Genome sequencing and general features

The C. militaris genome was shotgun sequenced to 147 × coverage and assembled into 33 scaffolds with an N50 of 4.6 Mb and a total genome size of 32.2 Mb. The genome is smaller than either the broad host range Metarhizium anisopliae (MAA) or the locust-specific pathogen Metarhizium acridum (MAC) that we sequenced previously (Table 1). The characteristic telomeric repeats (TTAGGG/CCCTAA) n were found at either 5' or 3' terminal of 13 scaffolds, including the terminal anchoring of two scaffolds, that is, the complete chromosomes. From mapping > 5,000 expressed sequence tags [12], the C. militaris genome was estimated to be > 99% complete. The genome was predicted to encode 9,684 protein genes, which is slightly fewer than M. anisopliae and M. acridum (Table 1). Consequently, many protein functional categories are smaller in Cordyceps than in Metarhizium spp. (Figure 2a). However, like M. anisopliae (17.6%) and M. acridum (15.1%), C. militaris has a higher proportion of its genes encoding putatively secreted proteins (15.9%) than other sequenced ascomycetes (5 to 10%) [10, 13, 14].

Table 1 Comparison of genome features among three insect pathogens Full size table

Figure 2 Comparative genomics analysis of three insect pathogens. (a) Functional classification and comparison of C. militaris (CCM), M. anisopliae (MAA) and M. acridum (MAC) proteins, showing that C. militaris has fewer genes in each category. Each circle represents the relative fraction of genes represented in each of the categories for each genome. (b) Reciprocal blast analysis of the predicted proteins among three insect pathogens. The cut-off E value is at ≤ 1e-5. Full size image

An InterproScan analysis identified 2,736 conserved protein families in C. militaris (containing 6,725 proteins), fewer than those in M. anisopliae (7,556 proteins in 2,796 families) or M. acridum (6,948 proteins in 2,746 families) [11]. In particular, the number of transposases is much fewer in C. militaris (4) than in Metarhizium spp. (148 in M. anisopliae and 20 in M. acridum) or other sequenced ascomycetes (15 to 426) (Table S1 in Additional file 1). The C. militaris genome lacks retrotransposase (Table S2 in Additional file 1), and has more than three-fold fewer pseudogenes than Metarhizium spp. (Table S3 in Additional file 1). About 16% of the predicted C. militaris genes (1,547) are putatively involved in pathogen-host interactions; this proportion is slightly lower than for Metarhizium spp. (17.3% in MAA and 16.5% in MAC) but is higher than four plant pathogens (10.8 to 15.5%; P = 0.0476; false discovery rate (FDR) = 0.0152) (Table S4 in Additional file 1).

More than 50% of M. anisopliae and M. acridum proteins have > 90% identity [11]. Although the rarely observed sexual stages of Metarhizium spp. have been identified as a Cordyceps species [1], the analysis revealed that < 2% of C. militaris genes were highly conserved in comparison with those from Metarhizium spp., that is, had Blast score ratio (BSR) values close to 1 (Figure 3a). A similar pattern was observed when comparing C. militaris, M. anisopliae and the plant pathogen Fusarium graminearum (Figure 3b). Comparative genomic analysis of the three insect pathogens found that the percentage of species-specific genes is much higher in C. militaris (13.7%) compared to M. anisopliae (4.8%) and M. acridum (3.5%) (Figure 2b). Based on the identities between orthologous proteins, C. militaris displays an average of approximately 63% amino acid identity with either M. anisopliae or M. acridum, slightly higher than with the plant pathogens F. graminearum (61.6%) and Magnaporthe oryzae (56.0%) (Table 2). Thus, the three insect pathogenic fungi are more highly diverged than F. graminearum, Fusarium oxysporum and Fusarium verticillioides, which share an average of 85% nucleotide sequence identity [14], and Aspergillus nidulans, Aspergillus fumigatus and Aspergillus oryzae, which share an average of 68% amino acid sequence identity [13], and Trichoderma reesei, Trichoderma virens and Trichoderma atroviride, which share an average of > 70% amino acid sequence identity [15].

Figure 3 Comparative genomics and evolutionary analysis of C. militaris. Scatter plots of Blast score ratio (BSR) analysis of (a) C. militaris (CCM), M. anisopliae (MAA) and M. acridium (MAC) genomes, and (b) CCM, MAA and F. graminearum (FG) genomes. The numbers in red at the lower left corners are the percentages of C. militaris species-specific sequences and the numbers at the upper left or lower right are the percentages of lineage-specific genes between pairs of genomes. (c) A maximum likelihood phylogenomic tree constructed using the Dayhoff amino acid substitution model showing the evolutionary relationship of C. militaris with different fungal species. Three insect pathogens are highlighted by the green shading. (d) Distribution of paralogous gene numbers with different levels of nucleotide similarity in C. militaris and other fungi. MY, million years. Full size image

Table 2 Genome-wide analysis of C. militaris gene sets. Full size table

The regions containing at least three contiguous open reading frames that are not present in the reference genome are designated as genomic islands (GIs) [16]. Whole genome reciprocal analysis of three insect pathogens demonstrated that, in comparison to Metarhizium spp., C. militaris has 52 GIs (2% coverage of its genome, harboring 21% of its species-specific genes), which is many more than M. anisopliae (8 GIs, 0.3%) or M. acridum (5 GIs, 0.2%) when referenced to C. militaris. As in aspergilli [17], many C. militaris species-specific gene-encoding proteins do not have conserved domains and the genes are clustered together to form GIs (Table 2). A phylogenomic analysis established that the Cordyceps lineage is more closely related to the wheat pathogen F. graminearum (divergence time of 200 to 260 million years ago (MYA)) than it is to Metarhizium spp. (26 to 34 MYA) (Figure 3c). Thus, the lineage leading to C. militaris appears to have diverged from plant pathogens around the Triassic-Jurassic boundary (200 MYA), while M. anisopliae and M. acridum diverged after the Cretaceous Extinction Event (65 MYA) [18]. Analysis of paralogous genes found only one pair of C. militaris genes with > 90% nucleotide sequence similarities (Figure 3d), which is similar to Neurospora crassa (one pair) [13] and F. graminearum (two pairs) [19]. Analysis of 24 paired C. militaris genes showing > 70% nucleotide identities found a strong overall C:G to T:A mutation bias (Figure S1 in Additional file 2), consistent with repeat-induced point mutations, the DNA methylation-linked processes that cause mutations of repeated fungal sequences [15, 20].

Protein family analysis

We identified gene family expansions for proteases, chitinases, lipases and protein kinases in C. militaris when compared with phytopathogenic fungi, whereas gene family contractions occurred for glycoside hydrolases (GHs; P = 0.0144; FDR = 0.02), cutinases (P = 0.0065; FDR = 0.0226) and pectin lyases (P = 0.0245; FDR = 0.0284) (Table S1 in Additional file 1). The largest family expansions were for proteases. The C. militaris genome contains 61 families of proteases but most of them were included in families of serine proteases (180/381) and metallopeptidases (108/381) (Table S5 in Additional file 1). Gene expansions within the subtilisin (P = 0.0109; FDR = 0.0189) and trypsin (P = 0.0077; FDR = 0.0178) families are consistent with their being virulence factors in insect pathogens [11]. However, different families of proteases are expanded in Metarhizium spp. and C. militaris, consistent with each lineage 'reinventing the wheel' during the evolution of entomopathogenicity. Thus, relative to Metarhizium spp., the S01 trypsin and S08 subtilisin subfamilies are smaller and the S53 subfamily is larger (Table S6 in Additional file 1). The C. militaris genome has 12 trypsin genes compared to 4 or less in plant pathogens. It lacks four subfamilies of trypsins present in M. anisopliae. Interestingly, the bacterial-like chymotrypsin identified in M. anisopliae [21] is absent in M. acridum [11] but present as two copies in C. militaris. The A01 aspartyl proteases are virulence factors of both mammalian and plant pathogens because of their ability to cleave an array of host proteins [22]. Compared to phytopathogenic fungi (average 17), their number is significantly (P = 0.0059; FDR = 0.0057) expanded in the three insect pathogens (average 24) (Table S4 in Additional file 1).

Compared to many plant pathogens, Metarhizium spp. and C. militaris have fewer cutinases for degrading plant cell walls (Table S1 in Additional file 1). They also have fewer (average 137, P < 0.05) GHs than plant pathogens (average 199), including the lack of 20 GH families used by most plant pathogens and saprobes to target plant cell walls - for example, GH6, GH7 and GH61 cellulases, GH10 and GH11 xylanases, GH28 pectinases and GH78 rhamnosidases (Table S7 in Additional file 1). There are also significant differences in the spectrum of enzymes produced by the entomopathogens. For example, compared to M. anisopliae, C. militaris has few xyloglucosyl transferases (GH16) for xyloglucan catabolism and lacks α-glucuronidases (GH115) active on xylan oligomers or polymeric xylan [23]. Consistent with this, C. militaris grows very poorly on xylose when compared with M. anisopliae (Figure S2 in Additional file 2). A phosphoketolase MPK1 involved in pentose metabolism is required for full virulence of M. anisopliae [24], but the homolog is absent in C. militaris. However, GH18 chitinases similar to those used by Metarhizium to degrade insect cuticles [11] are well represented in the C. militaris genome (20 in CCM versus 30 in MAA and 19 in MAC) relative to plant pathogens (average 11) (Table S7 in Additional file 1).

Cytochrome P450s (CYPs) play essential roles in fungal physiologies, including detoxification, degradation of xenobiotics and the biosynthesis of secondary metabolites [25]. C. militaris has only about half as many CYPs as Metarhizium spp., and most other fungi (Table S8 in Additional file 1). Seventy CYP subfamilies present in M. anisopliae and/or M. acridum are absent in C. militaris. Of particular interest, C. militaris lacks CYP55, CYP58 and CYP65. CYP55 is a nitric oxide reductase required for denitrification [25]. Thus, unlike most filamentous fungi, C. militaris may not respond to hypoxia through the bacterial ammonia fermentation mechanism. The absence of CYP58 (trichodiene oxygenase) and CYP65 (trichothecene C-15 hydroxylase) suggests that C. militaris will not produce the mycotoxin trichothecene [26]. M. anisopliae can efficiently metabolize insect epicuticle alkanes [27]. The CYP52 subfamily for alkane hydroxylation [25] is well represented in Cordyceps.

The major facilitator superfamily (MFS) and ATP-binding cassette (ABC) transporters are the two biggest families of fungal transporters. Members of the former typically function as nutrient symporters and drug antiporters, whereas the latter are more often implicated in defense against toxic metabolites [28]. C. militaris has approximately half (123) the number of these transporters as Metarhizium (269 in MAA and 236 in MAC) (Table S9 in Additional file 1). The MFS transporters that are underrepresented in Cordyceps include the carbohydrate symporters (37 in CCM versus 48 in MAA, 51 in MAC and an average of 58 in plant pathogens), vitamin B2 (riboflavin) transporters (2 in CCM versus 17 each in Metarhizium species and an average of 4 in plant pathogens) and multidrug antiporters (23 in CCM versus 110 in MAA, 77 in MAC and an average of 10 in plant pathogens). Consistent with their having many multidrug transporters, Metarhizium spp. are resistant to diverse antibiotics and fungicides [29]. Cordyceps has more ABC-type drug and metal resistant proteins than Metarhizium and plant pathogens (63 in CCM, 56 in MAA, 51 in MAC and an average of 54 in plant pathogens). The amino acid and dipeptide transporters are similarly represented in the three insect pathogens and other fungi (46 in CCM versus 53 in MAA, 49 in MAC and an average of 45 in plant pathogens).

Fungal G-protein coupled receptors (GPCRs) are required for pheromone/nutrient sensing and host recognition [11]. Thus, the Pth11-like GPCR of Magnaporthe mediates cell differentiation in responses to plant inductive cues [30]. C. militaris has fewer GPCRs than Metarhizium spp. and is particularly impoverished in Pth11-like GPCRs (Table S10 in Additional file 1). C. militaris has a similar number (167) of protein kinases as M. anisopliae (161) but less than M. acridum (192) (Table S11 in Additional file 1). Like other fungi, fungal specific transcription factors (TFs) and zinc finger TFs represent the two largest classes of TFs in C. militaris and their numbers are similar to those of other fungi (Table S1 in Additional file 1).

Mating-type and sexuality analysis

The fruiting bodies of Cordyceps spp. are the most commonly sold traditional Chinese medicine products [5]. However, the sexual cycle and fruiting of C. militaris is poorly understood. We only identified a MAT1-1 mating-type locus, including MAT1-1-1 and MAT1-1-2 genes, in the sequenced Cm01 strain, suggesting that C. militaris is heterothallic (Figure 4a). A single mating-type locus was also found in M. anisopliae (MAT1-1) and M. acridum (MAT1-2). Like aspergilli [10], the idiomorphic regions of the three insect pathogens are highly divergent (Figure 4a). The MAT1-1 locus of M. anisopliae contains a MAT1-1-3 gene but lacks the MAT1-1-2 gene present in C. militaris. Except for the mating-type locus region, most A. nidulans and N. crassa genes involved in mating, fruiting, karyogamy and meiosis are also present in insect pathogens (Table S12 in Additional file 1).

Figure 4 Comparative analysis of the C. militaris mating-type (MAT) locus. (a) Comparative analysis of the C. militaris MAT locus with those of sexually heterothallic and homothallic fungal species. Genes labeled in the same color have orthologous relationships. (b) Syntenic relationship of the MAT loci and their flanking regions between the three insect pathogens C. militaris (CCM), M. anisopliae (MAA) and M. acridum (MAC). Full size image

Strain Cm01 forms fruiting bodies on caterpillar pupae that lack perithecia and ascospores (Figure 5a-e). Thus, it is the first ascomycete species reported to fruit without an opposite mating-type partner. Other C. militaris isolates could also fruit sterilely with a single mating-type locus (Figure 6a, b). However, a hybrid strain, Cm06, with both MAT1-1 and MAT1-2 loci produced sexual perithecia and ascospores (Figure 5f, g). In addition, the sexual structures could be similarly re-formed after inoculation of the caterpillar pupae with different ratios of MAT1-1 and MAT1-2 isolate conidia (Figure 6c-e), confirming that C. militaris is heterothallic. PCR examination of 18 field-collected strains identified three containing both MAT1-1 and MAT1-2 loci (Figure 5h). However, 28 out of 30 single spore isolates of the Cm06 strain belonged to the MAT1-1 mating-type (Figure 5i). A similar unequal prevalence of mating types occurs in the dermatophyte fungus [31].

Figure 5 Fruiting body development, sexuality and mating-type analysis. (a-c) Chinese Tussah silkmoth pupae were inoculated with conidia from the C. militaris Cm01 strain and incubated for 14 days (a), 29 days (b) and 59 days (c) to produce nascent, mid-term and developmentally mature fruiting bodies. (d-g) The mature fruiting bodies of the Cm01 strain do not produce perithecia (d, e) but those of strain Cm06 are completely covered with protruded perithecia (f, g). (h) PCR examination of different strains (numbers labeled on the top) showed that strains Cm06, Pm36 and 80399 contain the MAT1-1-1, MAT1-1-2 and MAT1-2-1 genes while Cm01 and other strains lack the MAT1-2-1 gene. (i) PCR examination of 30 randomly selected single spore isolates from the hybrid strain Cm06 showed that only 2 out of 30 isolates contain the MAT1-2-1 gene. Full size image

Figure 6 Fruiting structures of different mating-type isolates. (a, b) Sterile fruiting bodies formed on caterpillar pupae after inoculation of MAT1-1 (a) and MAT1-2 (b) isolates acquired by single conidial spore isolation from a MAT1-1/MAT1-2 hybrid strain, Cm06. (c-e) Fertile fruiting structures formed on caterpillar pupae after inoculation of the mixed conidia of MAT1-1 (Cm01) and MAT1-2 (Cm06) at ratios of 1:9 (c), 1:1 (d) and 9:1 (e), respectively. The right panels represent close-up views of corresponding sterile (without protruded perithecia) or fertile (with protruded perithecia) fruiting bodies. After inoculation, the pupae were incubated at 22°C with a 12:12 hour light:dark cycle for 60 days. Full size image

Metabolism of medically active components and mycotoxins

One of the main pharmaceutically active components of C. militaris is cordycepin [5, 6], which is structurally similar to 2'-deoxyadenosine (Figure 7a). C. militaris possesses most of the genes required for metabolism of adenine and adenosine except for lacking a ribonucleotide trisphosphate reductase (RNR; converts ATP to dATP) and a deoxyadenosine kinase (converts deoxyadenosine to dAMP) (Figure 7b; Table S13 in Additional file 1). It has been suggested that the biosynthesis of cordycepin proceeds through a reductive mechanism as described for the formation of 2'-deoxyadenosine [32]. However, C. militaris resembles Metarhizium and other cordycepin non-producing fungi in having only two highly conserved subunits of class I RNRs (Figure S3 in Additional file 2). The substrates for class I RNRs are ADP, GDP, CDP and UDP but not TDP or nucleosides, and as the reductive reaction proceeds via a free radical mechanism [33], C. militaris RNRs will not be involved in cordycepin production.

Figure 7 Cordycepin analogues and the C. militaris adenine metabolic pathway. (a) The structures of cordycepin analogues. (b) The C. militaris adenine metabolic pathway. Abbreviations for different enzymes: ADA, adenosine deaminase; ADE, adenine deaminase; ADEK, adenylate kinase; ADK, adenosine kinase; ADN, adenosine nucleosidase; AMPD, AMP deaminase; APRT, adenine phosphoribosytransferase; DADK, deoxyadenylate kinase; DAK, deoxyadenosine kinase; NDK, nucleoside-diphosphate kinase; NT5E, 5'-nucleotidase; PK, pyruvate kinase; PNP, purine nucleoside phosphorylase; 3'-RNR, ribonucleotide triphosphate reductase. The red dashed lines show metabolic pathways present in other organisms but absent in C. militaris. Full size image

Contamination of food and feed by mycotoxins is a longstanding threat to the health of humans and animals [26]. C. militaris has been consumed for hundreds of years, implying safety, but the genome data allowed us to make the first comprehensive inventory of Cordyceps genes involved in biosynthesis of secondary metabolites for comparison with known mycotoxins. There are fewer secondary metabolite core genes in C. militaris relative to Metarhizium spp. or plant pathogens (Table 3). In comparison to Metarhizium spp., Cordyceps has fewer terpenoid synthases, polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs). Phylogenetic analysis of Cordyceps PKS and PKS-like genes using the ketoacyl CoA synthase (KS) domain sequences found that the C. militaris proteins grouped into different clusters compared to PKSs for known mycotoxins (Figure 8a). In addition, modular analysis indicated that, except for CCM_00603, which has a similar domain organization to the Aspergillus clavatus PatK gene for patulin biosynthesis, C. militaris PKSs are structurally different from mycotoxin PKSs (Figure 8b). The further survey showed that the CCM_00603 protein has only 27% identity with PatK and the gene cluster for patulin biosynthesis is absent in C. militaris (Table S14 in Additional file 1). This suggests that C. militaris PKSs do not produce patulin or other known human mycotoxins. Similarly, phylogenetic and modular analyses indicated that Cordyceps NRPSs had different protein structures than any NRPSs involved in production of known mycotoxins like enniatin, HC-toxin and gliotoxin (Figure S4 in Additional file 2).

Table 3 Numbers of core genes involved in the biosynthesis of secondary metabolites in different fungi Full size table

Figure 8 Phylogenetic and modular analysis of C. militaris polyketide synthases compared with those involved in the production of human mycotoxins. (a) A neighbor-joining tree showing the relationships of ketoacyl CoA synthase (KS) domain sequences. (b) Modulation and comparison of C. militaris PKSs with those involved in production of mycotoxins. The PKS-NRPS hybrid proteins CCM_04722, CCM_08261 and CCM_08018 are not included in the analysis. Domain definitions: ACP, acyl carrier protein domain; AT, acyltransferase domain; CYC, cyclase domain; DH, dehydratase domain; ER, enoyl reductase domain; KR, ketoreductase domain; MT, methyltransferase domain; TE, thioesterase domain. The accessions and references for different mycotoxins are provided in the Materials and methods. Full size image

The mycotoxin ergot alkaloids have a wide range of biological activities and are important in pharmaceuticals and agriculture [26]. Dimethylallyl tryptophan synthase (DMAT) catalyzes the alkylation of L-tryptophan, the first committed step in the ergot alkaloid biosynthetic pathway [34]. C. militaris has one putative DMAT gene (CCM_04410), in contrast to five in M. anisopliae and three in M. acridum (Table 3). A phylogenetic analysis showed that CCM_04410 is not clustered with the Claviceps DMAT clade involved in ergot alkaloid production (Figure S5 in Additional file 2). The trichothecenes T-2 toxin and deoxynivalenol (type B trichothecene) are natural fungal products that are toxic to both animals and plants [35]. Consistent with lacking CYP58 and CYP65, the C. militaris genome also lacks trichodiene synthase (Table S15 in Additional file 1). Thus, unlike Fusarium [26], C. militaris is not predicted to produce trichothecene mycotoxins. The presence of terpenoid cyclase, terpenoid synthase, fatty-acid synthase and geranylgeranyl diphosphate synthase genes in the C. militaris genome suggests that the fungus is capable of producing an array of metabolites, but the identity of these and their biological activities remain to be determined.

Transcriptional regulation of fruiting body development

To identify genes associated with C. militaris fruiting body development, we compared the expression profiles of undifferentiated mycelia from Sabouraud dextrose broth (SDB) culture with developmental stages on caterpillar pupae defined as nascent (14 days, termed as sample FB1), stalk formation (29 days, FB2) and mature fruiting bodies (59 days, FB3) (Figure 5a-c). Of the 9,684 genes, more than 63% were expressed during both undifferentiated hyphal growth and formation of fruiting bodies (Table S16 in Additional file 1). Relative to the growth in SDB, more than 900 genes were significantly (P < 0.05; FDR < 0.001) up-regulated while around 2,000 genes were down-regulated during fungal fruiting (Figure 9a). A Pearson correlation analysis indicated that transcriptional profiles at the different stages of fruiting body formation more closely resembled each other than they resembled the transcriptomes of undifferentiated mycelia (Figure S6a in Additional file 2). This is consistent with a Venn diagram analysis of the commonest co-expressed genes between different samples (Figure S6b in Additional file 2). Of the 100 most highly expressed genes in developing C. militaris fruiting bodies, 26 (FB1), 31 (FB2) and 37 (FB3) are functionally uncharacterized (Table S17 in Additional file 1). This suggests that the genes with unknown function are more likely to be stringently regulated and involved in developmental processes than orthologs of genes with known function. These genes are thereby the targets for future functional studies. In general, the genes involved in cell wall structure and biogenesis, detoxification, protein degradation and amino acid transportation were significantly up-regulated during formation of fruiting structures. In contrast, most of the genes specifically up-regulated by undifferentiated SDB cultures were involved in rapid growth and carbohydrate metabolism. Concomitant with fruiting structure maturation, the genes for cytoskeletal organization, cell cycle and secondary metabolism were up-regulated.

Figure 9 Differential gene expression by C. militaris in association with fruiting structure formation or growth in a liquid medium. (a) Estimation of significantly up- and down-regulated genes between different samples. (b) Heat map of protein kinases associated with the mitogen-activated and cAMP-dependent protein kinase pathways at different developmental stages. (c) Heat map of the highly expressed transcription factors at different developmental stages. Genes with expression values > 100 transcripts per million tags (TPM) are also indicated in red. Annotation information for the genes is provided in Table S19 in Additional file 1. DEG, differentially expressed gene. FB1, FB2 and FB3 are associated with nascent, stalk formation and mature developmental stages shown in Figure 5a-c, respectively. The transcriptome of undifferentiated mycelia harvested from SDB was included as a reference for gene expression analysis. Full size image

Unlike other fungi, C. militaris can fruit sterilely in the absence of a sexual partner (Figure 5a-c). Perhaps because of this, 31 of the 42 C. militaris orthologs of sex-related genes identified in other ascomycetes were not expressed or transcribed at low levels (< 10 transcripts per million tags (TPM)) in sterile fruiting bodies (Table S18 in Additional file 1). However, in some cases, C. militaris expresses paralogous genes to those employed by other fungi, suggesting they have co-opted different components of the same signal transduction pathways to fulfill similar functions. For example, GATA-type TFs are important for fruiting in both A. nidulans and N. crassa [36], but C. militaris fruiting structures expressed orthologs of these genes at very low levels or not at all (Table S19 in Additional file 1). In contrast, the Zn2Cys6-type TFs were highly transcribed during fruiting but not in undifferentiated fungal mycelia - for example, CCM_01809 and CCM_09644 (Figure 9b) - indicating that Zn2Cys6 type TFs are predominately involved in the major developmental switch of production of fruiting structures.

Pheromone receptors, that is, GPCRs, control fungal fruiting body formation and sexual cycle but not vegetative growth [36]. The pheromone receptor of C. militaris has not been identified. In comparison to undifferentiated mycelial growth, a putative pheromone receptor (CCM_01499) and a Pth11-like GPCR (CCM_03015) were significantly up-regulated (P < 0.05, FDR < 0.001), respectively, during initiation of fruiting body formation. Mitogen-activated protein kinase (MAPK) genes are required for fruiting in Aspergillus (AN1017) and Neurospora (NC02393) [36], but orthologous genes were not transcribed (CCM_04200 versus AN1017) or transcribed at low levels (CCM_01235 versus NCU02393) by C. militaris (Table S19 in Additional file 1). However, Cordyceps sharply up-regulated (P < 0.05, FDR < 0.001) a MAPK paralog (CCM_09637) as well as a calcium regulated kinase (CaMK, CCM_06085) (Figure 9c). These data, taken in conjunction with the single adenylate cyclase (CCM_06928) not being transcribed and the low level expression of both protein kinase A (PKA; CCM_03352) and Rap GTPase (CCM_01391), indicate that fruiting by C. militaris in the absence of a partner is more dependent on the MAPK pathway than the cAMP-dependent PKA pathway (Figure 10).