Parasites can be used as unique markers to investigate host evolutionary history, independent of host data. Here we show that modern human head lice, Pediculus humanus, are composed of two ancient lineages, whose origin predates modern Homo sapiens by an order of magnitude (ca. 1.18 million years). One of the two louse lineages has a worldwide distribution and appears to have undergone a population bottleneck ca. 100,000 years ago along with its modern H. sapiens host. Phylogenetic and population genetic data suggest that the other lineage, found only in the New World, has remained isolated from the worldwide lineage for the last 1.18 million years. The ancient divergence between these two lice is contemporaneous with splits among early species of Homo, and cospeciation analyses suggest that the two louse lineages codiverged with a now extinct species of Homo and the lineage leading to modern H. sapiens. If these lice indeed codiverged with their hosts ca. 1.18 million years ago, then a recent host switch from an archaic species of Homo to modern H. sapiens is required to explain the occurrence of both lineages on modern H. sapiens. Such a host switch would require direct physical contact between modern and archaic forms of Homo.

We reconstructed the evolutionary history of P. humanus and several outgroup taxa using both morphological and molecular data. First, we used louse morphological data to test for patterns of cospeciation between primate lice and their hosts. Then we collected molecular data from a subset of the same taxa and calculated divergence dates for nodes in the louse phylogeny. This broad phylogenetic approach allowed us to date the origin of the human louse, P. humanus, and to date the two divergent lineages within the species. Finally, we collected population genetic data for P. humanus to compare with population-level characteristics of extant humans. Taken together, our phylogenetic and population-level data provide a well-resolved picture of the evolutionary history of P. humanus, which can be used to indirectly infer human evolutionary history. Specifically, we compared three distinct models of modern human origins (Recent African Replacement without Introgression, Multiregional Evolution, and Diffusion Wave) to see which model best fits the data from human lice.

Recent molecular work by Leo et al. (2002) showed that, despite the ecological differences between head and body lice, the two forms are not genetically distinct. Kittler et al. (2003) confirmed this finding but also discovered two deeply divergent clades within P. humanus that are uncorrelated with the head and body louse forms. The divergent clades of lice stand in contrast to mitochondrial sequence data from extant human populations, which coalesce to a single lineage very rapidly. The shallow coalescence in human mitochondrial sequence data is likely the result of a recent population bottleneck and subsequent population expansion ( Rogers and Harpending 1992 ), which obscures much of the evolutionary history of humans prior to the bottleneck. The deep divergences within P. humanus have the potential to reveal aspects of human evolutionary history that cannot be recovered from human DNA markers.

Lice are obligate parasites of mammals or birds that complete their entire life cycle on the body of the host; they cannot survive more than a few hours or days off the host ( Buxton 1946 ). Mammal lice are closely tied to their hosts in both ecological ( Reed and Hafner 1997 ) and evolutionary ( Hafner et al. 1994 ) time. The lice found on primates are quite host specific, with most species occurring only on a single species of host ( Durden and Musser 1994 ). Host specificity is reinforced by the fact that primate lice require direct physical contact between hosts for transmission ( Buxton 1946 ; Durden 2001 ; Canyon et al. 2002 ; Burgess 2004 ). Host specificity often goes hand in hand with long-term coevolutionary patterns between hosts and parasites ( Page 2003 ), making primate lice excellent candidates for inferring host evolutionary history. Humans are parasitized by two species of lice: head/body lice (Pediculus humanus), the focus of this paper, and pubic lice(Pthirus pubis), which serve as a phylogenetic outgroup in this study. P. humanus is found in two forms (head and body lice) that are morphologically similar, but ecologically distinct. Body lice live primarily in clothing and move onto the skin to feed once or twice a day. Head lice are confined to the scalp and feed more frequently. Body lice vector the bacteria responsible for epidemic typhus, trench fever, and relapsing fever; head lice are not known to vector any agent of human disease under natural conditions ( Buxton 1946 ).

Fossils provide the only source of data available for most species of archaic humans and are therefore crucial to understanding the origin of modern humans. Unfortunately, missing taxa and fragmentary fossils limit our ability to reconstruct human evolutionary history based solely on fossil data. Molecular (DNA sequence) data have provided additional insight into the recent evolutionary history of humans, but these data are limited mainly to extant human populations. Ancient DNA was recently sequenced from H. neanderthalensis ( Krings et al. 1997 , 1999 , 2000 ) and a 24,000-year-old specimen of modern H. sapiens ( Caramelli et al. 2003 ), but even these ancient DNA studies do not agree on hypotheses of modern human origins ( Templeton 2002 ; Serre et al. 2004 ). Only a few ancient specimens have been examined molecularly, and additional sequences are slow to emerge. Furthermore, DNA may never be retrieved from some specimens because it is difficult, if not impossible, to liberate sequenceable DNA from poorly preserved ( Krings et al. 1997 ) or very old ( Paabo and Wilson 1991 ) fossil material. Therefore, the degree to which we can reconstruct human evolutionary history depends, in part, upon additional types of data.

One of the most intensely debated topics in evolutionary biology pertains to the origin of modern Homo sapiens. The debate concerns the precise manner in which anatomically modern humans arose from archaic ancestors. Empirical studies tend to support one of two prominent models of human origins, the Recent African Replacement model ( Stringer and Andrews 1988 ) or the Multiregional Evolution model ( Wolpoff et al. 1994 ). The Recent African Replacement model, as originally proposed, suggests that modern humans arose from an archaic ancestor in Africa ca. 130,000 years ago, and then replaced archaic humans in Asia, Africa, and Europe without introgression between archaic and modern humans. The Multiregional Evolution model (as proposed by Wolpoff et al. [1994] and revisited by Wolpoff et al. [2000] ) suggests that gene flow existed not only among populations of modern Homo sapiens, but also between modern H. sapiens and archaic forms of Homo (e.g., Homo neanderthalensis and Homo erectus), which led to some degree of regional continuity. Both models can be subdivided into many variants. There are two common variants of the Multiregional Evolution model. In one variant, the transition from archaic to modern humans occurs incrementally across a large geographic region (i.e., both within and outside Africa); in the other variant, the transition from archaic to modern humans arises first in Africa then spreads through gene flow outside of Africa. This latter variant is very similar to a Diffusion Wave model recently put forth by Eswaran (2002) . Both types of models of human origins (the Recent African Replacement and Multiregional Evolution models) have been examined with both human fossil and genetic data, but no single model or variant has been supported by all the data.

The age of the MRCA of P. humanus dates to 1.18 MYA (for mtDNA), which is roughly midway between the estimated ages of H. neanderthalensis (0.60 MY) and H. erectus (1.8 MY). We used a maximum likelihood (ML) analysis to test whether our two divergent lineages of lice could have diverged in tandem with H. sapiens and H. neanderthalensis (Neandertals). H. neanderthalensis is the only other species of Homo for which DNA sequence data are available ( Krings et al. 1999 ). The test evaluated whether relative branch lengths (scaled according to mutation rate) in the host tree, specifically for the branch between H. sapiens and H. neanderthalensis, are consistent with the parasite DNA sequence data ( Huelsenbeck et al. 1997 ). In cospeciating assemblages, host and parasite branch lengths are highly correlated due to a shared evolutionary history ( Page 1996 ). A likelihood ratio test (LRT) rejected (p < 0.0001) the H. sapiens/H. neanderthalensis split as a node of cospeciation with the two clades of P. humanus because the branch length between H. sapiens and H. neanderthalensis is far too short to explain the louse DNA sequence data. In other words, the split between H. sapiens and H. neanderthalensis is too recent to have been contemporaneous with the divergence of the two lineages of lice. If one artificially lengthens the branch between H. sapiens and H. neanderthalensis to approximate the split between H. sapiens and H. erectus (anywhere from 1.2 to 1.8 MYA), the LRT fails to reject this hypothesis of cospeciation.

The WW clade of P. humanus shows evidence of a recent population expansion (Fu and Li's D* = −2.80 [ Fu and Li 1993 ]; p < 0.02). We estimated the date of this population expansion from the mismatch distribution. The estimate was calculated by comparing the average pairwise difference within the WW clade of P. humanus (4.21 mutations) to the pairwise difference between P. humanus and P. schaeffi (220 mutations), which diverged 5.6 MYA. The population expansion of the WW clade is estimated to be 0.11 MYA, similar to the estimated date of population expansion of modern humans out of Africa ca. 0.10 MYA ( di Rienzo and Wilson 1991 ; Rogers and Harpending 1992 ; Harpending et al. 1993 ). In contrast, the NW clade of P. humanus does not exhibit the signature of a recent population expansion (Fu and Li's D* = 0.17), but instead shows a more stable population size.

We can calculate an expected date of mitochondrial coalescence for P. humanus if we assume for the moment that the entire population of lice mated at random (i.e., panmixia). The estimate of expected coalescence is based on the effective female population size (N ef ), which was estimated from the sample of all P. humanus specimens to be 1.1 million female lice from the equation Θ = 2N ef μ. The estimate of N ef provides an expected coalescence time for the two divergent mitochondrial lineages of P. humanus of 1.10 million generations or ca. 0.11 MYA, which is an order of magnitude younger than the observed divergence time of 1.18 MYA. In a large randomly mating population consisting of 1.1 million female lice, one would expect to maintain two distinct haplotypes for only ca. 0.11 million years (MY). This suggests that we can reject panmixia if we assume that N ef prior to the bottleneck was roughly similar to what we see today. If estimates of N ef were drastically higher (ca. 60 million female lice) prior to the bottleneck, then expected time to coalescence could be much longer. The F st value, a measure of genetic population differentiation, calculated for the WW and NW clades was 0.96, indicating substantial population structure, which also supports the rejection of panmixia.

The clades of P. humanus identified by Kittler et al. (2003) are nearly identical to those from our data, with the exception of their basal African clade, which was not represented in our data set. One clade contains both head lice and body lice and is WW in distribution. Another clade is comprised solely of head lice from the NW (our data) and Europe (samples from Kittler et al. 2003 ), and the most basal clade contains isolates 4, 18, and 33 from Kittler et al. (2003) , which are head lice from Africa. The size of the triangles representing the three clades are proportional in size to the number of taxa within the clade. This phylogeny is rooted with a divergent louse, Dennyus hirundinus, which is a bird louse in the suborder Amblycera. Note the placement of the Kittler et al. (2003) specimen of P. schaeffi, which falls outside all other primate lice and the rodent louse Fahrenholzia.

Our estimate of the age of the MRCA for P. humanus (1.18 MYA) is much older than that reported by Kittler et al. (2003) , which was only 0.53 MYA based on mtDNA. Their estimate of 0.53 MYA was determined using a mtDNA sequence from a specimen of the chimp louse, P. schaeffi, that is quite aberrant when compared to other primate lice. Phylogenetic analysis of the Kittler et al. Cytb data (downloaded from GenBank), combined with our own data, shows that the Kittler et al. sequence for P. schaeffi is 40% divergent from P. humanus and 40% divergent from our own sequence of P. schaeffi. Phylogenetic analysis places their specimen of P. schaeffi outside all other primate lice and even outside the rodent louse ( Figure 4 ), whereas our specimen of P. schaeffi is sister to P. humanus, based on both morphology and molecular data. We think that the Kittler et al. specimen has been attributed to the species P. schaeffi in error. In contrast to the mitochondrial data reported by Kittler et al. (2003) , our analysis of their nuclear elongation factor (EF1-alpha) sequences produces a MRCA for P. humanus that is ca. 2 MYA. Similarly, 18S rRNA sequences for P. humanus from Yong et al. (2003) , combined with an 18S rRNA sequence from P. schaeffi, provide a MRCA for P. humanus that is ca. 2 MYA (for GenBank accession numbers, see Supporting Information ). Together, these mitochondrial and nuclear markers support a MRCA for P. humanus greater than 1.18 MYA, which is an order of magnitude older than the MRCA for its human host.

This species exhibits distinct “head” and “body” forms, which differ in ecology, and slightly in size. Head lice (black lettering) are smaller than body lice (red lettering) and are confined to the scalp, whereas body lice live primarily in clothing. Haplotypes shown in green were found in both head and body lice. There are no fixed genetic differences between the head and body forms, suggesting a lack of reproductive isolation, despite the fact that the two forms can be distinguished using discriminant function analysis of morphological data. These results are consistent with experimental data showing that head lice can transform morphologically into body lice within a few generations ( Levene and Dobzhansky 1959 ). The Worldwide clade (red branches) shares a MRCA ca. 0.54 MYA and the geographically restricted New World clade (blue branches) has a much younger MRCA, ca. 0.15 MYA. Asterisks denote samples from Leo et al. (2002)

We used the date of 22.5 ± 2.5 MYA to calibrate the split between Pedicinus and Pthirus + Pediculus in the louse tree. This, in turn, yielded a divergence time of 11.5 MYA for the Pthirus/Pediculus split and 5.6 MYA for the split between Pediculus schaeffi and P. humanus ( Table 1 ). Our estimated divergence between chimp and human lice (5.6 MYA) is strikingly similar to the 5.5 MYA estimates for the chimp/human divergence based on both mitochondrial and nuclear sequence data ( Stauffer et al. 2001 ). To test the original calibration date of 22.5 MYA, we used the molecular estimate of the chimp/human split (5.5 MYA; Stauffer et al. 2001 ) to reverse calibrate the louse tree. This younger calibration point resulted in divergence estimates that were nearly identical to those from the previous calibration. For example, the 5.5 MYA calibration resulted in an estimated divergence of 22.65 MYA for the split between Pedicinus and Pthirus + Pediculus. Estimates of divergence time error were calculated from bootstrapped data sets ( Table 1 ). Other studies have shown that louse mitochondrial DNA (mtDNA) sequences evolve at a rate two to three times faster than that of host sequence rates ( Page 1996 ; Page et al. 1998 ). The lice in this study are evolving at ca. 2.3 times the rate of their primate hosts, when nucleotide substitutions are estimated under a best-fit model of sequence evolution.

Both the morphological and molecular data sets produced a single phylogenetic relationship for the louse species in Figure 1 . The phylogeny shows that Pediculus species on chimpanzees and humans are sister taxa, which together with Pthirus form a clade that is sister to Pedicinus, the most basal member of the ingroup ( Figure 1 ). Bootstrap support for these relationships is high. Reconciliation analysis using Treemap v. 2.0 (M. A. Charleston and R. D. M. Page, software distributed by authors) revealed significant congruence (p < 0.01) between the louse and primate phylogenies, thus validating the assumption of cospeciation ( Kittler et al. 2003 ). Reconciliation analysis using Treemap showed four cospeciation events and one host switch. One particular node of cospeciation determined that as cercopithecoid and hominoid primates diverged 20–25 million years ago (MYA) ( Benefit 1993 ; Leakey et al. 1995 ), Pedicinus diverged from the lineage leading to Pediculus and Pthirus. Since the nodes of cospeciation in congruent host and parasite trees are contemporaneous, the louse tree can be calibrated using the host tree.

Discussion

Morphological and molecular data agree that primates and their lice have been cospeciating for over 20 MY. Indeed, it is this cospeciation that permits us to use host fossil evidence to calibrate portions of the louse phylogenetic tree. This has resulted in the discovery of two extant lineages of human lice that diverged 1.18 MYA. This ancient divergence is surprising because humans, and presumably their lice, are thought to have passed through a population bottleneck ca. 0.05–0.10 MYA (Rogers and Harpending 1992). Such bottlenecks reduce genetic diversity by eliminating uncommon haplotypes, thereby making it less likely that multiple haplotypes survive bottleneck events. For example, mtDNA sequences from human populations coalesce to a single lineage very quickly (≤0.20 MYA), presumably the result of the population bottleneck. The deep divergences found in P. humanus could conceivably be the result of sequencing a nuclear copy of a mitochondrial gene. However, several lines of evidence strongly suggest otherwise. Because we amplified two different mitochondrial genes (COI and Cytb) that show the same divergent lineages and similar percent sequence divergences, copies of both mitochondrial genes would have had to enter the nucleus simultaneously, which is unlikely. In addition, we amplified each gene with a nested set of overlapping primers, and we never amplified more than one gene copy, even during bouts of cloning. Nucleotide base composition for our COI and Cytb data do not deviate from the mean values for all louse COI and Cytb sequences in GenBank (unpublished data), which would not be the case for a nuclear copy of a mitochondrial gene. Finally, the deep divergences seen in our mitochondrial genes are confirmed by preliminary analyses of nuclear data (EF1-alpha and 18S rRNA, unpublished data). Therefore, we are confident that the DNA sequences used in this study are mitochondrial in origin, and we must attempt to explain the occurrence of such ancient mitochondrial haplotypes in human lice.

Gene Trees and Ancient Polymorphisms Gene trees (e.g., mitochondrial lineages) can be considerably older than species trees, and therefore our louse mitochondrial lineages could predate the actual origin of the species P. humanus (i.e., its speciation time). It is useful to determine an expected time to coalescence from the estimated N ef of 1.1 million female lice, even though this estimate seems high for a parasite of humans, who themselves have had very small effective population sizes (as few as 10,000 individuals) and recently went through a population bottleneck (Rogers and Harpending 1992). Although we do not necessarily expect human and louse effective population sizes to be directly correlated, it is difficult to imagine that humans could have maintained such a large effective population of lice during a bottleneck event. Regardless, the expected time to coalescence was estimated to be 0.10 MYA, an order of magnitude younger than the observed divergence time of 1.18 MYA. The deeper gene tree that our data provide also could have been produced either by balancing selection or by subdivision of the louse population into several distinct groups with very limited gene flow. A Fu and Li test does not detect balancing selection when both lineages of P. humanus are evaluated together (p = 0.11); therefore, we must consider the alternative explanation of extensive population subdivision.

Population Substructure and Host Geographic Isolation Substantial isolation among populations of lice on modern H. sapiens could disrupt gene flow and allow the retention of very old lineages, making the age of P. humanus seem much older than it actually is. However, there is no evidence of such pervasive geographic isolation in the modern human hosts of these lice. Other species of lice have been shown to have substantial geographic substructure (i.e., isolation) even when hosts show no geographic isolation (Johnson et al. 2002). If populations of P. humanus are more highly subdivided than those of their hosts, then we might expect P. humanus to have retained ancient mitochondrial polymorphisms, even through host bottleneck events. One prediction of this hypothesis would be that both clades of P. humanus (the WW and NW clades) would show signs of the recent population expansion of humans during the last ca. 0.10 MY. However, only the WW clade shows evidence of this event, which very closely matches the timing of human population expansion. Because the WW clade is commonly found worldwide, and shows a population expansion concurrent with that of modern H. sapiens, we conclude that this lineage has a common evolutionary history with modern H. sapiens. In contrast, the NW clade appears to have diverged from the WW lineage 1.18 MYA, and has had a distinctly different evolutionary history. We are left unable to explain the retention of two ancient louse lineages, each with a different evolutionary history, within the confines of a single host, modern H. sapiens. Given the history of cospeciation between primate lice and their hosts, it is necessary to look beyond modern H. sapiens to determine whether the two divergent lineages of P. humanus are legacies of a more ancient divergence.

Contemporaneous Divergences in Pediculus and Archaic Homo spp. ML analyses rejected H. neanderthalensis as having diverged from H. sapiens contemporaneously with the two divergent lineages of lice. The mitochondrial MRCA of Neandertals and humans is 0.60 MYA (Krings et al. 1997), which is only about half as old as the MRCA of the two ancient lineages of P. humanus, 1.18 MYA. The same ML test failed to reject the codivergence of these lice with H. erectus and H. sapiens when their divergence was set anywhere between 1.2 and 1.8 MYA. Therefore, the deep divergence within P. humanus is entirely consistent with a cospeciation event within the genus Homo ca. 1.2–1.8 MYA, but not 0.60 MYA. Unfortunately, no DNA sequence data exist for H. erectus or any other archaic species of Homo to enable a more direct test of cospeciation. There is much debate regarding the past 2 MY of hominid evolution. However, one area of broad agreement is that, prior to 2 MYA, our ancestors were confined to Africa, then left the continent ca. 1.8 MYA. This first migration out of Africa resulted in archaic species of Homo that were widespread in distribution, and at times both contemporaneous with, and geographically isolated from, the lineage leading to modern H. sapiens (Figure 5). The 1.18 MY of isolation required to preserve the two ancient louse lineages must have occurred, in part, among these archaic species of Homo. It should be noted here that some interpretations of the Multiregional Evolution model do not necessarily consider modern H. sapiens to be a distinctly different species from archaic humans (e.g., H. erectus and H. neanderthalensis). We refer to them as “species” mostly for convenience of writing. Whereas the WW lineage has population genetic characteristics that are similar to those of modern H. sapiens, the geographically restricted NW lineage does not. It likely evolved on a now extinct species of Homo only to switch to modern H. sapiens very recently. For example, Figure 5 depicts one possible scenario where the NW lineage evolved on H. erectus and switched to modern H. sapiens. Interestingly, Hoberg et al. (2001) reported that two species of tapeworms of humans diverged ca. 0.78–1.71 MYA, and one of the two species, Taenia asiatica, is entirely restricted to Asia. This is consistent with the depiction in Figure 5, if one assumes that T. asiatica evolved on H. erectus. Although divergence dates are not available, it is intriguing that some Native American strains of HTLV have closer affinities to Asian primate strains than to other human strains of HTLV, suggesting an independent Asian origin of this virus in humans. One must still explain how these parasites came to be on modern H. sapiens, but taken together, the parasitological evidence (especially the deep divergences in tapeworms and lice) suggests that they might have evolved on H. erectus and switched recently to H. sapiens. If true, this implies that H. erectus was contemporaneous with modern H. sapiens in eastern Asia, as suggested by Swisher et al. (1996), and it begs a discussion of recent human origins. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 5. Temporal and Geographical Distribution of Hominid Populations Redrawn from Temporal and Geographical Distribution of Hominid Populations Redrawn from Stringer (2003) This figure depicts one view of human evolutionary history based on fossil data. Other interpretations differ primarily in the taxonomy and geographical distribution of hominid species. The temporal distribution of the two divergent lineages of P. humanus is superimposed on the hominid tree to show host evolutionary events that were contemporaneous with the origin of P. humanus. Whereas the NW lineage is depicted on H. erectus in this figure, several alternative hypotheses are consistent with our data when other evolutionary histories of hominids are considered (unpublished data). The WW clade is shown in red and the NW clade in blue (see text for descriptions of clades). https://doi.org/10.1371/journal.pbio.0020340.g005