Lysiphlebus testaceipes (Cresson) is a solitary endoparasitoid of aphids and is the primary parasitoid attacking cereal aphids in the Great Plains, especially Schizaphis graminum (Rondani). In a previous study, it was found that a Lincoln, NE, isolate of L. testaceipes had a much higher survivorship at cold temperatures than isolates from Stillwater, OK, and Corpus Christi, TX. This suggested that the Nebraska isolate was locally adapted to the northern environment and perhaps genetically divergent from southern populations. We tested for genetic differentiation of the above isolates by sequencing portions of the COI and 16S mtDNA genes. We also examined a Florida isolate reared from Toxoptera citricida (Kirkaldy) and L. fabarum Marshall as an outgroup. The Great Plains isolates (Nebraska, Oklahoma, and Texas) were homogeneous with 0% and 0Ð0.2% sequence divergence in the COI and 16S gene fragments, respectively. The Florida isolate differed from the Great Plains isolates in nucleotide sequence by 1.4% (COI) and 0.5Ð0.7% (16S). Phylogenetic analysis placed the Florida isolate of L. testaceipes basal to the Great Plains isolates with L. fabarum, suggesting a possible species complex within L. testaceipes

Elliott, N.C.

Jones, D.B.

Shufran, K.A.

Weathersbee, A.A., III

English

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Publications from USDA-ARS / UNL Faculty U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska 
2-18-2004 
Genetic Similarities Among Geographic Isolates of Lysiphlebus 
testaceipes (Hymenoptera: Aphidiidae) Differing in Cold 
Temperature Tolerances 
K.A. Shufran 
A.A. Weathersbee III 
D.B. Jones 
N.C. Elliott 
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MOLECULAR ECOLOGY AND EVOLUTION
Genetic Similarities Among Geographic Isolates of
Lysiphlebus testaceipes (Hymenoptera: Aphidiidae) Differing in
Cold Temperature Tolerances
K. A. SHUFRAN,1, 2 A. A. WEATHERSBEE III,3 D. B. JONES,4 AND N. C. ELLIOTT1
USDAÐARS, Plant Science and Water Conservation Research Laboratory, 1301 N. Western Road, Stillwater, OK 74075
Environ. Entomol. 33(3): 776Ð778 (2004)
ABSTRACT Lysiphlebus testaceipes (Cresson) is a solitary endoparasitoid of aphids and is the
primary parasitoid attacking cereal aphids in the Great Plains, especially Schizaphis graminum (Ron-
dani). In a previous study, it was found that a Lincoln, NE, isolate of L. testaceipes had a much higher
survivorship at cold temperatures than isolates from Stillwater, OK, and Corpus Christi, TX. This
suggested that the Nebraska isolate was locally adapted to the northern environment and perhaps
genetically divergent from southern populations. We tested for genetic differentiation of the above
isolates by sequencing portions of the COI and 16SmtDNA genes.We also examined a Florida isolate
reared from Toxoptera citricida (Kirkaldy) and L. fabarumMarshall as an outgroup. The Great Plains
isolates (Nebraska, Oklahoma, and Texas) were homogeneous with 0% and 0Ð0.2% sequence diver-
gence in the COI and 16S gene fragments, respectively. The Florida isolate differed from the Great
Plains isolates innucleotide sequenceby1.4%(COI) and0.5Ð0.7%(16S). Phylogenetic analysis placed
the Florida isolate of L. testaceipes basal to the Great Plains isolates with L. fabarum, suggesting a
possible species complex within L. testaceipes.
KEY WORDS parasitoid, aphid, biological control, geographic variation, cold tolerance
Lysiphlebus testaceipes (Cresson) is a solitary endo-
parasitoid of aphids and is native to the United States.
Its geographic range is Nearctic, Neotropical, and
Oceanic, and it is also found in Europe as a result of
intentional introductions (Mackauer and Stary´ 1967).
Ithas averybroadhost range,havingbeenreared from
over 120 aphid species (Mackauer and Stary´ 1967,
Stary´ et al. 1988, Pike et al. 2000, French et al. 2001,
Tang et al. 2002). In the American Great Plains, it is a
primary biological control agent of aphid pests in ce-
real and grain crops, especially wheat (Triticum aes-
tivum), barley (Hordeum vulgare), and sorghum (Sor-
ghum bicolor). Among the most common hosts for L.
testaceipes in these agriculture systems are Schizaphis
graminum (Rondani), Rhopalosiphum maidis (Fitch),
and R. padi L.
Despite the importance of L. testaceipes as a bio-
logical control agent, surprisingly little is known of its
ecology or population genetics. For a species with
such a wide geographic distribution and host range,
we would expect to Þnd genetic divergence. A few
studies have shown diversity within L. testaceipes.
Based on random ampliÞed polymorphic DNA and
allozyme data, constrained gene ßow was found
among L. testaceipes populations parasitizing Aphis
varians Patch on Þreweed (Epilobium angustifolium
L.) in subalpine mountain drainages (Hopkins 1998).
Variation in L. testaceipes has been noted in mtDNA
sequence divergence betweenWashington and Okla-
homa populations (Chen et al. 2002). Also, successful
parasitism of Aphis spiraecola (Patch) (a synonym of
A. citricola van der Groot) by L. testaceipes varies by
location, presumablybecauseof genetic differences in
the wasp (Costa and Stary´ 1988). Incomplete devel-
opment occurs in California, France, and Italy, but A.
spiraecola is the principal host ofL. testaceipes in Cuba
(Costa and Stary´ 1988). Royer et al. (2001) demon-
strated that a Nebraska population had a much higher
survivorship at cold temperatures than populations
from Oklahoma and Texas. This suggested local ad-
aptation to the environmental extremes and possible
genetic divergence of populations. Host adaptation
was not an issue in this case, because all three popu-
lations originated from S. graminum (Royer et al.
2001).
To test the hypothesis of geographical adaptation,
we explored whether there was parallel genetic vari-
ation inmtDNAgenes andcold tolerancebetween the
Mention of trade names or commercial products in this article is
solely for the purpose of providing speciÞc information and does not
imply recommendation or endorsement by the United States Depart-
ment of Agriculture.
1 USDAÐARS, Plant Science &Water Conservation Research Lab-
oratory, 1301 N. Western Road, Stillwater, OK 74075.
2 E-mail: kevin.shufran@ars.usda.gov.
3 USDAÐARS,U.S.HorticulturalResearchLaboratory, 2001S.Rock
Road, Fort Pierce, FL 34945.
4 Department of Entomology and Plant Pathology, 127 Noble Re-
search Center, Oklahoma State University, Stillwater, OK 74078.
three geographic isolates of Royer et al. (2001). We
also included another population fromFlorida, reared
fromToxoptera citricida (Kirkaldy), to test for possible
geographic and/or host variation.
Materials and Methods
Insect specimens were air-dried, laboratory-reared
female L. testaceipes of the three geographic isolates
(Lincoln, NE; Stillwater, OK; andCorpus Christi, TX)
examined by Royer et al. (2001). All three isolates
were originally collected from S. graminum on sor-
ghum and laboratory reared for their experiment on
cold tolerance. Each colony was established from 50Ð
100 females and reared 3Ð4 generations (Royer et al.
2001). Specimens of another L. testaceipes laboratory
population collected from T. citricida in Fort Pierce,
FL, also were used (Tang et al. 2002). The Florida
colony was established in November 1999 (Tang et al.
2002), and our specimens were removed from the
colony in April 2000 and preserved in 95% ethyl al-
cohol. An asexual form of L. fabarumMarshall, reared
from Aphis schneideri Bo¨rner in Germany, was the
outgroup (preserved in 95% ethyl alcohol) (Belshaw
et al. 1999). A wild population of L. testaceipes reared
from R. padi in Stillwater, OK (six females), also was
included. Voucher specimens are held by KAS.
We polymerase chain reaction (PCR)-ampliÞed
portions of the cytochrome oxidase subunit I (COI)
and16S rRNAmitochondrial genes for DNA sequenc-
ing. Three females in each isolate were used. Each
insect was ground in a buffer according to Black et al.
(1992), and 1Ð2 l of the homogenate was used as
DNA template per reaction. Three to four reactions
were conducted for each individual, isolate, and gene
combination.
The primers used for PCR ampliÞcation and se-
quencing were as follows: COI, C1-J-1718 and C1-N-
2191 (Simon et al. 1994); 16S, 16SWb and 16S.Sh
(DowtonandAustin1994,Chenetal. 2002).Reactions
were carried out in 50-l volumes with 20 mM Tris-
HCl, pH 8.4; 50 mM KCl; 0.4 mM dNTPs; 1.5 mM
MgCL2 for the COI and 3.0mMMgCL2 for the 16S; 20
pmol each primer; and 2.0 U Taq DNA polymerase
added at the Þrst annealing step. An MJ Research
PTC-100 Thermal Controller with “Hot-Bonnet” was
used with the following program steps: (1) 96C for 3
min; (2) 94C for 30 s; (3) 45C (16S) or 50C (COI)
for 30 s; (4) 72Cfor 1min; (5) cycle to step2, 29 times;
(6) 72C for 5 min; (7) 4C hold. PCR products were
gel-puriÞed using the Wizard PCR Preps DNA Puri-
Þcation System (Promega, Madison, WI) and were
directly sequencedusinganABIautomated sequencer
at the Sequencing and Genotyping Facility (Depart-
ment of Plant Pathology, Kansas State University,
Manhattan, KS).
The sequences were aligned with ClustalW
(Thompson et al. 1994) using the Lasergene Version
5.06 software package (DNASTAR, Madison, WI).
Distance and maximum likelihood (ML) methods
were used in phylogeny reconstruction with the Phy-
logenetic Analysis Using Parsimony (PAUP) version 4
statistical package (Swofford 2002). Distances were
estimated by the method of Tamura and Nei (1993)
using gamma correction factors of   0.0132 (16S)
and   0.0125 (COI) estimated by ML in PAUP.
Dendrograms were produced with neighbor-joining
(NJ) analysis (Saitou and Nei 1987) based on the
above estimated distances with 500 bootstrap replica-
tions.MLdendrogramswere producedby themethod
of Hasegawa et al. (1985) using a heuristic search
method. L. fabarum was declared the outgroup in all
tests. All sequences were submitted to GenBank (ac-
cession numbers: AY207558ÐAY207567).
Results and Discussion
PCR products for the COI and 16S genes were both
500 bp in length. However, we were able to obtain
only 428 and 438 usable nucleotides for the COI and
16S fragments, respectively, because of sequencing
errors at the 5 and 3 ends. The nucleotides adenine
and thiamine were abundant in both the COI (74.2%)
and 16S (82.3%) fragments and may have contributed
to sequencing error.
Each isolate was homogeneous, with no sequence
variation among individuals. Therefore, only one se-
quence for each isolate was used. Genetic distances
among L. testaceipes isolates and L. fabarum (in per-
cent nucleotide divergence) are shown in Table 1.
Despite a signiÞcant difference in L. testaceipes from
Nebraska to withstand cold temperatures compared
with those inOklahoma and southern Texas (Royer et
al. 2001), we found the mtDNA gene sequences of
these three populations to be homogeneous. There
were no differences in their COIDNA sequences, and
their 16S DNA sequences were minimally divergent.
Even though the 16S-NJ tree separated the Nebraska
isolatebranch fromtheOklahomaandTexasclade, the
separation had only 56% bootstrap support.
Despite the observed homogeneity of the Great
Plains L. testaceipes populations, there was weak evi-
denceprovidedby theNJ treebasedon16S sequences
(Fig. 1) that the Nebraska population may be diverg-
ing from the Oklahoma and Texas populations. How-
ever, each geographic isolate may not represent the
total variation that could be present in the wild pop-
ulation. In fact, homogeneity of each laboratory may
have been increased by the method of establishment
and rearing. However, a second population of L. testa-
ceipes from Stillwater, OK, had identical 16S and COI
sequences as theRoyer et al. (2001) isolate fromOkla-
Table 1. Percent nucleotide divergence among L. testaceipes
isolates and L. fabarum
NE OK TX FL L. fabarum
Nebraska * 0.2 0.0 0.5 4.4
Oklahoma 0.0 * 0.2 0.7 4.6
Texas 0.0 0.0 * 0.5 4.4
Florida 1.4 1.4 1.4 * 4.4
L. fabarum 7.4 7.4 7.4 8.1 *
Above diagonal, 16SmtDNA, 438 bp; below diagonal, COImtDNA,
428 bp.
June 2004 SHUFRAN ET AL.: GENETIC SIMILARITIES IN L. testaceipes POPULATIONS 777
homa (data not shown). Mitochondrial DNA se-
quences may not accurately reßect local geographic
adaptation caused by cold tolerance because of the
maternal nature ofmtDNA inheritance.NuclearDNA
markers, which are inherited bi-parentally, may be
more informative.
While we found little divergence in L. testaceipes
populations associatedwith cereal aphids in theGreat
Plains, genetic subdivision was recorded previously in
L. testaceipes populations in Colorado, albeit in a dif-
ferent plantÐaphid system (Hopkins 1998). Our anal-
yses indicated that the Florida population of L. testa-
ceipeswas divergent from those of the Great Plains. In
all of the dendrograms produced, it did not groupwith
theNebraska,Oklahoma,orTexaspopulationsbutwas
placed basal to these along with the outgroup L. faba-
rum. This may be evidence for a species complex in L.
testaceipes,which seems a tenablehypothesis basedon
its broad host and geographic ranges. Indeed, many
more individuals and populations from unique areas
and host species need to be examined to determine if
this is the case.
Acknowledgments
We thank A. Sitterly andD. Luter for technical assistance;
R.Belshaw for providing theLysiphlebus fabarum specimens;
K. Giles, R. Hufbauer, and Y. Cheng Zhu for peer review of
anearlierdraft of thispaper; andR.Erickson forproofreading
the Þnal draft. Additional comments and criticisms were
provided by G. Heimpel.
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778 ENVIRONMENTAL ENTOMOLOGY Vol. 33, no. 3


Genetic Similarities Among Geographic Isolates of Lysiphlebus testaceipes (Hymenoptera: Aphidiidae) Differing in Cold Temperature Tolerances

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Genetic Similarities Among Geographic Isolates of Lysiphlebus testaceipes (Hymenoptera: Aphidiidae) Differing in Cold Temperature Tolerances

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