NEW METHODS TO DETECT GHOST INTROGRESSION

197 words) 13 Although the possibility of introgression from ghost lineages (all unsampled extant and 14 extinct taxa) is now widely recognized, detecting and characterizing ghost introgression 15 remains a challenge. Here, we propose a combined use of the popular D-statistic method, 16 which tests for the presence of introgression, and the full-likelihood method BPP, which 17 determines which of the possible gene-flow scenarios, including ghost introgression, is truly 18 responsible. We illustrate the utility of this approach by investigating the reticulation and 19 bifurcation history of the genus Carya (Juglandaceae), including the beaked hickory Carya 20 ZHANG, W. P., DING, Y. M. , CAO, Y. , LI, P., YANG, Y. , PANG, X. X. , BAI, W. N. AND ZHANG, D. Y. sinensis. To achieve this goal, we generated two chromosome-level reference genomes 21 respectively for C. sinensis and C. cathayensis. Furthermore, we re-sequenced the whole 22 genomes of 43 individuals from C. sinensis and one individual from each of the 11 diploid 23 species of Carya. The latter dataset with one individual per species is used to reconstruct the 24 phylogenetic networks and estimate the divergence time of Carya. Our results 25 unambiguously demonstrate the presence of ghost introgression from an extinct lineage into 26 the beaked hickory, dispelling certain misconceptions about the phylogenetic history of C. 27 sinensis. We also discuss the profound implications of ghost introgression into C. sinensis for 28 the historical biogeography of hickory species. [BPP; Carya; D-statistic; gene flow; ghost 29 introgression] 30 INTRODUCTION 31 In recent years, increasingly available genome-scale data coupled with advances in analytical 32 methods have revealed that post-speciation gene flow between species, or simply 33 introgression, is ubiquitous across the tree of life (Mallet et al. 2016; Taylor and Larson 2019; 34 Edelman and Mallet 2021). Among many methods for detecting introgression, Patterson’s D35 statistic (Patterson et al. 2012), also known as ABBA-BABA test (Green et al. 2010; Durand 36 et al. 2011), is perhaps the most popular (Dagilis et al. 2022). The D-statistic considers 37 ancestral (A) and derived (B) alleles across the genomes of four taxa: three ingroup taxa and 38 one outgroup, with a ladder-like species tree (Fig. 1). Under the null hypothesis of no gene 39 flow among ingroup taxa, two particular allelic patterns ‘ABBA’ and ‘BABA’ should occur 40 equally frequent (Pamilo and Nei 1988). An excess of either ABBA or BABA, resulting in a 41 NEW METHODS TO DETECT GHOST INTROGRESSION D-statistic that is significantly different from zero, is usually interpreted as indicative of gene 42 flow between nonsister taxa, i.e., either between P2 and P3 (positive D-score) or between P1 43 and P3 (negative D-score) (Fig. 1a). However, an excess of ABBA or BABA can also arise 44 from a ghost lineage introgressing into P1 or P2 (Fig. 1b). More interestingly, Tricou et al.’s 45 (2022a) simulations demonstrate that most significant D-statistics are likely attributable to 46 ghost lineages, necessitating a rethink about the usual interpretation of the D-statistic. In 47 other words, failure to consider ghost introgression as a plausible working hypothesis will 48 probably lead to errors of inference in any study of introgression. 49