Polymorphism and evolutionary origins of accessory chromosomes in the basidiomycete Tremella fuciformis

Domain: chemistry
Published: 2026-02-27
Editorial outcome: accept

Abstract
Accessory chromosomes are non-essential for growth but poorly characterized in
basidiomycetes, unlike in Ascomycota. Here, we report whole-genome sequencing of 16 strains
of the basidiomycete Tremella fuciformis (silver ear fungus), generating 27 complete haplotypes
(5 monokaryons and 11 dikaryons, each contributing two distinct haplotypes). Genome size
varied by over one-third, driven by accessory chromosomes and repetitive sequences in core
chromosomes (essential for basic biology). Each strain harbored 8-10 core chromosomes
(polymorphic via fusion/fission) and 2-10 accessory chromosomes (total 108), whose
distribution reflects phylogeny and symbiotic specificity with the ascomycete Annulohypoxylon
stygium. Accessory chromosomes are small, transposon-rich, gene-poor, and exhibit higher
sequence similarity but more diverse structural variations than core chromosomes, with few
shared genes across phylogenetic branches. Both chromosome types show frequent copy number
variation during cell type transformation. Most accessory chromosome genes lack homologs in
core chromosomes or existing gene databases. Our study reveals basidiomycete accessory
chromosome diversity, suggesting an origin from unexplored species pre-dating T. fuciformis
speciation.

Introduction
With the advancement of next-generation sequencing technologies and
telomere-to-telomere (T2T) genome assembly techniques, an increasing number of complete
fungal genomes have been decoded1,2. These developments have facilitated a deeper
understanding of distinctive genomic characteristics exhibited by some fungi, such as the
two-speed genome phenomenon3. The genomic structure comprises core genome and accessory
genome. The core genome is defined as the set of core chromosomes (CCs) shared by all isolates,
encoding all essential functions required for survival under normal growth conditions4-6.
Accessory genome refers to the parts of fungal genomes that may consist of compartments from
CCs or entirely accessory chromosomes (ACs), which are also known as dispensable,
lineage-specific, supernumerary, and B chromosomes7. ACs are prevalent in animals, plants, and
microorganisms8. In fungi, research on accessory chromosomes has primarily focused on
pathogens within the Ascomycota phylum, such as species of Fusarium, Zymoseptoria,
Metarhizium, and Alternaria7,9-11. ACs in Fusarium, Metarhizium, and Alternaria contain
virulence factors, including effector proteins, secondary metabolite biosynthesis clusters, or other
metabolic gene clusters4,11,12. However, accessory chromosomes in Basidiomycetes are rarely
reported. To date, only a handful of species-including Sporisorium scitamineum13 and T.
fuciformis14 have been confirmed to harbor them, verifying their presence in this phylum.
Documented cases are far fewer than in Ascomycetes, underscoring the need for further
exploration in understudied taxa.

Fungal ACs are characterized by distinct sequence features that, while not entirely unique,
set them apart from core chromosomes. Typically smaller, ranging from 0.2 to 3.5 Mb15, ACs are
enriched with transposons or repetitive sequences and exhibit lower gene densities and distinct
codon usage compared to CCs16. These non-essential chromosomes evolve rapidly and often
harbor genes related to pathogenicity and adaptation17, highlighting their unique role in the
organism's biology. Predominantly heterochromatic, ACs are marked by significant enrichment
of histone modifications such as H3K9me3 and H3K27me318. H3K27me3 is enriched across
nearly the entire chromosome and is linked to the instability of accessory chromosomes, whereas
H3K9me3 is mainly associated with repetitive sequences and contributes to genome stability18-20.
While CCs can sometimes include compartments with low gene density, unique heterochromatin
structures, and high repeat content, these features are less pronounced and widespread than in
ACs. Fungal accessory chromosomes can sometimes be identified using (epi-)genomic
characteristics, though presence/absence polymorphism remains a key criterion for their
identification19,21.

The origin of fungal accessory chromosomes remains unresolved, with two main
non-exclusive hypotheses: they may originate from core chromosomes and diverge or degenerate
over time19, or they may be acquired through horizontal chromosome transfer from different
lineages or species22. In some species, the high similarity in sequence composition between core
and accessory chromosomes suggests that accessory chromosomes may have originated from
core chromosomes. Recent macrosynteny analyses in the rice blast pathogen Magnaporthe
oryzae have traced the emergence of accessory mini-chromosomes through structural
rearrangements and segmental duplication of core chromosomes23. In Zymoseptoria tritici,
genome sequencing of meiotic progenies has revealed that the accessory chromosomes primarily
originated from ancient core chromosomes through a degeneration process involving the
breakage-fusion-bridge cycle24. The horizontal chromosome transfer model is also supported by
some evidence. For instance, differences in codon usage between core and accessory
chromosomes suggest this possibility25. Phylogenetic analysis indicates that the accessory
chromosomes of Fusarium oxysporum f.sp. lycopersici have a different origin than the core
chromosomes4. Experimental evidence has demonstrated mechanisms that enable the horizontal
transfer of entire chromosomes between distinct lineages in species such as F. oxysporum21,26,
Alternaria spp.27, Metarhizium robertsii, and Colletotrichum gloeosporioides28.

Tremella fuciformis, commonly known as snow fungus or silver ear fungus, is a unique and
widespread fungus that thrives in tropical regions, often found on the dead branches of broadleaf
trees. It belongs to the order Tremellales and the family Tremellaceae29. The fruiting body
formation of T. fuciformis is extremely dependent on its companion fungi Annulohypoxylon
stygium in large-scale cultivation and natural environment30,31. Specifically, A. stygium maintains
a close association with T. fuciformis and functions as a key nutritional provider, supplying
essential nutrients to support the growth and development of the latter. T. fuciformis is notable
for its dimorphic nature, existing either as yeast-like cells that reproduce asexually through
budding or as filamentous cells that grow by apical extension14. Similar to Cryptococcus
neoformans32, the haploid phase in the life cycle of T. fuciformis exists exclusively in the yeast
form and cannot transition to the mycelial form, thus being unable to interact with A. stygium
hyphae as unicells or form fruiting bodies; in contrast, the heterokaryotic phase is dimorphic,
enabling such hyphal interactions and fruiting body development. In previous studies, three
accessory chromosomes have been identified in the genome of T. fuciformis Tr01, exhibiting
small size (<1 Mb), low gene density, and a high proportion of repetitive sequences. They may
be absent in its basidiospores or exist in multiple copies14. However, their distribution within
cells, genetic origin, functions, and impact on the growth and reproduction of T. fuciformis
remain unknown.

In this study, we intend to conduct whole-genome sequencing assembly and comparative
genomic analysis of 16 T. fuciformis strains from different regions. Our aim is to explore the
diversity of accessory chromosomes within T. fuciformis, explore their shared sequence
characteristics, and investigate their distribution and stability within cells. Additionally, we will
analyze their genetic origins and impact on the host cell genome. This research will enrich our
understanding of the genomic diversity of T. fuciformis and provide deeper insights into the
characteristics of accessory chromosomes in basidiomycete fungi.

Results
Haploid genome assemblies of 16 T. fuciformis strains
A total of 16 T. fuciformis strains were collected from the provinces of Fujian, Guangdong,
Sichuan, and Yunnan, China. Among these, two principal cultivated varieties (Tr01 and Tr21),
while the remaining 14 are wild isolates. The strain collection comprises 11 heterokaryotic
strains and 5 monokaryotic strains (Supplementary Table 1). Among these, 5 heterokaryotic
strains were found to harbor two distinct ITS sequences each, whereas the remaining 11 strains
contained only one ITS sequence. Phylogenetic analysis based on 21 target ITS sequences and 10
downloaded reference ITS sequences yielded a tree that clustered all tested strains into three
distinct clades (Supplementary Fig. 1), with all clades belonging to T. fuciformis. NCBI BLAST
analysis of the ITS sequence indicated that the isolates are more closely related to the known
species T. fuciformis. Additionally, their fruiting bodies exhibit the same morphological
characteristics as those of T. fuciformis (Supplementary Fig. 2). T. fuciformis strains within the
same cluster can mutually pair with their associated fungus, A. stygium, whereas those from
different clusters are unable to pair (Supplementary Fig. 3). It is suggested that the 16 strains
from these three clusters each belong to a distinct subspecies within the T. fuciformis species.

The PacBio sequel IIe system was utilized to conduct whole-genome sequencing on 15 T.
fuciformis strains, excluding Tr01 (Cluster III) with its known genome, using the circular
consensus sequencing (CCS) and continuous long read (CLR) mode. The sequencing process
produced between 40,120 and 575,272 HiFi reads, with an N50 length ranging from 12 Kb to 23
Kb. The total sequencing output was between 5.5 Gb and 10.5 Gb, resulting in a genome
coverage of 100× to 450× (Supplementary Table 2). Approximately 40 Gb of data was generated
from the Hi-C sequencing of both NDB and TF2206. HiFiasm with Hi-C mode was used to
assemble HiFi reads of NDB into 77 contigs. Using the Tr01 genome as a reference, these NDB
contigs were further assembled into 19 telomere-to-telomere gapless chromosomes, which were
assigned into two sets of haploid genomes by NuclearPhaser (Supplementary Fig. 4). Similarly,
the complete heterokaryotic genome of TF2206 was generated from 50 contigs, comprising 13
pairs of telomere-to-telomere, gapless chromosomes. Hi-C interaction heatmaps produced by
Hi-C Pro validated the assembled genomic architecture (Supplementary Fig. 5). Using the
genomes of TF2206, NDB, and Tr01 as references for Clusters I, II, and III, respectively, the
HiFi reads of the other 13 strains were assembled into either one complete haploid genome for
monokaryotic strains or two sets of complete haploid genomes for heterokaryotic strains. In total,
we have successfully obtained 27 complete haploid genomes, including two previously
established from the Tr01 strain (designated Tr01A and Tr01B, corresponding to its two distinct
haplotypes), to support subsequent research.

Divergence in genome size and chromosome number across genomic landscapes
Pan-genome analysis of the 27 haploid genomes identified 14,567 gene families, including
3,549 core genes (24.3% of the total), 9,638 accessory genes (66.2%), and 1,380 singletons
(9.7%). Using 3,549 core genes from the pangenome, a phylogenetic tree was constructed
(Fig.1a, column 1). This tree reveals three major clusters, which are consistent with the
phylogenetic tree based on ITS sequences and largely align with the three clades in the
mitochondrial genome-based phylogenetic tree-with the only exception being strain T0053,
which was reclassified from Cluster III to Cluster II (Supplementary Fig. 6).

The 27 haploid genomes of T. fuciformis exhibit significant size variation (Fig.1a, column
3). The largest genome is from strain NDB hapA, measuring 32.48 Mb, while the smallest is
from strain TF4-1 hapA, measuring 23.83 Mb, indicating a size difference of over one-third.
Additionally, there is substantial variation in their chromosome numbers, ranging from 11 to 19
(Fig.1a, column 2). Core chromosomes, identified as highly conserved and shared by all T.
fuciformis isolates (Supplementary Fig. 7), may vary in number across different strains due to
genetic events such as chromosomal recombination-for example, 8 in Tr01 HapA and 9 in Tr01
HapB14. Consistent with this observation, we found that the 27 haploid genomes harbor 8 to 10
core chromosomes, whereas the number of accessory chromosomes ranges from 2 to 10. The
size of the accessory chromosomes ranges from 1.4 Mb to 5.2 Mb and shows a positive
correlation with the total genome size (R² = 0.81) (Fig.1b top). In addition to the accessory
chromosomes, we also found significant differences in the core genome sizes among the haploid
genomes. By dividing the core chromosomes into repetitive and non-repetitive sequence regions,
we discovered that the size of the repetitive sequence regions shows a strong positive correlation
with the total genome size (R² = 0.90) (Fig.1b bottom), being the main source of variation in the
core genome size. In contrast, within the same heterokaryotic strain, the two sets of
chromosomes exhibit less differences in both number and size, with the variation in genome size
being less than 4%. There are significant differences in genome sizes among the three clusters.
The average size of the six haploid genomes in Cluster II is 31.79 Mb, which is larger than the
average size of the twelve genomes in Cluster III, measured at 27.33 Mb (P <0.0001).
Furthermore, the average genome size of Cluster III is significantly greater than that of the nine
genomes in Cluster I, which have an average size of 24.02 Mb (P <0.0001).

Three scenarios contribute to the differences in the number of core chromosomes among
these T. fuciformis species, and even between the two haploid genomes within the same
dikaryotic strain. In strain TF2206, Chr01-1 exhibited strong collinearity with the 5' end of
Tr01's Chr01 chromosome, while Chr01-2 showed similar collinearity with the 3' end. The
junction of the collinear sequences on Tr01's Chr01 chromosome was located at
Chr01:2,260,933-2,275,108, identified as a 14,175 bp DNA transposon T1 (Fig.1c). Hi-C
interaction heatmaps revealed that while Tr01's Chr01 chromosome was continuous at this
junction (Fig.1d top), the 3' end of Chr01-1 and the 5' end of Chr01-2 in TF2206 were not
connected and contained telomeric interaction hotspots (Fig.1d bottom), indicating accurate
chromosome assembly. Tr21 and Tr01 are the only two strains in a branch at the seventh node of
the phylogenetic tree, each having only one chromosome (Chr01) in this region, whereas the
other strains possess two chromosomes: Chr01-1 and Chr01-2 (Fig.1e). The chromosomal
rearrangement was further validated by mapping long HiFi and nanopore reads to the
corresponding regions of TF2206 and Tr01 (Supplementary Fig. 8). Similarly, strains in Cluster I
and Cluster II possess a single chromosome for Chr02, whereas strains in Cluster III consist of
two separate chromosomes (Supplementary Fig. 9 and 10). The third scenario involves the
published Tr01 genome14, where haplotypeB is similar to haplotypeA in genome size, gene count,
and proportion of repetitive sequences. However, chromosomal rearrangements are observed in
Chr01, Chr02, and Chr05, along with an additional core chromosome, Chr12B, resulting in a
difference in the number of core chromosomes in its heterokaryotic genome.

Regularity in the distribution of accessory chromosomes
The chromosomes of 27 haploid genomes were aligned with the core chromosomes
Chr01-Chr08 of the Tr01 hapA genome, resulting in the eight sets of homologous core
chromosome groups, designated as CC01-CC08. Similarly, 108 accessory chromosomes were
identified and categorized into 15 groups of homologous chromosomes, named AC01-AC15,
based on synteny. The distribution of the 15 sets of accessory chromosomes across the 27
haploid genomes was compared with the phylogenetic tree constructed from the core genes
(Fig.2). The homologous chromosomes of AC01 to AC07 are found in the genomes within
Cluster II, with the QSA and QSB genomes lacking AC03, and the NDBA and NDBB genomes
lacking AC06. The homologous chromosomes of AC09 to AC12 are located in the genomes of
Cluster I, where one or more genomes do not carry AC09, AC10, or AC11. The homologous
chromosomes of AC13 and AC14 are present in the genomes of Cluster III. The three largest
accessory chromosomes, AC01, AC12, and AC14, are each located on one of the three branches
of the phylogenetic tree. All strains in the genomes of each branch carry these chromosomes,
except for T0053. Synteny analysis of accessory chromosomes in the QCA and TF104A
genomes revealed that their homologous chromosomes AC10 to AC12 are nearly identical
(similarity >99.9%, coverage >99.3%; Supplementary Fig.11), while QCA lacks AC09. All
strains in Cluster I, except QTC and TF103, harbor AC09, suggesting this accessory chromosome
was originally present in the cluster but lost during evolution. The same pattern applies to other
chromosome losses. These findings demonstrate that the overall distribution pattern of these 15
accessory chromosomes in the haploid genome aligns with the three branches of the
phylogenetic tree. Each branch's heterokaryotic T. fuciformis strains could only pair with the
associated A. stygium strains of that branch and not with A. stygium strains from other branches,
indicating a specific pairing between T. fuciformis and A. stygium. It is evident that the
distribution pattern of accessory chromosomes matched that of the corresponding associated
fungus.

Common features of accessory chromosomes
By conducting a comparative analysis of the sequence similarities and differences between
the core chromosome groups CC01-CC08 and the accessory chromosome groups AC01-AC15,
the sequence characteristics of the accessory chromosomes in T. fuciformis were summarized.
The core chromosomes of the test strains all exceed 1 Mb in size, with the largest being the
CC01 homologous chromosome group, averaging 8.45 Mb in length. In contrast, the accessory
chromosomes are generally smaller. Except for the AC01, AC12, and AC14 groups, which have
average lengths of 2.57 Mb, 1.11 Mb, and 1.24 Mb, respectively, all other accessory
chromosomes are less than 1 Mb in length (Fig.3a). The core chromosomes carry an average of
33.86 to 39.26 genes per 100 Kb sequence. In contrast, the gene density of accessory
chromosomes is significantly lower, ranging from 10.29 to 21.71 genes per 100 Kb, with a
statistically significant difference (P-value =7.97E-15, Fig.3b). The average length of genes and
individual introns in accessory chromosomes is comparable to that of core chromosomes.
However, the average number of introns per gene in accessory chromosomes is significantly
lower, with only 2.1 introns per gene, which is notably less than the 3.3 introns per gene in core
chromosomes (P-value = 7.83E-13, Supplementary Fig. 12). The average proportion of repetitive
sequences in accessory chromosomes ranges from 40.16% to 80.98%, significantly higher than
that in core chromosomes, which ranges from 11.44% to 19.10% (P-value =3.87E-9, Fig.3c).
LTR/Ty3, LTR/Copia, TIR/Mutator, TIR/CACTA, helitron and repeat_fragment are the six most
abundant TE types in the T. fuciformis genome. Our comparative analysis of their proportional
abundances between ACs and CCs revealed that not all TEs are equally distributed between the
two chromosome types: relative to CCs, ACs show significantly reduced proportions of
TIR/Mutator and TIR/CACTA, while LTR/Ty3 and LTR/Copia are significantly enriched
(Supplementary Fig. 13). The average nucleotide identity (ANI) among homologous core
chromosomes in 27 haploid genomes ranged from 86.7% to 88.4%. In contrast, the similarity
between homologous accessory chromosomes is higher, with median ANI values ranging from
97.4% to 99.6%, showing a significant difference (P-value = 4.01E-21, Fig.3d).

The rapid structural variation of accessory chromosomes
Collinearity analysis of homologous accessory chromosomes revealed their rapid structural
variations, including inversion, terminal chromosome loss, insertion-deletion, and chromosome
fusion-splitting. In the phylogenetic tree, the NDBA genome is closest to Wuyi2015. In
Wuyi2015, the latter 1.0 Mb of the AC01 chromosome, from position 1,672,381 to the end, has
undergone an inversion (Fig.4a). Moreover, several smaller inversions are present throughout the
AC01 chromosome. Compared to Wuyi2015, the first half of the AC08 chromosome in NDBA
(positions 1 to 41,628 and 46,673 to 72,109) has experienced a deletion, accounting for one fifth
of its total length (Fig.4b). The sequence similarity between the homologous AC14
chromosomes of the A and B haplotypes in the dikaryotic B-1 strain is as high as 99.5%.
However, there are four insertion-deletion segments exceeding 50 Kb and one inversion of 107
Kb between them (Fig.4c). The AC14 of GDA is collinear with both AC13 and AC14 of Tr21A,
indicating a chromosome fusion-splitting event (Fig.4d). Additionally, the latter half of the AC13
chromosome in Tr21A (from position 231,466 to the end) has been lost, while the middle section
of the AC14 chromosome in GDA (from position 332,894 to 501,389) has also been lost.
Eight Starship giant transposons (41,942-605,797 bp) were identified across 6 genomes,
with 3 localized to core chromosomes and 5 to ACs. Notably, SS04 is positioned at a
chromosome fusion site (Fig. 4d), suggesting involvement in fusion events. Further, 6 Starships
formed 3 groups on homologous loci of heterokaryotic strains: SS02/SS03 (core Chr06) were
identical, while AC-localized pairs (SS06/SS07 on GD Chr09 and SS04/SS05 on C-1 Chr10)
showed structural variations (46 bp and 3,048 bp with 4 LTRs, respectively), supporting rapid
AC structural variation (Supplementary Table 3 and Supplementary Fig. 14).

High frequency of chromosomal copy number variation in both core and accessory
chromosomes
T. fuciformis can exist in either mycelial or yeast form. To illustrate its chromosomal copy
number variation (CNV) characteristics, we randomly selected dikaryotic strain TF2206 as a case
study and analyzed CNV in its three states. A single yeast-forming colony was expanded in
liquid medium for 3 days, then diluted and plated onto agar plates. Ten single colonies were
randomly selected for sequencing analysis, and copy number variations were found in six
chromosomes (Fig.5a). In the ten isolated colonies, the copy numbers of chromosomes Chr07,
Chr09, and Chr10 consistently varied, being 2.0×, 0.5×, and 0×, respectively. For chromosome
Chr01-2, only the YY3 colony exhibited a copy number change to 0.8×. In the case of
chromosome Chr05, all colonies except YY3 had a copy number of 1.5×, with YY3 having a
copy number of 1.1×. Regarding chromosome Chr11, the copy number in the YY1 colony
remained unchanged, while it was 0.9× in the YY8 strain and 0.5× in the other colonies. It can
be inferred that the chromosomal copy number of the initial single yeast-forming colony has
undergone variation. Specifically, the copy numbers of Chr05, Chr07, Chr09, Chr10, and Chr11
have changed to 1.5×, 2.0×, 0.5×, 0×, and 0.5× their original values, respectively. During the
expansion process, three colonies exhibited four instances of variation: Chr01-2 and Chr05 in
colony YY3, and Chr11 in colonies YY1 and YY8. We also conducted a chromosomal copy
number analysis on 10 mycelial colonies obtained from subculturing the same original parent
colony (Fig.5b). The results showed that the copy number of the core chromosome Chr06 in all
colonies had varied, reducing to 0.75×. Additionally, the three accessory chromosomes also
exhibited copy number variations, with Chr09 and Chr10 each having two types of copy number
variations, while Chr11 had four types. T. fuciformis mycelium can be transformed into yeasts,
and through streak purification techniques, we repeated the process 10 times, each time obtaining
one single-yeast forming colony. We conducted chromosomal copy number analysis using 10
single-yeast forming colonies and one mixed culture before purification. For these single-yeast
forming colonies (Fig.5c), copy number of three core chromosomes has undergone variation,
with Chr05 exhibiting one type of copy number variation, Chr08 having two types, and Chr02-2
having five types, all of which show an increase in copy number. Additionally, the three
accessory chromosomes also display copy number variations, with Chr09 having two types,
Chr11 having three types, and Chr10 having five types. We compared the normalized depth
distribution curves of Illumina sequencing between a representative single-yeast forming colony
(YY2, with stable genetic background) and a mixed yeast colony transitioning from mycelium
(Fig.5d). The results showed that the YY2 curve was relatively stable across most regions,
whereas the curve for the mixed colonies exhibited significant fluctuations. These fluctuations
might be attributed to the presence of numerous yeasts with varying sequencing depths within
the mixed colony.

Possible origins of accessory chromosomes
To investigate whether accessory chromosomes (ACs) originated from core chromosomes
or external sources, we took AC01 as a representative example. Among the 2340 genes in AC01,
1334 (57.0%) are shared by all six strains, 99 (4.2%) are unique to a single strain (Fig.6I-a), and
only 332 (14.2%) exhibit homology with core chromosome (CC) genes across the six haploid
genomes carrying this chromosome (Fig.6I-b). Of these 332 shared genes, 256 (77.1%) were
embedded within TE sequences (Fig.6I-c and Fig.6I-d). For AC02 to AC15, the majority of their
genes lack counterparts in core chromosomes (Fig.6I-e), and the limited shared genes are
associated with TE activity (Fig.6I-e). These findings collectively suggest that ACs are not
derived from the detachment of CCs but were acquired through horizontal gene transfer from
external sources.

Strains of Cluster I, II, and III carry 325, 891, and 590 AC genes respectively, totaling 1,806
genes (Fig.6II-a), among which 1,548 are AC-specific genes. Only 102 accessory genes (16.9%
on average) are homologous across all three clades, while the number of shared core genes
reaches 7,862 (accounting for 75.3%, Fig.6II-a). Among the 1,548 AC-specific genes, 1,156
(75%) failed to match any homologous sequences or conserved domains, and lacked functional
motifs, signal peptides, or transmembrane domains (Fig.6II-b). This not only indicates that the
majority of these genes have not been functionally characterized in previous studies, but also
strongly suggests they likely originate from unexplored or understudied species. Of the
remaining 392 genes, 232 showed matches to homologous regions but with unknown functions,
7 lacked homologous matches and encoded proteins bearing signal peptides, leaving only 153
with annotated functions. Among these 153 functionally characterized genes, the most abundant
were those encoding motor domains and members of the major facilitator superfamily-proteins
involved in transmembrane transport of nutrients, energy conversion, and molecular movement
(Fig.6II-c). Notably, 236 of the 392 genes with homologous matches exhibited the highest
sequence similarity to species within the Tremellales (Fig.6II-d).

The proportion of repetitive sequences in ACs varies significantly, ranging from 38% to
89% (Fig.6III-a). Among these repetitive sequences, RNA transposons are the most abundant
(34%-75%). The repetitive sequences in ACs of Cluster I and Cluster III share a higher
proportion with those in CCs, while the corresponding proportion for Cluster II ACs is less than
16% (Fig.6III-b). Taking AC01 as a case study, ACs and CCs contain 3 and 19 intact Type 1
LTR retrotransposons, respectively, among which 2 and 5 have sequence differences between the
two terminal LTRs. Sequence analysis of these differentially terminal LTRs shows that the LTR
retrotransposons originally existed in CCs and were first inserted into ACs 500,000 years ago.
AC01 harbors 11 types of intact LTR retrotransposons with copy numbers present in both ACs
and CCs. The earliest estimated time of genetic exchange between AC01 and CCs is 1 million
years ago (Type 3). By analogy, the approximate integration times of AC02 to AC15 range from
0.33 to a maximum of 1.69 million years ago, with 5 ACs inestimable due to insufficient intact
LTRs.

Methods
Tremella fuciformis strains
We analyzed a diverse set of 16 T. fuciformis strains (Supplementary Table 1). Fourteen of
these strains were isolated from various regions in China, specifically from the provinces of
Fujian, Guangdong, Sichuan, and Yunnan. Tr21 and Tr01 are the primary cultivated varieties of T.
fuciformis, accounting for over 99% of the total cultivation. Among these strains, 11 exhibit both
mycelial and yeast forms, while 5 exhibit only the yeast form. In this study, 15 T. fuciformis
strains were newly sequenced, and we included one publicly available complete genome
sequence (Tr01, which includes the complete haploid AR and haploid B genome sequences)14. All
these strains have been preserved at the Mushroom Germplasm Resource Preservation and
Management Center of Fujian Province.

Sample collection and Genome sequencing
We transferred the mycelial-form strains to PDA plates with a moist surface and incubated
them at 25°C for 3 days. During this period, some cells around the mycelia began to form into
yeast forms, which were then transferred to form yeast-form strains. All yeast-form strains were
inoculated into PDB medium and cultured with shaking at 150 rpm at 25°C for 3 days. Spores
were collected during the mid to late logarithmic growth phase. The yeasts were harvested by
centrifugation at 10,000 g for 10 minutes and washed twice with sterile double-distilled water.
The collected samples were used for PacBio CCS sequencing using the Sequel IIe platform,
aiming to obtain ~10 Gb of high-quality HiFi reads for whole-genome assembly. DNA extraction,
quantification and evaluation, library construction, and sequencing were all conducted by
Frasergen Bioinformatics Co., Ltd. (Wuhan, China).

Sixteen T. fuciformis strains were divided into three clusters, with the Tr01 strain located in
Cluster III, for which a complete genome sequence has been obtained. NDB and TF2206,
randomly selected as representatives of the Cluster I and Cluster II, were first subjected to
Hi-C-assisted genome assembly to obtain complete and accurately phased genomes. They then
served as reference genomes together with Tr01 to assist in the genome assembly of other strains
within their respective clusters. The samples used for Hi-C sequencing were collected in the
same way as those used for PacBio CCS sequencing. All steps of the Hi-C sequencing process,
including cell fixation, chromatin digestion, fragment ligation, cross-link reversal, DNA
purification, and high-throughput sequencing, were carried out by Frasergen Bioinformatics Co.,
Ltd. (Wuhan, China).

Using the TF2206 strain as an example, we analyzed the chromosomal copy number
variations in T. fuciformis during the proliferation of yeast-like cells, the proliferation of mycelial
cells, and the transition from mycelial to yeast-like cells. In the yeast-like cell proliferation
experiment, we selected a single yeast-like colony of TF2206, diluted it in sterile water, plated it,
and after cultivation, randomly picked 10 single yeast-like colonies. In the mycelial cell
proliferation experiment, we selected a single mycelial colony of TF2206, picked 10 mycelial
blocks, and transferred them to PDA plates to form 10 new mycelial colonies. In the experiment
of transitioning mycelial cells to yeast-like cells, we selected a single mycelial colony of TF2206,
picked 10 mycelial blocks, and transferred them to moistened PDA plates. During cultivation,
some cells around the mycelia began to form yeast-like structures, and single yeast-like colonies
were obtained through streak purification. The cultivation temperature for mycelial cells,
yeast-like cells, and the transition from mycelial to yeast-like cells was consistently maintained
at 25°C. These colonies were collected and sent to Annoroad Gene Technology Co., Ltd. (Beijing,
China) for Illumina high-throughput sequencing. The processes of DNA extraction, quality
control, library construction, and paired-end sequencing on the Illumina NovaSeq 6000 platform
(2×150 bp) were all completed by Annoroad Gene Technology Co., Ltd. (Beijing, China).

Genome assembly
For the TF2206 and NDB strains, we used the Hi-C mode of the HiFiasm (v0.15.3-r339)45
to assemble the sequenced HiFi reads with default parameters and ultimately obtaining two sets
of contigs, hap1 and hap2, each of which was further assembled individually. We manually
examine whether the 5' end of each contig contains a telomeric sequence with the repeat unit
TAACCCCC, and whether the 3' end contains a telomeric sequence with the repeat unit
TTAGGGGG. These telomeric sequences help determine if the contigs form complete
chromosomes and allow for a preliminary estimation of chromosome numbers. Contigs that do
not form complete chromosomes will be used for further analysis. The mitochondrial genome
sequence of T. fuciformis TF01 (GenBank accession: NC_036422.1) was downloaded from the
NCBI website. Using local BLAST, it was compared with the remaining contigs to identify those
related to the mitochondria. Since the mitochondrial genome of T. fuciformis is generally less
than 50 Kb46, a single contig can cover the entire mitochondrial genome sequence. The complete
mitochondrial genome is obtained by removing the repeat sequence at one end. These contigs
were then manually assembled into circular mitochondrial DNA. Each haploid genome contains
an rDNA region, composed of tandem repeats of approximately 10Kb rDNA units47, located in
the middle or at the end of a chromosome. The ITS sequence of T. fuciformis CBS 6970
(GenBank accession: NR_155936.1) was retrieved from nt database in NCBI website and
compared with the remaining contigs using local BLAST to identify contigs carrying the rDNA
region and exclude contigs that fall within the rDNA region. When two contigs carrying the
rDNA region are found, they are considered to be connected, forming a new scaffold. If only one
contig with the rDNA region is identified, the presence of rDNA at the chromosome end is
determined by searching for HiFi reads that contain both rDNA repeat units and telomere repeat
sequences. The number of tandem repeats of the rDNA unit is calculated as the ratio of the
sequencing depth of the rDNA unit to that of the single-copy sequence. Hi-C reads were utilized
to identify connections between contigs within a scaffold containing gaps, using the programs
HiC-Pro (v2.10.0)47 and HiCPlotter (v0.7.3)48. After completing the scaffolding process, manual
correction was employed to address the existing gaps. This involved trimming the ends of
adjacent contigs and performing overlapping alignments to achieve seamless connections
between the contigs. The phasing of the two sets of chromosomes was performed using program
NuclearPhaser. To rule out errors caused by genome assembly, the observed chromosomal
rearrangements were validated using orthogonal approaches, including long HiFi read mapping,
long nanopore read mapping, Hi-C interaction heatmaps, and/or long-range PCR assays.

For the remaining 13 strains, we used the HiFiasm in non-Hi-C mode to assemble their
sequenced HiFi reads into contigs using the default parameters. The determination of complete
chromosomes, preliminary estimation of chromosome numbers, assembly of the mitochondrial
genome, and rDNA-related analyses are all consistent with the analysis procedures of TF2206
and NDB. For the strains of Cluster I, II, and III, the complete genome sequences of TF2206,
NDB, and Tr01 were selected as references, respectively. Through synteny analysis, the
remaining contigs were assembled to the chromosomal level. Subsequently, sequence similarity
analysis was employed to classify all chromosomes of heterokaryotic strains into two genomic
sets.

Identification of accessory chromosomes
Accessory chromosomes are identified in this study by comparing 27 haploid genomes.
Chromosomes present in all strains are classified as essential, while the others are categorized as
accessory. The comparison of gene synteny between Tr01 hapA and the other 26 haploid
genomes was conducted using the MCScanX (https://github.com/wyp1125/MCScanX)49. The
annotation GFF files of each genome were first converted into standardized BED format using
the jcvi.formats.gff subroutine. Subsequently, local BLAST databases were constructed and
aligned to identify homologous genes across genomes. Based on the alignment results, the
MCScanX algorithm was employed to screen high-similarity anchor genes while precisely
determining their relative genomic positions, thereby detecting conserved syntenic blocks. To
visualize genome-wide collinearity patterns, conserved syntenic gene clusters with evolutionarily
stable arrangements were extracted using the group_collinear_genes.pl script from the MCScanX
pipeline, followed by the generation of high-resolution syntenic dot plots via the dot_plotter.java
program.

Gene annotation and TE prediction
Gene annotation was performed on 27 T. fuciformis genome assemblies using
FUNANNOTATE (v1.8.16)50. This tool integrated homologous protein sequences and full-length
transcript evidence from the reference genome Tr01 Parameters were set to specify the species as
T. fuciformis, with the closely related species Cryptococcus neoformans JEC21 selected as the
training set for Augustus.

To characterize the gene composition of accessory chromosomes, we first aligned
AC-encoded genes against a local non-redundant (nr) database using DIAMOND (v2.1.8)51 with
an E-value threshold of 0.1, and analyzed the taxonomic relationships of the top-scoring hits.
Additionally, InterProScan (v.5.67-99.0)52,53 was employed to query these genes against three
databases (Pfam 36.0, SMART 9.0, and MobiDBLite 2.0), facilitating the identification of
conserved protein domains, functional motifs, signal peptides, transmembrane domains,
low-complexity regions, and intrinsically disordered regions. Complementary predictions were
performed using DeepTMHMM (v.1.0)54 and SignalP 6.055 to determine the transmembrane
domains, signal peptides, and secretion types of AC-encoded proteins. Collectively, these
analyses yielded a comprehensive characterization of the genes within accessory chromosomes.

Transposable elements in each of the 27 haploid genomes were annotated using a
combination of tools: LTRharvest (GenomeTools) 1.6.556, LTRdigest57, RepeatModeler
(http://www.repeatmasker.org), and EDTA v2.2.258. All annotations were merged to construct a T.
fuciformis-specific TE database. This species-specific database was then used in conjunction
with RepeatMasker (v4.0)59 to perform genome-wide annotation of repetitive sequences across
the 27 haploid genomes.

Starship giant transposons in 27 haploid genomes were identified using Starfish (v.1.0.0)60
with multiple modules. Candidate tyrosine recombinase (YR) genes (potential captain genes)
were retrieved and filtered via the annotate module, leveraging the built-in YRsuperfams hidden
Markov model database (YRsuperfams.p1-512.hmm) and reference protein database
(YRsuperfamRefs.faa). The consolidate module integrated original genome annotations with YR
candidate annotations. The sketch module scanned YR genes and their 10 Kb flanking regions to
preliminarily define the boundaries and gene composition of potential Starship elements. Using
YR genes as anchors, the insert module performed similarity searches against a pre-constructed
BLAST database (parameters: ≥80% nucleotide identity, ≥750 bp HSP length) to determine
insertion start/end positions. Candidate Starship regions were confirmed by manual inspection.

Pan-genome analysis and phylogenetic tree construction
To identify orthologous groups (OGs), we employed OrthoFinder (v2.5.5)61 to analyze the
predicted proteins from the 27 haploid genomes. We used MAFFT (v7.525)62 to perform
homology alignment of the protein sequences from all genomes, with parameters set to default
mode. Subsequently, OrthoFinder was used to generate multiple sequence alignment files, which
were then integrated into a superalignment matrix using the -M msa parameter. OGs present in
all genomes were classified as core genes, those found only in some genomes were considered
accessory genes, and those existing in a single genome without clear orthologs were categorized
as singletons. The phylogenetic relationship among 27 T. fuciformis haploid genomes was
inferred using all core genes. Based on the alignment results, a maximum likelihood (ML)
phylogenetic tree was constructed with FastTree (v2.1.11)63, using the parameter -m set to MFP
and performing 1000 bootstrap resampling. Visualization was done with the web-based ChiPlot
(v2.6.1, https://www.chiplot.online/)64, where the tree layout was manually optimized and key
evolutionary branches were annotated.

The ITS sequence of T. fuciformis strain CBS 6970 was used as the query to align with the
PacBio sequencing reads databases of 16 strains via the local BLAST program. Fragments with
≥5 identical sequences in matches were screened, identified as ITS sequences of the
corresponding species, and truncated. These truncated ITS sequences served as new queries for
searching the online UNITE database (https://unite.ut.ee/schedule_analysis.php). The most
similar homologous sequences were downloaded for subsequent phylogenetic tree construction.
Sequence analysis was performed using MEGAN 11 (v11.0.13)65: All ITS sequences were aligned
via the built-in ClustalW tool with parameters set as gap opening penalty = 6.66 and gap
extension penalty = 15. A phylogenetic tree was constructed based on the ML method, with the
optimal tree structure searched via the Close Neighbor Interchange (CNI) strategy; its reliability
was evaluated using 1000 replicate bootstrap tests. Finally, the online tool ChiPlot was used for
phylogenetic tree visualization. The same alignment, tree-building, and visualization methods
were applied to construct the phylogenetic tree of mitochondrial genomes using the complete
genome sequences of the 16 strains.

Average nucleotide identity analyses
We utilized the Average Nucleotide Identity (ANI) method to analyze the differences
between phylogenetic relationships of homologous core chromosomes and those of homologous
accessory chromosomes in the T. fuciformis haploid genomes. The ANI values and coverage
were calculated using the online OrthoANIu (https://www.ezbiocloud.net/tools/ani)66, with
chromosome sequences as input. A single-cycle comparison approach was employed, where each
homologous chromosome was compared with all other homologous chromosomes, to assess the
phylogenetic relationships within the same set of homologous chromosomes.

Chromosomal copy number variation analyses
Illumina sequencing reads were aligned to the TF2206 hapA genome using BWA-MEM
(v0.7.18)67 with default settings for accurate mapping. Post-alignment, GATK (v4.0.11.0)68 was
employed for several processing steps: creating a sequence dictionary, rearranging SAM files,
sorting BAM files, and marking duplicates to avoid biases in variant calling. Samtools
(v1.15.1)69 converted SAM files to the more compact BAM format and calculated the
sequencing depth of the final BAM file, offering insights into genome coverage. Chromosome
copy number variations were identified by dividing each chromosome's sequencing depth by the
genome's average sequencing depth.

Chromosomal structural variation analyses
To analyze chromosomal structural variations, genomic data in GenBank format were used
as input files. Genome sequences were aligned using nucmer from the MUMmer (v4.0.0)70,
applying a length threshold of 2,000 to exclude shorter alignments and focus on significant ones.
Syntenic relationships and structural variations were visualized using PyGenomeViz (v1.5.0)
(https://github.com/moshi4/pyGenomeViz), which effectively illustrates complex genomic
rearrangements. The analysis focused on structural variations such as inversions, terminal
chromosome losses, insertions-deletions, and chromosome fusion-splitting events.

Analysis of accessory chromosome origins
To investigate the origins of ACs, we addressed three key questions through the following
analyses.
For distinguishing between core chromosome derivation and external acquisition, we
identified genes with homology to core chromosome genes via sequence alignment for each AC
(AC01 to AC15). Using AC01 as a representative, we analyzed all genes in both this accessory
chromosome and the core chromosomes of its corresponding strains, clustered them into
orthologous groups (OGs), and calculated the proportion of genes with homology. We then
mapped these homologous genes on the accessory chromosomes to determine whether they were
embedded in transposable element sequences and transferred between core and accessory
chromosomes via TE-specific loci71. Based on the results of these two analyses, we inferred
whether the AC01 originated from core chromosomes. This analytical framework was similarly
applied to AC02 to AC15.

For identifying potential donor species, we analyzed the all AC-specific genes by
conducting homology searches against public databases to classify genes as having known or
unknown functions; using InterProScan, DeepTMHMM, and SignalP 6.0 to annotate conserved
domains, functional motifs, signal peptides, transmembrane domains, low-complexity regions,
and intrinsically disordered regions; and evaluating the taxonomic distribution of top hits from
homology searches, with specific attention to sequence similarity to species within Tremellales
and other related taxa, complemented by functional enrichment analysis on annotated genes to
characterize overrepresented biological processes.

For estimating integration timing into T. fuciformis, we analyzed repetitive sequences in
AC01 to AC15, focusing on TEs and quantifying the proportion of TEs shared with core
chromosomes versus AC-specific TEs; for AC01, we characterized intact LTR retrotransposons
shared with core chromosomes, inferred their origins, and estimated insertion times using
sequence divergence-based method implemented in MEGA11 (Jukes-Cantor model)-specifically,
employing a neutral mutation rate (r = 2 × 10⁻⁹ substitutions per site per year) derived from
Cryptococcus neoformans72, with this approach replicated for AC02 to AC15 to approximate
their integration timing into the T. fuciformis genome.

Association experiments between T. fuciformis and A. stygium
Among the 16 T. fuciformis strains examined, 11 were able to form compatible associations
with their corresponding A. stygium strain. Based on the phylogenetic placement of T. fuciformis
(Clusters I, II and III), the 11 corresponding A. stygium strains were classified into three groups:
AF1, AF2 and AF3. Each A. stygium was inoculated onto a sawdust-based medium and
incubated at 25°C for 7 days until full mycelial colonization was achieved. The colonized
sawdust blocks were then transferred to fresh Petri dishes and gently compressed. The cultures
were incubated for an additional 3 days to allow for mycelial recovery and adaptation. A
single-cycle inoculation method was employed, in which all 11 T. fuciformis strains were
individually inoculated onto the same A. stygium -colonized sawdust substrate within each dish
for co-cultivation. The co-cultures were incubated at 25°C for 20 days. Successful associations
were identified by the appearance of dense, white, cotton-like mycelial tufts at the margins of the
T. fuciformis inoculation sites, indicating a compatible interaction between T. fuciformis and A.
stygium.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript “Exploring the polymorphism and origins of accessory chromosomes in Tremella fuciformis: insights into genome divergence and structural variation” by Jinxiang Zhang and co-authors presents a fascinating story on the evolution of accessory chromosomes (ACs) in Tremella basidiomycetes. Authors annotated 16 strains (27 haploid genomes) to assess the trends of ACs abundance among individual genomes and compare their similarity to the core chromosomes.\nThe manuscript presents several interesting and novel findings. It is worth mentioning that accessory chromosomes in fungi are known from Pezizomycotina and the report extends the taxonomic range of their occurrence to Basidiomycota/Agaricomycotina. The collection of ACs spans elements of diverse size (from 200kb to chromosome size objects), different degree of identity to the core chromosomes and conservation among isolates.\nAs expected the ACs are enriched in transposons and have a lower gene density than core chromosomes. However there are signs of AC coevolution with the core genome, namely closely related isolates harbored the same or almost the same set of ACs. Authors report a very small set of common genes in the pangenome (24.3%) and huge differences in genome size between the genomes 32.4 v 23.8Mb.\nThe study is well written, robust and straightforward. The main shortcoming is that the manuscript ends briefly, just before I expected to find an analysis of the gene and transposon content in accessory chromosomes and further interpretation of how these chromosomes originated and evolved.\nI would like to express only a few concerns /questions/invitations to extend the study:\nPlease provide a systematic analysis of the gene content in terms of gene function and properties of encoded proteins. If ACs follow the trends of the two speed genome it is expected to see repeat-rich, low complexity rich, disordered, effector-like and transposon-related sequences in the ACs more often than in the core chromosomes.\nWhich evolutionary scenario leads to the lower ANI observed in the ACs?\nStarships are shaping fungal genes and contribute to the moves of massive parts of the genomes. Have you found Starships in the Tremella genomes? If yes, how do they relate to the ACs? If you have not searched for them so far you might use the dedicated software starfish available on github.\nIs the pattern of intronization of genes similar between ACs and core chromosomes? Does the gene pool differ in terms of length, intronization, gene structure and function?\nPlease comment on the ACs gene and transposon content across the taxa. Are all TEs equally likely to be localized in ACs and core chromosomes?\nFig.4 lines 292-293 describe the location of the chromosome fusion. However there is no information about what exactly is there, is it a long transposon, repeat region or a genic region?\nThe description of TE content in lines 352-353 does not explain the diversity of TEs in the ACs compared to the core chromosomes. Please report which families of TEs are shared and which are unique to each of the chromosome types. Furthermore, please describe TE family proliferation and if possible date the events for LCR retrotransposon bursts.\nWhen delimiting fungal species it is recommended to use UNITE as a source of curated ITS references.\nThe description of gene content in lines 364-369 is not exhaustive. No hits in nr NCBI database does not necessarily show they are orphan genes. One possible path to describe these genes is to cluster the gene pool of the ACs to see how many are shared and how many are specific to an individual AC type. Another avenue is to map the genes on domain databases and assign them to protein families and folds. Having such annotation allows for a better prediction of protein function. Complementary to domain analysis it is recommended to use non-domain protein features such as secretion signal, cellular localization, transmembrane helices, coiled coils, disordered and low complexity regions to describe the encoded protein set in ACs relative to core chromosomes. I would suggest to perform all three annotation procedures to desribe in detail the content of ACs and compare them.", "reviewer_2": "Accessory chromosomes, also known as dispensable, lineage-specific, supernumerary or B-chromosomes, are thought to be non-essential for normal growth and/or development. These chromosomes occur in several eukaryotes, but their evolution and function has been extensively studied in ascomycete fungi, and especially in plant pathogens. Basidiomycetes are another major fungal lineage, comprising most of the mushroom forming species. In contrast to ascomycetes, however, the presence, function, and evolution of accessory chromosomes are not well documented.\nThe manuscript ‘Exploring the polymorphism and origins of accessory chromosomes in Tremella fuciformis: insights into genome divergence and structural variation’ by Zhang and colleagues reports on the genome sequencing and assembly of haplotype-resolved genome sequences of 16 strains of the basidiomycete fungus Tremella fuciformis. Using this resource, the manuscript studies their chromosomal makeup, identifying abundant accessory chromosomes. It provides an in-depth description of the unique genomic characteristic of accessory chromosomes and observed that most accessory chromosomes lacking identifiable homologs in other species. Based on these observations, the manuscript discusses potential origins of these accessory chromosomes as well as the implications for T. fuciformis’s biology.\nThe observation of abundant accessory chromosomes in a basidiomycete fungus is very interesting, broadening the common idea of their occurrence and importance solely in ascomycetes. Moreover, the generated genomic resources will be relevant for the fungal community. However, I have several concerns about the novelty of the observations, the robustness of the presented data, and the general clarity of the manuscript.\nMost importantly, nearly the same group of authors have previously published a manuscript in Genome Biology (https://doi.org/10.1186/s13059-023-03093-7) reporting on the presence of accessory chromosomes and detailed many of their characteristics. That manuscript had also already reported copy-number variation in core and accessory chromosomes as well as structural variations, alike much of the data presented here. However, the current manuscript is not transparent which observations are rather confirmatory to the previous work and what are truly unique and novel, and how these make a significant contribution to our understanding of accessory chromosomes in basidiomycetes. I do understand that the current submission is more exhaustive, providing data from different strains and haplotypes, but the true novelty is not apparent.\nThere are several experimental approaches that are not clearly explained, nor their impact is analyzed and/or discussed. Furthermore, some observations need more detailed explanations. For example, the manuscript reports the ITS tree for the 16 strains (L121ff), but it is not clear how the ITS is derived from dikaryotic strains that should have two ITS; also, NJ algorithm is used rather than more sophisticated tree reconstruction approaches. Hi-C data is generated for NDB and TF2206, however it is not explained why these strains were selected. Tr01 was used as a reference to place NDB contigs into chromosomes. Given the known chromosomal rearrangements (Deng et al. 2023), this could potentially introduce error in the assemblies; similarly, the Hi-C enhanced genomes were used to place contigs for other (L553). How was the potential issue addressed or corrected? Observed chromosomal rearrangements could for example be validated (e.g., via orthogonal approaches such as PCRs). It is not entirely clear how the core chromosomes were identified (L164), as I would expect a working definition would be that these need to be conserved in all strains. It seems that the number differs between eight and ten, while this is observation is only explained much later (and had been as far as I can tell reported previously already; Deng et al. 2023); the implication of the transposons at the breakpoints/junctions are not further detailed but could provide insights in the processes driving chromosome variation. For additional comments, see further details below.\nThe manuscript needs to overall improve in clarity. Just as an example, the abstract is illegible for non-expert readers. It is not explained what accessory chromosomes are in comparison to core chromosomes, what T. fuciformis is, or why 16 strains would yield 27 haplotypes (monokaryon vs dikaryons). The abstract mentions ‘homology similarity’ (L31), but this is not a common concept since homology is qualitative, but similarity is a quantitative trait. Similarly, parts of the introduction are superficial, repetitive (e.g., L59 vs L66 or L73 saying the same as few lines above), or plain wrong. For example, L48 implies that all fungi have unique characteristics such as the two-speed genome phenomenon, while there is plenty of evidence that this is not common to all fungi and differ significantly between even closely related fungi. In contrast to the statement of L59f, accessory chromosomes in Z. tritici have to my knowledge not been demonstrated to contain virulence factors or secondary metabolite clusters, and their functions remain enigmatic. Accessory genomic regions have been successfully identified using (epi-)genomic characteristics alone (in Magnaporthe and Verticillium) and thus the statement in L75 is also incorrect.\nThroughout the manuscript (e.g., L127), the companion fungus Annulohypoxylon stygium is mentioned several times by their significance to T. fuciformis remains unclear. It is suggested that there might be a link to the presence of a subset of accessory chromosomes (e.g., Fig 2), but the manuscript remains vague with respect to mechanisms and further implications for both partners. Moreover, the material & methods implies that the authors performed mating experiments, however, these seems to be rather association than true mating. The authors need to be much clearer on the presence, impact, and implication of the companion fungus to the genomics and biology of T. fuciformis.\nAdditional comments and suggestions:\nTitle: for the general audience, spell out what Tremella is. Moreover, the content before and after the colon seems to be redundant.\nL61: Does this imply that there are basidiomycetes with accessory chromosomes? If so, this needs to be introduced in much more detail.\nL120: What are ‘…principle cultivated varieties,…’? Please explain.\nL124: The sentence is unclear as it suggests that ITS sequences exhibit morphological characteristics. Please rephrase.\nL138: ‘… modern…’ not sure what this refers to, could this be an error?\nL202: The manuscript should be clearer when it reports on haploid genomes. Does it refer to a monokaryotic strain or to a dikaryotic strain where both haplotypes have been assembled? This distinction could also be clearer in the figures (e.g., the A and B in the strain name)\nL229: this pattern could also be due to horizontal transfer of the chromosome rather than loss. Could the authors perform additional analyses to clearly distinguish these scenarios?\nL262: What is driving the high similarity between accessory chromosomes? Is this mainly due to transposons or also over the protein-coding (non-TE) genes?\nL279: it needs to be clear that the two AC14 refer to the A and B haplotype rather than a copy-number variation of one of the two.\nL348: Why was C-1 used as a case study?\nL367: Additional information about the genes on accessory chromosomes are lacking. For example, are these expressed, are these predicted to be secreted, etc. Do they have other characteristics that makes them distinct from those on the core genome (e.g., number of introns, length, etc.). Moreover, the absence of any homology raises important questions about their origin that unfortunately remains unclear. I find it surprising that most genes do not even have homologs in other fungi and the explanation that we lack the corresponding species in the database seem unlikely given the ongoing sequencing efforts.\nL399: Does this sentence imply that this are plans? If so, please rephrase as this is not entirely clear.\nL351: I find it odd to read that transposons are treated similarly to genuine protein-coding genes. The abundance of these in the gene sets suggest that the structure gene annotation was not performed carefully, and transposons should be removed prior to any analyses\nL519: ‘…did not exist from the beginning…’ please rephrase\nL590: few details on the mitochondrial assemblies could be provided. Do they similarly display three distinct groups. Are phylogenies with mitochondrial markers concurrent to nuclear taxonomy?", "reviewer_3": "In this work the authors describe sequencing of 27 haplotypes from 16 strains of the basidiomycete Tremella fuciformis, and a subsequent pangenomics investigation of core and accessory chromosome statistics and distribution.\nMajor comments\nI think the primary critique involves the relationship between accessory chromosomes and A. stygium pairing, which is one of the more interesting biological aspects of this work. It is very interesting that the distribution of ACs correlate with the pairing groups, and this is a valid and potentially novel finding.\nHowever, supplementary table 1 introduces 4 strains that do not associate with A. stygium: Wuyi2014, Wuyi2015, Lys2020, and T0053. Firstly, the statements in the introduction (lines 101-103) seem to imply that all Tremella species associate with Annulohypoxylon, so it might be worthwhile to point out here (and potentially also in lines 127-129 in the results) that not all Tremella associate with Annulohypoxylon.\nBut overall, there is very little discussion about how the distinct accessory chromosome patterns relate to these four non-pairing strains, as they compare to their pairing-capable phylogenetic neighbors.\nFor example, Figure 4 shows structural variations in AC01 (inversion) and AC08 (deletion) between NDBA (a pairing strain) and Wuyi2015 (a non-pairing strain), but there is no discussion or speculation as to whether such differences may influence pairing.\nAdditionally, and specifically with respect to one of the major conclusions, that the three largest ACs (AC01, AC12, and AC14) correspond to the three distinct AF groups (AF2, AF1, AF3), there are nuances that are not addressed. For example, AC14 is missing from non-pairing strain T0053 but present in non-pairing LYS2020. Similarly, AC01 is present in both non-pairing strains Wuyi2014 and Wuyi2015.\nThere is also no discussion of other nuances observed in figure 2 and how they could relate to A. stygium pairing capabilities. For example, AC08 is primarily found in AF2, but also found in two AF3 strains LYS2020 and T0053, which are notably non-pairing. Similarly, AC06 and AC08 are absent in non-pairing Wuyi2014 but present in non-pairing Wuyi2015. Finally AC15 doesn't show exact group specificity, since it is present in both AF2 and AF3 (excluding B-1 and C-1).\nI think that at minimum, some inclusion of these observations in the discussion is warranted. I think it would be beneficial as well to include additional structural comparisons similar to Figure 4, between other closely related strains that differ in pairing ability. Or even a comparison and discussion of how the LYS2020 and T0053 AC08 chromosomes might differ from the AC08 chromosomes found in any of the AF2 strains might be informative.\nMinor comments:\n- Figure 1d: I don't see any black circles in either chart\n- Figure 2: \"Dots indicate the absence\" should probably be corrected to \"minus signs indicate the absence\"\n- Chromosomal copy number variation Section:\n- Why was only TF2206 selected? Many other strains exist in M/Y morphotypes\n- Why was only YY2 selected for the normalized depth distribution curve comparison (Figure 5d)?\n- The section states that \"Ten single colonies\" were selected for sequencing from strain TF2206. However the ten colonies are subsequently themselves referred to as \"strains\" (e.g. \"YY3 strain\", \"YY1 strain\"). To distinguish these from the broader biological strains (\"TF2206\", \"TF104\", etc) maybe they should be referred to as \"colonies\"?\n- Line 315-316: \"obtained from subculturing the same colony\" -> it was unclear which colony is \"the same\" colony?\n- In Figure 5: The \"M to Y\" label makes sense because this is described as coming from a mycelial to yeast transition. But \"Y to Y\" and \"M to M\" do not really make sense because these were isolated simply as yeast or mycelia, with no transitions involved.\n- Figure 6a\nThe Venn diagram is just the data from the C-1 row. So, why is CCs 8,653 in both cases, but ACs is 297 in Venn diagram and 318 in table? The value of CCs should be different between the Venn diagram and the table, depending on if 8,653 is the total count or the unique count."}, "author_response": "Core Revisions & Highlights\n1. Advanced understanding of T. fuciformis accessory chromosome (AC)\norigins: Expanded the prior simplistic analysis with comprehensive annotations of\nrepetitive sequences/gene composition across 27 haploid genomes and systematic core\nchromosome (CC) comparisons to address three key questions (origin, donor species,\nintegration timing)-the first dedicated in-depth analysis of AC origins, with evidence\nfor horizontal transfer from understudied taxa-supported by a newly generated Figure\n6 and fully rewritten corresponding Materials and Methods/Results sections.\n2. Novel insights into Starship giant transposons in T. fuciformis: Conducted\nsystematic identification of Starship giant transposons via Starfish software across 27\nhaploid genomes, revealing 8 elements (3 on core chromosomes, 5 on ACs) with\nfunctional implications-including potential mediation of chromosome fusion (SS04)\nand structural variation patterns that support rapid AC divergence-representing the first\nanalysis of Starships in this species and their association with AC dynamics.\n3. Rigorous validation of chromosomal rearrangements: Implemented multiple\northogonal approaches (HiFi/nanopore read mapping, Hi-C heatmaps, multi-strain\nsynteny analysis) to confirm rearrangements, addressing reference bias concerns and\nenhancing result reliability.\n4. Methodological optimization for ITS phylogenetics: Updated tree\nconstruction from NJ to Maximum Likelihood (ML) with 1000 bootstrap replicates,\nresolved heterokaryotic strain ITS ambiguity via PacBio read-specific identification,\nand utilized the UNITE database as curated ITS references for accurate fungal species\ndelimitation.\n5. Enhanced manuscript clarity, accessibility, and rigor: Revised the abstract\nto clarify key concepts (core/accessory chromosomes, T. fuciformis as silver ear fungus,\n27 haplotypes’ origin) for non-experts, corrected \"homology similarity\" to standard\n\"sequence similarity,\" and refined the Introduction by rectifying overgeneralizations\n(e.g., \"two-speed genome\" limited to \"some fungi\"), correcting incorrect claims about\nZ. tritici accessory chromosomes, resolving redundancy, and aligning identification-\nrelated statements with established literature.\nPoint-by-point responses to Reviewers’ comments.\nReviewer#1\nQ1:\nThe manuscript “Exploring the polymorphism and origins of accessory\nchromosomes in Tremella fuciformis: insights into genome divergence and structural\nvariation” by Jinxiang Zhang and co-authors presents a fascinating story on the\nevolution of accessory chromosomes (ACs) in Tremella basidiomycetes. Authors\nannotated 16 strains (27 haploid genomes) to assess the trends of ACs abundance\namong individual genomes and compare their similarity to the core chromosomes.\nThe manuscript presents several interesting and novel findings. It is worth\nmentioning that accessory chromosomes in fungi are known from Pezizomycotina and\nthe report extends the taxonomic range of their occurrence to\nBasidiomycota/Agaricomycotina. The collection of ACs spans elements of diverse size\n(from 200 Kb to chromosome size objects), different degree of identity to the core\nchromosomes (CCs) and conservation among isolates.\nAs expected the ACs are enriched in transposons and have a lower gene density\nthan CCs. However there are signs of AC coevolution with the core genome, namely\nclosely related isolates harbored the same or almost the same set of ACs. Authors report\na very small set of common genes in the pangenome (24.3%) and huge differences in\ngenome size between the genomes 32.4-23.8 Mb.\nThe study is well written, robust and straightforward. The main shortcoming is that\nthe manuscript ends briefly, just before I expected to find an analysis of the gene and\ntransposon content in accessory chromosomes and further interpretation of how these\nchromosomes originated and evolved. I would like to express only a few concerns\n/questions/invitations to extend the study:\nResponse:\nIn brief:\nWe sincerely appreciate your positive comments on our manuscript and the\nconstructive suggestions you have provided. We fully acknowledge that the final\nsection of the original manuscript was overly brief, which constituted a significant\nlimitation. Following your recommendations, we have conducted comprehensive\nannotations of the repetitive sequences and gene composition of ACs across all 27\nhaploid genomes, and systematically compared these features with those of CCs.\nThrough these analyses, we have addressed three key questions: 1) whether ACs\noriginated from CCs or were acquired from external sources; 2) the potential donor\nspecies of ACs; and 3) the timing of AC integration into T. fuciformis, with these\nfindings summarized in the newly added Figure 6.\nDetail:\nFor the analysis of repetitive sequences, we annotated TEs in each of the 27 haploid\ngenomes using LTRharvest/LTRdigest, RepeatModeler (for non-LTR TEs), and EDTA.\nThese annotations were merged to construct a species-specific TE database, which was\nthen used with RepeatMasker to annotate repetitive sequences in both CCs and ACs of\nthe haploid genomes.\nFor the analysis of gene composition, in addition to DIAMOND searches of AC\ngenes against the NCBI nr database, we used InterProScan to align these genes with\nthree databases (Pfam, SMART, and MobiDBLite), obtaining information on\nconserved protein domains, functional motifs, transmembrane domains, low-\ncomplexity regions, and intrinsically disordered regions. Furthermore, we performed\nDeepTMHMM and SignalP 6.0 analyses to predict transmembrane domains, signal\npeptides, and secretion types of the proteins encoded by AC genes. These analyses\ncollectively provide a comprehensive understanding of AC genes.\nTo address 1）whether ACs originated from core chromosomes or external sources：\nwe took AC01 as a representative example. Among the 2340 genes (518 OGs) carried\nby AC01 across 6 haploid genomes that harbor this chromosome, only 332 (14.2%)\nshowed homology with CC genes. Of these 332 shared genes, 256 were embedded\nwithin transposable element (TE) sequences indicating that these shared genes likely\narose through TE-mediated mobilization. Consistent observations were made for AC02\nto AC15: the majority of their genes lack counterparts in core chromosomes, and the\nlimited shared genes are associated with TE activity. These findings collectively\nsuggest that accessory chromosomes are not derived from the detachment of core\nchromosomes but were acquired through horizontal gene transfer from external sources.\nTo address 2) the origin of the species from which ACs were acquired: Among the\n1,548 AC-specific genes, 1,156 (75%) failed to match any homologous sequences or\nconserved domains, and lacked functional motifs, signal peptides, or transmembrane\ndomains. This not only indicates that the majority of these genes have not been\nfunctionally characterized in previous studies, but also strongly suggests they likely\noriginate from unexplored or understudied species. Of the remaining 392 genes, 232\nshowed matches to homologous regions but with unknown functions, 7 lacked\nhomologous matches and encoded proteins bearing signal peptides, leaving only 153\nwith annotated functions. Among these 153 functionally characterized genes, the most\nabundant were those encoding motor domains and members of the major facilitator\nsuperfamily-proteins involved in transmembrane transport of nutrients, energy\nconversion, and molecular movement. These functions are consistent with the role of\nAnnulohypoxylon in providing nutrients and energy for the growth and development of\nT. fuciformis. Notably, 236 of the 392 genes with homologous matches exhibited the\nhighest sequence similarity to species within the Tremellales. This suggests that the\naccessory chromosomes likely existed prior to the divergence of T. fuciformis as a\ndistinct species.\nTo address 3) when the ACs were integrated into T. fuciformis: We analyzed the\ntypes of repetitive sequences in AC01 to AC15, as well as the proportions of TEs shared\nwith CCs versus those specific to ACs. Taking AC01 as a case study, we conducted a\ndetailed analysis of the types of intact LTR retrotransposons shared between AC01 and\nCCs. For each type of intact LTR, we traced their origins and estimated their insertion\ntimes, thereby inferring the approximate timing of AC01’s integration into T. fuciformis.\nUsing the same approach, we estimated the approximate integration times of AC02 to\nAC15.\nRevisions in the Materials and Methods section: Revised the \"Gene annotation\nand TE prediction\" section (original Lines 626-631; revised Lines 720-724) to optimize\nTE annotation workflow for 27 haploid genomes. Inserted new content on accessory\nchromosome gene characterization at original Line 625 (revised Lines 725-736) using\nDIAMOND, InterProScan, DeepTMHMM, and SignalP 6.0. Revised the entire\n\"Analysis of accessory chromosome origins\" section (original Lines 696-711; revised\nLines 816-847) to include origin inference, donor species identification, and integration\ntiming estimation.\nRevision of the entire \"Possible Origins of Accessory Chromosomes\" section\nin the Results (original Lines 346-369; revised Lines 394-436): ACs are inferred to\noriginate from horizontal gene transfer (not CC detachment), supported by low\nhomology between AC and CC genes (e.g., 14.2% for AC01) and TE association of\nshared genes; among 1,806 AC genes across three clades, 1,548 are AC-specific, with\n75% lacking functional annotations and 236 showing highest similarity to Tremellales\nspecies, while AC repetitive sequences (38%-89%) are dominated by RNA transposons\nand LTR retrotransposon analysis estimates AC integration timing at 0.33-1.69 million\nyears ago.\nRevisions in the Discussion section (original Lines 497-523; revised Lines 579-\n600): <1/5 of AC genes have CC homologs (mostly transposons), supporting\ntransposon-mediated exchange rather than CC-derived origins; over 3/4 of 1,548 AC-\nspecific genes lack homologs/functional domains (implying understudied species\norigins); 102 AC genes are conserved across three T. fuciformis clades, with ~2/3 of\ndatabase-matched AC-specific genes aligning best with Tremellales species (indicating\npre-species AC existence); rapid structural variations (inversions, indels, etc.) reduce\nshared AC genes vs. CCs, reinforcing the hypothesis that ACs were acquired via pre-\nspeciation horizontal gene transfer and evolved independently as non-essential\nelements.\nQ2:\nPlease provide a systematic analysis of the gene content in terms of gene function\nand properties of encoded proteins. If ACs follow the trends of the two speed genome\nit is expected to see repeat-rich, low complexity rich, disordered, effector-like and\ntransposon-related sequences in the ACs more often than in the core chromosomes.\nResponse:\nFollowing your recommendations, we have conducted comprehensive annotations\nof the repetitive sequences and gene composition of accessory chromosomes (ACs)\nacross all 27 haploid genomes, and systematically compared these features with those\nof core chromosomes. Detailed responses can be found in Q1.\nQ3:\nWhich evolutionary scenario leads to the lower (higher) ANI observed in the ACs?\nResponse:\nThe higher average nucleotide identity (ANI) observed in accessory chromosomes\n(Acs) is likely shaped by two key evolutionary scenarios inferred from our data:\nFirst, ACs were acquired via horizontal gene transfer (HGT) at a specific node\nduring the evolution of T. fuciformis, an origin that contributes to their higher average\nnucleotide identity (ANI) relative to core chromosomes. Our findings reveal minimal\nhomology between ACs and core chromosomes, indicating they are not derived from\nthe latter-a stark contrast to core chromosomes, which have accumulated substantial\ndivergence through vertical inheritance over time. Freed from the prolonged divergent\nevolution associated with vertical transmission, these horizontally acquired ACs have\nretained relatively higher sequence conservation, further elevating their ANI. This\naligns with our discussion that \"The accessory chromosomes originated from\nunexplored species before the speciation of T. fuciformis.\"\nSecond, frequent exchanges between homologous ACs in heterokaryotic strains\n(e.g., TF2206) contribute to maintaining high sequence similarity. As noted,\nheterokaryons derived from different colonies of the same parent colony exhibit\nsubstantial variation in AC copy numbers across cells. This variability can lead to\nscenarios where ACs in one nucleus are lost, with subsequent replenishment from ACs\nof the other nucleus-a process that facilitates frequent sequence exchanges between\nACs.\nTo address this, we have inserted the following sentence in the Discussion section\nunder \"Extremely high frequency of chromosomal copy number variation in both core\nand accessory chromosomes\" (original Line 464): This improper allocation of\naccessory chromosomes during mitosis may further facilitate frequent exchanges\nbetween homologous accessory chromosomes in the heterokaryotic strain, resulting in\nhigh or even identical sequence similarity among them (revised Lines 542-545).\nQ4：\nStarships are shaping fungal genes and contribute to the moves of massive parts of\nthe genomes. Have you found Starships in the Tremella genomes? If yes, how do they\nrelate to the ACs? If you have not searched for them so far you might use the dedicated\nsoftware starfish available on github.\nResponse：\nIn brief:\nThank you for your valuable comment regarding the presence, roles of Starships in\nTremella genomes, and their potential association with accessory chromosomes (ACs).\nWe have systematically searched for Starship giant transposons in our dataset to address\nthis point, with key findings as follows: Eight Starship elements were identified across\nsix genomes, including 3 on core chromosomes and 5 on ACs; Starship SS04 maps to\na chromosome fusion site, implying potential involvement in mediating such events;\nNotably, while the core-localized Starship pair is identical, two AC-localized pairs\nexhibit distinct structural variations-supporting rapid AC structural divergence,\nconsistent with the dynamic nature of fungal accessory chromosomes reported\npreviously.\nDetail:\nWe have added these findings to the Revised Manuscript:\nAdded Starship identification methodology in the \"Gene annotation and TE\nprediction\" section of Materials and Methods in the manuscript (revised Lines 743-754):\nWe used Starfish (v1.0.0) to identify Starships in 27 haploid genomes, via YR gene\nretrieval (annotate module), boundary definition (sketch module), and BLAST\nsimilarity searches (insert module), with manual confirmation of candidates.\nAdditionally, we incorporated these Starship analysis results into the \"The rapid\nstructural variation of accessory chromosomes\" section in the manuscript (revised Lines\n321-328): 8 Starship giant transposons (41,942-605,797 bp) were identified across 6\ngenomes (3 on CCs, 5 on ACs). SS04 locates at a chromosome fusion site (Fig. 4d),\nimplying its role in fusion events. Among 3 Starship groups on heterokaryote\nhomologous loci: core-localized SS02/SS03 are identical, while AC-localized pairs\n(SS06/SS07, SS04/SS05) show structural variations (46 bp/3,048 bp differences),\nsupporting rapid AC structural divergence (newly added Supplementary Table 3 and\nSupplementary Figure 14).\nWe have also updated Fig. 4d to include the structural diagram of SS04, with the\nlegend revised to clarify the insertion of GDA sequences (356,504-531,280)\ncorresponding to SS04, which contains a typical captain gene, direct repeats (DRs), and\nterminal inverted repeats (TIRs).\nWe have added Supplementary Table 3, which lists the characteristics of the 8\nStarship giant transposons. Additionally, Supplementary Figure 14 has been included,\ndisplaying the synteny plots of SS02 and SS03, as well as those of SS04 and SS05.\nQ5：\nIs the pattern of intronization of genes similar between ACs and core chromosomes?\nDoes the gene pool differ in terms of length, intronization, gene structure and function?\nResponse\nThank you for your insightful questions regarding the intronization patterns and\ngene pool differences between accessory chromosomes (ACs) and core chromosomes\n(CCs).\nWe have conducted targeted comparative analyses to address these points: we\ncompared the average gene length, average intron length, and average number of\nintrons per gene between the two chromosome types, and further analyzed AC gene\nfunctions.\nOur results show that the average number of introns per gene in ACs is significantly\nlower than that in CCs, while there are no significant differences in average gene length\nor average intron length between them. These comparative data are presented in the\nnewly added Supplementary Figure 12. Regarding functional differences, as detailed in\nour response to Q1, most AC genes lack homologous sequences and functional domains\nin existing databases, which limits further direct functional comparisons with CC genes.\nWe inserted a sentence in original Line 256: The average length of genes and\nindividual introns in accessory chromosomes is comparable to that of core\nchromosomes. However, the average number of introns per gene in accessory\nchromosomes is significantly lower, with only 1.5 introns per gene, which is notably\nless than the 3.3 introns per gene in core chromosomes (P-value = 1.24E-7, newly added\nSupplementary Figure 12). (revised Lines 280-284)\nQ6：\nPlease comment on the ACs gene and transposon content across the taxa. Are all\nTEs equally likely to be localized in ACs and core chromosomes?\nResponse:\nThank you for your question regarding the gene and transposon content of\naccessory chromosomes (ACs) across taxa, as well as the differential localization of\ntransposable elements (TEs) between ACs and core chromosomes (CCs).\nRegarding TE localization, LTR/Gypsy, LTR/Copia, TIR/Mutator, TIR/CACTA,\nhelitron and repeat_fragment are the six most abundant TE types in the T. fuciformis\ngenome. Our comparative analysis of their proportional abundances between ACs and\nCCs revealed that not all TEs are equally distributed between the two chromosome\ntypes: relative to CCs, ACs show significantly reduced proportions of TIR/Mutator and\nTIR/CACTA, while LTR/Gypsy and LTR/Copia are significantly enriched (revised\nLines 287-293). This content has been inserted at original Line 259.\nFor AC gene content across taxa, as detailed in our response to Q1, most AC-\nspecific genes lack homologous sequences in existing databases and are poorly\nconserved across phylogenetic clades of T. fuciformis, with only 16.9% of accessory\ngenes shared among the three major clusters. This suggests taxon-specific divergence\nin AC gene content, consistent with the dynamic and lineage-specific nature of ACs.\nQ7：\nFig.4 lines 292-293 describe the location of the chromosome fusion. However there\nis no information about what exactly is there, is it a long transposon, repeat region or a\ngenic region?\nResponse:\nThis fragment consists of a single Starship giant transposon, which is inferred to\nbe potentially involved in chromosome fusion. Detailed responses are provided in Q3.\nQ8：\nThe description of TE content in lines 352-353 does not explain the diversity of\nTEs in the ACs compared to the core chromosomes. Please report which families of\nTEs are shared and which are unique to each of the chromosome types. Furthermore,\nplease describe TE family proliferation and if possible date the events for LTR\nretrotransposon bursts.\nResponse:\nWe have conducted comprehensive annotations of repetitive sequences in ACs\nacross all 27 haploid genomes and systematically compared their features with those of\ncore chromosomes. Specifically, we have analyzed the shared and unique transposable\nelement (TE) families between the two chromosome types, described the proliferation\npatterns of these TE families, and dated the burst events of LTR retrotransposons where\npossible. Detailed responses are provided in Q1.\nQ9：\nWhen delimiting fungal species it is recommended to use UNITE as a source of\ncurated ITS references.\nResponse:\nThank you for highlighting the importance of using UNITE as a source of curated\nITS references for fungal species delimitation. We fully agree with this\nrecommendation and have incorporated this into our revised analysis, as detailed in the\nMaterials and Methods section.\nSpecifically, after identifying and truncating the ITS sequences from our 16 strains\n(with validation of sequence consistency by screening fragments with ≥5 identical\nmatches), we used these truncated ITS sequences as queries to search the online UNITE\ndatabase (https://unite.ut.ee/schedule_analysis.php). We downloaded the most similar\nhomologous sequences from UNITE, which were then included as reference sequences\nin our phylogenetic analysis. This ensures that our species delimitation and\nphylogenetic clustering are anchored to curated, high-quality ITS references from\nUNITE, enhancing the reliability and comparability of our results.\nThe revised results, which integrate these UNITE-derived references, confirm that\nall tested strains cluster within three distinct clades of T. fuciformis, as shown in\nSupplementary Figure 1.\nWe have revised the content in the Materials and Methods section (original\nLines 662-668; revised Lines 771-786) to standardize the ITS sequence alignment,\nUNITE database query, MEGA 11-based ML phylogenetic tree construction (ClustalW\nalignment, CNI strategy, 1000 bootstraps), and ChiPlot visualization workflow.\nWe have revised the content in the Results section (original Lines 121-124;\nrevised Lines 127-135) to clarify strain composition (11 heterokaryotic, 5\nmonokaryotic; Supplementary Table 1), ITS sequence distribution (5 heterokaryotic\nstrains with two distinct ITS, 11 with one), and ML phylogenetic clustering of 21 target\n+ 10 reference ITS sequences into three T. fuciformis clades (Supplementary Figure 1).\nQ10：\nThe description of gene content in lines 364-369 is not exhaustive. No hits in nr\nNCBI database does not necessarily show they are orphan genes. One possible path to\ndescribe these genes is to cluster the gene pool of the ACs to see how many are shared\nand how many are specific to an individual AC type. Another avenue is to map the\ngenes on domain databases and assign them to protein families and folds. Having such\nannotation allows for a better prediction of protein function. Complementary to domain\nanalysis it is recommended to use non-domain protein features such as secretion signal,\ncellular localization, transmembrane helices, coiled coils, disordered and low\ncomplexity regions to describe the encoded protein set in ACs relative to core\nchromosomes. I would suggest to perform all three annotation procedures to desribe in\ndetail the content of ACs and compare them.\nResponse:\nWe fully agree that merely relying on the absence of hits in the NCBI nr database\nis insufficient to define orphan genes, and we have addressed your recommendations\nthrough in-depth analyses, as detailed in our revised manuscript and previous response\nto Q1.\nTo better characterize the gene pool of ACs, we have implemented two key\nstrategies: 1) Gene clustering and specificity analysis: We clustered genes from all ACs\n(AC01 to AC15) into orthologous groups (OGs) to distinguish shared genes across CCs\nfrom those specific to individual AC types. For example, in AC01, among 2340 genes\n(518 OGs) across 6 haploid genomes, only 14.2% showed homology with core\nchromosome genes, with most AC-specific genes lacking counterparts in core regions-\nsupporting their unique origin. 2) Comprehensive functional annotation beyond nr\ndatabase: We extended annotations by mapping AC genes to domain databases (Pfam,\nSMART, MobiDBLite) via InterProScan, assigning them to protein families and\nidentifying conserved domains, functional motifs, and structural features (e.g., low-\ncomplexity regions, intrinsically disordered regions). Complementarily, we analyzed\nnon-domain features using DeepTMHMM and SignalP 6.0, predicting transmembrane\nhelices, secretion signals, cellular localization, and secretion types. These analyses\nrevealed that 75% of AC-specific genes lack characterized domains or motifs, while the\nremaining 25% include functionally annotated genes (e.g., major facilitator superfamily\ntransporters), providing deeper insights into their potential roles.\nThese analyses have been integrated into the revised \"Gene annotation and TE\nprediction\" section (revised Lines 725-736) and \"Analysis of accessory chromosome\norigins\" section (revised Lines 819-838), ensuring a more exhaustive description of AC\ngene content. We believe these additions enhance the rigor of our functional predictions\nand address the gaps you identified.\nReviewer#2\nAccessory chromosomes, also known as dispensable, lineage-specific,\nsupernumerary or B-chromosomes, are thought to be non-essential for normal growth\nand/or development. These chromosomes occur in several eukaryotes, but their\nevolution and function has been extensively studied in ascomycete fungi, and especially\nin plant pathogens. Basidiomycetes are another major fungal lineage, comprising most\nof the mushroom forming species. In contrast to ascomycetes, however, the presence,\nfunction, and evolution of accessory chromosomes are not well documented.\nThe manuscript ‘Exploring the polymorphism and origins of accessory\nchromosomes in Tremella fuciformis: insights into genome divergence and structural\nvariation’ by Zhang and colleagues reports on the genome sequencing and assembly of\nhaplotype-resolved genome sequences of 16 strains of the basidiomycete fungus\nTremella fuciformis. Using this resource, the manuscript studies their chromosomal\nmakeup, identifying abundant accessory chromosomes. It provides an in-depth\ndescription of the unique genomic characteristic of accessory chromosomes and\nobserved that most accessory chromosomes lacking identifiable homologs in other\nspecies. Based on these observations, the manuscript discusses potential origins of these\naccessory chromosomes as well as the implications for T. fuciformis’s biology.\nThe observation of abundant accessory chromosomes in a basidiomycete fungus is\nvery interesting, broadening the common idea of their occurrence and importance solely\nin ascomycetes. Moreover, the generated genomic resources will be relevant for the\nfungal community. However, I have several concerns about the novelty of the\nobservations, the robustness of the presented data, and the general clarity of the\nmanuscript.\nQ1:\nMost importantly, nearly the same group of authors have previously published a\nmanuscript in Genome Biology (https://doi.org/10.1186/s13059-023-03093-7)\nreporting on the presence of accessory chromosomes and detailed many of their\ncharacteristics. That manuscript had also already reported copy-number variation in\ncore and accessory chromosomes as well as structural variations, alike much of the data\npresented here. However, the current manuscript is not transparent which observations\nare rather confirmatory to the previous work and what are truly unique and novel, and\nhow these make a significant contribution to our understanding of accessory\nchromosomes in basidiomycetes. I do understand that the current submission is more\nexhaustive, providing data from different strains and haplotypes, but the true novelty is\nnot apparent.\nResponse:\nWe sincerely appreciate your critical feedback regarding the novelty and\ndistinction of our current work relative to our previous study published in Genome\nBiology (https://doi.org/10.1186/s13059-023-03093-7). We acknowledge the need to\nclarify the unique contributions of this manuscript, and we provide a detailed\ncomparison below to distinguish confirmatory observations from novel findings:\nIn our prior Genome Biology study, we identified three accessory chromosomes\n(Chr09, Chr10, and Chr11) in Tremella fuciformis strain Tr01. These were\ncharacterized as mini-chromosomes (<1 Mb) with shared features: low gene density,\nhigh repeat content, low GC content, short gene lengths, absence of BUSCO genes, low\nheterozygosity in homologous regions, and deletions or copy number variations (CNVs)\nin meiotic offspring.\nThe current study extends this work by analyzing accessory chromosomes (ACs)\nacross an additional 15 T. fuciformis strains While we validate the core characteristics\nof ACs reported previously, we reveal several new features of T. fuciformis ACs that\nsignificantly advance our understanding of basidiomycete accessory chromosomes:\n1. ACs are not restricted to mini-chromosomes (<1 Mb): The largest AC (AC01 in\nstrain QS) reaches 2.7 Mb - exceeding the size of several core chromosomes (Fig. 3a),\nchallenging the prior assumption of ACs as uniformly small elements.\n2. Phylogenetically closely related strains harbor identical or nearly identical AC\nsets (Fig. 2), indicating co-evolutionary coupling between ACs and the core genome-a\npattern not observed in the single strain analyzed previously.\n3. ACs undergo rapid structural variation (Fig. 4), leading to higher sequence\nsimilarity among homologous ACs compared to core chromosomes (Fig. 3d)-even with\na substantially lower proportion of shared genes (Fig. 6 IA). This pattern provides\ncompelling evidence that accessory chromosomes evolve faster than core chromosomes.\n4. Beyond meiotic instability, ACs exhibit abundant CNVs in somatic cells during\nmitosis and the mycelium-to-spore transition (Fig. 5), highlighting their exceptional\ninstability across both sexual and asexual life cycles.\n5. In the revised manuscript, we performed comprehensive annotations of\nrepetitive sequences and gene composition for ACs across all 27 haploid genomes, and\nsystematically compared these features with those of core chromosomes. Through these\nanalyses, we addressed three key questions: (1) whether ACs originated from CCs or\nwere acquired from external sources; (2) the potential donor species of ACs; and (3)\nthe timing of AC integration into T. fuciformis. Summarized in the newly added Figure\n6, these findings represent the first dedicated analysis of T. fuciformis AC origins,\nproviding evidence that ACs likely derive from understudied taxa via horizontal gene\ntransfer-thus filling a critical gap in our understanding of accessory chromosome\nprovenance in basidiomycetes.\nCollectively, these novel findings expand the phenotypic and evolutionary\nspectrum of basidiomycete ACs, moving beyond descriptive characterization to reveal\ntheir dynamic evolution, life cycle-dependent instability, and exogenous origins. We\nbelieve these advances significantly enhance our understanding of accessory\nchromosome biology in fungi.\nQ2：\nThere are several experimental approaches that are not clearly explained, nor their\nimpact is analyzed and/or discussed. Furthermore, some observations need more\ndetailed explanations. For example, the manuscript reports the ITS tree for the 16 strains\n(L121ff), but it is not clear how the ITS is derived from dikaryotic strains that should\nhave two ITS; also, NJ algorithm is used rather than more sophisticated tree\nreconstruction approaches.\nResponse:\nWe appreciate your valuable feedback, which prompted us to address the critical\nconsideration of heterokaryotic strains potentially harboring two distinct ITS\nsequences. To address this, we have revised our approach to reconstructing the ITS-\nbased phylogenetic tree: using a known Tremella ITS sequence as the query, we\nperformed local BLAST alignments against the PacBio third-generation sequencing\nread databases of the test strains to identify both the number of ITS sequences per strain\nand their specific sequences. Given the high similarity among the analyzed sequences,\nwe constructed the phylogenetic tree using the Maximum Likelihood (ML) method,\nwith the optimal tree topology determined via the Close Neighbor Interchange (CNI)\nsearch strategy, and validated its reliability through 1000 bootstrap replicates.\nWe have revised the content in the Materials and Methods section (original\nLines 662-668; revised Lines 771-786) to standardize the ITS sequence alignment,\nUNITE database query, MEGA 11-based ML phylogenetic tree construction (ClustalW\nalignment, CNI strategy, 1000 bootstraps), and ChiPlot visualization workflow.\nWe have revised the content in the Results section (original Lines 121-124;\nrevised Lines 127-135) to clarify strain composition (11 heterokaryotic, 5\nmonokaryotic; Supplementary Table 1), ITS sequence distribution (5 heterokaryotic\nstrains with two distinct ITS, 11 with one), and ML phylogenetic clustering of 21 target\n+ 10 reference ITS sequences into three T. fuciformis clades (Supplementary Figure 1).\nQ3：\nHi-C data is generated for NDB and TF2206, however it is not explained why these\nstrains were selected. Tr01 was used as a reference to place NDB contigs into\nchromosomes. Given the known chromosomal rearrangements (Deng et al. 2023), this\ncould potentially introduce error in the assemblies; similarly, the Hi-C enhanced\ngenomes were used to place contigs for other (L553). How was the potential issue\naddressed or corrected? Observed chromosomal rearrangements could for example be\nvalidated (e.g., via orthogonal approaches such as PCRs).\nResponse:\nThank you for your insightful comments regarding the selection of strains for Hi-\nC sequencing and the potential errors introduced by using Tr01 as a reference amid\nknown chromosomal rearrangements. We appreciate your emphasis on validating\nchromosomal rearrangements, and we have thoroughly addressed these concerns\nthrough clear strategy clarification and multi-layered validation measures-details of\nwhich are reinforced below:\n1. Rationale for selecting NDB and TF2206 for Hi-C sequencing\nOur genome assembly strategy was designed to ensure high-quality, representative\nreferences for each phylogenetic clade: we selected one strain from each of the three\nidentified branches as a representative, with NDB and TF2206 randomly chosen as\nrepresentatives of Cluster I and Cluster II, respectively. These two strains, along with\nthe previously published Tr01 (representing Cluster III; Deng et al. 2023), were\nsubjected to combined Hi-Fi and Hi-C sequencing to generate complete, accurately\nphased genomes. These three clade-specific reference genomes were then used to assist\nin contig scaffolding for other strains within their respective clusters, ensuring assembly\nconsistency within each branch.\nTo clarify the rationale for this selection, we have revised the content (original\nLines 550-553; revised Lines 628-631).\n2. Addressing potential errors from Tr01 as a reference and validating chromosomal\nrearrangements\nWe fully acknowledge that chromosomal rearrangements (reported in Deng et al.\n2023) could introduce assembly errors when using Tr01 as a reference. To mitigate this\nrisk and rigorously validate observed rearrangements, we implemented multiple\northogonal validation measures-repeated across analyses to ensure reliability:\nFirst, in the Materials and Methods section, we have revised original Lines 607-\n610 to explicitly outline these validation strategies: \"To rule out errors caused by\ngenome assembly or reference bias, the observed chromosomal rearrangements were\nvalidated using multiple orthogonal approaches, including long HiFi read mapping,\nlong nanopore read mapping, Hi-C interaction heatmaps (generated via HiC-Pro and\nHiCPlotter), and multi-strain synteny verification. (revised Lines 690-693)\"\nSecond, in the Results section, we have strengthened empirical validation with\nexperimental data and supplementary figures:\nWe inserted at original Line 193: \"The chromosomal rearrangement was further\nvalidated by mapping Long HiFi and Nanopore reads to the corresponding regions of\nTF2206 and Tr01 (Supplementary Figure 8). (revised Lines 209-211)\" Supplementary\nFigure 8 illustrates Long HiFi reads that simultaneously map to the 3' end of Chr01-1\nand the 5' end of Chr01-2 in TF2206, as well as long HiFi/Nanopore reads mapping to\nthe syntenic region in Tr01-directly confirming the rearrangement.\nWe revised original Line 195 to reference “Supplementary Figure 9 and 10” and\nadded Supplementary Figure 10, which provides independent validation of another\nrearrangement: Long HiFi reads mapping to the junction of Chr02-1 and Chr02-2 in\nTF2206, with corresponding read mapping in Tr01 to confirm the structural difference.\nWe have added the data sources of PacBio and Nanopore sequencing data for strain\nTr01 to the Data Availability section.\nThese orthogonal validation measures-including independent Long-read mapping\n(HiFi and Nanopore), Hi-C interaction data, and multi-strain synteny analysis-\ncollectively address the potential error from reference-guided assembly and robustly\nconfirm the observed chromosomal rearrangements.\nQ4：\nIt is also not entirely clear how the core chromosomes were identified (L164), as I\nwould expect a working definition would be that these need to be conserved in all\nstrains. It seems that the number differs between eight and ten, while this is observation\nis only explained much later (and had been as far as I can tell reported previously\nalready; Deng et al. 2023); the implication of the transposons at the\nbreakpoints/junctions are not further detailed but could provide insights in the processes\ndriving chromosome variation. For additional comments, see further details below.\nResponse:\n1. Clarifying the definition and variable number of core chromosomes\nTo address the ambiguity in core chromosome identification, we have refined the\ndefinition to emphasize conservation across all isolates while explicitly explaining the\nobserved numerical variation (8-10) and its evolutionary basis.\nIn the Results section, we have revised the content (original Lines 161-165; revised\nLines 175-181).\nThis revision: (1) Provides a clear working definition (conserved across all isolates,\nwith synteny and gene content conservation); (2) Explicitly links numerical variation\nto evolutionary events (recombination, fusion/fission); (3) Integrates prior work (Deng\net al., 2023) to contextualize the observation; and (4) Eliminates confusion by clarifying\nthe 8-10 range as a strain-specific, evolutionarily driven feature.\n2. The implications of transposons (TEs) at chromosomal rearrangement\nbreakpoints/junctions\nWe have carefully followed your comment to conduct a detailed analysis of the\nsequence composition at these breakpoints/junctions across all chromosomal\nrearrangements.\nOur analysis revealed that the TE-derived fragments are incomplete, highly\ntruncated, and account for only a small fraction (~7.7%) of the junction sequences.\nDue to their fragmented nature and low abundance, these TE-derived sequences lack the\nstructural integrity required to support functional inferences (e.g., active transposition,\nrecombination mediation) or to clearly link TEs to the mechanisms driving\nchromosome variation. Given the absence of compelling evidence for TEs playing a\ndirect role in these rearrangements, we have opted to weaken the relevant content in the\nmanuscript and removed the TE-related annotations from the corresponding figures to\navoid overinterpretation.\nQ5：\nThe manuscript needs to overall improve in clarity. Just as an example, the abstract\nis illegible for non-expert readers. It is not explained what accessory chromosomes are\nin comparison to core chromosomes, what T. fuciformis is, or why 16 strains would\nyield 27 haplotypes (monokaryon vs dikaryons). The abstract mentions ‘homology\nsimilarity’ (L31), but this is not a common concept since homology is qualitative, but\nsimilarity is a quantitative trait.\nResponse:\nThank you for your valuable comments on improving the clarity of the abstract.\nWe have carefully addressed each concern as follows:\n1. Clarification of key concepts for non-expert readers\nAccessory chromosomes vs. core chromosomes: We have explicitly\ndistinguished the two in the revised abstract. Core chromosomes are defined as essential\nfor basic biological processes, while accessory chromosomes are non-essential for\nnormal growth and development-this distinction helps non-specialists grasp their\nfunctional differences.\nTremella fuciformis: We added its common name “silver ear fungus” and\nspecified it as a basidiomycete, providing immediate context for readers unfamiliar with\nthe species.\n16 strains yielding 27 haplotypes: We clarified that the 27 haplotypes derive from\n5 monokaryotic and 11 dikaryotic strains. Each dikaryon contributes two distinct\nhaplotypes, resolving the discrepancy between the number of strains and haplotypes.\n2. Correction of the “homology similarity” concept\nIn the revised abstract, we have replaced “homology similarity” with “sequence\nsimilarity,” a standard academic term that accurately reflects the quantitative\ncomparison of accessory and core chromosome sequences.\n3. Overall clarity enhancement\nWe further refined the abstract structure to improve readability: simplified\nredundant expressions, adjusted sentence logic for smoother flow, and retained only\nnecessary parenthetical notes. These revisions ensure the abstract is accessible to both\nexperts and non-experts while preserving core scientific information.\nWe have revised the entire Abstract (original Lines 19-41; revised Lines 20-36).\nQ6：\nSimilarly, parts of the introduction are superficial, repetitive (e.g., L59 vs L66 or\nL73 saying the same as few lines above), or plain wrong. For example, L48 implies that\nall fungi have unique characteristics such as the two-speed genome phenomenon, while\nthere is plenty of evidence that this is not common to all fungi and differ significantly\nbetween even closely related fungi. In contrast to the statement of L59f, accessory\nchromosomes in Z. tritici have to my knowledge not been demonstrated to contain\nvirulence factors or secondary metabolite clusters, and their functions remain\nenigmatic. Accessory genomic regions have been successfully identified using (epi-\n)genomic characteristics alone (in Magnaporthe and Verticillium) and thus the\nstatement in L75 is also incorrect.\nResponse:\nWe have carefully addressed each concern to improve accuracy, reduce\nredundancy, and enhance rigor:\n1. Overgeneralization of the \"two-speed genome\" phenomenon (L48):\nWe acknowledge the imprecision in implying this phenomenon is universal across\nall fungi. As noted, evidence confirms it is not common to all fungi and varies even\namong closely related species. We have revised original Line 48 to: \"These\ndevelopments have facilitated a deeper understanding of distinctive genomic\ncharacteristics exhibited by some fungi, such as the 'two-speed genome' phenomenon.\n(revised Lines 42-44)\"\n2. Incorrect claim about Zymoseptoria tritici accessory chromosomes (L59, L59f):\nWe appreciate pointing out the inaccuracy. The original statement incorrectly\nattributed virulence factors or secondary metabolite clusters to Z. tritici accessory\nchromosomes, which is not supported by current evidence. We have revised the text to\nspecify that such features are observed in Fusarium, Metarhizium, and Alternaria\n(consistent with literature) and removed Zymoseptoria from this context, clarifying that\nits accessory chromosomes have enigmatic functions as noted.\n3. Redundancy and incorrect statement on accessory chromosome identification\n(L66, L73, L75):\nTo resolve redundancy between descriptions of accessory and core chromosome\nfeatures (original Line 66 vs. original Line 73), we have streamlined the text to highlight\ndistinct characteristics of accessory chromosomes while concisely noting overlapping\ntraits in core chromosomes, avoiding repetitive phrasing.\nRegarding the incorrect claim that accessory chromosomes cannot be identified by\n(epi-)genomic characteristics alone (original Line 75), we have revised this to: \"Fungal\naccessory chromosomes can sometimes be identified using (epi-)genomic\ncharacteristics, though presence/absence polymorphism remains a key criterion for\ntheir identification. (revised Lines 73-75)\" to align with established evidence.\nQ7：\nThroughout the manuscript (e.g., L127), the companion fungus Annulohypoxylon\nstygium is mentioned several times by their significance to T. fuciformis remains\nunclear. It is suggested that there might be a link to the presence of a subset of accessory\nchromosomes (e.g., Fig 2), but the manuscript remains vague with respect to\nmechanisms and further implications for both partners. Moreover, the material &\nmethods implies that the authors performed mating experiments, however, these seems\nto be rather association than true mating. The authors need to be much clearer on the\npresence, impact, and implication of the companion fungus to the genomics and biology\nof T. fuciformis.\nResponse:\nWe fully agree that the original manuscript was vague regarding these aspects, and\nwe have revised sections of the introduction, materials and methods and discussion to\naddress this gap comprehensively:\n1. Revisions to introduction\nTo clarify the role of A. stygium in the biology of T. fuciformis, we inserted a\nsentence at original Line103: \"Specifically, A. stygium maintains a close association\nwith T. fuciformis and functions as a key nutritional provider, supplying essential\nnutrients to support the growth and development of the latter. (revised Lines 100-102)\"\n2．Revisions to materials and methods\nWe fully agree that the interactions described here represent symbiotic associations\nrather than true mating. To address this, we have revised the section title from \"Mating\nbetween T. fuciformis and A. stygium (original Line 712)\" to \"Association experiments\nbetween T. fuciformis and A. stygium\" and replaced all instances of \"mating\" and\n\"pairings\" with \"associations\" to accurately reflect the nature of the interaction.\n2．Revisions to discussion\n1）Strengthening the significance of A. stygium: We explicitly emphasize that A.\nstygium is an obligate symbiont of T. fuciformis, providing essential nutrients for\nfruiting body development. This contextualizes why symbiotic specificity between the\ntwo partners matters for T. fuciformis’ survival and productivity.\n2) Clarifying the mechanistic hypothesis: We now propose a specific, testable\nmechanism: T. fuciformis ACs likely encode genes involved in interspecific recognition\nor metabolic complementation that mediate compatible association with A. stygium.\nThis fills the previous gap in explaining how ACs might influence symbiosis.\n3) Outlining future experiments: We provide concrete directions for validating the\nhypothesis, including targeted AC knockouts, transcriptomic analysis of AC-borne\ngenes during interaction, and identification of interspecific protein interactions.\nThese experiments will directly test the mechanistic role of ACs in symbiotic specificity.\nWe have revised the Discussion section (original Lines 406-438; revised Lines\n485-516)\nQ8:\nTitle: for the general audience, spell out what Tremella is. Moreover, the content\nbefore and after the colon seems to be redundant.\nResponse:\nWe fully agree with your suggestion to enhance readability for the general audience\nand have revised the title as recommended. The revised title is: \"Exploring the\npolymorphism and origins of accessory chromosomes in basidiomycete Tremella\nfuciformis: insights into genome divergence and structural variation\"\nRevision Explanations:\nSpell out T. fuciformis for general audience: We added the taxonomic class\n\"basidiomycete\" before T. fuciformis to explicitly define the fungal group of the\nresearch object. This helps readers unfamiliar with T. fuciformis quickly grasp its\nbiological classification, addressing the need for clarity for the general audience while\nretaining the precise scientific name.\nAddress potential redundancy: The original title’s structure was carefully reviewed.\nWe confirmed that the phrase before the colon focuses on the core research objects\n(polymorphism and origins of accessory chromosomes in T. fuciformis), while the part\nafter the colon clarifies the broader scientific implications (genome divergence and\nstructural variation). To avoid redundancy without losing key information, we only\nsupplemented the common name instead of restructuring the title, as the two segments\nlogically complement each other (specific research content general scientific insights)\nand align with the standard format of academic titles in fungal genomics research.\nQ9:\nL61: Does this imply that there are basidiomycetes with accessory chromosomes? If so,\nthis needs to be introduced in much more detail.\nResponse:\nWe acknowledge the original statement’s vagueness and have revised original Line\n61 to clarify accessory chromosomes’ existence in Basidiomycetes with specific\nexamples, as following: \"However, accessory chromosomes in Basidiomycetes are\nrarely reported. To date, only a handful of species-including Sporisorium scitamineum\nand T. fuciformis-have been confirmed to harbor them, verifying their presence in this\nphylum. Documented cases are far fewer than in Ascomycetes, underscoring the need\nfor further exploration in understudied taxa. (revised Lines 55-60)\"\nQ10:\nL120: What are ‘…principle cultivated varieties,…’? Please explain.\nResponse:\n“two are principal cultivated varieties” has been revised to \"two principal\ncultivated varieties (Tr01 and Tr21) \"\nQ11:\nL124: The sentence is unclear as it suggests that ITS sequences exhibit\nmorphological characteristics. Please rephrase.\nResponse:\nWe have revised the sentence to clarify the separate lines of evidence (molecular\nand morphological) supporting the species identification, as following: \"NCBI BLAST\nanalysis of the ITS sequence indicated that the isolates are more closely related to the\nknown species T. fuciformis. Additionally, their fruiting bodies exhibit the same\nmorphological characteristics as those of T. fuciformis. (revised Lines 134-136)\"\nQ12:\nL138: ‘… modern…’ not sure what this refers to, could this be an error?\nResponse:\nWe have corrected \"modern\" to \"mode\" (a typo).\nQ13:\nL202: The manuscript should be clearer when it reports on haploid genomes. Does\nit refer to a monokaryotic strain or to a dikaryotic strain where both haplotypes have\nbeen assembled? This distinction could also be clearer in the figures (e.g., the A and B\nin the strain name)\nResponse:\nThe manuscript and figures have been revised to clarify genome naming rules,\ndistinguishing haploid genomes from monokaryotic and dikaryotic strains, as following:\nOriginal Lines 148-150 has been revised to \" In total, we have successfully\nobtained 27 complete haploid genomes, including two previously established from the\ndikaryotic strain Tr01 (designated Tr01A and Tr01B, corresponding to its two distinct\nhaplotypes), to support subsequent research. (revised Lines 158-161)\"\nAdd the following sentence to the legend of Fig. 1: Haploid genomes of\nmonokaryotic strains are named directly after the strain, while those of dikaryotic\nstrains are labeled \"strain name + A/B\" to distinguish the two haplotypes.(revised Lines\n220-222)\nQ14:\nL229: this pattern could also be due to horizontal transfer of the chromosome rather\nthan loss. Could the authors perform additional analyses to clearly distinguish these\nscenarios?\nResponse:\nGiven that more strains in Cluster I harbor AC09, we propose that AC09 was\noriginally present in QCA but lost during evolution-this is more plausible than\nhorizontal transfer. Corresponding revisions have been made to the manuscript as\nfollows.\nSynteny analysis of accessory chromosomes in the QCA and TF104A genomes\nrevealed that their homologous chromosomes AC10 to AC12 are nearly identical\n(similarity >99.9%, coverage >99.3%; Supplementary Figure 11), while QCA lacks\nAC09. All strains in Cluster I, except QC and TF103, harbor AC09, suggesting this\naccessory chromosome was originally present in the cluster but lost during evolution.\n(revised Lines 247-252)\nQ15:\nL262: What is driving the high similarity between accessory chromosomes? Is this\nmainly due to transposons or also over the protein-coding (non-TE) genes?\nResponse:\nThe higher high similarity observed in ACs is likely shaped by two key\nevolutionary scenarios inferred from our data:\nFirst, accessory chromosomes (ACs) were acquired via horizontal gene transfer\n(HGT) at a specific node during the evolution of T. fuciformis, an origin that contributes\nto their higher average nucleotide identity (ANI) relative to core chromosomes. Our\nfindings reveal minimal homology between ACs and core chromosomes, indicating\nthey are not derived from the latter-a stark contrast to core chromosomes, which have\naccumulated substantial divergence through vertical inheritance over time. Freed from\nthe prolonged divergent evolution associated with vertical transmission, these\nhorizontally acquired ACs have retained relatively higher sequence conservation,\nfurther elevating their ANI. This aligns with our discussion that \"The accessory\nchromosomes originated from unexplored species before the speciation of T.\nfuciformis.\"\nSecond, frequent exchanges between homologous accessory chromosomes (ACs)\nin heterokaryotic strains (e.g., TF2206) contribute to maintaining high sequence\nsimilarity. As noted, heterokaryons derived from different colonies of the same parent\ncolony exhibit substantial variation in AC copy numbers across cells. This variability\ncan lead to scenarios where ACs in one nucleus are lost, with subsequent replenishment\nfrom ACs of the other nucleus-a process that facilitates frequent sequence exchanges\nbetween ACs.\nTo address this, we have inserted the following sentence in the Discussion section\nunder \"Extremely high frequency of chromosomal copy number variation in both core\nand accessory chromosomes\" (original Line 464): This improper allocation of\naccessory chromosomes during mitosis may further facilitate frequent exchanges\nbetween homologous accessory chromosomes in the heterokaryotic strain, resulting in\nhigh or even identical sequence similarity among them.(revised Lines 542-545)\nQ16:\nL279: it needs to be clear that the two AC14 refer to the A and B haplotype rather\nthan a copy-number variation of one of the two.\nResponse:\nWe have revised the sentence to explicitly state this distinction, and the updated\nversion reads as follows:\n\"The sequence similarity between the homologous AC14 chromosomes of the A\nand B haplotypes in the dikaryotic B-1 strain is as high as 99.5%. (revised Lines 313-\n315)\"\nQ17:\nL348: Why was C-1 used as a case study?\nL367: Additional information about the genes on accessory chromosomes are\nlacking. For example, are these expressed, are these predicted to be secreted, etc. Do\nthey have other characteristics that makes them distinct from those on the core genome\n(e.g., number of introns, length, etc.). Moreover, the absence of any homology raises\nimportant questions about their origin that unfortunately remains unclear. I find it\nsurprising that most genes do not even have homologs in other fungi and the\nexplanation that we lack the corresponding species in the database seem unlikely given\nthe ongoing sequencing efforts.\nResponse:\nThank you for your insightful comments, which have helped us improve the\ncompleteness and depth of our analysis on accessory chromosomes. We fully\nacknowledge that the final section of the original manuscript was overly brief, which\nconstituted a significant limitation. We have conducted comprehensive annotations of\nthe repetitive sequences and gene composition of ACs across all 27 haploid genomes,\nand systematically compared these features with those of CCs. Through these analyses,\nwe have addressed three key questions: 1) whether ACs originated from CCs or were\nacquired from external sources; 2) the potential donor species of ACs; and 3) the timing\nof AC integration into T. fuciformis. For detail information, please see our response to\nreviewer #1, Q1.\nBesides, we added statistical comparisons of intron number and gene length. AC\ngenes have fewer introns (average 1.5 vs. 3.3 in core genes, revised Lines 280-284),\nwhich is consistent with the structural characteristics of rapidly evolving or newly\nacquired genes. For detail information, please see our response to reviewer #1, Q5.\nTo address this comment, we have revised the manuscript accordingly: we have\nadded a new Figure 6, rewritten the last section of the Results (revised Lines 395-436，\nThe content related to \"C-1 as a case study\" has been removed from the revised\nmanuscript), and updated the relevant content in the Materials and Methods (revised\nLines 816-847) as well as the Discussion sections (revised Lines 579-600).\nIn addition, we analyzed the expression profiles of these genes. Taking strain Tr21\nas an example, we selected transcriptome sequencing data from its yeast-like stage,\nhyphal stage, and different fruiting body development stages. After assembly with\nTrinity software, a total of 66,868 transcripts were obtained. BLAST alignment of these\ntranscripts against the 294 predicted accessory chromosome genes of Tr21A showed\nthat 254 genes had a sequence coverage of >80% (accounting for 86.4%), confirming\nthe expression activity of the predicted genes. Due to the difficulty in naturally\nintegrating this part of the results into the main framework of the paper, it was not\nincluded in the main text.\nQ18:\nL399: Does this sentence imply that this are plans? If so, please rephrase as this is not\nentirely clear.\nResponse:\nThis sentence refers to our future research plans. We have rephrased it to explicitly\nclarify that these in-depth analyses are intended to be conducted in subsequent studies,\nas shown below.\n\"Future in-depth analyses will deepen our understanding of these chromosomes,\nfocusing on their stability and interactions in heterokaryotic states, as well as the\npotential roles of accessory chromosomes in dimorphic transitions and fruiting body\nmorphogenesis.(revised Lines 479-482)\"\nQ19:\nL351: I find it odd to read that transposons are treated similarly to genuine protein-\ncoding genes. The abundance of these in the gene sets suggest that the structure gene\nannotation was not performed carefully, and transposons should be removed prior to\nany analyses\nResponse:\nThank you for your valuable comment, which has helped us clarify an ambiguous\nexpression in the original manuscript.\nWe would like to clarify that our initial description was imprecise-our actual\nintention was to highlight that some transposable elements (TEs) harbor genes, rather\nthan treating TEs themselves as genuine protein-coding genes. To address this\nambiguity, we have revised the relevant content in the revised manuscript: we classified\nthe shared genes on accessory chromosomes into two categories based on their genomic\nlocations-those located within TEs and those outside TEs.\nThis revised classification and corresponding descriptions are now integrated into\nthe text, and the detailed distribution results are presented in Figure 6I-C for clear\nvisualization.\nWe apologize for the confusion caused by the unclear original expression and\nappreciate your careful review, which has enhanced the accuracy and rigor of our\nmanuscript.\nQ20:\nL519: ‘…did not exist from the beginning…’ please rephrase\nResponse:\nWe have rephrased the phrase to enhance clarity and academic precision, and the\nrevised sentence is provided below.\n\"In summary, we hypothesize that unlike core chromosomes, the accessory\nchromosomes of T. fuciformis did not originate concomitantly with the species. Instead,\nthey were acquired through horizontal gene transfer from an unexplored species prior\nto the species’ formation, and as non-essential elements, underwent rapid structural\nvariations post-acquisition. \"\nQ21:\nL590: few details on the mitochondrial assemblies could be provided. Do they similarly\ndisplay three distinct groups. Are phylogenies with mitochondrial markers concurrent\nto nuclear taxonomy?\nResponse:\nWe have supplemented relevant details and verified the consistency of\nmitochondrial phylogeny with nuclear taxonomy as suggested:\nWe added detailed mitochondrial genome assembly methods to the \"Genome\nassembly\" section (inserted at original Line 590; revised Lines 668-671).\nIn the \" Pan-genome analysis and phylogenetic tree construction\" method section,\nwe clarified that the same analytical pipeline of ITS sequences was applied to\nmitochondrial genomes (inserted at original Line 668): \"The same alignment, tree-\nbuilding, and visualization methods were applied to construct the phylogenetic tree of\nthe 16 strains.(revised Lines 784-786)\"\nIn the Results section, we updated the relevant finding (original Line 154; revised\nLines 165-170) to confirm consistency.\nThese revisions confirm that the mitochondrial genomes of the 16 strains form\nthree distinct groups, with the mitochondrial marker-based phylogeny largely\nconsistent with nuclear-based taxonomy (core genes and ITS sequences)-the only\ndiscrepancy being strain T0053, which shifts from Cluster III to Cluster II in the core\ngene tree relative to the mitochondrial tree.\nReviewer#3\nIn this work the authors describe sequencing of 27 haplotypes from 16 strains of\nthe basidiomycete Tremella fuciformis, and a subsequent pangenomics investigation of\ncore and accessory chromosome statistics and distribution.\nQ1:\nMajor comments\nI think the primary critique involves the relationship between accessory\nchromosomes and A. stygium pairing, which is one of the more interesting biological\naspects of this work. It is very interesting that the distribution of ACs correlate with the\npairing groups, and this is a valid and potentially novel finding.\nHowever, supplementary table 1 introduces 4 strains that do not associate with A.\nstygium: Wuyi2014, Wuyi2015, Lys2020, and T0053. Firstly, the statements in the\nintroduction (lines 101-103) seem to imply that all Tremella species associate with\nAnnulohypoxylon, so it might be worthwhile to point out here (and potentially also in\nlines 127-129 in the results) that not all Tremella associate with Annulohypoxylon.\nBut overall, there is very little discussion about how the distinct accessory\nchromosome patterns relate to these four non-pairing strains, as they compare to their\npairing-capable phylogenetic neighbors. For example, Figure 4 shows structural\nvariations in AC01 (inversion) and AC08 (deletion) between NDBA (a pairing strain)\nand Wuyi2015 (a non-pairing strain), but there is no discussion or speculation as to\nwhether such differences may influence pairing.\nAdditionally, and specifically with respect to one of the major conclusions, that\nthe three largest ACs (AC01, AC12, and AC14) correspond to the three distinct AF\ngroups (AF2, AF1, AF3), there are nuances that are not addressed. For example, AC14\nis missing from non-pairing strain T0053 but present in non-pairing LYS2020.\nSimilarly, AC01 is present in both non-pairing strains Wuyi2014 and Wuyi2015.\nThere is also no discussion of other nuances observed in figure 2 and how they\ncould relate to A. stygium pairing capabilities. For example, AC08 is primarily found\nin AF2, but also found in two AF3 strains LYS2020 and T0053, which are notably non-\npairing. Similarly, AC06 and AC08 are absent in non-pairing Wuyi2014 but present in\nnon-pairing Wuyi2015. Finally AC15 doesn't show exact group specificity, since it is\npresent in both AF2 and AF3 (excluding B-1 and C-1).\nI think that at minimum, some inclusion of these observations in the discussion is\nwarranted. I think it would be beneficial as well to include additional structural\ncomparisons similar to Figure 4, between other closely related strains that differ in\npairing ability. Or even a comparison and discussion of how the LYS2020 and T0053\nAC08 chromosomes might differ from the AC08 chromosomes found in any of the AF2\nstrains might be informative.\nResponse:\nWe appreciate the insightful comments regarding the relationship between\naccessory chromosomes (ACs) and A. stygium pairing, as well as the need to clarify the\nstatus of non-pairing strains. Here, we address these points based on the biological\ncharacteristics of T. fuciformis:\nSimilar to Cryptococcus neoformans, the haploid phase in the life cycle of T.\nfuciformis exists exclusively in the yeast form and cannot transition to the mycelial\nform, thus being unable to interact with A. stygium hyphae as unicells or form fruiting\nbodies; in contrast, the heterokaryotic phase is dimorphic, enabling such hyphal\ninteractions and fruiting body development.\nThe four strains (Wuyi2014, Wuyi2015, Lys2020, and T0053) listed in\nSupplementary Table 1 are in the haploid phase. Due to their inability to form mycelia,\ntheir accessory chromosomes were not further investigated for roles in A. stygium\ninteractions, which we have now clarified in the Introduction section.\nIn the Introduction section, we have inserted at original Line 103: \"Similar to\nCryptococcus neoformans, the haploid phase in the life cycle of T. fuciformis exists\nexclusively in the yeast form and cannot transition to the mycelial form, thus being\nunable to interact with A. stygium hyphae as unicells or form fruiting bodies; in contrast,\nthe heterokaryotic phase is dimorphic, enabling such hyphal interactions and fruiting\nbody development. (revised Lines 104-109)\"\nRegarding another strain TF104 with haploid genome: As noted in Supplementary\nTable 1, samples used for genomic analysis were derived from a single purified yeast\ncolony of heterokaryotic mycelia, confirmed to be haploid (presumably transitioning\nfrom the heterokaryotic to haploid state). In contrast, samples employed for association\nassay with A. stygium were heterokaryotic mycelia. We have added this information to\nthe note of Supplementary Table 1.\nQ2:\nFigure 1d: I don't see any black circles in either chart\nResponse：\nBlack circles have been added to Figure 1d\nQ3：\nFigure 2: \"Dots indicate the absence\" should probably be corrected to \"minus signs\nindicate the absence\"\nResponse：\nWe have corrected Figure 2’s legend to \"Minus signs indicate the absence\".\nQ4:\nChromosomal copy number variation Section:\nWhy was only TF2206 selected? Many other strains exist in M/Y morphotypes\nResponse：\nGiven the substantial genome sequencing workload required for analyzing copy\nnumber variation (CNV) across all strains, we randomly selected T. fuciformis TF2206\nas a representative case study to investigate chromosomal CNV in its different\nmorphological states. This approach allows us to illustrate the CNV characteristics of\nT. fuciformis while balancing research feasibility.\nOriginal Lines 299-300 has been revised to \"T. fuciformis can exist in either\nmycelial or yeast form. To illustrate its chromosomal copy number variation (CNV)\ncharacteristics, we randomly selected dikaryotic strain TF2206 as a case study and\nanalyzed CNV in its three states. (revised Lines 344-346)\"\nQ5:\nWhy was only YY2 selected for the normalized depth distribution curve\ncomparison (Figure 5d)?\nResponse：\nYY2 was selected as a representative single-yeast forming strain (stable genetic\nbackground) to contrast with the mixed yeast colony, ensuring clear visualization of\ndepth fluctuation differences associated with mycelium-to-yeast transition.\nTo this end, we have revised the sentence original Lines 329-331 to \"We compared\nthe normalized depth distribution curves of Illumina sequencing between a\nrepresentative single-yeast forming strain (YY2, with stable genetic background) and a\nmixed yeast colony transitioning from mycelium (Fig.5d). (revised Lines 376-378)\"\nQ6:\nThe section states that \"Ten single colonies\" were selected for sequencing from\nstrain TF2206. However the ten colonies are subsequently themselves referred to as\n\"strains\" (e.g. \"YY3 strain\", \"YY1 strain\"). To distinguish these from the broader\nbiological strains (\"TF2206\", \"TF104\", etc) maybe they should be referred to as\n\"colonies\"?\nResponse：\nWe agree that precise terminology is important for clarity. In the revised\nmanuscript, we have consistently referred to the ten isolates derived from TF2206 as\n\"colonies\" (e.g., \"YY3 colony\", \"YY1 colony\") to distinguish them from the broader\nbiological strains such as TF2206 and TF104. This revision ensures accurate\ndifferentiation between the original strains and their derived single-colony isolates.\nQ7:\nLine 315-316: \"obtained from subculturing the same colony\" -> it was unclear\nwhich colony is \"the same\" colony?\nResponse：\nTo clarify, \"the same colony\" refers to the original parent colony from which the\n10 mycelial colonies were subcultured. We have revised original Lines 315-316 to: \"We\nalso conducted a chromosomal copy number analysis on 10 mycelial colonies obtained\nby subculturing the same original parent colony (Fig.5b). (revised Lines 361-363)\" to\navoid confusion.\nQ8:\nIn Figure 5: The \"M to Y\" label makes sense because this is described as coming\nfrom a mycelial to yeast transition. But \"Y to Y\" and \"M to M\" do not really make sense\nbecause these were isolated simply as yeast or mycelia, with no transitions involved.\nResponse：\nWe agree that \"Y to Y\" and \"M to M\" may misleadingly imply transitions. In the\nrevised Figure 5, we have replaced \"to\" with \"-\" for these labels, changing them to \"Y-\nY\" and \"M-M\" to accurately reflect that these samples represent stable yeast or mycelial\nstates without transitions, while retaining \"M to Y\" for the transition group to maintain\nconsistency with the described mycelium-to-yeast process.\nQ9:\nFigure 6a\nThe Venn diagram is just the data from the C-1 row. So, why is CCs 8,653 in both\ncases, but ACs is 297 in Venn diagram and 318 in table? The value of CCs should be\ndifferent between the Venn diagram and the table, depending on if 8,653 is the total\ncount or the unique count.\nResponse：\nThank you for your careful observation and valuable comment on Figure 6a.\nWe would like to clarify the data discrepancy: in the original table, 8,653 and 318\nrepresent the total number of genes on core chromosomes (CCs) and accessory\nchromosomes (ACs), respectively, while 21 denotes the number of shared genes\nbetween the two chromosome types. The Venn diagram was intended to illustrate these\nshared genes, but the CCs value (8,653) in the diagram was incorrectly used as the total\ncount rather than the unique count (which should be 8,653 - 21 = 8,632).\nWe have redrawn Figure 6 in the revised manuscript, and the problematic Venn\ndiagram has been removed. The updated figure now presents the gene distribution data\naccurately and clearly.\nWe apologize for the confusion caused by the initial data misrepresentation and\nappreciate your meticulous review, which has helped improve the accuracy of our\nfigures.\nPoint-by-point responses to Reviewers’ comments.\nReviewer #1\nThe revised version of the manuscript \"Exploring the polymorphism and origins of accessory\nchromosomes in basidiomycete Tremella fuciformis: insights into genome divergence and\nstructural variation\" provides new findings. One of the most exciting results is the description\nof eight Starships integrated both to core and accessory chromosomes, including one exactly\nat the junction locus. Authors answered all the reviewers questions, introduced requested\nchanges and performed additional analyses which resulted in a significant improvement of the\nmanuscript.\nQ1. Line 292 TIR/Mutato should be TIR/Mutator\nLTR/Gypsy should be replaced along the manuscript with LTR/Ty3 because of the\ndiscriminatory character.\nR1:\nRevised accordingly.\nQ2. data availability:\nannotated assemblies should be deposited; the BioProject: PRJNA1247727 stores the\nraw reads\nR2:\nThe nuclear and mitochondrial genome sequences of 15 Tremella fuciformis strains have been\ndeposited at the National Genomics Data Center (NGDC) under the accession number\nPRJCA052213 [https://ngdc.cncb.ac.cn/search/all?q=PRJCA052213].\nReviewer #3 (Remarks to the Author):\nThe revised manuscript by Jinxiang Zhang and co-authors thoroughly addresses the\ncomments I made in my original review. In addition, the manuscript has been carefully and\nsubstantially improved with enhanced methods, new and expanded analyses, and refined\nclarity in the text. I have no further comments to offer."}]

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