Tamarisks (Tamarix spp., Tamaricaceae), also known as saltcedars, are exotic weeds that have invaded riparian areas across western North America causing extensive economic and ecological damage (Shafroth et al. 2005). Five species of tamarisks have become invasive in North America and there are several known instances of hybridization, with hybrids of Tamarix ramosissima Ledeb. and Tamarix chinensis Lour. comprising a large share of the tamarisk invasion in western North America (Gaskin and Kazmer 2009; Gaskin and Schaal 2002). The severity and widespread nature of the tamarisk invasion led to development of a biological control program, which in 2001 resulted in the first open field releases of a tamarisk feeding leaf beetle Diorhabda elongata Brullé (Chrysomelidae). Beetles were initially released at two locations in Nevada and one location in each of four states: Wyoming, Utah, California, and Colorado (DeLoach et al. 2004). Since 2001 the tamarisk beetles have become widely established and they have provided control over large areas of the tamarisk infestation (Carruthers et al. 2008). Even so, the beetles failed to establish or did poorly in some areas, especially in the southern portion of the invaded range of tamarisks (Texas, New Mexico, Southern California) and in coastal California, where the primary invader is Tamarix parviflora DC. (Carruthers et al. 2008; Dalin et al. 2009; Lewis et al. 2003).

At the outset of the tamarisk biological control program the leaf beetles were classified as a single species, D. elongata, with a range across North Africa and Eurasia into China and Mongolia (Tracy and Robbins 2009), and in all regions they were known to be specialists on the genus Tamarix. The initial collections of D. elongata sensu lato for tamarisk biological control were from the interior of central Asia: one near the town of Chilik in Kazakhstan, and the other near the city of Fukang in the Xinjiang Province of northwestern China (DeLoach et al. 2003). These populations did well against T. ramosissima and T. chinensis and their hybrids in the northern interior states of Nevada, Utah, Colorado, and Wyoming but failed to thrive in coastal California, Texas, or southern New Mexico (DeLoach et al. 2004). This was because of several factors, including mismatches in the photoperiodic requirements for reproduction and diapause at more southern latitudes, which restricted the normally multivoltine beetles to a univoltine life history (Bean et al. 2007a; Lewis et al. 2003). Another factor was the mismatch of target species in areas where T. parviflora was dominant because it appears to be a suboptimal host for central Asian Diorhabda (Dalin et al. 2009). The presence of predators, especially ants, was also shown to inhibit establishment of tamarisk beetles (Herrera 2003). Recognizing these problems, additional ecotypes of D. elongata were imported for use in the Tamarix biological control program, beginning in 2002 (DeLoach et al. 2004; Milbrath and DeLoach 2006a; Tracy and Robins 2009). Because D. elongata is widely distributed in the native range, multiple ecotypes could be found to match the diverse ecological conditions encountered in North America, including host plant species, climate, and latitude.

New ecotypes were collected from sites in the Mediterranean basin, Uzbekistan, and China (DeLoach et al. 2004). Beetles from new collections were considered to be novel ecotypes and were initially held under quarantine conditions, tested, and treated in much the same way as if they were distinct species. Host range testing was done for each ecotype (Milbrath and DeLoach 2006a, b; Herr et al. 2009) and they were found to have the same or similar host range properties as the original ecotypes; that is they were all tamarisk specialists. In addition to safety testing, the new ecotypes were screened for traits that might make them more effective in areas where the original releases had been ineffective. New ecotypes were found to be better adapted to southern Tamarix phenology, as well as to photoperiod regimes found in the southernmost ranges of Tamarix in North America (Dalin et al. 2010; Dudley et al. 2006; Milbrath et al. 2007). The ecotype collected from Crete, Greece, was shown to establish on T. parviflora whereas the Fukang, China, ecotype had failed (Carruthers et al. 2008; Thomas et al. 2009). The new ecotypes displayed an array of traits that were extremely useful for the Tamarix biological control program but it became clear that genetic and taxonomic relationships among ecotypes needed further evaluation. Characterization of the Diorhabda ecotypes was initiated and coordinated under the auspices of the Saltcedar Consortium, a group of scientists, weed managers, government agencies, and other stakeholders with an interest in tamarisk biological control (DeLoach et al. 2004). Characterization included the molecular genetic and hybridization investigations described in this study as well as a taxonomic revision of the tamarisk-feeding leaf beetles based on morphology (Tracy and Robbins 2009).

The Diorhabda elongata species complex was defined and described using morphological characteristics, primarily of the genitalia, in a study that also included extensive biogeographical information (Tracy and Robbins 2009). Tracy and Robbins divided the tamarisk-feeding Diorhabda into five species, including four species that had been considered ecotypes within the tamarisk biological control program. Central Asian ecotypes formerly known as Diorhabda elongata deserticola, became Diorhabda carinulata Desbrochers, with the common name of northern tamarisk beetle. Western Asian ecotypes, including beetles collected in Uzbekistan, became Diorhabda carinata Faldermann or the larger tamarisk beetle. Ecotypes from the northeastern Mediterranean basin, including beetles collected in Crete and Possidi, Greece, remained D. elongata, now known as Mediterranean tamarisk beetle whereas the North African and western Mediterranean ecotypes became Diorhabda sublineata Lucas or the subtropical tamarisk beetle. The fifth species, not currently used in the biological control program, was classified as Diorhabda meridionalis Berti and Rapilly with a range centered in southern and western Iran. There are a few subtle differences in outward appearance between the newly designated species but the characteristics that define them are found in the hidden sclerites of the endophallus and to a lesser extent in sclerites and palpi of the female reproductive system (Tracy and Robbins 2009), making these essentially cryptic species.

This study presents the molecular genetic component of the ecotype characterization project in order to determine the relationships among the Tamarix-feeding members of the Diorhabda elongata sensu lato species complex and examines concordance of molecular genetic traits with morphological, behavioral, and ecological traits. Data on interspecific hybridization within the species complex and within species is also presented, along with molecular genetic assays to assess hybridization and genetic introgression between species and ecotypes. This information will benefit the tamarisk biological control program by enabling better identification of Diorhabda species and by providing precise methods for measuring gene flow between ecotypes in the field or under laboratory conditions.

Materials and Methods

The insects used in this study were originally collected on Tamarix in Eurasia and North Africa (Figure 1). Cultures were maintained at four facilities in North America: the U.S. Department of Agriculture Agricultural Research Service (USDA ARS) Western Regional Research Center, Exotic and Invasive Weeds quarantine facility in Albany, CA (USDA Albany); the USDA ARS Grassland Soil and Water Research Laboratory quarantine facility, Temple, TX (USDA Temple); the New Mexico State University Entomology, Plant Pathology and Weed Science quarantine facility, Las Cruces, NM (NMSU Las Cruces); and the Palisade Insectary, Biological Pest Control Program, Colorado Department of Agriculture, Palisade, CO (CDA Palisade). Beetles were cultured on live cuttings of Tamarix, including T. ramosissima, T. chinensis, and their hybrids (Gaskin and Schaal 2002), which served as the primary culturing material at USDA Temple, NMSU Las Cruces, and CDA Palisade. Tamarix parviflora was the primary species used to culture insects at USDA Albany. Beetles were reared in well-ventilated plastic containers under day lengths of at least 16 h to prevent diapause induction. More detailed culturing methods are described elsewhere (Bean et al. 2007b, Petersen 2007). Cultures of D. carinulata were derived from beetles collected near the cities of Fukang, China (44.17°N, 87.98°E; elevation 552 m [1,811 ft]); Turpan, China (42.86°N, 89.22°E; elevation 70 m below sea level); and Chilik, Kazakhstan (43.6°N 78.25°E; elevation 662 m). They are referred to as the Fukang, Turpan, and Chilik ecotypes of D. carinulata. The Chilik ecotype was released near the town of Delta, UT, and cultures used in this study were derived from beetles collected there in 2003. Cultures of D. carinata used in this study originated from collections made in 2002 near Karshi (Qarshi), Uzbekistan (38.86°N, 65.72°E; elevation 350 m). Cultures of D. elongata originated from near Possidi, Greece (39.96°N, 23.36°E, elevation 5 m) and from near Sfakaki, Crete, Greece (35.83°N, 24.6°E; elevation 7 m) and were called the Possidi and Crete ecotypes, respectively. Cultures of D. sublineata originated near the town of Sfax, Tunisia (34.66°N, 10.67°E; elevation 10 m). Beetles from all sites were originally identified as D. elongata (DeLoach et al. 2004; Milbrath and DeLoach 2006 a, b).

Figure 1.

Origin of cultures and samples. Stars represent locations of towns and cities nearest collection sites for samples used in this study. Shading represents approximate ranges of Diorhabda species (taken from Tracy and Robbins 2009). Black  =  Diorhabda sublineata, gray stippled  =  Diorhabda elongata, hatching  =  Diorhabda carinata, and gray  =  Diorhabda carinulata. (Color for this figure is available in the online version of this paper.)

Beetles used for DNA analysis originated from the sites listed above or were collected from four additional sites, including Bukhara, Uzbekistan (39.82°N, 64.39°E); Ashgabat, Turkmenistan (37.95°N, 58.67°E); Kyparissia, Greece (37.26N° 22.65°E); and Astros, Greece (37.44°N, 22.75°E). Collection sites are described in more detail elsewhere (Tracy and Robbins 2009).

Results

Mitochondrial DNA Sequence Analysis.

The mtDNA region flanked by primers C1-J-1718 and TL2-N-3014 produced sequences 1,270 base pairs in length; 240 base positions were parsimony-informative. The maximum parsimony tree is shown in Figure 2. Three individuals from Bukhara, Uzbekistan, and two from Karshi, Uzbekistan,had identical sequences and are represented by accession M148. All four individuals from Crete, Greece, had identical sequences and are represented by accession M155. Three individuals from Fukang, China, with identical sequences are represented by accession W5. Four individuals from Turpan, China, with identical sequences are represented by accession M154. Three individuals from Ashgabat, Turkmenistan, are represented by accession M559.

Figure 2.

Maximum parsimony 50% majority consensus cladogram of 288 trees of cytochrome c oxidase subunit 1 mtDNA region for Diorhabda spp. and outgroups. Tree is 518 steps in length, representing 1,270 base pairs of aligned sequence data. Bootstrap values are shown above branches. Major clades are indicated by capitalized letters A to E and shaded regions. City, country, and accession number are at tips of tree. Diobrotica specimen is followed by GenBank accession number.

Five major clades are present within Diorhabda (Figure 2). Three of the clades—A, C, and E—are, respectively, D. carinulata, D. sublineata, and D. carinata. The remaining two clades are D. elongata.

AFLP Analysis.

The four primer pairs produced 115 variable loci from the 64 insects included in the analysis, which resulted in the dendrogram shown in Figure 3. Four major groups, each corresponding to a morphospecies, are present. Unlike the mtDNA COI sequence results, the AFLP data assemble D. elongata sensu stricto specimens into a single group.

Figure 3.

Unweighted pair group method with arithmetic mean dendrogram of 64 Diorhabda specimens from amplified fragment length polymorphism data. Bootstrap values (> 50%) shown above branches. Accession numbers, origins, and species designations are listed to right of dendrogram.

Egg Viability from Crosses within Species of Diorhabda.

Crosses within D. carinulata between the Fukang ecotype and the Chilik ecotype yielded eggs with high viability (88 and 94%) with no significant viability differences between eggs from the F₁ (hybrid) crosses and eggs from the parental crosses (P  =  0.5392, Table 1). Hybrid adults were either crossed with each other or backcrossed into the parental strains and eggs produced from these crosses had viabilities ranging from 89 to 100%. These viability values were not significantly different from those from parental ecotype crosses (P  =  0.4537, Table 1). Eggs from crosses within D. carinulata and between the Fukang ecotype and the Turpan ecotype had viabilities > 90% and viability values were not significantly different from those from parental crosses (P  =  0.7212, Table 1). Hybrid F₁ adults were crossed with each other and backcrossed into both parental ecotypes. Eggs from those crosses had viabilities ranging from 88 to 99% with no significant differences amongst the F₂ and first-generation backcrossed (BC₁) groups (P  =  0.5219, Table 1). Parental ecotype crosses were not done in this experiment and so the hybrid crosses could not be directly compared with parental crosses. Crosses within D. elongata, between the Possidi ecotype and the Crete ecotype, yielded egg viabilities that were not significantly different from crosses within ecotypes (P  =  0.2304 for F₁ egg viability and P  =  0.1161 for F₂ egg viability).

Table 1.

Egg viability following crosses between ecotypes^a of Diorhabda carinulata and their F₁ hybrids^b or first-generation backcrosses (BC₁).

F₂ and BC₁ Egg Viability from Hybrids between Species of Diorhabda.

Crosses between D. carinulata (Fukang ecotype) and the other three species yielded hybrids that produced low-viability F₂ or BC₁ eggs (Figures 4–Figure 5.6). In all cases the viability of F₂ or BC₁ eggs was significantly lower (P < 0.0001) than egg viability within parental species crosses. In the case of D. carinulata × D. elongata (Possidi ecotype) there were no viable eggs produced when F₁ interspecific hybrid males were backcrossed into either parental strain or crossed with F₁ interspecific hybrid females (Figure 4). This pattern was nearly identical with D. carinulata × D. elongata (Crete ecotype) interspecific F₁ hybrids: the F₁ cross was sterile, backcrosses involving F₁ males also produced no viable eggs, and the F₁ females produced few viable eggs when backcrossed into the parental strains (data not shown). Overall the F₁ males from a D. carinulata × D. elongata were sterile and the F₁ females produced eggs of low viability (< 22%) when they were backcrossed into the parental strains (Figure 4, and data not shown for the Crete ecotype).

Figure 4.

Egg hatch from crosses within Diorhabda carinulata (Cr) and Diorhabda elongata (El), reciprocal backcrosses of F₁ interspecific hybrids (Hy) to parental strains, and crosses of F₁ interspecific hybrids (Hy/Hy). Species of the male is indicated first, the female second. Error bars represent SE; n  =  7 pairs for each group. Values for all crosses involving hybrids are significantly different from the parental species values (P < 0.0001). No viable eggs were produced when hybrid males were part of the cross. Diorhabda carinulata were the Fukang ecotype and D. elongata were the Possidi ecotype.

Figure 5.

Egg hatch from crosses within Diorhabda carinulata (Cr) and Diorhabda carinata (Ca), reciprocal backcrosses of F₁ interspecific hybrids (Hy) to parental strains, and crosses of F₁ interspecific hybrids (Hy/Hy). Species of the male is indicated first, the female second. Error bars represent SE; n  =  7 pairs for each group. Values for all crosses involving hybrids are significantly different from the parental cross values (P < 0.0001). Diorhabda carinulata were the Fukang ecotype.

Figure 6.

Egg hatch from crosses within Diorhabda carinulata (Cr) and Diorhabda sublineata (Su), reciprocal backcrosses of F₁ interspecific hybrids (Hy) to parental strains, and crosses of F₁ interspecific hybrids (Hy/Hy). Species of the male is indicated first, the female second. Error bars represent SE; n  =  10 pairs except for Hy/Cr, which had 13 pairs. Values for all crosses involving hybrids are significantly different from the parental cross values (P < 0.0001). Diorhabda carinulata were the Fukang ecotype.

Crosses between D. carinulata and D. carinata yielded F₁ interspecific hybrids that produced low-viability F₂ eggs (< 4%) or low-viability BC₁ eggs (< 15%) resulting from backcrosses into either parental species (Figure 5). All crosses involving F₁ interspecific hybrids had a significantly reduced egg viability compared to the parental crosses (P < 0.0001).

Crosses between D. carinulata and D. sublineata yielded F₁ interspecific hybrids that produced low-viability F₂ or BC₁ eggs (< 15%) (Figure 6). F₂ and BC₁ egg viabilities were significantly reduced compared to those of the parental crosses (P < 0.0001).

Crosses between D. elongata (Crete ecotype) and D. sublineata yielded F₁ interspecific hybrids that produced high-viability F₂ eggs (> 85%) or high-viability BC₁ eggs in cases where the F₁ interspecific hybrid was female (Figure 7). Egg viability was reduced in the two backcrosses of F₁ interspecific hybrid males with females from either parental species (Figure 7). In these cases egg viability was significantly lower than with crosses within the parental strains (P < 0.0001). In another experiment, the D. sublineata × D. elongata hybrids were shown to produce stable cultures for three generations and these produced F₄ eggs with a viability of 75% (data not shown).

Figure 7.

Egg hatch from crosses within Diorhabda elongata (El) and Diorhabda sublineata (Su), reciprocal backcrosses of F₁ interspecific hybrids (Hy) to parental strains, and crosses of F₁ interspecific hybrids (Hy/Hy). Species of the male is indicated first, the female second. Error bars represent SE; n  =  16 pairs for each group. Values for crosses between male hybrids and the parental populations are significantly different from the parental cross values (P < 0.0001). Diorhabda elongata were the Crete ecotype.

Hybrids resulting from D. carinata × D. elongata produced eggs with a mean viability of 67% (n  =  10 egg collections, made every other day) whereas hybrids resulting from D. carinata × D sublineata produced eggs with a mean viability of 42% (n  =  10 egg collections, made every other day). These data are not directly comparable to data from other crosses because the pairs were pooled in these experiments and individual pairs were used in the other experiments. It is clear, though, that F₂ egg viability was higher in these crosses than from the three interspecific crosses involving D. carinulata where F₂ egg viability ranged from 0 to 3% (Figures 4–Figure 5.6).

The viability of F₂ and BC₁ eggs was used to estimate the likelihood of gene flow between species as they encounter each other in the field. F₂ egg viability of 40% or higher, as seen in crosses involving D. carinata, D. sublineata, and D. elongata, was seen as an indication that gene flow would be likely if these species encountered each other. In crosses involving D. carinulata, the F₂ and BC₁ egg viabilities were well below 30%, which would make gene flow less likely but still a distinct possibility. Some crosses involving D. carinulata also revealed male hybrid sterility and mortality due to genital mismatch. Therefore we considered the possibility of gene flow to be low in these crosses (Figure 8). These designations do not take into account the likelihood of prezygotic reproductive isolation, as described below in the Discussion.

Figure 8.

Relative likelihood of gene flow based on F₂ egg viability results.

Mortality in D. carinulata Crosses.

Crosses between D. carinulata and the other three species resulted in high mortality in D. carinulata females (Table 2). In the most extreme example 100% female mortality was observed when D. elongata males were crossed with D. carinulata females. Sometimes pairs were unable to uncouple, indicating severe genital mismatch, which had been previously noted (Tracy and Robbins 2009). In most cases, uncoupling occurred but females ceased laying eggs and died, indicating internal damage.

Table 2.

Adult survival during hybridization experiments^a in which Diorhabda carinulata was crossed with the other three Diorhabda species.^b

Detection of Hybrids using AFLP PCOA.

The four AFLP primer pairs produced 52 variable loci from the 57 insects included. The resulting PCOA is shown in Figure 9. PCOA axis 1 accounts for 58% of the variance and PCOA axis 2 accounts for 6% of the variance. This analysis provides clear separation of D. elongata (Crete ecotype), D. sublineata, and laboratory-generated F₁ interspecific hybrids between the two (Figure 9a). The F₁ interspecific hybrids have intermediate PCOA scores along PCOA axis 1. This result is expected as AFLP markers are dominant and therefore the F₁ hybrids exhibit AFLP markers of each parental type.

Figure 9.

Principal coordinates analysis of amplified fragment length polymorphism data from 57 Diorhabda specimens including the following: (a) Diorhabda elongata (Crete ecotype), Diorhabda sublineata, and known D. elongata × D. sublineata F₁ hybrids; (b) inclusion of specimens from laboratory colonies including D. elongata (Crete ecotype) from the U.S. Department of Agriculture Agricultural Research Service Grassland Soil and Water Research Laboratory quarantine facility, Temple, TX (USDA Temple) and D. sublineata from the USDA Temple facility, the Colorado Department of Agriculture's Palisade Insectary,, and the New Mexico State University Entomology, Plant Pathology and Weed Science quarantine facility in Las Cruces, NM; and (c) inclusion of specimens collected in the field from release sites near the towns of Encino and Kingsville in southern Texas that were suspected to be D. elongata × D. sublineata F₁ hybrids.

Additional PCOA results are shown in Figure 9b for specimens from several laboratory colonies that were suspected of containing hybrids, beginning in 2006. Three specimens from an indoor colony of D. elongata (Crete ecotype) at Temple, TX, have PCOA scores similar to that of the parental type and were not suspected to be hybrids based on morphology (J. Tracy, personal communication). All other specimens, thought to be D. sublineata, have PCOA scores intermediate to the known F₁ hybrids and the Crete ecotype of D. elongata. Six of the 10 specimens from the Temple, TX, outdoor colony of D. sublineata were suspected hybrids based on morphology (J. Tracy, personal communication). The NMSU Las Cruces colony of D. sublineata was founded using individuals from the Temple, TX, outdoor colony of D. sublineata and the CDA Palisade D. sublineata colony was subsequently founded using insects from the NMSU Las Cruces colony.

Additional PCOA results are shown for specimens from two field release sites, one near Kingsville, TX, and a second near Encino, TX (Figure 9c). The hybridization status of these populations was questioned after it was established that hybridization was occurring in some of the laboratory colonies. The PCOA scores for the Kingsville population are generally within the PCOA score range of the Crete ecotype of D. elongata. Four specimens from the Kingsville population were identified as D. elongata based on morphology (J. Tracy, personal communication). Two specimens identified morphologically as D. elongata had PCOA axis 2 scores lower than other Crete ecotype specimens but in general there is little evidence of hybridization in the Kingsville population. PCOA scores for the specimens from the Encino population are closer to those of known F₁ hybrid specimens than to either D. sublineata or D. elongata.

Discussion

Members of the Diorhabda species complex currently used in the North American Tamarix biological control program separate into four distinct clades based on AFLP analysis of genomic DNA (Figure 3). These results support the conclusions of Tracy and Robbins (2009) who have divided the Diorhabda species complex into five related species, four of which are used in the Tamarix biological control program. Mitochondrial COI sequence analysis yielded five clades instead of four, with D. elongata separating into two groups: one from eastern Greece and Crete and the other from southern and western Greece (Figure 2). Incongruence between nuclear- and mitochondrial-based DNA phylogenies is not unusual (e.g., Sota and Vogler 2001) and can be traced to differences in DNA metabolism and inheritance patterns between the two genomes (Ballard and Whitlock 2004; Moore 1995; Rubinoff and Holland 2005). Because the incongruence between mitochondrial and genomic DNA is relatively minor (see Sota and Vogler 2001 for an example of a major incongruence in a beetle), we conclude that the molecular genetic data strongly support the morphological data.

There are no apparent genetic barriers between ecotypes of D. carinulata (Table 1) or between ecotypes of D. elongata, and molecular genetic analysis did not provide delineation of these ecotypes within species. This is evidence that the Chilik ecotype of D. carinulata will be compatible with the Fukang ecotype of that species as they meet across wide boundaries in the western United States. This is the only case in which two ecotypes of the same species are currently being used in the tamarisk biological control program, although the Crete and Possidi ecotypes of D. elongata have both been released in the field. The Crete ecotype is well established but the Possidi ecotype apparently failed to establish, although beetles overwintered and a small population persisted for 3 yr following release (Tracy and Robbins 2009).

Hybridization studies revealed the potential for gene flow between the four Diorhabda species, although in combinations involving D. carinulata there are substantial barriers to hybridization including F₁ male sterility (Figure 4), lethal mismatches in genitalia (Table 2; lock-and-key incompatibility, see Shapiro and Porter 1989), and low F₂ and BC₁ egg viability (Figures 4–Figure 5.6). Crosses between D. carinulata and D. elongata resulted in male sterility in the F₁ interspecific hybrids (Figure 4; data not shown for the Crete ecotype of D. elongata). Male sterility is a strong but not absolute barrier to hybridization in the field as hybrid females were capable of producing a low percentage of viable eggs when backcrossed into the parental strains (Figure 4) and it has been shown in other beetle species that male hybrid sterility does not completely eliminate hybridization and interspecific genetic introgression in natural populations (Brouat et al. 2006; Streiff et al. 2005).

The genital mismatches noted between D. carinulata and the other three species resulted in high mortality in females (Table 2). These results are consistent with the genital lock-and-key hypothesis, which states that rapidly evolving genital structures enforce genetic isolation between closely related species. The hypothesis is compelling because rapid evolutionary divergence is often observed in genital structures (Shapiro and Porter 1989), including within the Diorhabda species complex (Tracy and Robbins 2009). Nonetheless, examples of lock-and-key mismatch are rare (Gröning and Hochkirch 2008; Shapiro and Porter 1989;). One example was documented in the beetle genus Ohomopterus (Nagata et al. 2007; Sota and Kubota 1998) and Diorhabda may provide a second example in a beetle. It has been noted that uncoupling of mating pairs may not occur when D. carinulata are crossed with the other species (Tracy and Robbins 2009) but the nature of the physical damage has yet to be shown, as it was in Ohomopterus (Sota and Kubota 1998).

Crosses between D. carinulata and the other three species resulted in significantly decreased F₂ and BC₁ egg viability (P < 0.0001). This, as well as male hybrid sterility and genital incompatibility, will limit gene flow between D. carinulata and the other three species. The importance of these observations is magnified by the widespread distribution of D. carinulata in North America and the high likelihood that this species will eventually come into contact with D. elongata and D. sublineata in the field.

In the three crosses involving D. sublineata, D. elongata, and D. carinata we found no evidence of male sterility or genital mismatch, and these crosses yielded higher-viability F₂ eggs, and BC₁ eggs in the case of D. sublineata and D. elongata (Figure 7), than in the three crosses involving D. carinulata (Figures 4–Figure 5.6). These results point to a greater likelihood of hybridization in the field for these species combinations (Figure 8). Diorhabda sublineata × D. elongata showed a significantly reduced BC₁ egg hatch (P < 0.0001) when hybrid males were backcrossed into the parental stains (Figure 7). In spite of this, stable hybrid cultures of D. sublineata × D. elongata were established and maintained for four generations under laboratory conditions (data not shown) and genetic introgression occurred under the artificial conditions experienced in the laboratory and in field cages, with interesting patterns revealed by AFLP analysis (Figure 9). For instance, the PCOA scores of the apparent hybrids in Figure 9b are intermediate to the known F₁ hybrids and the Crete ecotype of D. elongata, suggesting two things. First, hybridization may have been ongoing for two or more generations. Second, the Crete ecotype has the more “dominant” genome. This could occur through selection in hybrid colonies for traits of the Crete ecotype that favor its persistence in colonies, more frequent invasion of the colonies by the Crete ecotype, or a higher rate of backcrossing of hybrids to the Crete ecotype rather than D. sublineata.

These data indicated that the colonies of D. sublineata were a mixture of D. sublineata and D. elongata. Subsequent to these studies all laboratory and field colonies of D. sublineata were destroyed and new collections were made from Sfax, Tunisia, in the spring of 2008. All D. sublineata used after 2008 for field releases and for laboratory studies were derived from beetles collected in 2008.

In the native range of Diorhabda there are regions where two or more species are sympatric or parapatric (Figure 1) yet intermediate forms have not been found, in spite of extensive examination of museum and freshly collected specimens (Tracy and Robbins 2009). In some cases where species are parapatric or narrowly sympatric there could be relatively small and undetected hybrid zones. In the case of D. carinulata and D. carinata there is a vast region in western Asia where they are sympatric and where both species have been collected in close proximity, even from the same plant (Tracy and Robbins 2009), yet no hybrids have been detected. The behavioral, mechanical, and genetic barriers appear to be sufficient to maintain species integrity.

Prezygotic isolation mechanisms may prevent hybridization in the native ranges of the Diorhabda species complex and these could also function to keep the species distinct in North America. Diorhabda carinulata utilizes a male-produced aggregation pheromone consisting of two components released in a ratio of approximately 1 ∶ 1 (Cossé et al. 2005). The other three species have the same components, released in different ratios and amounts (R. Bartelt and A. Cossé, personal communication) which could result in species sorting during aggregation and mating (for a more detailed discussion of reproductive isolation via differences in aggregation pheromone see Tracy and Robbins 2009). In addition there are probably other chemical, visual, and tactile cues that bring about pair formation (Cossé et al. 2005) and could act as barriers between species in the field. Such barriers would probably not be detected in our experiments because artificial conditions are well known to interfere with prezygotic isolation mechanisms (Gröning and Hochkirch 2008).

Hybrids in general are considered to be less fit because the genetic combinations that result from hybridization have not been subject to natural selection and evolution (Barton 2001). However, in new ecological settings where parental species could be at a disadvantage, hybrids may provide a rich source of novel genetic combinations for natural selection (Seehausen 2004). The Diorhabda species complex and the new ecological settings they encounter in western North America may provide ideal starting material for hybridization, hybrid zones, and gene flow. Hybrid zones are known to form between beetle congeners (Nagata et al. 2007; Sota 2002), including chrysomelids (Gatto et al. 2008; Peterson et al. 2005), and these may (Sota 2002; Sota and Vogler 2001) or may not (Peterson et al. 2005) result in genetic introgression. In the case of the Diorhabda species complex it is probable that there will be formation of hybrids in the field and that their fate will be a function of selection for new genetic combinations.

Hybridization and gene flow between Diorhabda species could result in new and stable genetic combinations in North America as has been documented with the host plant genus Tamarix. Tamarix ramosissima and T. chinensis don't hybridize in the native range, yet they form hybrids in North America (Gaskin and Schaal 2002, 2003) with introgression into the parental strains resulting in mixed genomes (Gaskin and Kazmer 2009). Tamarix chinensis contributes more heavily to the genetic composition of plants in the southern range of the North American distribution whereas T. ramosissima contributes more heavily to the genetic composition of plants in the northern range of the invasion (Gaskin and Kazmer 2009). This pattern may be an artifact of founding events, or may have arisen from differential natural selection across a latitudinal gradient (Friedman et al. 2008; Gaskin and Kazmer 2009), which could also happen with Diorhabda as the newly released species adapt to ecological settings very different from those in the native range.

The revision of the Diorhabda species complex based on morphological (Tracy and Robbins 2009) and now molecular traits has helped provide a biological basis for host specificity (Dalin et al. 2009; Herr et al. 2009) and other developmental and physiological differences among Diorhabda species used in the tamarisk biological control program (Dalin et al. 2010). Molecular methods will now be essential to track hybridization and introgression in the field. As shown in this study (Figure 9), AFLP analysis can be used to detect hybridization and introgression between D. sublineata and D. elongata, which will be important because both species have been introduced in Texas and will soon become sympatric in at least one area along the Rio Grande (C. Ritzi, personal communication). It will be instructive to see if they hybridize in the field, to what extent introgression occurs, and if beetles with new combinations of genes perform better in novel ecological settings. There is a need for molecular protocols that can be used to follow hybridization and new genetic combinations in the field within the Diorhabda species complex. Although D. sublineata × D. elongata was the most critical and timely of the possible crosses to characterize at the molecular level, the others may eventually become of equal or greater importance as populations expand and species meet in the field. Information on genetic composition and field performance within the Diorhabda species complex is a necessary component of the tamarisk biological control program.

Molecular analysis should have been incorporated early in the development of Diorhabda as a potential biological control agent for tamarisk. Clarification of the relationships between ecotypes, including the discovery of potential cryptic species, would have allowed a more targeted approach to agent development, including host range testing, and a more informed evaluation of other biological parameters. As it was, each ecotype was tested for host range (Herr et al. 2009; Milbrath and DeLoach 2006a, b) but this was done as a safety precaution and not with the knowledge that Diorhabda elongata was in fact a species complex. Molecular genetic analysis has become critical in decision-making within biological control programs (e.g., Madeira et al. 2001, 2006; Rauth et al. 2011) and this study is another example of the benefits that molecular genetic analysis can provide to biological control programs.