Comparative genomic analysis reveals contraction of gene families with putative roles in pathogenesis in the fungal boxwood pathogens Calonectria henricotiae and C. pseudonaviculata

Boxwood blight disease caused by Calonectria henricotiae and C. pseudonaviculata is of ecological and economic significance in cultivated and native ecosystems worldwide. Prior research has focused on understanding the population genetic and genomic diversity of C. henricotiae and C. pseudonaviculata, but gene family evolution in the context of host adaptation, plant pathogenesis, and trophic lifestyle is poorly understood. This study applied bioinformatic and phylogenetic methods to examine gene family evolution in C. henricotiae, C. pseudonaviculata and 22 related fungi in the Nectriaceae that vary in pathogenic and saprobic (apathogenic) lifestyles. A total of 19,750 gene families were identified in the 24 genomes, of which 422 were rapidly evolving. Among the six Calonectria species, C. henricotiae and C. pseudonaviculata were the only species to experience high levels of rapid contraction of pathogenesis-related gene families (89% and 78%, respectively). In contrast, saprobic species Calonectria multiphialidica and C. naviculata, two of the closest known relatives of C. henricotiae and C. pseudonaviculata, showed rapid expansion of pathogenesis-related gene families. Our results provide novel insight into gene family evolution within C. henricotiae and C. pseudonaviculata and suggest gene family contraction may have contributed to limited host-range expansion of these pathogens within the plant family Buxaceae.

and defoliation under conducive environmental conditions. Few effective boxwood disease management practices have been identified and current research efforts are focused on developing host-plant resistance strategies as multiple fungicide applications are costly and unsustainable for the year-round occurrence of this disease [15].
Boxwood blight is caused by the ascomycete fungi Calonectria henricotiae (Che) and C. pseudonaviculata (Cps), two closely related sister species in the family Nectriaceae. Cps also infects two additional genera in the plant family Buxaceae, Pachysandra and Sarcococca [7]. When boxwood blight disease was first discovered in the 1990s in Europe, Cps was the only known causal agent. However, in 2005, a second species-Che was identified from diseased boxwood in the UK and continental Europe [24]. Several studies have shown limited genetic diversity from natural populations of Che and Cps, consistent with the hypothesis of predominant asexual reproduction and introduced clonal lineages [8,37,38]. Malapi-Wight et al. (2019) determined that populations of Cps have a single mating type idiomorph (MAT1-1) compared to populations of Che which possess the MAT1-2 idiomorph [44]. Separate studies demonstrated that pathogen populations have low genetic diversity and no evidence of sexual recombination, suggesting limited opportunities for mating with predominately clonal asexual reproduction [8,37]. Despite possessing opposite mating types and a sympatric geographic distribution in Europe and the UK, successful mating between Che and Cps has not been observed in nature or under laboratory conditions [7,37,44]. Additional population genomic analyses of Che and Cps have also shown limited gene flow between the two species and absence of shared genetic polymorphisms [38].
The genus Calonectria includes more than 160 described species that inhabit a broad range of ecological habitats and lifestyles globally [41]. In addition to Che and Cps, several other species of Calonectria are plant pathogens and the causal agents of diseases on approximately 335 plant species across 100 plant families [13,42]. For example, C. ilicicola infects at least 70 plant species in multiple families while C. gordoniae is a pathogen of a single host plant species native to the southeastern US, loblolly bay (Gordonia lasianthus) [21]. Despite the incredible diversity of lifestyles employed by Calonectria species, little is known about the mechanisms that these fungi utilize to successfully infect their hosts and extract nutrients. Two comparative genomic and transcriptomic studies were recently conducted on the Eucalyptus pathogen C. pseudoreteaudii (Cpr) to elucidate pathogenesis mechanisms. In these studies, enzymes involved in secondary (specialized) metabolite biosynthesis were up-regulated in Cpr mycelia grown in Eucalyptus tissue culture medium [75]. These authors identified expanded gene families of Major Facilitator Superfamily (MFS) transporters that enhance pathogenicity suggesting that MFS proteins may provide an adaptive mechanism for degrading and transporting compounds produced by Eucalyptus that are toxic to the fungus. Ye et al. (2017) analyzed Cpr gene expression profiles at three temporal stages of Eucalyptus infection and disease symptom development. The authors identified differentially expressed genes involved in plant cell wall degradation, detoxification of phytoalexins, toxin synthesis, iron uptake, and reactive oxygen species scavenging. Genes encoding cutinase enzymes, which are crucial for plant pathogenic fungi that penetrate through the host cuticle, were also up-regulated during plant pathogenesis and expressed earlier than other cell wall degrading enzymes [76]. An additional report of secondary metabolites as virulence factors was observed in C. iliciola and production of the PF1070A phytotoxin was correlated with increased disease symptom expression in 17 isolates examined [49]. The extracellular proteomes of Che and Cps were recently examined and revealed 124 putative effectors produced by both species which are hypothesized to be involved in plant pathogenesis [74]. However, to date, gene expression profiles of Che and Cps during boxwood blight disease development have not been the subject of comprehensive investigation in a gene family evolution framework.
During genome evolution, gene duplication and gene loss events can contribute to contraction and expansion of gene families [17]. Genome changes can be linked to evolutionary processes that result in environmental niche adaptation [17]. Studying changes in gene family contraction and expansion can provide useful insight into organismal, ecological, and lifestyle transitions of plant pathogenic fungi. In many disease-causing fungal species, changes in gene family size have been linked to observed variation in host adaptation, pathogenesis, and virulence [54,61]. Rapid expansion of gene families in plant pathogenic fungi associated with host cell wall degradation, secondary, and carbohydrate metabolism is providing insight into pathogenesis and virulence processes [40,48,64]. Rapid contraction of gene families involved in similar processes have also been linked to biotrophy (obligate parasitism) and ecological lifestyle [65,78]. For example, in insect and plant pathogenic fungi, contraction of gene families was associated with cuticle and cell wall degradation and limited (narrow) host range [3,71]. In plant and insect systems, analysis of gene family evolution has elucidated different aspects of pathogen biology and ecology. In the Northern California black walnut (Juglans hindsii), a plant species native to the western US, contraction of gene families involved in abiotic stress and disease were associated with resistance to Armillaria root rot disease [69]. In another recent study, identification of rapidly evolving gene families led to the development of novel strategies for managing blood-feeding insects [23].
In this study, we deployed comparative phylogenomic tools to characterize and identify rapidly evolving gene families within the genomes of Che, Cps, and 22 additional fungal taxa. Using multiple analytical methods, we generated annotations for protein sequences within rapidly evolving gene families to determine putative functional classes. Further annotation of putative pathogenicity factors and secreted effectors within rapidly evolving gene families of Che, Cps, closely related plant pathogenic and saprobic (apathogenic) species of Calonectria, and less-aggressive pathogens of hosts in Buxaceae Pseudonectria buxi (Pbu), P. foliicola (Pfo), and Coccinonectria pachysandricola (Cpa), were conducted to identify shared patterns in gene family evolution associated with fungal-host plant adaptation, pathogenesis, and virulence. We hypothesized that gene families important for host infection and pathogenesis have expanded in Che and Cps, relative to other pathogenic and saprobic species of Calonectria and closely related non-Calonectria Buxaceae pathogens. Here we report on (1) the quantity and predicted functional classes of rapidly contracting and expanding gene families in 24 fungal taxa in the Nectriaceae that vary in pathogenic and saprobic lifestyle; and (2) the predicted functional annotation and comparison of putative pathogenicity factors and secreted effectors within rapidly evolving gene families of Che, Cps, and closely and distantly related species of Calonectria and non-Calonectria pathogens of hosts in Buxaceae.

Gene family identification based on time-calibrated phylogenetic analyses
Genome assemblies and predicted proteomes for each of the 24 fungal taxa showed high levels of completeness based on BUSCO scores of 95% or higher (Additional file 1). Overall, 95% of the predicted protein sequences across all the taxa were assigned to a gene family, with a total of 19,750 gene families identified. The average number of proteins in a gene family was 16.7 and 2154 gene families had single copy proteins found in all 24 taxa. Construction of a maximum likelihood phylogenetic tree using protein sequence data from 2154 single copy genes showed 100% confidence in tree topology (Additional file 2).

Identification of rapidly evolving gene families
Across the time-calibrated phylogeny of the 24 fungal taxa examined in this study, CAFE4 identified 422 gene families evolving at a non-random rate (rapidly evolving) (p ≤ 0.01; Additional file 3, Additional file 4). In total, 17,596 gene families experienced a change in size across the phylogeny, either through expansion or contraction. To provide a measure of rapid gene family evolution that each species experienced relative to total changing (both rapidly evolving and randomly evolving) gene families, calculations of the percent of rapidly evolving gene families per total changing gene families were performed (Fig. 1). For 17 species, rapidly expanding gene families accounted for ≥ 1% of total expanding gene families. Rapidly contracting gene families accounted for ≥ 1% of total contracting gene families in seven species. However, randomly contracting gene families were more numerous than randomly expanding gene families across the 24 taxa and may partially explain the generally lower observed percentages.
The percentage of rapidly evolving gene families showed variation among the 24 fungal taxa. For example, Cpa, which has the smallest assembled genome among the 24 taxa (26.4 Mb), contained the largest number of total contracting gene families (11,967 gene families) and mean gene losses (-8.4 genes) but was one of five species to have < 10 total rapidly evolving gene families (six gene families) (Fig. 1, Additional file 1, and Additional file 5). Surprisingly, Che exhibited the highest and second highest percentages of rapidly contracting and expanding (respectively) gene families, despite having the second and first smallest totals (contracting and expanding, respectively) ( Fig. 1). Similar to Che, Cps experienced comparable trends in both total changing and total rapidly evolving gene families ( Fig. 1 and Additional file 5). The proportion of rapidly expanding gene families compared to rapidly contracting gene families for each species showed that each of the 24 taxa exhibited distinct patterns of gene family evolution directionality, with either more rapid gene family contraction or more rapid gene family expansion (Fig. 2).
Among species of Calonectria, Che and Cps were the only species to undergo more rapid gene family contractions than expansions and had considerably fewer total expanding and contracting gene families than Cmu, Cna, Cle, and Cpr (Figs. 1, 2, and Additional file 5). The saprobic species Cmu and Cna exhibited nearly exclusive rapid gene family expansion. For example, Cna had the greatest number of total expanding gene families across all 24 taxa (Figs. 1, 2, and Additional file 5). Compared to the non-Calonectria Buxaceae pathogens (Cpa, Pbu, and Pfo), Che and Cps experienced more rapid gene family expansions and contractions than Cpa, Pbu, and Pfo. However, Che and Cps had a similar number of total contracting and expanding gene families compared to the two species of Pseudonectria examined in this study.
For example, Pseudonectria species had ~ 11,000 fewer total contracting gene families than Cpa, which had the most total contracting gene families among the 24 taxa examined (Figs. 1, 2, and Additional file 5). The non-Calonectria pathogens of plants in the family Buxaceae consistently placed in the bottom five species with the fewest total rapidly evolving gene families (Pbu, Pfo, and Cpa had one, two and six total rapidly evolving gene families, respectively; Additional file 5).
To identify rapidly evolving gene families shared between Che and Cps and the other Calonectria species and non-Calonectria pathogens of species in Buxaceae (Cpa, Pbu, and Pfo), a series of UpSet plots were generated (Additional file 6). For rapidly contracting and expanding gene families, Che and Cps shared two or fewer gene families with saprobic Calonectria species, pathogenic Calonectria species, and non-Calonectria pathogens of plants in the family Buxaceae (Additional file 6). Che and Cps shared the most rapidly contracting and rapidly expanding gene families with the pathogenic Calonectria species (Cle and Cp) (Additional file 6). Che and Cps did not share any rapidly evolving gene families within the "rapidly expanding" or "rapidly contracting" categories but did share three gene families that were rapidly evolving in opposite directions in each species (OG0000649, OG0001150, and OG0007608). Individually, Che and Cps experienced rapid expansion and rapid contraction of three gene families (OG0000150, OG0000440, and OG0000796 in Che, and OG0000026, OG0000101, and OG0000854 in Cps) that were not rapidly evolving in any of the other 22 additional fungal taxa.

Annotation of rapidly evolving gene families
While 422 gene families were identified by CAFE4 as rapidly evolving across the phylogeny, only those rapidly evolving at terminal taxa were characterized (403 gene families). Of the 7221 protein sequences grouped into the 403 extant gene families, 5912 received a COG annotation (sequences that received an 'NA' or not annotated designation by eggNOG were removed from subsequent analyses) ( Table 1). The 5912 annotated sequences were used to classify 332 rapidly evolving gene families into a putative functional category which were annotated with between one and 11 (1.56 ± 1.09; Mean ± SD) COG categories based on the annotations of protein sequences within each gene family. Approximately 68% (225 gene families) of the COG-annotated gene families were annotated with a single COG category. For Pfam annotation,   Table 1). Of the 115 Pfam targets (13.6 ± 27.4 proteins per target) spanning the S-categorized gene families, heterokaryon incompatibility protein (HET; PF06985.12; 253 proteins) was the most frequently observed and was found in seven S-categorized gene families. The second most frequently observed Pfam target was NACHT domain (PF05729.13; 97 protein sequences), which is associated with programmed cell death and heterokaryon incompatibility (HET) loci. Together, HET and NACHT domain Pfam targets spanned 13 S-categorized gene families which were rapidly evolving in 14 of the 24 taxa (Che, Cmu, Cna, Cps, Cp, Dactylonectria macrodidyma, Fusarium fujikuroi, F. oxysporum 4287, F. oxysporum Fo47, F. solani, Neonectria hederae, N. punicea, Stachybotrys chartarum, and S. chlorohalonata) (Fig. 3). Excluding F. oxysporum 4287 and Fo47, and S. chlorohalonata, each species experienced either exclusive rapid expansion or contraction of these gene families. After S-categorized gene families, Q-categorized gene families (secondary metabolism, biosynthesis, and catabolism) were the second most frequently observed COG category with 40 gene families (709 protein sequences). Pfam targets within Q-categorized gene families spanned a narrower range than in S-categorized gene families of 56 targets (12.7 ± 35.1 proteins per target) with the most frequently observed targets being cytochrome P450 (PF00067. 23 gene families ( Table 1). The comprehensive range of Pfam functional targets within each COG-annotated gene family are presented in Additional file 7.

Identification of putative pathogenicity factors and secreted effectors
To further screen rapidly evolving gene families in the genus Calonectria and non-Calonectria pathogens of hosts in Buxaceae for potential roles in plant pathogenesis, protein sequences in these gene families were compared to accessions in the Pathogen Host Interactions (PHI) database. The PHI database catalogues pathogenicity, virulence, and effector genes that have been experimentally tested in pathogen-host interactions of fungal, oomycete, and bacterial pathogens with animal, plant, fungal, and insect hosts [70]. In total 2682 sequences were searched against the PHI database and 1566 sequences spanning 112 rapidly evolving gene families received hits with e-values ≤ 1e−5. Protein sequences from all species except the non-Calonectria Buxaceae pathogen Pfo received PHI hits. To identify PHI-annotated sequences putatively involved in virulence and pathogenicity, sequences homologous to proteins with annotated mutant phenotypes of reduced virulence (RV), loss of pathogenicity (LOP), or effector (E) in other pathogens were identified from the dataset. Of 1566 sequences with similarity to sequences in the PHI database, 738 sequences matched these criteria and spanned 64 rapidly evolving gene families ( Fig. 4 and Additional file 8). The same set of sequences used for PHI annotation were also classified into secreted and other (no signal peptide identified) protein categories using SignalP v5.0 [1]. Of 2682 sequences, 123 sequences were classified as secreted proteins and spanned 35 rapidly evolving gene families ( Fig. 5 and Additional file 8). All species except for Pbu and Cpa had sequences classified as secreted proteins in rapidly evolving gene families. Sequences classified as secreted proteins were further classified into effector and non-effector categories using EffectorP v2.0 (Fig. 5) [66]. The predominant PHI phenotype for annotated sequences within rapidly evolving gene families was RV followed by LOP and E ( Fig. 4 and Additional file 8). Several shared gene families identified with the UpSet plots between Che, Cps, sapbrobic Calonectria species, non-Buxaceae Calonectria pathogens, and non-Calonectria pathogens of species in Buxaceae also contained PHI-annotated sequences ( Fig. 4 and Additional file 6). Of 123 sequences classified as secreted proteins by SignalP, seven were classified as   (Fig. 5). Gene families containing secreted effectors were not rapidly evolving in Che. Gene families rapidly evolving within saprobic Calonectria species Cmu and Cna that contained PHI-annotated and/or predicted secreted effectors experienced exclusive rapid expansion, while gene families rapidly evolving in Che and Cps that contained sequences with similar annotations experienced predominant rapid contraction (Figs. 4, 5). COG and Pfam annotation information for gene families that contained PHI annotated sequences and/or secreted effectors that were rapidly evolving in Che and Cps are presented in Table 2. These data showed that 10 gene families belonged to the secondary metabolism (Q), carbohydrate metabolism (G), and intracellular trafficking and secretion (U) COG categories. Among the 10 families, Che and Cps experienced rapid contraction in eight gene families within these categories (Table 2).

Discussion
Since the global emergence of boxwood blight disease in the 1990s, research on the evolution of Che and Cps has focused primarily on understanding factors influencing pathogen population genetic and genomic diversity. However, gene family evolution in Che and Cps and its putative role in plant pathogenesis has not been studied. For this study, we identified and annotated rapidly evolving gene families in the genomes of Che  Among the pathogenic and saprobic species of Calonectria examined in this study, only Che and Cps exhibited predominant rapid contraction of gene families with putative involvement in host plant infection based on combined use of multiple annotation analyses. Gene families were assumed to play putative roles in plant pathogenesis based on examination of proteins that displayed similarity to proteins documented in the PHI database or contained predicted secreted effectors. While rapid expansion of gene families involved in pathogenesis has been reported for fungal plant pathogens, rapid contraction of pathogenesis-related gene families has been linked to biotrophy (obligate parasitism) and a restricted host range [3,62,65,71,78]. Given that infection by Che and Cps results in necrosis of diseased leaves and stems and because Che and Cps can be readily cultured on artificial nutrient medium, it is unlikely that they are obligate biotrophs. However, it remains unknown whether Che and Cps possess an initial biotrophic phase to obtain nutrients from living cells upon entry into plant tissues during infection, and whether these fungi exhibit hemibiotrophic or necrotrophic behaviors. Better studied fungal plant pathogens in the Nectriaceae including Neonectria spp. and Fusarium spp. have been characterized as hemibiotrophs [43,57,68]. Without knowledge of the trophic behavior and lifestyle of Che and Cps, it is challenging to interpret how the mechanism(s) of nutrient acquisition influence gene family evolution in these species. Future comprehensive investigation on the trophic behaviors and lifestyles of Che and Cps is warranted to provide additional insight into the relative contribution of  these behaviors on contraction of gene families involved in plant pathogenesis in these species. To date, Cps has been isolated and identified from environmental samples of diseased leaves on plants in three genera in the family Buxaceae: Buxus, Pachysandra, and Sarcococca [21]. This restricted host range provides at least one plausible explanation for predominant rapid contraction of pathogenesis-related gene families. Che causes disease primarily on species of Buxus in nature, although artificial inoculation experiments in the laboratory indicate that Pachysandra terminalis 'var. Compacta' is a host [4]. Differences in Che and Cps host range may explain observed differences in gene family evolution between these closely related species. Interestingly, contraction of gene families involved in trophic behavior and plant/animal host recognition among 45 fungal genomes sampled in the order Hypocreales were associated with a restricted host range [78]. Comparative genomic analyses of fungal insect pathogens in the Ophiocordyceps unilateralis species complex also revealed contraction of gene families involved in cuticle degradation and other insect-host interactions and specificity [71]. The contraction of gene families involved in cell-wall degradation and secondary metabolite biosynthesis associated with plant pathogenesis in fungal plant pathogens is also well documented [65]. In other plant pathogens, the absence or presence of pathogenicity-related genes have been shown to be strong determinants of plant host range for lineages of Magnaporthe oryzae, Verticillium dahliae, Leptosphaeria maculans-Leptosphaeria biglobosa species complex, and closely related Zymoseptoria species [12,16,25,26]. For example, effector genes were hypothesized to have emerged after speciation and contributed to differences in host specificity in the closely related sister species Zymoseptoria pseudotritici and Z. ardibiliae [25]. Perhaps this is the case for Che and Cps and explains the limited overlap in rapidly evolving, pathogenesisrelated gene families between these species. Based on these observations, our hypothesis that gene families involved in plant pathogenesis are expanding in Che and Cps was rejected.
Three gene families were rapidly evolving in both Che and Cps, but none had putative roles in plant pathogenesis. Instead, these gene families received COG annotations of unknown function, coenzyme transport, and cytoskeleton and did not contain proteins that received PHI annotations or secreted effector classifications. Individually, Che and Cps contained three rapidly evolving gene families each that were not rapidly evolving in any of the additional 22 fungal taxa investigated. In Cps, one Table 2 Rapidly evolving gene families in the genomes of the boxwood blight pathogens Calonectria henricotiae (Che) and C. pseudonaviculata (Cps) that contain predicted proteins with pathogen host interactions (PHI) annotations and/or putative secreted effectors (C) and (E) indicate rapid contraction and expansion of each gene family, respectively of three uniquely evolving and rapidly contracting gene families contained a top Pfam target of CFEM domain (PF05730.12) which has a proposed role in fungal pathogenesis and contained putative secreted proteins [34].
In Che, none of the three uniquely evolving gene families had putative roles in pathogenesis. Che and Cps are closely related sister species with minor genetic and genomic differences, but exhibit phenotypic differences in thermotolerance, fungicide sensitivity, and secretome composition [24,37,74]. Additionally, the geographic distribution of Cps is more widespread than Che which has not expanded its range beyond the UK and continental Europe [38]. Differences in geographic range may contribute to the rapid evolution of different gene families in these species. However, the relationship between genetic diversity and biogeography are not well documented in fungal species except in certain pathogenic, mushroomforming, and arbuscular mycorrhizal fungi [2,18,60,79]. One isolate genome of Che and Cps was included for comparing rapidly evolving gene families between these species since pathogen populations of Che and Cps have been shown to be clonal in nature and display limited genetic diversity [37]. However, complete genome analyses of asexual pathogens like Verticillium dahliae have revealed that this plant pathogenic species can harbor substantial numbers of accessory genes, which can be enriched in candidate effectors not shared between strains [58]. Future studies should confirm similar trends in gene family evolution across genetically different isolates of Che and Cps. Two of the closest known relatives of Che and Cps included in this study were the apathogenic soil saprobes, Calonectria multiphialidica (Cmu) and C. naviculata (Cna), which exhibited nearly exclusive rapid gene family expansion and had the greatest number of rapidly evolving gene families among Calonectria species investigated. The greatest number of rapidly expanded gene families in Cmu and Cna were classified into the unknown function, secondary metabolite biosynthesis, and carbohydrate metabolism COG categories. Expanded gene families involved in plant cell wall degradation and secondary metabolite biosynthesis are commonly observed in saprobic fungal genomes due to their involvement in nutrient degradation and defense against competing microorganisms, respectively [36,39]. Mutualistic ecto-and endomycorrhizal fungi are also known to produce a variety of secreted PCWDEs and effectors known as mycorrhiza-induced small secreted proteins (MiSSPs) that allow them to initiate their symbiotic association with plants [45]. Interestingly, many rapidly expanding gene families in Cmu and Cna contained proteins similar to those characterized in pathogen-host interactions and were the same gene families rapidly contracting in Che and Cps. While there are no published reports of plant diseases caused by Cmu and Cna, rapid expansion of pathogenesis-related gene families suggests that these species may be evolving in a similar manner to plant pathogenic fungi [40,48,64]. Saprobic fungi have been shown to produce and secrete large repertoires of effectors similar in sequence to those produced by plant pathogenic fungi. However, the function of these effectors in a saprobic lifestyle remains unclear [22]. Functional annotation of gene families involved in pathogenesis in plant pathogenic Calonectria species of non-Buxaceae hosts, Calonectria leucothoes (Cle) and Calonectria pseudoreteaudii (Cp), were also performed in this study. Cle and Cpr are well documented pathogens of Leucothoe spp. and Eucalyptus spp. in the Ericaceae and Myrtaceae plant families, respectively [21]. Based on these reports, Cle and Cpr have a similar size and restricted host range to Che and Cps. However, the same observation of predominant rapid contraction of gene families involved in plant pathogenesis was not observed. Compared to Che and Cps, Cle and Cpr had fewer total rapidly evolving gene families and experienced predominant expansion of rapidly evolving gene families including those involved in plant pathogenesis. Based on these observations, Cle and Cpr displayed the most typical trends in gene family evolution observed in other fungal plant pathogens compared to the other Calonectria species examined [40,48,64]. A valuable and future comparison of gene family evolution between the restricted plant host range Calonectria species examined in this study and a species of Calonectria with a broader host range is warranted. At the time of initiating our study, relatively few Calonectria genomes were publicly available for this comparison. Lastly, we compared rapidly evolving gene families in Che and Cps to non-Calonectria Buxaceae pathogens Pseudonectria buxi (Pbu), P. foliicola (Pfo), and Coccinonectria pachysandricola (Cpa). Pbu, Pfo, and Cpa are the causal agents of Volutella leaf blight on different hosts in the family Buxaceae and are considered non-aggressive pathogens that typically occur on plants experiencing abiotic stress compared to Che and Cps [56,73]. Pbu causes disease on species of Buxus, while Pfo causes disease on both Buxus and Sarcococca spp., and Cpa causes disease on Sarcococca and Pachysandra spp. [21]. Pbu, Pfo, and Cpa clustered with Aquanectria penicillioides and Thelonectria rubi with the fewest total rapidly evolving gene families while Cpa shared two rapidly contracting gene families involved in pathogenesis with Che. Because of the relatively low quantity of rapidly evolving gene families in Pbu, Pfo, and Cpa comparisons with Che and Cps were limited. However, the relatively low number of rapidly evolving gene families may suggest that these species are experiencing different selective pressures than Che and Cps [17].
In addition to the functional annotation analyses of pathogenesis-related gene families within Calonectria species and non-Calonectria Buxaceae pathogens, we performed broad functional annotation of gene families rapidly evolving across all 24 fungal taxa selected. Approximately, one third of rapidly evolving gene families across the 24 taxa were classified into the unknown function COG category. Within unknown function gene families, the predominant Pfam targets were heterokaryon incompatibility protein (HET; PF06985.12) and NACHT domain (PF05729.13). Both HET and NACHT domains are subunits of proteins commonly found in the HNWD gene family that allow fungi to recognize self from non-self for successful cell and cytoplasmic fusion [10]. Cell and cytoplasmic fusion are essential and fundamental processes in fungi that allows them to transition from unicellular to multicellular organisms and form hyphal networks for maximizing nutrient acquisition. HET genes have been shown to be highly polymorphic and contribute to the rapid evolution of members within HNWD gene families [10,33,53]. Constant rapid evolution of HET genes and their associated gene families allows fungi to maintain genome integrity and evade mycoparasitic exploitation or mycovirus infection, which is critical for fungal species success, irrespective of trophic behavior and lifestyle [52]. This would partially explain why the greatest number of rapidly evolving gene families contained proteins important for heterokaryon (vegetative) incompatibility across the 24 taxonomically diverse taxa examined in this study. Among species of Calonectria, heterokaryon incompatibility and HET loci have not been well studied. However, HET loci in Che and Cps likely have a similar function to other previously examined fungi in the Ascomycota where an incompatible (cell death) reaction is initiated when there are allelic differences at HET loci of two interacting fungal isolates of the same species.

Conclusions
In this study, we used comparative phylogenomic methods to identify and characterize gene families that are rapidly evolving in Che and Cps and other closely related fungi to better understand adaptation and pathogenesis mechanisms for infection of hosts in the plant family Buxaceae by these pathogens. Our work highlights and provides new information on the evolutionary trajectories of Che and Cps and their close relatives that suggest a restricted host range in Che and Cps and gene family evolution trends in saprobic species Cmu and Cna that are analogous to many plant pathogenic fungi. Our results serve as a framework for future studies examining Che and Cps during infection and pathogenesis on Buxaceae hosts that may be used to develop novel disease management strategies. This research also raises new questions about the complex involvement of gene family evolution in the trophic lifestyles of Calonectria species and provides further evidence for an evolutionarily relevant role of pathogenesis-related gene families in fungi with saprobic lifestyles.

Genome selection and assembly quality assessment
Twenty-four fungal taxa representing 10 genera in the family Nectriaceae and two outgroup species of Stachybotrys (Stachybotryaceae) were selected for this study (Additional file 1). Genome assemblies were obtained from NCBI GenBank for all taxa except Calonectria multiphialidica. References and accession numbers for genome assemblies are shown in Additional file 1. Genome data for C. multiphialidica were generated using Illumina sequencing technology and assembled as previously described [38]. Predicted protein sequence data were also downloaded from NCBI GenBank where available; otherwise, proteins were predicted using the Funannotate v1.8.1 pipeline [51]. Completeness of all predicted proteomes and underlying genome assemblies were assessed using BUSCO v3.1.0 using the fungalspecific gene set 'Fungi odb9' [63]. No plant material was used in this study.

Estimation of gene families and construction of time-calibrated phylogeny
Clusters of orthologous genes were identified with OrthoFinder v2.2.7 using the "diamond" option for sequence alignment and "msa" option for gene-tree inference [20]. Single copy orthologs identified using OrthoFinder were concatenated into an alignment and poorly conserved regions were filtered with Gblocks v0.91 [6]. The best-fit model of protein evolution was identified using ProtTest v3.4.2 [14]. The protein sequence alignment was used to construct a maximum-likelihood phylogeny with RAxML v8.2.12 using the JTT model of protein evolution and 100 bootstrap replicates to assess confidence in tree topology [67]. The program r8s v1.81 was used to generate a time-calibrated ultrametric tree from the RAxML phylogeny using an estimated 244 MYA median divergence between Stachybotrys chartarum and Fusarium graminearum determined from the TimeTree database [35,59]. The time-calibrated phylogeny and orthogroup data were used to measure changes in gene family size and identify rapidly evolving gene families.

Identification and annotation of rapidly evolving gene families
Rapidly evolving gene families were identified using CAFE v4.2.1 which models gene family evolution through time using a stochastic birth and death model and identifies gene families that have experienced a significant change in size [28]. Input data were represented by the orthogroups (gene families) identified with OrthoFinder and the time-calibrated phylogeny representing evolutionary relationships among the 24 fungal taxa. A birthdeath parameter (lambda, -s option) of 0.00353252 was estimated for the phylogeny using an optimization algorithm that maximizes the log likelihood of the data for all gene families. A default significance level of 0.01 (-p option) was used to calculate Viterbi p-values to assess rapid (significant) contraction or expansion of gene families along each branch. A custom Python script was developed to extract gene families that were rapidly evolving in extant species to perform additional analyses (Additional file 5).
Protein sequences within rapidly evolving gene families for each species were annotated to determine putative functional classes of gene families. Broad functional annotation was performed using eggNOG-mapper v2 which identifies Clusters of Orthologous Groups (COG) functional categories for each sequence using an e-value threshold of 1e−3 [30,31]. The most frequently observed COG category within rapidly evolving gene families was used to categorize the gene families for further analysis. Gene families with equivalent frequencies of more than one COG category were classified to the first of the tied categories, which were ordered alphabetically for a given gene family. Protein sequences from Aquanectria penicillioides (20 total protein sequences) and Pfo (eight total protein sequences) did not receive COG annotations and were not used in gene family characterization. Additional annotation of protein sequences within gene families was performed by searching sequences against the Pfam-A database v33.1 using HMMER 3 with an e-value threshold of 1e−5 [19,46]. For each annotation procedure, hits with the lowest e-value were selected for annotation of protein sequences that matched multiple subject sequences. Interspecific gene family annotation summaries and analyses were conducted in R v4.0.3 [55] using the following packages: tidyverse v1.3.0, UpSetR v1.4.0, ggtree v2.4.1, seqinr v4.2-5, Biostrings v2.58.0 [9,11,50,72,77].

Identification of putative pathogenicity factors and secreted effectors
Protein sequences within rapidly evolving gene families from Che, Cps, two saprobic Calonectria species (C.