How to Tackle Phylogenetic Discordance in Recent and Rapidly Radiating Groups? Developing a Workflow Using Loricaria (Asteraceae) as an Example

A major challenge in phylogenetics and -genomics is to resolve young rapidly radiating groups. The fast succession of species increases the probability of incomplete lineage sorting (ILS), and different topologies of the gene trees are expected, leading to gene tree discordance, i.e., not all gene trees represent the species tree. Phylogenetic discordance is common in phylogenomic datasets, and apart from ILS, additional sources include hybridization, whole-genome duplication, and methodological artifacts. Despite a high degree of gene tree discordance, species trees are often well supported and the sources of discordance are not further addressed in phylogenomic studies, which can eventually lead to incorrect phylogenetic hypotheses, especially in rapidly radiating groups. We chose the high-Andean Asteraceae genus Loricaria to shed light on the potential sources of phylogenetic discordance and generated a phylogenetic hypothesis. By accounting for paralogy during gene tree inference, we generated a species tree based on hundreds of nuclear loci, using Hyb-Seq, and a plastome phylogeny obtained from off-target reads during target enrichment. We observed a high degree of gene tree discordance, which we found implausible at first sight, because the genus did not show evidence of hybridization in previous studies. We used various phylogenomic analyses (trees and networks) as well as the D-statistics to test for ILS and hybridization, which we developed into a workflow on how to tackle phylogenetic discordance in recent radiations. We found strong evidence for ILS and hybridization within the genus Loricaria. Low genetic differentiation was evident between species located in different Andean cordilleras, which could be indicative of substantial introgression between populations, promoted during Pleistocene glaciations, when alpine habitats shifted creating opportunities for secondary contact and hybridization.

Parallel introgression, not recurrent emergence, explains apparent elevational ecotypes of polyploid Himalayan snowtrout

The recurrence of similar evolutionary patterns within different habitats often reflects parallel selective pressures acting upon either standing or independently occurring genetic variation to produce a convergence of phenotypes. This interpretation (i.e. parallel divergences within adjacent streams) has been hypothesized for drainage-specific morphological ‘ecotypes’ observed in polyploid snowtrout (Cyprinidae: Schizothorax ). However, parallel patterns of differential introgression during secondary contact are a viable alternative hypothesis. Here, we used ddRADseq ( N = 35 319 de novo and N = 10 884 transcriptome-aligned SNPs), as derived from Nepali/Bhutanese samples ( N = 48 each), to test these competing hypotheses. We first employed genome-wide allelic depths to derive appropriate ploidy models, then a Bayesian approach to yield genotypes statistically consistent under the inferred expectations. Elevational ‘ecotypes’ were consistent in geometric morphometric space, but with phylogenetic relationships at the drainage level, sustaining a hypothesis of independent emergence. However, partitioned analyses of phylogeny and admixture identified subsets of loci under selection that retained genealogical concordance with morphology, suggesting instead that apparent patterns of morphological/phylogenetic discordance are driven by widespread genomic homogenization. Here, admixture occurring in secondary contact effectively ‘masks’ previous isolation. Our results underscore two salient factors: (i) morphological adaptations are retained despite hybridization and (ii) the degree of admixture varies across tributaries, presumably concomitant with underlying environmental or anthropogenic factors.

Taxonomic Uncertainty and the Anomaly Zone: Phylogenomics Disentangle a Rapid Radiation to Resolve Contentious Species (Gila robusta complex) in the Colorado River

Species are indisputable units for biodiversity conservation, yet their delimitation is fraught with both conceptual and methodological difficulties. A classic example is the taxonomic controversy surrounding the Gila robusta complex in the lower Colorado River of southwestern North America. Nominal species designations were originally defined according to weakly diagnostic morphological differences, but these conflicted with subsequent genetic analyses. Given this ambiguity, the complex was re-defined as a single polytypic unit, with the proposed ‘threatened’ status under the U.S. Endangered Species Act of two elements being withdrawn. Here we re-evaluated the status of the complex by utilizing dense spatial and genomic sampling (N = 387 and >22k loci), coupled with SNP-based coalescent and polymorphism-aware phylogenetic models. In doing so, we found that all three species were indeed supported as evolutionarily independent lineages, despite widespread phylogenetic discordance. To juxtapose this discrepancy with previous studies, we first categorized those evolutionary mechanisms driving discordance, then tested (and subsequently rejected) prior hypotheses which argued phylogenetic discord in the complex was driven by the hybrid origin of Gila nigra. The inconsistent patterns of diversity we found within G. robusta were instead associated with rapid Plio-Pleistocene drainage evolution, with subsequent divergence within the ‘anomaly zone’ of tree space producing ambiguities that served to confound prior studies. Our results not only support resurrection of the three species as distinct entities, but also offer an empirical example of how phylogenetic discordance can be categorized within other recalcitrant taxa, particularly when variation is primarily partitioned at the species-level.

Extensive Genome-Wide Phylogenetic Discordance Is Due to Incomplete Lineage Sorting and Not Ongoing Introgression in a Rapidly Radiated Bryophyte Genus

The relative importance of introgression for diversification has long been a highly disputed topic in speciation research and remains an open question despite the great attention it has received over the past decade. Gene flow leaves traces in the genome similar to those created by incomplete lineage sorting (ILS), and identification and quantification of gene flow in the presence of ILS is challenging and requires knowledge about the true phylogenetic relationship among the species. We use whole nuclear, plastid, and organellar genomes from 12 species in the rapidly radiated, ecologically diverse, actively hybridizing genus of peatmoss (Sphagnum) to reconstruct the species phylogeny and quantify introgression using a suite of phylogenomic methods. We found extensive phylogenetic discordance among nuclear and organellar phylogenies, as well as across the nuclear genome and the nodes in the species tree, best explained by extensive ILS following the rapid radiation of the genus rather than by postspeciation introgression. Our analyses support the idea of ancient introgression among the ancestral lineages followed by ILS, whereas recent gene flow among the species is highly restricted despite widespread interspecific hybridization known in the group. Our results contribute to phylogenomic understanding of how speciation proceeds in rapidly radiated, actively hybridizing species groups, and demonstrate that employing a combination of diverse phylogenomic methods can facilitate untangling complex phylogenetic patterns created by ILS and introgression.

Ecological Specialisation and Evolutionary Reticulation in Extant Hyaenidae

During the Miocene, Hyaenidae was a highly diverse family of Carnivora that has since been severely reduced to four species; the bone-cracking spotted, striped, and brown hyenas, and the specialised insectivorous aardwolf. Previous studies investigated the evolutionary histories of the spotted and brown hyenas, but little is known about the remaining two species. Moreover, the genomic underpinnings of scavenging and insectivory, defining traits of the extant species, remain elusive. Here, we generated an aardwolf genome and analysed it together with the other three species to reveal their evolutionary relationships, genomic underpinnings of their scavenging and insectivorous lifestyles, and their respective genetic diversities and demographic histories. High levels of phylogenetic discordance suggest gene flow between the aardwolf lineage and the ancestral brown/striped hyena lineage. Genes related to immunity and digestion in the bone-cracking hyenas and craniofacial development in the aardwolf showed the strongest signals of selection, suggesting putative key adaptations to carrion and termite feeding, respectively. A family-wide expansion in olfactory receptor genes suggests an acute sense of smell was a key early adaptation. Finally, we report very low levels of genetic diversity within the brown and striped hyenas despite no signs of inbreeding, putatively linked to their similarly slow decline in Ne over the last ∼2 million years. High levels of genetic diversity and more stable population sizes through time are seen in the spotted hyena and aardwolf. Taken together, our findings highlight how ecological specialisation can impact the evolutionary history, demographics, and adaptive genetic changes of an evolutionary lineage.

Ecological specialisation and evolutionary reticulation in extant Hyaenidae

During the Miocene, Hyaenidae was a highly diverse family of Carnivora that has since been severely reduced to four extant genera, each of which contains only a single species. These species include the bone-cracking spotted, striped, and brown hyenas, and the specialised insectivorous aardwolf. Previous genome studies have analysed the evolutionary histories of the spotted and brown hyenas, but little is known about the remaining two species. Moreover, the genomic underpinnings of scavenging and insectivory, defining traits of the extant species, remain elusive. To tackle these questions, we generated an aardwolf genome and analysed it together with those from the other three species. We provide new insights into the evolutionary relationships between the species, the genomic underpinnings of their scavenging and insectivorous lifestyles, and their respective genetic diversities and demographic histories. High levels of phylogenetic discordance within the family suggest gene flow between the aardwolf lineage and the ancestral brown/striped hyena lineage. Genes related to immunity and digestion in the bone-cracking hyenas and craniofacial development in the aardwolf showed the strongest signals of selection in their respective lineages, suggesting putative key adaptations to carrion or termite feeding. We also found a family-wide expansion in olfactory receptor genes suggesting that an acute sense of smell was a key early adaptation for the Hyaenidae family. Finally, we report very low levels of genetic diversity within the brown and striped hyenas despite no signs of inbreeding, which we putatively link to their similarly slow decline in Neover the last ∼2 million years. We found much higher levels of genetic diversity in both the spotted hyena and aardwolf and more stable population sizes through time. Taken together, these findings highlight how ecological specialisation can impact the evolutionary history, demographics, and adaptive genetic changes of a lineage.

Species delimitation in the grey zone: introgression obfuscates phylogenetic inference and species boundaries in a cryptic frog complex (Ranidae: Pulchrana picturata)

ABSTRACTAs molecular methods continue to elucidate genetic structure at increasingly finer resolutions, delimiting species in the grey zone of the speciation continuum is becoming more relevant in biodiversity research, especially in under-studied biodiversity hotspots such as Southeast Asia where new species are being described at an unprecedented rate. Obvious species at both ends of the speciation continuum have mostly been described and attention is now turning towards the “grey zone:” an intermediate stage in which species criteria are in conflict and boundaries between populations and species are less clear. This study demonstrates that widely-used criteria (phylogenetic placement, genetic divergence, phylogeny- and distance-based species delimitation methods) can overestimate species diversity/boundaries when introgression is present. However, a comprehensive species delimitation framework that considers spatial and genetic population structure, introgression, and the use of species delimitation methods based on parameter estimation, can provide a more accurate characterization of species boundaries in this grey zone. We applied this approach to a group of Southeast Asian frogs from the Pulchrana picturata Complex that exhibits continuous morphological variation and high genetic divergences. Results showed that introgression was rampant among Bornean populations, which led to phylogenetic discordance and an overestimation of species. We suspect that our results do not form an isolated case; and that introgression among cryptic populations, occurring continuously across a wide geographic area (e.g., the topographically complex island of Borneo, and Earth’s major continents), may be more common than previously thought.

Taxonomic Uncertainty and the Anomaly Zone: Phylogenomics Disentangle a Rapid Radiation to Resolve Contentious Species (Gila robusta complex) in the Colorado River

Species is an indisputable unit for biodiversity conservation, yet their delimitation is fraught with both conceptual and methodological difficulties. A classic example is the taxonomic controversy surrounding the Gila robusta complex in the lower Colorado River of southwestern North America. Nominal species designations were originally defined according to weakly diagnostic morphological differences that conflicted with traditional genetic analyses. Consequently, the complex was re-defined as a single polytypic unit, with the proposed ‘threatened’ status of two being withdrawn at the federal level. Here, we utilized dense spatial and genomic sampling (N=387 and >22k loci) to re-evaluate the status of the complex, based on SNP-based coalescent and polymorphism-aware phylogenetic models. In doing so, all three species were supported as evolutionarily independent lineages, despite widespread phylogenetic discordance. To understand this discrepancy with past studies, we categorized evolutionary mechanisms driving discordance. We tested (and subsequently rejected) prior hypotheses suggesting that phylogenetic discord in the complex was hybridization-driven. Instead, we found the G. robusta complex to have diverged within the ‘anomaly zone’ of tree space and, as such, have accumulated inconsistent patterns of diversity which have confounded prior studies. After extending these analyses with phylogeographic modeling, we propose that this is reflective of a rapid radiation promoted by Plio-Pleistocene tectonism. Our results not only support resurrection of the three species as distinct entities, but also offer an empirical example of how phylogenetic discordance can be categorized in other recalcitrant taxa where variation is primarily partitioned at the species-level.

Recombination rate variation shapes barriers to introgression across butterfly genomes

ABSTRACTHybridisation and introgression can dramatically alter the relationships among groups of species, leading to phylogenetic discordance across the genome and between populations. Introgression can also erode species differences over time, but selection against introgression at certain loci acts to maintain post-mating species barriers. Theory predicts that species barriers made up of many loci throughout the genome should lead to a broad correlation between introgression and recombination rate, which determines the extent to which selection on deleterious foreign alleles will affect neutral alleles at physically linked loci. Here we describe the variation in genealogical relationships across the genome among three species of Heliconius butterflies: H. melpomene, H. cydno and H. timareta, using whole genomes of 92 individuals, and ask whether this variation can be explained by heterogeneous barriers to introgression. We find that species relationships vary predictably at the chromosomal scale. By quantifying recombination rate and admixture proportions, we then show that rates of introgression are predicted by variation in recombination rate. This implies that species barriers are highly polygenic, with selection acting against introgressed alleles across most of the genome. In addition, long chromosomes, which have lower recombination rates, produce stronger barriers on average than short chromosomes. Finally, we find a consistent difference between two species pairs on either side of the Andes, which suggests differences in the architecture of the species barriers. Our findings illustrate how the combined effects of hybridisation, recombination and natural selection, acting at multitudes of loci over long periods, can dramatically sculpt the phylogenetic relationships among species.