{"paper_id":"27cd8539-a587-477e-9e24-1a408228f5cd","body_text":"1 \nTitle: Comparative functional and evolutionary analysis of essential germline stem cell 1 \ngenes across the genus Drosophila and two outgroup species 2 \n 3 \nLuke R. Arnce *1, Jaclyn E. Bubnell1, and Charles F. Aquadro1 4 \n 5 \n*First author and Correspondence: 6 \nLuke R Arnce, la424@cornell.edu 7 \n 8 \nDepartment of Molecular Biology and Genetics 9 \n233 Biotechnology Building 10 \n526 Campus Rd. Ithaca, NY 14853 11 \n 12 \n1Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY , USA 13 \n 14 \n 15 \n 16 \n 17 \n 18 \n 19 \n 20 \n 21 \n 22 \n 23 \n 24 \n 25 \n 26 \nAbstract 27 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n 2 \nIn Drosophila melanogaster, bag of marbles (bam) encodes a protein essential for 28 \ngermline stem cell daughter (GSC) differentiation in early gametogenesis. Despite its 29 \nessential role in D. melanogaster, direct functional evaluation of bam in other closely 30 \nrelated Drosophila species reveal this essential function is not necessarily conserved. In 31 \nD. teissieri, for example, bam is not essential for GSC daughter differentiation. Here, we 32 \ngenerated bam null alleles using CRISPR-Cas9 in a species more distantly related to D. 33 \nmelanogaster, D. americana, to interrogate whether bam’s essential GSC differentiation 34 \nfunction is novel to the melanogaster species group or a function more basal to the 35 \nDrosophila genus. To further characterize the extent of the functional flexibility of other 36 \nGSC regulating genes, we generated a gene ortholog dataset for 366 GSC regulating 37 \ngenes essential in D. melanogaster across 15 additional Drosophila and two outgroup 38 \nspecies. We find that bam’s essential GSC function is conserved between D. 39 \nmelanogaster and D. americana and therefore originated prior to the formation of the 40 \nmelanogaster species group. Additionally, we find that ~8% of the 366 GSC genes 41 \nessential in D. melanogaster are absent in at least one of the 17 species in our ortholog 42 \ndataset. These results indicate that developmental systems drift (DSD), in which the 43 \nspecific genes regulating a function may change, but the final phenotype is retained, 44 \noccurs in stem cell regulation and the production of gametes across Drosophila species.  45 \n 46 \nArticle summary 47 \nResults from CRISPR induced bam null mutants in D. americana and comparative 48 \northolog analysis of essential GSC regulating genes indicate that the evolutionary origin 49 \nof bam’s essential GSC differentiation function is likely basal to the Drosophila genus, 50 \nand there is functional flexibility in at least ~8% of the 366 GSC regulating genes across 51 \nthe 17 included species.   52 \n 53 \nKeywords: bam, germline stem cells, comparative functional analysis, CRISPR 54 \n 55 \n 56 \n 57 \n 58 \nIntroduction 59 \nProper production of gametes is critical for reproduction, and in Drosophila it begins with 60 \nthe asymmetric division of germline stem cells (GSCs) to both self-renew, maintaining 61 \nthe germline, and the ultimate differentiation into sperm and eggs (Kahney et al. 2019). 62 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n 3 \nMis-regulation of this highly sensitive process can quickly lead to sterility, so 63 \ncoordination of these early cellular divisions might be presumed to be highly conserved 64 \n(Gleason et al. 2018). However, recent results have demonstrated that several GSC 65 \nregulating genes essential for reproduction in D. melanogaster show signs of positive 66 \nselection through rapid amino acid diversification, are nonessential for fertility, and/or 67 \nare completely absent in non-melanogaster Drosophila species (Civetta et al. 2006; 68 \nBauer DuMont et al. 2007; Bubnell et al. 2022), DuMont et al. 2021, Choi et al. 2015, 69 \nFlores et al. 2015.  70 \nOne of these GSC regulating genes is bag-of-marbles (bam). The encoded protein Bam 71 \nis 442 amino acids with several known functions that are executed in complexes with 72 \nother protein partners in D. melanogaster. The most well characterized of Bam’s 73 \nfunctions is as the switch for GSC daughter differentiation, but Bam also has 74 \ndocumented roles in the   maintenance of gut integrity, and as a switch for preventing 75 \npremature differentiation of hematopoietic progenitor cells (McKearin and Spradling 76 \n1990; Insco et al. 2009; Tokusumi et al. 2011).  77 \nBam specifically acts as the switch gene for GSC differentiation for D. melanogaster 78 \nfemales and is necessary for terminal differentiation of spermatogonia in males 79 \n(McKearin and Spradling 1990). In females, bam is repressed in GSCs and bam 80 \nexpression causes differentiation by binding to several protein partners including benign 81 \ngonial cell neoplasm (Bgcn) in order to repress the production of self-renewal factors 82 \nNanos and elF4a. The resultant differentiating cystoblast undergoes several mitotic 83 \ndivisions (Shen et al. 2009, Ohlstein et al. 2000, Li et al. 2013, Li et al. 2009). 84 \nSimultaneously, Bam concentrates at the fusome, which connects the cysts, and Bam 85 \nand Bgcn function together to regulate the timing of mitotic divisions between cells. In 86 \nmales, Bam is expressed in GSCs, and as differentiation continues, expression 87 \nincreases (McKearin and Spradling 1990, Sgromo et al. 2018, Pan et al. 2014, Ji et al. 88 \n2017). Once bam expression reaches a threshold in the early spermatogonia, Bam 89 \nbinds to Bgcn and tumorous testis (tut) (Ting et al. 2013, Insco et al. 2009, Insco et al. 90 \n2012). Binding represses mei-P26 and ends proliferation, triggering terminal 91 \ndifferentiation and beginning meiosis (Shen et al. 2009, Ohlstein et al. 2000, Li et al. 92 \n2013, Li et al. 2009). Loss of bam function prevents differentiation in both sexes and 93 \ncauses over-proliferation of GSCs in females and spermatogonia in males, leading to 94 \ntumors and sterility in D. melanogaster (Shivdasani et al. 2003, Ohlstein 1997). 95 \nThough bam plays an essential role in GSC regulation in D. melanogaster, recent 96 \nresults show bam’s sequence and function vary considerably across Drosophila or 97 \noutgroup species (Bubnell et al. 2022). The total 442 amino acid Bam protein in D. 98 \nmelanogaster differs by 60 fixed amino acid differences (~14%) with its sibling species 99 \nD. simulans. Bam sequences differing from D. melanogaster by up to 308 (67%) of 100 \namino acids in other Drosophila species and up to 87% in outgroup species (Arnce, 101 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n 4 \nBubnell, Aquadro 2025). Statistical tests of selection for D. melanogaster and D. 102 \nsimulans bam suggest that 94% and 72% of fixed amino acid differences respectively 103 \nwere driven by natural selection between the two current species and their common 104 \nancestor (Bubnell et al. 2022). Additional signals of positive selection at bam were 105 \ndetected in other Drosophila lineages across the genus leading to D. yakuba, D. 106 \nananassae, and D. rubida for example while other lineages, despite evaluation, showed 107 \nno evidence of positive selection for protein diversification (Bubnell et al. 2022). This 108 \ndata suggests a striking level of sequence divergence, much of which potentially driven 109 \nby natural selection, in orthologs of a gene critical for ensuring fertility in a D. 110 \nmelanogaster.  111 \nAdditionally, recent functional studies of bam using complete loss-of-function (null) 112 \nalleles in Drosophila species (Bubnell et al. 2022) revealed divergent roles across D. 113 \nsimulans, D. melanogaster, D. teissieri, D. yakuba, and D. ananassae. In D. teissieri 114 \nbam null mutants showed no germline stem cell (GSC) differentiation defects in either 115 \nsex, suggesting bam lacks its canonical role as a critical differentiation regulator in this 116 \nspecies. While in D. ananassae bam null females were sterile, males exhibited normal 117 \nspermatogenesis. These results demonstrate evolutionary divergence in bam sequence 118 \nand function, despite its conserved essential role in GSC regulation in other Drosophila 119 \nlineages. 120 \nAnalyses of DNA polymorphism and divergence among other GSC regulatory genes 121 \nwithin the Drosophila melanogaster species group have revealed distinct signatures of 122 \nadaptive evolution across lineages (Bubnell et al. 2022). Notably, some core 123 \ncomponents of GSC regulatory networks exhibit dynamic evolutionary trajectories, 124 \nincluding lineage-specific gene loss. For example, Yb—a gene expressed in somatic 125 \ncap cells of the germarium and essential for GSC maintenance and transposable 126 \nelement suppression in D. melanogaster (Szakmary et al. 2009)—shows pronounced 127 \namino acid divergence between D. melanogaster and D. simulans (Flores et al. 2015). 128 \nWhile Yb null mutants in D. melanogaster result in female sterility (Swan et al. 2001), 129 \npreliminary evidence from our lab suggested that orthologs of Yb are absent in the D. 130 \npseudoobscura, D. persimilis, and D. miranda lineages raising the possibility of 131 \nfunctional redundancy or network rewiring. These findings underscore both the 132 \nevolutionary plasticity of GSC regulation and the potential for critical gene turnover 133 \nwithin conserved developmental pathways. 134 \nA persistent challenge in comparative functional genetics lies in the widespread 135 \nassumption of ortholog functional conservation, despite limited empirical validation. 136 \nMost studies focus on single-species models, leaving ortholog activity across taxa 137 \nlargely inferred rather than experimentally confirmed (Tekaia 2016). Systematic 138 \nassessment of the mechanistic basis and degree of functional conservation is crucial for 139 \nreconstructing the evolution of gene networks. This limited validation of ortholog 140 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n 5 \nconservation introduces significant limitations when interpreting comparative genomic 141 \ndata: non-conserved ortholog functions imply divergent evolutionary pressures across 142 \nlineages, thereby confounding hypotheses about ancestral genetic architectures or 143 \ndrivers of molecular evolution. Recent studies in bacteria and Diptera have begun to 144 \nevaluate conservation of functional genes across closely related species and have 145 \nidentified surprising variability in ortholog functional conservation (Carranza et al. 2018, 146 \nBergmiller et al. 2012, Leeuwen et al. 2020, Deng et al. 2024, Hopkins et al. 2024, 147 \nZakerzade et al. 2025). Lineage-specific ortholog loss of functional reproductive genes 148 \nhas also been observed between humans and nonhuman primates (Carlisle et al. 149 \n2024).  Altogether, these results suggest a more comprehensive comparative functional 150 \nanalysis of bam and other essential GSC regulating genes across the genus Drosophila 151 \ncould provide insight into the functional evolutionary history and extent of network 152 \nflexibility of bam and essential GSC genes more broadly. 153 \nWe executed our comparative functional analysis of D. melanogaster-essential GSC 154 \nregulating genes using a two-pronged approach: generating a bam null allele in an 155 \nadditional divergent Drosophila species and evaluating ortholog presence or absence 156 \nacross 15 diverse Drosophila and two outgroup species (sheep blowfly Lucillia cuprina 157 \nand house fly Musca domestica.  158 \nWe chose to generate a bam null mutant in D. americana as this species represents a 159 \nmajor, more divergent outgroup lineage to the D. melanogaster species group within the 160 \nDrosophila genus and has previously been successfully edited with CRISPR/Cas9 161 \n(Vankuren et al. 2018, Lamb et al. 2020). We evaluated cytology and fertility in this null 162 \nmutant using the strategy from Bubnell et al (2022) to evaluate bam’s function in GSC 163 \ndifferentiation. This analysis adds broader evolutionary scope to our knowledge of bam 164 \nfunction and provides additional insight into whether bam’s essential role in GSC 165 \ndifferentiation is likely basal to all Drosophila species and was lost in specific lineages or 166 \nwhether bam’s critical role was a gained function within the D. melanogaster species 167 \ngroup. Defining bam null phenotypes also provide a broader context for understanding 168 \nthe relationship between bam function and positive selection. 169 \nNull mutants are effective for performing comparative analyses of function for genes, 170 \nlike bam, with orthologs across species of interest. However, generating null mutants is 171 \nexpensive and time consuming in non-model species. Identifying ortholog absences in 172 \nother species for GSC regulating genes essential in D. melanogaster provides an 173 \nalternative rapid and cost-effective strategy for identifying potential functional 174 \ndifferences in the genes and interaction networks that regulate germline stem cell 175 \nmaintenance, self-renewal and differentiation. To investigate ortholog functional 176 \nconservation via ortholog presence or absence, we started with a set of 366 GSC 177 \nregulating genes determined to be essential in D. melanogaster from a functional RNAi 178 \nscreen (Yan et al. 2014) along with two of bam’s close interacting partners that are also 179 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n 6 \nessential for fertility, bgcn and Yb (Szakmary et al. 2009, Li et al. 2009). We then used 180 \northology tools in conjunction with a custom reciprocal blast best hit (RBBH) pipeline to 181 \nidentify ortholog presences/absences across 17 diverse Drosophila and two outgroup 182 \nspecies. We also used the custom pipeline to identify physical and genetic GSC 183 \ninteracting gene orthologs and validate ortholog absences using a combination of 184 \nlocalized PCR and sequencing when possible. Finally, we integrated functional 185 \ncategories as identified in Yan et al. (2014) for the 366 included GSC regulating genes. 186 \nThis generated gene ortholog dataset enables evaluation of the extent and 187 \ncharacteristics of essential GSC regulating gene network flexibility as well as informed 188 \npredictions regarding their functional evolutionary histories. 189 \nHere, we report that both female and male D. americana bam null mutants are sterile 190 \nwith GSC regulation defects, indicating bam’s essential GSC regulating function is not 191 \nnovel to the melanogaster species group, is likely basal to the genus Drosophila, and 192 \nprovides additional evidence that species in which bam is not necessary for 193 \ngametogenesis (e.g., D. teissieri and D. ananassae) represent lineage-specific 194 \nfunctional losses. Our comparative ortholog analysis of GSC regulating genes reveals 195 \nthat there is additional functional flexibility beyond bam with ~8% (30 of 366) of the 196 \ngenes absent in one or more of the 17 included species and ~3% absent in one or more 197 \nof the 15 included Drosophila species. Surprisingly, we find that Yb is from D. 198 \npseudoobscura and D. obscura species, although it is necessary for GSC function and 199 \ndevelopment, and therefore fertility, in D. melanogaster. Ortholog conservation does not 200 \nnecessarily indicate conservation of function (e.g., bam), so this represents the 201 \nminimum functional flexibility in essential GSC regulating genes. These results 202 \naltogether are consistent with other recent studies showing genes essential in one 203 \nspecies for critical functions, like fertility, do not necessarily have the same essential 204 \nfunction even among closely related species (Leeuwen et al. 2020). Bam functional 205 \nvariation and the absences of several essential GSC regulating gene across Drosophila 206 \nare potential examples of developmental systems drift (DSD) or divergence in genetic 207 \nsystems that underpin a conserved phenotype (Weiss and Fullerton 2000, True and 208 \nHaag 2001). 209 \n 210 \n 211 \nMaterials and methods 212 \nFly stocks and rearing  213 \nWe raised fly stocks on standard cornmeal-molasses food at room temperature, and we 214 \nused yeast-glucose food for fertility assays. We acquired lines with sequenced genomes 215 \nfor 15 Drosophila and two outgroup species: Drosophila simulans (strain: w501), 216 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n 7 \nDrosophila sechellia (strain: sech25), Drosophila teissieri (strain: GT53w), Drosophila 217 \nyakuba (strain: Tai18E2), Drosophila takahashii (strain: IR98-3 E-12201), Drosophila 218 \nelegans (strain: 14027-0461.03), Drosophila serrata (strain: Fors4), Drosophila 219 \nananassae (strain: 14024-0371.14) , Drosophila pseudoobscura (strain: MV2-25), 220 \nDrosophila obscura (strain: BZ-5 IFL), Drosophila willistoni (strain: 14030-0811.24), 221 \nDrosophila mojavensis (strain: 15081-1352.22), Drosophila virilis (strain: 15010-222 \n1051.87), Drosophila grimshawi (strain: 15287-2541.00), Drosophila rubida (strain: PH 223 \n161), Musca domestica (strain: aabys), Lucilia cuprina (strain: Lc7/37) (S1 Table A). We 224 \nalso acquired D. simulans, sechellia, yakuba, serrata, willistoni, mojavensis, virilis,and 225 \ngrimshawi from the National Drosophila Species Stock Center (NDSSC) 226 \n(http://blogs.cornell.edu/drosophila/), D. takahashii from Kyorin-fly Drosophila species 227 \nstock center (https://shigen.nig.ac.jp/fly/kyorin/), D. teissieri, D. pseudoobscura, the 228 \nhouse fly Musca domestica, and the sheep blowfly Lucillia cuprina as gifts from Daniel 229 \nMatute, Andy Clark, Jeffrey Scott, and Max Scott, respectively. D. elegans and D. 230 \nananassae were gifts from Artyom Kopp. D. obscura and D. rubida were gifts from 231 \nDmitri Petrov. We acquired the D. americana white eye mutant line (Lamb et al. 2020) 232 \nfor null generation as a gift from Trisha Wittkopp (S1 Table A). 233 \n 234 \nStrategy for generating a bam null phenotype in D. americana 235 \nWe generated the bam null disruption by targeting the first exon of bam and introducing 236 \nan early stop codon in the coding sequence using CRISPR/Cas9 gene editing. Because 237 \nnull homozygotes are sterile and thus cannot be maintained, we developed two bam 238 \ndisruption lines (one marked by 3x3P-Dsred and the other by 3xP3-YFP) which can be 239 \nmaintained as heterozygous lines (S1 Tables B-F). In order to phenotype the null 240 \nhomozygote, we crossed bam3xP3-Dsred/bamwt and bam3xP3-YFP/bamwt flies to create a 241 \nbam disruption null homozygotes (bam3xP3-Dsred/bamYFP) which we then identified via 242 \nfluorescent eye screen.  243 \n 244 \nBam null construct cloning 245 \nYasir Ahmed gifted us bam nucleotide sequences for D. americana (now on NCBI as 246 \nG96 accession: PRJNA475270), and we performed cloning design in Geneious (S1 247 \nTable D). We generated PCR products using the NEB Q5 High Fidelity 2x master mix, 248 \nthen gel extracted and purified using the NEB Monarch DNA gel extraction kit. For PCR, 249 \nsequencing, and cloning, we used IDT primers. We also generated donor plasmids for 250 \nboth the 3xP3-YFP and 3xP3-Dsred bam disruption lines using the strategy outlined in 251 \nBubnell et al. (2022). We prepared and purified plasmids for embryo injections with the 252 \nQiagen plasmid plus midi-prep kit followed by phenol-chloroform extraction for further 253 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n 8 \nRNase removal and then sequenced plasmids with whole plasmid sequencing 254 \n(Plasmidsaurus).  255 \n 256 \nCRISPR/Cas9 and gRNA selection 257 \nWe used Geneious to select gRNAs with no predicted off-targets in the reference 258 \ngenomes for D. americana (S3). We generated these as synthetic gRNAs (sgRNAs) 259 \nfrom Synthego and used up to two gRNAs per injection to improve the likelihood of 260 \nsuccessful CRISPR events (S1 Table F). 261 \n 262 \nEmbryo injections 263 \nGenetivision performed CRISPR/Cas9 injections including the appropriate plasmid 264 \ndonor, sgRNAs, and Cas9 protein (Synthego) into the D. americana line. We screened 265 \nbam disruption lines for eye color to identify CRISPR/Cas9 mutant flies in-house using a 266 \nNightsea fluorescence system with YFP (cyan) and DsRed (green) filters. We 267 \nbackcrossed positive flies to generate lines which were maintained as heterozygous 268 \nstocks. We confirmed CRISPR insertions by linear sequencing (Plasmidsaurus). 269 \n 270 \nFertility assays  271 \nWe executed following the strategy from Flores et al. (2015) for all female fertility 272 \nassays. We collected and aged virgin females to sexual maturity (3-4 days for D. 273 \namericana) (Markow and O’Grady 2006). We collected all generated genotypes from 274 \neach bottle to control for bottle effects. Wildtype virgin males for each species were also 275 \naged until sexual maturity and distributed from different bottles across female 276 \ngenotypes. We crossed single females with two males, allowed to mate for nine days, 277 \nthen flipped onto new vials for nine more days, and then finally cleared from the vials 278 \nwhile the offspring develop. We counted progeny daily and cleared to get total adult 279 \nprogeny per female. We also conducted male fertility assays with wildtype females and 280 \nmales of all generated genotypes. We executed fertility assays for D. americana on 281 \nyeast-glucose food and all fertility experiments were kept at room temperature 282 \n(approximately 21 degrees C). 283 \nFertility assay statistics 284 \nWe used estimation statistics to assess fertility assay mean difference (effect size) in 285 \nnumber of adult progeny between the wildtype bam genotype and the bam null 286 \nhomozygote and heterozygote genotypes.  We generated estimation statistics and 287 \nshared control Cumming plots using www.estimationstats.com (Ho 2019) 288 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n 9 \n(github.com/lukearnce/bam_null_ortholog). We used estimation statistics to enable 289 \ndetermination of the size of the impact of the bam genotype on fertility via a non-290 \nparametric methodology. We reported significance as an effect size outside the 95% 291 \nconfidence interval (github.com/lukearnce/bam_null_ortholog).  292 \n 293 \nImmunostaining 294 \nWe used the following primary antibodies: anti-Hts-1B1 (mouse, 295 \nAB_528070, Developmental Studies Hybridoma Bank, concentrate 1:40) and anti-vasa 296 \n(rat, AB_760351, DSHB, concentrate 1:20. We also used the following secondary 297 \nantibodies: Alexaflour goat anti-rat 488 and goat anti-mouse 568 (Invitrogen) at 1:500. 298 \nWe performed immunostaining as described in Bubnell et al. (2022). In short, we 299 \ndigested ovaries and testes in cold 1x PBS and pipetted up and down to improve 300 \nantibody permeability, fixed tissues in 4% paraformaldehyde, washed in PBST (1X PBS, 301 \n0.2% Triton-X 100), blocked in PBTA (1X PBS, 0.2% Triton-X 100, 3% BSA) (Alfa 302 \nAesar), and next, incubated in the appropriate primary antibody in PBTA overnight. We 303 \nwashed (PBST), blocked (PBTA), and incubated tissues in the appropriate secondary 304 \nantibody for two hours. Tissue was then washed again (PBST) and finally mounted in 305 \nmounting media with DAPI (Prolong glass antifade with NucBlue, Invitrogen) for 306 \nimaging.  307 \n 308 \nMicroscopy 309 \nWe imaged ovaries and testes on a Zeiss i880 confocal microscope with 405, 488, and 310 \n568 nm laser lines at 40X (Plan-Apochromat 1.4 NA, oil) (Cornell BRC Imaging Core 311 \nFacility). We analyzed and edited images using Fiji (ImageJ). 312 \n 313 \nMcDonald-Kreitman test of selective neutrality for the D. americana lineage. 314 \nWe tested D. americana bam for significant departures from neutrality via McDonald-315 \nKreitman test (MKT) (McDonald and Kreitman 1991) using polymorphism data gifted by 316 \nYasir Ahmed and the bam sequence from D. virilis as an outgroup. We implemented the 317 \nstrategy for MKT analysis of D. americana bam from Bubnell et al. (2022). In brief, we 318 \naligned bam sequences using PRANK (version) with the -codon and -F parameters 319 \nusing the PRANK tree guide. We used the codeml package from PAML (version 4.9) 320 \n(Yang 1997, 2007) to generate the predicted common ancestor sequences for 321 \ncalculating lineage-specific divergence for bam with the MKT. We used PRANK 322 \nalignments and trees as inputs to codeml with control file parameters (noisy=9, 323 \nverbose=2, runmode=0, seqtype=1, CodonFreq=2, clock=0, aaDist=0, model=0, 324 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n 10 \nNSsites=0, icode=0, getSE=0, RateAncestor=1, Small_diff=.5e-6, cleandata=0, 325 \nmethod=1) (github.com/lukearnce/bam_null_ortholog). We conducted an MKT 326 \ncomparing nonsynonymous and synonymous changes (Egea et al. 2008) using 327 \nthe http://mkt.uab.cat/mkt/mkt.asp webtool. We excluded polymorphic sites at less than 328 \n12% frequency, classified as slightly deleterious alleles not yet removed by purifying 329 \nselection (Charlesworth and Eyre-Walker 2008). We used the predicted common 330 \nancestral species sequence to calculate lineage-specific divergence. We recorded 331 \nvalues for the contingency table, the P-value of the χ2, alpha, and proportion of fixations 332 \npredicted to be due to positive selection (Eyre-Walker 2006) 333 \n(github.com/lukearnce/bam_null_ortholog). 334 \n 335 \nGenomes used in analysis 336 \nWe downloaded genomes from NCBI for 15 Drosophila and two outgroup species: 337 \nDrosophila simulans (assembly: Prin_Dsim_3.1), Drosophila sechellia (assembly: 338 \nASM438219v2), Drosophila teissieri (assembly: Prin_Dtei_1.1), Drosophila yakuba 339 \n(assembly: Prin_Dyak_Tai18E2_2.1), Drosophila takahashii (assembly: ASM1815269), 340 \nDrosophila elegans (assembly: ASM1815250), Drosophila serrata (assembly: Dser1.1), 341 \nDrosophila ananassae (assembly: ASM1763931v2) , Drosophila pseudoobscura 342 \n(assembly: UCI_Dpse_MV25), Drosophila obscura (assembly: ASM1815110v1), 343 \nDrosophila willistoni (assembly: UCI_dwil_1.1), Drosophila mojavensis (assembly: 344 \nASM1815372v1), Drosophila virilis (assembly: Dvir_AGI_RSII-ME), Drosophila 345 \ngrimshawi (assembly: ASM1815329v1), Drosophila rubida (assembly: ASM3504616v1), 346 \nMusca domestica (assembly: Musca_domestica.polishedcontigs.V.1.1), Lucilia cuprina 347 \n(assembly: ASM2204524v1) (S1 Table A). 348 \n 349 \nEnsembl ortholog analysis 350 \nWe used the Ensembl Compara online ortholog tool (Kersey et al. 2010 and Accessed 351 \ndate: June 2022) to collect ortholog predictions for 366 GSC regulating genes for the 10 352 \nDrosophila species that were available in Ensemble Compara (D. melanogaster, D. 353 \nsimulans, D. sechellia, D. yakuba, D. ananassae, D. pseudoobscura, D. willistoni, D. 354 \nmojavensis, D. virilis, D. grimshawi) and two outgroup species (L. cuprina and M. 355 \ndomestica) (S2 Tables A-B). This tool catalogs relevant information about each ortholog 356 \nincluding sequence alignment, target percent ID (percentage of orthologous sequence 357 \nmatching the Drosophila melanogaster sequence), query percent ID (percentage of 358 \nDrosophila melanogaster sequence matching the orthologous sequence), gene order 359 \nconservation score (evaluating synteny), and high or low ortholog confidence 360 \n(calculated using results from other categories) (Kersey et al. 2010). Predicted orthologs 361 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n 11 \nwere included as confident predictions if sequence alignment is equal to or greater than 362 \n25% identity (github.com/lukearnce/bam_null_ortholog).  363 \n 364 \nReciprocal Best Blast Hit (RBBH) ortholog pipeline 365 \nTo execute our RBBH ortholog analysis, implemented a multi-stage pipeline: 366 \n1. Initial RBBH 367 \nWe first downloaded highly contiguous, long-read full genome sequences from NCBI for 368 \nall included species (15 Drosophila species plus the two outgroup species). D. teissieri, 369 \nD. takahashii, D. elegans, D. serrata, D. obscura, D. rubida, M. domestica , and L. 370 \ncuprina were added to the initial set of species from Ensembl Compara. Then, we used 371 \ncustom scripts to perform forward and reverse BLASTp searches to identify potential 372 \northologs and filter ortholog hits for GSC regulating genes as well as GSC gene 373 \ninteraction network genes (S2, github.com/lukearnce/bam_null_ortholog).  374 \n2. RBBH + syntenic evaluation 375 \nFor genes with predicted absences, we conducted forward and reverse BLASTn 376 \nsearches as well as reciprocal BLASTp searches for the gene predicted absent and the 377 \nsyntenic genes (three on each side) flanking the GSC gene in D. melanogaster. We 378 \nexecuted this to search for genes that are actually present but may have been predicted 379 \nabsent due to location at the end of contigs (S2, S3, 380 \ngithub.com/lukearnce/bam_null_ortholog).   381 \n3. Direct validation of predicted gene ortholog absences 382 \nWe evaluated predicted GSC gene ortholog absences with retained syntenic blocks 383 \ndirectly via sequencing. We developed primers (IDT) for PCR amplification the 384 \nsequence between retained syntenic genes. We then gel extracted, purified, and 385 \nsequenced PCR products were to directly verify gene absence (S2, S3, 386 \ngithub.com/lukearnce/bam_null_ortholog). 387 \n 388 \nInteraction networks and functional information 389 \nWe cataloged physical interactors and genetic interactors from Flybase datasets for 390 \nGSC regulating genes with predicted absences and evaluated interaction network 391 \ngenes for orthologs across included species using the same RBBH pipeline (Choi JY 392 \nand Aquadro CF 2015) as well as ortholog predictions from Li et al. (2021). Annotated 393 \nmolecular functions (Choi and Aquadro 2015), functional categories identified by 394 \ncomplex-enrichment analysis of the 366 GSC genes, and defect type, defined by the 395 \nobserved phenotypic effect of RNAi knockdown, were also incorporated from Yan et. al 396 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n 12 \n(2014). Defect types include GSC loss (cell viability), GSC loss (agametic), 397 \ndifferentiation defect, and oocyte-specific phenotypes/late oogenesis (S3). 398 \n 399 \n 400 \n 401 \n 402 \n 403 \n 404 \n 405 \n 406 \n 407 \n 408 \n 409 \n 410 \n 411 \n 412 \n 413 \n 414 \nResults 415 \nBam shows a lineage-specific signature of positive selection in D. americana  416 \nWe detected a significant departure from neutrality suggesting positive selection 417 \nfavoring accelerated amino acid substitutions via the McDonald-Kreitman (1981) test for 418 \nbam in the D. americana lineage. Contingency table values (Neutral Polymorphism: 42, 419 \nNeutral Divergence: 0, Non-neutral Polymorphism: 27, and Non-neutral Divergence: 3) 420 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n 13 \nshow the ratio of nonsynonymous to synonymous variation between species is greater 421 \nthan the same ratio within species, consistent with expectations for positive selection (χ2 422 \n= 4.390 P-value = 0.036) and 100% of fixations estimated to be due to positive selection 423 \n(alpha = 1.00) (github.com/lukearnce/bam_null_ortholog). This raised the possibility that 424 \nthere may have been a functional change at D. americana bam. 425 \n 426 \nBam is necessary for female and male fertility and germ cell differentiation in D. 427 \namericana 428 \nWe used CRISPR/Cas9 gene editing to generate bam null alleles in D. americana, a 429 \nrepresentative of a lineage divergent from D. melanogaster in the genus Drosophila.  430 \nWildtype (+/+) and bam null heterozygotes, (bam3xP3-Dsred/bam+) and (bam3xP3-YFP/bam+), 431 \nare all fertile in both males and females (Fig. 1). However, bam null homozygotes, 432 \n(bam3xP3-Dsred/bam3xP3-YFP), were completely sterile in males and females (P < 0.0001, 433 \npermutation test, Fig. 1a&d) (github.com/lukearnce/bam_null_ortholog). As in D. 434 \nmelanogaster, D. simulans, D. yakuba, and D. ananassae (Bubnell, et al. 2022), one 435 \ncopy of wildtype bam is sufficient to rescue the bam null sterility phenotype in D. 436 \namericana.  437 \n 438 \n 439 \n 440 \n 441 \n 442 \n 443 \n 444 \n 445 \n 446 \n 447 \nFigure 1. 448 \nFertility and cytological analyses of bam function in adult D. americana. Fertility is 449 \npresented as adult D. americana progeny per fly for each bam genotype and 450 \npresented separately for females (a) and males (d). The raw data as progeny per 451 \nfly is plotted on the upper axes with the mean difference for the three genotype 452 \ncomparisons against the shared control wildtype illustrated in the Cumming 453 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n 14 \nestimation plots on the lower axes. Mean differences are plotted as bootstrap 454 \nresampling distributions. Each mean difference is depicted as a dot, and 95% 455 \nconfidence intervals are indicated by the vertical black bars. Immunostaining of 456 \novaries (b and c) and testes (e and f) of wildtype (b and e) and null bam 457 \ngenotypes (c and f). Composite Z-projections for ovaries and testes show 458 \nstaining for the germline (vasa), fusome (1B1), and nuclei (DAPI) with separate 459 \nsingle channels for each image illustrated in the side panels (i. vasa, ii. DAPI, iii. 460 \n1B1).  Wildtype tissue phenotypes are indicated with arrows and mutant tissue 461 \nphenotypes are indicated with arrowheads. 462 \n 463 \nTo confirm the bam null sterile fertility phenotype was due to defects in GSC function as 464 \nexpected if bam function is conserved between D. melanogaster and D. americana, we 465 \nevaluated the cytology of D. americana bam null ovaries and testes (Fig. 1). We imaged 466 \n3-5 day-old ovaries from D. americana bam wildtype and bam null females that we 467 \nimmunostained with antibodies to vasa and 1B1 and mounted with DAPI. (Lavoie et al. 468 \n1999). Homozygous bam null cytology recapitulated the classic bag of marbles 469 \nphenotype, with over-proliferation of small, undifferentiated GSC-like cells in the ovaries 470 \n(Fig. 1c) and testes (Fig. 1f) in contrast to bam wildtype ovaries (Fig 1b) and testes (Fig 471 \ne) which consist of cysts made of larger differentiating germline cells. Our cytological 472 \ndata reveal that bam is necessary for early germ cell differentiation in D. americana, 473 \nconsistent with fertility assay results (Fig. 1a & d). 474 \n 475 \nEssential GSC gene ortholog absences across species 476 \nWhile functional genetic analyses for bam across diverse lineages in the genus 477 \nDrosophila revealed some striking variation in bam’s role in fertility and germ cell 478 \ndifferentiation, the financial and time costs required to generate null mutants for other 479 \nGSC genes across diverse species are prohibitive at this point. Therefore, we next 480 \nchose an alternative, albeit less sensitive, approach to evaluate the functional 481 \nconsistency of the roles of GSC regulating genes that are essential in D. melanogaster. 482 \nFocusing on the experimentally defined set of GSC regulating genes determined to be 483 \nessential in D. melanogaster (Yan et al. 2014), we defined a functional difference in 484 \nGSC regulation pathways between species as the absence of an essential ortholog in 485 \nD. melanogaster in any other Drosophila and/or outgroup species.  486 \nOur initial Ensembl ortholog analysis predicted absences for 311 of 366 GSC regulating 487 \ngenes for nine Drosophila and two outgroup species (S2 Tables A-B, 488 \ngithub.com/lukearnce/bam_null_ortholog). Number of absences per species ranged 489 \nfrom 27 (7.38%) in D. simulans to 222 (60.66%) in the outgroup species Lucillia cuprina 490 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n 15 \n(sheep blowfly). Next, we used our Reciprocal Best Blast Hit (RBBH) ortholog 491 \nassessment that revealed predicted absences for 79 of 366 GSC regulating genes 492 \nacross 15 Drosophila and two outgroup species. Number of absences per species 493 \nranged from two (0.5%) in D. simulans to 39 (10.7%) in D. rubida. Finally, with our most 494 \nstringent approach combining RBBH and syntenic evaluation of orthologs, we predicted 495 \nabsences for 30 of 366 GSC regulating genes across 15 Drosophila and two outgroup 496 \nspecies (Fig. 3) (S3, github.com/lukearnce/bam_null_ortholog). Our most stringently 497 \nassessed number of absences per species ranged from one (0.27%) in D. simulans to 498 \n13 (3.6%) in the outgroup species Musca domestica (house fly).  499 \n 500 \n 501 \n 502 \n 503 \n 504 \n 505 \n 506 \n 507 \n 508 \n 509 \n 510 \n 511 \n 512 \n 513 \n 514 \n 515 \n 516 \n 517 \n 518 \n 519 \n 520 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n 16 \n 521 \n 522 \n 523 \n 524 \n 525 \n 526 \n 527 \n 528 \n 529 \n 530 \n 531 \n 532 \n 533 \n 534 \n 535 \nFigure 2. 536 \nPredicted absences of 366 GSC regulating genes essential in D. melanogaster 537 \n(predicted by Yan et al. 2014) by species plus bgcn and Yb using different 538 \northolog detection strategies. 539 \na.  Percentage of GSC genes predicted absent by species for three ortholog 540 \nidentification strategies. Light grey bars represent ortholog predictions using 541 \nEnsembl, dark grey represent predictions with RBBH alone, and black represent 542 \nRBBH plus syntenic evaluation. The bar below the species indicates divergence 543 \ntime from D. melanogaster (MY). 544 \nb. Presence and absence of GSC genes across 15 Drosophila and two outgroup 545 \nspecies that are predicted absent in at least one species after RBBH and 546 \nsyntenic evaluation. Blue indicates gene presence and black indicates gene 547 \nabsence. 548 \n 549 \nVerification of gene absences with retained syntenic blocks 550 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n 17 \nTo experimentally confirm the predicted GSC regulating gene ortholog absences we 551 \nfound with RBBH + syntenic evaluation with retained syntenic blocks, we PCR amplified 552 \nand sequenced the syntenic block spanning the expected gene absence. The 553 \nsequencing results verified all predicted absences with retained synteny therefore our 554 \nRBBH + syntenic computational predictions represented real absences in the amplified 555 \nregions (github.com/lukearnce/bam_null_ortholog). Using our final ortholog presence 556 \nand absence results, we then sought to identify potential patterns in gene presence or 557 \nabsence across the included species. Primarily, we evaluated whether and in what ways 558 \nessential gene conservation varied across gene functional categories and interaction 559 \nnetwork size (S3). 560 \n 561 \nVariable GSC gene conservation across species and functional categories 562 \nWe found that GSC regulating genes with predicted absences are unequally 563 \nrepresented across functional categories identified by complex enrichment. At the 564 \nextremes, there are zero predicted absences in the proteasome functional category (15 565 \ngenes) and two of six genes (33%) in the Kinetochore and spindle functional category 566 \n(Fig. 3). Absences categorized by defect types show less variation ranging from 7.14% 567 \nfor GSC loss (cell viability) (12 of 168) to 11.11% for genes with oocyte-specific 568 \nphenotypes/late oogenesis (five of 45). 569 \n 570 \n 571 \n 572 \n 573 \n 574 \n 575 \n 576 \n 577 \n 578 \n 579 \n 580 \n 581 \n 582 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n 18 \n 583 \n 584 \n 585 \n 586 \n 587 \n 588 \n 589 \nFigure 3. 590 \nPercentage of GSC genes absent by functional category. Categories are pulled 591 \nfrom the gene-interaction network generated in Yan et al. (2014). The “No 592 \nsubcategory (105)” group includes genes represented in the interaction network 593 \nmap without clear categorical associations and the “No functional category (57)” 594 \nincludes GSC genes that do not appear in the interaction network map. 595 \n 596 \nVariability in absent GSC gene interaction network size and absences 597 \nOf the 30 GSC regulating genes (from the 366 in Yan et al. (2014)) with predicted 598 \nabsences across included species, 23 genes have physical and/or genetic interactions 599 \n(Fig. 4, S3). 13 of these 23 genes also have predicted absences in their interaction 600 \nnetworks, and most of these interaction network absences are in the same species as 601 \ntheir related absent GSC regulating gene (Fig. 5, S3).  602 \n 603 \n 604 \n 605 \n 606 \n 607 \n 608 \n 609 \n 610 \n 611 \n 612 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n 19 \n 613 \n 614 \n 615 \n 616 \nFigure 4. 617 \nInteraction network size and absences in absent GSC genes. Genes with at least 618 \none absence in the included species are listed with white bars representing the 619 \nsize of their interaction networks including genetic and physical interactors. The 620 \nnumber of interaction network genes absent are represented with black bars. 621 \n 622 \n 623 \n 624 \n 625 \n 626 \n 627 \n 628 \n 629 \n 630 \n 631 \n 632 \n 633 \n 634 \n 635 \n 636 \n 637 \n 638 \n 639 \n 640 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n 20 \n 641 \n 642 \n 643 \n 644 \n 645 \n 646 \n 647 \n 648 \n 649 \n 650 \n 651 \n 652 \n 653 \n 654 \n 655 \nFigure 5.  656 \nGSC gene and associated network absences across species. GSC genes with 657 \nabsences in at least one included species are highlighted in dark grey. 658 \nAssociated interaction network (I.N.) genes are indented, italicized, and 659 \nhighlighted in light grey. Gene presence is indicated by light blue and absence is 660 \nindicated by black. First, GSC genes with no absences in their interaction 661 \nnetworks are arranged by increasing interaction network size. Next, GSC genes 662 \nwith absences in their interaction networks are arranged in the same manner. 663 \nGSC genes with no interaction networks (six genes) are excluded from this 664 \nfigure. 665 \n 666 \n 667 \n 668 \n 669 \n 670 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n 21 \n 671 \n 672 \n 673 \n 674 \n 675 \n 676 \n 677 \n 678 \n 679 \n 680 \n 681 \n 682 \n 683 \nDiscussion 684 \nFertility and germline cytological assays of bam null mutants in D. americana reveal that 685 \nbam is essential for GSC differentiation in both males and females. This demonstrates 686 \nthat bam’s essential function in GSC differentiation is not novel to the D. melanogaster 687 \nspecies group, and its evolutionary origin in this function likely occurred just prior to or 688 \njust after the origin of the genus Drosophila. Bam functional differences in D. teissieri 689 \nand D. ananassae likely represent lineage-specific GSC functional losses. Similar 690 \nlosses may exist among the many other non-tested taxa in this species-rich genus. 691 \nThese analyses of bam function show that high amino acid sequence variability 692 \nbetween species does not necessarily imply functional divergence. D. americana bam 693 \nand D. melanogaster bam share only 35% sequence identity, yet both execute the same 694 \nessential function in GSC daughter differentiation. In contrast, D. teissieri bam and D. 695 \nmelanogaster bam share 75% sequence identity and they are functionally distinct. 696 \nConservation of sequence does not necessarily imply conservation of function and 697 \ndivergence of sequence does not necessarily imply a divergence in function. A classic 698 \nexample of this dynamic includes cytochrome c (Garrido et al. 2006). Cytochrome c is 699 \none of the most conserved proteins among eukaryotes with an amino acid sequence 700 \nconservation of 70-90% between species as divergent as yeast and mammals. In many 701 \nyeasts and lower eukaryotes, cytochrome c functions solely as an electron carrier in the 702 \nmitochondrial respiratory chain while in mammals the same (or nearly identical) 703 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n 22 \ncytochrome c also acquired an additional role as an apoptotic signal by helping to 704 \ntrigger caspase activation and programmed cell death. This extra “moonlighting” 705 \nfunction in apoptosis is a dramatic divergence in biological role despite a highly 706 \nconserved structure (Garrido et al. 2006).  707 \nThe resolution and depth of our analysis was made possible due to the large collection 708 \nand phylogenetic density of high-quality genomes for a growing number of species in 709 \nthe genus Drosophila in combination with the depth of functionally defined genes within 710 \nD. melanogaster. Large-scale gene ortholog identification is mostly not possible in 711 \nspecies with only low-quality genomes available, and, potentially as a result, there are 712 \ncurrently very few large-scale comparative analyses of gene orthologs, essential or 713 \nnonessential (Carranza et al. 2018, Bergmiller et al. 2012, Leeuwen et al. 2020, Deng et 714 \nal. 2024). However, as more high-quality genomes are produced and published for more 715 \nspecies, this type of comparative ortholog analysis becomes more feasible. Even when 716 \nusing only species with available high-quality genomes for comparative analysis, efforts 717 \nmust be made to ensure potential orthologs and absences are properly identified. For all 718 \nof the GSC regulating genes in D. melanogaster that we analyzed (366 from Yan et al. 719 \n(2014) plus bgcn and Yb), ortholog predictions vary dramatically based on identification 720 \nstrategy. Using Ensembl alone, we predicted 85% (311 of 366) of GSC regulating genes 721 \nabsent in at least one included species while we only predicted ~8% (30 of 366) absent 722 \nbased on the more detailed evaluation incorporating RBBH and synteny. Due to the 723 \nsignificant discrepancy in predictions, the predicted presence or absence of orthologs 724 \nvia Ensembl must be further interrogated by additional strategies including RBBH and 725 \nsyntenic evaluation before confidently characterizing predictions as orthologs that are 726 \ntruly absent. As an example, benign gonial cell neoplasm (bgcn) is crucial gene that is 727 \nbroadly conserved, including a human ortholog, that was predicted to be absent in the 728 \ninitial RBBH evaluation. However, using follow-up BLASTn and syntenic evaluation we 729 \nfound the bgcn ortholog split over short contigs that made initial identification difficult. In 730 \ncontrast, Yb, an additional essential GSC regulating gene in D. melanogaster was 731 \ncomputationally predicted absent in D. obscura and D. pseudoobscura. Follow-up 732 \nsyntenic evaluation and sequencing confirmed these absences of Yb from their syntenic 733 \nregions in these two species. 734 \nThe 30 GSC (out of 366) GSC regulating genes that are confidently predicted to be 735 \nabsent represent significant flexibility in essential GSC regulating genes beyond bam 736 \nacross species. These results add to a growing body of evidence that suggests the 737 \ngeneral ortholog assumption of shared function is not universal and provide examples of 738 \ndevelopmental systems drift (DSD) in a process critical for early reproduction. These 30 739 \ngenes are also not equally distributed across functional categories. While there are no 740 \npredicted absences in proteasome genes, two of six (33%) genes involved in the 741 \nkinetochore and spindle have predicted absences. This variable flexibility across 742 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n 23 \nfunctional categories suggests there may be some networks that are more flexible in 743 \ntheir regulation while others are particularly intolerant.  Additionally, further analysis of 744 \nthese 30 genes with absent orthologs shows that these genes generally have 745 \ninteraction networks including genetic and physical interactors. Absences in orthologs of 746 \ninteraction network genes also generally occur in the same species that their related 747 \nGSC gene is absent. This could potentially indicate that having a network of interacting 748 \npartners more easily enables gene loss by compensating for the function of the absent 749 \ngene through redundancy or alternative regulation while essential genes without large 750 \ninteraction networks could generally be more difficult to replace (Albalat 2016, Wagner 751 \n2008, Carroll 2008). One gene, Prosalpha1, is absent in the most species of the 752 \nincluded GSC genes. In addition to having a large interaction network (47 genes), it also 753 \nhas a very similar gene, Prosalpha1R, within the surrounding syntenic region. 754 \nProsalpha1R could possibly facilitate alternative execution of Prosalpha1’s essential 755 \nfunction. 756 \nOur findings here provide additional evidence that orthologous genes do not necessarily 757 \nfunction identically even when critical for regulating essential systems. Despite the 758 \nessential nature of proper gamete development, what has been described as 759 \ndevelopmental systems drift (DSD) is occurring in genes critical for regulating the 760 \nearliest stages of this process across Drosophila and closely related outgroups. 761 \nSpermatogenesis regulating genes further downstream of GSC regulation have also 762 \nbeen evaluated across some Drosophila species. Results showcase similar functional 763 \nflexibility with duplications or losses of sperm nuclear basic proteins as well as the 764 \nemergence of many functional de novo genes (Lee U 2025, Chang C 2023). Some of 765 \nthe particular genes involved in the process change, but the final phenotype is 766 \nmaintained across species. 767 \nWe conclude that ortholog function must be evaluated in a species-specific manner. 768 \nThese 30 GSC genes and Yb that show absences in some species represent the 769 \nbaseline of functional flexibility for the D. melanogaster essential GSC regulating genes, 770 \nbut many other GSC regulating genes still present, like bam, could have variable 771 \nfunction across species. The extent of developmental systems drift occurring beyond 772 \nGSC regulating genes in these 17 species is largely unknown, but further evaluation of 773 \northolog functional flexibility focusing on different organisms and phenotypes could 774 \nreveal differences in the key genes regulating any number of traits across species. 775 \nResults from this dataset suggest some potential patterns related to ortholog loss of 776 \nessential genes, but more extensive analysis needs to be executed to make more 777 \nconcrete assessments of the prevalence and characteristics of ortholog functional 778 \nflexibility. 779 \n 780 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n 24 \n 781 \nData Availability 782 \nAll raw and filtered data are available in a public repository at 783 \nwww.github.com/lukearnce/bam_null_ortholog. These data include the following:  784 \nMcDonald-Kreitman test for D. americana bam 785 \nPRANK inputs and control parameters for the D. americana bam MK test with 786 \ncontingency table values, the P-value of the χ2, alpha, and proportion of fixations 787 \npredicted to be due to positive selection.  788 \nD. americana bam CRISPR Null fertility assays 789 \nD. americana male and female estimation statistics and significance as an effect size 790 \noutside of the 95% confidence intervals 791 \nGSC ortholog identification 792 \nInitial RBBH ortholog analysis results, Full genomes, custom scripts to execute forward 793 \nand reverse BLASTp, filtered ortholog hits, interaction network genes, RBBH with 794 \nsyntenic evaluation gene hits, and sequence verification of predicted ortholog absences 795 \nAcknowledgements 796 \nWe thank Jolie Carlisle for her thoughtful comments on an earlier version of the 797 \nmanuscript. 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It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint \n\n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.04.30.651540doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}