Disparate Rates of Molecular Evolution in quence data. In cases where different methods yielded different results, we re Cospeciating Hosts and Parasites tained all host and parasite trees for to pological comparison in order to deter Mark S. Hafner, Philip D. Sudman, Francis X. Villablanca, mine whether the inference of cospecia Theresa A Spradling, James W. Demastes, Steven A Nadler tion is warranted and, if so, whether the inference is sensitive to the method of DNA sequences for the gene encoding mitochondrial cytochrome oxidase I in a group of analysis. rodents (pocket gophers) and their ectoparasites (chewing ~ice) provide evidence for All analyses of the pocket gopher se cospeciation and reveal different rates of molecular evolution in the hosts and their quence data (using different models of parasites. The overall rate of nucleotide substitution (both silent and replacement chang DNA sequence evolution) yielded trees es) is approximately three times higher in lice, and the rate of synonymous substitution that were very similar in overall branch (based on analysis of fourfold degenerate sites) is approximately an order of magnitude ing structure. For example, phylogenetic greater in lice. The difference in synonymous substitution rate between lice and gophers analysis (I 1) of the COl sequence data for correlates with a difference of similar magnitude in generation times. pocket gophers yielded two most-parsimo nious trees of equal length (1423 steps). One of these trees (Fig. ZA) was topolog ically identical to the tree generated by a Chewing lice of the genera Geom:ydoecus in this host-parasite assemblage. We se maximum-likelihood analysis of the same and Thomom:ydoecus are obligate ectopara quenced and compared homologous regions data ( 12). The other most-parsimonious sites of pocket gophers (Fig. 1 ). Because the of the gene encoding the mitochondrial tree showed only minor differences ( 13) entire life cycle of these lice occurs exclu cytochrome c oxidase subunit I (COl) in from the tree shown in Fig. 2A. The sively in the fur of the host, and because both groups (8). Of the 379 nucleotides general structure of the gopher parsimony different host species rarely interact, each sequenced for each taxon, 134 positions tree (Fig. ZA) also was supported by species of louse is normally restricted to a were variable in pocket gophers and 178 Fitch-Margoliash (Fig. ZB) and neighbor single host species (1). As a result, there is positions were variable in chewing lice (Ta joining (14) analyses of genetic distances close correspondence between gopher tax ble 1). ( 12). Differences among the trees gener onomic boundaries and louse taxonomic The cospeciation hypothesis predicts ated by the parsimony, maximum-likeli boundaries (2). When viewed over large that the branching structure of the host hood, Fitch-Margoliash, and neighbor geographic and temporal scales, this re and parasite phylogenies will be similar to joining analyses of the pocket gopher data stricted distributional pattern of chewing a degree beyond that expected to occur by were judged nonsignificant by a likeli Lice on pocket gophers has resulted in phy chance. This prediction can be evaluated hood ratio test (15). Accordingly, all four logenetic histories of lice and gophers that statistically (3, 4). For any particular trees were retained for topological com are remarkably similar (3-5). host-parasite assemblage, confidence in parison with the parasite trees. The basic AJthough well-documented cases of the test of cospeciation can be no stronger structure of these trees and, in particular, host-parasite cospeciation are rare (3 , 6), than confidence in the phylogenies under relations within the genera Onhogeom:ys they are of interest because they permit comparison. Thus, it is essential that the and Geom:ys, also are supported by inde- comparative study of organisms with a long host and parasite phylogenies accurately history of parallel evolution. The temporal estimate the evolutionary history of each Table 1. Obselved percent of difference (mean component of parallel phylogenesis (in group. There are many methods for esti :t1 SD) in various elements oftheCOl nucleic acid sequence from pocket gophers and their acto which lineages of hosts and their parasites mating phylogenies from sequence data parasitic chewing lice. speciate repeatedly at approximately the (9), each of which uses a different model same time) permits examination of relative of nucleotide evolution and potentially Percent of sequence rates of evolution in the two groups by yields a phylogenetic hypothesis (a tree) difference (:t 1 SO)' comparison of the amount of change each that differs from that estimated with other has undergone during their parallel histo methods ( 10). To consider the effects of Gophers Lice ries. Because the life histories of hosts and different evolutionary models on our esti their parasites are often profoundly differ mates of phylogeny, we applied multiple Rrstposition 1.43(0.49) 2.09(0.62) transitions ent, studies of molecular evolution in host methods of analysis (II, 12) to our se F1rst position 0.00(0.25) 0.46(0.34) parasite assemblages can help answer a transversions broad spectrum of questions relating to the Seoond position 0 .24 (0.18) 0.38(0.37} possible effects of generation time, metabol transitions ic rate, and other life history parameters on / -~ Seoond position 0 .00(0.00) 0.16 (0.17} rates of mutation and evolutionary change. transversions ' Third position 8.74 (1.65) 9.59 (1 .51) We examined DNA sequence variation transitions in 14 species of pocket gophers and their Third position 5.01 (1 .67} 7.68 (1.99) chewing lice (7) to test for cospeciation and transversions to investigate rates of molecular evolution Total difference 15.64 (3.20) 20.75 (2.31) Silent nucleotide 14.75 (3.09) 17.66 (2.32) differences Replacement 0.89 (0.71) 2.67 (1.05) nucleotide
Fig. 1. Chewrlg lice (Geomydoecus texanus, In differences set) are wingless insects that are obligate ecto· Amino acid 2.40 (1.83) 6.85(2.62) parasites of pocket gophers (Thomomys bottae is differences shown here). The entire life cycle of the chewing ·Meanand standard delliation based on alpaiiWise com louse occurs in the fur of these fossorial rodents. parisons.
pendent phylogenetic studies based on and parasites (3), corroborates indepen maximum-likelihood distance matrices for morphology, allozymes, comparative im dent evidence for cospeciarion in several cospedating gophers and lice using Man munology, karyology, and nucleotide se genera of pocket gophers (Orchogeomys, tel's test (25), which showed a high ly sig quence data (3, 5, 16-18). Geomys, and Thomomys) and their lice nificant (P < 0.01) association between All analyses of the chewing Louse se (3-5). genetic distances in corresponding hosts quence data likewise yielded trees with Given evidence for cospecianon, it is and parasites. This test demonstrates that similar branching structures. Phyloge possible to test the null hypothesis that evolutionary rates in gophers and lice are netic analysis of the louse COl data yield pocket gophers and chewing lice have un significantly correlated, regardless of cree ed three most-parsimonious trees of equal dergone equivalent amounts of genetic dif structure. To tesr for equality of rates be length (4208 steps). One of these trees ferentiation during their parallel histories. tween gophers and lice, we compared max (Fig. 2A) was topologically identical to 1c is appropriate that th is test be restricted imum-likelihood branch lengths for all pos the tree generated by a maximum-likeli to hosts and parasites that have cospeciated, sible combinations of cospeciating raxa (24, hood analysis of the same data. The two because time since divergence can be as 26). In all cases, Wilcoxon sign-rank tests remaining parsimony trees showed only sumed to be equal only for host-parasite showed thal louse branches were signifi minor differences (involving one spec1es pairs that show cospeciarion (9 host-para cantly longer than gopher branches (P < in each case) from the tree in Fig. 2A sire pairs in Fig. 2A and 10 host-parasite 0.003 in each case). Given this significant (19). The Fitch-Margoliash analysis (Fig. pairs in Fig. 2B) (24). We first compared difference, we used Model II regression 28) and the neighbor-joining analysis (ZO) yielded louse trees very similar to those generated by the parsimony analy A sis. Differences among the trees generated o. ~ ...... a.~ 0. chetrlli 0 0 0 0 0 0 00 •• 00 0 0 0 0 0 0 G. chetrlli by the parsimony, maximum-likelihood, 0 . IJIId«wocd 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G. AfUtl Fitch-Margoliash, and neighbor-joining L-...;- O. c::e~ ····· ·· ·····- G. ~ analyses of the louse data were judged '--...... ,\~- 0. hllplcM ...... ··-··-····· G. dvpW ---.,;::--' nonsignificant by a likelihood ratio tesr 1----- Z lrt;hoput ....•...... G. trlt:ht:f1l (15). Accordingly, all five louse trees '------P. bcilltf· ·················-· · G. n.llerl were retained for topological comparison c. ~ ··············· G.....,_ -----, c.mem.trW ·. 0 •• G. «:ff.Jool with the host trees. The basic structure of ...--.:'!:,...-- G. penotWIIII :\···········'· G.,_.. the louse trees and, in particular, relations '------~ G.br~Mc~p~J ...:·., ....,.::". .... G. ewing/ among lice hosted by species of Orchogeo G. bclrNtiu. (a) .:·.,..-: G. ~ mys and Geomys , also are supported by I G.~(b} •/::•._. ...• a.~ independent phylogenetic studies of alloz ...... \.~(;_= ymes (3, 5, 21). K T. bo111N ./..,.,·::·....••....• T. m/rto( • The COMPONENT program (22) de '------!'.';;- T. lll~ ..- ·...... T. b8ttWnN 1------' termined if the fit between observed par asite and host trees was significantly bet ter than the fit between the parasite tree ~. 8 and trees drawn at random from the set of 0. /NJttlffdll oo .... oo. G. C05tllli:>IUsll all possible host trees (4). For each of 20 0 . chetrlli 0 0 0 0 0 0 0 0 0 00 0 0 0 --.- G. chetrlli pairwise compansons (four host trees and 0 . ll"ltieft1loo6 0 0 0 0 0 • • • 0 0 0-0 0 G. a«zetf 0. cal'llltlr ···· ·· · ·· · · · · G.~ five parasite trees), the observed de{!ree of 1-"'""""- o.hifpldc.- ...... G.dvpW -...,..,.----' fir between the gopher and louse trees was ,....---- Z trfchopul ··...... :G, lhomomyul significantly better (P < 0.01) than the P. bcilltf ...... •..)::::.-.G. trlt:ht:f1l fir between the louse tree and I0,000 c. ceatanopf. 0 0 0 0 0 . 0 : G. ,.,., randomized gopher trees (23). These re C. ITIMrlaml •• /""""G.~ sults, which are robust to the method of ,....---- G. ~ ·:...··-i-······· G. rex.anu. L-.---i G. bmhpf 0 0 0 :-• ..:····-·-···G. ewing~ phylogenetic inference and to the evolu G.~(•) f.·., a.~
rionary models used, falsify the null hy G. ~(b) ;..•:,__: 0 ·G. QeOtiiJdt pothesis of chance similariry between the hosr and parasite crees. Although this ev T.IDiae ..,jt.:::::::·~~:: ------.....1 idence is consistent with the hypothesis L------fO..' T.lll~ !...... T. beltllnle of cospeciarion, the concordant phylog o.n enies might instead result from dispersal, Fig. 2. Phylogenies of pocket gophers Oeft) and chewing lice (nght) generated by analysis of DNA extinction, or incomplere sampling of sequences of the gene encoding COl. In each set of trees, coexis!Jng hosts and parasites are linked by closely related taxa (4). However, only dotted 6nes (each parasite examined was taken directly from the fur of the host individual exaffi108d). (A) the cospeciation hypothesiS predicrs tem Host and parasite treesgenerated byparsimonyandmaximum-likelihood analyses of the sequence data. poral congruence of host and parasire spe (B) Trees generated by Frt:ch-MargofJa$11 analyses of the host and parasite data. Pocket gopher taxa ciation events, which (given roughly examined include the genera Orthogeomys, Zygogeomys, Pappogeomys. Cratogeomys, Geomys, and time-dependent molecular change in each Thomomys. Chewing louse taxa include the genera Geomydoecus and Thomomydoecus. The louse G. group) wou lu resu lt in a significant rela setzeri also has been reported from 0. cherrfel in areas where the range of 0. cheniel abuts that of 0. tion between measures of molecular dif underwood!. Relative rates of molecular evolution were investigated by comparison of expected numbers ferenriarion In the host and parasite trees. of substitutions persite for hosts and parasites. Maximum-likelihood branch lengths (X100) based on all nucleotide substitutions are indicated lor one of four possible branch combtnations 111 Fig. 2A and for one We demonstrate below rhar our molecular of the two possible combinatlons in FIQ.28(24). Letters above branches Indicatebranches compared in data are consistent with this prediction. FIQ. 3. Comparisons were restricted to parasites whose phylogenetic hiStory iS topologically IdentiCal to This finding, which requires no assump that of thelr hosts. Because most of the uncertainty in the phylogenetlc analyses involved branches near tions about rare similarity between hosts the base of the trees, only terminal and subterminal branches were compared between gophers and lice.
analysis (through rhe origin) and deter then change at these sites should fit a nucleotide substitutions ar fourfold de mined chat the slopes of the regressions molecular clock model (30). Accordingly, generate sites indicates that rates ofsilent (Fig. 3A) ranged from 2.60 to 2.83 (with a we rested all possible combinations of substitution in this gene region are rough mean of 2.74). This indicates chat che over cospeciating gophers (Orthogeomys only) ly 11 rimes greater in lice than in gophers all rate of nucleotide substitution in lice is and their lice (24) for significant depar (Fig. 3B). The fact rhar this 11-fold rate approximately three times higher than in ture from clocklike behavior, usmg the difference is not evident when all substi gophers (27). log-likelihood rario test (I 2). In all cases, tutions are considered is probably the re To estimate rates of synonymous sub che data were consistent with molecular sult of selective constraints on replace stitution in gophers and lice, we restricted clock assumptions, which indicates that menr substitutions. High levels of func our analysis of the COl sequences to four substitutions within gophers and within tional constraint on the COl enzyme fold degenerate sires (sites at which ~~ II lice accumulate in a roughly time-depen have been reponed in other organisms base substitutions are silem). We focused dent fashion. Wilcoxon sign-rank tests (28). on the largest group of closely related showed that louse branches were signifi The 11-fold difference in rates of syn species (Orthogeomys species and their cantly longer than gopher branches in onymous substitution in Orthogeomys go lice) because these species are sufficiendy four of the six possible comparisons (P < phers and their lice (Fig. 3B) cannot be closely related to ensure that corrections 0.05 in each case). We used Model II explained by transition bias or nucleotide for multiple mutations are effective (28). regression analysis (through the origin) to frequency bias. Because silent substitu The numbers of variable fourfold degen quantify the relation between gopher and tions at che fourfold degenerate sites show erate sites in gophers and lice were ap louse branch lengths. Slopes of the regres clocklike behavior, it is likely that they proximately equal (67 and 69, respective sions (Fig. 38) ranged from 9.88 to 11.83 are neutral or nearly neutral (30). Several ly). We used a maximum-likelihood mod (with a mean of 11.04 ), which indicates possible mechanisms could account for el of evolution to infer branch lengrhs that the estimated rate of silent subsmu this rare difference, including mutation based solely on substitutions at these sires tion for this gene region is approxnnately rare differences caused by possible differ (24). The model included corrections for an order of magnitude greater in chewing ences in gene order char affect vulnera observed transitional bias (a maximum Lice than in pocket gophers. Evidence for bility to mutation (32), differences in 4: l bias in gophers; LO: I bias in lice) and a higher rare of substitution in lice ap metabolic rare or general metabolic phys fQr significantly different nucleotide com pears to be independent of the evolution iology, generation-time differences, or positional biases in rhe rwo data sets (P < ary model employed, although the magni other factors correlated with body stze 0.05) (29). These corrections are neces tude of the rate difference is sensitive to (33). Alternatively, this rate difference sary to estimate evolutionary rates (28), certain parameters of the model (31 ). could be caused by mechanisms rhat are and spurious re5ults should nor occur Sim Viewed together, the analysts of all independent of mutation rare, such as ply because of differences in the number nucleotide substitutions (Fig. 3A) and the codon bias and other constraints on the of characters compared or differences in analysis ofsubstitutions at fourfold degen translational apparatus, or differences in mutational dynamics (and resultant satu erate sites (Fig. J!j) provide insight into DNA repair efficiency. It is perhaps im ration levels) in the rwo groups being the dynamics of molecular evolution for portant char this 11-fold rate difference Is compared. In theory, rates of synonymous this gene region in the species studied. accompanied by a similar cllfference in substitution are proportional ro mutation The analysis of all substitutions indicates generation time between gophers and lice rates (30), bur our estimates cannot be that the overall rate of evoluttonary (approxunarely 1 year in gophers and 40 considered dtrect measures of mutation change IS approximately three ttmes days in lice) (34). If the observed rare rates because we have not controlled for greater in chewing lice than in their hosts difference results from an underlying dif possible constraints on che translational (Fig. 3A). Likewise, the means of all pair ference in mutation rare, then generarion apparatus, such as codon bias and second wise replacement differences for nucleoti time may explain this difference. Howev ary structure of mRNAs. des and amino acids are approximately er, mutation rates are more likely to be If nucleotide substitutions at fourfold three times greater in lice than in gophers influenced directly by nucleotide genera degenerate sites arc selectively neutral, (Table 1). In contrast, the analysis of tion time than by organismal generation time (33). As such, ourstudy suggests that each organismal ge neration is equivalent Fig. 3. Companson of rates of molecular change A B w equal numbers of nucleotide genera in a 379-bp region of the gene encoding COl in 8 tions in pocket gophers and chewmg lice. cospeciated pocket gophers and chewing lloe. E 2.o All Sllbstitvtions 4.0 Silent substiM!ons If the l J-fold rare difference reflects Letters refer to branches labeled in Rg. 2A. (A) B • o; 1.8 F a similar difference in mutation rate, Comparison of maximum-likelihood branch 3.5 F lengths (based on all nucleotide substitutions) lor 8. 1.6 then these findings are consistent with 3.0 the phy1ogeny shown in Rg. 2A (one of six possi the neutral theory of molecular evolution ble combinations of cospeciating taxa) (24). 2.5 (30), because once rhe data are corrected Slopes of Model II regressions (through the ongln) for the difference in generation time, ranged from 2.60 to 2.83 (with a mean of 2.74), 2.0 they suggest equal rates of mutation per which indicates that the overall rate of nucleotide 1.5 c generation m distantly related groups of substitution 1n hoe is approximately three times animals. nigher than in gophers (27). (B) ComparisOn of 1.0 D maximum-likelihood branch lengths based solely REFERENCES AND NOTES on nucleotide substitutions at fourfold degenerate sites for Orthogeomys gophers and their lice. 1. S.A Nadler, M.S. Hafner. J. C. Hafner, D. J Hafner, Slopes of Model II regressions (through theorigin) EvolutJoo 44, 9¢2 (1990). ranged from 9.88 to 11 .83 (with a mean of 11.04), 2. A. D. Price and K C. Emerson. J. Med. Entomol. 8, which indicates that the rate of synonymous sub· 228(1971). 3. M.S. Hafnor and S. A. Nadler. ~- Zool. 39. 192 stitut1011 in th1s gene region 1s approximately an (1990). Nature 332, 258 (1988). order of magnitude greater In chewing lice than In pocket gophers. 4. A. D. M Page, Sysr. ZOO!. 39. 205 (1990).
5. J. W. Oemastes and M. S. Hafner, J. Mammal. 74, P. Huelsenbeck,J. Hered. 83, 189(1992)). Gophers in ciating taxa in Fig. 2A [that is, the nine-taxon gapher 521 (1993). the genusThornomys and lice ofthe genus Thomomy· tree can include either 0. underwood! or 0. cavator 6. R. D. M. Page. Int. J. Parasltol. 23. 499 (1993). doecus were designated as out~oups. The outgroup (but not both] and G. breviceps crG. personatus (but 7. Voucher specimens are deposited in the Museum of status ol these taxa is supported by previous motpho not both)). There are 2 possible combinations or 10 Natural Science, Louisiana State University (LSUMZ) loglcaJ and molecular analyses (16, 77) [R. A. Hellenthal cospeciating taxa In Fig. 26 (that is, the tree can cr the New Mexico Museum of Natural History andR. D. Price,J. Kans. Entomol. SOc. 57,231 (1984); include either G. breviceps orG. personatus. but not (NMMNH) and are as follows: Orthogeomys under S. A. Naclerand M.S. Hafner,Ann. Entomo/. Soc. Am. both). Because most of the uncertainty in the phylo· woodi (l.SUMZ 29493), 0. hispidus (LSUMZ 29231). 82, 109 (1989)]. geneticanalyses involved branches near the base of 0. cavator (LSUMZ 29253), 0. cherriel (LSUMZ 12. We used PHYLIP [Phylogeny Inference Package, the trees, only terminal and subterminal branches 29539). 0. heterodus (LSUMZ 29501), Geoolys 3.5c, J. Felsenstein, Department ofGenetics. Uni were compared between gophers and lice. breviceps (LSUMZ 33940). G. personatus (LSUMZ versity of Washington. Seattle] tor maximum-like 25. N. Mantel, CancerRes. 27, 209 (1967). We calculat 31400), G. bursarius halli (LSUMZ 31463; destgnat lihood, Fitch-Margollash (W. M. Fitch and E. Mar ed distance matrices using the maximum-likelihood ed "a" in FJQ. 2). G. b. majusculus (LSUMZ 31448; goliash, Science 155, 279 (1967)], and neighbor model (12). For the 9 cospeclating taxa ln Fig. 2A, t designated "b" in Ftg. 2), Cratogeomys castanops joining (N. 5aitou and M. Nei, Mol. Bioi. Evol. 4, ~ 4.073, P < 0.01 . For the 10 cospeciatlng taxa in (LSUMZ 31455), C. merriamt (LSUMZ 34343). Pap· 406 (1987)] analyses of the sequence data. Empir Ag. 2B, t ~ 4.289, P < 0.01. Degrees of freedom pogeomys bullerl (LSUMZ 34338), Zygogeomys Ical base frequencies and maximal observed tran were adjusted to n - 1, where n =number of taxa trichopus (LSUMZ 34340). Thomomys bottae sition biases ( 10: 1 for gophers and 17: 1 for lice) compared (3). (LSUMZ 29320 and 29569). and T. ca/poides were used in the maximum-likelihood analysis. 26. Trees for cospeciating taxa (24) were input as user (NMMNH 1634 and 1637). The Fitch-Margoliash and neighbor-joining analy trees into PHYLIP (12) in order to estimate maxi 8. Pocket gophers were trapped, killed, and Immedi ses were based on distance matrices corrected mum-likelihood branch lengths. Empirical esti· ately brushed to recover lice. Gopher tissues and with the Kimura two-parameter model (substitut mates of transition bias, nucleotide frequency lice were stored at - 7o•c. DNA extractions from Ing maximal observed transition bias values tor the bias, and positional bias (first:second:third codon gophers followed the phenol-chloroform tech· default (2:1 ) value (M. Kimura, J. Mol. Evol. 16, position: 5:1:40 for gophers and 5:1 ;21 for lice) nique (D. M. Hillls, A. Larson, S. K. Davis, E. A. 11 1 (1980)]). We repeated each analysis at least were incorporated into the maxlmum-likehhood Zimmer. in Molecular Systematics, D. M. Hillis and 10 times, varying the input order of taxa using the model (12). We recognize that the parameters C. Moritz, Eds. (Sinauer, Sunderland. MA 1990), jumble option of PHYLIP. used in maxjmum-likellhood models will influence pp, 318-370]. DNA extractions from lice followed 13. In the alternative parsimony tree for pocket gophers, rate estimates [K. Fukami-Kobayashl and Y. a modification of the protocol described by H. Uu the phylogenetic positions of P. bulleri and Z. tricho Tateno, J. Mol. Evol. 32, 79 (19g1)J. Our models and A. T. Beckenbach (Mol. Phylogenet. Evol. 1. pus were reversed and the two species otCratogeo use empirical estimates of these parameters (rath 41 (1992)] and used two lice from each gopher. mys were not depicted as sister taxa. er than arbitrary or constant values) to obtain a One 11-lof the extraction solution was used for DNA 14. The neighbor-joining tree differed fr9m the Rtch more realistic approximation of the actual mota· amplification In a 50· JJ.I reaction. A 379-base patr Margoliash tree (FIQ. 1B) only In the placement ofP. tionaI dynarnics of the gene region being surveyed (bp) region of the mitochondrial COl gene was bulleri(immediately basal to the five-taxon clade con (72, 28). amplified by polymerase chain reaction (PCR) with taining 0. hispidus) and Z. trichopus (basal to the 27. We used Model II regression analysis (reduced two degenerate primers: L6625 (5'-CCGGATC eight-taxon clade containing C. castanops, C. mer major axis model). which does not permit conven CTTY!'GRTlYTlYGGNCAYCC-3') and H7005 riam!. and P. bulferi). tional tests of significance [R. R. Sakal and F. J. (5'-CCGGATCCACNACRTART ANGTRTCRTG 15. H. K'IShino and M. Hasegawa, J. Mol. Evol. 29, 170 Rohtr, Biomelty (W. H. Freeman, San Francisco, 3'). Primer names refer to the 3' position of each (1989), as used in PHYUP (72). 1969)]. However, the nonparametric Mantel's test primer relative to the human mitochondrial ge 16. R. S. Russell, Univ. Kans. Pub/. Mus. Nat. Hist. 16, documents a significant relation between genetic nome (S. Andersonetai.,Nature 290. 457 (1981)]. 473 (1968): R. L. Honeycutt and S. L Williams. J. distances in gophers and lice (25), and the non Double-stranded amplifications were done by four Mammal. 63. 208 (1982); M. S. Hafner, ib1d. 72, 1 parametric Wilcoxon sign-rank test shows that cycles of 1 min of denaturation (95°C), 1 min of (1991). louse branches are significantly longer than go annealing (45•CJ, and 1 min of extension (72•C), 17. M. S. Hafner. Z. Zoo!. Syst. Evotutionsforsch. 20. pher branches. When the regression analyses fOllowt~d I.Jy 30 cycles at reduced denaturation 118 (198.2). Wt>ltl not constrained through the origin, none or temperatures (93.C) and increased annealing tem 18. P. D. Sudman and M. S. Hafner, Mol. Phytogenet. the y-intercepts was significantly different frorn peratures (6Q•C). Methods for production of sin Evol. 1. 17 (1992). zero. gle-stranded templates and tor sequencing ofsin 19. In the second parsimony tree. the louse G. actuosl 26. R. Kondo, S. Horai, Y. sana. N. Takahata. J. Mol. gle-stranded products are given elsewhere (78). was linked with the ol