IntroductionThe brown dog tick, Rhipicephalus sanguineus (Latreille) is one of the most widely distributed ticks in the world, found globally between 50°N and 30°S latitudes (Walker et al. 2000). This cosmopolitan tick infests domestic and wild mammals, and incidentally man. Its immature stages may infest rodents and other small mammals (Hoogstraal 1985), but canines and especially domestic dogs are the preferred hosts. In addition to being prolific and a menacing ectoparasite of domestic dogs, Rh. sanguineus is a vector of several veterinary and human pathogens including Babesia vogeli, Ehrlichia canis, Hepatozoon canis, Rickettsia conorii, Rickettsia massiliae and Rickettsia rickettsii (reviewed by Dantas-Torres 2008).
Rhipicephalus sanguineus is, however, the most controversial tick species in the genus Rhipicephalus (Farid 1996). Following its original description, a number of morphologically and biologically similar species and subspecies have been described around the world. Rh. sanguineus remains the type species of the so-called “Rh. sanguineus complex” or the Rh. sanguineus group, which includes at least five closely related Old World species (Walker et al. 2000). Their differentiation and delineation remain subjects of an ongoing debate (Feldman-Muhsam 1952; Feldman-Muhsam 1968; Pegram et al. 1987a, b, 1989; Farid 1996; Zahler et al. 1997; Baker 1998; Oliveira et al. 2005; Szabo et al. 2005). Morphological identification of brown dog ticks is complicated by intraspecific variability of morphological traits and close similarity to those in related species (Farid 1996; Oliveira et al. 2005) with the result that they are often misidentified (Pegram et al. 1987b; Ioffe-Uspenskiy et al. 1997). Some 18 of the separately described Rhipicephalus species have been eventually synonymized with Rh. sanguineus (Pegram et al. 1987a, b; Walker et al. 2000). However, recent studies suggested that the resulting combined taxon may actually represent more than one species based on phylogenetic analyses that segregated European and African Rh. sanguineus into distinct clades (Szabo et al. 2005; Burlini et al. 2010, Moraes-Filho et al. 2011). These authors also found that Rh. sanguineus from Brazil and Asia are genetically closer to tick specimens morphologically identified as Rhipicephalus turanicus, than to Rh. sanguineus from either North America or Mediterranean.
Yet, the accurate discrimination of species within the Rh. sanguineus group is crucial for understanding of the epidemiology and etiology, as well as for control, of the pathogens they transmit. Closely related tick species, and possibly even different populations within a tick species, can differ in their ability to transmit pathogens (Baker 1998; Anderson 2002; Matsumoto et al. 2005) and in the frequency of contacts with humans and domestic animals. This affords medical and veterinary importance to the accurate identification and delineation of tick species. In cases where separation of sibling tick species based solely on morphological traits is difficult, hybridization experiments and molecular markers may be necessary to differentiate between them (Hafez et al. 1981; Zahler et al. 1995; Zahler and Gothe 1997; Baker 1998; Labruna et al. 2009).
Herein, we assess the conspecificity of brown dog ticks (Rh. sanguineus) from Africa, Mediterranean, and North America by cross-breeding ticks from representative populations and comparing results of ticks’ hybridization to their molecular phylogeny.
ResultsIn the three purebred colonies, reproductive parameters did not differ significantly between the parental and F1 generations. Therefore, data for all individual pure-bred females in those two generations were combined to increase the statistical power of the analysis (Tables 3 and 5). The duration of larval and nymphal feeding was similar in F1 progenies of all purebred and hybrid crosses with larvae completing engorgement within 3–5 days and nymphs—in 4–7 days. Therefore, these parameters were not used for further comparison of crosses.
Purebred linesWhen female Rh. sanguineus from three different colonies mated with sympatric males (purebred crosses), the reproductive parameters differed between the three studied populations (Tables 3 and 4). Ticks from the North American colony all engorged between days 7 and 10 after being placed on rabbits. Those from the Israeli and African colonies on average took 1–1.5 days longer to feed to repletion completing their engorgement between days 7 and 13 (Table 3). The overall feeding success of purebred adult ticks from the American and Israeli colonies was similar (95–100 %) and significantly higher than in ticks of African origin (P = 0.0056). The weights of replete females in North American, Israeli, and African colonies fluctuated within the ranges of 0.21–0.39, 0.19–0.42, and 0.16–0.25 g, respectively. On average, the engorgement weight of a Rh. sanguineus female from the African population was approximately 30 % lower than female engorgement weights in North American and Israeli colonies (P<0.0001). The BMCI did not differ significantly between the three purebred colonies (Table 3; P>0.01). Consequently, African females oviposited smaller egg-masses than those of North American and Israeli origins.
All purebred engorged females began laying eggs within four to fourteen days after repletion regardless of their origin or the engorgement weight. Eggs laid by individual females from the American colony hatched after 32–38 days and those produced by Israeli and African ticks required 35–48 days of incubation. On average, the incubation period under 22 °C was significantly shorter in the American colony than in the African colony (Table 3; P<0.01). Eclosion of larvae was observed in every egg batch, but the average proportion of fertile eggs varied significantly between colonies—from 78 % among ticks of Israeli origin to above 90 % among the American Rh. sanguineus (P = 0.0039) (Table 3).
Following engorgement on NZW rabbits, 97–99 % of purebred larvae successfully molted into the nymphal stage (Table 4). However, purebred nymphs engorging on the same host species had low molting success. The molting success of nymphs from the Israeli colony was significantly lower than those of American origin (P = 0.0011) and almost twice as high as nymphs in the African colony (P<0.0001) (Table 4). The male/female ratio among purebred adult ticks did not diverge significantly from the expected 50/50 proportion (Table 4). This confirms that viable progenies in the purebred colonies developed from fertilized eggs, and no parthenogenesis was observed.
Hybrid linesWhen North American female Rh. sanguineus were fed with Israeli male ticks (US–IS cross), 19 out of 20 females fed to repletion. On average, they required 2 days longer to complete their engorgement (P<0.0001) than those mating with sympatric males (Table 3). Means of engorgement weight, duration of the preoviposition period, the weight of an egg batch and BMCI, as well as proportions of ovipositing females were similar between American females crossbreeding with male ticks of Israeli origin and the purebred colony (Table 3). The average egg hatchability in the US–IS cross was lower than in the purebred American colony but not significantly different (P = 0.0137). Larval molting success in the F1 progeny of the US–IS cross was twice as low as that in the purebred parental colonies (P<0.0001); but survival of engorged nymphs was higher, i.e. P = 0.0270 in comparison with the American colony, and P<0.0001 if compared to the Israeli colony. The proportion of males in the F1 progeny of the US–IS cross did not deviate from the expected 50/50 ratio (Table 4) confirming that cross-mating resulted in successful fertilization of eggs, and crossbred females did not reproduce parthenogenetically. The resulting F1 adult ticks from the US–IS cross fed successfully and oviposited within the time frame typical for the purebred parental colonies (Table 5). The average weights of both the engorged females and the egg-masses they produced were a little lower than in the parental colonies (P>0.01), but normal viable F2 larvae eclosed from the absolute majority of eggs in every batch (Table 5). These hybrid larvae remained alive and active in an incubator for over four months.
North American female Rh. sanguineus mated with African males (US–AF cross) fed to repletion within the same time as period as those in the purebred American colony (Table 3). However, their overall feeding success was low with only 16 out of 20 females successfully engorging (P = 0.0034). The average engorgement weight, average duration of the preoviposition period, and the proportion of ovipositing females did not differ significantly from the parameters of a purebred colony (Table 3). On the other hand, BMCI in the US–AF cross was significantly lower (P = 1.9 × 10−6), and consequently, egg batches produced in the US–AF cross were significantly smaller (P<0.0001). Moreover, a significantly lower (P = 0.0003) proportion of those eggs produced viable larvae (Table 3). Molting success of immature stages in the US–AF cross was high (Table 4). In fact, significantly more engorged nymphs survived through the molt in the US–AF cross than in either purebred colony (P<0.0001). The proportion of males in the F1 progeny of US–AF cross did not deviate significantly from the expected 50/50 ratio (Table 4). The resulting hybrid adults successfully fed to repletion within normal time and all engorged females oviposited (Table 5). Although the weights of the engorged females and the egg batches they produced were somewhat lower than in the corresponding purebred colonies, these were the same as in the crossbred parental females (Table 4) and in the US–IS cross (Table 5). The resulting US–AF egg batches, however, did not produce viable F2 progeny; no larvae eclosed from the 20 egg batches.
When females from the Israeli Rh. sanguineus colony were mated with male ticks from the North American colony (IS–US cross), 19 out of 20 females fed to repletion within the same time period as those mating with sympatric males (Table 3). Seventeen (89.5 %) of the engorged females produced egg batches within 7–11 days after repletion. The average engorgement weight of these ticks was significantly lower than in the purebred Israeli colony (P = 0.0006). The BMCI was also low, and the average size of resulting egg batches was reduced by almost 50 % compared to the purebred Israeli females (P = 2.8 × 10−5) (Table 3). However, the average proportion of hatching eggs in the IS–US cross was similar to that in the purebred Israeli colony (P = 0.26). Larval molting success in the F1 progeny of the IS–US cross was significantly lower than in the purebred parental colonies (P<0.0001); but survival of engorged nymphs was the same as in the Israeli colony. The proportion of males in the F1 progeny of IS–US cross did not deviate from the expected 50/50 ratio indicating that the IS–US cross did indeed produce fertilized eggs (Table 4). The resulting F1 adult ticks from the IS–US cross successfully fed on a rabbit and oviposited within the time frame typical for the purebred parental colonies (Table 5). All other reproductive parameters of these hybrids were also almost identical to those in the purebred Israeli colony (P>0.20), including a normal size of produced egg batches and a high proportion of hatching eggs (Table 5). IS–US hybrid larvae remained alive and active in an incubator for over four months.
Out of 20 Israeli Rh. sanguineus females cross-mated with African males (IS–AF cross), only 14 fed to repletion and took 10–14 days to feed. On average, they completed engorgement approximately three days later than those in the purebred Israeli colony (P = 8.3 × 10−6) (Table 3). Nevertheless, they reached similar engorgement weights to the Israeli females mating with sympatric males, and produced egg batches of the same size. All the remaining reproductive parameters in the parental generation were also similar to the purebred colony (Table 3). Molting success of IS–AF hybrid larvae was as high as that of their purebred counterparts, and survival of engorged nymphs exceeded the nymphal molting success in either of the parental purebred colonies (Table 4). The male/female ratio in the F1 progeny of IS–AF cross (73/98) was not significantly different from the expected 50/50 ratio indicating that eggs produced by Israeli females were successfully fertilized by males of African origin (Table 4). The resulting F1 females mating with males from the same cross fed to repletion within the normal time period without a significant delay noticed in the parental generation (Table 5). After engorgement, all females produced egg batches whose average size was only a little smaller than that in the purebred parental colonies. However, these US–AF egg batches did not produce viable F2 progeny (Table 5). Only four individual larvae hatched among the 20 egg batches; and those all died within a few days after eclosion.
When African female ticks were placed on rabbits with male ticks from either the American (AF–US cross) or Israeli colony (AF–IS cross), their feeding success, duration of engorgement, engorgement weights, BMCI, and the average size of egg batches were all similar to those in the purebred African colony (Table 3). High proportions of eggs hatched in all hybrid batches within normal time periods. The molting success of engorged hybrid larvae in both AF–US and AF–IS crosses was lower than in either parental colonies, but the nymphal molting success was much higher than expected for Israeli and especially African ticks (Table 4). Male/female ratios in AF–US and AF–IS two hybrid lines were 96/62 and 97/72, respectively, which confirmed that African females were fertilized by either American or Israeli males and their progeny was not a result of parthenogenetic production.
In the F1 progeny of the AF–US cross, all 20 females placed on a rabbit fed to repletion but their feeding lasted for 11–14 days—on average 2.5–4 days longer than that of purebred African or American females (P = 6.3 × 10−7) (Table 3). The engorgement weights of these ticks were lower than in parental colonies, though the difference did not reach the level of statistical significance (P = 0.043) (Table 5). All engorged females oviposited, yet the BMCI was much lower than in the African colony (P = 0.0078), which resulted in production of very small egg batches (P = 0.0064) (Table 3). All eggs produced by AF–US hybrid females were infertile as no larvae hatched from the 20 egg batches within 12 weeks after oviposition.
In the F1 progeny of the AF–IS cross, the length of female engorgement was not significantly delayed in comparison with either African or Israeli purebred colonies (P = 0.26) (Table 5). All 20 hybrid females successfully fed and reached engorgement weights intermediate between the parental African and Israeli colonies (P = 0.0098 and P = 0.0016, respectively). The average BMCI was also intermediate between the two parental colonies and all engorged hybrid females laid egg batches of normal size (Table 5). However, these eggs were infertile as only a single larva was observed to attempt eclosion, which was unsuccessful.
Phylogenetic analysis of the purebred ticksThe phylogenetic analysis of partial (287 bp) sequences of the 12S rDNA gene showed 3.2 % divergence between Rh. sanguineus from the US and Israeli populations, while the same gene of African ticks differed by 9.2 % from either of those. As a result, North American and Israeli ticks clustered close on the neighbor-joining tree and ticks of African descent represented a separate clade with 74 % bootstrap support (Fig. 1). Rh. sanguineus IS grouped together with Rh. sanguineus from California (Los Angeles—HM014443.1) and Arizona (HM138903.1), as well as from Portugal (FJ536553.1) with 76 % bootstrap support. Rh. sanguineus Oklahoma, US (HM138900.1) appeared closely related to Rh. sanguineus from Italy (HM014442.1) and Rh. turanicus from France (AY947467.1). Rh. sanguineus AF, on the other hand, clustered with Rh. sanguineus from Brazil (AY559842.1) and Thailand (AY987377.1), as well as with African ticks from Zambia (DQ849232.1) and Zimbabwe (AF150017.1) morphologically identified as Rh. turanicus (Fig. 1). Noticeably, ticks from different countries and continents morphologically identified as Rh. sanguineus were intermixed in various phylogenetic clades with those identified as Rh. turanicus. This suggests high variability of morphological features in either species and underscores the difficulty of their delineation based solely on morphology.
DiscussionDelineation of species within the Rh. sanguineus group has been a controversial and confusing issue with many morphologically similar ticks described in different locations under different names only to be later synonymized with the Rh. sanguineus sensu stricto (Pegram et al. 1987a, b, 1989; Walker et al. 2000). Ticks within this group are known to be competent vectors of viral, bacterial, and protozoan pathogens of humans and domestic animals. As different vector species vary considerably in their ability to acquire, maintain, and transmit particular pathogens, the proper identification of vectors is a prerequisite for studies in biology, ecology, and epidemiology of any vector-borne pathogen. Therefore, clarification of the status and relationships between the members of the Rh. sanguineus group presents more than just academic interest.
In accordance with the biological species concept (Mayr 1970), successful interbreeding between organisms from different populations resulting in fertile progeny indicates their conspecificity. Conversely, inability to produce fertile progeny is a result and a sign of reproductive separation between species. Reproductive separation is not usually expected among members of the same species while fruitful hybridization is rare between representatives of different biological species.
Most studies in the possibility of hybridization between closely related tick species have confirmed this concept of reproductive separation. For example, no viable F1 hybrids resulted from cross-mating between Dermacentor variabilis and Dermacentor andersoni (Oliver et al. 1972), Amblyomma americanum and Amblyomma maculatum (Gladney and Dawkins 1973), Rhipicephalus (Boophilus) microplus and Rhipicephalus (Boophilus) decoloratus (Spickett and Malan 1978), or between Dermacentor marginatus and Dermacentor reticulatus (Zahler and Gothe 1997). Interspecific matings producing infertile F1 hybrids have been reported for Rhipicephalus (Boophilus) annulatus and R. (B.) microplus (Graham et al. 1972, Davey et al. 1983), Amblyomma variegatum and Amblyomma hebraeum (Rechav et al. 1982; Clarke and Pretorius 2005), Ixodes ricinus and Ixodes persulcatus (Balashov et al. 1998), Ornithodoros papillipes, Ornithodoros tartakovskyi and Ornithodoros verrucosus (Balashov 1970). Moreover, reproductive incompatibility between geographically different populations of Amblyomma cajennense and R. (B.) microplus suggested that these taxa actually represent complexes of different species (Labruna et al. 2009, 2011).
Published examples of hybridization between otherwise “recognized” tick species include an early report from the former U.S.S.R. where Rh. sanguineus and Rh. turanicus were observed to interbreed and produce fertile progeny (Pervomaisky 1950); and a recent study showing that Amblyomma gemma and A. hebraeum were genetically compatible producing fully fertile hybrids (Clarke and Pretorius 2005). In both studies, successful cross-mating rendering a fertile hybrid was interpreted as an indication of possible conspecificity of the respective ticks. In addition, conspecificity of Rh. sanguineus and Rh. turanicus has been suggested based on the results of DNA analysis when ITS2 sequences of Rh. sanguineus from both Azerbaijan and Burkina Faso were found to be identical to that of Rh. turanicus from Turkmenistan (Zahler et al. 1997).
It was also proposed that interbreeding might theoretically be possible between recently delineated closely related and morphologically similar ticks (Farid 1996). Rh. appendiculatus and Rh. zambeziensis are morphologically very similar (Walker et al. 2000) although molecular analysis confirmed that they are separate species (Mtambo et al. 2007). Fertile hybrids were produced by Rh. zambeziensis females mating with Rh. appendiculatus males, whereas a reciprocal cross resulted in a sterile F1 hybrid (Zivkovic et al. 1986).
Herein, we tested conspecificity of Rh. sanguineus from three geographically distant populations by assessing the genetic diversity between them, studying reproduction and survival of ticks under standard laboratory conditions, and comparing the reproductive performance of their crosses.
Recorded reproductive parameters differed between the three tick colonies. Most likely, the noted variations in the duration of egg incubation period and the nymphal molting success reflect how much our standard laboratory conditions diverge from the natural environmental conditions to which particular tick populations have adapted. Inversely, it indicates that environmental requirements vary between the three geographically distant populations, with the African ticks more different in their requirements from ticks of either North American or Israeli origin than those two from each-other.
When pairing female ticks from distinct geographic locations with allopatric males, we considered a possibility of parthenogenetic reproduction. With exception of a few species, females of ixodid ticks feeding in the absence of conspecific males increase their feeding period, have smaller engorgement weights and oviposit none or only a small number of fertile eggs when compared to inseminated females (Oliver 1989). Moreover, parthenogenesis in ticks inevitably results in production of only female offspring. In our study, we did observe delayed engorgement, decreased engorgement weights, and smaller egg masses in some instances of interbreeding. However, all viable F1 progenies (hybrid as well as purebred) contained both males and females, and no gynandromorphs were observed. This shows that all variants of cross-mating in the parental generation resulted in successful egg fertilization with no evidence of parthenogenesis.
Interestingly, results of interbreeding between males from one population with females from another differed from those when origins of males and females were reversed. Although high proportions of cross-mating females in both US–IS and IS–US crosses fed to repletion, engorgement weights in the IS–US cross were lower than in the reciprocal US–IS cross (Table 3). Overall, the American females mating with Israeli males performed almost as well as those fertilized by sympatric males. On the other hand, the decreased engorgement weights in combination with low BMCI and relatively poor egg hatchability resulted in a much smaller number of F1 progeny in the IS–US cross. The US–IS and IS–US crosses also differed in survival and reproductive parameters of F1 hybrids. Molting success of hybrid larvae decreased in both cross-breeding combinations, but in the US–IS cross it was reduced to a greater extent—by more than 50 %—compared to the purebred colonies (Table 4). Notably, the survival of hybrid nymphs in the US–IS cross was higher than in the parental colonies or in the reciprocal IS–US hybrids. Nonetheless, most of the reproductive parameters of US–IS and IS–US hybrid adults of the F1 generation were similar to those in purebred colonies, and both crosses produced normal numbers of F2 larvae (Table 5), which remained alive for over 4 months after eclosion.
Reciprocal cross-mating between North American and African ticks also resulted in successful fertilization. Although the feeding success of crossbreeding females was lower than in those paired with allopatric males, the fed females achieved normal (for the respective source colony) engorgement weight. Reproductive parameters of crossbred African females were essentially the same as in the purebred African colony, while fertilization of American females by African males resulted in significantly reduced fecundity. Survival of F1 hybrid larvae in both crosses decreased, but the molting success of F1 hybrid nymphs again was much higher than in purebred colonies. Contrary to differences between the two crosses observed in parental generation, F1 adults resulting from the US–AF cross blood-fed faster and better than those from the AF–US cross and produced larger egg masses. However, the 40 egg masses laid by the US–AF and AF–US hybrid females produced no viable progeny.
Observed effects of interbreeding between Israeli and African ticks were similar to those described for US–AF and AF–US crosses. Overall, the African females mating with Israeli males performed as well as in the purebred African colony while fertility and fecundity in the reciprocal cross (IS–AF) were reduced. However, neither parameter decreased as significantly as when North American females cross-mated with African males. All reproductive parameters of F1 hybrids in the IS–AF and AF–IS crosses were similar; they also were at the midway between their parental purebred colonies. And again, no viable F2 progeny resulted from any of the total of 40 hybrid egg masses.
Overall, interbreeding of Rh. sanguineus ticks from three geographically distant populations in most cases resulted in reduction of their fecundity, although heterosis apparently caused significant improvement of nymphal molting success in hybrid lines. Nonetheless, hybridization between North American and Israeli Rh. sanguineus resulted in fully fertile offspring, while all crosses of either American or Mediterranean ticks with those of African origin invariably resulted in complete infertility of the F1 generation. It appears that in all four F1 hybrid progenies involving parents (either males or females) from the African colony, females remained capable of producing eggs, but the eggs were either not fertilized or failed to develop.
Results of our hybridization experiments confirm conspecificity of Mediterranean and North American populations of Rh. sanguineus, which corresponds with conclusions from recent molecular studies (Szabo et al. 2005, Burlini et al. 2010). Reproductive incompatibility is not expected to occur between different populations of a single species. Thus, our results demonstrate that ticks from Reunion belong to a species different from Rh. sanguineus of North American and Israeli origins. Akin to our study, crossbreeding between R. (Boophilus) microplus from Australia with those from Africa or South America produced viable F1, but F1 adults were infertile (Spickett and Malan 1978, Labruna et al. 2009). This indicated that R. (Boophilus) microplus ticks from Africa and S. America represent the same species, while ticks from Australia may be a separate species.
Phylogenetic analysis of partial (287 bp) mitochondrial 12S rDNA gene sequences supports the separation between the brown dog ticks from Reunion and the other two populations by demonstrating deep divisions on a molecular level (Fig. 1). Rh. sanguineus AF comprises a separate clade on the phylogenetic tree together with Rh. sanguineus from Brazil and Thailand as well as with Rh. turanicus from Zambia and Zimbabwe. On the other hand, North American and Israeli Rh. sanguineus ticks cluster together with Rh. sanguineus from Portugal, France, Uruguay, and Argentina and with Rh. turanicus from France. These results concur with a recently published study in phylogeny of the Rh. sanguineus group based on mitochondrial 16S rDNA sequences (Moraes-Filho et al. 2011). For our study, we chose to use the mitochondrial 12S rDNA gene because it has been demonstrated to be a good marker for the establishment of genetic relationships among closely related tick species (Szabo et al. 2005; Ketchum et al. 2009; Labruna et al. 2009; Burlini et al. 2010), while deep divergences of the 16S rDNA gene may arise within a single species (Leo et al. 2010). Taken together, sequence analyses of both genes indicate that the taxon Rh. sanguineus might represent more than one species.
Thus, we evaluated the reproductive compatibility between three geographically distant populations of Rh. sanguineus and found that heterologous crosses between Israeli and North American ticks resulted in highly fertile progeny. The complete reproductive compatibility exhibited in cross-breeding experiments provides an additional evidence of conspecificity between the Mediterranean and North American Rh. sanguineus populations.
Our results also showed that ticks from the Reunion population are reproductively incompatible with either the Mediterranean population from Israel or the North American population from Oklahoma. The molecular phylogenetic analysis similarly separated the Reunion population from American and Israeli ticks. Jointly, these results imply that this particular African population represents a separate species or subspecies. Rh. sanguineus population from Reunion was chosen for this study because no other tick species belonging to the Rh. sanguineus group have been identified on the island (Walker et al. 2000; Müller et al. 2004). A degree to which the Reunion population is representative of brown dog ticks of the African continent in general requires further detailed assessment. Yet, close relatedness between both the 12S rDNA and 16S rDNA gene sequences of ticks from Reunion to those from Brazil, Thailand, Iran, Zambia, and Zimbabwe indicate that the purported taxon has wide geographical distribution.
Results of our experiments support a hypothesis that at least two different species currently share the name Rh. sanguineus. These species need to be redescribed and delineated. Studies involving large number of tick strains from different geographic locations and combining crossbreeding with the molecular approach are needed to accomplish this task.