Journal of Heredity Advance Access originally published online on April 5, 2006
Journal of Heredity 2006 97(3):290-293; doi:10.1093/jhered/esj027
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Brief Communications |
Genetic Divergence of Connecticut Melanoplus femurrubrum Populations
From the School of Forestry and Environmental Studies, Yale University, New Haven, CT 06520 (Lee and Schmitz); and Yale Institute for Biospheric Studies—Molecular Systematics and Conservation Genetics Laboratory, Department of Ecology and Evolutionary Biology, Yale University, 21 Sachem Street, New Haven, CT 06520 (Marshall and Caccone)
Address correspondence to A. Caccone at the address above, or e-mail: adalgisa.caccone{at}yale.edu.
We surveyed Melanoplus femurrubrum populations within the state of Connecticut for genetic diversity at multiple genetic markers, including three mitochondrial [cytochrome oxidase subunit 1 (COI), reduced form of nicotinamide adenine dinucleotide dehydrogenase subunit 2 (ND2), and AT rich] and one nuclear [internal transcribed spacers of the ribosomal DNA cluster (ITS1)] gene regions. All markers were variable, and the AT-rich gene showed the highest sequence divergence. Analysis of molecular variance (AMOVA), fixation index (Fst) analysis, and phylogeographic patterns showed little divergence between northern and southern regions. Estimates of genetic diversity (
) showed higher mitochondrial diversity in the northern region but nearly equal diversity for the ITS1 gene. This study shows for the first time in Melanoplus genetic variation for the ND2, AT rich, and ITS genes within a small geographic area. Our methods and results should be useful for other researchers interested in conducting population-level studies on closely related species.
An important focus in ecology and conservation is to understand the consequences of loss of species diversity to vital, life-sustaining ecosystem services. It has recently been suggested that focusing on species as the unit of conservation concern may not be the appropriate scale at which diversity should be measured (Hughes et al. 1997; Luck et al. 2003). If gene flow among populations within a species is restricted, then traits important to a species' interaction in an ecosystem may become geographically structured. So, local populations will only represent a subset of the range of ecosystem services exhibited within an entire species (Hughes et al. 1997). In such cases, each population may only provide an incremental amount toward an ecosystem function or service. The implication of this finding is that simply conserving species without regard to population structure may fail to conserve the full spectrum of functions provided by that species. Consequently, it becomes critical to understand the nature of population structure of species.
In some model species, such as Drosophila, selecting appropriate loci for phylogenetic or phylogeographic analysis is relatively easy. However, this is not always the case for many less studied insect species. One such case is Melanoplus femurrubrum, a generalist grasshopper found in abandoned farming fields, typically 3–10 ha, across the eastern United States. Previous studies have examined the phylogenetic relationships among species of Melanoplus in North and South America and Eurasia (Chapco et al. 2001) and among Melanoplus species in the western United States (Knowles 2000). Some phylogeographic studies have been done at the population level within several Melanoplus species using the COI mtDNA gene (Knowles 2001a). Notably, extensive phylogeographic research has been done with Melanoplus oregonensis using both the COI mtDNA gene (Knowles 2001b) and amplified fragment length polymorphism (Knowles and Richards 2005) data sets.
Molecular markers such as mitochondrial and nonrecombining nuclear genes are useful to population geneticists as their variable rates of evolution provide tools that can be geared to the level of resolution required, and unlike traditional allozyme allele frequency, data a priori delimitation of population boundaries are not a requisite (Avise 2000). The many well-known advantages of mitochondrial genes (for example, high resolution and quick elimination of ancestral polymorphism) have triggered a near dominance of their use in population and phylogeographic studies. However, one limitation of mitochondrial genes is that they represent only a single marker. Nuclear genes represent multiple independent markers, and many of their previous limitations are being solved by rapidly advancing technologies and analytical tools (Zhang and Hewitt 2003). To this end, the primary purpose of this study is to provide a preliminary survey of the genetic diversity present in the COI mtDNA gene within the species M. femurrubrum and to identify and characterize additional mitochondrial and nuclear markers that are variable at the intraspecific level within the genus Melanoplus.
For this study, we extracted and sequenced three mitochondrial and one nuclear DNA regions. Because the mtDNA genes are linked, we treat them as a single marker when estimating geographic variation; however, we treat each mitochondrial gene individually when illustrating its genetic variability and potential utility for phylogenetic and phylogeographic utility. The mitochondrial regions include two protein-coding genes, the cytochrome oxidase subunit I (COI) and the reduced form of nicotinamide adenine dinucleotide dehydrogenase subunit 2 (ND2), and one noncoding fragment, the AT-rich region. The nuclear region is one of the internal transcribed spacers of the ribosomal DNA cluster (ITS1). We selected these fragments because they have been widely used in phylogeographic studies in other organisms, and information is available on their evolutionary rates and inheritance modes.
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Materials and Methods |
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Twenty-five M. femurrubrum individuals were collected from five populations (three northern—N1, N2, and N3, and two southern—S1 and S2) in the state of Connecticut (CT) (Figure 1A). Additionally, W. Chapco generously donated two samples from western British Columbia (BC), Canada (roughly 5,000 km west of CT). The sex for each sample was visually determined, indicating slight excess of females in our samples. Each rear leg pair was cut off and stored at –70°C in the laboratory.
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Total DNA was extracted from five individuals per population from leg muscles using a DNEasy Tissue kit (Qiagen, Valencia, CA). Polymerase chain reactions (PCR) for the mitochondrial COI, ND2, AT-rich, and nuclear ITS1 regions were performed in a final reaction volume of 50 µl containing Tris-HCl (50 mM, pH 8.3), MgCl2 (3 mM for COI and ITS1, 4 mM for ND2, and 2.5 mM for AT rich), KCl (50 mM), deoxynucleoside triphosphate (400 µM for each nucleotide), 0.25 U of Taq polymerase, 10 pmoles of each primer, and 5 ng of template DNA. Cycling conditions for COI, ND2, and ITS1 were 2 min at 94°C, then 35 cycles of 1 min at 94°C, 1 min at 50°C (COI and ND2) or 53.5°C (ITS1), and 1 min at 72°C, with a final extension of 5 min at 72°C. The conditions for the AT-rich gene PCR cycle were 2 min at 94°C, then 25 cycles of 1 min at 94°C, 30 s at 59°C, and 2 min at 72°C, with a final extension of 5 min at 72°C. Primer pairs for the COI, ND2, AT-rich region, and ITS1 were MD6-MD1 (COI; Litzenberger and Chapco 2001a), ND2A-ND2B (ND2; Litzenberger and Chapco 2001b), J1-J6 (AT-rich region; Zhang et al. 1995), and ITS1A-ITS1B (ITS1; Kuperus and Chapco 1994), respectively. Optimal annealing temperatures were used for each gene. Product bands were then cut from agarose gels, purified with GENECLEAN III (BIO 101, Irvine, CA), and strands sequenced in both directions in ABI 3100 DNA SEQUENCER (Kimbolton, Cambs, UK) following manufacturer's protocols. Sequences were edited using SEQUENCER 4.1 (Gene Codes Corporation, Ann Arbor, MI) and aligned by eye.
We analyzed nucleotide diversity () and haplotype diversity (Hd) within four groups: (1) all individuals from CT, (2) all individuals from CT and BC, (3) all individuals from northern CT populations, and (4) all individuals from southern CT. Diversity estimates were calculated using DNASP 4.10.1 (Rozas et al. 2003) and are based on equations 10.5, 8.4, and 8.12 of Nei (1987). Levels of genetic divergences between populations were calculated using the Fst index (Excoffier et al. 1992) and permutation test statistics estimated using DNASP 4.10.1 (Rozas et al. 2003). Additionally, we performed an analysis of molecular variance (AMOVA, Weir and Cockerham 1984) to reveal the partitioning of variation among populations and geographic regions. These were computed in ARLEQUIN (Schneider et al. 2000). Evolutionary relationships were examined by constructing a haplotype network with the DNA sequences based on the statistical parsimony method of Templeton et al. (1992). The analysis was performed in TCS (Clement et al. 2000).
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Results and Discussion |
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TopMaterials and MethodsResults and DiscussionReferences |
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Table 1 shows estimates of sequence (
) and Hd for each gene for all the previously defined groups. Within the CT samples, the AT-rich gene consistently had the highest sequence and haplotype diversity estimates, with the COI gene having the next highest estimates, then ND2, and ITS1 the lowest. Comparing northern and southern CT regions, we see both genetic and sequence divergence estimates higher in the north. The inclusion of the BC samples also increases diversity estimates but not considerably. Of interest, all four genes did show considerable levels of variation for such a small geographic area. Although sequence divergence comparisons between species can be difficult due to unequal sampling, different sampling strategies, and varying mobility of species, in general overall intraspecific sequence divergence of M. femurrubrum was within the range of other insect species or spiders. The sequence divergence of COI for damselfly, aphid, lice, and spider was 
1.1%, 0.6%, 0.8%, and 3.96%, respectively (Brown et al. 2000; Favret and Voegtlin 2004; Johnson et al. 2002; Paquin and Hedin 2004). The sequence divergence for ND2 in honeybees is 1.22% (Arias and Sheppard 1996).
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We estimated Fst indices between the northern and southern CT regions and the BC population for all mitochondrial genes as a combined marker. Nonsignificant values (P > 0.05) were obtained for most comparisons except between N2 and S1 (Fst = 0.13155) and N2 and S2 (Fst = 0.10463) and all BC comparisons (Fst = 0.48–0.53). Only two samples were available for sequencing from BC, and these samples each carried unique haplotypes. The same analysis using the ITS data set resulted in a similar pattern of Fst values. Large Fst values resulted when comparing this population with all CT populations, but the extremely limited sampling is cause for restraint in interpreting these results. Based on the results from comparisons within the CT samples, the N2 population is arguably the most divergent. However, again given the low sample sizes of the populations used in this study, this result may be due to sample error.
Little evidence supports the genetic differentiation between northern and southern populations in CT. Figure 1B,C show TCS haplotype networks estimated for the ITS and mitochondrial markers. In the ITS network, most haplotypes are shared at nearly equal proportions between northern and southern CT populations, and the BC haplotypes are not shared but closely related. In the mtDNA network, most individuals contained unique haplotypes. One exception was the most common and inferred ancestral haplotype (represented by a square) that was found in both southern and northern CT populations. None of the haplotypes from a single population (including the BC population) grouped uniquely in any region of the network. Results from the AMOVA analyses for CT populations were similar for the ITS and mtDNA data sets. Both indicate that most of the genetic variation is accounted for within populations rather than between populations or regions. For example, the analysis of the mtDNA data set indicated that the percentage of variation accounted for among regions was 10.73% compared with 92.15% between regions. These results indicate little structure between northern and southern CT populations but again small sample size merit caution in interpretations. Because we were unable to sequence the AT-rich gene for the BC samples, the ITS data set was used for an AMOVA analysis comparing the BC population with all the samples from CT as a single population. Nearly equal amount of variation was accounted for within populations (49.53%) and among populations (50.47%). Again given the low sample sizes of the populations (BC = 2), we caution against overinterpretation of these results.
This study shows that the CT M. femurrubrum populations, even at a reduced geographic scale, harbor genetic variation at several loci. Including distant samples from BC did increase genetic variation but not proportionally. This should be useful for other researchers interested in doing population-level studies on this or closely related species. Although we could not see differentiation between northern and southern regions or between most pairwise population comparisons, we do note a trend toward increased genetic diversity in the northern region. Larger and more geographically dispersed samples are needed to confirm this trend and to better estimate the amount of divergence between samples from CT and BC.
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Acknowledgments |
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We would like to acknowledge William Chapco for generously providing the BC sample and David Call for advice and training. We would also like to thank three anonymous reviewers for their very helpful comments. The work was supported by funds from Yale School of Forestry and Environmental Studies to O.J.S., YIBS to A.C., and Postdoctoral Fellowship Program of Korea Science & Engineering Foundation to J.L.
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Footnotes |
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Corresponding Editor: L. Lacey Knowles
Received May 6, 2005
Accepted February 16, 2006
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References |
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