Phylogenetic analyses of bat-associated bugs (Hemiptera: Cimicidae: Cimicinae and Cacodminae) indicate two new species close to Cimex lectularius

Background Bats are regarded as the primary (ancestral) hosts of bugs of the family Cimicidae. The historically and economically most important species in the family is the common bedbug (Cimex lectularius), because of its worldwide occurrence and association with humans. This molecular-phylogenetic study was initiated in order to expand the knowledge on the phylogeny of cimicid bugs of bats, by investigating samples from Hungary, Romania (representing central-eastern Europe) and two further countries (South Africa and Vietnam). Results Altogether 216 cimicid bugs were collected (73 Ci. lectularius, 133 Ci. pipistrelli, nine Cacodmus ignotus and one Ca. sparsilis). Members of the Cimex lectularius species group were found both in the environment of bats (only Myotis emarginatus, which is a cave/attic-dwelling species) and on three crevice-dwelling bat species (two pipistrelloid bats and M. bechsteinii). On the other hand, Ci. pipistrelli always occurred off-host (near M. myotis/blythii, which are cave/attic-dwelling species). In addition, two Cacodmus spp. were collected from Pipistrellus hesperidus. The morphological characters of these specimens are illustrated with high resolution pictures. Analysis of cytochrome c oxidase subunit 1 (cox1) sequences generated from 38 samples indicated relative genetic homogeneity of Ci. pipistrelli, while the Ci. lectularius group had two haplotypes (collected from pipistrelloid bats in Hungary and Vietnam) highly divergent from other members of this species group. These results were confirmed with molecular and phylogenetic analyses based on the internal transcribed spacer 2 (ITS2). Bat-associated bugs morphologically identified as Ca. ignotus and Ca. sparsilis were different in their cox1, but identical in their ITS2 sequences. Conclusions Molecular evidence is provided here on the existence of two new genotypes, most likely new species, within the Ci. lectularius species group. The relevant specimens (unlike the others) were collected from pipistrelloid bats, therefore the association of Ci. lectularius with different bat host species (pipistrelloid vs myotine bats) should be evaluated further as a possible background factor of this genetic divergence. In addition, Ca. ignotus is reported for the first time in South Africa.


Background
Cimicid bugs (Hemiptera: Cimicidae) include approximately 110 described species [1], which are obligate, haematophagous ectoparasites of birds and mammals. Bats are regarded as the primary (ancestral) hosts of bugs in the Cimicidae, with subsequent switches to other hosts, including birds and humans [2][3][4]. This is well reflected by the fact that the majority (approx. twothirds) of cimicid species are bat-associated [2,5].
The historically and economically most important species in the family is the common bedbug (Cimex lectularius), because of its worldwide occurrence and preference of human environment. While the number of reported cases of bedbug infestations had been showing a decline until the middle of twentieth century, during the last two decades Ci. lectularius became an emerging pest even in highly developed countries [6]. This is the most important aspect that may illustrate the growing significance of this species, but not the only one. Cimex lectularius is the potential vector of at least 65 pathogens [6], but its vector competency awaits verification.
The morphology of cimicid bugs is frequently a matter of controversy. For instance, in the Ci. pipistrelli group, the morphological characters intended to delineate species were shown to vary significantly enough to ascribe progeny of the same female to different species [5,7]. Accordingly, the taxonomy of bat-associated bugs is currently in a state of transition. While some formerly distinguished Cimex species are suggested to be synonymous (as exemplified by Ci. pipistrelli and Ci. dissimilis, see [5]), new species are also discovered/described [8]. The genus Oeciacus (associated with birds) has been transferred to Cimex [4]. These and other examples highlight the importance of molecular phylogenetic studies focusing on cimicid bugs, which recently have started to expand [5,9].
Bat-associated bugs, as well as other cimicid bugs, are temporary ectoparasites, which spend most of their life offhost. On the other hand, bat-associated bugs are unable to fly, therefore strictly rely on their hosts for colonization of new habitats, as well as for distribution over large distances [5,10]. Cimicid bug species associated with bats show different host ranges [2,11]. Adaptation to host species has been suggested to be a driver of morphological rather than genetic diversification in the case of Ci. pipistrelli [5]. At the same time, host preference will also influence the small-scale (habitat-related) as well as large-scale (geographical) distribution of bat-associated bugs [9,11,12].
While Ci. lectularius, as a man-associated parasite, has a worldwide geographical distribution [6,9], accounts of its bat-related lineages concentrate in the Western Palaearctic [9,11]. In this region a recent phylogeographic study on Ci. lectularius revealed that this species is currently undergoing lineage divergence through host association [9]. However, in the latter survey Hungary was underrepresented, and in another comprehensive survey on bat-associated bugs [5] the Balkans and other regions of the Old World had not been included. This molecular-phylogenetic study was initiated in order to expand the knowledge on the phylogeny of cimicid bugs of bats, by investigating samples from Hungary, Romania (the latter representing the Balkans) and two further countries (South Africa and Vietnam).

Sample collection and study design
Bat-associated cimicid bugs were collected in Hungary (six locations), Romania (two locations), South Africa (one location) and Vietnam (one location) between 2011 and 2016 ( Table 1). The bats were caught for monitoring and ringing purposes at cave entrances from sunset to dawn, using harp traps or Ecotone mist-nets (Gdynia, Poland) with standard 12 m length, 2.5 m height and 14 × 14 mm mesh size. Bats were released immediately after parasite removal and recording data (date and place of collection, bat species). Alternatively, the close environment of nursing bat colonies with different habitat types (Table 1) was checked for the presence of Cimicidae. The bugs were immediately placed into and stored in 96% ethanol.
Concerning study design, because the size of structures important for morphological identification of cimicid bugs was shown to exhibit significant intraspecific variation [2,5], detailed measurements were not taken, and only discrete morphological characters were considered for species identification.
Morphological identification of adult bugs was carried out under a stereomicroscope (SMZ-2 T, Nikon Instruments, Japan) by using standard keys [2], focusing on the pronotum, paragenital sinus (Cimex spp.) or the paramere (Cacodmus spp.). Concerning samples (one from Hungary and two from Vietnam), which showed high degrees of genetic divergences from other members of their phylogenetic group, their conspecificity with Ci. emarginatus (reported in Bulgaria) and Ci. insuetus (reported in Thailand), respectively, was excluded based on descriptions of the latter species [8,13]. Cacodmus sp. females were identified according to the cox1 sequences of morphologically identified males. Pictures were made with a VHX-5000 (Keyence Co., Osaka, Japan) digital microscope.

DNA extraction and molecular analyses
DNA was extracted from individual bugs with the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instruction, including an overnight digestion in tissue lysis buffer and Proteinase-K at 56°C. Molecular phylogenetic analysis was attempted from 44 samples, including 1-10 adult bugs from each location.
PCR products were visualized in 1.5% agarose gel. Purification and sequencing were done by Biomi Inc. (Gödöllő, Hungary). Obtained sequences were manually edited, then aligned and compared to reference GenBank sequences by nucleotide BLASTN program (https://blast.ncbi.nlm.nih.gov). Representative sequences (including identical haplotypes from different locations) were submitted to GenBank (accession numbers: MF161520-MF161531 for cox1, and MF161532-MF161540 for ITS2). Phylogenetic analyses were conducted by using MEGA version 6.0, with the Maximum Likelihood method and the model (Tamura 3) selected by the program.

General morphology and host species of bat-associated bugs
Altogether 216 cimicid bugs were collected from the bodies or roosts of seven bat species of three genera (Table 1). Bugs morphologically most closely related to Cimex lectularius were found both in the environment of bats and on the bat species Pipistrellus pipistrellus, Myotis bechsteinii and Hypsugo pulveratus. On the other hand, Ci. pipistrelli occurred only off-host ( Table 1).
The Cimex lectularius species group was represented by 73 specimens. These showed similar general morphological  (Fig. 1) or from bats in Hungary (Figs. 2 and 3b), including the shape of the pronotum (breadth to length ratio ≥ 2.5, broad lateral lobes), paragenital sinus (cleft with bristles) and external spur on coxa III (with a broad basis). However, while the pronotum and coxal spur were similar in the case of specimens from Hungary (Fig. 3a-c) and Vietnam (Fig. 3d), the paragenital sinus of the female bug from Vietnam was rounded (Fig. 4d). Cimex pipistrelli was represented by 133 specimens ( Table 1). All of these from Hungary and Romania shared the shape of the pronotum (breadth to length ratio < 2.5, narrow lateral lobes) and of the paragenital sinus (cleft and naked) (Fig. 5).
Cacodmus sp. males from South Africa had either evenly curved and tapering, apically straight, medium to long paramere (Fig. 6c-d), or long paramere bent laterally at the tip (almost sinuate at apex) (Fig. 6b). On this basis specimens were assigned to Ca. ignotus and Ca. sparsilis, respectively.

Sequence comparison and phylogeny of bat-associated bugs
The cox1 gene fragment was successfully amplified and sequenced from 38 samples (Table 1). Bugs morphologically most closely related to Ci. lectularius (15 samples) had four cox1 haplotypes in Hungary. The majority of these exhibited up to five nucleotide differences from each other, corresponding to 99.2-100% sequence similarity (626-631/631 bp). However, a Cimex sp. from P. pipistrellus (Hungary) showed 46 nucleotide differences from the Ci. lectularius reference sequence (MF161520: from Hungary), i.e. only 585/631 bp (92.7%) sequence similarity. The cox1 gene fragment of another Cimex sp.
The ITS2 fragment was successfully amplified and sequenced from 16 samples (Table 1). In general, this nuclear marker showed a much lower degree of intraspecific divergence compared to cox1. Members of the Cimex lectularius group from Hungary had only two different ITS2 haplotypes. However, the Cimex sp. from P. pipistrellus (Hungary) showed only 96.7% (622/643 bp) sequence similarity in its longest region of continuous alignment with the ITS2 reference sequence (MF161534: from Hungary). In addition, the Cimex sp. from Vietnam showed even lower, 88.3% (580/657 bp) sequence similarity in its longest region of alignment with the ITS2 reference sequence.
The phylogenetic relationships of cox1 and ITS2 sequences are shown in Figs Similarly, the within-group separation of Cimex sp. from Vietnam received high (99%) support based on its ITS2 haplotype (Fig. 8), but only low (59%) support based on its cox1 haplotype (Fig. 7). All Ci. pipistrelli cox1 haplotypes belonged to the same group with other conspecific isolates (Fig. 7), and this was confirmed in the phylogenetic analysis based on ITS2 sequences (Fig. 8). Bugs identified morphologically as Ca. ignotus and Ca. sparsilis were well separated from each other (with moderate, 87% bootstrap value) in the cox1 phylogenetic tree (Fig. 7).

Discussion
This study provides molecular data of bat-associated cimicid bug species from three distant regions of the Old World (i.e. central-eastern Europe, south-eastern Asia and South Africa). One of the studied bug species, Ci. lectularius is the most significant member of Cimicidae, taking into account its association with humans, global distribution, historical and economic impact, recently witnessed emerging character and potential health hazards [6]. In a recent study on Ci. lectularius [9] mostly central and western Europe were represented by sampling sites, therefore results shown here can be regarded as complementary to that study, introducing samples from more locations in Hungary, as well as samples from Romania and Vietnam into the phylogenetic analysis of this species group. In addition, molecular analyses of Cacodmus spp. from South Africa have not yet been reported.
In this study all Ci. pipistrelli, and the majority of Ci. lectularius were collected in roosting places of Myotis spp., which can be regarded as their principal hosts [11]. Only one Ci. lectularius (from M. bechsteinii), the Cimex spp. (from Hungary, Vietnam) and Cacodmus spp. were found on hosts, in particular on four bat species, three of which are pipistrelloid bats (including Hypsugo [formerly Pipistrellus] pulveratus). According to literature data, bat species (such as Pipistrellus and Nyctalus spp.), which roost in narrow spaces (rock crevices or tree holes) and switch these places quite often, are more likely to carry bat-associated bugs on their wing membrane [12]. This is confirmed by the data presented here, taking into account the roosting behavior of the four bat species, which were found bug-infested (Table 1). In addition to P. pipistrellus, P. hesperidus colonies can also be found in narrow cracks and dead trees [17]. Myotis bechsteinii is a tree-dwelling bat species; its females establish their maternity colonies in tree holes and switch their day-roosts regularly [18,19].
In the present study two new genotypes (belonging to the Ci. lectularius group, but highly divergent from its other members) were identified. Both of the relevant specimens were collected from pipistrelloid bat hosts. The first of these specimens, collected from P. pipistrellus in b a c d Hungary, showed the morphology of Ci. lectularius and was different from Ci. emarginatus (e.g. in the parameters of its head, palpal segments, posterior bristles and the shape of paragenital sinus). The second specimen, collected from H. pulveratus in Vietnam, was also similar to Ci. lectularius based on its coxal spur and some aspects of its pronotum (which was 2.5 times broader than long, unlike that of Ci. insuetus). However, the paragenital sinus of the latter female was different from that in Ci. lectularius, i.e. it was rounded, which is a character of Neotropical species of the genus Cimex, not found in the Old World [2]. In addition, taking into account that in case of both of these new variants the cox1 genetic difference exceeded 7% in comparison with Ci. lectularius (and this value was 5.8-6.4% between Ci. lectularius and members of the Ci. hemipterus or Ci. pipistrelli species groups, see [4]), they probably represent new species. In order to clarify the precise taxonomical status of these new genotypes, they will Fig. 7 Phylogenetic tree based on the cox1 gene including sequences obtained in this study (indicated in red and with GenBank accession numbers in bold) and representative sequences from GenBank. Species identification is provided as in the GenBank database, although Oeciacus spp. were recently transferred into the genus Cimex [4]. Branch lengths represent the number of substitutions per site inferred according to the scale shown have to be compared by including more specimens and analyses. While Ci. lectularius from roosts of Myotis spp. yielded multiple haplotypes within the same major haplogroup [9], in the present study Ci. lectularius-related specimens from pipistrelloid bats showed highly divergent cox1 and ITS2 haplotypes at both small and large geographical scales (i.e. in Hungary and Vietnam, respectively). This phenomenon is similar to the one suggested in the case of bat-associated bugs of the Ci. pipistrelli group, which were also shown to have different host ranges [2], although the association of Ci. pipistrelli with different host species is thought to be a driver of morphological (rather than genetic) variability [5].
The present results extend the geographical range of Ca. ignotus (hitherto only reported from Uganda, see [2]) to South Africa. This can be explained by the occurrence of P. hesperidus (from which it was collected in the present study) in much of East Africa, from Ethiopia to South Africa [20]. However, it was unexpected to find that batassociated bugs identified here on a morphological basis as Ca. ignotus and Ca. sparsilis had highly (6.8%) different cox1, but identical ITS2 sequences. This observation is similar to that reported previously in the Western Palaearctic region on Ci. pipistrelli, which had only limited variability in ITS2 sequences (and none in other nuclear markers), despite the separation of corresponding cox1 haplogroups [5]. In general, the resolution of cox1 analysis to assess the degree of divergence between closely related species is known to be much higher compared to ITS2 (ticks: 6.1 vs 2.3%; mites: 3.0-4.0% vs < 0.5%, respectively) [21,22]. Nevertheless, in the present case, the identity of ITS2 sequences between individuals of two Cacodmus spp. could have resulted from genetic introgression or hybridization.

Conclusions
Bugs of the Ci. lectularius group associated with different bat host species (myotines vs pipistrelloid bats) were found to belong to different genetic lineages. Sequence comparisons and phylogenetic analyses of cox1 and ITS2 sequences of specimens from pipistrelloid bats (collected in Hungary and Vietnam) suggest that they may belong to new species. In addition, Ca. ignotus is reported for the first time in South Africa.
Abbreviations cox1: Cytochrome c oxidase subunit 1; ITS2: Internal transcribed spacer 2 Fig. 8 Phylogenetic tree based on the ITS2 including sequences obtained in this study (indicated in red and with GenBank accession numbers in bold) and representative sequences from GenBank. Species identification is provided as in the GenBank database, although Oeciacus spp. were recently transferred into the genus Cimex [4]. Branch lengths represent the number of substitutions per site inferred according to the scale shown