Species Concepts as Applied to the Whitefly Bemisia tabaci Systematics: How Many Species Are There?

Journal of Integrative Agriculture

February 2012

2012, 11(2): 176-186

REVIEW

Species Concepts as Applied to the Whitefly Bemisia tabaci Systematics: How Many Species Are There? LIU Shu-sheng1, John Colvin2 and Paul J De Barro3 Key Laboratory of Agricultural Entomology, Ministry of Agriculture/Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, P.R.China 2 Natural Resources Institute, University of Greenwich, Kent ME4 4TB, United Kingdom 3 CSIRO Ecosystem Sciences, Brisbane QLD 4001, Australia 1

Abstract The worldwide distribution and extensive genetic diversity of the whitefly, Bemisia tabaci, has long been recognized. However, the levels of separation within B. tabaci and the nomenclature of the various genetic groups have been a subject of debate. Recent phylogenetic analyses indicate that B. tabaci is a complex composed of

28 morphologically

indistinguishable species. In this article, we first review the debate and difficulties associated with B. tabaci’s taxonomy and systematics, and argue for the need to apply the biological species concept in order to elucidate B. tabaci’s systematics. We summarize the accumulated genetic and behavioural data on reproductive incompatibilities evident amongst phylogenetic mtCOI groups of B. tabaci. Crossing studies have been conducted with 14 of the 28 putative species covering 54 reciprocal inter-species pairs, and observations on mating behaviour have been conducted for seven species pairs. Data from both crossing trials and behavioural observations indicate a consistent pattern of reproductive isolation among the putative species. We then discuss the technical and conceptual complexities associated with crossing experiments and behavioural observations designed to reveal reproductive incompatibility. Finally, we elaborate on a strategy for further clarifying the pattern of reproductive isolation between B. tabaci groups and propose future research directions on the systematics of this complex. Key words: whitefly systematics, biological species, cryptic species, phylogenetic species, reproductive isolation, mating behavior, taxonomy

OVERVIEW AND PERSPECTIVES The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodoidea) has a global distribution and extensive genetic diversity (Dinsdale et al. 2010; De Barro et al. 2011). Some groups within B. tabaci are important pests of a range of agricultural, horticultural and ornamental crops. The pest status of B. tabaci has risen considerably in the past 20 years, due to the widespread invasions by what have been referred to as the B and Q Received 1 March, 2011

biotypes of the whitefly (Brown et al. 1995; Liu et al. 2007; De Barro et al. 2011). While the genetic complexity and economic importance of B. tabaci has been recognized, its species status has been the subject of debate for decades despite the considerable effort in investigating its taxonomy and systematics. The history of studies on B. tabaci taxonomy and systematics, as well as the current species status of this whitefly, can be found in the recent review by De Barro et al. (2011). Two issues are particularly relevant here. First, investigations on classifi-

Accepted 13, October, 2011

Correspondence LIU Shu-sheng, Tel: +86-571-86971505, E-mail: [email protected]

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Species Concepts as Applied to the Whitefly Bemisia tabaci Systematics: How Many Species Are There?

cation of B. tabaci have been unproductive due to the lack of reliable morphological variability either in the fourth instar nymphs/pupae or in the adults (Mound 1963; Rosell et al. 1997; Calvert et al. 2001; Gill and Brown 2010), and have been characterized by repeated descriptions of new species and synonymization (Russel1 1958; Mound and Halsey 1978; De Barro et al. 2011). Second, in the last 20 years we have seen a proliferation of B. tabaci biotype designations throughout the world, and these designations have been based primarily on genetic markers, whether protein or DNA, and not on biological data or any between-biotype boundaries. This practice has led to a misuse of the term biotype (Downie 2010), at least 36 of which have been designated. In many cases genetic groups that are apparently isolated reproductively were named as biotypes, and in other cases haplotypes were named as biotypes without any demonstration of associated biologically differentiating traits; by definition, a biotype is a biologically differentiated form of a purported single species (Gullan and Cranston 2005). Based on phylogenetic analysis and pairwise com-

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parisons of genetic distance between genetic groups of B. tabaci worldwide, Dinsdale et al. (2010) provided a framework to suggest that B. tabaci is a cryptic (or sibling) species complex containing 11 higher genetic groups and at least 24 morphologically indistinguishable species. Following the ‘phylogenetic’ species bounds proposed by Dinsdale et al. (2010), Hu et al. (2011) recently added four more species, increasing the total number of cryptic species to 28 (Table 1). Field surveys conducted in India indicate that more putative species may be added to the list (Chowda-Reddy et al. 2012). While phylogenetic species themselves have limited biological significance, because their criteria and degrees of difference are intrinsically subjective (Avise 2000), the presence of a good delineation of phylogenetic species within a cryptic species complex provides a realistic structure against which the existence of biological species can be tested (De Barro et al. 2011). Members of a cryptic species complex are indistinguishable morphologically and so ultimate differentiation between them comes from evidence of reproductive

Table 1 Putative species groups and species of the whitefly Bemisia tabaci species complex as revealed by phylogenetic analysis in Dinsdale et al. (2010) and Hu et al. (2011)1) Species group Africa/Middle East/Asia Minor

New World Uganda Sub-Saharan Africa

Italy Asia II India Asia II

Asia III China

Australia Australia/Indonesia Asia I 1)

Species Mediterranean Middle East-Asia Minor 1 Middle East-Asia Minor 2 Indian Ocean New World Uganda Sub-Saharan Africa 1 Sub-Saharan Africa 2 Sub-Saharan Africa 3 Sub-Saharan Africa 4 Italy Asia II 8 Asia II 1 Asia II 2 Asia II 3 Asia II 4 Asia II 5 Asia II 6 Asia II 7 Asia II 9 Asia II 10 Asia III China 1 China 2 China 3 Australia Australia/Indonesia Asia I

Biotype designations associated Q, J, L, and Sub-Saharan Africa Silverleafing B and B2 MS A, BR, C, D, F, Jatropha, N, R, and Sida

S

T K, P, PCG-1, PK1, SY, and ZHJ2 ZHJ1 G Cv

ZHJ3

AN H, M, NA, and PCG-2

The species groups in the table are listed in the order indicated in the phylogenetic trees.

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isolation. A combined analysis on molecular phylogenetics and reproductive incompatibility has proved to be a productive approach to studying systematics of cryptic species complexes (Suatoni et al. 2006; Heraty et al. 2007). In this review, we present the argument that supports the necessity of applying the biological species concept to the study of B. tabaci systematics and then summarize the genetic and behavioural evidence that has accumulated on the reproductive incompatibility among different B. tabaci mtCOI genetic groups. We will then discuss the technical and conceptual complexities associated with crossing experiments and behavioural observations conducted to reveal reproductive incompatibility. Finally, we present what we believe to be a productive strategy for further clarifying the pattern of reproductive isolation within the B. tabaci species complex and discuss the prospects of discovering morphological differences associated with the new classification.

EVIDENCE OF REPRODUCTIVE ISOLATION Crossing experiments Xu et al. (2010) collated the data of crossing experiments that were conducted from various parts of the world from 1993 to 2009, and identified 30 reciprocal inter-species crosses among 11 putative species as identified by Dinsdale et al. (2010). Recently, Wang et al. (2010, 2011) conducted trials for all possible crosses among six cryptic species collected in China. The reproductive incompatibility between the two globally invasive members of the species complex, Mediterranean (MED) and Middle East-Asia Minor 1 (MEAM1), were further examined in detail by Elbaz et al. (2010) and Sun et al. (2011b). During the literature search for this review, we found that a crossing experiment between the MED and Italy putative species was conducted by Demichelis et al. (2005), which was overlooked by Xu et al. (2010). Table 2 summarizes all the published data of 54 reciprocal inter-species crosses involving 14 putative species within the complex. Whiteflies, including B. tabaci, are haplodiploid, producing male progeny from unfertilized eggs and female progeny from fertilized eggs. Reproductive compat-

ibility between putative species of B. tabaci can thus be examined by comparison of realized fecundity and fertility between intra- and inter-species crosses. A failure to produce female progeny or a substantial reduction in fecundity and/or proportion of females in the progeny indicates reproductive incompatibility. In all the 14 species examined, all intra-species controls had 50-70% females in their progeny. In contrast, in the 54 reciprocal inter-species crosses, 44 produced no F1 hybrid females, only 10 produced low proportions of F1 females, mostly in the range of 1-5% compared to over 50% in the intra-species controls, and the F1 hybrid females, where their fertility and/or viability was tested, were shown to be either sterile or substantially less viable (Table 2).

Behavioural observations Observations on mating behaviour have been conducted for seven species pairs (Table 3). In all species pairs observed, males and females from different species exhibited extensive courtships. However, they very rarely achieved copulation. In a study on patterns of speciation in Drosophila, Coyne and Orr (1989) proposed an index of prezygotic isolation (Pre), an index of postzygotic (Post), and then an index of total isolation (T), as follows: Pre=1-Frequency of heterospecific matings/Frequency of homospecific matings Post=1-Frequency of fertile and viable sexes in heterospecific tests/Frequency of fertile and viable sexes in both homospecific tests T=Pre+(1-Pre)×Post Applying the index of prezygotic isolation here, Pre equaled or approached to 1, i.e., complete isolation, in all species pairs except in the cross MEAM1 ×Asia II 1 where Pre was relatively low (Table 3). However, in this latter cross, no F1 hybrid females were produced, indicating that either no zygotes were produced or the zygotes failed to develop (Luan and Liu 2012). It should be noted that all the observations on B. tabaci interspecies pairs were conducted in a no choice setting, i.e., adults were offered access only to individuals of an opposite sex from another species. In the observations on MEAM1×MED, no copulation was observed when the observations were conducted for a few hours (Elbaz

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Table 2 Summary of reproductive incompatibility among 14 putative species of the Bemisia tabaci cryptic species complex from published crossing studies Female source1) (biotype name associated) 1 MED (Q) 2 MEAM1 (B) 3 Sub-Saharan Africa 1 4 Sub-Saharan Africa 2 (S) 5 Uganda 6 Italy (T) 7 Asia I (H) 8 Australia (AN) 9 Asia II 1 (ZHJ2) 10 Asia II 3 (ZHJ1) 11 Asia II 5 (G) 12 Asia II 7 (Cv) 13 China 1 (ZHJ3) 14 New world (A) 1) 2)

1

2

3

4

5

6

Male source2) 7 8

9

10

11

12

13

14

The codes of male source correspond to those of female source listed in the first column. Symbols indicating levels of reproductive incompatibility. , complete reproductive compatibility; , no F1 hybrid females were produced; , low number of F1 hybrid females was produced in both directions of cross but the hybrids were sterile and/or characterized by reduced viability and fertility; , low number of F1 hybrid females was produced in one direction of cross, but the hybrids had reduced viability and fertility; , low number of F1 hybrid females was produced but the fertility of F1 females was not tested.

Table 3 Summary of pre-copulation barrier among seven putative species pairs of the Bemisia tabaci cryptic species complex from published behavioural observations Species pairs (biotype name associated) MEAM1 ×MED (B×Q) MEAM1 ×MED (B×Q) MEAM1 ×New World (B×A) MEAM1 ×New World (B×A) MEAM1 ×Asia II 1 (B×ZHJ2) MEAM1 ×Asia II 1 (B×ZHJ2) MEAM1 ×Australia (B×AN) MEAM1 ×Australia (B×AN) MEAM1 ×Asia II 2 (B×ZHJ1) MEAM1 ×Asia II 2 (B×ZHJ1) MED ×Asia II 1 (Q×ZHJ2) MED ×Asia II 1 (Q×ZHJ2) MED ×Asia II 3 (Q×ZHJ1) MED ×Asia II 3 (Q×ZHJ1) 1)

Pre 1)

References

0.99-1.00 0.99-1.00 1.00 1.00 0.98 0.74 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.99

Elbaz et al. 2010; Sun et al. 2011b Elbaz et al. 2010; Sun et al. 2011b Perring et al. 1993 Perring et al. 1993 Luan and Liu 2012 Luan and Liu 2012 Liu et al. 2007 Liu et al. 2007 Zang and Liu 2007; Liu et al. 2007 Zang and Liu 2007; Liu et al. 2007 Wang et al. 2010 Wang et al. 2010 Wang et al. 2010 Wang et al. 2010

In the original definition of Pre, frequencies of matings are values observed in choice situation, i.e., adults are offered potential mates simultaneously from both species; for the data in this table, heterospecific matings were observed with no choice situation, i.e., adults were offered only potential mates from another species, and frequencies of homospecific matings were taken from control treatments of intra-species. It is likely that Pre values derived this way may underestimate the level of isolation, see text for discussion.

et al. 2010) or 3 d (Sun et al. 2011b), the two species achieved copulation very occasionally only when adults of opposite sex of the two species were enclosed together in a small arena for 5 d (Sun et al. 2011b). If the observations were conducted in a choice setting, it is likely that the probability of inter-species copulation would be further reduced.

Field observations By definition, a species is a group of natural interbreed-

ing populations and so reproductive incompatibility between putative species requires field confirmation by observations on gene flow. Moya et al. (2001) analyzed the composition of B. tabaci populations from six locations in the southern Iberian Peninsula where both MEAM1 and MED co-occurred and did not detect any hybrids. MEAM1 and MED have showed sympatric distribution in Israel for many years, and field sampling in this country has never detected hybrids (Elbaz et al. 2010). In the last several years, sympatric distribution of MEAM1 and MED has occurred in large

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areas in China, extensive field sampling indicated displacement of MEAM1 by MED in some areas, but has never detected any hybrids (Chu et al. 2010; Hu et al. 2011; Rao et al. 2011). These records suggest that MEAM1 and MED are genetically isolated in the field. Hu et al. (2011) revealed the distribution of 13 indigenous B. tabaci species in addition to the alien MEAM1 and MED in China. Sympatric distribution of indigenous species as well as alien and indigenous species seems common. However, hybrids in the field have never been detected. Circumstantial evidence indicates that widespread sympatric distribution of the alien MEAM1 and indigenous species have occurred in the USA and Australia, but no occurrence of hybrids (Perring 1996; Liu et al. 2007). Interestingly, the only evidence for inter-species introgression in the field comes from that between the closely related alien MEAM1 and indigenous MS species on the Indian Ocean Island of La Reunion although the frequency of introgression was generally low (Delatte et al. 2006, 2011). Further analysis showed that hybridization between these two species in the field is non-random and may be manipulated by some secondary symbionts in the whiteflies (Thierry et al. 2011). The global Bayesian phylogenetic analysis of the B. tabaci complex by Dinsdale et al. (2010), which used a break in the K2P sequence divergence frequency distribution to identify the point at which a genetic group is translated into a species, showed that MED and MEAM1 are sibling species, and these two species and MS seem to be directly descended from a common ancestor. As we will discuss late, the literature shows that it is not unusual for closely related species to occasionally produce hybrids. As many of the species in the B. tabaci complex seem to show partially sympatric or parapatric distributions (Hu et al. 2011), one may expect that hybridizations may occasionally occur between some of them. The rarity of hybridization detected so far may suggest that interspecies introgression seldom occurs in the field, although it is likely that incidences of occasional hybridization between some other species in the field may be detected if more intensive field sampling is conducted using more effective methods for hybrid detection.

TECHNICAL AND CONCEPTUAL COMPLEXITIES Technical complications in crossing trials Although it is often stated that only virgin females were used in inter-species crossing experiments, much confusion has occurred in this regard, because the methodologies used did not separate insects before emergence. In 1989, Li et al. (1989) observed the mating behaviour of a Bemisia species, most probably the New World, and reported that newly emerged adult females were not attractive to males for about 10 h. Females accepted courting males 20-24 h after emergence. Although that article does not report in detail how the timing of pre-mating intervals were observed and determined, some subsequent studies have used its information to design procedures for collecting virgin adults: cohorts of 4th instar nymphs/pupae on plants or detached plant shoots were kept, and newly emerged adults were collected at 6-18 h intervals for crossing trials (Perring et al. 1993; De Barro and Hart 2000; Maruthi et al. 2001, 2004; Perring and Symmes 2006). Similar protocols have been used in other studies, although the precise intervals for collecting newly emerged adults were not reported (Costa et al. 1993; Bedford et al. 1994; Caballero and Brown 2008). With the video recording system developed by Ruan et al. (2007), Liu et al. (2007) and Luan et al. (2008) were for the first time able to observe the mating activities of B. tabaci on live plants continuously for several days. They isolated test insects prior to their emergence and placed individual adults 1-2 h post emergence together to initiate observations. Their behavioural data on four species, i.e., MEAM1, Australia, Asia II 1, and Asia II 3 showed that 60-80% newly emerged adults in each of the species would copulate at least once within 12 h post emergence, and some individuals would copulate as early as 2 h post emergence. Although variation occurred between species, for example, MEAM1 achieved higher probability of copulation than the indigenous species during the same time interval post emergence, the detailed data warn that test insects must be isolated prior to emergence to ensure that only virgin females are used in

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Species Concepts as Applied to the Whitefly Bemisia tabaci Systematics: How Many Species Are There?

crossing trials (Luan et al. 2008; Xu et al. 2010; Wang et al. 2010, 2011). If mature pupae were placed together and adults were collected at 6-18 h intervals, as practiced in some of the earlier studies, female and male adults may have stayed together for any length of time between 0-18 h depending on their time of emergence and some of them could have already mated. Studies on reproductive compatibility between the same species by different authors have shown disparity (Costa et al. 1993; Perring et al. 1993; Caballero and Brown 2008), which in some cases may have been caused by the failure to exclude mated parental females from crosses. Recently Li et al. (2012) examined the effects of space dimension and temperature on the crossing mating of three species of the B. tabaci complex, i.e., MEAM1, MED, and Asia II 7, and found that neither of the two factors affected the lack and very low incidence of hybridization among the three species.

Technical complications in behavioural observations Observations on mating behaviour of B. tabaci have often been conducted on detached leaves and viewed with the aid of a stereo microscope (Perring et al. 1993; Perring and Symmes 2006; Roditakis et al. 2009; Elbaz et al. 2010). This protocol creates two problems. First, observations using a stereo microscope allow only a tiny field of vision, which necessitates the use of an arena with the very small diameter of 6-8 mm in which whitefly movement is greatly restricted. Second, mating activities on detached leaves are reduced compared to those on intact leaves (Zang and Liu 2007; Sun et al. 2011a). Sun et al. (2011a) observed that the reduction in mating activity on detached leaves was associated with decreased oviposition on this substratum compared with that on intact leaves.

Complexities in interpreting laboratory crossing trial data While by definition gene flow between species in nature is restricted or does not occur, species have evolved and so are not discrete natural units. Thus, occasional occurrence of hybrids between closely related species

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in nature is not uncommon. In nature, at least 25% of plant species and 10% of animal species, mostly the youngest species, are involved in hybridization and potential introgression with other species (Mallet 2005). In addition, numerous studies have demonstrated that many species coexist without interbreeding in nature readily hybridize in the artificial settings of the laboratory or zoo (Coyne and Orr 1989; Benirschke and Kumamota 1991; Wang and Dong 2001; Mayr 2002; Wang 2007), indicating that captivity and benign laboratory environments may mask both pre- and post-zygotic isolations that exist in the wild. This complexity brings up the issue of how to interpret laboratory crosses where only the opposite gender of another putative species is present, i.e., ‘no-choice conditions’, yet some hybridization occurs. In a comprehensive analysis on the pattern of speciation in Drosophila, Coyne and Orr (1989) showed that the mean level of total isolation among 44 sympatric species pairs was 0.907±0.026, with a lower bound of 0.85 as defined by the 95% confidence interval. Because this level of reproductive isolation has been sufficient to maintain the distinctiveness of sympatric species, Coyne and Orr (1989) suggest that total isolation of 0.85 is enough to prevent the fusion of allopatric taxa upon secondary contact. In the 54 species pairs examined in the B. tabaci complex, the indices of total isolation in all cases reached one or nearly so (Tables 2 and 3). If the criterion of reproductive isolation among Drosophila species can be applied to the B. tabaci species complex, the data in Tables 2 and 3 would indicate the putative species as defined by Dinsdale et al. (2010) and Hu et al. (2011) are real biological species, at least for the 14 species for which crossing trials have been conducted. Captivity and benign laboratory environments have been shown repeatedly to promote interbreeding that does not occur in the wild, and so far no case studies indicate the opposite, i.e., two species that do not interbreed in captivity do so in the wild. It seems reasonable, therefore, to infer genetic groups as different species if they do not interbreed under favourable laboratory conditions. However, when different genetic groups can achieve some interbreeding in the laboratory, interpretation of the data requires caution. Until a comprehensive analysis on patterns of speciation in the subject

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species group has been conducted and a criterion of reproductive isolation for species differentiation has been derived, as has been done for Drosophila (Coyne and Orr 1989), interpretation of such data may require some qualitative judgment. This is not unusual, as virtually all methods used for delimiting species require researchers to make some kind of qualitative judgment (Sites and Marshall 2004).

FUTURE PROSPECTS Recognition of cryptic species is critical to meaningful biological research and effective pest management (Rosen 1977; Adler 1988; Bush 1993). Bemisia tabaci cryptic species differ in host range (Zang et al. 2006; Xu et al. 2011), insecticide resistance (Costa et al. 1993; Horowitz et al. 2005; Luo et al. 2010), behaviour (Liu et al. 2007; Crowder et al. 2010a, b; Wang et al. 2010), virus transmission (Bedford et al. 1994; Li et al. 2010; Liu et al. 2010) and interactions with viruses and host plants (Colvin et al. 2006; Jiu et al. 2007; Liu et al. 2009; De Barro and Bourne 2010). The review above shows that we have made substantial progress in solving the conundrum of B. tabaci species complex, but much remains to be done before the species status is resolved to a level acceptable to most whitefly workers around the world (De Barro 2012). So far 28 putative cryptic species have been suggested (Table 1). While the crossing trials have involved 14 of the 28 putative species and collectively they indicate a consistent pattern of reproductive isolation among them (Table 2), more crossing studies are required. The 28 putative species would produce 756 inter-species crosses. However, it is neither possible nor necessary to conduct all the possible inter-species crosses. The phylogenetic trees derived by Dinsdale et al. (2010) and Hu et al. (2011) may be consulted to design the most productive crosses. One strategy would be to conduct more crosses between older and younger species as well as between the younger species themselves as shown by the trees. It would also help to reveal further the pattern of reproductive isolation if effort were made to involve each of the putative species in some crossing trials. Considering the widespread reproductive isolation observed among B. tabaci cryptic species, we may

LIU Shu-sheng et al.

speculate that fine morphological variations, possibly in the genitalia of adults, may exist among the species. Thus, some effort should be made to find stable morphological variations among the putative species, although separation of Bemisia species based on morphology has long been a difficult and daunting task (Gill and Brown 2010). In addition to the intrinsic difficulties in identifying morphological variation in the whiteflies, we must be prepared to face a number of uncertainties. First, B. tabaci and its relatives may have evolved over time without morphological changes, so we may fail eventually to classify the putative species on morphological grounds. Second, even if we are able to find some morphological variations among the putative species in one geographic region, the plasticity of phenotypes in response to host plant probably means that one set of morphological characteristics may not be applicable to all species. In the event that we are unable to make progress in finding stable morphological variations, an alternative approach would be to use congruence between phylogenetic and biological species to nominate the cryptic species, as indentified by molecular markers (Dinsdale et al. 2011; Hu et al. 2011) in association with data of crossing trials and observations on mating behaviour (Tables 2 and 3). If we are able to separate tabaci from its relatives on morphological grounds, we may use one set of morphological characters, probably in both adult and nymphal stages, to distinguish all species in the B. tabaci complex from their relatives. We may then use consensus DNA sequences to indentify each of the cryptic species within the complex. If this data set matches the biological species concept data, then this should provide the confidence to nominate name-bearing types using molecular markers within the complex (De Barro et al. 2011). With development of effective DNA barcoding methods, it will be more practical to identify the species on the basis of molecular differentiation than the use of morphological characters, which will almost certainly require a high level of morphological taxonomic skill and are likely to be more time consuming. In the event that we are able to nominate the cryptic species by the above approach, we still face the challenge of fulfilling the requirements of priority in the International Code of Zoological Nomenclature, because

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Species Concepts as Applied to the Whitefly Bemisia tabaci Systematics: How Many Species Are There?

over the last century 22 species names have been given to B. tabaci. As Gill and Brown (2010) have pointed out, ‘many original descriptions are woefully inadequate by modern standards, adults are known for only a very small percentage of described species, and in many cases type material is not available or adequate for further study’. Under these circumstances, one feasible approach is for the whitefly science community to jointly write and submit an appeal to the International Commission on Zoological Nomenclature aimed at abolishing all previous types. The above discussions have assumed that the bounds of >3.5% divergence in COI sequences (Dinsdale et al. 2010) are valid for species separation across the whole of B. tabaci. De Barro et al. (2005) compared both mtCOI and ITS1 and showed that they produced the same genetic groupings, and thus added credibility to the species delineations by mtCOI. In spite of this accumulating evidence, there are dangers in relying on mtDNA data alone to identify basal taxa, and DNA sequence from a single region (DeSalle et al. 2005) or an arbitrary level of divergence (Avise 2000) cannot define species boundaries reliably. It is generally accepted that additional data from nuclear genes, ecology or crossing experiments should also be used to substantiate such boundaries (King et al. 2008). Given the genetic diversity and complexity across the whole of B. tabaci, it is probable that species separation may be more variable than that defined by mtCOI divergence alone (De Barro 2012). We need to consider genetic groups that diverge less than 3.5% in mtCOI sequence. For example, in the phylogenetic analysis by Dinsdale et al. (2010), all genetic groups from the New World are defined as a single species. Real biotypes, i.e., host races of B. tabaci were first identified from different populations from the New World (De Barro et al. 2011). Crossing trials between plant-host adapted populations that differ by less than 3.5% mtCOI divergence are needed for many of the mtCOI phylogenetic groups, but particularly so for the New World. While biologists use species as the primary biological units for our work, we have always been troubled by the philosophical question, ‘what is a species?’ (Merrell 1981; Mayr 2002; Sites and Marshall 2004). As we have encountered in the studies on the B. tabaci complex, genetically and reproductively distinct, but

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morphologically indistinguishable groups of closely related species may represent an unsolvable challenge to classical taxonomy that relies on morphological ‘types’ of each species. In order to progress, therefore, we probably need to find new ways of describing and identifying such species, as we have proposed above for classification of the species within the B. tabaci complex. One of the purposes of this paper is to consider future challenges in B. tabaci systematics. With the increasing weight of evidence and acceptance by entomologists that B. tabaci is indeed a species complex composed of numerous closely related cryptic species, the question arises of whether or not these might constitute a previously unrecognized genus. There are three generally agreed criteria for delimiting a genus, which are: monophyly, reasonable compactness and distinctness. The molecular frameworks reviewed here show clearly that all descendants of an ancestral B. tabaci can be grouped together. The name “Bemisia tabaci”, therefore, currently encompasses a compact and discrete group of species, which share many distinct ecological and biological characteristics. All B. tabaci, for instance, transmit begomoviruses, but other recognized whitefly species in the genus Bemisia do not. The three criteria do appear to fit our current perception of B. tabaci and so it is not inconceivable that with the progress that is likely to be made on the species and genus level diagnostics in whiteflies in the years to come, the Bemisia tabaci species complex could one day also be considered to be a “good” genus. B. tabaci researchers have made considerable progress in resolving the systematic complexity present in this whitefly complex, and we have every reason to expect greater resolution of this conundrum in the near future.

Acknowledgements This study was funded by the National Basic Research Program of China (2009CB119203), the National Natural Science Foundation of China (30730061), and the China Agriculture Research System (CARS-25-B-08).

References Adler P H. 1988. Ecology of black fly sibling species. In: Kim K C, Merritt R W, eds., Black Flies: Ecology, Population Management, and Annotated World List. Pennsylvania State University Press, University Park,

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