Nonrandom Patterns of Genetic Admixture Expose the Complex Historical Hybrid Origin of Unisexual Leaf Beetle Species in the GenusCalligrapha

Abstract

Many unisexual animal lineages supposedly arose from hybridization. However, support for their putative hybrid origins mostly comes from indirect methodologies, which are rarely confirmatory. Here we provide compelling data indicating that tetraploid unisexual Calligrapha are true genetic mosaics obtained via analysis of mitochondrial DNA (mtDNA) and allelic variation and coalescence times for three single-copy nuclear genes (CPS, HARS, and Wg) in five of six unisexual Calligrapha and a representative sample of bisexual species. Nuclear allelic diversity in unisexuals consistently segregates in the gene pools of at least two but up to three divergent bisexual species, interpreted as putative parentals of interspecific hybridization crosses. Interestingly, their mtDNA diversity derives from an additional yet undiscovered older evolutionary lineage that is possibly the same for all independently originated unisexual species. One possibly extinct species transferred its mtDNA to several evolutionary lineages in a wave of hybridization events during the Pliocene, whereby descendant species retained a polymorphic mtDNA constitution. Recent hybridizations, in the Pleistocene and always involving females with the old introgressed mtDNA, seemingly occurred in the lineages leading to unisexual species, decoupling mtDNA introgression (and inferences derived from these data, such as timing and parentage) from subsequent acquisition of the new reproductive mode. These results illuminate an unexpected complexity in possible routes to animal unisexuality, with implications for the interpretation of ancient unisexuality. If the origin of unisexuality requires a mechanism where (1) hybridization is a necessary but insufficient condition and (2) multiple bouts of hybridization involving more than two divergent lineages are required, then the origins of several classical unisexual systems may have to be reassessed.

Phylogeographic assessment of mtDNA paraphyly and the evolution of unisexuality inCalligrapha(Coleoptera: Chrysomelidae)

“…In the case of Wg data, ambiguous positions of heterozygotes were resolved in most cases by reference to known alleles in the population as observed in homozygotic individuals in the alignment or, in a second round of inference, considering the new alleles deduced in the previous step. Moreover, relevant in the case of unisexual species of known hybrid origin, these data were aligned and compared with the homologous sequences of putative parental Calligrapha species from Montelongo and Gómez‐Zurita (). Information on sample diversity, including segregating sites, haplotype and nucleotide diversity, and theta, as a measure of divergence, as well as measures of heterozygosity in the case of Wg , were obtained using DnaSP 5.10.1 (Librado & Rozas, ).…”

“…More recently, the analysis of allelic diversity of three single‐copy nuclear genes in five of the six unisexual Calligrapha species and a representative sample of bisexual species allowed validating this hypothesis. Indeed, individuals of unisexual species proved to be genetic mosaics sharing alleles with two and up to three bisexual species, extant representatives of the lineages involved in independent historical interspecific hybridization events (Montelongo & Gómez‐Zurita, ). Interestingly, this study also revealed some unexpected and intriguing findings.…”

“…In fact, this mtDNA lineage, the U‐clade, also included representatives of the bisexual species recognized as being involved in the interspecific crosses that led to each of the unisexual species, generalizing the idea of species mtDNA paraphyly in this group. The most parsimonious explanation for this complex pattern required a single ancestral species that introgressed its mtDNA some 2–3 Ma ago via hybridization into several lineages of Calligrapha and without fixation in the populations, so that they remained polymorphic for their mtDNA, as observed today (Montelongo & Gómez‐Zurita, , ). Later, already in the Pleistocene, species in these lineages were involved in new waves of hybridization events, and when these crosses involved females carrying the introgressed DNA, the resulting hybrid lineages would persist and eventually become unisexual.…”

Parthenogenesis and developmental constraints

“…Such disruptions will usually cause aneuploidy, abortive development, and sterility. Both successful lab experiments and careful genomic analyses have shown that interspecific hybridization followed by back-crossing events can indeed result in parthenogenetic reproduction with restoration of ploidy that retains heterozygosity (e.g., Choleva et al, 2012;Drosopoulos, 1978;Lunt et al, 2014;Lutes, Baumann, Neaves, & Baumann, 2011;Montelongo & Gómez-Zurita, 2015;Schultz, 1973;White, Contreras, Cheney & Webb, 1977;Xu et al, 2013Xu et al, , 2015. It is thought that a rare balance between similarity and dissimilarity is necessary (the balance hypothesis, Janko et al, 2018;Moritz et al, 1989;Vrijenhoek, 1989).…”

“…Hence it is vital that there should not be too much dissimilarity. Importantly single interspecific hybridization events are apparently not sufficient to initiate parthenogenesis and complex hybridization patterns appear to be usually necessary (Janko et al, 2018;Lunt et al, 2014, Montelongo & Gómez-Zurita, 2015Moritz et al, 1989;Xu et al, 2015). Both successful lab experiments and careful genomic analyses have shown that interspecific hybridization followed by back-crossing events can indeed result in parthenogenetic reproduction with restoration of ploidy that retains heterozygosity (e.g., Choleva et al, 2012;Drosopoulos, 1978;Lunt et al, 2014;Lutes, Baumann, Neaves, & Baumann, 2011;Montelongo & Gómez-Zurita, 2015;Schultz, 1973;White, Contreras, Cheney & Webb, 1977;Xu et al, 2013Xu et al, , 2015.…”