Survey of Early-Diverging Lineages of Fungi Reveals Abundant and Diverse Mycoviruses

Survey of Early-Diverging Lineages of Fungi Reveals Abundant and Diverse Mycoviruses J. M. Myers, a A. E. Bonds, a R. A. Clemons, a N. A. Thapa, a D. R. Simmons, a D. Carter-House, b J. Ortanez, b P. Liu, b A. Miralles-Durán, c A. Desirò, d J. E. Longcore, e G. Bonito, d J. E. Stajich, b J. W. Spatafora, f Y. Chang, f L. M. Corrochano, c A. Gryganskyi, g I. V. Grigoriev, h,i T. Y. James a a University of Michigan, Department of Ecology and Evolutionary Biology, Ann Arbor, Michigan, USA b University of California—Riverside, Department of Microbiology and Plant Pathology, Riverside, California, USA c University of Seville, Department of Genetics, Seville, Spain d Michigan State University, Department of Plant Soil and Microbial Sciences, East Lansing, Michigan, USA e University of Maine, School of Biology and Ecology, Orono, Maine, USA f Oregon State University, Department of Botany and Plant Pathology, Corvalis, Oregon, USA g L. F. Lambert Spawn Co., Coatesville, Pennsylvania, USA h U.S. Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, California, USA i University of California—Berkeley, Department of Plant and Microbial Biology, Berkeley, California, USA ABSTRACT Mycoviruses are widespread and purportedly common throughout the fungal kingdom, although most are known from hosts in the two most recently diverged phyla, Ascomycota and Basidiomycota, together called Dikarya. To augment our knowledge of mycovirus prevalence and diversity in underexplored fungi, we conducted a large-scale survey of fungi in the earlier-diverging lineages, using both culture-based and transcriptome-mining approaches to search for RNA viruses. In total, 21.6% of 333 isolates were positive for RNA mycoviruses. This is a greater proportion than expected based on previous taxonomically broad mycovirus surveys and is suggestive of a strong phylogenetic component to mycoviral infection. Our newly found viral sequences are diverse, composed of double-stranded RNA, positive-sense single-stranded RNA (ssRNA), and negative-sense ssRNA genomes and include novel lineages lacking representation in the public databases. These identified viruses could be classified into 2 orders, 5 families, and 5 genera; however, half of the viruses remain taxonomically unassigned. Further, we identified a lineage of virus-like sequences in the genomes of members of Phycomycetaceae and Mortierellales that appear to be novel genes derived from integration of a viral RNA- dependent RNA polymerase gene. The two screening methods largely agreed in their detection of viruses; thus, we suggest that the culture-based assay is a costeffective means to quickly assess whether a laboratory culture is virally infected. This study used culture collections and publicly available transcriptomes to demonstrate that mycoviruses are abundant in laboratory cultures of early-diverging fungal lineages. The function and diversity of mycoviruses found here will help guide future studies into mycovirus origins and ecological functions. IMPORTANCE Viruses are key drivers of evolution and ecosystem function and are increasingly recognized as symbionts of fungi. Fungi in early-diverging lineages are widespread, ecologically important, and comprise the majority of the phylogenetic diversity of the kingdom. Viruses infecting early-diverging lineages of fungi have been almost entirely unstudied. In this study, we screened fungi for viruses by two alternative approaches: a classic culture-based method and by transcriptome-mining. The results of our large-scale survey demonstrate that early-diverging lineages have higher infection rates than have been previously reported in other fungal taxa and that laboratory strains worldwide are host to infections, the implications of which Citation Myers JM, Bonds AE, Clemons RA, Thapa NA, Simmons DR, Carter-House D, Ortanez J, Liu P, Miralles-Durán A, Desirò A, Longcore JE, Bonito G, Stajich JE, Spatafora JW, Chang Y, Corrochano LM, Gryganskyi A, Grigoriev IV, James TY. 2020. Survey of earlydiverging lineages of fungi reveals abundant and diverse mycoviruses. mBio 11:e02027-20. https://doi.org/10.1128/mBio.02027-20. Editor John W. Taylor, University of California, Berkeley Copyright © 2020 Myers et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to J. M. Myers, [email protected]. [This article was published on 8 September 2020. There were errors in the affiliations. This was corrected in the version posted on 3 December 2020.] Received 26 July 2020 Accepted 5 August 2020 Published RESEARCH ARTICLE Host-Microbe Biology crossm September/October 2020 Volume 11 Issue 5 e02027-20 ®mbio.asm.org 1 8 September 2020 Downloaded from https://journals.asm.org/journal/mbio on 20 July 2022 by 150.214.182.32. are unknown. The function and diversity of mycoviruses found in these basal fungal lineages will help guide future studies into mycovirus origins and their evolutionary ramifications and ecological impacts. KEYWORDS mycovirus, dsRNA virus, Chytridiomycota, Blastocladiomycota, Neocallimastigomycota, Zoopagomycota, Mucoromycota, double-stranded RNA virus, mycoviruses Fungal viruses (mycoviruses) have been reported from all major fungal taxonomic groups (1, 2). However, the overwhelming majority of mycoviruses have been identified in hosts belonging to just two phyla—Ascomycota and Basidiomycota (known together as “Dikarya”)—though the kingdom Fungi is comprised of at least eight phyla (3–5)(Fig. 1). Known mycovirus infections in non-Dikarya are limited to an early report of virus-like particles from ultrastructure studies of Allomyces arbusculus (6) and sequence-based identification in Rhizopus oryzae (7), Rhizopus microsporus (8), six isolates of arbuscular mycorrhizal fungi (9, 10), Umbelopsis ramanniana (11), and Entomophthora muscae (12, 13). While it has become clear that mycoviruses are widespread, disproportionate sampling across the fungal kingdom has resulted in an incomplete understanding of mycovirus diversity and prevalence. By sampling unexplored and phylogenetically diverse fungal lineages, we predicted to find equally diverse viruses that could empower meaningful inquiries into the origins and ecological functions of mycoviruses. The conventional approach to mycovirus discovery exploits the observation that double-stranded RNA (dsRNA) genomes predominate among known mycoviruses (1). The most common method uses cellulose chromatography to purify dsRNA from total RNA extracts and effectively isolate mycoviruses with dsRNA genomes and the dsRNA replicative intermediates of single-stranded RNA (ssRNA) genomes (14). This approach is quick and inexpensive; however, its exclusive use ignores DNA viruses and reinforces the bias that mycoviral genomes are predominantly composed of RNA. Furthermore, viral RNA is detected visually by gel electrophoresis (see Fig. 2), which could result in false negatives in instances of low-titer infections. To our knowledge, estimates of the proportion of false negatives based on chromatography have not been reported. We tested the relative accuracy of cellulose chromatography for identifying fungal isolates with viral infection and compared the results to more indirect sequence-based alternative approaches. In silico approaches to mycovirus discovery have become more common (15–17). These approaches search transcriptomic data for sequences similar to RNA-dependent RNA polymerase (RdRp), which is the only conserved gene among RNA viruses and is diagnostic of infection. Conceptually, these approaches should be more sensitive to low titer viral infections because they do not rely on gel visualization and high transcriptome sequencing depth is readily achieved. Indeed, RNA sequencing has revealed cryptic coinfection in isolates previously thought to be singularly infected (18) (J. Myers, unpublished data). As sequencing costs have dropped, the availability of transcriptome (RNA) sequencing data has grown, which presents a novel opportunity to probe existing data sets to characterize viruses for the first time. Gilbert et al. (17) developed a data-mining pipeline for mycovirus identification, applied it to public transcriptomes of fungi in the subphylum Pezizomycotina, and discovered 52 novel mycoviruses, demonstrating the utility of this approach. However, such approaches have yet to be applied widely to non-Dikarya lineages. Our primary goal was to improve understanding of prevalence and sequence diversity of RNA mycoviruses in the early-diverging lineages of Fungi, specifically Mucoromycota, Zoopagomycota, Chytridiomycota, Blastocladiomycota, Neocallimastigomycota, and Cryptomycota/Rozellomycota. We screened early-diverging lineages with both a culture-based chromatography approach (“in vitro”) and a transcriptome data-mining approach (“in silico”). When possible, we compared methods by screening the same isolates used for transcriptome-generation with the in vitro method. In total, Myers et al. ® September/October 2020 Volume 11 Issue 5 e02027-20 mbio.asm.org 2 Downloaded from https://journals.asm.org/journal/mbio on 20 July 2022 by 150.214.182.32. we screened 333 hosts, of which 72 (21.6%) harbored viruses. These results demonstrate that mycoviruses are abundant in the early-diverging lineages of the fungal kingdom, including in research laboratory cultures around the world. RESULTS AND DISCUSSION Viral prevalence. Through deep-sequencing approaches, recent studies have revealed the abundance of viruses in pathogenic fungal species, marine environments, FIG 1 Bar plot showing the number of unique hosts of exogenous mycoviruses, per phylum, as represented in GenBank before this study (A) and with the data from this study added (B). FIG 2 Agarose-gel electrophoresis image of the purified dsRNAs of six isolates of Cladochytrium sp. with varied banding patterns, flanked by 1-kb ladders. The first sample (lane 2) shows dsRNA of Cladochytrium sp. JEL861, which was sequenced in this study. Mycoviruses in Early-Diverging Fungi ® September/October 2020 Volume 11 Issue 5 e02027-20 mbio.asm.org 3 Downloaded from https://journals.asm.org/journal/mbio on 20 July 2022 by 150.214.182.32. and soils (15, 19, 20). We build on this momentum by exploring viruses hosted in species from the deep branches of kingdom Fungi. In doing so, we more completely characterized the abundance of viruses across the kingdom and uncovered novel viral sequence diversity linked to specific fungal hosts. We determined the first sequences of mycoviruses in the particularly under-explored phyla Blastocladiomycota, Neocallimastigomycota, and Chytridiomycota (Fig. 1). In total, we screened 333 fungi spanning six phyla by either or both in vitro (cellulose chromatography) and in silico (transcriptomemining) methods (see Table S1 in the supplemental material) and found one or more viruses in 72 isolates (21.6%), 65 of them not previously known as mycoviral hosts (Fig. 1). Of the 36 hosts for which we obtained viral sequence data—either by assembling from host transcriptomes or direct sequencing—we generated 154 complete or partial viral sequences which, using the criteria described above, we conservatively reduced to 85 unique viruses. All sampled phyla were hosts to viruses except the poorly sampled Cryptomycota. Infection prevalence at the phylum level ranged from 15.9 to 40.0% and 0 to 89% at the subphylum and order levels but was highest in Glomeromycotina, Entomophthoromycotina, and Cladochytriales (Table 1 and Fig. 3). Glomeromycotina are also notorious hosts of endosymbiotic bacteria which may have originated by the invasion of freeliving bacteria following hyphal damage by herbivores or other fungi (21); it is possible that mycoviruses similarly originated in these fungi by invasion through damaged cell walls. In the zoosporic fungi, we predicted that polycentric organisms would be disproportionately virally infected since this mode should support viral transmission more frequently than monocentric growth. In monocentric development a zoospore encysts on a substrate and develops into a single zoosporangium—the structure which will ultimately cleave into many zoospores. Polycentric development, on the other hand, involves a single zoospore producing multiple zoosporangia with cytoplasmic continuity via aseptate rhizomycelium. Thus, a single viral infection in a polycentric organism could lead to many spores being infected, while monocentric development may allow for clearing of viral infection through selection between zoospores. Within TABLE 1 Percent viral prevalence based on combined in vitro and in silico screening, by phyla and subphyla, or order Taxonomic group Sample size (n) Virus prevalence (%) Mucoromycota 107 27.1 Glomeromycotina 9 88.9 Mucoromycotina 71 19.7 Mortierellomycotina 27 25.9 Zoopagomycota 25 40.0 Entomophthoromycotina 11 54.5 Kickxellomycotina 11 27.3 Zoopagomycotina 3 33.3 Blastocladiomycota 44 15.9 Blastocladiales 42 16.7 Physodermatales 2 0 Chytridiomycota 146 16.4 Monoblepharidales 5 0 Cladochytriales 21 47.6 Synchytriales 2 0 Chytridiales 40 10.0 Lobulomycetales 2 0 Rhizophydiales 35 14.3 Rhizophlyctidales 6 0 Spizellomycetales 36 13.9 Neocallimastigomycota 8 25.0 Cryptomycota 20 Total 333 21.6 Myers et al. ® September/October 2020 Volume 11 Issue 5 e02027-20 mbio.asm.org 4 Downloaded from https://journals.asm.org/journal/mbio on 20 July 2022 by 150.214.182.32. the Chytridiomycota, the percentage of polycentric organisms that were viral positive (46.2%, n⫽26) was significantly greater (P⬍0.00004, Fisher exact test) than monocentric organisms (9.2%, n⫽120). Ten of the twelve positive polycentric isolates were from the order Cladochytriales, however, and consequently we were unable to determine whether this trend is a result of the morphological trait or susceptibility traits that track the host phylogeny without additional data from non-Cladochytrialean polycentric organisms. Although the sample size is inadequate for definitive results regarding Neocallimastigomycota, viral infection did not favor polycentric over monocentric organisms in this phylum (polycentric n⫽4 and monocentric n⫽4, 25% of each was viral positive). Early in the study of mycoviruses, Bozarth (22) estimated 10 to 15% infection prevalence for fungal cultures predominantly made up of dikaryotic lineages but including some organisms in the Mucoromycota and Blastocladiomycota. A recent in silico survey of mycoviruses in Pezizomycotina (Ascomycota) revealed infection prevalence ranging up to 50% in some classes but an overall rate of about 8% (47/569) for the subphylum (17). Our estimate is approximately 22% (Table 1) across the earlydiverging phyla. Interestingly, we also found substantial variation across taxa (Table 1 and Fig. 3). Among the phyla with the highest infection rates, Mucoromycota and Zoopagomycota, viral prevalence at the subphylum level ranged from 19.7 to 88.9% (Table 1), a surprising difference from the 8% prevalence found for the same taxonomic level, Pezizomycotina, in Ascomycota. These data sets were not explicitly controlled for FIG 3 Cladogram of the organisms screened for viruses by both methods in this study. The size of the collapsed clades is proportional to the number of isolates screened. Pie charts indicate the proportions of isolates in each taxon that were viral positive (darker shade); pie charts are sized according to number of isolates screened. Whole pies had a 0% infection rate. Mycoviruses in Early-Diverging Fungi ® September/October 2020 Volume 11 Issue 5 e02027-20 mbio.asm.org 5 Downloaded from https://journals.asm.org/journal/mbio on 20 July 2022 by 150.214.182.32. geographic origin of isolates, which could influence prevalence if, for example, some geographic regions had a higher proportion of viruses relative to others. However, the isolates in our study predominantly originate from distinct populations across the globe (see Table S1 in the supplemental material). Our results indicate higher prevalence in some subphyla of basal fungal lineages compared to Pezizomycotina. From our comparison of viral prevalence at the subphylum level between Pezizomycotina (Ascomycota) versus the early-diverging lineages, it is tempting to speculate on differing viral prevalence at the phylum level. However, a taxonomically thorough and geographically controlled study of viral prevalence in the other subphyla of Ascomycota and in Basidiomycota is needed in order to make a direct comparison. In addition, anecdotal evidence suggests mycoviruses are commonly lost through culturing. Our estimates from the in vitro assays are thus perhaps underestimates of the true infection load in nature. Nonetheless, it is compelling to consider the implications of years or decades of stable maintenance of mycoviruses in culture on how the viruses affect the fungi. Future studies are needed to better characterize the effects of these mycoviruses on these phylogenetically diverse hosts. Taken together, these findings indicate a strong phylogenetic component to viral prevalence. The earlier-diverging fungal lineages surveyed in this study typically share the trait of coenocytic mycelia, which may benefit the transmission of obligate symbionts and thus facilitate mycoviral infection; if true, it is possible that septa evolved, at least in part, as a viral defense mechanism to limit the spread of viruses throughout a mycelium. Comparison of screening approaches. The results of screening the same isolate by chromatography and transcriptome-mining were mostly in agreement (i.e., at least one band of dsRNA was present by chromatography and RdRp sequence(s) were identified in silico or no dsRNA was present by chromatography, and no RdRp sequences were identified) (85.7%, n⫽21). Of the three isolates where the two methods varied in detection, two were found positive in vitro but negative in silico, and one with the inverse result. For Mortierella humilis (PMI 1414), where the in silico method revealed viral sequences but the in vitro method did not, RT-PCR confirmed viral presence in our culture. From the transcriptome data, we determined the virally derived sequences made up 2.58 transcripts per million. If we consider this a proxy of viral titer, the low viral abundance in the host likely accounts for the initial negative result obtained with the in vitro method. From Umbelopsis nana TLT204, which was positive by in-house chromatography but negative in silico, we sequenced purified dsRNA and assembled a toti-like viral contig containing a complete RdRp domain and an L-A virus major coat-protein domain, confirming a viral presence in our culture. Most likely, the virus was lost through subculture of this isolate in the laboratory in which transcriptome analysis was performed. Paired comparison of screening approaches suggests minimal discrepancies, but more viruses overall were detected by the in silico method (33.3% positive) than in vitro (17% positive). The in silico method is likely to be more sensitive than the in vitro method, but it is possible that the higher virus detection rate in silico was a result of phylogenetic skews of the data sets rather than methodology. Approximately 73% of the cultures screened in vitro were in the Chytridiomycota or Blastocladiomycota, the lesser-infected phyla. Accordingly, 64% of the isolates screened in silico were in the Mucoromycota or Zoopagomycota, the greater-infected phyla. Thus, we recommend screening by transcriptome-mining when the data are available, but the chromatography approach is a good alternative for very cost-effective initial screening. Mycoviral diversity. We found mycoviruses in all five “branches” of the Riboviria (RNA virus) tree published by Wolf et al. (23)(Fig. 4 to 8). Many of these newly found viruses will need to be described as new taxa at the levels of genus, family, and order. In addition, some viruses, including a group of related, endogenous virus-like sequences, represent novel diversity such that they were unable to be assigned to a branch, though they show sequence similarity to other unplaced mycoviruses (Fig. 9). Myers et al. ® September/October 2020 Volume 11 Issue 5 e02027-20 mbio.asm.org 6 Downloaded from https://journals.asm.org/journal/mbio on 20 July 2022 by 150.214.182.32. FIG 4 Maximum-likelihood tree of new mycovirus RdRps with top blast hits (included in tree) to viruses in “branch 1” of Riboviria. The best model of amino acid substitution for this model was determined to be LG⫹G according to Prottest v3.4. Host taxonomy (Continued on next page) Mycoviruses in Early-Diverging Fungi ® September/October 2020 Volume 11 Issue 5 e02027-20 mbio.asm.org 7 Downloaded from https://journals.asm.org/journal/mbio on 20 July 2022 by 150.214.182.32. Coinfection of a single host by multiple viruses was very common (mean and median number of viruses per host ⫽2.4 and 1.5, respectively). Even after filtering by our conservative criteria, a single host, Kickxella alabastrina (Kickxellomycotina), was found to harbor at least 11 unique viruses. Such a result would be unsurprising in macrobes, such as humans but is staggering for a microfungus. (i) Riboviria: “branch 1.” Branch 1 (Fig. 4) is composed of positive-sense ssRNA viruses, including the bacterium-infecting Leviviridae, the mitochondrion-infecting mitoviruses (Narnaviridae), and the cytoplasmic narnaviruses (Narnaviridae) and ourmiaviruses (Botourmiaviridae). In total, 22 viruses were assigned to branch 1 based on blast similarity. A complete RdRp gene from the transcriptome of Chaetocladium brefeldii was most similar to viruses in family Leviviridae. These viruses are currently only known from bacteria, so it is possible, and perhaps likely, that this contig derives from the viruses of a bacterial endosymbiont of C. brefeldii. Interestingly, the new viruses in this group are mostly hosted by fungi in the Mucoromycota or Zoopagomycota. Previous evidence suggests the origin of mitoviruses was a common ancestor of Mucoromyota and Zoopagomycota, which was followed by cospeciation of host and mitoviruses and horizontal transmission to plants (13, 24). Our findings support this hypothesis, but sequence data are still limited for viruses in Chytridiomycota, Blastocladiomycota, and Neocallimastigomycota. Additional sequencing of the viral positives in those groups found by chromatography in this study will more strongly support or refute this hypothesis. (ii) Riboviria: “branch 2.” The picornavirus supergroup makes up branch 2 (Fig. 5), which also includes Partitiviridae,Amalgaviridae,Barnaviridae, and Potyviridae. In all, nine viruses were assigned to branch 2 based on BLAST similarity. The hosts of these contigs represent the Mucoromycota, Zoopagomycota, Neocallimastigomycota, and Chytridiomycota. A virus of Entomophthora muscae nested within the picorna-like viruses, specifically in the genus Iflavirus, which is only known to infect insects. Thus, it may have originated by cross-kingdom horizontal transfer. A virus of the arbuscular mycorrhizal fungus Rhizophagus irregularis (Glomeromycotina) is nested within the plant-infecting Potyviruses and likely also arose by horizontal transmission given the tight mutualism and nutrient exchange between R. irregularis and its plant hosts. (iii) Riboviria: “branch 3.” Branch 3 (Fig. 6) includes the alphavirus and flavivirus supergroups. In total, six contigs were assigned to this branch based on BLAST similarity. We found three new tombusvirus-like viruses, hosted by Mortierella selenospora (Mucoromycota), Syncephalis fuscata (Zoopagomycota), and Anaeromyces sp. (Neocallimastigomycota). Two arbuscular mycorrhizal fungi, R. irregularis and Paraglomus brasilianum, contained viruses related to the predominantly plant-hosted viruses in Virgaviridae. Another Glomeromycotina fungus, Geosiphon pyriformis, was host to a tymo-like virus positioned basally in the order Tymovirales and may represent a new family in this order. (iv) Riboviria: “branch 4.” The largest number of new contigs were assigned to branch 4 (Fig. 7), which includes Totiviridae,Chrysoviridae,Quadriviridae, and Reoviridae. In total, our analysis assigned 41 contigs to this group. Hosts are largely represented by Chytridiomycota and Blastocladiomycota, including substantial coinfection within the same individual, but also by Mucoromycota and Zoopagomycota. Ten of these viruses group within the genus Totivirus, and three new viruses grouped within the genus Victorivirus, both genera belonging to the family Totiviridae. By our analysis, these two genera are composed of fungal viruses truly spanning the Kingdom, including hosts in Chytridiomycota, Blastocladiomycota, Zoopagomycota, Mucoromycota, Ascomycota, and Basidiomycota. This finding may align with the cospeciation hypothesis for this viral family: Göker et al. presented evidence that FIG 4 Legend (Continued) is indicated by branch symbols, and viral taxonomic groupings are indicated by a shaded background. Solid blue circles indicate well-supported nodes with ⱖ70% bootstrap support. New sequences are indicated by purple text. Myers et al. ® September/October 2020 Volume 11 Issue 5 e02027-20 mbio.asm.org 8 Downloaded from https://journals.asm.org/journal/mbio on 20 July 2022 by 150.214.182.32. Totiviridae speciate through codivergence with their hosts (25). The predominance of totiviridae-like sequences in early-diverging lineages supports this hypothesis and suggests that an early fungal ancestor harbored ancestors of Totiviridae. However, this origin hypothesis does not preclude occasional horizontal transfer, many instances of which are suggested by our phylogenetic tree. Further, this viral family also include two genera, Leishmaniavirus and Giardiavirus, that infect protozoa; since protozoans and Fungi are polyphyletic, ancient horizontal transfer between host groups is likely. Basal to Victorivirus, a clade formed including viruses from K. alabastrina,Operculomyces laminatus, the ampicomplexan Eimeria tenella, and multiple-diatom colonies. This finding aligned with our prediction of close-relatedness between formerly reported diatom-associated viruses and the newly reported sequences from Chytridiomycota, a group that contains diatom parasites, but lacked bootstrap support. Viruses from multiple isolates of Allomyces sp. form a clade sister to Botybirnavirus, likely a new FIG 5 Maximum-likelihood tree of new mycovirus RdRps with top blast hits (included in tree) to viruses in “branch 2” of Riboviria. The best model of amino acid substitution for this model was determined to be VT⫹I⫹G according to Prottest v3.4. Host taxonomy is indicated by branch symbols, and viral taxonomic groupings are indicated by a shaded background. Solid blue circles indicate well-supported nodes with ⱖ70% bootstrap support. New sequences are indicated by purple tip labels. Mycoviruses in Early-Diverging Fungi ® September/October 2020 Volume 11 Issue 5 e02027-20 mbio.asm.org 9 Downloaded from https://journals.asm.org/journal/mbio on 20 July 2022 by 150.214.182.32. L.M.C., A.M.-D., A.D., A.G., I.V.G., and T.Y.J. All authors provided helpful comments on the manuscript. 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