Evolution, 44(8), 1990, pp, 2113-2134

SYSTEMATICS AND POPULATION GENETICS OF FIRE (SOLENOPSIS SAEVISSIMA COMPLEX) FROM ARGENTINA

KENNETH G. Ross' AND JAMES C. TRAGER2 'Department ofEntomology, University ofGeorgia, Athens, GA 30602 USA -Depanmeni ofBiology, University ofMissouri-St. Louis, St. Louis, MO 63121 USA

Abstract. - Specimens ofseven fire collected from their native ranges in Argentina were studied by protein electrophoresis and morphological analysis. Concordance between the genetic and morphological character sets is strong (96% agreement on identifications), suggesting that recognition ofreproductively isolated populations and partitioning ofintra- and interspecific vari­ ation can in most cases be achieved using appropriate characters ofeithertype in this taxonomically difficult group. Genetic differentiation between native (Argentina) and introduced(USA) conspecific populations oftwo species, Solenopsis invicta and S. richteri, is rather typical ofthe differentiation existing between conspecific populations found within either country. Furthermore, there appears to have been little reduction of variability (heterozygosity) at enzyme loci following colonization by either species ofthe , although some rare alleles have been lost in the introduced populations. Hybridization is rare between S. invicta and S. richteri where their native ranges overlap in central Argentina, in contrast to the extensive hybridization of these species in the UnitedStates, suggesting that prezygotic barriers to gene flow have been compromised in introduced populations. Phylogenetic analysis ofthe seven species indicates that S. invicta and S. richteri are relatively distantly related within the S. saevissima complex. Given that hybrids between these species in the United States suffer little apparent loss offitness, genomic incompatibilities generally may be insufficient to create effective postzygotic barriers to interspecific gene flow in this group ofants.

Received August 29, 1989. Accepted April 18, 1990.

The ants (family Formicidae) represent a 1989). Resolution of the taxonomic diffi­ remarkably successful lineage of ecologi­ culties created by such patterns of clado­ cally diverse, highly eusocial species, the genesis may in many instances require the systematics of which has suffered relative use of new character sets (e.g., molecular neglect given the ecological and economic markers) to define genetically isolatedgroups importance ofthe group. This neglect stems and to assess the distribution ofgenetic and in large part from considerable taxonomic morphological variability within and be­ difficulties at the species level. For instance, tween species. Because ant species com­ intraspecific geographic variability often prising taxonomically difficult groups are transcends interspecific variability in ants likely to be evolutionarily young, compar­ (e.g., Ward, 1984, 1989), leading to diffi­ ative population genetic and systematic culties in deciding the taxonomic status of studies of such groups may be particularly closely related populations when traditional informative with respect to the processes characters are studied in a limited number leading to reproductive isolation. of specimens. Furthermore, morphologi­ One group of ants in which the species­ cally cryptic species, that is, reproductively level has posed continuing prob­ isolated populations in which morphologi­ lems is the Solenopsis saevissima species cal divergence is not evident, are sufficiently complex (subfamily Myrmicinae), mem­ common that most routine electrophoretic bers of which are commonly known as fire surveys have revealed previously unde­ ants. This complex, which includes rela­ scribed species (Crozier, 1977a, 1981; Ward, tively large-bodied forms with well devel­ 1980; Halliday, 1981; Crozier et al., 1986; oped worker , is distributed Heinze, 1989; see also Ross et al., 1987a). naturally over a large part of the South These findings have been taken to suggest American continent east ofthe Andes. The that many groups of ants are in phases of taxonomy ofthe group has a history ofcon­ active radiation and that the process ofspe­ fusion and disagreement, with the number ciation may often be decoupled from sig­ offormally recognized taxa (species or sub­ nificant morphological evolution in these species) ranging from two to 17 (Buren, (see Crozier, 1977b; also Larson, 1972). A recent resurgence ofinterest in the 2113 2114 K. G. ROSS AND J. C. TRAGER

FIG. 1. Distribution ofintroduced fire ants (Solenopsis saevissima complex) in the United States (modified from Diffieet al., 1988). Dotted line indicates approximate northernandwestern limits ofexpansion ofintroduced populations. Locations of three study populations of Ross et al. (1987a) resampled for this study are also indicated (Mor, Tur, Ala). systematics of these ants has come about (in an order characterized by male haploi­ for several reasons. First, fire ants intro­ dy), and loss of premating isolating mech­ duced to the United States have emerged as anisms associated with interspecific hybrid­ significant pests (Lofgren et aI., 1975). Sec­ ization (Brian, 1983 p. 259; Tschinkel and ond, the introduced species provide one of Nierenberg, 1983; Ross and Fletcher, 1985a; the few well-documented examples of an Ross et aI., 1987b). hybrid zone forming in historical Fire ants of the S. saevissima complex times (Vander Meer et aI., 1985; Ross et aI., were introduced to southern Alabama, in 1987b). Third, fire ants have become im­ the early part ofthis century and have since portant model systems for studying the evo­ become widespread throughout the south­ lution of social organization, the origin of eastern and south-central parts ofthe Unit­ reproductive isolation, and the relationship ed States (Fig. 1). Introduced populations, between these two facets ofevolution in ad­ originally assigned to the single variable vanced eusocial (e.g., Wilson species Solenopsis saevissima (Creighton, and Brown, 1958; Crozier, 1979; Ross et 1930; Ettershank, 1966), have been consid­ aI., 1987a, 1988; Ward, 1989). Continued ered since the revision of Buren (1972) to progress ofresearch on all fronts will depend comprise two species, S. invicta and S. rich­ on an improved understanding ofthe taxo­ teri. The work ofBuren (Buren, 1972; Buren nomic status and phylogenetic affinities of et aI., 1974), based on morphological anal­ introduced and native populations, yses of specimens from the United States as can be obtained through an integrated and central , suggested that systematics/population genetics approach. S. invicta and S. richteri are phenotypically Furthermore, comparative population ge­ distinctive and internally quite uniform. netic data can shed light on the extent to Buren (1972) further concluded that hy­ which population bottlenecks have influ­ bridization between the two species is rare enced genetic structure in introduced pop­ or absent, despite the abundant opportunity ulations, an important consideration given for gene exchange between introduced pop­ that founder effects have been invoked to ulations in the United States. explain the origin ofsuch intriguing features A recent challenge to Buren's view has ofintroduced fire ants as (multiple come from the data of Vander Meer et aI. queens per colony), frequent male diploidy (1985). These authors, using gas chromato- FIRE ANT SYSTEMATICS AND POPULATION GENETICS 2115 graphic analyses of diagnostic micromo1e­ in the United States; 5) to obtain evidence cu1ar characters (hydrocarbons and venom bearing on the possibility of gene flow be­ ), provided evidence that hybrid­ tween fire ant species in their native ranges, ization commonly occurs between S. invicta particularly with reference to S. invicta and and S. richteri in east-central Mississippi. S. richteri; and 6) to infer phylogenetic re­ This has since been confirmed using elec­ lationships among the species. A related trophoretic markers (Ross et al., 1987b), and study in which morphological characters the zone ofhybridization between these in­ provide the basis ofa formal revision ofthe troduced forms is now known to encompass S. saevissima complex and allied taxa is pre­ a large area extending from eastern Missis­ sented elsewhere (Trager, 1990). sippi to northwestern Georgia (Diffie et al., 1988; see Fig. 1). Genetic analyses of the MATERIALS AND METHODS zone at its eastern and western extremes have revealed that genotype distributions at Sample Collections informative loci do not depart substantially Samples were collected from 201 nests of from distributions expected under Hardy­ Solenopsis distributed over 48 sites in Weinberg equilibrium and that a diverse ar­ northern Argentina (Buenos Aires, Santa Fe, ray ofrecombinant multilocus genotypes is Corrientes, Chaco, Formosa, Santiago del present (Ross et al., 1987b; Ross and Rob­ Estero, and Cordoba Provinces; see Fig. 2). ertson, 1990). These findings are consistent Wherever possible a series of nests (up to with panmixia in introgressed populations eight) ofthe same species was sampled at a and suggest that there is no large-scale single site (preliminary morphological iden­ breakdown in hybrid viability or . tification was made in the field). Several These results from hybrid populations of winged female sexuals and workers were introduced S. invicta and S. richteri again taken from each nest, the former for elec­ raise questions concerning our concepts of trophoresis and the latter for subsequent de­ species in fire ants and highlight our igno­ tailed morphological analysis. Collected rance ofevolutionary and genetic relation­ specimens were placed immediately in a dry ships among morphologically recognizable cryogenic refrigerator for transport to the forms comprising the S. saevissima com­ Athens laboratory, where they were trans­ plex. However, because interactions be­ ferred to an ultra-low-temperature freezer tween introduced fire ant populations may (- 60°C) for storage. have been influenced by their colonization Collection sites were situated so that geo­ ofnovel or by the genetic effects of graphic variability in S. invicta (and to a recent population bottlenecks, systematic lesser extent in S. richteri and S. quinque­ uncertainties surrounding this group ofants cuspis) could be studied (Fig. 2). Samples of cannot be resolved by exclusive study of other species were collected opportunisti­ populations in the United States. Thus, the cally. Because introduced S. invicta and S. comparative genetic study of native popu­ richteri hybridize in North America, a spe­ lations reported here was undertaken to ac­ cial effort was made to locate any areas in complish the following objectives: 1) to de­ South America where the ranges of these termine the extent of congruence between two species overlap. Such an area was found species assignments (concepts) based on in southern Santa Fe Province, Argentina morphology and those based on genetic (near Rosario), so collecting activities were markers for fire ants in their native South concentrated in this area. A total of 100 S. American ranges; 2) to identify the extent invicta, 57 S. richteri, 26 S. quinquecuspis, of geographically based genetic variability 6 S. interrupta, and a single S. macdonaghi within these fire ant species; 3) to link ge­ colony were sampled. A single colony ofS. netically the native populations ofS. invicta electra (a member ofthe S. saevissima com­ and S. richteri with fire ant populations es­ plex but placed in a different subcomplex tablished in the United States; 4) to com­ than the above species-Trager, 1990) was pare levels of genetic variability in native included to serve as a representative out­ populations ofS. invicta and S. richteri with group taxon to the other species. Among the levels in conspecific introduced populations remaining samples, either species identifi- 2116 K. G. ROSS AND J. C. TRAGER

• S. i nv i c t a *S. r i c h t e r i o s. q u i n q u e c u s p i s as. m a c d o n a g h i • S. i n t e rru p t a c S. eleelra

FIG. 2. Locations ofcollection sites of Solenopsis in Argentina. Not every collection site is figured in areas with a high density ofsites. Location of the city ofRosario is indicated by '@: colonies) or these samples likely represent and a key for identification of the species previously undescribed species (six colo­ can be found in Trager (1990). nies) (see below). Genetic Analyses Morphological Analyses Electrophoresis was conducted on 14% Initial identification of specimens made starch gels using procedures outlined in Ross in the field was later confirmed in the lab­ et al. (1987 a). The products of 26 pre­ oratory by examining morphological fea­ sumptive loci were studied, these being tures ofthe major workers under a dissect­ identical to the markers studied in Ross et ing microscope at 25 x with bright al. (1987a) with the following exceptions: incandescent illumination. Morphological 1) Aid was omitted because of poor reso­ determinations were made without knowl­ lution ofbands; 2) a new polymorphic locus edge of the results of the genetic analyses. (Pro-O) visualized with a general protein Useful characters for identification include stainwas added; 3) previously unrecognized color patterns, head shape and proportions, variability was discerned for the product of and shape and surface sculpture ofthe post­ Pgm by altering running conditions. Be­ petiole. Representative voucher specimens cause variability at the latter two markers of the material examined are deposited in was not characterized previously for S. in­ the Museum of Comparative Zoology at victa from the United States, three of the Harvard University. Detailed descriptions study populations ofRoss et al. (1987a) from FIRE ANT SYSTEMATICS AND POPULATION GENETICS 2117

Georgia and Florida were resampled (30 nests each from the Mor, Tur, and Ala pop­ ulations; see Fig. I) and allele frequencies estimated for Pro-O and Pgm. Mendelian inheritance of the products of eight of the study loci (Agp-l, Est-Z, Est-s, Gpi, Ldh-l, Mpi, Odh, Pgm) has been demonstrated (Ross and Fletcher, 1985b; Ross et al., 1987b, 1988; K. Ross and D. Shoemaker, unpubl.). For analyses ofgenetic data only a single genotype per colony was used because of the high correlation (non-independence) of genotypes within nests previously deter­ mined for several fire ant populations in the United States (Ross and Fletcher, 1985b; Ross et al., 1988). Comparisons ofgenotype frequencies at sufficiently polymorphic loci Argentina with those expected under Hardy-Weinberg equilibrium were conducted for S. richteri and S. invicta (total data sets or broken down regionally ifsample size permitted) using x2 tests with Levene's (1949) correction for FIG. 3. Locations of pooled regional collections small sample sizes. For polymorphic loci ('populations') for analysis ofgeographic variability in with extreme allele frequencies, rare alleles S. invicta and S. richteri. Location ofthe city ofRosario were collapsed intoa single class and Emigh's is indicated by •®.' (1980) continuity-corrected x2 test was em­ ployed. The fixation indices FIs and FIT> for three U.S. populations distributed on a which assess the magnitude of departures similar spatial scale along a north-south from Hardy-Weinberg genotypic propor­ transect in Georgia and Florida (see above tions regionally and over the entire collec­ and Fig. 1). Sample sizes from Argentina tion, were estimated using the methods of were not sufficiently large to examine geo­ Weir and Cockerham (1984; see below) to graphic variability in species other than S. complement the x2 tests. invicta and S. richteri. To assess the extent of intraspecific geo­ Intraspecific genetic diversity (expected graphically based genetic variability in na­ heterozygosity, H exp ) was estimated from tive S. invicta and S. richteri, the eastern observed allele frequencies using equation collections from Argentina were pooled into 8.4 in Nei (1987) for S. invicta, S. richteri, five 'populations' for S. invicta, each con­ and S. quinquecuspis. Diversity was com­ sisting of 18 colonies, and two 'popula­ pared between species in Argentina, as well tions' for S. richteri, consisting of 33 and as between U.S. and Argentine populations 24 colonies (Fig. 3). The fixation index F ST of S. invicta and S. richteri, using paired­ (standardized allele frequency variance), sample t tests on the angular-transformed which measures the extent ofgenetic differ­ single-locus heterozygosity data (see Archie, entiation among populations, was estimat­ 1985). ed using the methods of Weir and Cocker­ Genetic distances (Nei's D-Nei, 1987 pp. ham (1984) for all alleles at polymorphic 222-227; see also Tomiuk and Graur, 1988) loci present at frequencies greater than 0.01 between the species and between conspecific (see Table 1). A single bias-corrected esti­ populations ofS. invicta and S. richteri were mator and its variance were obtained by estimated using a jackknife procedure im­ jackknifing over loci, with confidence limits plemented in the program of Sattler and (95%) generatedassuming the t distribution. Hilburn (1985). This procedure corrects for Values ofFST for Argentine S. invicta pop­ limited sample size and numberofloci stud­ ulations were compared to values obtained ied as well as unequal rates of evolution. 2118 K. G. ROSS AND J. C. TRAGER

TABLE 1. Allele frequencies at all loci exhibiting variability in S. invicta and S. richteri in Argentina and the United States (data for U.S. samples from Ross et al., 1987a). 'N is the number ofdiploid genotypes studied (one genotype per nest). Dashes indicate the allele has not been found in this species.

s.invicta s. richteri Argentina Argentina (all) (northern)" u.s. Argentina u.s. N~ 100 N= 36 N=421 N~ 57 N= 58 Agp-1 125 O.OlD 0.014 0 100 0.960 0.903 0.669 1.0 0.991 41 0.030 0.083 0.331 30 0 0.009 Est-2 116 0.035 0 0 0.008 0 110 0.025 0 0 0.868 0.612 100 0.890 1.0 1.0 0.009 0 84 0.Ql8 0 80 O.OlD 0 0 0.070 0.388 60 0.015 0 0 0.009 0 47 0.025 0 0 0.Ql8 0 Est-4 155 0.308 0.300 0.414 100 0.692 0.700 0.586 1.0 1.0 Dia 113 0 0 0.001 100 0.995 1.0 0.999 1.0 1.0 69 0.005 0 0 Gapdh 138 0.018 0 130 0 0 0.001 100 1.0 1.0 0.999 0.982 1.0 Gpi 128 0.005 0 0 113 0.005 0 0 107 0.005 0 0 0.991 1.0 100 0.975 1.0 1.0 0.009 0 79 O.OlD 0 0 Lap 113 0 0 0.006 100 1.0 1.0 0.994 1.0 1.0 Ldh-1 121 0.005 0.014 0 100 0.985 0.986 1.0 1.0 1.0 63 0.010 0 0 Ldh-2 150 0.009 0 100 1.0 1.0 1.0 0.991 1.0 Mdh-2 100 0.995 1.0 0.999 1.0 1.0 87 0.005 0 0.001 Mpi 100 1.0 1.0 0.999 1.0 1.0 95 0 0 0.001 Odh 110 0.005 0 0 0 0 100 0.980 0.986 1.0 0.321 0.008 86 0.679 0.992 FIRE ANT SYSTEMATICS AND POPULATION GENETICS 2119

TABLE 1. Continued.

s. invicta s. richter; Argentina Argentina (all) (northern)" U.S. Argentina U.S. N~ 100 N~ 36 N~421 N~ 57 N~ 58 Odh 69 0.005 0.014 0 52 O.OlD 0 0 Pep(gl) 121 0.009 0 100 1.0 1.0 1.0 0.982 1.0 79 0.009 0 Pgd-2 433 0.005 0 0 100 0.995 1.0 1.0 1.0 1.0 Pgm 105 0.111 0 0 0.009 0 100 0.702 0.861 0.922 0.991 1.0 95 0.141 0.111 0.078 85 0.046 0.028 0 Pro-O 100 0.995 0.986 0.989 1.0 1.0 94 0.005 0.014 0.011 Pro-1 107 0.005 0 0.001 100 0.995 1.0 0.999 1.0 1.0 Pro-3 111 0 0 0.005 100 1.0 1.0 0.995 0.991 0.983 92 0.009 0.017 • Based on samples from two northernmost populations only.

Phylogenetic Analyses PHYSYS distributed by J. S. Farris and M. Putative phylogenetic relationships F. Mickevich) was employed to generate the among the species were studied using three 50 best-fitting trees, that is, those trees for approaches to tree-building: 1) a phyloge­ which the derived matrix ofdistance values netic procedure for unrestricted fitting of most closely resembled the observed dis­ branch lengths to the matrix ofgenetic dis­ tance matrix (as assessed by the percent tances; 2) a parsimony-based phylogenetic standard deviation statistic). character analysis in which each locus was For the first phylogenetic character anal­ treated as a character, and allele frequency ysis, the modified Wagner method ofSwof­ arrays were regarded as continuous char­ ford and Berlocher (1987) for analyzing al­ acter states; and 3) a parsimony-based phy­ lele frequency data was used. This procedure logenetic character analysis in which each (implemented in the program FREQPARS locus was treated as a character, and allelic distributed by D. L. Swofford) is a parsi­ complements at the locus were encoded as mony method for finding the tree with the discrete character states. The undirected least amount of allele frequency change trees produced using all of these analyses (minimum total branch length) given the were rooted by specifying S. electra as the constraint that allele frequencies at each outgroup. locus sum to one in hypothetical ancestors. The modified distance Wagner procedure Allele frequencies at 10 informative loci of Farris (1981), which does not assume (weighted means over all conspecific pop­ constant evolutionary rates, was chosen for ulations in Argentina) constituted the input analysis of the genetic-distance data. This data set (Table 2). procedure (implemented in the program For the second character analysis, the TABLE 2. Input data set (allele frequencies) for phylogenetic characteranalysis ofArgentine fire ants (Solenopsis) using the modified Wagner method ofSwofford and Berlocher (1987).

S. richter; S. quinquecuspis S. invicta S. macdonaghi S. interrupta 'Species x' S. electra Agp-I 150 0 0 0 0 0 I 0 125 0 0 0.010 0 0 0 0 100 I I 0.960 I I 0 1 41 0 0 0.030 0 0 0 0 Agp-2 125 0 0 0 I I 0 0 100 I I I 0 0 I I Dia 106 0 0 0 0 0 0 I 100 I I 0.995 I I 0 69 0 0 0.005 0 0 0 0 Est-2 121 0 0 0 0 I 0 0 116 0.008 0 0.035 0.500 0 0 0 110 0.868 I 0.025 0 0 0 0 100 0.009 0 0.890 0.500 0 I 0 84 0.018 0 0 0 0 0 0 80 0.070 0 0.010 0 0 0 0 60 0.009 0 0.015 0 0 0 0 47 0.Ql8 0 0.025 0 0 0 0 16 0 0 0 0 0 0 I Est-4 155 0 0 0.308 0 I 0 0 100 I I 0.692 I 0 I 0 56 0 0 0 0 0 0 I Gpi 128 0 0 0.005 0 0 0 0 113 0 0 0.005 0 0 0 0 107 0.991 0 0.005 0 0 0 0 100 0.009 0.981 0.975 I I I I 79 0 0 0.010 0 0 0 0 10 0 0.019 0 0 0 0 0 Gr 109 0 0 0 0 0 0.100 0 103 0 0 0 0 0 0.900 0 100 I I I I 0 0 0 94 0 0 0 0 I 0 0 88 0 0 0 0 0 0 I Odh 110 0 0 0.005 0 0 0 0 100 0.321 I 0.980 0 I I 0 86 0.679 0 0 0 0 0 0 81 0 0 0 I 0 0 0 69 0 0 0.005 0 0 0 0 52 0 0 0.010 0 0 0 0 null 0 0 0 0 0 0 I Pgd-2 433 0 0 0.005 0 0 0 0 133 0 0.019 0 0 0 0 0 100 I 0.981 0.995 I I I I Pgm 105 0.009 0 0.111 0 0 0 I 100 0.991 I 0.702 I 0.750 I 0 95 0 0 0.141 0 0 0 0 87 0 0 0 0 0.250 0 0 85 0 0 0.046 0 0 0 0 FIRE ANT SYSTEMATICS AND POPULATION GENETICS 2121

TABLE 3. Input data set (character state values) for phylogenetic character analysis of Argentine fire ants (Solenopsis) in which allelic combinations were encoded as discrete states. Characters for which two equally parsimonious character state orderings exist were re-coded as two characters with the subscripts 'a' and 'b'. The weight given to each character appears in parentheses beside it.

s. S. richter; quinquecuspis S. invicta S. macdonaghi S. interrupta 'Species x' S. electra Agp-2 (100) 0 0 0 2 2 0 0 Est-2. (25) 0 I 7 8 3 7 5

Est-2b (25) 0 I 7 8 5 7 3 Est-4. (35) I I 0 I 3 1 5 es-», (35) 1 I 0 I 5 1 3 Gr (95) 0 0 0 0 1 3 2 Odh; (30) 0 I 1 3 1 1 5 oan; (30) 0 I I 5 I 1 3 Pgm (60) 4 4 2 4 5 4 0 combinations ofalleles observed at a locus at a locus were ordered according to the in each of the taxa were encoded so as to minimum allele turnover model of Mick­ define discrete character states (Mickevich evich and Mitter (1983). This ordering and Mitter, 1983; Buth, 1984). This dis­ scheme minimizes both gains and losses of cretization of the data was done without alleles in a single-locus transformation se­ regard to the frequencies of constituent al­ ries. In cases where two character state or­ leles, except that alleles present at frequen­ derings were equally parsimonious, the lo­ cies less than 0.10 (weighted mean over all cus was recoded as two characters receiving con specific populations in Argentina) were halfthe weight ofthe original character (re­ excluded. Character states were coded ad­ sulting in nine characters in the input data ditively (with the exception of Gr), the dif­ set; see Table 3). Loci were weighted orig­ ferences in integer values between two states inally in inverse proportion to the observed reflecting the necessary numberofallelic gain extent ofintraspecific polymorphism (Kluge andloss steps separating them when all states and Farris, 1969). Most parsimonious trees

TABLE 4. Frequencies of diagnostic and informative alleles for identification of seven species of Solenopsis from Argentina.

s. S. richter; quinquecuspis S. invicta S. macdonaghi S. interrupta 'Species x' S. electra Agp_1150 0 0 0 0 0 1.0 0 DialO6 0 0 0 0 0 0 1.0 Est-186 0 0 0 0 0 0 1.0 Est-2121 0 0 0 0 1.0 0 0 Est-2l lO 0.868 1.0 0.025 0 0 0 0 Est-216 0 0 0 0 0 0 1.0 Est_4155 0 0 0.308 0 1.0 0 0 Est-4lO0 1.0 1.0 0.692 1.0 0 1.0 0 Est-456 0 0 0 0 0 0 1.0 Gpi lO7 0.991 0 0.005 0 0 0 0 GpilOO 0.009 0.981 0.975 1.0 1.0 1.0 1.0 GrlO9 0 0 0 0 0 0.100 0 GrlO3 0 0 0 0 0 0.900 0 Gr94 0 0 0 0 1.0 0 0 G,s8 0 0 0 0 0 0 1.0 Mpi93 0 0 0 0 0 0 1.0 Odh81 0 0 0 1.0 0 0 0 Odhnull 0 0 0 0 0 0 1.0 Pgml O5 0 0 0.111 0 0 0 1.0 pgm95 0 0 0.141 0 0 0 0 pgm85 0 0 0.046 0 0 0 0 2122 K. G. ROSS AND J. C. TRAGER

TABLE 5. Values ofFST (±SE) for populations ofS. invicta from Argentina (based on five or three populations; see text) and the United States, and for S. richteri from Argentina.

Argentine Argentine u.s. Argentine S. invicta (5) S. invicta (3) S. invicta S. richteri 0.035 ± 0.031 0.036 ± 0.026 0.079 ± 0.030 0.004 ± O.Q1S constructed using a branch and bound al­ ognized as being distinct on the basis oftheir gorithm implemented in the program morphology. The latter most likely repre­ HENNIG86 (distributed by J. S. Farris). A sent colonies of heretofore unrecognized single tree was generated from the resulting species. One of these unique colonies in­ multiple trees ofminimal length 1) by strict cluded only females homozygous for the (Nelson) consensus criteria and 2) by suc­ Peptpapl-Z'" allele, in contrast to all other cessive approximations character weighting ants from this study which were homozy­ (e.g., Carpenter, 1988). gotes for thePepipapl-Z'?" allele. More com­ pelling as an example ofa new cryptic spe­ REsULTS cies are the five colonies monomorphic for Species Identifications the Agp_1150 allele and possessing the Gr J0 9 A high degree of concordance exists be­ and GrJ03 alleles, all ofwhich are unique to tween the assignment ofsamples to species these colonies (see Table 4; 'species x'). on the basis ofmorphological characters and These five colonies, collected in northern their identification from genetic markers, Buenos Aires and southern Santa Fe Prov­ with 96% agreement between the methods inces at sites where S. richteri and S. quin­ over all of the samples. Each of the mor­ quecuspis were also collected, were identi­ phologically recognizable species, with the fied initially as hybrids between these two exception of S. invicta, is characterized by species on the basis oftheir morphology. monomorphism for unique alleles at one or more loci, or by possession ofunique com­ Intraspecific Genetic Variability and binations of alleles over two or more loci Relationship ofNative to (Table 4). For instance, S. richteri is unique Introduced Populations in being effectively monomorphic for the Comparisons of genotype distributions Gpi107 allele, S. macdonaghi is defined by observed with those expected under Hardy­ possession ofthe Odh81 allele, S. interrupta Weinberg equilibrium were conducted for is fixed for the unique Est-2121 and Gr 94 two loci in S. richteri and for 11 locus/pop­ alleles, and S. electra is distinguished by ulation combinations in S. invicta. For S. J0 6 93 possession ofthe Dia , Mpi , and Odhv" richteri, observed genotypes at both loci oc­ alleles (among others). Solenopsis quinque­ cur at frequencies virtually identical to those cuspis is defined by joint possession (in ef­ expected (both P > 0.25), and values ofFIT fectively monomorphic condition) of the and F IS do not differ significantly from zero. Est-2JJ O and Gpi JOO alleles. Solenopsis invic­ For S. invicta, homozygotes generally occur ta is characterized by the absence of these in excess ofthe frequency expected at both alleles or allelic combinations diagnostic of the species and regional levels, with hetero­ the other species, and by possession ofchar­ zygote deficiencies significant in two of 11 acteristic polymorphisms at Est-4 and Pgm (18%) of the cases (both P < 0.025), both (Table 4). of these at the species level. On the other Discordance between morphological hand, the 95% confidence limits for FITand identifications and those based on the ge­ FIsfor S. invicta encompass zero, suggesting netic data involve one colony each of S. that overall departures from Hardy-Wein­ invicta and S. interrupta that can be iden­ berg ratios are unlikely to be ofimportance tified readily from their genotypes but were at either level. judged as ambiguous in terms of morphol­ Geographically based intraspecific genet­ ogy, and six colonies that possess novel al­ ic variability in Argentine S. invicta was leles at one or two loci but were not rec- studied by examining allele frequency dis- FIRE ANT SYSTEMATICS AND POPULATION GENETICS 2123

TABLE 6. Pairwise genetic distance values (Nei's D) USA* e.sa1.001 for S. invicta populations in Argentina and the United States. 0.ll6

0.94 Argentina u.s. Pop. 4 Pop. 3 Pop. 2 Pop. I Pops. 0.• 2 -.,..----.---,.---.---.,----I i , , , Pop. 1 Pop. 2 Pop. 3 Pop . .( Pop. 5 POp. 5 0 0.0012 0.0028 0.0023 0.0016

32 Pop. 4 0.0007 0.0031 0.0014 0.0038 Agp_1 41 USA* POp. 3 0 0 0.0065 0.24 Pop. 2 0. 1 0.0009 0.0087 0.16 Pop. I 0.0077

0.08 0.00 L;===:===::=~:=-_..,..- __ Pop. 1 Pop. 2 Pop. 3 Pop. 4 Pop. 5 was done to facilitate comparison of geo­ graphic structure in native and introduced 100 pgm USA* S. invicta, as the U.S. samples were col­ O•• 0.8 j lected from relatively discrete localities sep­

0.7 arated by 240-290 km (see Ross et al.,

0.8 1987a). The value ofFST for S. richteri from Argentina, based on only two populations, 0.5 -.,..----.---.,----.---.,----, Pop. 5 is very close to zero. The modest geographically based vari­

0.24 ability characterizing Argentine S. invicta appears, in the case ofseveral alleles, to be 0.16 pgm105 clinally distributed (Fig. 4), with the more 0.08 USA northerly samples most closely resembling 0.00 introduced conspecific populations in the Pop. 1 Pop, 2 Pop. 3 Pop. " Pop. 5 * United States. This resemblance is due to relatively greater similarities between northern Argentina and U.S. populations in USA* 0.•'.OJ allele frequencies at several loci (see Fig. 4, Tables 1, 6), but is especially significant for 0.8 the Agp_141 allele. This is a common allele throughout the range of S. invicta in the 0.7 '---.----r--.,..----r--,.---, Pop. 5 United States (p = 0.331), but in our Ar­ FIG. 4. Frequencies of five informative alleles for gentine samples it is present only in ants five S. invicta populations located along a north-south collected from Formosa Province (Popula­ transect extending from southern Santa Fe Province, tion 5), where it occurs at a frequency of Argentina, to the Paraguayan border (see Fig. 3). Mean frequencies ofthese alleles in U.S. populations are also 0.167. Among our samples, specimens ofS. indicated (asterisks). invicta from northern Argentina also are most similar to introduced S. invicta in the United States in terms ofcolor patterns and tributions and estimating F ST for the five head shape (see also Trager, 1990). groups of colonies ('populations') distrib­ Despite some geographic heterogeneity, uted on a north-south transect along the Rio S. invicta and S. richteri from Argentina Parana and Rio Paraguay from southern exhibit high overall genetic similarity to Santa Fe Province to the Paraguayan border conspecific populations introduced to the (Fig. 3). Values of F ST are quite low and United States, in comparison with their statistically indistinguishable from zero similarity to their closest heterospecific rel­ (Table 5). No difference in F ST is evident atives. For both species genetic distances between the Argentine and U.S. popula­ between native and introduced populations tions ofS. invicta, regardless ofwhether all fall close to or within the range ofdistances five Argentine populations are included in estimated between S. invicta populations the analysis or only populations 1, 3, and 5 within Argentina or within the United States are included (Table 5). The latter analysis (Fig. 5). In S. invicta, all alleles present at a 2124 K. G. ROSS AND J. C. TRAGER

6.0 TABLE 7. Heterozygosity (Hexp, gene diversity) in native and introduced populations ofSolenopsis (data 5.0 for introduced populations and S. geminata from Ross ;:::- et al., 1987a). 'N' is the number ofdiploid genotypes + 4.0 studied (one genotype per nest). '"0 ... Native Introduced )( 3.0 populations populations 9- S. richteri 0.032 0.021 2.0 .: (N = 57) (N = 58) S. invicta (all) 0.052 0.044 1.0 (N = 100) (N = 421) S. invicta (northern)* 0.036 (N = 36) Argentina USA Argentina S. quinquecuspis 0.003 vs, (N= 26) USA inter- S. geminata 0.048 ---~----specific intraspecific (N= 30) FIG. 5. Intra- and interspecific genetic distances (In • Based on samples from two northernmost populations only. [D x 103 + 1]) for the S. saevissima complex. In the first column are intraspecific distances for S. invicta (dots) and S. richteri (square) in Argentina. In the sec­ tween introduced and native populations ond column are intraspecific distances for three S. in­ statistically significant. Values for S. invicta victa populations in the United States. In the third column are distances between native Argentine and are even more similar when only the north­ introduced U.S. populations ofS. invicta (dot) and S. ernmost Argentine samples are compared richteri (square). Interspecific distances for pairs ofS. with the U.S. populations. Among the Ar­ saevissima complex species in Argentina (excluding S. gentine species, S. quinquecuspis exhibits the electra) are shown in the fourth column. lowest and S. invicta the highest heterozy­ gosity (the difference is significant at P = within Argentina or within the United States 0.014). Solenopsis quinquecuspis is unique (Fig. 5). In S. invicta, all alleles present at a among all fire ants studied to date for which frequency greater than 0.05 in the two reasonable sample sizes have been obtained northernmost Argentina populations are in that it is effectively monomorphic (0.95 present also in U.S. populations (see Table criterion) at every locus surveyed. I). Furthermore, allele frequencies in these The rather insignificant effect of coloni­ northern populations at the two robustly zation on overall loss ofallozyme variabil­ polymorphic loci Agp-I and Est-4 are sim­ ity in S. invicta and S. richteri is further ilar to frequencies found in several U.S. reflected in the similar distributions for populations (Ross et al., 1987a). In S. rich­ numbers of effective alleles (ne) at the 26 teri, the two most common alleles (out of marker loci in native and introduced pop­ seven total) at the locus Est-2 in Argentine ulations (Fig. 6). The great preponderance samples are the only alleles present in the ofloci in all ofthese populations has fewer U.S. population (Table I). At the locus Odh, than 1.05 effective alleles. the only other robustly polymorphic locus While genetic variability as assessed by in this species (i.e., frequency of the most measures of allelic evenness (Hexp , ne) ap­ common allele <0.95), two alleles are pres­ pears little affected by colonization in the ent in Argentine populations but the most two species, consideration ofthe total num­ common one (p = 0.679) is virtually fixed bers ofalleles and distributions oftheir pop­ in the U.S. population (p = 0.992). ulation frequencies (Fig. 7) suggests thatrare and S. quinquecuspis in Table 7, along with alleles present in the native ranges were lost an estimate ofthis parameter for a Florida during colonization, leading to reduced al­ population ofS. geminata (see Ross et al., lelic richness in the introduced populations. 1987a), a fire ant species thought to be na­ For S. invicta in Argentina, over 40% ofthe tive to the United States. Heterozygosity is observed alleles are present at a frequency little diminished in introduced S. invicta and of 0.05 or less, but only half of this per­ S. richteri relative to native conspecific pop­ centage ofrare alleles is found in the United ulations, with neither ofthe differences be- States. The distribution ofallele frequencies FIRE ANT SYSTEMATICS AND POPULATION GENETICS 2125

S. invicta

25

20 Argentina N =1 00 15

10

5

S. richteri

25 25 Northern Samples '(3 Argentina 20 (Argentina) U 20 0 0 -I N =57 15 N =36 -I 15 -0 10 -0 10 ... 5 ... 5 CI) CI) .c .c E E j j Z Z

25 25 USA 20 20 N =58 15 15

10 10

5 5

1.05 1.25 1.05 1.25 1.15 >1.3 1.15 >1.3

Number of Effective Alleles FIG.6. Number ofeffective alleles at 26 marker loci for S. invicta and S. richteri in Argentina and the United States (data for U.S. samples from Ross et aI., 1987a). 'N' is the number of diploid genotypes studied (one genotype per nest). Labels on the abscissa indicate interval midpoints, except for the final column, which includes all values greater than 1.3.

in the two northernmost Argentine samples introduced S. richteri, the proportional rep­ is similar to that for the U.S. populations resentation of rare alleles is only one-third (Fig. 7), suggesting that the relative absence that in native populations, based on nearly of rare alleles in introduced S. invicta may identical sample sizes from each area. be due more to an effect of the geographi­ cally restricted sampling of colonists than to a population bottleneck per se (see also Evidence for Reproductive Isolation Table 1). On the other hand, the small sam­ between S. invicta and S. richteri ple size for the northernmostcollections can The low level ofintraspecific genetic dif­ be expected to result in underrepresentation ferentiation found among geographic pop­ of rare alleles through sampling error. For ulations ofS. invicta and S. richteri in Ar- 2126 K. G. ROSS AND J. C. TRAGER

S. invicta

Argentina 0.4 0.3 n=51 N=100 0.2 S. richteri 0.1

0.6 0.7 O. Argentina Northern Samples 0.6 (Argentina) 0.4 n=40 0.5 0.3 N=57 0.4 n=34 0.2 N=36 0.3 0.1 0.2 0.1 -o -o c c 0.8 o o USA :e 0.7 0.7 o :eo Q. 0.6 Q. 0.6 n=30 USA N=58 e 0.5 e D. D. 0.4 n=37 N=421 0.3 0.2 0.1

o 0.2 0.4 0.6 0.8 1.0 o 0.2 0.4 0.6 0.8 1.0 Allele Frequency Allele Frequency FIG. 7. Distributions ofall alleles ofS. invicta and S. richteriin Argentina and the United States by allele frequency class (data for U.S. samples from Ross et aI., 1987a). For each panel, on' is the total number ofalleles observed at the 26 marker loci and 'N' is the number ofdiploid genotypes studied (one genotype per nest).

gentina contrasts sharply with the genetic in 30 km of Rosario (almost all within 20 differentiation existing between these spe­ km) constitute the focal sample for deter­ cies and, indeed, with that occurring among mining the extent ofreproductive isolation all of the species studied. This is reflected between S. invicta and S. richteri in this in the substantial gap between intra- and zone of parapatry. Twenty-three of these interspecific genetic distance values (Fig. 5), colonies were identified as S. richteri from as well as by the fact that the species gen­ the morphology, 13 as S. invicta, and three erally are characterized by diagnostic alleles as uncertain, the latter including specimens or allelic combinations (see above). suspected ofbeing hybrids between S. rich­ An effort was made to identify areas where teri andeitherS. invicta or S. quinquecuspis. the ranges ofS. invicta and S. richteri come That these may indeed represent products into contact and, thus, where hybridization ofhybridization is further suggested by het­ between these two taxa is possible. Such an erozygosity at individual loci typically fixed area exists in southern Santa Fe Province for species-diagnostic alleles or by unusual in the city ofRosario and its environs, where combinations ofalleles over two such loci. colonies of both species are common (see Frequencies ofsix informative alleles for Fig. 2). Thirty-nine colonies collected with- ants from the zone of parapatry are pre- FIRE ANT SYSTEMATICS AND POPULATION GENETICS 2127

TABLE 8. Frequencies of six informative alleles in the area of contact between S. invicta and S. richteriin southern Santa Fe Province, Argentina. 'N' is the number ofdiploid genotypes studied (one genotype per nest).

ESI_211O Odh86 Est_4155 Pgm J05 Pgm85 GpiJ07 S. invicta (N = 13) 0 0 0.269 0.154 0.193 0.038 S. richteri" (N = 26) 0.731 0.673 0 0 0.019 0.942 S. richteri (N = 23) 0.783 0.717 0 0 0 * Includes three colonies considered hybrid S. richteri/invicta or S. richterirquinquecuspis on the basis of morphology.

sented in Table 8, both with the three pu­ which is diagnostic for S. richteri, is absent tative hybrid colonies included with S. rich­ from S. quinquecuspis (Table 4). However, teri and with these colonies excluded. In one colony collected near Rosario was iden­ either case four of the six alleles occur at tified morphologically as a probable S. rich­ moderate to high frequencies in colonies of teri/quinquecuspis hybrid, and the geno­ one ofthe species, yet they are absent from types observed in this colony are consistent colonies of the other. On the other hand, with this interpretation. the Pgm" allele (which accounts for one­ Ofwhat significance is the limited amount fifth of the alleles at the locus Pgm in S. of interspecific gene flow detected between invicta in Rosario but is absent from all of S. invicta, S. richteri, and S. quinquecuspis? the other species) is present in heterozygous To answer this question, F ST was estimated condition in one of the putative hybrids, as above by treating samples ofeach ofthese which bears S. richteri alleles at two other species collected within 30 km of Rosario loci. Also, in one suspected hybrid colony as populations, with the putativehybrid col­ and one S. invicta colony from the zone of onies included with S. richteri. Assuming parapatry are individuals heterozygous for neutrality and an infinite-island model of 7 Gpi/0 , an allele effectively fixed in S. richteri gene flow (Wright, 1931; Slatkin, 1985, and absent from S. invicta throughout their 1987), FST can be used to estimate Nm, ranges in Argentina and in the United States where N is the effective population size and (e.g., Tables 1and 4). From these allelic data m is the rate ofgenetic migration, from the it appears that reproductive isolation be­ following formula: tween S. invicta and S. richteri in Argentina Nm = (11F - 1)/4. is not absolute in that a small amount of ST gene exchange most likely occurs between An island model is appropriate for such these species where their ranges overlap. analysis because the small number of spe­ The range of S. invicta also appears to cies and their adjacent or overlapping dis­ abut that of S. quinquecuspis in southern tributions mean that each species is prob­ Santa Fe Province, and the latter species is ably equally accessible to immigrants from frequently associated with S. richteri in east­ either of the other species. central Argentina (the two were collected An unbiased estimator ofNm (Nm*) and together at six sites; Fig. 2). Thus it is of its variance can be obtained using a jack­ interest to know how strong the barrier to knife procedure over loci (Johnson et al., gene flow is between S. quinquecuspis and 1988). Taking 0.5 to be a threshold value the other species. The available data indi­ for N m below which gene flow is insufficient cate a situation similar to that for S. invicta to prevent the differentiation ofpopulations and S. richteri. For instance, S. quinque­ by drift alone (Wright, 1931; Slatkin, 1985), cuspis is monomorphic for the allele Est­ the estimate ofNm' = 0.046 ± 0.068 (SE) 21/0, an allele that is absent from S. invicta we obtain for these fire ant species indicates collected near Rosario, while the allele Est­ that gene flow occurring among them ap­ 4155 , present at moderate frequencies in all parently is insignificant in terms of influ­ S. invicta populations, is absent from S. encing population gene dynamics. Essen­ quinquecuspis (see Table 8 for S. invicta, tially identical results are obtained if only also Table 4). Similarly, the allele Gpi'?', S. invicta and S. richteri are included in the 2128 K. G. ROSS AND J. C. TRAGER

TABLE 9. Matrix ofgenetic distances (Nei's D) for seven species of Solenopsis from Argentina.

S. quinquecuspis S. invicta S. macdonaghi S. interrupta 'Species x' S. electra S. richteri 0.061 0.105 0.162 0.237 0.189 0.413 S. quinquecuspis 0.042 0.120 0.165 0.120 0.359 S. invicta 0.094 0.149 0.083 0.351 S. macdonaghi 0.165 0.176 0.414 S. interrupta 0.211 0.414 'Species x' 0.414 analysis. These estimates ofgene flow may All ofthe trees generated from the genetic be high given the small number (n) ofpop­ data agree in recognizing the broadly sym­ u1ations and the fact that Nm should be patric S. richteri and S. quinquecuspis as devalued by the factor [(n - l)/nF in the sister species. An interesting additional fea­ n-is1and model (Slatkin, 1985). In any case ture of the trees based on character state such estimates have a substantial degree of analysis is that S. richteri and S. invicta con­ uncertainty when only a few populations are sistently are placed in different sister line­ involved (Nei et al., 1977). ages, the common ancestor ofwhich occurs at the base ofthe S. saevissima subcomp1ex Extent ofGenetic Differentiation and (=S. saevissima complex without S. elec­ Putative Phylogenetic Relationships of tra). Thus these two species apparently are Argentine Fire Ant Species not closely related within the complex even Pairwise genetic distances for all of the though they hybridize readily in the south­ fire ant species studied are presented in Ta­ ern United States. ble 9, while those for all species except S. electra are graphed in relation to genetic DISCUSSION distances between conspecific populations Statements regarding the systematics and in Figure 5. evolution offire ants ofthe Solenopsis sae­ For the phylogenetic distance analyses, vissima complex have been fraught with un­ no pronounced gap in goodness-of-fit ofthe certainty, both because of the absence ofa 50 trees identified by the distance Wagner thorough taxonomic revision of the group procedure is evident, and a strict (Nelson) based on extensive samples from native consensus tree for these is completely un­ populations, and because ofthe absence of resolved. The best fitting of the 50 trees, comparative population genetic data from which is separated from the second best tree native and introduced populations. In con­ by only 0.4 percent standard deviation, is junction with a recent revision based on the shown as Figure 8a. The first phylogenetic morphology of the ants (Trager, 1990), the character analysis, that in which allele fre­ genetic data presented here establish a foun­ quencies constitute continuous character dation for understanding evolutionary pat­ states, leads to the single tree shown as Fig­ terns in the S. saevissima complex and for ure 8b. The second character analysis, that relating these to population-level processes in which allelic combinations are treated as occurring in areas where these ants are new­ discrete character states, results in three trees ly introduced. ofminima11ength; the single trees produced An important conclusion ofthis study is by applying Nelson consensus procedures that there is substantial concordance be­ and successive approximations weighting tween the genetic data and results derived appear as Figures 8c and 8d, respectively. from analysis of traditional taxonomic Placement ofthe roots on the trees derived characters. All ofthe species previously rec­ from phylogenetic character analysis (Figs. ognized by virtue of their morphological 8b-d) was done using S. electra as the out­ distinctiveness (Trager, 1990) can also be group, in accord with its formal placement identified on the basis of unique single- or in a different subcomp1ex than the other multi-locus genotypic profiles. Further­ species on the basis ofmorphology (Trager, more, the clinally structured intraspecific 1990). genetic variability characterizing S. invicta FIRE ANT SYSTEMATICS AND POPULATION GENETICS 2129

.------electra in Argentina is paralleled by a cline in mor­ ~------macdonaghi phological character states (Trager, 1990), .------interrupta with the northernmost Argentine samples invicta most closely resembling U.S. populations in both their morphology and allozymes. A a 'species x' significant aspect ofthis concordance ofthe richteri character sets is that it suggests that genetic quinquecuspis and morphological variability in this com­ plex of ants can be effectively partitioned into intra- and interspecific components on the basis ofeither type ofdata, and thus the .------electra morphological characters are validated as suitable markers ofgenetic continuityamong ...... ------invicta populations in most situations. As is often ...... ----'species x' the case in taxonomically difficult groups, b macdonaghi however, the electrophoretic markers are in interrupta some instances capable ofdistinguishing re­ richteri productively isolated populations where the quinquecuspis morphological characters cannot. Geographic populations of S. invicta in Argentina exhibit only modest genetic dif­ ferentiation (as apparently does S. richteri), with this genetic structure similar in mag­ .------electra nitude to that found in introduced S. invicta interrupta in the United States. This result is perhaps macdonaghi surprising in view ofthe presumed long his­ I-----invicta tory of occupation of the South American range, contrasted with the recent explosive c L-.----'species x' colonization of North America. Neverthe­ richteri less, average levels ofgene flow appear suf­ quinquecuspis ficiently high on both continents to over­ come effects of local adaptation or genetic drift, a conclusion that is consistent with the reported high vagility of these ants. ..------electra Winged adult reproductives of both sexes take part in mass flights during which .------interrupta they may ascend to heights greater than 300 macdonaghi m. Matedfemales typically disperse less than L-.-+---invicta two km from their natal nests but occasion­ d 'species x' ally are transported far greater distances on richteri air currents (Markin et al., 1971). This ef­ quinquecuspis fective natural means of dispersal, com­ bined with human-aided transport, has re­ FIG.8. Trees ofputative phylogenetic relationships sulted in rates of spread of 10-50 km per for Argentine fire ants (Solenopsis saevissima complex) year (generation) by S. invicta into unin­ generated using a distance Wagner procedure (a) or fested regions in the United States (Vinson using parsimony analyses with loci treated as charac­ ters (b--d). The parsimony analyses involve direct use and Greenberg, 1986; K. Ross and D. of allele frequency data (b) or the encoding ofthese as Fletcher, unpubl.). discrete character states (c, d). The tree in (a) has a percent standard deviation of 3.072 and two negative branch lengths. The tree in (b) has a length of 34.4. The tree in (c) is the consensus tree for three equally parsimonious trees with consistency indices of77, re­ ;- tention indices of 50, and lengths of2,350 (calculated character weighting of these three most parsimonious according to the weights shown in Table 3). The tree trees; the consistency index for this tree is 92, the re­ in (d) is generated from successive approximations tention index is 92, and the length is 170. 2130 K. G. ROSS AND J. C. TRAGER

Although geographically based genetic as there is only a modest (not statistically variability in Argentine S. invicta is modest significant) drop in heterozygosity in intro­ in extent, the variability that exists at sev­ duced S. richteri. Distributions ofnumbers eralloci is clinally distributed along a lati­ of effective alleles are similar between na­ tudinal gradient in the area we sampled. This tive and introduced populations of both gradual transition in the genetic composi­ species. Our finding of minimal effects of tion of S. invicta populations is of special the founding events on heterozygosity is in note in that the more northerly populations keeping with the theoretical results of Nei most closely resemble S. invicta in the Unit­ et aI. (1975), who demonstrated that loss of ed States. Northern Argentina populations heterozygosity in such situations is greatly often lack alleles present further south, al­ ameliorated if there is rapid population leles thatare also absent in the United States. growth following a bottleneck. Given the Most importantly, however, a common al­ high intrinsic reproductive rates offire ants lele in U.S. populations (Agp_14J) is present and their rapid spread throughout the only in samples taken near the Paraguayan southern United States, there can be little borderin the north. Evidence from the mor­ doubt that the original founding popula­ phology indicates that, among specimens tions experienced growth of the sort that collected from across the range ofS. invicta would preserve most of the variability at in South America, those found along the allozyme loci, with only relatively rare al­ Rio Paraguay drainage near the Argentina! leles being lost during this process. The ap­ Paraguay frontier bear the strongest resem­ parent greater loss of heterozygosity in S. blance to the ants in the United States (Tra­ richteri, if real, may reflect a lower repro­ ger, 1990). Buren (1972) earlier placed the ductive rate and/or reduced effective pop­ possible source area for the North American ulation size compared to S. invicta, the far colonists further north along this drainage, more successful of the two colonizing spe­ in central , on the basis of his mor­ cies (e.g., Fig. 1). This apparent difference phological studies. Future collections for ge­ in loss of heterozygosity between the two netic analysis from the northern sections of species could also explain why the genetic the drainage will be required to more ac­ distance between native and introduced S. curately pinpoint the location ofthe source richterifalls toward the high endofthe range population(s). ofintraspecific differentiation (Fig. 5), as one This differential resemblance of regional effect ofa loss ofheterozygosity following a populations aside, introduced S. invicta and bottleneck is to increase genetic distance be­ introduced S. richteri are very similar ge­ tween populations (Chakraborty and Nei, netically to their native conspecific popu­ 1977). lations. In both species some rare alleles While the effects offounding ofthe U.S. present in native populations have been lost populations appear to be modest or insig­ in the colonizing populations (as expected nificant with regard to the simple enzyme­ with finite founder population size), and in encoding loci used as markers here, effects S. richteri one allele common in Argentina ofthe same events on more complex genetic is all butabsent in the United States (Odh JOO). systems, such as those involving overdom­ Nonetheless, estimates of genetic distance inance or epistasis, may be quite different between native and introduced populations (e.g., Yokoyama and Nei, 1979; Goodnight, of both species fall close to or within the 1987). A relevant example here is the hy­ range of expected intraspecific distances pothesized perturbation of the genetic sex­ based on population surveys in both coun­ determining system of S. invicta following tries. introduction of this species to the United There is little evidence from our study of States. Sex determination in S. invicta and any meaningful loss of allozymic diversity othersocial Hymenopteraappears to be me­ (in terms of measures of allelic evenness) diated by heterozygosity at one or more ma­ associated with colonization of the United jor sex loci, such that individuals hemizy­ States. Values for expected heterozygosity gous or homozygous at the sex locus (loci) are virtually identical between native and develop into males (reviewed in Crozier, introduced populations ofS. invicta, where- 1977b). Although the occurrence of male FIRE ANT SYSTEMATICS AND POPULATION GENETICS 2131 diploidy in native S. invicta has not been Demonstration ofsimilar values for het­ studied, the unexpectedly high frequency of erozygosity at enzyme genes in native and mated queens in the United States that pro­ introduced S. invicta and S. richteri is of duce diploid male progeny (15-20%) has further significance as it bears on the more been viewed as evidence ofa substantial loss general issue oflevels ofenzyme-gene vari­ of allelic diversity (and concomitant het­ ability in eusocial Hymenoptera. Native erozygosity) at the sex locus (loci) in intro­ populations of all four fire ant species for duced populations (Ross and Fletcher, which we have adequate samples are char­ 1985a). This hypothesis is not necessarily acterized by values of H exp of 0.05 or less, contradicted by the above data for enzyme with S. quinquecuspis essentially mono­ markers because ofthe differential effects of morphic at all 26 loci studied. The mean bottlenecks on the two types of loci. For value for these four species (0.034) is iden­ highly polymorphic loci such as the sex loci tical to the mean value for all other ant spe­ in Hymenoptera (at which 10 or more al­ cies for which similar surveys have been leles are routinely present at roughly equal conducted (Graur, 1985), butis three to four frequencies [yokoyama and Nei, 1979; Ross times lower than typical values estimated and Fletcher, 1985a]), loss ofallelic diver­ for non-hymenopterous insects (Hexp = sity is critically dependent on bottleneck size, 0.116 for 127 species excluding Drosophila; whereas at less robustly polymorphic loci, Graur, 1985). Thus the suggestion that eu­ such as those we have studied electropho­ social Hymenoptera are relatively deficient retically, the loss ofvariability may be more in genetic variability as determined from dependent on the nature ofsubsequent pop­ electrophoresis (e.g., Metcalf et al., 1975; ulation growth (Nei et al., 1975; also Ma­ Pamilo and Crozier, 1981; Graur, 1985) re­ ruyama and Fuerst, 1985). Thus a scenario ceives further support from this study. in which relatively few colonists established Our data indicate that S. saevissimacom­ a rapidly growing population presumably plex species are discrete, recognizable en­ could explain differing effects ofthe found­ tities that maintain their genetic integrity ing event on the two types ofgenes. even though the ranges ofmany ofthe spe­ These remarks are pertinent also with re­ cies are more or less broadly overlapping. gard to other features ofintroduced fire ant Close examination of the strength of bar­ populations suggested to have arisen as a riers to gene flow between S. invicta and S. result of loss of genetic variability during richteri in a zone ofparapatry and between colonization, including polygyny (multiple S. richteri and S. quinquecuspis over their queens per colony) (Tschinkel and Nieren­ broadly sympatric ranges reveals that these berg, 1983) and interspecific hybridization barriers are notabsolute; rather, they appear (Ross et al., 1987b). Depending on the na­ to be slightly 'leaky' boundaries, permitting ture of the genetic architectures involved, small amounts ofinterspecific nuclear gene the nestmate- and mate-recognition sys­ flow. Nonetheless, the extent of such gene tems of these ants (which mediate social flow appears insufficient to degrade the in­ organization and premating isolation) may ternal cohesion of these species or to pre­ or may not have survived the founding vent their further differentiation in the face events relatively intact (see Crozier [1987] of genetic drift or divergent selective re­ for a review ofthe genetic bases ofnestmate gimes (e.g., Templeton, 1989). Thus the recognition and Templeton [1980] and Gid­ members ofthis complex are effectively in­ dings and Templeton [1983] for discussion dependent evolutionary entities that war­ of bottleneck effects on mate-recognition rant species status (cf. Szymura and Barton, systems). That polygynous social organi­ 1986). zation in S. invicta in the United States did This finding of insignificant levels of in­ not arise solely as a result of founder-in­ terspecific gene flow in native fire ant pop­ duced disruption ofrecognition capabilities ulations is especially important in view of is suggested by the discovery ofpolygynous the extensive hybridization that occurs be­ populations ofS. invicta, S. richteri, and S. tween S. invicta and S. richteri populations quinquecuspis in Argentina (Jouvenaz et al., introduced to the United States. Hybrid 1989; Ross and Trager, unpubl.). populations of these species feature a full 2132 K. G. ROSS AND J. C. TRAGER array of recombinant genotypes occurring sophila, which suggest that prezygotic bar­ in proportions approaching Hardy-Wein­ riers typically develop in advance ofpostzy­ berg expectations (Ross et al., 1987b), and gotic barriers in parapatric or sympatric hybrid individuals experience only slight species pairs (Coyne and Orr, 1989). Ifit is disruption ofdevelopmental regulation rel­ assumed that hybrid viability is likely to ative to the parental species, as determined decrease monotonically with time since di­ from analyses offluctuating bilateral asym­ vergence, the formation of viable fire ant metry (Ross and Robertson, 1990). Thus hybrids between two of the more distantly postzygotic barriers to gene flow between related members ofthe complex, taken to­ these two species clearly are poorly devel­ gether with the modest genetic distances be­ oped. Our phylogenetic character analyses tween species (D = 0.212), suggest that this suggest that S. invicta and S. richteri are is a youthful group in a phase ofactive ra­ members of two different lineages consti­ diation. Future studies of the ecology, tuting the S. saevissima subcomplex, so that breeding habits, and biogeography in South the time since their divergence from a com­ America may shed light on the nature and mon ancestor (during which genetic incom­ origin of prezygotic isolating mechanisms patibilities have accumulated [e.g., Coyne and the possibility that their reinforcement and Orr, 1989]) is likely to be as great as is involved in the process of speciation in that for any species pair in the subcomplex. these ants. Postzygotic barriers may thus be minimal between many of the species. From this it ACKNOWLEDGMENTS follows that reproductive isolation ofnative We thank J. H. Cane, J. M. Carpenter, sympatric populations may typically be en­ and P. S. Ward for comments on the manu­ forced by prezygotic mechanisms which, in script and help with the analyses; H. A. Cor­ the case ofS. invicta and S. richteri, appear do, R. K. Vander Meer, and J. T. Costa for to have been compromised in some way logistical assistance in Argentina and the during colonization of the United States. United States and T. D. Canerday for sup­ One possibility here is that such prezygotic port throughout the project. This work was isolating mechanisms are well developed in funded in part by NSF Grant BSR-8615238 areas ofnatural parapatry between these two to the senior author. species, such as in central Argentina, but poorly developed in areas where they do not LITERATURE CITED come into contact (presuming continuing ARCHIE, J. W. 1985. Statistical analysis of hetero­ zygosity data: Independent sample comparisons. selection for reinforcement occurs in para­ Evolution 39:623-637. patry and intraspecific gene flow is not over­ BRIAN, M. V. 1983. 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