pine.

Literature cited 1.  Strong, D. R., Lawton, J. H., & Southwood, S. R. (1974). Insects on plants.

Community patterns and mechanisms. Blackwell Scientific Publications. 2.  Mitter, C., Farrell, B., & Wiegmann, B. (1988). The phylogenetic study of

adaptive zones: has phytophagy promoted insect diversification?. American Naturalist, 107-128.

3.  Matsubayashi, K. W., Ohshima, I., & Nosil, P. (2010). Ecological speciation in phytophagous insects. Entomologia Experimentalis et Applicata, 134(1), 1-27.

4.  Schluter, D. (2001). Ecology and the origin of species. Trends in Ecology & Evolution, 16(7), 372-380.

5.  Carvajal-Rodriguez, A., & Rolan-Alvarez, E. (2006). JMATING: a software for the analysis of sexual selection and sexual isolation effects from mating frequency data. BMC Evolutionary Biology, 6(1), 40.

6.  Z Test Calculator for 2 Population Proportions. (n.d.). Retrieved April 27, 2015, from http://www.socscistatistics.com/tests/ztest/.

7.  Microsoft Excel for mac 2011. Microsoft, Redmond, WA. Computer software.

Future Work •  Results in these experiments may have been

influenced by the use of seedlings in the performance assays. We will repeat preference and perfomance analyses using mature hosts to assess this influence.

•  To identify potential sources of divergent selection between sawfly populations, we are currently measuring differences in needle architecture and chemical content.

•  Genetic analyses of gene flow and population structure are forthcoming.

Figure 3: Percentage of females that mated during no-choice assays. Isolation index (IPSI) and p-values were calculated using JMating5. We did not detect significant sexual isolation between SL and P (A), SL and VA (B), or P and VA (C). In all cases, 30 pairs were attempted per combination.

Figure 4A. Proportion of mated females that laid eggs in no-choice cages. P females were more willing to oviposit in their natal host; but VA females avoid pitch. 4B. Number of eggs laid by females in no-choice cages. Females showed no significant difference in the number of eggs between hosts. 4C. Weight of female cocoons. Females achieve significantly different cocoon weights on all hosts, with P females heaviest on their natal host. For all graphs, groups marked with different letters are statistically different in Z-tests6 or t-tests7 (p < 0.05).

Some evidence of ecological divergence in oviposition preference and larval performance.!

Figure 5. Sawfly eggs laid within the needle

of the host tree.

No evidence of pre-mating reproductive isolation between populations. !

0

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SL ♀ x SL ♂ P ♀ x P ♂ SL ♀ x P ♂ P ♀ x SL ♂

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IPSI = -0.0197 p = 0.85

IPSI = -0.0992 p = 0.43

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IPSI = 0.024 p = 0.85

C!

Figure 2A. Screenshot of reproductive isolation assays. 2B.Sawfly cocoons can be easily sexed, as female cocoons

are considerably larger than males.

A.! B.!

♀ !♂ !

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% fe

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Female Line

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Virginia

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(n = 12-16) (n = 6-12) (n = 12)

(n = 10-12) (n = 4-7) (n = 6-11)

(n = 132-200) (n = 93-212) (n = 39-295)

SL ♀ P ♀ VA ♀

SL ♀ P ♀ VA ♀