Chromosomal Evolution and Apomixis in the Cruciferous Tribe Boechereae

The mustard family (Brassicaceae) comprises several dozen monophyletic clades usually ranked as tribes. The tribe Boechereae plays a prominent role in plant research due to the incidence of apomixis and its close relationship to Arabidopsis. This tribe, largely confined to western North America, harbors nine genera and c. 130 species, with >90% of species belonging to the genus Boechera. Hundreds of apomictic diploid and triploid Boechera hybrids have spurred interest in this genus, but the remaining Boechereae genomes remain virtually unstudied. Here we report on comparative genome structure of six genera (Borodinia, Cusickiella, Phoenicaulis, Polyctenium, Nevada, and Sandbergia) and three Boechera species as revealed by comparative chromosome painting (CCP). All analyzed taxa shared the same seven-chromosome genome structure. Comparisons with the sister Halimolobeae tribe (n = 8) showed that the ancestral Boechereae genome (n = 7) was derived from an older n = 8 genome by descending dysploidy followed by the divergence of extant Boechereae taxa. As tribal divergence post-dated the origin of four tribe-specific chromosomes, it is proposed that these chromosomal rearrangements were a key evolutionary innovation underlaying the origin and diversification of the Boechereae in North America. Although most Boechereae genera exhibit genomic conservatism, intra-tribal cladogenesis has occasionally been accompanied by chromosomal rearrangements (particularly inversions). Recently, apomixis was reported in the Boechereae genera Borodinia and Phoenicaulis. Here, we report sexual reproduction in diploid Nevada, diploid Sandbergia, and tetraploid Cusickiella and aposporous apomixis in tetraploids of Polyctenium and Sandbergia. In sum, apomixis is now known to occur in five of the nine Boechereae genera.

The mustard family (Brassicaceae) comprises several dozen monophyletic clades usually ranked as tribes. The tribe Boechereae plays a prominent role in plant research due to the incidence of apomixis and its close relationship to Arabidopsis. This tribe, largely confined to western North America, harbors nine genera and c. 130 species, with >90% of species belonging to the genus Boechera. Hundreds of apomictic diploid and triploid Boechera hybrids have spurred interest in this genus, but the remaining Boechereae genomes remain virtually unstudied. Here we report on comparative genome structure of six genera (Borodinia, Cusickiella, Phoenicaulis, Polyctenium, Nevada, and Sandbergia) and three Boechera species as revealed by comparative chromosome painting (CCP). All analyzed taxa shared the same seven-chromosome genome structure. Comparisons with the sister Halimolobeae tribe (n = 8) showed that the ancestral Boechereae genome (n = 7) was derived from an older n = 8 genome by descending dysploidy followed by the divergence of extant Boechereae taxa. As tribal divergence post-dated the origin of four tribe-specific chromosomes, it is proposed that these chromosomal rearrangements were a key evolutionary innovation underlaying the origin and diversification of the Boechereae in North America. Although most Boechereae genera exhibit genomic conservatism, intra-tribal cladogenesis has occasionally been accompanied by chromosomal rearrangements (particularly inversions). Recently, apomixis was reported in the Boechereae genera Borodinia and Phoenicaulis. Here, we report sexual reproduction in diploid Nevada, diploid Sandbergia, and tetraploid Cusickiella and aposporous apomixis in tetraploids of Polyctenium and Sandbergia. In sum, apomixis is now known to occur in five of the nine Boechereae genera.

INTRODUCTION
Geographically well-defined clades provide ideal study systems for understanding the role of whole-genome duplications (WGDs, polyploidy) and chromosomal rearrangements in speciation and diversification. Frequently, a group of species confined to an island, mountain range, or (sub)continent is assumed to have originated in this region, perhaps following an earlier dispersal event from another part of the world (e.g., Linder and Barker, 2014;Givnish et al., 2016). With the advent of molecular phylogenetics, in many cases, the inferred monophyly of a group has confirmed its geographical determinant and helped to elucidate its origin as well as directionality of later migrations and dispersals (e.g., Cowie and Holland, 2008;Ogutcen and Vamosi, 2016;Huang et al., 2018;Carter et al., 2019;Kim et al., 2019). A geographically restricted clade might be supported by different synapomorphies, such as morphological traits, specific metabolic pathways, pollination syndromes, or a shared WGD, some falling in the category of rare genomic changes (RGCs, Rokas and Holland, 2000). Structural chromosomal changes may underlie incipient reproductive isolation inducing species splits and the evolution of separate gene pools, i.e., cladogenesis (Faria and Navarro, 2010). Dysploidal (i.e., chromosome number changes caused by fusions and fissions) as well as non-dysploidal (i.e., deletions, duplications, inversions, and translocations) chromosomal rearrangements can modify recombination frequency, gene expression, the duration of cellular processes (replication, mitosis, and meiosis), and the degree of infertility of heterozygous hybrids. Thus, some chromosomal rearrangements may precipitate lineage splitting yet occur within a monophyletic clade (Freyman and Höhna, 2017).
Here we focus on the tribe Boechereae which harbors c. 130 species. The vast majority of Boechereae taxa occurs only in North America, with one of these extending to Greenland and three species being endemic to the Russian Far East (Alexander et al., 2013;Doudkin and Volkova, 2013). Molecular studies (Beilstein et al., 2010;Nikolov et al., 2019) using various chloroplast and nuclear gene markers support the Boechereae (with a shared chromosome base number of x = 7) as a monophyletic clade sister to the New World tribe Halimolobeae (x = 8; 39 species in five genera, Al-Shehbaz, 2012). Alexander et al. (2013) recognized nine genera of Boechereae, seven of which (Anelsonia J. F. Macbride and Payson, Cusickiella Rollins, Nevada N. H. Holmgren, Phoenicaulis Nuttall, Polyctenium Greene, Sandbergia Greene, and Yosemitea P. J. Alexander and Windham) are mono-or bispecific, and, except for Sandbergia whitedii (Piper) Greene, restricted to the western United States. Boechera Á. Löve and D. Löve is by far the most diverse genus of the tribe, largely confined to the western part of the North American continent (Alexander et al., 2013). One group of eight species often assigned to Boechera was transferred to the genus Borodinia N. Busch by Alexander et al. (2013). This species group has the most discrete geographic range, apparently restricted to eastern North America and the Russian Far East [Borodinia macrophylla (Turcz.) O. E. Schulz]. Despite their largely allopatric distributions, Boechera and Borodinia species have hybridized in nature to produce one widespread sexual tetraploid and a series of apomictic triploids and tetraploids that erase any morphological distinctions between the two genera (Windham et al., 2014). When subsumed within Boechera, these lineages are informally designated the "western" and "eastern" clades, respectively.
The species now assigned to Boechera (x = 7) were originally included in Arabis L. (tribe Arabideae; x = 8), but a series of molecular analyses (Koch et al., 2000;Beilstein et al., 2010;Nikolov et al., 2019) has shown that these genera belong to different major lineages of Brassicaceae. Boechera is phylogenetically closely related to the model genus Arabidopsis Heynh. (Figure 1A) and is best known for its classic agamic complex consisting of numerous, morphologically diverse, facultative, and obligate gametophytic apomicts. These are generally of hybrid origin, arising from a diverse array of sexual diploids with more restricted habitats. The genus is named for Danish botanist Tyge Böcher, who first documented apomixis in Boechera holboellii (Hornem.) Á. Löve and D. Löve (Böcher, 1951). The relatively close relationship of these species to Arabidopsis, combined with its diversity of ploidies (Alexander et al., 2015) and apomixis types, at both the diploid and polyploid levels (Carman et al., 2019), have made the genus a major focus for apomixis research (e.g., Naumova et al., 2001;Schranz et al., 2005;Lee et al., 2017;Kliver et al., 2018;Rojek et al., 2018;Brukhin et al., 2019). Until recently, apomixis within Boechereae was thought to be confined to the large genus Boechera. However, Mandáková et al. (2020) documented the occurrence of apomixis at the diploid, triploid, and tetraploid levels in one of the smaller genera of Boechereae (Phoenicaulis), raising the possibility that apomixis might also occur in other Boechereae genera.
In flowering plants, apomixis can be defined as asexual seed formation where clonal embryos originate either from unreduced eggs produced in unreduced female gametophytes (gametophytic apomixis) or from somatic cells of the ovule wall without an intervening unreduced gametophyte generation (sporophytic apomixis). In sporophytic apomixis, a reduced gametophyte forms, which supports the clonal embryo while it develops. The reduced gametophyte may or may not contain a sexually derived embryo (Asker and Jerling, 1992;Hand and Koltunow, 2014). Gametophytic apomixis is a prominent mode of reproduction  in Boechera (Böcher, 1951;Roy, 1995;Naumova et al., 2001;Schranz et al., 2005), where it has greatly increased the diversity of genotypes and phenotypes by stabilizing the products of reticulate evolution (Beck et al., 2012;Carman et al., 2019). Three types of gametophytic apomixis occur in Boechera, and these are differentiated based on where the unreduced gametophyte forms: (i) if from a megasporocyte [megaspore mother cell (MMC)], it is referred to as Antennaria type diplospory, (ii) if from an apomeiotic dyad member of a first division meiotic restitution event, it is referred to as Taraxacum type diplospory, and (iii) if from a nucellar or parietal cell, it is referred to as Hieracium type apospory (Carman et al., 2019). The Antennaria type appears to be an oddity in Boechera that has been observed only rarely in plants that otherwise reproduce by Taraxacum type diplospory. In contrast, Taraxacum type diplospory and apospory are more commonly encountered in natural populations of Boechera than is sexual reproduction (Carman et al., 2019).
In the light of the fragmentary knowledge of genome evolution and reproductive modes in the Boechereae, we embarked on comparative cytogenetic and embryological analysis of several taxa representing the tribal diversity. We followed several aims: (1) To expand cytogenomic sampling of the species and genera of Boechereae to provide a more complete understanding of chromosomal evolution in the tribe, (2) to determine whether diversification within the tribe was accompanied by clade-specific chromosomal rearrangement, (3) to test whether an ancestral n = 7 genome inferred for Boechera and Phoenicaulis (Mandáková et al., 2015b(Mandáková et al., , 2020 provides an accurate reconstruction of the ancestral genome of the tribe as a whole, and (4) to gain insights into the reproductive modes of previously unsampled Boechereae genera by conducting embryological analyses on most of the species analyzed cytogenomically.

Species Analyzed
Ten Boechereae species were selected to span the phylogenetic diversity of the tribe (Figure 1). These included Boechera formosa (Greene) , Boechera gracilipes , and S. whitedii (2n = 21). As the tribe Halimolobeae was repeatedly retrieved as a sister clade to Boechereae (e.g., Beilstein et al., 2010;Couvreur et al., 2010;Alexander et al., 2013), Pennellia micrantha (A. Gray) Nieuwland (2n = 16) was selected to represent the Halimolobeae clade outgroup. The origins of the analyzed populations are listed in Supplementary Table S1; multiple individuals were analyzed from each population.
The analyzed plants were either collected in the wild or grown in a growth chamber from seeds collected in the wild. Young inflorescences of the analyzed plants were collected and fixed in freshly prepared fixative (ethanol: acetic acid, 3: 1) overnight, transferred to 70% ethanol and stored at −20 • C.

DNA Probes
The BAC clone T15P10 (AF167571) of Arabidopsis thaliana (L.) Heynh. bearing 35S rRNA gene repeats was used for in situ localization of nucleolar organizer regions (NORs), and the A. thaliana clone pCT4.2 (M65137), corresponding to a 500 bp 5S rDNA repeat, was used for localization of 5S rDNA loci. For Comparative Chromosome Painting (CCP), 674 chromosomespecific BAC clones of A. thaliana (The Arabidopsis Information Resource, TAIR 2 ) were used to establish contigs corresponding to the 22 genomic blocks (GBs) and eight chromosomes (AK1-AK8) of the Ancestral Crucifer Karyotype (ACK; Lysak et al., 2016). See Supplementary Tables S2-S8 for the list of BAC clones used to identify the 22 GBs on chromosomes of the Boechereae species. To determine and characterize inversions and split GBs, some BAC contigs were split into smaller subcontigs and differentially labeled (e.g., Aa, Ab, Ca, Cb, see Supplementary  Tables S3-S8). All DNA probes were labeled with homemade biotin-dUTP, digoxigenin-dUTP, or Cy3-dUTP by nick translation as described by Mandáková and Lysak (2016b).

Comparative Chromosome Painting (CCP)
DNA probes were pooled to follow the design of a given experiment, ethanol precipitated, dried, and dissolved in 20 µl of 50% formamide and 10% dextran sulfate in 2 × SSC. The 20 µl of the dissolved probe were pipetted on a chromosomecontaining slide and immediately denatured on a hot plate at 80 • C for 2 min. Hybridization was carried out in a moist chamber at 37 • C overnight. Post-hybridization washing was performed in 20% formamide in 2 × SSC at 42 • C three times (5 min each time). Hybridized probes were visualized either as the direct fluorescence of Cy3-dUTP or through fluorescently labeled antibodies against biotin-dUTP and digoxigenin-dUTP following Mandáková and Lysak (2016b). Chromosomes were counterstained with 4 ,6-diamidino-2-phenylindole (DAPI, 2 µg/ml) in Vectashield antifade. Fluorescence signals were analyzed and photographed using a Zeiss Axioimager epifluorescence microscope with a CoolCube camera (MetaSystems). Images were acquired separately for all four fluorochromes using appropriate excitation and emission filters (AHF Analysentechnik). The four monochromatic images were pseudocoloured, merged, and cropped using Photoshop CS (Adobe Systems) and ImageJ (National Institutes of Health).

Embryological Analyses
Clusters of pre-anthesis staged floral buds were fixed in 3:1 fixative for 48 h and stored in 70% EtOH. Ovaries were excised, cleared, measured, and mounted following Mandáková et al. (2020). An Olympus (Center Valley, PA, United States) BX53 microscope with differential interference contrast (DIC) optics and equipped with a DP74 digital camera with cellSens Dimension 1 software (Olympus) was used to investigate parietal cell, MMC, and female gametophyte origins.

RESULTS
Based on the ACK, and on previously analyzed Boechereae species (Mandáková et al., 2015a(Mandáková et al., , 2020, detailed comparative cytogenetic maps were constructed by CCP for each of the 10 Boechereae species and for P. micrantha (Figure 2 and Supplementary Tables S2-S8 Table S2). The ACK-like Pennellia genome further corroborrated the earlier assumption (Mandáková et al., 2015b(Mandáková et al., , 2020 that the Most Recent Common Ancestor (MRCA) of Boechereae and Halimolobeae had eight chromosomes and structurally resembled the ancestral genome of crucifer lineage I.

Overall Structural Stasis of Boechereae Genomes
Comparative chromosome painting with painting probes designed according to the structure of the seven linkage groups in Boechera (Mandáková et al., 2015a) and Phoenicaulis (Mandáková et al., 2020), were effective in identifying all seven or 14 chromosome pairs among the 10 Boechereae species analyzed. All 10 genomes had a very similar organization (Figure 2), except for a few species-specific chromosomal rearrangements (see below). The overall structural genome similarity among different Boechereae genera allowed us to reconstruct the genome of the MRCA of Boechereae.
CCP chromosome painting analyses allowed us to reconstruct the origin of the four Boechereae-specific chromosomes. The origin of chromosomes Boe1 and Boe2 ( Figure 4A) most likely included an initial 0.52-Mb pericentric inversion on ancestral chromosome AK1 with breakpoints within GBs A [between BAC clone F13B4 (At1g13620) and T16N11 (At1g15410)] and C [between F8L10 (At1g53170) and F12M16 (At1g53160)]. The size of this inversion and other documented rearrangements were inferred from the physical length (Mb) of A. thaliana BAC contigs spanning these chromosome regions. The inversion-bearing AK1 chromosome underwent a whole-arm translocation with paleochromosome AK2 resulting in chromosomes Boe1 (GBs Aa, Ca, and D) and Boe2 (Cb, Ab, B, and E). Chromosome Boe3 (F, G, W, and X) originated by a whole-arm translocation between paleochromosomes AK3 and AK8 ( Figure 4B). The second translocation chromosome (GBs V and H) was involved in an end-to-end translocation with chromosome AK5, mediating the chromosome number reduction (8 → 7) in Boechereae. The collinearity of GBs K-L and M-N and the absence of the original centromere suggest that the "chromosome fusion" was accompanied or followed by a removal of the AK5 paleocentromere ( Figure 4B). Remnants of the AK5 paleocentromere, apparent as heterochromatic knobs and/or unpainted chromosome segments, were not observed in any of the analyzed species (Figures 3, 4B).

Clade-Specific Chromosomal Rearrangements
The ancestral Boechereae genome remained conserved in the diploid B. gracilipes (Figures 2, 3 and Supplementary Table S3) and the tetraploid P. fremontii (Figure 2 and Supplementary Table S3). Given that both Polyctenium subgenomes had identical chromosome structure and the pachytene chromosomes formed quadrivalents (Figure 5), the analyzed accession of P. fremontii was most likely of autotetraploid origin.
Chromosome Boe4 of tetraploid C. douglasii also displayed inversions (Figure 2 and Supplementary Table S6). A 2.06-Mb pericentric inversion, spanning the whole short arm (GB I), converted the chromosome from acrocentric to telocentric. Boe4 was also modified by a 5.35-Mb paracentric inversion [breakpoint between blocks I and J, and within block Jbetween T28M21 (At2g40090) and T3G21 (At2g40240)]. The Cusickiella population analyzed for this study was most likely of autotetraploid origin given that both subgenomes were structurally similar, including a reshuffling of Boe4 (data not shown). Finally, in B. formosa and B. oxylobula, Boe5 was altered by a 9.83-Mb pericentric inversion with breakpoints within block V [between K14A3 (At5g47175) and MQL5 (At5g47150)] and K-L [between MQP15 (At3g30655) and MED5 (At3g30663)], converting the chromosome from acrocentric to metacentric (Figures 2, 6 and Supplementary Table S7).
Borodinia missouriensis had the most reshuffled genome encountered among the taxa analyzed (Figures 2, 6 and Supplementary Table S8). A whole-arm translocation between Boe1 (GBs Aa, Ca, and D) and Boe3 (F, G, W, and X) produced two B. missouriensis-specific translocation chromosomes. The  As subgenomes of the polyploid species/cytotypes have the same structure, only a single (sub)genome is shown for triploids (P. cheiranthoides, S. whitedii) and tetraploids (C. douglasii, P. cheiranthoides, P. fremontii). Color coding of 22 genomic blocks (A to X) reflects their position on the eight ancestral chromosomes (AK1-AK8) in the Ancestral Crucifer Karyotype . Blocks split into two segments are labeled as "a" and "b." Downward-pointing arrows denote the inverse orientation of GBs compared to their position in the ACK represented here by the P. micrantha genome. Black arrows mark the Boechereae-specific inversions which occurred prior to the divergence of the tribe, whereas red arrows denote genus-and species-specific inversions that occurred after the divergence of Boechereae. I pe : pericentric inversion; I pa : paracentric inversion; t: whole-arm translocation. All ideograms are drawn to scale, whereby the size of GBs corresponds to the size of homeologous blocks in the Arabidopsis thaliana genome (The Arabidopsis Information Resource, TAIR; http://www.arabidopsis.org). Genome structure of P. cheiranthoides was adopted from Mandáková et al. (2020).   (Hal1-Hal8) and Boechera gracilipes (Boe1-Boe7). The ancestral Boechereae genome (n = 7) originated from a Halimolobeae-like genome (n = 8) through descending dysploidy mediated by an end-to-end translocation accompanied by a centromere inactivaton, two additional reciprocal translocations and a pericentric inversion (see Figure 4 for more details). In the CCP images, different colors correspond to the eight ancestral chromosomes (AK1-AK8) in the Ancestral Crucifer Karyotype , whereas capital letters refer to 22 genomic blocks (A to X). A. thaliana BAC clones defining each BAC contig/painting probe in Pennellia and Boechera are listed in Supplementary Tables S2, S3, respectively. Chromosomes were counterstained by DAPI. The fluorescence signals of the painting probes were captured as black and white photographs, and the signals were then pseudocolored to match the eight chromosomes of the ACK. Scale bars, 10 µm. species also shared a 2.55-Mb whole-arm pericentric inversion spanning block V of Boe5 with N. holmgrenii. In B. missouriensis, this was followed by a small 0.57-Mb pericentric inversion splitting block V into Va and Vb and placing the Boe5 centromere close to the chromosome terminus between BACs K24F5 (At5g43211) and MNL12 (At5g43190).

The Inactive Centromere Between Genomic Blocks K-L and M-N in Boechera stricta
The B. stricta genome contains an inactive centromere between GBs K-L and M-N on chromosome Boe5. To characterize this region at the sequence level, we compared scaffold 556 in the B. stricta assembly with orthologous genes on homeologous chromosome 3 and 5 in A. thaliana and A. lyrata, respectively ( Figure 7A). The centromeric region is delimited by genes of the Peroxidase superfamily (loci At3g32980) and genes of the Transducin family (loci At3g33530). In B. stricta, the site of the eliminated (AK5) paleocentromere corresponds to a 13-kb region between orthologs Bostr.0556s0638 and Bostr.0556s0640. This region contains a single gene (Bostr.0556s0639), which is presumably paralogous to gene Bostr.13158s0074 (homology of 84.9%), located in the distant part of block M-N on the same chromosome. In the Arabidopsis genomes, orthologs of Bostr.13158s0074 are located within M-N on chromosomes At3 (At3g60740; At3: 22,447,453,364) and Al5 (scaffold_503309.1; Al5: 20,028,781−20,034,751), respectively. Additionally, the A. lyrata genome possesses a paralog, Al_scaffold_0002_1021, located on Al2 in GB E (position 9,412,104-9,422,712). No remnants of tandem repeats were detected within the 13-kb region ( Figure 7B). Comparable distribution of transposable elements and their remnants was observed within the former centromeric region, in upstream and downstream 20 kb regions ( Figure 7C) and along the whole Boe5 pseudo-chromosome. The absence of repeat segment enrichment within the short 13-kb region supports an almost complete  Table S3). Chromosomes were counterstained by DAPI. Scale bars, 10 µm.
removal of the AK5 paleocentromere after the end-to-end "chromosome fusion" (Figure 4B).

High Frequency Apospory Occurs in Polyctenium and Sandbergia
Six accessions (Supplementary Table S1), representing five species distributed across four genera, were embryologically analyzed (Figure 8). Tetraploid C. douglasii, diploid N. holmgrenii, and diploid S. perplexa, were sexual. Tetraploid P. fremontii (MDW 2055, ES 1078) and triploid S. whitedii were highly aposporous. In the P. fremontii population ES 1078, the dyad to tetrad ratio was high (Figure 8), which in Boechera would generally indicate diplospory (Carman et al., 2019). However, three of the four observed gametophytes were forming from nucellar cells (apospory), and the fourth was forming from the surviving megaspore of a sexual tetrad. While this sample size is too small to rule out diplospory, our observations indicated that apospory initiates early during ovule development with meiosis regularly terminating as early as the sexual dyad to early tetrad stages. Such termination may have inflated the dyad to tetrad ratio (Figure 8).
Parietal cell frequencies were ≥ 70% for five of the six taxa studied (Figure 8). These frequencies are similar to those observed in Phoenicaulis (Mandáková et al., 2020), but they are much higher than those generally observed in Boechera (<50%) (Naumova et al., 2001;Carman et al., 2019). The ES 1078 P. fremontii sample was too small to determine this frequency. Parietal cells form from the distal daughter cell of the mitotic division of the archesporial cell. In these cases, the MMC forms from the proximal daughter cell (Figures 9A,B). In ovules of tenuinucellate species, the opposite normally occurs, i.e., the MMC differentiates distally, and the proximal cell is considered nucellar (Johri et al., 1992). In this respect, the Boechereae show tendencies toward crassinucellate development, with parietal cells sometimes undergoing further division to produce a parietal tissue that positions the meiocyte deeper within the ovule (Figures 9C-G).
Sexual and aposporous gametophyte formation were of the eight-nucleate Polygonum type (Figures 9H-J). In the aposporous taxa, one or more nucellar cells, and sometimes parietal cells, initiated vacuolate gametophyte formation as early as MMC differentiation (Figures 9K,L). As described above, this may have terminated meiosis prior to M II and caused the abnormally high sexual dyad to tetrad ratio observed for the P. fremontii population ES 1078 (Figure 8). Nucellar, parietal, and nucellar epidermis cells degenerated quickly in front of the rapidly growing sexual or aposporous gametophytes (Figures 9H-N).

The Ancestor of the Boechereae Had Seven Chromosomes and Descended From an ACK-Like Genome With Eight Chromosomes
Using BAC-based CCP, we analyzed 14 samples of Boechereae representing seven of the nine genera recognized by Alexander et al. (2013). These analyses revealed a high level of genomic stasis across the tribe, with the earliest diverging genus Polyctenium (Figure 1; Beilstein et al., 2010;Couvreur et al., 2010) and B. gracilipes (a member of the derived "western Boechera clade, " Figure 1) showing identical chromosome structures (Figure 2). Our analysis corroborrated our earlier hypothesis (Mandáková et al., 2015b(Mandáková et al., , 2020 of an ancestral Boechereae genome with seven chromosomes (n = 7) derived from an older n = 8 genome by descending dysploidy. This precursor n = 8 genome structurally resembled the ACK, an ancestral genomic arrangement present in many tribes of crucifer lineage I . Indeed, analysis of the P. micrantha genome from the Halimolobeae, sister group to the Boechereae, confirmed that the eight chromosomes of Halimolobeae genomes (Bailey et al., 2007) are homeologous to the eight chromosomes of the ACK. While the eight ancestral chromosomes remained conserved in Halimolobeae, the divergence of extant Boechereae appears to coincide with or follow a descending dysploidal change from n = 8 to n = 7.

Tribe-Specific Descending Dysploidy
Given that all sampled Boechereae exhibit a chromosome base number of x = 7, it is likely that the descending dysploidy event documented above occurred prior to the initial divergence of the extant members of the tribe approximately 8 million years ago (Couvreur et al., 2010). This reduction of chromosome number did not result from a "simple fusion" of two chromosomes. The first step involved the origin of a transient translocation (AK3/AK8) chromosome by a wholearm translocation, followed by an end-to-end translocation between AK3/AK8 and AK5 (→ chromosomes Boe3, Boe5). The chromosome-arm collinearity of the resulting Boe5 points to inactivation or loss of the AK5 centromere. Interestingly, this paleocentromere has disappeared from many other crucifer genomes independently, and it is the most frequently inactivated centromere detected so far among the tribes of lineage I (Camelineae: Lysak et al., 2016;Cardamineae: Mandáková et al., 2016;Microlepidieae, Mandáková et al., 2010, 2017b. As tribal divergence post-dates the origin of four tribe-specific chromosomes, we propose that the three translocations involving five out of eight ancestral chromosomes were a key evolutionary innovation underlying the origin and diversification of the Boechereae in North America.

Genus-and Species-Specific Inversions
Although reciprocal translocations clearly played a major role in the origin of the tribe, the only additional translocation noted within the tribe was a unique whole-arm transfer between Boe1 and Boe3 that produced two structurally unique chromosomes in B. missouriensis (Figure 2). However, chromosomal inversions proved to be common within the group, as they are in other mustards (e.g., Mandáková et al., 2015a;Fransz et al., 2016) and land plants in general (Hoffmann and Rieseberg, 2008). Autapomorphic pericentric inversions on chromosome Boe5 were observed in both Borodinia and Nevada, while Cusickiella exhibited unique pericentric and paracentric inversions on Boe4 (Figure 2).
Our analyses identified three potentially synapomorphic chromosomal rearrangements within the tribe: (1) a 9.83-Mb pericentric inversion on Boe5 shared by B. formosa and B. oxylobula, but not by B. gracilipes, (2) a 2.55-Mb pericentric inversion on Boe5 shared by N. holmgrenii and Borodinia missouriensis, and (3) a 8.24-Mb paracentric inversion on Boe4 shared by N. holmgrenii, all three ploidies of P. cheiranthoides, and both species of Sandbergia (Figure 2). Support for the synapomorphic status of these three chromosomal rearrangements is equivocal in the few phylogenetic analyses of Boechereae published to date.
With respect to the 9.83-Mb pericentric inversion apparently shared by B. formosa and B. oxylobula, the only phylogenetic analysis with appropriate taxon sampling is the concatenated nuclear gene tree of Alexander et al. (2013). This tree shows very weak support for a clade encompassing B. oxylobula and B. gracilipes but excluding B. formosa. A close relationship Bostr FIGURE 7 | Sequence comparison of the AK5 centromeric region on homeologous chromosomes in Arabidopsis thaliana, A. lyrata and Boechera stricta. (A) The functional centromere on chromosome 3 and 5 in A. thaliana and A. lyrata, respectively, is located between genomic blocks K-L and M-N, between At3g32980 and At3g33530. The 13-kb region corresponding to the inactive AK5 centromere is located between genes Bostr.0556s0638 and Bostr.0556s0640 on chromosome Boe5. (B) A dot-plot self-comparison of the 13-kb region on Boe5 showing the absence of tandem repeat arrays. (C) Annotation of the B. stricta scaffold 556 including the 13-kb region and regions 20 kb upstream and downstream. Transposable elements and their remnants do not exhibit a higher abundance within the 13-kb region.
between B. oxylobula (which has the inversion) and B. gracilipes (which does not) is congruent with morphology in this case, and it argues against the 9.83-Mb pericentric inversion being synapomorphic.
Concerning the 2.55-Mb pericentric inversion shared by N. holmgrenii and B. missouriensis, the two published analyses with appropriate taxon sampling yielded conflicting topologies. The concatenated nuclear gene tree of Alexander et al. (2013) provides very weak support for a sister relationship between Nevada and Borodinia, which would favor the interpretation of the 2.55-Mb inversion as a synapomorphic character. However, the Boechereae phylogeny presented by Beilstein et al. (2010) places Nevada as sister to Phoenicaulis not Borodinia, the latter being sister to a clade comprising Boechera s.s. and Anelsonia (Figure 1B). If this phylogeny is correct, the 2.55-Mb inversion apparently shared by Nevada and Borodinia would have originated independently.
The final potential chromosomal synapomorphy to be considered is the 8.24-Mb paracentric inversion shared by N. holmgrenii, all three ploidies of P. cheiranthoides, and both species of Sandbergia (Figure 2). Among previously published phylogenies, the Beilstein et al. (2010) topology hypothesizes a sister relationship between Nevada and Phoenicaulis (Sandbergia was not sampled). This would be congruent with the 8.24-Mb inversion on Boe4 being interpreted as synapomorphic. However, the concatenated nuclear gene tree of Alexander et al. (2013) hypothesizes a sister relationship (very weakly supported) between Nevada and Borodinia and provides no resolution of inter-generic relationships for Phoenicaulis or Sandbergia. This creates a conflict between the only two inversions that could be a synapomorphy. If the Beilstein et al. (2010) phylogeny is correct, then the 8.24-Mb inversion discussed here could be synapomorphic but the 2.55-Mb inversion mentioned previously would not. On the other hand, if the Alexander et al. (2013) topology is correct, then the 2.55-Mb inversion could be synapomorphic but the 8.24-Mb inversion would not. A better resolved and supported phylogeny will be needed to assess the homology of the chromosomal rearrangements documented herein. Chromosomal inversions appear to be relatively common in Boechereae, but the existence of breakage "hotspots" on several chromosomes can make it difficult to infer homology. In our dataset, most of the inversions detected are pericentric and occur in just one or two samples or taxa. The only inversion that appears to have any time depth is the 8.24-Mb paracentric inversion on Boe4 (shared by six samples representing four species and three genera). The others appear to be more recent, like the 8.4-Mb paracentric inversion on chromosome Bs1 that distinguishes the West genotype of B. stricta from other populations of the species (Lee et al., 2017). Some of these young inversions likely originated since the last glacial maximum, suggesting that this type of chromosomal rearrangement may be an ongoing and important contributor to reproductive isolation and speciation within the Boechereae.

The Boechereae n = 8 Ancestor in a Phylogeographic Context
Eurasia, specifically the Irano-Turanian floristic region, is believed to be the cradle of crucifer origin (Franzke et al., 2009). Although long-distance dispersal events contributed to plant migrations from Eurasia to North America and in the opposite direction (e.g., Wang et al., 2015;Huang et al., 2018), the Bering land bridge played the key role for the spreading of seed plants, including crucifers, from Eurasia to the North American subcontinent (e.g., Carlsen et al., 2010;Wen et al., 2010;Karl and Koch, 2013;Jiang et al., 2019). Among the clades belonging to crucifer lineage I, some are endemic to the New World (Halimolobeae and Physarieae), others have a bi-continental distribution in Asia and America (Camelineae, Cardamineae, Crucihimalayeae, Descurainieae, Erysimeae, and Smelowskieae), but the Boechereae are confined to North America with only three species occurring in the Russian Far East. The bi-continental distribution of several tribes in the lineage I, and tribes of other crucifer lineages (e.g., Arabideae: Karl and Koch, 2013), makes the Bering land bridge the most plausible colonization route for several (and perhaps all) crucifer clades to North America.
Since the Halimolobeae are consistently retrieved as the sister clade to Boechereae (e.g., Beilstein et al., 2010;Nikolov et al., 2019) and Halimolobeae genomes resemble the ACK genome with n = 8, it is likely that the ancestral genomes of Boechereae and Halimolobeae arose from this common ancestor. The present-day geographic ranges of the two tribes differ, with Boechereae concentrated in the United States and Canada (barely extending into Mexico) and Halimolobeae generally more southern in distribution. In fact, Halimolobeae has two distinct centers of diversity, one extending from central Mexico to the southwestern United States, and the other in the Andes, from Ecuador to central Argentina (Bailey et al., 2007). The geographic ranges of the two tribes overlap in a narrow band stretching east to west along the United States/Mexico border. As both tribes have the closest phylogenetic affinity to the primarily Eurasian clades of lineage I, we propose that the MRCA of Boechereae/Halimolobeae reached North America via the Bering land bridge. Couvreur et al. (2010) estimated that the Boechereae and Halimolobeae diverged c. 8 mya (late Miocene) and their MRCA diverged from its predominantly Eurasian sister lineage during mid to early Miocene. The Bering land bridge connected northeastern Asia and northwestern North America from the Cretaceous until the Pliocene (Gladenkov et al., 2002;Jiang et al., 2019). In the latter study, rates of dispersal from Eurasia to North America were significantly elevated throughout the Oligocene and early Miocene (c. 34 to 16 mya), particularly around 26 to 24 mya. These time estimates broadly coincide with the origin and diversification of Brassicaceae lineage I in early Miocene (∼23 to 18 mya; Hohmann et al., 2015), which likely used the Bering land bridge to disperse from northeastern Asia to North America.

Apomixis-Related Chromosomes in the Boechereae
Two heterochromatic chromosomes (Het and Del) have been previously described in Boechera apomicts (Kantama et al., 2007;Mandáková et al., 2015a). In eudiploid apomicts (2n = 14), a Het chromosome was identified as one of the Boe1 homologs (GBs A, C, and D). In aneuploid apomicts (2n = 15, 22), a centric fission partitioned Het chromosome to a larger Het' (GBs A and C) and to a smaller Del (block D) chromosome (Mandáková et al., 2015b). In P. cheiranthoides, apomictic triploids and tetraploids contained a heterochromatic Het (GBs A, C, and D), but it was absent in diploid apomicts (Mandáková et al., 2020). This observation indicates that aposporic reproduction in Phoenicaulis is not associated with the presence of a Het chromosome. This is further corroborated herein by the apparent absence of a Het chromosome in two other aposporous apomicts, tetraploid P. fremontii and triploid S. whitedii.

Apomixis Originated Several Times Independently During Brassicaceae and Boechereae Diversification
Following the pioneering work on apomixis in B. holboellii [as Arabis holboellii Hornem. in Böcher (1951)], Mulligan (1966) reported its occurrence in Erysimum L. (Erysimeae) and Mosquin and Hayley (1966) documented possible asexual seed production in Parrya R. Brown (Chorisporeae). Mulligan and Findlay (1970) identified several species of Draba L. (Arabideae) that they inferred to be apomictic, and subsequent embryological and single seed flow cytometry analyses of one of these species (Draba oligosperma Hook.) were suggestive of apospory (Jordon-Thaden and Koch, 2012). Detailed cytological studies of Draba, Erysimum, and Parrya are needed to verify the regular occurrence of asexual seed production and the specific pathway involved. However, the available anecdotal evidence for this reproductive pathway occurring in four distantly related crucifer tribes (and its induction by hybridization in a fifth tribe, Brassiceae; Ellerstrom, 1983) suggests that apomixis has evolved independently multiple times within the family.
The Boechereae appear to be a "hotspot" for the origin and diversification of apomictic taxa. Our earlier publications have embryologically confirmed apomixis (either diplospory or apospory) in more than 20 diploid and triploid taxa of Boechera Carman et al., 2019), and apospory in diploid, triploid, and tetraploid Phoenicaulis (Mandáková et al., 2020), and in diploid Borodinia laevigata (Muhl. ex Willd.) P. J. Alexander and Windham [as Boechera laevigata (Muhl. ex Willd.) Al-Shehbaz in Carman et al., 2019]. Here, two new genera are added to the list of aposporous apomicts, Polyctenium (tetraploid) and Sandbergia (triploid). Two genera of Boechereae (Anelsonia and Yosemitea) remain unsampled, and the exclusive occurrence of sexual reproduction in Nevada and Cusickiella must be confirmed by more extensive sampling. In summary, apomixis is now known to occur in five of the nine genera of Boechereae, and in two of these (Phoenicaulis and Polyctenium) it is the only reproductive pathway documented to date. Based on current sampling, Sandbergia exhibits equal proportions of apomictic (S. whitedii) and sexual reproduction (S. perplexa). Among Boechera species, apomictic taxa of hybrid origin greatly outnumber their sexual diploid progenitors (Li et al., 2017), while in Borodinia, sexual populations appear to predominate.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.