size in a variety of ways. Tandem duplications of control region sequences are ... ture founded with collections from Gainesville, Florida, and. Tully, New York.
Copyright 0 1989 by the Genetics Society of America
Mitochondrial DNA in the Bark Weevils: Size, Structure and Heteroplasmy Thomas M. Boyce, Michael E. Zwick and Charles F. Aquadro Section of Genetics and Development, Cornell University,Ithaca, New York 14853-2703 Manuscript receivedJune 13, 1989 Accepted for publication August 18, 1989
ABSTRACT Mitochondrial DNA of higher animals has been described as an example of extreme efficiencyin genome structure and function. Where exceptionally large size molecules have beenfound (>20 kb), most have occurred as rare variants within a species, suggesting that these variants arise infrequently and do not persist for long periodsin evolutionary time. In contrast, all individuals of at least three species of bark weevil(Curculionidae:Pissodes)possess a mitochondrial genomeof unusually large size (30-36 kb). The molecule owes its large size to a dramatically enlargedA+T-rich region (9-13 kb). Gene content andorder outside of this region appear to be identical to that found in Drosophila. A series of 0.8-2.0-kbrepeated sequences occur adjacentthe to large A + T rich region and have perhaps played a role in the generationof the large size as well as an unprecedented frequencyof size variant heteroplasmy. Every weevil sampled in all three species (n = 219) exhibits anywhere from two to five distinct size classes of mtDNA. The persistence of this large amount of size polymorphism through two speciation events combined with the abundantsize variation within individuals suggests that these molecules may not be subject to strong selection for small overall size and efficiency of replication. This pattern of variation contrasts stronglywith the conservation of gene content and arrangement in the coding region of the molecule.
A
NIMAL mitochondrial DNA is generally known for its conservation of gene content and size across a variety of taxa (MORITZ, DOWLINGand BROWN1987; SEDEROFF 1984). T h e majority of animal mtDNAs encode 13 protein genes, 22 transfer RNAs, and two ribosomal RNAs. Most taxa studied have mtDNA molecules of similar size ranging from 15 to20 kb. Coding sequencesare tightly abutting if not overlapping, and lack introns. Thus, it has been thought that mtDNAsize has been constrainedin the evolutionary history of animals. Recently, however, mitochondrial genomesof relatively large size (greater than ca. 20 kb) have been found in several vertebrate taxa (MORITZand BROWN1987), in thedeep sea scallop, Placopecten magellanicus (SNYDER et a l . 1987) and in the nematode Romanomermis culicivorax (PowERS, PLATZERand HYMAN 1986). These molecules havegainedtheirlarge size in a variety of ways. Tandem duplications of control region sequencesare responsible for size increases in newts and fishes, whereascodingsequenceshavebeenduplicated in lizards (MORITZ and BROWN1986; MORITZ,DOWLING andBROWN1987). T h e large size of the scallop mtDNA molecule is in part the result of the addition of ca. 11 kb of unknown sequence, over and above the approximately 14to16 kbpresent in typical mitochondrial genomes. T h e mtDNA of the nemaThe publication costs of this article were partly defrayed by the payment ofpage charges. Thisarticle must therefore be hereby marked"advertisement" in accordance with 18 U.S.C. $1734 solely to indicate this fact. Genetics 123: 825-836 (December, 1989)
tode, R. culicivorax, owes its large size to many dispersed repeats (POWERS, PLATZER and HYMAN 1986). In addition to these large sizes, heteroplasmy for size variants may be relatively common in many organisms other than mammals (BERMINGHAM,LAMB and AVISE1986; HARRISON 1989). Minor size variation, ca. 500 base pairs(bp) or less and typically localized to the control region,is common within and among lower vertebrates and insects studied to date (MORITZ,DOWLINGand BROWN1987). Tandem repetition of a 1.2-kb element of unknown sequence is responsible for large size variants in the sea scallop (SNYDER et al. 1987) and a similar pattern with a 1.5kb repeat is found in the american shad (BENTZEN, LEGGETand BROWN1988). Nevertheless, in most taxa, large size mtDNA molecules appear only as rare variants within species, suggesting that these variants do not persist for long periods in evolutionary time. This observation is consistent with the view that mtDNA size is somewhat constrained in animals (HALE andSINGH1986; SEDEROFF 1984; MORITZ,DOWLING and BROWN1987). Experiments tracingsize variant frequency changesin isofemale lines of Drosophila mauritiana (SOLIGNAC et al. 1984) and heteroplasmic female crickets and their progeny (RAND andHARRISON 1986), while not conclusive, have suggested that smaller sized molecules are more frequently transmitted. A simple model for such an advantage stems from the potential kinetic advantage a smaller molecule may have during repli-
826
T. M.Zwick Boyce, E. M.
cation (RAND andHARRISON1986, 1989; WALLACE 1989). Larger size molecules are viewed to be at a selective disadvantage simply because they take longer to replicate, leaving fewer copies to be transmitted. Under this hypothesis, the largest molecules should be underthe most intense selection and have the shortest residency time in a population if the transmission rates of different size variants are equal. We have been using mtDNA polymorphisms in three species of bark weevil (Curculionidae: Pissodes) to assess the degree and natureof genetic differentiation of various pest populations of these insects. As groundwork for that study,we have characterized the gene content, order and size variation ofPissodes mitochondrial genomes and found them to be uniformly large (30-36 kb) in three species. Furthermore, across the three species all individuals sampled (n = 219) are heteroplasmic for up to five major size classes. These size variants appear to be the result of different copy numbers of a tandemly repeated region, itself varying in sizefrom 0.8 to 2.0 kb. Through the use of restriction site mapping, transfer-hybridization, and cloning of Pissodes mtDNA, we determined the nature and extent of the size increase and heteroplasmy. For comparison,we have characterized the mitochondrial genomes of two other species of weevil, the boll weevil (Anthonornus grandis) and the palesweevil (Hylobius pales). Furthermore, we have aligned thecomplete Drosophila genetic mapof mtDNA with the Pissodes restriction site map. Our findings imply that while selection appears to be acting to maintain relative gene order andcompact arrangement of the major coding regions, the large size of the Pissodes mtDNA molecule and extensive heteroplasmy suggest that there must not be strong constraints on total size of the mtDNA molecule in weevils, in contrast to patterns found in most animal taxa studied to date. MATERIALSANDMETHODS
Isolation of mtDNA: Forpreparation of purified mtDNA, live Pissodes nemorensis were obtained from a culture foundedwith collections from Gainesville, Florida, and Tully, New York. Live P. strobi were excavated from white pine shoots from Ithaca, New York, and liveAnthonomus grandis were provided from a culture at the University of Mississippi. mtDNA was isolated using the protocolof LANSMAN et al. (1 98 1) with the following modifications. Homogenization was carried out in 0.21 M mannitol/0.07 M sucrose/0.05 M Tris (pH 7.5)/0.003 M CaC12/0.01 M EDTA using a Tekmar Tissumizer. Nuclei and debriswere pelleted and the supernatant resuspended in fresh homogenization buffer. T h e sample was rehomogenized and debris removed again. Samples were pooledand mitochondria were pelleted, resuspended in 0.21 M mannitol/0.07 M sucrose/0.05 M Tris (pH 7.5)/0.01 M EDTA and repelleted. T h e mitochondria wereresuspended in 100 mM NaC1/50 mM Tris-HCI/lO mM EDTA and lysed with SDS. T h e SDS was precipitated from solution with CsCl salt and the supernatant made 0.6
and C. F. Aquadro mM in propidium iodide. Covalently closed circular DNA was isolated through CsCl gradients (density 1.578 g/ml, refractive index of 1.878). T h e propidium iodide was extracted with saturated CsCkisopropanol ( l : l , v:v), the volume adjusted with T E [ 10 mM Tris/l mM EDTA (pH = 7.6)], the DNA ethanol precipitated, and resuspended in TE. Isolation of total genomic DNA: Total genomic DNA was isolated from individual weevils via a modification of the protocol of DOYLEand DOYLE(1987). Individuals of Pissodes, H. pales, and A. grandis were collected live and frozen at -70" until use (see Table 1 for collection data). Species identity was confirmed by morphology and host data. Weevils were quick-frozen in liquid nitrogenthen quickly placed in an Eppendorf tube and shattered with a small plastic pestle. 500 pl of 2X CTAB buffer [O. 1 M TrisHC1/0.02 M EDTA/1.4 M NaC1/0.2% /3-mercaptoethanol (v:v)/0.05 M hexadecyltriethylammoniumbromide (pH 8.3)] was added and the homogenategently suspended in it. T h e tube was incubated at 65" for 4560 tomin then centrifuged in an Eppendorf 54 14 centrifuge for 15 sec. T h e supernatant was decanted andsaved. T h e pellet was resuspended in gently rehomogenized, 300 pl of fresh CTABbuffer, brought to 65" for 10 min then centrifuged for 10 min. This supernatant was pooled with the first and extracted once with an equal volume of ch1oroform:isoamyl alcohol (24: 1) and precipitated with ice-cold isopropanol. T h e DNA was pelleted, washed with 70% ethanol, dried, and resuspended in TE. Cloning of P. strobi mtDNA: Purified mtDNA from P. strobi collected as adults at white pine in Ithaca, New York, was digested to completion with either Hind111 or combinations of BamHI with PstI and XbaI. These fragmentswere cloned into appropriately digested pBS vector DNA (Stratagene, Inc.). Recombinant plasmid DNA was isolated from bacterial culture and characterized through restriction enzyme digestion and electrophoresis. Plasmids thus obtained containing P. strobi mtDNA fragments are designated as, for example,Plasmid E. strobi &amHI-XbaI number 28 &e., pPSBX.28). Restriction site mapping of weevil mtDNA Restriction site positions were determined throughcomplete single and double digests of total genomic DNAs from individual weevils. Digested fragments were electrophoresed in 0.8% agarose and transferred to nylon membranes (WESTNEAT et al. 1988). Purified weevil mtDNA was labeled with "Pby randompriming (FEINBERGand VOGELSTEIN1983)and hybridized to nylon membranes in 7% SDS/525 mM NaP04/1 mM EDTA/1.0% BSA (w:v) at 55". Membranes were washed atroomtemperature in 1% SDS/40 mM NaP04/1 mM EDTA for 5 min, then twice for 45 min at
55".
T o determine homology of fragmentsamong species, purified mtDNA of P. nemorensis wasdigested to completion with BglII and the fragments recovered via electrophoresis in low melting point agarose. T h e membranes were then separately hybridized to probes made fromeach fragment. Cloned regions of P. strobi mtDNA (see above) were also used as probes across species. Indirect end-labeling of partial digestions of total genomic DNA was used to confirm restriction fragment lengths and order as follows: DNA was first cutto completion with BamHI, which cuts only once, or with PstI, which cuts on either side of the BamHI site (Figure l), then partially digested with an enzyme of choice. A range of partial digestion was ensured by digesting with 0.1, 1.O, 2.0, and 10.0 units of enzyme for 15 min at 37". Thesedigests were then electrophoresed, transferred, and probed with clones
Weevils mtDNA in Bark
827
TABLE 1 Collection data for individualsused in characterizing PissodesmtDNA Species
Localities
Stage collected
Date
N
Danby, New York strobus Pinus Brevard, North Carolina strobus Pinus Terrace, British Columbia, Canada Picea sitchensis Golden, British Columbia, Canada Picea sp. Picea sitchensis Waldport, Oregon Flathead National Forest, Montana engelmannii Picea
Adults/larvae Larvae Larvae Larvae Larvae Larvae
5/88-8188 8/87
31 9 27 19
P. nemorensis Germar New
Danby, New York Tully, York Gainesville, Florida
Adults/larvae Adults Adults
5/88-8188 25 7/88 3/86
19 10
P. terminalis Hopping
Hobart Mills, California
7/87
23
P . strobi (Peck)
Host tree species
strobus Pinus resinosa Pinus elliottii Pinus contorta Pinus
of P. strobi mtDNA that extendin either direction from the BamHI site (clones pPSH.6.1, pPSBX.28, and pPSBX.2). Gene content and order: Mitochondrialgenecontent and order were determined by hybridization to probes representing specific genes or sets of genes in Drosophila mtDNA prepared from Drosophila yakuba mtDNA cloned in pBR322 (CLARYand WOISTENHOLME1985),D. melanogaster mtDNA clonedintopBR322 (GARESSE1988; DEBRUIJN1983), or from purified mtDNA prepared from D. melanogaster (Table2). Forfragments from purified D. melanogaster mtDNA, the desired fragments were separated via electrophoresisin low melting point agarose, excised and labeled as describedabove.Additionally, a 382-bp XbaI restriction fragment of D. melanogaster mtDNA within the 12s rRNA gene (SOLIGNAC, MONNEROT,and MOUNOLOU 1986) was cloned into appropriately digested pBS vector DNA (Stratagene, Inc.) andrecombinantplasmid DNA prepared as above. Hybridizationof Drosophila mtDNA to weevil DNA was carried out as described above but under reduced stringency (hybridization and washesat 42"). RESULTS
Restriction enzyme site maps and size: MtDNA restriction site maps of Pissodes nemorensis, P. strobi, P. terminalis,Hylobius pales (the pales weevil) and Anthonomus grandis (the boll weevil) were constructed for several enzymes (Figure 1). Consistent restriction site maps for Pissodes species were obtainable only through a combinationof techniques. Single and double restriction enzyme digests provided relative positions of sites in most cases. However, several fragments 0.8-2.0 kb long were evidently repeatedas judged by darker intensity on autoradiographs, leadingto some ambiguity in site positions. Digestions with BamHI revealed that it consistently cleaves the mtDNA of all Pissodes species at only one conserved site. Likewise, PstI cleaves the molecules at two invariant sites on either side of the BamHI site (Figure 1). Fragment sizes and their relative placements for particular enzymes were obtained via indirect end-labeling of partial digests of DNA previously cut with BamHI or PstI. This technique revealed the order in which fragments are arranged extending from the BamHJ site and confirmed that all bands arise from a closed, circular molecule (Figure 2).
Larvae
7/87 7/87 7/87 7/86
37 19
The restriction site maps forthethree Pissodes species show a ca. 16-kb region where most mapped enzymes cleave, a 9-1 3-kb fragmentthat is not cleaved by mostenzymes used, and a series of repeated fragments (Figure 1). Cleavage patterns for 2 19individual weevils were examined and all revealed sizes for the threespecies of pine weevil in the range of 30 kb to 36 kb, althoughP. strobi typically hasthe largest genome and P. terminalis the smallest of the three species. Percent nucleotide sequence divergence (T; NEI and TAJIMA 1981) among single representative mtDNA types of three species of Pissodes was estimated using the presence or absence of mapped restriction sites in the nonduplicated regions of each species. The estimated divergence between P. strobi and P. nemorensis is 2.87%; between P. strobi and P. terminalis, 3.66%; and between P. nemorensis and P. terminalis, 1.83%. Hylobius and Anthonomus restriction enzyme maps are too divergent to align to those of Pissodes with confidence and hence values of divergence were not estimated. Remarkably, every weevil examined inall three species was heteroplasmic for several distinct size classes of mtDNA (Figure 3). These size classes most often differed by multiples of the average repeat unit size (ca. 1.5 kb). Particular digestion patterns of repeated fragments could not be consistently correlated with particular heteroplasmic profiles, suggesting that any particular size class may be arrived at through a number of different combinations of repeated fragments. Verification of mtDNA as weevil DNA: The unusual size and configuration of the mtDNA raised the concern that the DNA analyzed may not have been of weevil origin. In particular, the possibility that the DNA being analyzed was fromgut endosymbionts needed to beruledout.Fungi, yeast, and various other organisms are known to be endosymbionts of wood feeding insects, including Pissodes (BUCHNER 1965) and may have large mtDNA genomes (SEDEROFF 1984). T o thatend, several experiments were performed.
T. M. Boyce, M. E. Zwick and C. F. Aquadro
828
TABLE 2 Restriction fragmentsof D. rnelanogaster and D. yakuba used in determining gene order in Pissodes mtDNA
rence description' Fragment/clone Fragment"
A
382-bp XbaI fragment of D.melanoEaster SOLIGNAC, MONNEROTand MOUNOLOU(1986), cloned into pBS, clone pDMX.26 and this paper 896-bp EcoRI fragment of D.melanogasterGARESSE (1988) cloned into pBR322 (7245-8141) 2198-bp MspI fragment of purified mtDNA GARESSE (1988) of D.melanogaster (5374-7572) 3970-bp SstI fragment of purified mtDNA GARESSE(1988) of D.melanogaster (2283-6253) 37 19-bp MspI fragment of purified mtDNA of D.melanogaster (1654-5373)
GARESSE(1988)
1772-bp EcoRI fragment of D.melanogaster cloned into pBR322 (86-1858)
GARESSE (1988)
970-bp XbaI fragment of D.yakuba, clone CLARY and WOLSTENHOLME (1985) pDYHB(3753-4723) 600-bp SstI fragment of D.yakuba, clone CLARY and WOLSTENHOLME COI (1985) pDYHB of CLARY and WOLSTENHOLME (1735-2334) 4868-bp Hind111 fragment of D.melanoDE BRUIJN(1983) gaster cloned into pBR322 (1-4869)
12s rRNA 16s rRNA, tRNA'"', ND-1 16s rRNA, tRNA1'", ND-1, Cyt b Cyt b, ND-6, tRNAP'", tRNAthr,ND-4L, ND-4, ND-5 Cyt b, ND-6, tRNAP'", tRNAthr,ND-4L. ND-4 tRNAhBs, ND-5 ND-5, tRNAph', tRNAg'", tRNA"', tRNAas", tRNA"'g, tRNA"'", ND-3, tRNAg'y,COIII tRNAiY', ATPase 6 , ATPase 8
COII, ATPase 6,ATPase 8, tRNA"'p, tRNAIYS, COII, tRNA"", COI, tRNA'y', tRNA''', tRNA", ND-2
Letters refer to fragments diagrammed in Figure 1. The numbers in parentheses refer to the base pairs contained in the fragment as determined from DNA sequences published in the appropriate reference.
First, the purified Pissodes mtDNA was digested to completion with HindIII, electrophoresed in 0.8% agarose,stained with ethidiumbromide, andthe banding pattern compared to that obtained through probing of HindIII digested total DNA with Drosophila and presumed Pissodes mtDNA under normal stringency (55" incubation and washes). The band patterns were identical, indicating no contamination by more than one source species in the DNA being analyzed and also that the DNA is likely to be insect mtDNA. Second, to test for gut endosymbiont contamination, thoracic muscle and complete gut tissues were dissected from live individual weevils of P. strobi and P. nemorensis and their separate DNAs prepared. Endosymbionts of Pissodes species are known to inhabit the mycetomes of theanterior mid-gut (BUCHNER 1965) and thelikelihood of endosymbionts occurring in thoracic muscle is remote. Gut tissue DNAs were prepared without evacuating the gut and included as much of thegut as could beremovedintact(the anterior esophagus to the anus). Thoracic tissues were removed beforedissection of the gut. HindIII banding patterns of these DNAs were then compared through probing with purified mtDNA. Species specific band patterns were observed with no loss or gain of bands between DNAs from thoracic muscle and gut tissue,
indicating that there was little likelihood of endosymbiont contamination. Insect pathogens may also contain large mtDNA genomes displaying size variation (POWERS,PLATZER and HYMAN1986). Dissected weevils displayed no obvious pathology (e.g., fungal or nematode infection) that might have led to identical band patterns in the two DNAs. Furthermore, the probability is remote that all individuals across the wide seasonal and geographic rangesof allthree species would contain levels of fungal or nematode infection sufficient to mimic mtDNA of the host. Third, total genomic DNA of D. melunoguster was digested and probed with purified Pissodes mtDNA at normal stringency ( 5 5 O incubation and washes) and the restriction fragment patterns compared to published data. The purified mtDNA hybridized to Drosophila mtDNA under the conditions used, except for A+T-rich regions; again suggesting that the DNA is insect mtDNA. Taken together, the results of these experiments sufficiently rule out the possibility that the DNA being analyzed is of endosymbiont or pathological origin and strongly indicate that it is mtDNA of Pissodes. Gene order and genome organization: The restriction enzyme map of Pissodes species obtained through double digestion profiles and indirect end labeling was wholly consistent with the map obtained through
829
mtDNA in Bark Weevils
=A-B -C
ED-
Drosophila fragments used
in determining gene order
F-
P. nemorensis
'Ix
H
'Ix
~
I
I
A
HM
I I HM
I
A
M
I
A
II I
xI
I
B
II
P H H
XB
nI
B
I
M
II
BH
BA
u
M
I
n
P. terminalis
I
I
I
I
n
I
n
I
n
I
n
H
n
I
I
n
n
I
I
H
n
a
Hylobius pales I
I
B
I
B X
X
l
l
H
P
n
I
X
"
I
xn
E
"
H
B
10
~
I E
I
I
I1
n
B
E S
I 1 1 1 1 1 B B H I1 H X
0 kb
I I
1
Anthonomus grandis
II I Xtl H
~
l
~
20
l
~
~
~
30
~
~
FIGURE 1 .-Linear restriction site and genetic maps of Pissodes species, Hylobius pales, Anthonomus grandis and Drosophila melanogaster. Gene contentand orderwere determined with probes made from the Drosophila fragments shown (described in Table 2). Individual transfer RNA genes were not localized and arenot shown. Cloned segments of Pissodes mtDNA are as described in the text. Restriction enzyme sites are identified by letters as follows: A = Haelll, B = BglII, E = EcoRI, H = HindIII, M = Mspl, P = PstI, S = SacII, X = Xbal.
probing with specificfragments ofDrosophila mtDNA andthrough probings of Drosophilamelanogaster mtDNA with Pissodes mtDNA clones. In no case did a Drosophila fragment probe two discontinuous Pissodes fragments, indicating that there has not been any major duplication or extensive rearrangement of gene sequences. Drosophila genes all mapalonga single, contiguous segment of Pissodes mtDNA; gene order appears identical inPissodes and Drosophila (Figure 1). T h e region of Pissodes mtDNA containing the coding sequences is the region cut most often by restriction enzymes, while the large, typically uncut region and the repeated sequences in Pissodes mtDNA map to the A+T-rich region of Drosophila mtDNA. Probings of mapping digests for the three Pissodes species with particularfragments of P. nemorensis mtDNA allowed the alignment of homologous restriction sites. Probings of mapping digests of H. pales and A. grandis with the 4.8-kb HindIII fragment of D. yaRuba and the Pissodes clone pPSBX.28 aligned the
restrictionsite maps of these weevilswith that of Pissodes and Drosophila. T h e mtDNA of A. grandis is of a more common size, 18.2 kb. The restriction site map of H . pales reveals that it is slightly larger (ca. 26 kb). N o obvious repeatedsequence motifs are observed in the restriction site maps of these two species. Characterizationandmapping of repeated sequences: T h e presence of variable numbers and sizes of repeated sequencesin Pissodes mtDNA can be seen in restriction enzyme digestion patterns for HindIII, XbaI, andHaeIII. Mapping digests probed with a clone of the repeat unit (pPSH.2) show that several fragments in the 0.8-2.0-kb size range as well as the very large,uncutfragmentcontaintheserepeated sequences (Figure 4). Indirect end-labeling of partial digestions with these enzymes (as described above) reveal that the repeat units are arranged tandemly (Figure 2). None of the other three clones of Pissodes mtDNAcontainrepeated sequences. MtDNA from H. pales and A. grandis, and D. melanogaster do not
~
T. M. Boyce, M. E. Zwick and C . F. Aquadro
830
A std B
FIGURE2.-Fragment size and order via indirect end-labeling of Pissodes mtDNA. Total genomic DNA from an individual with P. nemorensis mtDNA was digested to completion with Pstl then partially digested with Hindlll, electrophoresed and transferred to a nylon filter. The filter was probed in successionwith pPSBX.28 (lane A) and pPSH.6.1 (lane B). Thus, the two band patternsrepresent the opposite orders of Hindlll restriction sites. Note that this map corresponds with that of P. nemorensis diagrammed in Figure 1. Distances between bands correspond to fragment lengths except for Pstl/Hindlll double digestion products as indicated at the bottom of each lane. The 1.8-kb PstI fragment appears in both lanes A and B, and is proportionately darker since it is a complete digestion product. Size standard is Bethesda Research Laboratories 1-kb ladder in middle lane (std). Standard band sizes are (from top to bottom in kb) 12.2, 11.2, 10.2.9.2, 8.1, 7.1, 6.1, 5.1, 4.1, 3.1, 2.0, 1.6. 1.0, 0.5, 0.4. The 1.6, 0.5, and 0.4 kb standardfragmentsshare sequence with the vector used(pBS) and thus appear exceptionally dark, partially masking the 2.0-kb standard fragment just above it.
containsequencessimilar enough to the repeated region in Pissodes to be probed under the conditions of reduced stringency used (42"). DISCUSSION
The mtDNA of these species of Pissodes is unusual in the followingrespects: (1) it is uniformlynearly
(2) the large twice aslarge as most metazoan mtDNAs, sizeis accompanied by extensivesizevariation and heteroplasmy, and (3) these characteristicsare shared by all observed individuals in the three species examined. These three species of Pissodesare members of the Pissodes strobi species group, and are considered to be closely related (PHILLIPS 1984). Yet, the mtDNA
mtDNA in Bark Weevils
P. nemorensis
P. strobi
1 2 3 4 5 6 7 8 9 I
83 1
P. terminalis
10 11 12 13 14 15 16 17 18
b (kb) 12.2= 10.2-
-
8.1-
6.15.14.13.0-
l9 2o 21 22 25
26 27
FIGURE3.-Heteroplasmy in Pissodes nemorensis (lanes 2-9). P. strobi (lanes 10-16 and 18). and P. lerminalis (lanes 19-22 and 24-27). Individualweevilstypicallydisplayseveral size classes of the largest Bglll digestion product indicated by arrow. BglI1 restriction sites lie outside the size variable region for all three species (Figure 1). Close examination of the autoradiographs and digestion patterns with other enzymes confirm that weevils appearing homoplasmic in this figure, e.g., lanes 19 and 27, are, in fact, heteroplasmic. Size standards (lanes 1, 17,and23) are Bethesda Research Laboratories 1-kb ladder.
2.0-
A
.
..
.
.. _ .
B -8.1 -
-
-5.1 -4.1
-3.0
-2.0 -1.6
-1
1
2
3
4
5
6
1
2 3
4
5
.o
6std(kb)
FIGURE 4.-Size variation and distribution of repeated sequences. A and B are thesame mapping digests of an individual P. nrmorensis probed w i t h (A) clone pPSH.2 containing repeated sequences and (B) with complete Pissodes mtDNA. Digestion patterns are from digestions with; lane 1. HindlI1; lane 2, Hindlll and Haelll; lane 3. Haelll; lane 4, HindIIl; lane 5 , Hindlll and Hinfl; lane 6, Hinfl. The cloned fragmentcorresponds to the 1.6-kb Hindlll fragment identified by the arrow. Note that several bands (400 bp to ca. 2 kb) and the largest band (ca. 16+ kb) are probed by the clone. Fragments not probed by the cloned repeated sequences map to the Pissodes coding region.
lineages of the three are distinct, differingin sequence by as much as3.6% in the codingportion of the molecule. Nevertheless, the mitochondrialgenomes of the three species retain a similar structure. The very large size mtDNA of these weevils is not apparent in the other weevil taxa studied (Figure 5 ) . A.grandis displays a typical size of 18.2 kb. T h e of mtDNA of H. pales, while notaslargeasthat Pissodes, is ca. 6 kb larger than typical. Whether a
tendency for large mtDNA size (and/or heteroplasmy) is a characteristic of the clade of which Pissodes is a member will require more extensive characterization of several genomes in the clade and therest of Pissodes as well. Gene content and order: Through theuse of genespecific clones and restriction enzyme fragments from Drosophila mtDNA, we were able to determine that the mtDNA of Pissodes is similar to thatof Drosophila in that it is a closed, circular molecule and contains the same set of gene sequences. No duplications of codingsequenceweredetected. Furthermore,the major coding regions of Pissodes mtDNA occurin the same order as in Drosophila and display the same contiguous arrangement. As the Diptera and Coleoptera lineages diverged at least 250 million years ago (RIEK1970), this observation further reinforces other work suggesting that major gene rearrangementsmay occur only rarely, with relatively few (two to six) rearrangements accounting for different gene orders among major phyla (JACOBS et al. 1988; butsee YANG and ZHOU 1988). Transfer-RNA genes, however, appear to be more labile to rearrangement than the major protein coding genes in some taxa (JACOBS et al. 1988; CANTATORE et al. 1987; MORITZ,DOWLING and BROWN1987). In insects, HAUCKE andCELLISSEND (1988) have found the order of the tRNA genes coding for lysine and aspartate to be reversed in Locusta migratoria (Orthoptera) as comparedto Drosophilayakuba (Diptera). Within the Diptera, the tRNA genes for arginineand alanine in the mosquito Aedes albopictus are reversed relative to D. yakuba,and thedirection of transcription has been reversed forthe serinetRNA(DUBIN, HSUCHENand TILLOTSON 1986). Because tRNA codingsequences areshort, they may not have been detectable with the probes and hybridization conditions employed and we cannot completely rule out
Mammals Buds Reptiles Fish Echinoderms Other Arthropods
16-20Kb
.
Scallops: 32 - 40 Kb\
\
such rearrangements in Pissodes mtDNA. Similarly, while the mtDNA of Pissodes appears to contain all of the major coding regions of Drosophila mtDNA, we cannot completely ruleoutthe possibility that additional novel coding sequences exist that result in the large size ofthe molecule. For reasons cited below, however, this possibility seems unlikely. The large size of Pissodes mtDNA results from the addition to the molecule of a large sequence (9-1 3 kb) and many tandemly repeated sequences (0.8-2.0 kb in length), over and above sequences required for mtDNA coding functions. This pattern is similar to that found in the sea scallop where ca. 11 kb has been added to the 14 to 16 kb necessary for the coding sequences of a typical mitochondrial genome. In Pissodes, we have found thatthese two additional regions map genetically to the presumed A+T-rich region of the molecule. The majority of the added sequencehas characteristics expected of an A+T-rich region, including a rarity of sites for the restriction enzymes used. The repeated sequences in Pissodes all occur at one end of the presumed A+T-rich region, between the 12s rRNA gene and thelarge sequence addition. Repeated sequences in Cnemidophorus lizards also occur at one end of the control region of the molecule, adjacent to the rRNA genes, and in fact contain partial coding sequences of the rRNA genes (MORITZ and BROWN 1986). However,as judged by lack of cross hybridization to Drosophila mtDNA and their location in the Pissodes restriction site map, the repeated sequences in Pissodes do not show any sequence similarity to either of the rRNA genes, nor to any other known mtDNA coding region.
FIGURE5.-Phylogenetic distribution of mitochondrial genome sizes. All three species of Pissodes examined to date display the large mitochondrial genome size. Within the weevils, Hylobius pales does display a slightly larger mitochondrial genome than the more distantly related Anthonomus grandis, but the large genome size does not appear to be characteristic of the weevils or beetles as a whole. Size data forother animal taxa are taken from MORITZ, DOWLINGand BROWN(1987), except for sea scallops (SNYDER et al. 1987) and Dineutus assimilis (B. NURENBURGER personal communication). The phylogenetic arrangement of the weevil taxa is taken from G . KUSCHEL (unpublished manuscript). Hash marks on the tree indicate unknown patristic distances between taxa and the presence ofmany intervening branches not shown.
Size variation and repeated sequences: The magnitude of size variant differences, the number of size classes possiblewithin single individuals, and the abundance of mtDNA heteroplasmy in all individuals of three species of Pissodes is unprecedented. Most taxa exhibit size class heteroplasmy at population frequencies much lower than observed in Pissodes, although heteroplasmy involving small scale size differences (0.2 kb or less)may be very frequent (e.g., Rana esculenta; MONNEROT, MOUNOLOU and SOLIGNAC 1984). Pissodes mtDNA is exceptional in that 100% of the 2 19 individuals surveyed are heteroplasmic for size variants differing by at least 0.8 kb, and may contain as many as five distinct size classes differing by as much as 7.5 kb (Figure 3). These sizeclasses apparently correspond to the presence of different copy numbers of the repeated sequences in different sized molecules. The repeated sequences in Pissodesthemselves vary in size (0.8-2.0 kb), most often by discrete units of ca. 200 bp, indicating that duplication of the sequence is not always complete, or that smaller insertions/deletions are common within thelargerrepeating sequence. Insertion and deletion of both largeand small fragments may be the result of intermolecular recombination among different mtDNA molecules of the same individual, or the result of intramolecular recombination (RANDand HARRISON 1989). Slipped mispairing (either within or between molecules) during replication has been implicated as a significant force in DNA sequence evolution (LEVINSON and GUTMAN 1987) and could play a role in the generation of size variation in Pissodes. Slipped mis-
mtDNA in Bark Weevils
pairing involves the generation of insertions and deletions through the mispairing of complementary sequences of tandemrepeats. Smallsize variants in mtDNA may be the result of this mechanism operating et al. 1984) and on homopolymer tracts (HAUSWIRTH larger variants may be the result of similar mechanisms operatingonrepeat sequences separated by several hundred base pairs. Repeats of a symmetric dyad sequence have been found in cricket mtDNA at the bounds of a repeated 206-bp sequence responsible for size heteroplasmy (RAND and HARRISON1989). Insightinto the mechanism by which so many repeated sequences ofPissodes have beengenerated may be gainedthrough a similar analysis of the repeat (clone pPSH.2). That the cloned repeat sequence hybridizes to the large, typically uncut fragmentindicates sequence similarity between the two regions. T h e repeated sequences may be implicated in the generation of the overall large size of the molecule via additions to the A+T-rich region throughmechanisms of duplication/ deletion coupled with high mutation rates in A+Trich sequences. If the mechanism of duplication/deletion is dependent on the presence of highly similar sequences from repeat to repeat, mutation in those sequences could protect a region from furtherduplication and deletion. Repeat units changed in this way would presumably not beable to formsecondary structures necessary for excision of theunit,and would remainas extra sequence in theA+T rich region. Extra sequencewould continue to accumulate as repeats adjacent to the region lose the ability to excise. T h e amount of divergence necessary to prevent deletionof a memberof the repeatedfamily may not be much. The frequency of deletion of a 1.5-kb repeat in bacteriophage f l was significantly reduced by the change of only a single base pair in a 14-bp invertedrepeatsequence (ALBERTINIet al. 1982). Thus, the large, typically uncut A+T rich region in Pissodes mtDNA may consist of older repeat unitsno longer able to be excised. That the extra sequences have accumulated only on one side of the repeated regions may be related to thedirection of replication; presumably the repeated regions are the last to be replicated (see below). If this mechanism is responsible for the size increase, sequence similarity between repeat units and the large fragment should decrease as distance from the 12s rRNA gene region increases. The high degree of insertion/deletion polymorphismmay also berelatedtothe location of the repeated sequences relative to the sites of initiation and termination of replication. RANDand HARRISON (1986) have suggested that the presence of multiple initiation sites for replication could be a mechanism generating significant frequencies of large size variants. The repeated region of Pissodes mtDNA lies
a33
adjacent to the 12s rRNA gene, near or at the presumed originof replication (Figure 1).If the repeated region contains the initiation site for replication, multiple copies of the region mayallow for multiple replication forks, each producing a potentially different sized molecule. This mechanism would still require several duplication events to initially generate multiple sites for initiation of replication since the products of multiple replication forks could only be as large or smaller than the original template molecule. Alternatively, if the origin of replication is not repeated, the formation of secondary structures involved in slipped mispairing, and thus the generation of larger size variants, could be facilitated by the delay in light strand replication. Drosophila mtDNA initiates heavy strandreplication justpriortothe 12s rRNA gene and the initiation of light strand replication is typically delayed until heavy strand replication is nearly complete(GODDARDand WOLSTENHOLME 1978). If the location of the repeating units lies just prior to the initiation site for heavy strand replication, secondary structures important for duplication and deletion (RANDand HARRISON 1989) may be able to form during thedelay between heavy and light strand synthesis, promoting the rapid formationof different size classes. Population geneticsof heteroplasmy and mtDNA size: There are a variety of mechanisms that could maintain the high degree of heteroplasmy observed in the three species depending on the degreeof selection acting on size variants and the demographics of Pissodes species. These mechanisms can be classified as to whether stochastic (drift of neutral variants) or deterministic forces (selection) are expected to dominate the fateof size variants. Mitochondrial DNA size variants have usually not been viewed as strictly neutral (HALE and SINGH1986; MACRAEand ANDERSON 1988; RAND andHARRISON 1986; SEDEROFF1984; WALLACE1989).It is often argued that mtDNA is under selection that acts to keep the molecule compact, perhaps because smaller molecules are more efficiently replicated. Evidence for the selective advantage of smaller genomes has been reported in D.mauritiana (SOLIGNAC et al. 1984) and in the cricket Gryllusfirmus (RAND and HARRISON 1986). Thisposes a dilemma in explaining the patterns of size variation in Pissodes since there appears to be ample size variation andno clearindication that smaller genomes are selectively favored. If smaller molecules have a significant selective advantage, one would expect the smallest sizeclass to be most frequent, if not fixed in most individuals as is seen in a world-wide survey of D. melanogaster mtDNA size classes (HALE and SINGH1986). In therepresentatives of Pissodes examined to date, the smallest size classes
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T. M . Boyce, M. E. Zwick and C. F. Aquadro
are not the most frequent (Figure 3 and T. BOYCE, unpublished data). CLARK (1988) has shown that a balance of mutation, sampling, and selection can produce globally stable equilibrium frequencies of heteroplasmy under conditions that are not particularly stringent. In particular,wheremutation is bidirectional, as among size classes generated via duplication and deletion of repeated elements, mutationand sampling are sufficeint for the maintenance of heteroplasmy. If smaller mtDNA molecules in Pissodes are in fact at a selective advantage, then themutation rate to larger size classesmust be high enoughto effectively counter the effects of such an advantage. An additional factor that may act to help maintain significant frequencies of heteroplasmy and large size variants in Pissodes may be related to maternal age effects. SOLIGNAC et al. (1987) have shown that the frequency of long mtDNA size variants in progeny increased with female age at oviposition in D. melanogaster. Pissodes adults may live in nature for up to 4 years andreproduce in each of those seasons (MCMULLENand CONDRASHOFF 1973). If a similar age-dependent bias in size classtransmission occurs in Pissodes as in D. melanogaster, population processes that effect timetoreproduction or age-dependent fecundity could significantly contribute to the retention of larger mtDNA size variants. The persistence of the large size through the divergence of the three species suggests that the large size is not at a strong disadvantage. If the size variants observed in Pissodesare strictly neutral, then thelevel of heteroplasmy becomes a function of the effective population size (which removes variantsdue togenetic drift) and the mutation rateto new size classes (which generates new variants). RAND andHARRISON (1 989) used theorydeveloped by BIRKY,MARUYAMAand FUERST(1983) in a hierarchical analysis of size class variation in two species of crickets and found that the balance of mutation rate and drift was such that size class frequency changeswere significant from individual to individual due to theeffects of drift within cell lineages, but that the mutation rate was high enough to maintain a substantial frequency of heteroplasmy and little differentiation among populations. Qualitatively, the distribution of size class variation among individuals of Pissodes is similar to that described for crickets in that individuals usually contain unique profiles of size classes, indicating some influence of drift within lineages. However, the overall incidence of heteroplasmy within populations is substantially higher in Pissodes than in crickets (100% vs. 61.5%). While not conclusive, these observations suggest that mutation rates to different size classes and/ or mitochondrial effective population sizesmay be significantly larger, or rates of vegetative segregation lower in Pissodesrelative to most other taxa displaying
mtDNA heteroplasmy for size variants (BIRKY, FUERSTand MARUYAMA1989). These results may be particularly relevant to the use of mtDNA markers in studying population structure in Pissodes since high levels of heteroplasmy may lead to significant errors in estimations of population subdivision andgene diversity (BIRKY,FUERST and MARUYAMA1989).A larger analysis of heteroplasmy in Pissodes, partitioning size variation among individuals, populations and species will be necessary to more completely understand the population genetics of size variation in these species. An additional, and not necessarily exclusive explanation for the high level of heteroplasmy in Pissodes is paternal leakage of mtDNA. It is commonly assumed that mtDNA is maternally inherited,either because there is no parental contribution at fertilization, or because such contributions would necessarily be a small fraction of mtDNA in the developing embryo and would most likely not become fixed in the germ line (AVISEand VRIJENHOEK 1987; LANSMAN, AVISEand HUETTEL1983; GYLLENSTEIN, WHARTON and WILSON1985).However,recentstudies have suggested paternal contributions of mtDNA in Drosophila and marine mussels(SATTA et al. 1988; W. BROWN, personal communication).Significant paternal leakage or outright equal contribution to mtDNA polymorphism would increase the mitochondrial effective population size, and if mtDNA length variants are strictly neutral,larger effective population size would allow the accumulation of a greater number of mtDNA size variants. Furthermore, a significant paternal contribution would allow new combinations of different sizeclassesin progeny. Following the frequencies of size variants from parents through progeny of specific crosses among lines with distinct mtDNA types will be necessary to determine if significant paternal leakage is occurring in these species. Whatever balance of selection, mutation and drift that may be responsible, the ubiquitous heteroplasmy for size variants differing by as much as 7.5 kb, and the large overall size of mtDNA in all three species of Pissodes is unique among animals studied so far. If,as has been suggested from other studies, smaller sized mtDNA molecules are atsome replicative or selective advantage, some mechanism is present in Pissodes that has generated an unusually large size molecule in the face of whatever selective disadvantages mightbe associated with large size. The large mtDNA of Pissodes has existed since before the divergence of the three species examined,andnot as a rare variant within a species. Likewise, heteroplasmy involving an apparently high mutation rate for mtDNA variants of substantially different size has probably persisted over the same period of time. The high frequency of substantial size differences within and amongspecies sug-
mtDNA in Bark Weevils
gests that, at least in Pissodes, smaller size molecules may not be at any strong selective advantage. Moreover, the extensive size variationcontrastsstrongly with the conservation of order and structure within the coding region of the molecule, suggestingthat the two regions of the molecule experience significantly different selective regimens. We thank T . W. PHILLIPS for providing specimens and invaluable advice on collections and Pissodes biology. S. G. and H. A. BOYCE, J. DEWEY,T . KOERBER, J. MCLEAN,R. CLOWER,E.L. CUEBASINCLE,L. A. MART~NEZ also provided specimens or aided in their collection. R. GARMEkindly provided clones of D. melanogaster mtDNA, and D. WOLSTENHOLME clones of D.yakuba mtDNA. W. NOON,R. HARRISON and J. LEEprovided laboratory assistance. We J. DOYLE,R. HARRISON, D. RAND and two anonthank A. CLARK, ymous reviewers for their comments. This work was supported by the U.S. Department of Agriculture and by the National Institutes of Health.
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Communicating editor: A. G. CLARK
