Invasion genetics of the longhorn crazy ant: the global expansion of a double-clonal reproduction system

Reproduction mode represents a key determinant for success of biological invasion as it influences the genetic variation and evolutionary potential of introduced populations. The world’s most widespread invasive ant, Paratrechina longicornis, was found to display an unusual double-clonal reproduction system, whereby both males and queens were produced clonally, while workers are produced sexually. Despite its worldwide distribution, the origin of this ant species and the prevalence of the double-clonal reproductive system across the ant’s geographic range remain unknown. To retrace the evolutionary history of this global invasive species and its reproductive system, we examined genetic variation and characterized the mode of reproduction of P. longicornis sampled worldwide using both microsatellite genotyping and mitochondrial DNA sequencing approaches. Analyses of global genetic variations indicate that the Indian subcontinent is a genetic diversity hotspot of this species, suggesting that this geographic area is at least part of its native range. Our analyses revealed that inferred native and introduced populations both exhibit double-clonal reproduction. Remarkably, queens and males worldwide belong to two separate, non-recombining clonal lineages. Workers are highly heterozygous and first-generation inter- lineage hybrids, a pattern strongly supportive of a strict worldwide prevalence of double clonality. By maintaining heterozygosity in the worker force, this unusual genetic system allows P. longicornis to avoid inbreeding during colonization bottlenecks and may have acted as an adaptive trait linked to the species’ invasion success.


INTRODUCTION
from 2 localities), suggesting that they developed from unfertilized eggs with random segregation 152 of maternal alleles as is typical for most hymenopteran males (arrhenotokous parthenogenesis). 153 (iii) Five males with more than one allele per marker were found in another 3 localities. It is unclear 154 whether these males were diploid individuals that were homozygous at the complementary sex  The production of each type of male was not confined to particular localities as multiple 158 male types could be found within several localities (Data S2_Global_Genotypes). To gain insights 159 into the relative frequency of each type, we genotyped males produced in two laboratory colonies. 160 Out of 223 males, 80 males were inferred to be androgenetic, 127 were inferred to be 161 arrhenotokous, and 16 had more than one allele per locus, suggesting that these were diploids or 162 chimeras. To investigate whether arrhenotokous males are functional, we inspected their 163 reproductive system and genotyped the spermathecal contents of queens. Live sperm was observed 164 in seminal vesicles of arrhenotokous males (N=20), but all dissected queens carried sperm with a 165 single allele from M lineage at each locus, suggesting that they had mated with androgenetic males 166 only (N=86). 167 To summarize, workers sampled in 252 localities across four continents were first-168 generation hybrids of the same two nuclear lineages. The two lineages appeared to be maintained 169 genetically distinct over generations through clonal reproduction of each sex: the genome of M 170 lineage is transmitted through androgenesis from males to males. In contrast, the genome of Q 171 lineage is parthenogenetically transmitted from queens to queens. Although males carrying Q 172 alleles occur regularly, they do not seem to readily reproduce. Altogether, our results suggest that  (Table S1 and Supplementary Text).

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The analysis of the 257 queen and 256 male genotypes across the 256 localities surveyed 182 revealed that the allelic diversity of the M lineage was 2.7 times greater than that of the Q lineage 183 (N = 253 genotypes per lineage, after reduction to one genotype per locality per lineage; Table S2). androgenetic males analyzed. A median-joining network of the genotypes revealed a star-like 198 structure with lower-frequency genotypes at the tips of the branches (Fig. 3d)

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In P. longicornis, new males and queens mate within their natal nest and dispersion occurs 206 primarily through colony budding (Passera, 1994). If no dispersion occurs among colonies, the 207 queen and male genomes should evolve in parallel leading to an association between queen clones, 208 maternal mtDNA and male MLLs. No strong correlation between the and male nuclear variations 209 was observed and no one-to-one association between the 15 most common males' MLLs and the 210 13 most common maternal mtDNA haplotypes was found. Yet, some male MLLs appeared to 211 mate more frequently with one queen clone than the other (Table S3). For example, less than one  Variation in mtDNA 224 We identified 55 mtDNA haplotypes. Of these, 15 were common haplotypes found in more than 225 one geographic region and appeared to be randomly distributed ( Fig. 3e-f). The two queen clones  In most organisms, mtDNA is maternally deposited. To determine if androgenetic males 240 also inherit mtDNA from their mother, we compared the mtDNA haplotypes of androgenetic males, queens and workers in nine localities. Analyses indicated that all castes shared the same haplotype 242 within each of the localities studied consistent with a shared mode of maternal mitochondrial DNA 243 transmission among castes (Data S3_colony-mthaplotype). These results suggest that androgenetic 244 males are cytonuclear hybrids carrying paternal nuclear DNA and maternal mtDNA.  (Table 2).  pool of a native range. Rarefaction analyses indicated that the Indian subcontinent harbored the 267 highest haplotype/allele richness for both mtDNA (Fig. 4a) and microsatellites (Fig 4b). Therefore, 268 our results suggest that the Indian subcontinent is a genetic diversity hotspot for P. longicornis and 269 likely constitutes the native range of the species.   Remarkably, our phylogeographic analyses revealed that all clonal queens and clonal males of P.  (Table 1).

387
Our sampling revealed three different types of males (see Results). To investigate the 388 evolutionary significance of these males, two colony fragments consisting of ~50 reproductive 389 queens and a few thousand workers were collected in two neighboring Spanish populations 390 (Malaga) and maintained in laboratory conditions. Some of the emerging males were randomly 391 dissected to assess whether they produced sperm and were preserved for genetic analyses. The     Numbers of longhorn crazy ant localities, numbers of genotyped males, queens and workers, and inferred male and queen haplotypes/genotypes for each surveyed geographic region.

Genotyped individuals
Inferred genotypes