The consensus tree ( Figure 1 ) of five DNA sequence segments of four genes (1) shows that the Cimicidae are monophyletic and firmly placed within the Cimicomorpha []; (2) provides robust resolutions of other debated relationships ( Figure 1 ), including the paraphyly of the groups that parasitize swallows (martin bugs, previously genus Oeciacus) []; and robustly identifies Primicimex+Bucimex (3) as a monophyletic group ( Figure 1 ) supporting morphological arguments [] and a concurrent investigation [], and (4) as the sister of the remaining extant Cimicidae, solving a long-standing problem in insect systematics []. (5) The biogeographical distribution ( Figure 1 ) shows continent-restricted ranges of higher cimicid taxa that may relate to either occasional cross-continent host dispersal or geological events. Consistent with the latter, ancestral clades (Primicimicinae and Afrocimicinae) are restricted to continents that developed from western Gondwana []. India may not have been colonized until about until 75 mya (Cacodminae), when it started to collide with Asia, and Europe not before 50 mya (Cimicinae), following the collision with the African plate. Wallace’s line represents a distribution border. Consistent with dispersal by hosts is some degree of host conservatism (see results below) and a (near) cosmopolitan distribution only for human-associated species. On the other hand, it is striking that a continuous form of cross-continent dispersal by hosts has not altered the biogeographic distribution of those cimicids that parasitize swallow species: neither the North American C. vicarius nor the European C. hirundinis have been recorded in the winter grounds of their hosts, South America and Sub-Saharan Africa, respectively.

Bayesian consensus tree based on five gene sequences showing the biogeographical distribution (branch colors) and classical taxonomy at the subfamily level. Photographs show morphologically typical representatives of each subfamily. Numbers beside the nodes indicate posterior probability values. The branch lengths scale represents the number of estimated nucleotide substitutions per site. For species, sample codes, collection details, and sequences of outgroups (boxed in shaded gray and taken from GenBank), see Table S1

Thus, the stem species of bedbugs evolved 115–122 mya, well before the Cretaceous–Tertiary (K-T) mass extinction boundary, a key event in vertebrate diversification. The identity of the ancestral host(s) from which bats were colonized repeatedly is unknown.

All four ancient bedbug lineages predate the evolution of bats ( Figure 2 ) but were reconstructed to ancestral bat hosts ( Figure 3 A). This suggests that bats were colonized several times independently, unless the evolutionary origin of bats [] has been grossly underestimated.

Mirror trees showing (A) systematic host groups and (B) their classification as putative host specialist or generalist (see main text for classification; Figures S1 and S2 ). In (A), colors indicate different host types reported [] ( Table S1 ). In (B), putatively specialized (black) or generalist (white) host uses were reconstructed with (unordered) parsimony. Separate analyses with MrBayes [] confirmed specialized host use as the ancestral state for all Cimicidae. The result did not change if the two lineages with the highest uncertainty about their ancestral state, i.e., Cimicinae and Cacodminae + Haematosiphoninae were analyzed separately by setting all other clades to an unknown state of G or S (probabilities of bats as ancestral host 98% and ancestral specialist 85% for the Cimicinae and 96% and 98%, respectively, for the Cacodminae + Haematosophinae). Leptocimex duplicatus was analyzed as Leptocimex spec. to demonstrate human host use in this genus. Results were identical if ancestral analysis host and specialization employed bats or bats + human. The results also did not change if classes of the exact number of currently known host genera were employed (see also Figures S1 and S2 ). Comparing host and parasite mean ages shows that diversification is not generally driven by co-speciation with hosts on bat or bird hosts; see also Figures S1–S3 and Table S1

New family host and records of Acanthocrios furnarii (Cordero & Vogelsang, 1928) (Hemiptera: Cimicidae) from Argentina, and implications in the transmission mechanism of cimicid bugs among birds’ nests.

Independently dating the phylogenetic tree using a fossil from the related family Vetanthocoridae (152 mya) [] rejects the widely held view [] that the Cimicidae evolved on bat hosts. Our mean estimate of 115 mya (74–170; 95% highest posterior density [HPD] interval) for the stem of the Cimicidae supports the idea of a minimum age of the group of 100 mya based on fossil evidence []. The origin of the Cimicidae crown group with a mean of 93.8 (56–137; 95% HPD) mya is placed 30–50 Ma before the earliest known bats [] ( Figure 2 ) and 20 Ma before the earliest inferred bats (73 [64–81; 95%] mya) []. Our estimate appears robust: employing the oldest known cimicid fossil as an additional calibration point places the stem species at 122 mya (111–150 mya; 95% HPD; relaxed molecular clock estimation of lineage divergence points within the family) and the crown divergence at 102 mya (91–114 mya; 95% HPD; Figure 2 ). Our estimate is also robust against previous suggestions that the Vetanthocoridae might be the sister group to all Cimicoidea []. Using the Cimicoidea + Nabidoidea divergence as calibration point produces very similar results, dating the Cimicidae ancestor to 127 mya and the first divergence of the crown group to 103 mya. However, the latter approach devaluated the support for some of the clades that were well supported from unconstrained phylogenetic estimates, and the topological constraint for the calibration changed the position of Afrocimex (but also with low support values; https://doi.org/10.5281/zenodo.2642215 ).

Bayesian consensus tree of the Cimicidae and selected outgroup taxa in relation to geological age (mya; x axis). A relaxed clock model [] was used to date the tree based on two calibration points, fossil Vetanthocoridae (152 mya) [] and the oldest known fossil cimicid (100 mya) []. Numbers below nodes represent Bayesian posterior probability values; blue bars represent 95% highest posterior density intervals of the time estimates in mya. Scale is in millions of years. The Cimicidae are boxed in shaded blue. “gr.” stands for group, a taxonomic aggregate. The time estimates returned a mean age of 103 Ma for the crown group of the Cimicidae and are robust against alternate taxonomic assumptions of the Vetanthocoridae ( https://doi.org/10.5281/zenodo.2642215 ).

Our phylogeny does not support ancestral host generalism (G) in cimicids ( Figure 3 B), so we propose the commonly assumed evolution of hematophagy from facultative blood feeding by ancestral predators [] did not occur. This result is robust against variation in the definition of species along the host specialist (S) and host G axis, depending on the specialization metrics or recording intensity []. For example, technically, all specialists are “putative specialists” until additional hosts may eventually be found. In any case, the derived state of G holds true if the number of currently known host genera is used ( Figure S2 ) and if G are defined by the phylogenetic distance of their hosts [], i.e., as using more than one of the four major, phylogenetically deeply diverged host groups of waterfowl (Galloanseres) and other birds (Neoaves), as well as bats (Chiroptera) and humans ( Figure 3 A). It also holds true for a definition of G accounting for variability within taxonomic groups [] as being those parasites recorded from more than three host genera ( Figure S1 ). Therefore, hematophagy likely evolved within the true bugs (Heteroptera), in insects that were already specialists and gave rise to the Cimicidae. This result is compatible with the view that the specialist blood-sucking Polyctenidae is the sister group of the Cimicidae [].

How specialists can be generalists: resolving the “parasite paradox” and implications for emerging infectious disease.

Is specialization an evolutionary dead end? Testing for differences in speciation, extinction and trait transition rates across diverse phylogenies of specialists and generalists.

Specialization and generalization in the diversification of phytophagous insects: tests of the musical chairs and oscillation hypotheses.

Pattern of Host Shifts

1 Usinger R.L. Monograph of Cimicidae (Hemiptera-Heteroptera). Of the 29 species on our tree that allow a classification, most (24/29; 83%) are S (broadly defined; Figure 3 A) or 55% (15/27), using tighter definitions ( Figures 3 B and S2 ). Five cimicid species on our molecular tree are G (broadly defined) [].

31 Drummond A.J.

Suchard M.A.

Xie D.

Rambaut A. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Host shifts between bat taxa were common by ancient specialists because most extant bat-parasitic cimicid lineages evolved before their extant hosts’ lineages. For example, comparing means of the phylogenetic age of host and parasite (95% lower–upper highest posterior distribution inferred by BEAST) [], we found that Afrocimex (103–77) evolved 67 Ma before its current host Roussettus (26–18), Bucimex (42–13) 6 Ma before Myotis (25–16), Primicimex (42–13) 4 Ma before Tadarida (27–17), or the Cimicinae + Cacodminae + Haematosiphoninae clade (94–65) 26 Ma before the Vespertillionidae (60–50; Figure 3 ). Host switches from bats to birds also occurred; we identified at least three such independent events ( Figure 3 A). For bird hosts, the Haematosiphoninae diverged around 50 mya, the bird-parasitic Paracimex (around 15 mya) or Cimex vicarius (around 18 mya)—long after their respective swift or swallow host groups had appeared in the early Eocene ( Figures 2 and 3 ).

14 Hoberg E.P.

Brooks D.R. A macroevolutionary mosaic: episodic host-switching, geographical colonization and diversification in complex host-parasite systems. 15 Agosta S.J.

Janz N.

Brooks D.R. How specialists can be generalists: resolving the “parasite paradox” and implications for emerging infectious disease. 37 Hafner M.S.

Sudman P.D.

Villablanca F.X.

Spradling T.A.

Demastes J.W.

Nadler S.A. Disparate rates of molecular evolution in cospeciating hosts and parasites. Our host reconstruction indicates that parasite diversification is not generally driven by co-speciation with hosts [] (but see []) for either bat or bird hosts ( Figure 3 ). Together, these observations suggest that the extant pattern of G/S distribution in cimicids is the result of evolutionarily dynamic host transitions.

When examining host transitions at all 31 subterminal nodes on our tree that are classifiable as G or S, we found the highest number (9/31 or 29%) involved host specialists switching host but staying specialist (S→S). Two nodes were G→S transitions (6%), and five (16%) were S→G transitions (or 7/31 [23%] if specialists are defined more strictly; Figures 3 B and S2 ).

16 Park A.W.

Farrell M.J.

Schmidt J.P.

Huang S.

Dallas T.A.

Pappalardo P.

Drake J.M.

Stephens P.R.

Poulin R.

Nunn C.L.

Davies T.J. Characterizing the phylogenetic specialism-generalism spectrum of mammal parasites. 9 Futuyma D.J.

Moreno G. The evolution of ecological specialization. 10 Poulin R.

Krasnov B.R.

Shenbrot G.I.

Mouillot D.

Khokhlova I.S. Evolution of host specificity in fleas: is it directional and irreversible?. 11 Janz N.

Nylin S. The oscillation hypothesis of host-plant range and speciation. 12 Hardy N.B.

Otto S.P. Specialization and generalization in the diversification of phytophagous insects: tests of the musical chairs and oscillation hypotheses. 14 Hoberg E.P.

Brooks D.R. A macroevolutionary mosaic: episodic host-switching, geographical colonization and diversification in complex host-parasite systems. 16 Park A.W.

Farrell M.J.

Schmidt J.P.

Huang S.

Dallas T.A.

Pappalardo P.

Drake J.M.

Stephens P.R.

Poulin R.

Nunn C.L.

Davies T.J. Characterizing the phylogenetic specialism-generalism spectrum of mammal parasites. The paucity of G→S transitions departs from the general pattern in mammalian parasites [] and indicates that the “resource efficiency” hypothesis (where host S evolve from G by fitness advantages on specific hosts) [] does not appear to apply to cimicids. An extension of this idea, the “oscillation” hypothesis, proposes that genetic variation or phenotypic plasticity maintained in S species allows them to add hosts to their portfolio (and so become G again), depending on ecological opportunities []. Although this hypothesis allows for any number of S and G transitions, S→G transitions should be evenly distributed across evolutionary time if they are regularly oscillating. This prediction was rejected: all seven S→G transitions occurred in a short period, between 10 and 20 mya (cf. Figures 2 and 3 ).

14 Hoberg E.P.

Brooks D.R. A macroevolutionary mosaic: episodic host-switching, geographical colonization and diversification in complex host-parasite systems. 5 Ueshima N. Cytology and bionomics of Primicimex cavernis Barber. Acceptance of unusual hosts under ecological opportunities (such as laboratory-forced host feeding) can serve as an indicator of plasticity or genetic variation in host preference []. Such propensity to switch hosts has only been recorded in G ( Figures 3 and S2 ), but not in S species [] (K.R., R.N., and M.T.S.-J., unpublished data; O.B., unpublished data; S.R., unpublished data)—which the oscillation hypothesis requires—but few experimental tests exist. Anecdotal acceptances of unusual hosts outside the laboratory suggested to mimic ecological opportunities created by humans have been reported during guano mining, chicken breeding, or pet keeping, again, however, in G, or unscorable, but not in S species. Unless future systematic screening of such events would reveal a massive usage of unusual hosts by S species, there is little current evidence to suggest that S species commonly oscillate to evolve into G species or that host specialization in the Cimicidae is driven by selection for resource efficiency.

12 Hardy N.B.

Otto S.P. Specialization and generalization in the diversification of phytophagous insects: tests of the musical chairs and oscillation hypotheses. 12 Hardy N.B.

Otto S.P. Specialization and generalization in the diversification of phytophagous insects: tests of the musical chairs and oscillation hypotheses. 14 Hoberg E.P.

Brooks D.R. A macroevolutionary mosaic: episodic host-switching, geographical colonization and diversification in complex host-parasite systems. 15 Agosta S.J.

Janz N.

Brooks D.R. How specialists can be generalists: resolving the “parasite paradox” and implications for emerging infectious disease. 16 Park A.W.

Farrell M.J.

Schmidt J.P.

Huang S.

Dallas T.A.

Pappalardo P.

Drake J.M.

Stephens P.R.

Poulin R.

Nunn C.L.

Davies T.J. Characterizing the phylogenetic specialism-generalism spectrum of mammal parasites. S→S transitions (host switches without extensions in host breadth, or so-called “musical chairs” pattern) [] are the common pattern in cimicids. The musical chairs hypothesis makes no further predictions [], but S→S transitions can, like S→G, be based on the ecological opportunities new hosts present [], such as after major (e.g., intercontinental) dispersal events []. In support, for example, two of the three bat-to-bird host shifts in cimicids concerned the Haematosiphoninae and Paracimex, where bird hosts replaced bats rather than having been added ( Figure 3 ). Both examples simultaneously involved the colonization of another continent (South America and Southeast Asia). However, other S→S transitions are not related to intercontinental shifts.

38 Talbot B.

Balvín O.

Vonhof M.J.

Broders H.G.

Fenton B.

Keyghobadi N. Host association and selection on salivary protein genes in bed bugs and related blood-feeding ectoparasites. 39 Balvín O.

Roth S.

Talbot B.

Reinhardt K. Co-speciation in bedbug Wolbachia parallel the pattern in nematode hosts. The only temporary association of cimicids with the host body would be expected to increase opportunities for alternative host use and hence generalism (such as in mosquitoes). However, the widespread and ancient specialization reported here (predicted for parasites with tight host associations that cannot readily exploit new hosts, such as lice) finds a parallel in selection on salivary proteins [] and divergence in endosymbionts [], both of which aid hematophagy.

In conclusion, several bedbug lineages specialized on bats in ancient times, but subsequent host shifts were frequent, and the switches (and expansions of host portfolio) that can be explained by current models of host specialization are related to the ecological opportunities that human activity or intercontinental dispersal provided. As general models of host specialization only had limited ability to predict patterns of host use in cimicids, we examined more specific ideas developed for their colonization of human hosts.