Phylogeny Reconstruction Research Articles

Plastid phylogenomic insights into relationships of all flowering plant families

BackgroundFlowering plants (angiosperms) are dominant components of global terrestrial ecosystems, but phylogenetic relationships at the familial level and above remain only partially resolved, greatly impeding our full understanding of their evolution and early diversification. The plastome, typically mapped as a circular genome, has been the most important molecular data source for plant phylogeny reconstruction for decades.ResultsHere, we assembled by far the largest plastid dataset of angiosperms, composed of 80 genes from 4792 plastomes of 4660 species in 2024 genera representing all currently recognized families. Our phylogenetic tree (PPA II) is essentially congruent with those of previous plastid phylogenomic analyses but generally provides greater clade support. In the PPA II tree, 75% of nodes at or above the ordinal level and 78% at or above the familial level were resolved with high bootstrap support (BP ≥ 90). We obtained strong support for many interordinal and interfamilial relationships that were poorly resolved previously within the core eudicots, such as Dilleniales, Saxifragales, and Vitales being resolved as successive sisters to the remaining rosids, and Santalales, Berberidopsidales, and Caryophyllales as successive sisters to the asterids. However, the placement of magnoliids, although resolved as sister to all other Mesangiospermae, is not well supported and disagrees with topologies inferred from nuclear data. Relationships among the five major clades of Mesangiospermae remain intractable despite increased sampling, probably due to an ancient rapid radiation.ConclusionsWe provide the most comprehensive dataset of plastomes to date and a well-resolved phylogenetic tree, which together provide a strong foundation for future evolutionary studies of flowering plants.

Monophyletic blowflies revealed by phylogenomics

BackgroundBlowflies are ubiquitous insects, often shiny and metallic, and the larvae of many species provide important ecosystem services (e.g., recycling carrion) and are used in forensics and debridement therapy. Yet, the taxon has repeatedly been recovered to be para- or polyphyletic, and the lack of a well-corroborated phylogeny has prevented a robust classification.ResultsWe here resolve the relationships between the different blowfly subclades by including all recognized subfamilies in a phylogenomic analysis using 2221 single-copy nuclear protein-coding genes of Diptera. Maximum likelihood (ML), maximum parsimony (MP), and coalescent-based phylogeny reconstructions all support the same relationships for the full data set. Based on this backbone phylogeny, blowflies are redefined as the most inclusive monophylum within the superfamily Oestroidea not containing Mesembrinellidae, Mystacinobiidae, Oestridae, Polleniidae, Sarcophagidae, Tachinidae, and Ulurumyiidae. The constituent subfamilies are re-classified as Ameniinae (including the Helicoboscinae, syn. nov.), Bengaliinae, Calliphorinae (including Aphyssurinae, syn. nov., Melanomyinae, syn. nov., and Toxotarsinae, syn. nov.), Chrysomyinae, Luciliinae, Phumosiinae, Rhiniinae stat. rev., and Rhinophorinae stat. rev. Metallic coloration in the adult is shown to be widespread but does not emerge as the most likely ground plan feature.ConclusionsOur study provides the first phylogeny of oestroid calyptrates including all blowfly subfamilies. This allows settling a long-lasting controversy in Diptera by redefining blowflies as a well-supported monophylum, and blowfly classification is adjusted accordingly. The archetypical blowfly trait of carrion-feeding maggots most likely evolved twice, and the metallic color may not belong to the blowfly ground plan.

An Information-Entropy Position-Weighted K-Mer Relative Measure for Whole Genome Phylogeny Reconstruction.

Alignment methods have faced disadvantages in sequence comparison and phylogeny reconstruction due to their high computational costs in handling time and space complexity. On the other hand, alignment-free methods incur low computational costs and have recently gained popularity in the field of bioinformatics. Here we propose a new alignment-free method for phylogenetic tree reconstruction based on whole genome sequences. A key component is a measure called information-entropy position-weighted k-mer relative measure (IEPWRMkmer), which combines the position-weighted measure of k-mers proposed by our group and the information entropy of frequency of k-mers. The Manhattan distance is used to calculate the pairwise distance between species. Finally, we use the Neighbor-Joining method to construct the phylogenetic tree. To evaluate the performance of this method, we perform phylogenetic analysis on two datasets used by other researchers. The results demonstrate that the IEPWRMkmer method is efficient and reliable. The source codes of our method are provided at https://github.com/ wuyaoqun37/IEPWRMkmer.

Genomic insights into the biosynthesis and physiology of the cyanobacterial neurotoxin 2,4-diaminobutanoic acid (2,4-DAB)

Cyanobacteria are an ancient clade of photosynthetic prokaryotes, whose worldwide occurrence, especially in water, presents health hazards to humans and animals due to the production of a range of toxins (cyanotoxins). These include the sometimes co-occurring, non-encoded diaminoacid neurotoxins 2,4-diaminobutanoic acid (2,4-DAB) and its structural analogue β-N-methylaminoalanine (BMAA). Knowledge of the biosynthetic pathway for 2,4-DAB, and its role in cyanobacteria, is lacking. The aspartate 4-phosphate pathway is a known route of 2,4-DAB biosynthesis in other bacteria and in some plant species. Another pathway to 2,4-DAB has been described in Lathyrus species. Here, we use bioinformatics analyses to investigate hypotheses concerning 2,4-DAB biosynthesis in cyanobacteria. We assessed the presence or absence of each enzyme in candidate biosynthesis routes, the aspartate 4-phosphate pathway and a pathway to 2,4-DAB derived from S-adenosyl-L-methionine (SAM), in 130 cyanobacterial genomes using sequence alignment, profile hidden Markov models, substrate specificity/active site identification and the reconstruction of gene phylogenies. In the aspartate 4-phosphate pathway, for the 18 species encoding diaminobutanoate-2-oxo-glutarate transaminase, the co-localisation of genes encoding the transaminase with the downstream decarboxylase or ectoine synthase – often within hybrid non-ribosomal peptide synthetase (NRPS)-polyketide synthases (PKS) clusters, NRPS-independent siderophore (NIS) clusters and incomplete ectoine clusters – is compatible with the hypothesis that some cyanobacteria use the aspartate 4-phosphate pathway for 2,4-DAB production. Through this route, in cyanobacteria, 2,4-DAB may be functionally associated with environmental iron-scavenging, via the production of siderophores of the schizokinen/synechobactin type and of some polyamines. In the pathway to 2,4-DAB derived from SAM, eight cyanobacterial species encode homologs of SAM-dependent 3-amino-3-carboxypropyl transferases. Other enzymes in this pathway have not yet been purified or sequenced. Ultimately, the biosynthesis of 2,4-DAB appears to be either restricted to some cyanobacterial species, or there may be multiple and additional routes, and roles, for the synthesis of this neurotoxin.

Mitochondrial Genomes, Phylogenetic Associations, and SNP Recovery for the Key Invasive Ponto-Caspian Amphipods in Europe.

The Ponto-Caspian region is the main donor of invasive amphipods to freshwater ecosystems, with at least 13 species successfully established in European inland waters. Dikerogammarus spp. and Pontogammarus robustoides are among the most successful, due to their strong invasive impact on local biota. However, genomic knowledge about these invaders is scarce, while phylogeography and population genetics have been based on short fragments of mitochondrial markers or nuclear microsatellites. In this study, we provide: (i) a reconstruction of six mitogenomes for four invasive gammarids (D. villosus, D. haemobaphes, D. bispinosus, and P. robustoides); (ii) a comparison between the structure of the newly obtained mitogenomes and those from the literature; (iii) SNP calling rates for individual D. villosus and D. haemobaphes from different invasion sites across Europe; and (iv) the first time-calibrated full mitogenome phylogeny reconstruction of several Ponto-Caspian taxa. We found that, in comparison to other gammarids, the mitogenomes of Ponto-Caspian species show a translocation between the tRNA-E and tRNA-R positions. Phylogenetic reconstruction using the mitogenomes identified that Ponto-Caspian gammarids form a well-supported group that originated in the Miocene. Our study supports paraphyly in the family Gammaridae. These provided mitogenomes will serve as vital genetic resources for the development of new markers for PCR-based identification methods and demographic studies.

An assembly-free method of phylogeny reconstruction using short-read sequences from pooled samples without barcodes

A current strategy for obtaining haplotype information from several individuals involves short-read sequencing of pooled amplicons, where fragments from each individual is identified by a unique DNA barcode. In this paper, we report a new method to recover the phylogeny of haplotypes from short-read sequences obtained using pooled amplicons from a mixture of individuals, without barcoding. The method, AFPhyloMix, accepts an alignment of the mixture of reads against a reference sequence, obtains the single-nucleotide-polymorphisms (SNP) patterns along the alignment, and constructs the phylogenetic tree according to the SNP patterns. AFPhyloMix adopts a Bayesian inference model to estimate the phylogeny of the haplotypes and their relative abundances, given that the number of haplotypes is known. In our simulations, AFPhyloMix achieved at least 80% accuracy at recovering the phylogenies and relative abundances of the constituent haplotypes, for mixtures with up to 15 haplotypes. AFPhyloMix also worked well on a real data set of kangaroo mitochondrial DNA sequences.

Complete genome sequence of camellia virus A, a tentative new member of the genus Waikavirus.

A new positive-strand RNA virus genome was discovered in Camellia japonica plants. The complete genome of the virus is 12,570nt in size, excluding the poly(A) tail, and contains one large open reading frame (ORF1) and two small open reading frames (ORF2, ORF3). ORF1 and ORF2 are homologous to sequences of waikaviruses, while ORF3 has no relatives in the databases. ORF1 encodes a putative polyprotein precursor that is putatively processed into eight smaller proteins, as in typical waikaviruses. Comprehensive analysis, including BLAST searches, genome organization and pairwise sequence comparisons, and phylogeny reconstructions, invariably placed the virus with the waikaviruses. Furthermore, due to lower amino acid sequence identity to known waikaviruses than the threshold species demarcation cutoff, this virus may represent a new species in the genus Waikavirus, family Secoviridae, and we have tentatively named it "camellia virus A" (CamVA). Finally, a field survey was conducted to assess the occurrence of CamVA in camellias and its associated symptoms.

Sympatric and independently evolving lineages in the Thoropa miliaris – T. taophora species complex (Anura: Cycloramphidae)

Species delimitation can be challenging and affected by subjectivity. Sibling lineages that occur in sympatry constitute good candidates for species delimitation regardless of the adopted species concept. The Thoropa miliaris + T. taophora species complex exhibits high genetic diversity distributed in several lineages that occur sympatrically in the southeastern Atlantic Forest of Brazil. We used 414 loci obtained by anchored hybrid enrichment to characterize genetic variation in the Thoropa miliaris species group (T. saxatilis, T megatympanum, T. miliaris, and T. taophora), combining assignment analyses with traditional and coalescent phylogeny reconstruction. We also investigated evolutionary independence in co-occurring lineages by estimating gene flow, and validated lineages under the multispecies coalescent. We recovered most previously described lineages as unique populations in assignment analyses; exceptions include two lineages within T. miliaris that are further substructured, and the merging of all T. taophora lineages. We found very low probabilities of gene flow between sympatric lineages, suggesting independent evolution. Species tree inferences and species delimitation yielded resolved relationships and indicate that all lineages constitute putative species that diverged during the Pliocene and Pleistocene, later than previously estimated.

Accounting for the Biological Complexity of Pathogenic Fungi in Phylogenetic Dating.

In the study of pathogen evolution, temporal dating of phylogenies provides information on when species and lineages may have diverged in the past. When combined with spatial and epidemiological data in phylodynamic models, these dated phylogenies can also help infer where and when outbreaks occurred, how pathogens may have spread to new geographic locations and/or niches, and how virulence or drug resistance has developed over time. Although widely applied to viruses and, increasingly, to bacterial pathogen outbreaks, phylogenetic dating is yet to be widely used in the study of pathogenic fungi. Fungi are complex organisms with several biological processes that could present issues with appropriate inference of phylogenies, clock rates, and divergence times, including high levels of recombination and slower mutation rates although with potentially high levels of mutation rate variation. Here, we discuss some of the key methodological challenges in accurate phylogeny reconstruction for fungi in the context of the temporal analyses conducted to date and make recommendations for future dating studies to aid development of a best practices roadmap in light of the increasing threat of fungal outbreaks and antifungal drug resistance worldwide.

Diversification and distribution of gall crabs (Brachyura: Cryptochiridae: Opecarcinus) associated with Agariciidae corals

Coral reefs are home to the greatest diversity of marine life, and many species on reefs live in symbiotic associations. Studying the historical biogeography of symbiotic species is key to unravelling (potential) coevolutionary processes and explaining species richness patterns. Coral-dwelling gall crabs (Cryptochiridae) live in obligate symbiosis with a scleractinian host, and are ideally suited to study the evolutionary history between heterogeneous taxa involved in a symbiotic relationship. The genus Opecarcinus Kropp and Manning, 1987, like its host coral family Agariciidae, occurs in both Indo-Pacific and Caribbean seas, and is the only cryptochirid genus with a circumtropical distribution. Here, we use mitochondrial and nuclear DNA gene fragments of Opecarcinus specimens sampled from 21 Indo-Pacific localities and one Atlantic (Caribbean) locality. We applied several species delimitation tests to characterise species diversity, inferred a Bayesian molecular-clock time-calibrated phylogeny to estimate divergence times and performed an ancestral area reconstruction. Time to the most recent common ancestor (tMRCA) of Opecarcinus is estimated at 15−6 Mya (middle Miocene—late Miocene). The genus harbours ~ 15 undescribed species as well as several potential species complexes. There are indications of strict host-specificity patterns in certain Opecarcinus species in the Indo-Pacific and Atlantic, however, a robust phylogeny reconstruction of Agariciidae corals—needed to test this further—is currently lacking. The Indo-West Pacific was inferred to be the most probable ancestral area, from where the Opecarcinus lineage colonised the Western Atlantic and subsequently speciated into O. hypostegus. Opecarcinus likely invaded from the Indo-West Pacific across the East Pacific Barrier to the Atlantic, before the full closure of the Isthmus of Panama. The subsequent speciation of O. hypostegus, is possibly associated with newly available niches in the Caribbean, in combination with genetic isolation following the closure of the Panama Isthmus.

Cuban history of CRF19 recombinant subtype of HIV-1.

CRF19 is a recombinant form of HIV-1 subtypes D, A1 and G, which was first sampled in Cuba in 1999, but was already present there in 1980s. CRF19 was reported almost uniquely in Cuba, where it accounts for ∼25% of new HIV-positive patients and causes rapid progression to AIDS (∼3 years).We analyzed a large data set comprising ∼350 pol and env sequences sampled in Cuba over the last 15 years and ∼350 from Los Alamos database. This data set contained both CRF19 (∼315), and A1, D and G sequences. We performed and combined analyses for the three A1, G and D regions, using fast maximum likelihood approaches, including: (1) phylogeny reconstruction, (2) spatio-temporal analysis of the virus spread, and ancestral character reconstruction for (3) transmission mode and (4) drug resistance mutations (DRMs). We verified these results with a Bayesian approach. This allowed us to acquire new insights on the CRF19 origin and transmission patterns. We showed that CRF19 recombined between 1966 and 1977, most likely in Cuban community stationed in Congo region. We further investigated CRF19 spread on the Cuban province level, and discovered that the epidemic started in 1970s, most probably in Villa Clara, that it was at first carried by heterosexual transmissions, and then quickly spread in the 1980s within the “men having sex with men” (MSM) community, with multiple transmissions back to heterosexuals. The analysis of the transmission patterns of common DRMs found very few resistance transmission clusters.Our results show a very early introduction of CRF19 in Cuba, which could explain its local epidemiological success. Ignited by a major founder event, the epidemic then followed a similar pattern as other subtypes and CRFs in Cuba. The reason for the short time to AIDS remains to be understood and requires specific surveillance, in Cuba and elsewhere.

Whole-Genome Duplication and Purifying Selection Contributes to the Functional Redundancy of Auxin Response Factor (ARF) Genes in Foxtail Millet (Setaria italica L.).

Auxin response factors (ARFs) play crucial roles in auxin-mediated response, whereas molecular genetics of ARF genes was seldom investigated in Setaria italica, an important crop and C4 model plant. In the present study, genome-wide evolutionary analysis of ARFs was performed in S. italica. Twenty-four SiARF genes were identified and unevenly distributed on eight of the nine chromosomes in S. italica. Duplication mode exploration implied that 13 SiARF proteins were originated from whole-genome duplication and suffered purifying selection. Phylogeny reconstruction of SiARFs by maximum likelihood and neighbor-joining trees revealed SiARFs could be divided into four clades. SiARFs clustered within the same clade shared similar gene structure and protein domain composition, implying functional redundancy. Moreover, amino acid composition of the middle regions was conserved in SiARFs belonged to the same clade. SiARFs were categorized into either activators or repressors according to the enrichment of specific amino acids. Intrinsic disorder was featured in the middle regions of ARF activators. Finally, expression profiles of SiARFs under hormone and abiotic stress treatment not only revealed their potential function in stress response but also indicate their functional redundancy. Overall, our results provide insights into evolutionary aspects of SiARFs and benefit for further functional characterization.

Population Genetic Structure and Contribution of Philippine Chickens to the Pacific Chicken Diversity Inferred From Mitochondrial DNA.

The Philippines is considered one of the biodiversity hotspots for animal genetic resources. In spite of this, population genetic structure, genetic diversity, and past population history of Philippine chickens are not well studied. In this study, phylogeny reconstruction and estimation of population genetic structure were based on 107 newly generated mitochondrial DNA (mtDNA) complete D-loop sequences and 37 previously published sequences of Philippine chickens, consisting of 34 haplotypes. Philippine chickens showed high haplotypic diversity (Hd = 0.915 ± 0.011) across Southeast Asia and Oceania. The phylogenetic analysis and median-joining (MJ) network revealed predominant maternal lineage haplogroup D classified throughout the population, while support for Philippine–Pacific subclade was evident, suggesting a Philippine origin of Pacific chickens. Here, we observed Philippine red junglefowls (RJFs) at the basal position of the tree within haplogroup D indicating an earlier introduction into the Philippines potentially via mainland Southeast Asia (MSEA). Another observation was the significantly low genetic differentiation and high rate of gene flow of Philippine chickens into Pacific chicken population. The negative Tajima’s D and Fu’s Fs neutrality tests revealed that Philippine chickens exhibited an expansion signal. The analyses of mismatch distribution and neutrality tests were consistent with the presence of weak phylogeographic structuring and evident population growth of Philippine chickens (haplogroup D) in the islands of Southeast Asia (ISEA). Furthermore, the Bayesian skyline plot (BSP) analysis showed an increase in the effective population size of Philippine chickens, relating with human settlement, and expansion events. The high level of genetic variability of Philippine chickens demonstrates conservation significance, thus, must be explored in the future.

Premolar enamel thickness and distribution of a Miocene hominid Lufengpithecus hudienensis compared with Pleistocene and extant hominids

Lufengpithecus hudienensis is a Late-Miocene hominid from the Yuanmou basin of southwestern China. Previous studies link Lufengpithecus to either the Sivapithecus-orangutan clade or a derived branch from the basal stem of the Hominidae. Despite a rich fossil assemblage, the taxonomy of L. hudienensis and its phylogenetic relationship with other hominids is still unclear. Enamel thickness in fossil and modern hominids can provide insights into taxonomy, phylogeny, and dietary reconstructions. In this study, 24 upper and lower L. hudienensis premolars were imaged using high-resolution microcomputed tomography. Three-dimensional average enamel thickness and relative enamel thickness (RET) indices, as well as whole-crown enamel distribution patterns were recorded for the L. hudienensis specimens and compared with a total of 113 specimens of fossil and extant apes and recent modern humans (RMH): fossil Pongo (n = 36), Gigantopithecus blacki (n = 21), Pan troglodytes (n = 11), Gorilla gorilla (n = 7), and RMH (n = 38). RET of the premolars of L. hudienensis was found to be similar to that of fossil Pongo, P. troglodytes, and G. gorilla, but less than Gi. blacki and RMH values. The distribution of enamel thickness shows a taxon-specific pattern for L. hudienensis, generally characterized by thicker enamel on the lateral wall than on the cusp apex, on the lingual cusp of the upper and buccal cusp of the lower premolars, with some differences with respect to fossil Pongo, Gi. blacki, P. troglodytes, and G. gorilla patterns. Additional characterizations of enamel thickness distribution patterns of the molars and other dental endostructural morphologies are needed to further explore the phylogenetic relationships of L. hudienensis with other hominids.

Genome-wide identification and expression analysis of aquaporin family in Canavalia rosea and their roles in the adaptation to saline-alkaline soils and drought stress

BackgroundCanavalia rosea (Sw.) DC. (bay bean) is an extremophile halophyte that is widely distributed in coastal areas of the tropics and subtropics. Seawater and drought tolerance in this species may be facilitated by aquaporins (AQPs), channel proteins that transport water and small molecules across cell membranes and thereby maintain cellular water homeostasis in the face of abiotic stress. In C. rosea, AQP diversity, protein features, and their biological functions are still largely unknown.ResultsWe describe the action of AQPs in C. rosea using evolutionary analyses coupled with promoter and expression analyses. A total of 37 AQPs were identified in the C. rosea genome and classified into five subgroups: 11 plasma membrane intrinsic proteins, 10 tonoplast intrinsic proteins, 11 Nod26-like intrinsic proteins, 4 small and basic intrinsic proteins, and 1 X-intrinsic protein. Analysis of RNA-Seq data and targeted qPCR revealed organ-specific expression of aquaporin genes and the involvement of some AQP members in adaptation of C. rosea to extreme coral reef environments. We also analyzed C. rosea sequences for phylogeny reconstruction, protein modeling, cellular localizations, and promoter analysis. Furthermore, one of PIP1 gene, CrPIP1;5, was identified as functional using a yeast expression system and transgenic overexpression in Arabidopsis.ConclusionsOur results indicate that AQPs play an important role in C. rosea responses to saline-alkaline soils and drought stress. These findings not only increase our understanding of the role AQPs play in mediating C. rosea adaptation to extreme environments, but also improve our knowledge of plant aquaporin evolution more generally.

Abstract LB020: Epigenomic tumor evolution modeling with single-cell methylation data profiling

Abstract The heritability of methylation patterns in tumor cells, as shown in recent studies, suggests that tumor heterogeneity and progression can be interpreted and predicted in the context of methylation changes. To elucidate methylation-based evolution trajectory in tumors, we introduce a novel computational method for methylation phylogeny reconstruction leveraging single cell bisulfite treated whole genome sequencing data (scBS-seq), incorporating additional copy number information inferred independently from matched single cell RNA sequencing (scRNA-seq) data, when available. We validate our method with the scBS-seq data of multi-regionally sampled colorectal cancer cells, and demonstrate that the cell lineages constructed by our method strongly correlate with original sampling regions. Our method consists of three components: (i) noise-minimizing site selection, (ii) likelihood-based sequencing error correction, and (iii) pairwise expected distance calculation for cells, all designed to mitigate the effect of noise and uncertainty due to data sparsity commonly observed in scBS-seq data. In (i), we present an integer linear program-based biclustering formulation to select a set of CpG-sites and cells so that the number of CpG-sites with non-zero coverage in the selected cells is maximized. This procedure filters out cells with read information in too few sites and CpG-sites with read information in too few cells. In (ii), we address the sequencing errors commonly encountered in currently available platforms with a maximum log likelihood approach to correct likely sequencing errors in scBS-seq reads, incorporating CpG-site copy number information in case it can be orthogonally obtained. Given the copy number and read information for a site in a cell, together with the overall sequencing error probability, we compute the log likelihood for all possible underlying allele statuses. If the mixed read statuses at the CpG-site for the cell are more likely due to sequencing error on homozygous alleles as opposed to the presence of alleles mixed methylation statuses, we correct the reads of the minority methylation status to the majority one. In (iii), we introduce a formulation to estimate distances between any pair of cells. As scBS-seq data is typically characterized by shallow read coverage, there is rarely read count evidence for two (or more, depending on CNV status) alleles at a CpG-site. Since allele-specific methylation has been shown to have increased frequency in cancer tissues, given the reads at a CpG-site, it is especially important to consider the possibility of unobserved alleles and their methylation status when determining the CpG-site's possible methylation zygosities. Our method incorporates copy number information when available, and for each CpG-site in a cell, we compute a probability distribution across all possible methylation zygosities. Then, given specific distance values between pairs of distinct zygosities and the likelihood of each possible zygosity for each shared CpG-site in both cells, we compute the expected total distance between any pair of cells as the mean of expected distances across all shared CpG-sites. We leverage such pairwise distances in methylation phylogeny construction. Citation Format: Xuan C. Li, Yuelin Liu, Farid Rashidi, Salem Malikic, Stephen M. Mount, Eytan Ruppin, Kenneth Aldape, Cenk Sahinalp. Epigenomic tumor evolution modeling with single-cell methylation data profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB020.

Abstract LB019: Trisicell: Scalable Tumor Phylogeny Reconstruction and Validation Reveals Developmental Origin and Therapeutic Impact of Intratumoral Heterogeneity

Abstract Emerging sets of single-cell sequencing data makes it appealing to apply existing tumor phylogeny reconstruction methods to analyze associated intratumor heterogeneity. Unfortunately, tumor phylogeny inference is an NP-hard problem and existing principled methods typically fail to scale up to handle thousands of cells and mutations observed in emerging single-cell data sets. Even though there are greedy heuristics to build hierarchical clustering of cells and mutations, they suffer from well-documented issues in accuracy. Additionally even when “optimal” solutions are feasible, existing approaches only provide a single “most likely” tree to depict the evolutionary processes that may result in an observed collection of cells and mutations. To make matters worse, the vast majority of single-cell sequencing data sets are transcriptomic and as a result, suffer from considerable variation in coverage across mutational loci. In this paper, we introduce Trisicell, a computational toolkit for scalable tumor phylogeny reconstruction and validation from single-cell genomic, exomic or transcriptomic sequencing data. Trisicell has three components: (i) Trisicell-DnC, a new tumor phylogeny reconstruction method from genotype matrices derived from single-cell data, (ii) Trisicell-ConT a new algorithm for constructing the consensus for two or more tumor phylogenies - which may be built through the use of different data types on the same set of cells, or built through the use of different methods on the same data, and (iii) Trisicell-PF, a new partition function method for assessing the likelihood of any user-defined subtree/set of cells to be seeded by a given set of mutations in the phylogeny. Collectively, these tools provide means of identifying and validating robust portions of a tumor phylogeny, offering the ability to focus on the most important (sub)clones and the genomic alterations that seed the associated clonal expansion. We applied Trisicell to a panel of clonal sublines derived from single-cells of a parental mouse melanoma model on which we performed both whole exome and whole transcriptome sequencing. The tumor phylogenies of the clonal sublines built on exomic and transcriptomic mutations by Trisicell-DnC, were shown by Trisicell-ConT to be highly similar and the subtrees comprised of phenotypically similar clonal sublines were shown to be strongly associated by Trisicell-PF to their seeding mutations. In addition, we applied Trisicell to single-cell whole transcriptome sequencing data from a tumor derived from the same parental melanoma cell line, which was subjected to anti-CTLA-4 immunotherapy. The phylogenies generated from both studies featured distinct subtrees, strongly associated with phenotypes including cell differentiation status, tumor growth and therapeutic response. These results suggest that Trisicell can be used for scalable tumor phylogeny reconstruction and validation through both single-cell and clonal-subline sequencing data, which may reveal strong phenotypic associations. In particular, they suggest that the developmental status and phenotypic intratumoral heterogeneity of melanoma originates from observable subclonal variation. Citation Format: Farid Rashidi Mehrabadi, Salem Malikić, Kerrie L. Marie, Eva Pérez-Guijarro, Erfan Sadeqi Azer, Howard H. Yang, Can Kızılkale, Charli Gruen, Huaitian Liu, Christina Marcelus, Aydın Buluç, Funda Ergün, Maxwell P. Lee, Glenn Merlino, Chi-Ping Day, S. Cenk Sahinalp. Trisicell: Scalable Tumor Phylogeny Reconstruction and Validation Reveals Developmental Origin and Therapeutic Impact of Intratumoral Heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB019.

Defining Coalescent Genes: Theory Meets Practice in Organelle Phylogenomics.

The species tree paradigm that dominates current molecular systematic practice infers species trees from collections of sequences under assumptions of the multispecies coalescent (MSC), that is, that there is free recombination between the sequences and no (or very low) recombination within them. These coalescent genes (c-genes) are thus defined in an historical rather than molecular sense and can in theory be as large as an entire genome or as small as a single nucleotide. A debate about how to define c-genes centers on the contention that nuclear gene sequences used in many coalescent analyses undergo too much recombination, such that their introns comprise multiple c-genes, violating a key assumption of the MSC. Recently a similar argument has been made for the genes of plastid (e.g., chloroplast) and mitochondrial genomes, which for the last 30 or more years have been considered to represent a single c-gene for the purposes of phylogeny reconstruction because they are nonrecombining in an historical sense. Consequently, it has been suggested that these genomes should be analyzed using coalescent methods that treat their genes-over 70 protein-coding genes in the case of most plastid genomes (plastomes)-as independent estimates of species phylogeny, in contrast to the usual practice of concatenation, which is appropriate for generating gene trees. However, although recombination certainly occurs in the plastome, as has been recognized since the 1970's, it is unlikely to be phylogenetically relevant. This is because such historically effective recombination can only occur when plastomes with incongruent histories are brought together in the same plastid. However, plastids sort rapidly into different cell lineages and rarely fuse. Thus, because of plastid biology, the plastome is a more canonical c-gene than is the average multi-intron mammalian nuclear gene. The plastome should thus continue to be treated as a single estimate of the underlying species phylogeny, as should the mitochondrial genome. The implications of this long-held insight of molecular systematics for studies in the phylogenomic era are explored. [c-gene; coalescent gene; concatalescence; organelle genome; plastome; recombination; species tree.].

Single-Cell Joint Methylomics and Transcriptomics Define the Epigenetic Evolution and Lineage Histories of Chronic Lymphocytic Leukemia

Genetic and epigenetic intra-tumoral heterogeneity cooperate to shape the evolutionary course of cancer progression and relapse. In chronic lymphocytic leukemia (CLL), we have previously uncovered a fundamental property of the CLL epigenome: stochastically disordered DNA methylation (DNAme), which reflects ongoing changes in the DNA methylome (‘epimutation‘). Bulk bisulfite sequencing revealed that DNAme epimutation is significantly higher in CLL compared to normal B cells. Yet, bulk analysis does not allow to phase epimutations across distant genomic loci within the same single cells. As such, it cannot answer whether epimutations occur homogenously across the cell population, or rather affect a minority of cells with highly disordered methylation.We therefore developed multiplexed single-cell reduced representation bisulfite sequencing (MscRRBS) and applied it to 382 normal B cells from four healthy donors and 324 CLL cells from three CLL patients (Fig A). We achieved bisulfite conversion rates of 99.7%±0.001 (median±SEM) and recovered on average ~500K unique CpG per cell. The overall epimutation rate was higher across CLL cells population compared to single normal B cells, in agreement with our prior bulk RRBS analysis. Uniquely, the single-cell data allowed us to measure cell-to-cell variation in epimutation rate and revealed that CLL samples, irrespective of their IgVH mutational status, exhibit minimal cell-to-cell variability, while normal B cells showed widely variable epimutation rates (Fig B).This finding is consistent with the hypothesis that epimutation serves as a cellular molecular clock, reflecting the number of generations in the cell's history. Whereas the common clonal CLL origin results in uniform epimutation rate, the variable rate in normal B cells reflects an admixture of cells with different replicative histories. In support of these findings, MscRRBS of sorted naïve and memory B cells revealed aged memory B cells exhibit higher epimutation rate than naïve B cells (Fig C).Our prior analyses quantified DNAme stochastic epimutation by assessing the concordance of adjacent CpGs recovered within the same sequencing read. As MscRRBS enables the phasing of CpGs even if not contained within the same read, we extended our analysis to calculate the DNAme concordance between any two given CpGs as function of their genomic distance. Consistent with a stochastic epimutation model, we observed faster concordance decay as a function of genomic distance in CLL compared with normal B cells. Indicative of positive selection, this phenomenon was magnified in regions with major roles in gene regulation in CLL (e.g., promoters of tumor suppressor genes such as TP53/TP63 and their targets) (Fig D).Lastly, the heritable information captured through MscRRBS epimutation data also allowed us to accurately reconstruct phylogenetic relationships between CLL cells, providing a native lineage tracing system. Lineage trees were inferred with maximum likelihood-based phylogeny reconstruction and were found to be 3-fold more robust than simple parsimony trees that ignore the phylogenetic structure. By comparing topological features (maximum tree depth and patristic distance) of normal and CLL phylogenetic trees, we have retraced the evolutionary histories of CLL populations, and found that they exhibit early branching, consistent with rapid drift after the initial malignant transformation. In contrast, normal B cell clades appear to follow a pattern that resembles typical normal B cell differentiation with late branching and deeper tree topology (Fig E, F).To validate lineage relationships inferred from single-cell DNA methylomes and enable coupling of phylogenetic relationships with transcriptional profiles, we performed parallel MscRRBS and single-cell transcriptome sequencing on 42 individual CLL cells. We found that transcriptional similarity between cells decreases as a function of their phylogenetic distance. Specifically, cells separated by one node (siblings) were ~10% more similar than cells separated by 2 nodes (cousins), providing a direct measurement of the heritability of the transcriptional profile (Fig G). To our knowledge, these measurements offer the first direct comparison of lineage and transcriptional distances between individual cells in primary patient samples, and provide further insight into the epigenetic dimension of CLL evolution. [Display omitted] DisclosuresWu:Neon Therapeutics: Consultancy. Landau:Pharmacyclics: Membership on an entity's Board of Directors or advisory committees.

Two new species of the Balkan genus Paladilhiopsis Pavlović, 1913 (Caenogastropoda, Moitessieriidae).

The Balkan Peninsula is inhabited by the worldwide most diverse subterranean gastropod fauna. This fauna is still poorly studied, since its habitats are not easily accessible, and its sampled populations are mostly not rich in specimens’ numbers. Often only empty shells are known, but the shell is hardly useful, not only in phylogeny reconstruction, but even in species determination. The exclusively obligatory subterranean family Moitessieriidae is especially poorly studied. Representatives of the genus Paladilhiopsis Pavlović, 1913 (Moitessieriidae) collected at three localities, distributed in Croatia and Bosnia & Herzegovina, were studied. The pigmentation of their shells and soft parts, as well as the female and male reproductive organs in one taxon, are presented. The partial sequences of the molecular markers mitochondrial cytochrome oxidase subunit I (COI) and nuclear histone 3 (H3) were used to infer their systematic status and phylogenetic relationships. Two species new to science are described. For one of them, also studied anatomically, 15 specimens were sequenced for COI, and all show the same haplotype.