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Mountains of the Balkan Peninsula are significant biodiversity hotspots with great species richness and a large proportion of narrow endemics. Processes that have driven the evolution of the rich Balkan mountain flora, however, are still insufficiently explored and understood. Here we focus on a group of Cardamine Brassicaceae perennials growing in wet, mainly mountainous habitats. It comprises several Mediterranean endemics, including those restricted to the Balkan Peninsula. We used target enrichment with genome skimming Hyb-Seq to infer their phylogenetic relationships, and, along with genomic in situ hybridization GISH , to resolve the origin of tetraploid Cardamine barbaraeoides endemic to the Southern Pindos Mts. We also explored the challenges of phylogenomic analyses of polyploid species and developed a new approach of allele sorting into homeologs that allows identifying subgenomes inherited from different progenitors. We obtained a robust phylogenetic reconstruction for diploids based on 1, low-copy nuclear genes, which suggested both allopatric and ecological speciation events. In addition, cases of plastid—nuclear discordance, in agreement with divergent nuclear ribosomal DNA nrDNA copy variants in some species, indicated traces of interspecific gene flow. Our results also support biogeographic links between the Balkan and Anatolian—Caucasus regions and illustrate the contribution of the latter region to high Balkan biodiversity. An allopolyploid origin was inferred for C. Overall, our study demonstrates the importance of a thorough phylogenomic approach when studying the evolution of recently diverged species complexes affected by reticulation events at both diploid and polyploid levels. We emphasize the significance of retrieving allelic and homeologous variation from nuclear genes, as well as multiple nrDNA copy variants from genome skim data. Processes that have given rise to such biodiversity hotspots at a finer scale are complex and reflect interactions of climatic, geological, and biogeographic history of the Mediterranean region Hewitt, ; Nieto Feliner, ; Thompson, Mountains, however, are not just reservoirs, but also cradles of diversity. Great habitat diversity over short geographic distances and high topographic complexity of the mountains creates opportunities in which both adaptive and nonadaptive speciation may occur Harrison and Noss, ; Perrigo et al. These factors also favored the evolution of narrow endemism in the Mediterranean Thompson, In addition, range or niche shifts in response to geological and climatic events may bring vicariant taxa into contact and cause hybridization, with or without a ploidy level increase Nieto Feliner, Although hybridization and polyploidization are recognized as significant processes for plant evolution and speciation Soltis and Soltis, ; Soltis et al. Despite extensive botanical explorations and well-described endemism patterns in this area, speciation processes that have driven the evolution of the rich mountain flora are still not sufficiently explored. High species diversity in this area may also be connected with adjacent Anatolia, which is recognized as a center of lineage diversification in several plant genera and a possible source for the colonization of the Balkan Peninsula e. Cardamine L. The target group of species studied here comprises approximately 30 taxa, both at species and subspecies levels, and includes a few widespread taxa distributed across Europe, several endemics confined to Southern Europe, and also some species from SW Asia mainly the Anatolian and Caucasus regions. In contrast to this traditional, morphology-based delimitation, phylogenetic reconstructions suggested the existence of only two complexes resolved as respective monophyletic clades, one comprising the C. The crown group ages of both clades have been dated back to the Pliocene approximately 3—4 Mya , and divergence of the extant species likely occurred during the Pleistocene Huang et al. Most of the species diversity of these complexes is concentrated in Mediterranean mountains, which host several diploid and polyploid endemics Marhold et al. In addition, tetraploid populations from the Pindos Mts. It is a species with an uncertain circumscription and unknown polyploid origin Marhold et al. High-throughput DNA sequencing has brought excellent opportunities to improve phylogenetic inferences, particularly when facing difficult evolutionary cases, such as rapid radiations or recent speciation characterized by low genetic divergence and presence of incomplete lineage sorting ILS often complicated by hybridization and polyploidy Schmickl et al. Disentangling reticulate and polyploid evolution, however, has been a difficult task, and phylogenomic studies on polyploids have lagged behind Oxelman et al. Recent advances in this respect see, e. Approaches that account simultaneously for ILS and reticulation have been developed and improved Oberprieler et al. Those network methods can provide significant insights into the evolution of polyploids based on multilocus sequence data e. Still, standard practice when assembling sequencing reads is to generate a single consensus sequence per locus and individual, which represents a strong violation for allopolyploid genomes. The outcome of such consensus assembly is a mix of sequences retrieved from different homeologs parental subgenomes and chimeric sequences. Therefore, the crucial steps to resolve in polyploid phylogenetics are to separate sequencing reads originating from different subgenomes, assemble haplotype allele sequences, assign them to the subgenomes, and trace the parental origin of these subgenomes by multilabeled species tree or network inference methods Rothfels, A few recent studies have explored different ways how to accomplish these steps, either via mapping and categorization of the sequence reads to the reference diploid genomes Page et al. Nevertheless, the assignment of alleles to parental subgenomes has been critical and difficult to achieve readily for hundreds of loci typically recovered by target enrichment techniques. Some statistical methods for this task are under development and appear promising Freyman et al. In this article, we employ target enrichment with genome skimming Hyb-Seq using genus-specific probes to capture hundreds of orthologous low-copy nuclear loci target exons with flanking intronic and intergenic regions , along with obtaining the complete plastid genome and high-copy nuclear ribosomal DNA Weitemier et al. Here we develop a novel computational approach to sort alleles obtained from polyploids into parental subgenomes, utilizing genetic distances among alleles, and employ it to reconstruct the origin and parentage of tetraploid C. In detail, we aimed to 1 resolve phylogenetic relationships among Balkan Cardamine species and determine major factors affecting endemism patterns in mountains of the Balkan Peninsula; 2 reconstruct the origin of tetraploid C. They grow in wet habitats from lowlands up to the alpine belt, in or nearby running or standing water, usually along river and stream banks, in springs, wet meadows and pastures, in flood-plain to montane forests. Morphologically, they are characterized by pinnate basal leaves, pinnate to pinnatisect stem leaves, and white, pale pink to purple flowers arranged in racemes e. In the Balkan Peninsula, they include mostly endemics C. Presl et C. Apart from tetraploid records for C. Only diploid representatives have so far been reported from the adjacent Anatolian—Caucasus region Marhold et al. The three species, C. The position of C. Tan, or as C. Figure 1. Distribution of the Cardamine taxa under study, based on data compiled from floras, herbarium specimens, previous studies, and our own records. The western borders of the area of Cardamine amara subsp. Cardamine matthioli and C. The area of Cardamine uliginosa extends further to the south and southeast, reaching the mountains of Iran and Lebanon. The occurrence of the taxa along the elevational gradient is indicated in brackets as follows: l, lowland; mt, montane; subal, subalpine; al, alpine belt. Circles indicate our sample sites; see Supplementary Data Sheet 1 for details on the populations sampled. Here, we included all taxa occurring in the Balkan Peninsula, plus diploids from adjacent areas, C. Altogether, we sampled 46 populations representing nine species 13 taxa , which were used for ploidy level and genome size measurements by flow cytometry accessions , polymerase chain reaction PCR amplification of the nuclear ribosomal DNA nrDNA ITS region 48 accessions , and Hyb-Seq analyses 22 accessions capturing target nuclear genes, plastid DNA, and nrDNA. The tetraploid C. In addition, four diploids representing phylogenetically divergent lineages following the genus phylogeny, Carlsen et al. The list of the populations sampled and accessions analyzed is given in Supplementary Data Sheet 1. Chromosomes of C. Chromosome spreads were prepared following Marhold et al. These measurements were performed to confirm that the ploidy level of the analyzed populations and accessions is uniform and agrees with the known records, as well as to determine genome size differences between the species. Each individual was analyzed separately for precise relative or absolute nuclear DNA content values , or up to three individuals were pooled for ploidy level inference only; see Supplementary Data Sheet 1. Sample preparation followed the protocols described by Marhold et al. Relative nuclear DNA content 2C value given in arbitrary units was calculated as the ratio between the positions of the G1 peaks of the sample and the standard. Absolute nuclear DNA content 2C value given in pg was calculated from the ratio of the respective G1 peaks and the known 2C value of the standard. Polymerase chain reaction amplification, molecular cloning, and Sanger sequencing of the ITS region of nrDNA were employed to explore the diversity of ITS variants within and between individuals, both diploid and tetraploid, as well as to compare this approach with the accuracy and efficiency of retrieving different ITS variants from high-throughput genomic reads. It was included among the target genes in the Hyb-Seq approach, and therefore, the sequenced CHS clones were used to verify and optimize the assembly of allele sequences by read-backed phasing and the procedure of allele sorting into parental homeologs in tetraploid accessions see below. Multiple clones per sample were sequenced see Supplementary Data Sheet 1 for details. The pooled library was size-selected using SPRIselect beads as above and measured again with the Qubit 2. Genome skim hits were assembled into larger contigs, which were filtered for length and uniqueness, and compiled as probe sequences for bait synthesis. In total, 14, mer biotinylated RNA baits, capturing 2, exons from 1, genes, were synthesized by MYcroarray now Arbor Biosciences. Demultiplexed reads were trimmed of adapters and low-quality bases using Trimmomatic v. Read ends with quality below Q20 were discarded, and the remaining part of the read was trimmed if average quality in a 4-bp sliding window was below Q Finally, any reads trimmed to less than 50 bp were discarded. Consensus target sequences were assembled using HybPiper version 1. HybPiper generates a single consensus sequence per individual, with potentially heterozygous bases called as the nucleotide with the highest read frequency. Alignments were inspected visually, and misassemblies were removed. In addition to using the consensus supercontig sequences, the allele sequences were inferred with read-backed phasing described in detail below in Extracting Allele Sequences and Identifying Homeologs Inherited From Different Parents using WhatsHap Martin et al. Both consensus and allele data sets were used in further analyses. The recovered sequences of the target nuclear genes were analyzed using the following workflow. First, we performed phylogenomic analyses of diploid taxa only with both the consensus and allele sequence alignments , to provide a robust phylogenetic framework, using both concatenation of assembled genes and species tree inference under the multispecies coalescent model. As next, we analyzed diploids together with the tetraploid C. Considering that the tetraploid genome consists of two subgenomes that may be more or less differentiated, and thus potentially conveys conflicting phylogenetic information, we used here multiple approaches. To gain initial insights into the tetraploid genome, we used consensus supercontig sequences and applied methods that can detect and visualize conflict caused by potential discordance between consensus supercontigs retrieved from independent genes. In allopolyploids, the consensus sequences may comprise different homeologs or even consist of artificial, chimeric sequences. The analyses included supernetwork and species network calculations based on the gene trees obtained from the assembled consensus sequences, as well as single-nucleotide polymorphisms SNPs calling followed by Bayesian clustering of the SNP datasets. Finally, when the conflict between the subgenomes of the tetraploid became apparent, we derived allele sequences of the exons by read-backed phasing also from the tetraploids see below in Extracting Allele Sequences and Identifying Homeologs Inherited From Different Parents. Up to four different alleles obtained from the exons of tetraploid C. The resulting allele alignments were submitted to coalescent-based species tree inference. Branch support of the best ML trees was estimated by bootstrap BS replicates. The quartet sampling method Pease et al. PhyloNet was employed to infer a species network evaluating reticulate evolutionary relationships in individual gene trees. SuperQ v. This pipeline uses target sequences those used for probe synthesis as a reference and calls variants with respect to ploidy. To ensure that no linkage existed between sites, datasets were produced by drawing a single random SNP site from each gene containing at least 10 SNPs across the samples. The approach of Evanno et al. If the phased sequences were divided into multiple blocks, only the longest phase block for each individual was retained, and the remaining interallelic variant sites were masked by using Ns on those positions. The alleles obtained from the tetraploid C. The first step was to find two pairs of alleles, in which the alleles are closest to each other within the pairs while more distant between the pairs. Interallelic distances were estimated from the branch lengths of the corresponding exon or gene ML trees computed by cophenetic function of package stats, R Core Team, The optimal threshold for unequivocal allele sorting was set to 4 for more details about searching for the optimal threshold value, see Supplementary Text 1. This means that if an average distance between alleles within the proposed two pairs was more than four-time shorter than the average distance between alleles within any other possible arrangement, these pairs of alleles were considered unequivocally different and attributable to different homeologs see also Supplementary Text 2. If the allele sorting did not pass the desired threshold, two options were followed. Either the interallelic SNPs were masked by using Ns on those positions such unsorted, masked exons were used for further concatenation into gene alignments, see below or the sample was removed from the alignment for exon-based analyses. As next, the allele pairs were attributed to different homeologs and labeled by calculating their distances to the alleles of all diploid species. The allele pair that was closer to C. Gene alignments were also assembled, in which the phased alleles of the respective exons were concatenated to genes to obtain longer alignments with potentially stronger phylogenetic signal. After exon concatenation, the allele sorting into two homeologs was verified for each gene, with the same threshold as set for the exons above, to confirm unambiguity or to remove the equivocal sample from the gene alignments. The labeled homeologs, representing the two subgenomes within C. To explore the significance of phylogenetic placements of the A and B homeologs of C. This approach accounts for gene tree estimation error and evaluates the relative support for specific alternative hypotheses. First, the hypotheses to be tested are defined by performing constrained ML gene tree searches with enforced monophyly of the examined clades in RAxML. Here we considered three different topologies for both A and B homeologs, following the results of PhyloNet analyses and exon- and gene-based species trees inferred from phased sequences see Results for details. The topology test was then performed for each nuclear gene or exon i. The AU test performs simultaneous comparisons of multiple trees and estimates a P value for each topology. The trees are then ranked according to the P values, and the results are visualized as plots of the cumulative number of constrained gene trees and their AU test P values for each topology. R10 Kearse et al. The sequences recovered from HybPiper were also proceeded further to read-backed phasing to retrieve multiple nrDNA variants, as described above. This pipeline utilizes Trimmomatic v. Two chloroplast DNA cpDNA alignments were generated and used for phylogenetic tree reconstructions, one comprising the complete sequences of the LSC, SSC, and IRb regions, including intergenic spacers, and the other consisting of the concatenated sequences of annotated genes only. Although it has been widely assumed that plastid genes are inherited as a single locus, favoring their concatenation before phylogenetic analyses, some recent studies have indicated that these genes may not be as tightly linked as expected and may experience different evolutionary histories. Genomic in situ hybridization was performed in C. Mitotic chromosome spreads of C. The immunodetection of hapten-labeled probes was performed as follows: biotin-dUTP was detected by avidin—Texas red Vector Laboratories and amplified by goat anti-avidin—biotin Vector Laboratories and avidin—Texas red; digoxigenin-dUTP was detected by mouse antidigoxigenin Jackson ImmunoResearch and goat anti-mouse—Alexa Fluor Invitrogen. Images were acquired separately for the three fluorochromes using appropriate excitation and emission filters AHF Analysentechnik. Flow cytometry confirmed the tetraploid level in all five sampled populations 27 individuals in total; Supplementary Data Sheet 1. Ploidy level screening within the other studied species showed consistent results, supporting a single, diploid level. Only few exceptions were identified, such as one apparently triploid individual of C. The diploid species displayed a wide range of 2C values, and most of the species differed from each other in their nuclear DNA content Supplementary Data Sheet 1 , Figure 2. Populations of C. UD, northwestern Turkey and the Caucasus Mts. AM, Armenia showed markedly different values in accordance with their genetic divergence, see below and were kept as two separate entities. The smallest genome sizes were observed in C. In accordance with the tetraploid level, the largest nuclear DNA content was measured in C. Figure 2. Genome size variation of the Cardamine species under study, based on flow cytometric analyses. In the tetraploid Cardamine barbaraeoides marked by asterisk , however, DNA content of the meiotically reduced genome corresponding to the 2 x level is assessed and presented. Boxplot graphs show the 25th and 75th percentiles boxes , median values vertical lines within boxes , and minimum to maximum values whiskers. Orange color is used for species of the Cardamine amara group, blue for the other diploids, and black for the tetraploid C. The number of analyzed individuals per species is indicated. See Supplementary Data Sheet 1 for more details and population-level values. The sequencing process yielded, on average, 1. Adapter trimming, quality filtering and deduplication resulted in an average loss of 1. Of the remaining reads, Mean coverage of the plastid genome fluctuated widely among samples, from Of the 2, exons from 1, genes, targeted by the designed RNA baits, 1, The length of the exon alignments ranged from 63 to 3, bp bp on average , whereas the gene length ranged from 72 to 8, bp, with a mean of 1, bases. The concatenated alignment of all genes was 1,, bp long. Maximum likelihood analysis of the diploid taxa, based on the concatenated dataset of all 1, loci consensus supercontigs from 1, nuclear genes, resulted in a tree with two major well-supported clades Figure 3A , Supplementary Figure 1. One clade comprised accessions of C. Because the two geographically distant accessions of C. The species trees inferred using ASTRAL from 1, ML gene trees, based either on consensus sequences Figure 3B or phased allele sequences results not shown , showed identical topologies and branch support. These trees were also fully congruent with the ML tree of the concatenated dataset. Two branches that received lower QC values in the ML tree, congruently, showed slightly decreased local posterior probabilities in the species trees. Figure 3. Phylogenetic trees inferred from the complete dataset of 1, nuclear genes, based on consensus supercontig sequences of diploid Cardamine accessions. Orange color is used for the clades and branches of the Cardamine amara group, whereas blue is used for the remaining diploids resolved in the sister position. Multiple individuals per species are shown collapsed see Supplementary Figure 1 for the fully labeled version of the tree including bootstrap support. Branch support is indicated by quartet concordance QC values colored circles in the nodes. Branch support is indicated by pie charts, depicting three local posterior probabilities for the given branch dark blue for the main topology as resolved here and light blue for the alternative ones; not shown for the fully supported branches when the local posterior probability for the present topology equals 1. The SuperQ network derived from 1, ML gene trees based on consensus sequences displayed two well-differentiated groups of diploid taxa corresponding to the two major clades as resolved above and strong conflict in the placement of the tetraploid accessions Figure 4A. The species network analysis PhyloNet based on the same set of ML gene trees suggested a hybrid origin of C. Interestingly, some of the repeated PhyloNet runs indicated a reticulation event also for C. Figure 4. Phylogenetic analyses indicating the hybrid allopolyploid origin of the tetraploid Cardamine barbaraeoides. Orange color is used for the accessions from the Cardamine amara group C. A Supernetwork representation of quartets generated in SuperQ, which was derived from 1, nuclear gene trees estimated in RAxML and based on consensus supercontig sequences. B Species network inferred in PhyloNet from the same set of 1, nuclear gene trees. Inheritance probabilities are shown along the branches indicating the origin of C. The coloring in the graph indicates the sample assignment to the two genetic clusters. Thick vertical lines separate different species. Single-nucleotide polymorphisms calling utilized genes, which harbored at least 10 SNPs across the samples. Thus, all these analyses showed strong conflict in the consensus supercontig sequences of the tetraploid and suggested an allopolyploid origin of C. Read-backed phasing yielded two alleles per exon for diploids and four alleles for tetraploids. In diploids, the level of heterozygosity varied widely from Allele phasing in the tetraploid C. Homozygous The complete set of 1, targeted exons of C. The optimized threshold for allele sorting invalidated They definitely regarded the homozygous exons and partially heterozygous one those with the alleles in the ratio and part of the other heterozygous exons Supplementary Table 1. Alleles from all three samples of C. At the gene level with concatenated exons , attempts to sort the alleles into two different homeologs succeeded in Subsequently, for species tree inferences in ASTRAL, we assembled multiple datasets that were derived from phased exon- and gene-based alignments. For exons, they included the following: No. The species trees inferred from all three datasets recovered the same topology and differed only in some branch support values Figure 5A , Supplementary Figures 4A—C. As for the diploid taxa, the topology was largely congruent with that of the trees derived from the diploid sequence data only Figure 3 , see above , differing only in the placement of the species pair C. The position of this species pair, however, received a relatively low QC value in the tree of diploids Figure 3A. The A homeolog of C. The B homeolog of C. Figure 5. Phylogenetic analyses based on phased allele sequences of exons A or genes B , for which alleles of all three accessions of Cardamine barbaraeoides were successfully phased and sorted into A and B homeologs. Branch support is indicated by pie charts, depicting three local posterior probabilities for the given branch not shown for the fully supported branches. Tree branches are colored according to the group membership; branches in black show C. The plots depict the cumulative number of constrained gene trees, which support the given topology, and their P values obtained from the approximately unbiased AU tests. The tested topologies were as follows: homeolog A, red: C. Species trees and GGI plots from the alternative datasets allowing for missing tetraploid accessions are presented in Supplementary Figure 4. Similarly, as for the exons, three datasets of phased gene-based alignments were assembled: No. The species trees recovered the same topology for all three datasets, with differences only in branch support Figure 5B , Supplementary Figures 4D—F , and were almost identical to those inferred from exon-based data. The only difference was in the placement of the A homeolog of C. When computing distances between the alleles retrieved from C. Topology tests based on the GGI analyses yielded robust and highly congruent results both from the exon- and gene-based datasets, when considering the set of trees in which alleles from all three accessions of C. The GGI results clearly favored the topology in which C. Two alternative topologies, i. As for the placement of the B homeolog of C. The second topology, with C. Slightly different and also equivocal GGI results in some cases were obtained when including also the exons or genes, in which one or two accessions of C. In those datasets, the two topologies with C. The placement of homeolog A in the dataset of 1, exons also remained equivocal, with similar support given for its sister position to either C. In the dataset of genes, the same topology for C. The ITS alignment obtained from molecular cloning was bp long and comprised sequences from 48 ingroup individuals. It contained variable sites High intraspecific and even intraindividual diversity of the ITS variants ribotypes was revealed in the diploid taxa Supplementary Data Sheet 1. Nevertheless, the ribotypes observed within individuals and within species were mostly similar and clustered together, with the exceptions of rare divergent ribotypes found in a single accession of C. In accordance with the data from the target nuclear loci, genetic differentiation was observed within C. In the tetraploid C. Three ribotypes i. The rest of the ribotypes Figure 6. Cloning: dataset obtained from molecular cloning and Sanger sequencing; phasing: dataset obtained from read-backed phasing of Hyb-Seq reads, with multiple nrDNA variants retrieved per sample; consensus: consensus assembly with the majority rule base calls from Hyb-Seq reads; ambiguity: ambiguous assembly from Hyb-Seq reads with intraindividual SNPs replaced by IUPAC codes. See Supplementary Figure 6 for the complete ML trees obtained from these datasets. The ITS alignment from the consensus assembly of the reads mapping to the reference sequence comprised 20 ingroup sequences with 53 variable 8. The alignment of the ambiguous assembly contained 68 ambiguous bases and 43 variable 6. Read-backed phasing of the assembled ITS sequences resulted in 1 to 4 ITS variants per individual, and the alignment comprised 47 different ingroup sequences with 77 variable The topologies of the ML trees obtained from the consensus and ambiguous datasets were largely congruent Figure 6 , Supplementary Figure 6 , except of the position of C. In the consensus dataset, C. The former topology agreed with the position of all but one ribotype resolved in C. In both the consensus and ambiguous datasets, the tetraploid C. Phasing revealed the presence of divergent nrDNA variants in both C. The rare ribotypes clustering with C. The alignment of the concatenated annotated genes was 96, bp long and included 74 protein-coding genes and 31 tRNA and four rRNA genes. The ML trees inferred from the two alignments showed high congruence Supplementary Figure 7. Topological differences were found only in clades that displayed very short branches and low BS support. Two major clades with high BS were retrieved in both ML trees, which corresponded to those resolved by nuclear genes. One comprised C. The other major clades in the ML trees comprised two well-differentiated and supported subclades. One subclade consisted of C. The other subclade comprised C. Except of the last two species, resolved in a well-supported sister position, the relationships within this subclade received only low support and differed between the two cpDNA datasets. Two internal branches, which determined the positions of C. This agrees with the topological differences between the ML trees from the concatenated data and thus suggests that the placement of these two species is uncertain. Figure 7. Branch support is shown by pie charts, depicting three local posterior probabilities for the given branch not shown for the fully supported branches. Tree branches are colored according to the group membership; branches in black show Cardamine barbaraeoides. DAPI staining of mitotic chromosomes in C. The L chromosomes carried terminal heterochromatin knobs Figure 8B , which were previously reported in the C. The gDNA probes of the three tested accessions from the C. The gDNA probes of the other five accessions tested C. Although quantification of hybridization signals is problematic, we observed stronger fluorescence of the gDNA probes of C. These two major groups of species differ in their genome size see above in Chromosome Numbers and Genome Size Variation and Figure 2. This difference is reflected by bigger chromosomes, more pericentromeric heterochromatin, and terminal heterochromatic knobs within the C. Figure 8. B Close-up view of a heterochromatic knob-bearing chromosome the terminal knob marked by arrowheads. Uncovering phylogenetic relationships within recently diverged plant groups can be challenging even at the diploid level. Persistence of ancestral polymorphisms, low genetic divergence between species, and both past and contemporary interspecific gene flow hamper robust phylogenetic inferences Naciri and Linder, ; see, e. In the present study, we applied a target enrichment approach, recently shown to provide high resolution also at low phylogenetic levels between the closest relatives Villaverde et al. Indeed, using custom, genus-specific probes, we were able to retrieve sequences from more than 1, nuclear genes from each sample. At the diploid level, the topologies of the ML tree obtained from concatenation of targeted loci and the coalescent-based species tree were fully congruent, which suggests a low degree of ILS, in accordance with high support in the species tree Figure 3. Using either consensus or phased allele sequences, here we obtained the same species tree topology, which additionally supports the robustness of our data. Some topological conflicts, however, appeared between the nuclear- and plastome-derived phylogenetic trees Figure 7. This plastid—nuclear discordance among the diploids, described in more detail below, can identify traces of interspecific gene flow and thus shed further light onto the evolutionary histories of Cardamine species. With more extensive sampling in the future, including all representatives of the studied species complex across Europe, this approach has great potential to infer their phylogeny comprehensively. Here we provide our first insights from the perspective of Balkan species. Cardamine lazica , a species from the Pontic mountains and western Caucasus, was identified here as a sister species to C. Furthermore, the present data supported the monophyly of C. One accession of C. Furthermore, both nuclear and plastid data congruently revealed a sister species relationship between C. On the other hand, the population of C. Previous studies have already indicated that it is a highly polymorphic species Marhold et al. The relationships between the other four species, C. Both ongoing and past gene flows, the latter probably facilitated by range shifts in glacial-interglacial periods, have been inferred to occur between C. The lowland species C. Interestingly, the phylogenetic placements of its homeologs do not favor a very recent i. Alleles retrieved from two subgenomes appeared differentiated from those observed in present-day diploids, suggesting that the parental species of C. Still, the former hypothesis of an older allopolyploidization event and a relict character of this species is favored also by the plastome tree, which confirmed the same phylogenetic placement of C. Based on a recently published tribe-wide dated phylogeny Huang et al. The highly restricted occurrence of C. S Pindos remains intriguing. We may speculate whether the present occurrence is only a remnant of a previously wider range, or whether the allopolyploidization event took place within the current area and the species never expanded much beyond it. The Cardamine taxa under study exhibit parapatric to allopatric distributions, and all occupy similar wet habitats, partly with different elevational preferences Figure 1. From the presented phylogenetic reconstructions, we can infer that they likely evolved via both allopatric and ecological speciation processes, which have also been affected by interspecific gene flow. The studied species complexes comprise numerous endemics not only in the Balkan Peninsula but also across the other parts of the Mediterranean Marhold et al. These patterns may reflect past range fragmentation in response to Pleistocene climatic oscillations Nieto Feliner, , as well as spatially restricted gene flow and species dispersal, which may be the two principal causes, acting in concert, of the current high endemism rate in these species complexes. Lowland-alpine species pairs, such as C. Ecological niche analyses in four species of the C. It appears that divergent ecological requirements may play a certain role in the evolution of these species complexes but probably do not constitute a strong constraint that would significantly hamper range expansion and explain the high incidence of endemics. With the present results, we provide additional support for the prominent role of Mediterranean mountains both as cradles and reservoirs of species and genetic diversity and, more specifically, for the contribution of polyploid speciation to the origin of biodiversity hotspots. Indeed, the Southern Pindos range, the area of C. Quaternary climatic oscillations have led to species range shifts, repeated range fragmentation, and reduction followed by expansion, and these processes have facilitated contacts between previously isolated lineages and brought opportunities for hybridization Nieto Feliner, ; Marques et al. The great ecological and topographic heterogeneity of Mediterranean mountains has likely favored not only hybridization events, but also the establishment and persistence of newly formed allopolyploids. Several examples of polyploid endemics confined to some mountains of the Balkan Peninsula e. Our present study revealed cases in which Balkan taxa have their phylogenetically closest counterparts in the Anatolian or Caucasus regions, in support of the known biogeographic links between these areas Strid, ; Bilgin, ; Thompson, However, they may have been penetrated especially in colder periods at the Pliocene—Pleistocene transition and during Pleistocene glaciations Strid, ; Ansell et al. One of the common phylogeographic patterns recognized in Anatolia suggests a genetic break within Anatolia, differentiating populations in western Anatolia and the Balkan Peninsula from those in eastern Anatolia Bilgin, This pattern resembles the present case of high affinity between C. Furthermore, closer evolutionary relationships and traces of hybridization between C. The employment of low-copy nuclear genes in phylogenetic studies, especially when polyploids are involved, is crucial. Nuclear genes show biparental inheritance and typically retain evidence of a reticulate history e. Still, it is known that individual gene trees may show discordant histories that do not match the true evolutionary history, because of various processes related to the complexity of nuclear genomes, such as high allelic variation and ILS, nonhomologous recombination, gene duplication, and gene loss Maddison, ; Small et al. Therefore, the use of multiple unlinked loci has been strongly advised Naciri and Linder, Target enrichment techniques that may capture hundreds of unlinked orthologous loci are promising in resolving the origins and evolutionary histories of polyploid species with much greater confidence Kamneva et al. Assembly of short sequence reads, however, remains a challenge for allopolyploid genomes, because a mixture of reads belonging to both homologous and homeologous loci is obtained Kyriakidou et al. Most phylogenetic studies have used consensus assembly e. This means, however, that sequences from different homeologs parental subgenomes , as well as chimeric sequences, may be retrieved. Allopolyploid speciation is then commonly inferred by network analyses, which account for both ILS and hybridization Crowl et al. In the present study, we employed the network analyses based on the consensus sequences, which, in congruence with the SNP data analyses, identified conflicting signal within the data and suggested allotetraploid origin of C. Nevertheless, as a significant step further, we proceeded to allele assembly and sorting. Some approaches or tools for assembling allele sequences and distinguishing among homeologs have recently been proposed for polyploids Page et al. Several previous studies used parallel amplicon sequencing to analyze polyploid species, but capturing only a low number of loci up to 12 loci and with manual homeolog identification and sorting Brassac and Blattner, ; Rothfels et al. Here we propose a novel approach in which hundreds of loci obtained from target enrichment techniques can be analyzed simultaneously and allele sorting does not require manual inspection and labeling. We inferred phased alleles based on available tools and developed a bioinformatics procedure to sort them into homeologs. Allele sorting is based on calculating distances between alleles, obtained from branch lengths of corresponding gene trees, first between alleles from a given polyploid to identify allele pairs and then from its diploid relatives. Homeolog labeling is based on allele pair distances to the suspected maternal species, as identified by plastome analyses. The phylogenetic positions of the obtained homeologs, representing two parental subgenomes in the polyploid, are then explored by a species tree inference. This approach is most straightforward when the maternal species is at least approximately determined, but could be applied even if this information is unknown, in the case of missing cpDNA data, a possibly extinct or an unsampled maternal parent. Under such scenarios, one of the most closely related species, a possible progenitor of the investigated polyploid, could be identified from the network analyses inferred from the consensus sequences and subsequently used for homeolog labeling. Two shortcomings may potentially limit the efficiency of our approach. Successful allele sorting in polyploids, namely, depends on both parental genome divergence and the informativeness phylogenetic signal of target loci. Alleles from some genes may not be unequivocally sorted into homeologs, because of low phylogenetic signal and low sequence divergence. Still, when employing a large set of target loci during sequence capture and including also more variable flanking intronic and intergenic regions as is achieved via the Hyb-Seq approach; Weitemier et al. Here we demonstrate that with several reduced datasets, allowing either missing accessions or loci, we obtained the same topologies of the species trees, and the same allopolyploid scenario was inferred. The second obstacle is related to the short length of sequence reads obtained from the Illumina platform, which throws down a challenge to allele phasing software. The shorter length of sequence reads more often causes sequence splitting into multiple phase blocks. Variant sites are phased with other sites within the given block but cannot be phased with respect to variants in the other blocks because of insufficient read data between the blocks see Kates et al. If multiple phase blocks occurred, phased alleles were retained only in the largest phase block, and the remaining intraindividual variants were masked Concatenation of exon sequences to genes has a dual partially contradicting effect. The sequence length has a positive effect on the resolution of the phylogenetic tree. On the other hand, concatenation involved both sorted and unsorted with masked interallelic SNPs exons, which means that interallelic variation was partly homogenized. To investigate the impact of this issue on phylogenetic reconstruction, we compared two datasets that differed in the length of the loci used and the amount of masked SNP variants: shorter exon-based and longer gene-based datasets. Only a single topological difference was observed between the species trees inferred from these datasets, inspected in more detail by running GGI topology tests Arcila et al. Overall, we demonstrate that allele phasing and distinguishing homeologous copies are crucial for determining the origin of polyploids and for resolving reticulate evolution of polyploid complexes. Genome skimming, performed as part of the Hyb-Seq approach Weitemier et al. Because high-throughput sequencing recovers reads from all potential repeat variants within and among nrDNA loci, we explored different possibilities how to deal with such intraindividual polymorphisms. We compared the two most commonly used coding schemes for such polymorphisms, the consensus majority one and the ambiguous one Vargas et al. Indeed, as we revealed in the cases of C. On the other hand, really rare variants, such as those in C. By contrast, with the amplicon sequencing approach, Tkach et al. Therefore, although genome skim data are easy to obtain and provide huge amounts of data from both organellar and nuclear DNA high-copy fractions, they should be considered with caution especially in groups with reticulate evolutionary histories e. With the recently increasing efforts to develop target enrichment probes specific to relatively narrow focus groups e. Our study demonstrates the importance of a thorough phylogenomic approach when studying the evolution of recently diverged species complexes affected by reticulation events at both the diploid and polyploid levels. We emphasize the significance of retrieving allelic and homeologous variation from nuclear genes, as well as divergent nrDNA copy variants from high-throughput genomic data. Along with the employment of multiple analysis methods, they all, in concert, allow to resolve the origins of polyploids, detect cases of interspecific gene flow, and explain plastid—nuclear phylogenetic discordance. We suggest that despite recent advances in phylogenomic data analyses, significant improvements are needed especially in processing and analyzing sequence data from polyploid and hybrid genomes. 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