The invasive red imported fire ant, Solenopsis invicta Buren, has successfully dispersed across many countries from its South American homeland and now has reached the US-Mexico border (e.g., Matamoros, state of Tamaulipas, México), where it now coexists with native fire ants, Solenopsis geminata, Solenopsis xyloni, and others. The morphological identification of Solenopsis spp. workers is difficult, particularly small ones. We examined the sequence of the cytochrome oxidase I (COI) mitochondrial gene (mtDNA) as a marker for fire ants collected at several Mexican localities. PCR products from this locus yielded unique sequences and restriction patterns that allowed distinguishing between S. invicta, S. geminata, and specimens harboring S. xyloni sequences. The S. invicta sequences obtained were 99% identical to sequences reported from Florida and New Mexico specimens. The S. xyloni sequences obtained were 96% identical to New Mexico sequences. The S. geminata sequences were similar (93% identity) to those from Florida, and shared a Hinf I restriction site with some but not all Florida sequences. The S. xyloni sequences were detected in S. geminata/S. xyloni hybrids identified by morphology; along with other characters, the marker allows their characterization.
The invasive red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae) is considered the most destructive ant species in the world (Landcare Research 2007). Solenopsis invicta has multiple impacts, including changing food webs (Vinson 1997), threatening endangered species (Wojcik et al. 2001), and negatively impacting human and animal health (Jetter et al. 2002). Red imported fire ants were reported to have the ability to feed on any insect species, and in general will attack and consume any invertebrate species that is not capable of defending itself or escaping attack (Ricks & Vinson 1970). After arriving accidentally to the Gulf coast region of the United States, this species has been enormously successful ecologically; by 1999, it infested more than 121 million hectares in the United States and Puerto Rico (Wojcik et al. 2001). Sénchez-Peña et al. (2005) published the first reports of S. invicta in Mexico, at several locations along the USA-Mexico border, all within 1 km from the Rio Grande in the Mexican states of Tamaulipas and Coahuila. In 2007, infestations were observed 3 km south of the Rio Grande, east of Matamoros, Tamaulipas (Sánchez-Peña unpublished observations). All these identifications of S. invicta in Mexico were based on morphology. The municipality of Matamoros includes the most extensive S. invicta populations detected in Mexico so far (Sanchez-Peña, unpublished observations) and the area contains the most favorable habitats for unimpeded expansion of S. invicta further south into Mexico (Phillips & Thorvilson 1992).
In addition to the numerous urban, medical, veterinarian, and agricultural implications of the S. invicta invasion (Jetter et al. 2002), there are several sites of prime ecological importance in Northeastern Mexico that could be severely impacted by this species. One such site is the sea-turtle egg-laying region (e.g., Rancho Nuevo beach) in central and southern Tamaulipas state. Predation by S. invicta at Rancho Nuevo could impact the Kemp's ridley sea turtle, (Lepidochelys kempii (Garman)), considered one of the most endangered vertebrates in the world. This location is the only site worldwide where large nesting aggregations of this sea turtle are known to occur (Johnson et al. 1999). Parris et al. (2002) documented S. invicta predation of sea turtle hatchlings (Caretta caretta) in Florida, and considered S. invicta to be a serious threat. The unique ecosystems at Cuatrociénegas (state of Coahuila) and El Cielo Biosphere Reserve (state of Tamaulipas) also may be impacted by S. invicta.
Fire ants in the Solenopsis saevissima species-group generally appear to be a morphologically homogeneous clade, making species identification quite difficult. Native fire ants (4 described species: Solenopsis amblychila Wheeler, Solenopsis aurea Wheeler, Solenopsis geminata (F.), and Solenopsis xyloni McCook) and several hybrids between these species are (or were) sympatric over a large portion of the current range of S. invicta in North America; S. invicta has caused large local extinctions of S. geminata and S. xyloni (Trager 1991; Wojcik et al. 2001). All 4 native species have been collected in northern Mexico (Trager 1991; Helms Cahan & Vinson 2003). The identification of these polymorphic North American fire ant species by morphology is most easily accomplished by examination of the largest (major) workers; species identification becomes considerably more difficult as worker size diminishes (Trager 1991).
Populations of the tropical fire ant, S. geminata exist in Northeastern Mexico. It is a key-stone predator in some Latin American agroeco-systems, and it has an important effect on the composition of arthropod fauna (Risch & Carroll 1982; Perfecto 1991). The key morphological characteristic for identification of S. geminata is soldiers (major workers) with disproportionately enlarged heads (Trager 1991). However, these soldiers comprise a very minor proportion of the workers in colonies, they rarely forage outside the nest, and they tend to retreat deeper into soil when the colony is disturbed (Trager 1991). Thus, samples of S. geminata colonies commonly lack soldiers making morphological identification more difficult.
Jacobson et al. (2006) remarked on the difficulty of identifying all size castes of S. invicta from those of S. xyloni, the southern fire ant, which is an extremely similar native species. They developed a PCR assay for the identification of S. xyloni and S. invicta, involving specific amplification in the maternally-inherited cytochrome oxidase I (COI) mitochondrial locus, followed by digestion of PCR products with the restriction enzyme Hinf I. This method identified unambiguously all S. invicta samples tested.
Identification of native fire ant species is not straightforward either, as mentioned. Trager (1991) groups the very similar North American species S. xyloni, S. aurea, and S. amblychila (i.e., non-S. geminata native fire ants) into the “S. xyloni subcomplex”. Although among major workers, eye size and ommatidia number are reported to be adequate characters for separating S. xyloni from S. aurea and S. amblychila (Trager 1991; E. LeBrun personal communication), the morphological discrimination of some samples of S. xyloni, S. aurea, and S. amblychila is very difficult or almost impossible (Trager 1991). Our preliminary observations indicate that in Northeastern Mexico these native, non-S. geminata fire ants species do not include bicolored forms (with brownish-black gaster) but mainly rather uniform yellow-reddish forms (Trager 1991; Cook & O'Keefe 2002). Considering the similarity of these 3 species, the lack of comprehensive fire ant taxonomical studies in this region, and the lack of information on COI sequences for S. aurea (although Shoemaker et al. (2006) reported having sequenced the COI gene of S. amblychila), in this report we take a conservative approach and for purely practical reasons we use the term “S. xyloni subcomplex” sensu Trager (1991) regarding our collections of non-S. geminata native fire ants. Thus, it is possible that they might correspond to either S. xyloni, S. aurea, or S. amblychila. In this respect it must be mentioned that the cladistic analysis and phylogenetic study of Pitts et al. (2005) failed to support the splitting of the Solenopsis fire ants into subordinate divisions; we acknowledge that the “S. xyloni subcomplex” of Trager (1991) might not be a phylogenetically supported clade despite the apparent relatedness of its members.
In this study, we adapted the PCR assay of Jacobson et al. (2006) to identify Solenopsis spp. samples in northeastern Mexico, initially to verify the presence of S. invicta in the municipality of Matamoros. A PCR detection assay could be a valuable tool for identification of Solenopsis spp. in northern Mexico.
MATERIALS AND METHODS
Collection and Morphological Identification of Fire Ants
Table 1 lists the samples analyzed and their origin; localities are shown in Fig. 1. At all sites, samples were obtained by either (1) directed searches of nests or (2) baiting with 5-cm hotdog pieces placed on the ground and collection when Solenopsis fire ants were present.
Solenopsis invicta were collected at the cities of Nuevo Laredo and Matamoros, state of Tamaulipas, within 1 km of the Rio Grande river (USA-Mexico border) (Sánchez-Peña et al. 2005). Native fire ants were collected at the following states and municipalities (Table 1): CHIHUAHUA: Chihuahua; COAHUILA: Torreón; NUEVO LEON: Monterrey, Cadereyta, Pesquería (town of Santa María la Floreña); TAMAULIPAS: Matamoros. Ants were transferred to 96% ethanol, stored at room temperature, and processed for molecular analysis within 1 month of collection. Solenopsis spp. were identified according to Trager (1991).
Total genomic DNA was extracted from 1 to 5 worker ants stored in 96% ethanol with the lithium chloride method for insects (Huang et al. 2000). PCR amplification of cytochrome oxidase I (COI) regions were carried out as described by Jacobson et al.(2006). Reactions were carried out in a volume of 25 µL consisting of 2.5 µL of 10X buffer, 0.75 µL of 50mM MgCl2 1.25 µL of 2.5mM dNTPs, 1.0 µL of genomic ant DNA, 1.3 µL of CIJ2195 primer (13.6 µM), 1 µL Jerry Garcia-CI primer (15.2 µM) (Jacobson et al. 2006), 0.3 µL of Taq DNA polymerase (5U/µL) (all reagents from Invitrogen, Carlsbad CA), and 16.9 µL of MilliQ water (Millipore Corp., Billerica, MA). PCR conditions were 95°C (1 min), 40 cycles of 94°C (1 min each), 43°C (1 min), 68°C (2 min), and 72°C (2 min). Amplification products were visualized on 1.2% agarose gel stained with ethidium bromide. They were digested Hinf I enzyme; standard restriction enzyme digestions were carried out in a 10 µL volume consisting of the following: PCR product (2 µL), 0.3 µL Hinf I enzyme (10U/µL), 1 µL of 10X buffer, and 6.7 µL MilliQ water. Each reaction mixture was incubated at 37°C overnight and PCR digestion was visualized on 1.2% agarose gel stained with ethidium bromide to compare the size of the bands obtained with Msp I-digested pBlueScript as molecular marker.
Nucleotide sequencing of PCR fragments were performed by cloning into the PGEM-T vector system (Promega, Madison, WI). Recombinant plasmids were characterized with Pvu II enzyme (New England Biolabs, Ipswich, MA) and purified by the Wizard SV Gel and PCR Clean-up System (Promega, Fitchburg, WI). PCR fragments were sequenced with pUC/M13 reverse and forward sequencing primers in a Perkin Elmer/Applied Bio-systems Model 3730 sequencer at the Universidad Nacional Autonoma de Mexico (UNAM) Sequencing Facility, Mexico City.
The standard genetic analysis of raw sequences data were performed at the National Center for Biotechnology Information web site ( http://www.ncbi.nlm.nih.gov/) with the BLAST site ( http://www.ncbi.nlm.nih.gov/BLAST/). Hinf I sites were verified for all PCR products with New England Biolabs NEBcutter version 2.0.
RESULTS AND DISCUSSION
Identification according to Trager (1991) indicated that specimens collected in our study included the invasive S. invicta, and the following native taxa: S. geminata; members of the S. xyloni subcomplex of species, and S. geminata/S. xyloni hybrids (Fig. 2). It was possible to identify specimens as native (non-invicta) fire ants based on morphology and color. Identification of native fire ants to species was sometimes not possible because some samples lacked major workers; fire ant morphological taxonomy is mainly based on these workers (Trager 1991, and references therein).
Only sample 2 from the city of Chihuahua, state of Chihuahua (North-Central Mexico) included S. xyloni workers (Trager 1991) very similar to S. invicta in pigmentation and color pattern (Jacobson et al. 2006). This S. xyloni sample from Chihuahua is removed from S. geminata distribution sites (Trager 1991; Mackay & Mackay 2002; Helms Cahan & Vinson 2003), reducing the chances that this sample includes S. xyloni/S. geminata hybrids (Helms Cahan & Vinson 2003). Morphological examination of the remaining native fire ant samples indicated that some were typical S. geminata, and others S. geminata/S. xyloni hybrids. We also collected samples similar to the hybrids but lacking major workers, thus restricting morphological identification.
Fire Ant Molecular Markers Detected
Analysis of PCR products of all our samples yielded 3 main patterns (Fig. 3). These PCR products, digestions and sequences are described below. Regarding S. invicta markers, sequencing of the PCR product from sample 3 from Nuevo Laredo, Mexico and homology search (BLAST) in GenBank sequences indicated highest identity (99%) with 18 S. invicta COI accessions (i.e., EF620559 from the Wu-Chuan, China population). Comparison of our PCR product with one of these 99% identical sequences (Genbank accession DQ153014), submitted by Jacobson et al. (2006) indicated that both possess the diagnostic Hinf I restriction site, splitting the PCR product into 2 fragments (691 and 249 bp in this work; 687 and 252 bp in Jacobson et al. (2006)) (Fig. 3). The PCR-Hinf I digestion of COI in our samples (Table 1) confirms the presence of S. invicta populations across hundreds of kilometers in Mexico (Nuevo Laredo and Matamoros), and supports the use of this molecular marker as an identification tool for Mexican populations of S. invicta when fire ant morphological identification is limited.
Fire ant samples, localities, and morphological and molecular identification.
Native Fire Ants with the S. xyloni COI Marker
Most of our samples identified as native fire ants harbored the S. xyloni marker (i.e., COI digestion pattern) described by Jacobson et al. (2006). These samples were the following.
(1) the Chihuahua S. xyloni specimens (sample 2). These were morphologically typical S. xyloni, with bicolored major workers having a lighter, brown-reddish head and mesosoma, and uniformly dark gaster lacking light reddish or brown areas, in a pattern very similar to that of U.S. populations of S. invicta (Trager 1991; MacKay & MacKay 2002). We did not observe these bi-colored majors of S. xyloni in the samples from Nuevo Léon and Tamaulipas described herein, nor in the survey of Sánchez-Peña et al (2005). Trager (1991) reports that S. xyloni is very rare or absent along the Gulf of Mexico coast.
(2) Samples identified as S. geminata/S. xyloni hybrids by morphology. Some native ant samples had large workers that match the description of Trager (1991) for the S. geminata/S. xyloni hybrids: larger workers with bilobed head having posteriorly divergent sides; head width ≥1.50 mm (larger than that of the parental queens, unlike S. xyloni major workers, but smaller than that of S. geminata soldiers), and poorly developed propodeal carinae; they are yellowish-red, in a rather uniform color pattern, with a darkened gaster tip. Alate queens from these colonies belonged to the S. xyloni subcomplex sensu Trager (1991) (Fig. 3). The color of these putative hybrid workers is similar to, but usually clearly paler than, the orange color of sympatric S. geminata. The following comparisons can be made across the hybrid and the parental species S. geminata and S. xyloni; they might be useful for hybrid identification if the following castes are available. As described in Trager (1991), the head of the majors of these putative hybrids workers' is clearly wider that that of S. geminata queens; these were identified morphologically and they have the S. geminata COI marker as well (see below). S. xyloni major's heads are not wider than those of S. geminata queens (Trager 1991).
(3) We also found the S. xyloni marker in native fire ant samples lacking major workers and sexuals; these samples resembled the hybrid samples in coloration; and they were collected within meters of hybrids. As mentioned, fire ant taxonomy is based on the major workers (Trager 1991; Jacobson et al. 2006); to our knowledge, there are no descriptions of non-major workers of the S. geminata/S. xyloni hybrid and morphological identification is limited at this point.
The sequence of sample 18 from Monterrey (identified as a S. geminata/S. xyloni hybrid by morphology) was 96% identical to the S. xyloni COI sequence (Genbank accesion DQ153015) from New Mexico (Jacobson et al. 2006). Both sequences had an identical restriction pattern as indicated by virtual and actual Hinf I digestion of our samples. Bands generated are 18, 108, 138, 327 and 347 kb, resulting in a 2-band pattern on gel (347/327 kb) and (138/108 kb) (Jacobson et al. 2006; Fig. 3). Our S. xyloni sequence (sample 18) had a maximum of 88% identity with our S. geminata marker (see next section), and with Genbank COI accessions of U.S. (Florida) S. geminata (Ross et al. 2003) and South American accessions of the Solenopsis saevissima species group.
A third band pattern (Fig. 3) was obtained from samples morphologically identified as typical reddish S. geminata (Trager 1991). These were sample 1 from Torreon, sample 4 from Matamoros and sample 27 from Cadereyta (Table 1). This pattern was obtained also from samples lacking the typical S. geminata soldiers (likely due to limited sampling) from Monterrey (sample 19) and Matamoros (sample 8). The PCR product sequence from S. geminata (sample 4) from Matamoros was 938 bp long. The 15 S. geminata CO1 accessions in Genbank originate from 2 Northern Florida populations (Alachua and Leon Counties), about 1000 km NE from Matamores (Ross et al. 2003); these accessions are 795 bp because they were generated with primers different from the ones we used, and they are nested within our longer sequence. In the overlapping regions, our S. geminata sample had 93% sequence homology with GenBank accession AY254488, the most similar. Our S. geminata sequence shares a Hinf I restriction site on position 545 with 4 out of the aforementioned 15 S. geminata Genbank accessions (haplotypes 1, 12, 14, and 15). All 15 Genbank accessions have a Hinf I site on position 436; our S. geminata samples (1, 4, 6, 19 and 27) lack this site as shown by analogous digestion (Fig. 2). Eleven Genbank accessions have only 1 site at position 436. Haplotype 1 is restricted to a polygyne form and haplotypes 12, 14 and 15 are variants from monogyne colonies (Ross et al. 2003). We do not know if our samples come from polygyne or monogyne S. geminata. Our Sample 4 of S. geminata and the Genbank accessions from Ross et al. (2003) of this species had 88% identity with our S. xyloni (sample 18). The sequences obtained from all these Mexican populations of fire ant species will be accessed in GenBank.
The molecular characterizations described in this work have implications for regional fire ant identification. Considering the wide geographical distribution of this S. invicta marker (i.e., New Mexico, Jacobson et al. 2007); Southeastern USA (Shoemaker et al. 2006); and this report) it is probably useful for general S. invicta identification. Jacobson et al. (2006) emphasized the difficulties in accurately discriminating between S. invicta and S. xyloni from New Mexico. They suggested that several morphological characteristics should be used to distinguish S. xyloni from S. invicta, a task to be performed by “taxonomists familiar with Solenopsis”. Although somewhat time-consuming and expensive, the molecular method described herein makes use of methodologies from standard assays in Drosophila genetics that might be available to a considerable number of researchers. Molecular methods constitute a significant tool for species identification when certainty is critical (Jacobson et al. 2006).
Regarding S. geminata, our samples spanned more than 600 km; therefore, this marker can probably be used for identification of local samples. However, considering our data and the Genbank sequences from Florida, S. geminata appears to be a variable species regarding this marker.
In conclusion, the modification described to the assay of Jacobson et al. (2006) can be a useful tool for identification of S. invicta, S. geminate, and the S. xyloni subcomplex from northeastern Mexico. The mtDNA polymorphism in S. geminata and particularly the widespread presence of the S. geminata/S. xyloni hybrid in this area (Helms Cahan & Vinson 2003) adds a facet to fire ant identification. This assay might be particularly useful for accurate hybrid identification, because yellow-orange major hybrid workers with conspicuously enlarged heads and black mandibles (Fig. 3) could rather easily be misidentified as S. geminata. It must be considered, however, that widely distributed fire ant morphospecies might comprise cryptic species or species complexes with diagnostic genetic regional differences reflected in their mtDNA haplotype. Ross et al. (2007) show evidence of this in S. invicta; S. geminata could be a similar case considering the mtDNA polymorphism detected. Thus, although the reported bioassay (and that of Jacobson et al. 2006) are probably useful for the geographical areas they analyzed, we cannot be sure that they are universally useful in all areas where the nominal species might co-occur without further documentation of the mtDNA diversity in these species.
Our morphological and molecular identifications of native fire ants are consistent with the report of Helms Cahan & Vinson (2003) regarding native fire ant populations from central and extreme south Texas. Using allozyme and microsatellite markers, they reported that native fire ant workers collected in these regions were S. geminata and S. geminata/S. xyloni hybrids only. Solenopsis xyloni is very rare or absent along the Gulf of Mexico coast in the USA (Trager 1991); the Matamoros site herein is about 10 km from the Gulf coast, and very close to the Texas, USA border.
According to Helms Cahan & Vinson (2003), the S. geminata/S. xyloni hybrid workers from these populations are all F1 and heterozygotic; however they possess the S. xyloni mitochondrial genome almost exclusively, because this hybrid is unidirectional and the result of a S. geminata male/S. xyloni female cross. Helms Cahan & Vinson (2003) found that in Texas the frequency of hybrid colonies relative to that of S. geminata increased in a north-south direction, from 51% in Central Texas (Austin, Travis county) up to 82% at the Mexican border (Weslaco, Hidalgo county); this last location is about 50 km west (inland) from the Matamoros site reported herein. Likewise, most (89%, n = 9) of our hybrid-looking samples (most lacking major workers) had the xyloni marker. See Helms Cahan & Vinson (2003) for the description of social hybridogenesis in these Solenopsis populations.
Accurate identification of S. invicta will support management strategies to mitigate the environmental, urban and economic impact of this invasive ant species in Mexico. Also, regarding native fire ant populations, a more complete knowledge of their genetic markers will contribute to our understanding of local biodiversity. Our analysis of the COI marker appears to confirm the very restricted gene flow previously reported between S. geminata and S. xyloni sexuals, thus supporting their validity as “good” biological species. These Solenopsis hybrids are still abundant and geographically widespread (Hung & Vinson 1977; Trager 1991; Helms Cahan & Vinson 2003). However, native Solenopsis species and their hybrids have been greatly reduced or eliminated from large areas (such as the southeastern USA) after the S. invicta invasion in the 60s and 70s (Trager 1991; Wojcik et al. 2001; Helms Cahan & Vinson 2003). The clarification of the identity of invasive and native fire ant species and of their hybrids will contribute towards our understanding of their ecology and their interactions, like displacement of native ants by the S. invicta.
We thank Carlos Blanco (USDA-ARS, Stoneville), Edward LeBrun (University of Texas at Austin), Natalie; Hummel (Louisiana State University) and Omaththage Perera (USDA-ARS, Stoneville) and 2 anonymous reviewers for helpful editorial comments. Collecting was partially supported by L. Gilbert (University of Texas at Austin). We thank the support from both Universidad Autonoma Agraria Antonio Narro (Directión de Investigación, UAA-012-32) and Universidad Autónoma de Nuevo León.