Molecular Population Genetics of Accessory Gland Protein Genes and Testis-Expressed Genes in Drosophila Mojavensis and D. Arizonae
Wag, Bradley J., Begun, David J., Genetics
Molecular population genetic investigation of Drosophila male reproductive genes has focused primarily on melanogaster subgroup accessory gland protein genes (Acp's). Consistent with observations from male reproductive genes of numerous taxa, Acp's evolve more rapidly than nonreproductive genes. However, within the Drosophila genus, large data sets from additional types of male reproductive genes and from different species groups are lacking. Here we report findings from a molecular population genetics analysis of male reproductive genes of the repleta group species, Drosophila arizonae and D. mojavensis. We find that Acp's have dramatically higher average pairwise K^sub a^/K^sub s^ (0.93) than testis-enriched genes (0.19) and previously reported melanogaster subgroup Acp's (0.42). Overall, 10 of 19 Acp's have K^sub a^/ K^sub s^ > 1 either in nonpolarized analyses or in at least one lineage of polarized analyses. Of the nine Acp's for which outgroup data were available, average K^sub a^/ K^sub s^ was considerably higher in D. mojavensis (2.08) than in D. arizonae (0.87). Contrasts of polymorphism and divergence suggest that adaptive protein evolution at Acp's is more common in D. mojavensis than in D. arizonae.
MOLECULAR studies in a diverse array of animal taxa suggest that genes involved in reproduction evolve at an accelerated rate relative to other genes (reviewed in SWANSON and VACQUIER 2002). Positive selection has been inferred for some proteins (SwANSON and VACQUIER 1995; METZ and PALUMBI 1996; SUTTON and WILKINSON 1997; WYCKOFF et al. 2000; TORGERSON et al. 2002), although population genetic data are sufficiently sparse to leave unresolved the question of the relative importance of directional selection vs. genetic drift in reproduction-related proteins compared to other protein classes. In any case, rapid phenotypic/ molecular evolution of reproductive characters/genes is consistent with the notion that male-male and malefemale interactions may contribute to the rapid divergence between populations and the evolution of reproductive isolation (EBERHARD 1996; RICE 1998).
Molecular evolutionary investigation of Drosophila reproduction has focused on male accessory gland protein genes (Acp's) of melanogaster subgroup species. The number of putative Acp's in these species is on the order of 83 (SwANSON et al. 2001), although <20 have extensive experimental support (SCHAFER 1986; DiBENEDETTO et al. 1987; CHEN et al. 1988; MONSMA and WOLFNER 1988; WOLFNER et al. 1997). Genetic analysis has shown that Acp's contribute to proper sperm storage (NEUBAUM and WOLFNER 1999; TRAM and WOLFNER 1999; CHAPMAN et al. 2000), normal ovulation and oviposition (HERNDON and WOLFNER 1995; HEIFETZ et al. 2000), increased egg-laying rates, and reduced female receptivity (CHEN et al. 1988; AIGAKI et al. 1991; KALB et al. 1993; CHAPMAN et al. 2003; LIU and KUBLI 2003). Acp's show higher rates of protein divergence (AouADÉ 1997, 1998,1999; TsAURand Wu 1997; TSAURétal. 1998; BEGUN et al. 2000; SWANSON et al. 2001) and protein polymorphism (COULTHART and SINGH 1988; BEGUN et al. 2000) compared to "average" proteins in Drosophila melanogaster and D. simulans (e.g., BEGUN et al. 2000). Less energy has been devoted to population genetic investigation of male reproductive genes primarily expressed in testes (but see DUVERNELL and EANES 2000; PARSCH et al. 200Ia). However, a few analyses suggest that Drosophila testis-expressed genes evolve quickly (PARSCH et al. 2001b; MEIKLEJOHN et al. 2004; RICHARDS et al 2005) and may sometimes be associated with evolution of novel function (LONG and LANGLEY 1993; NURMINSKY et al. 1998; BETRAN and LONG 2003).
Because our current population genetic understanding of Drosophila is dominated by data from melanogaster subgroup species, we have no way of knowing whether the patterns of polymorphism and divergence or the functional biology of reproduction-related proteins will be similar in other Drosophila species (WAGSTAFF and BEGUN 2005). Given the hypothesis that the dynamics of certain male reproduction-related proteins may be driven by male-male and male-female postcopulatory interactions, one strategy for furthering our understanding of the evolution of these proteins is to investigate Drosophila species having different reproductive biology from D. melanogaster and D. simulans. D. arizonae and D. mojavensis are cactophilic fly species within the mulleri complex of the repleta group. As members of the subgenus Drosophila, these desert Drosophila are ^4O60 million years diverged from D. melanogaster and other Sophophora subgenus flies (POWELL and DESALLE 1995).
A major difference in the reproductive biology of desert Drosophila vs. D. melanogaster is that remating occurs more frequently in desert Drosophila. Within 24 hr of an initial mating, 95% of D. arizonae and D. mojavensis females tend to remate, while only 2% of D. melanogaster females remate in this same time period (MARKOW 1982, 1996). Frequent remating favors competition between male ejaculates, whereas infrequent remating would be more likely to favor genotypes successfully obtaining initial access to females (e.g., MARKOW 2002). Data from Drosophila species suggest that there is a positive correlation between high female remating rates and exaggerated ejaculates in the form of either sperm gigantism or excessive ejaculate donation to female tissues (MARKOW 2002). Although both desert Drosophila species discussed here contribute large ejaculate donations to ovaries, D. arizonae and D. mojavensis contribute small and large donations, respectively, to female somatic tissues (PITNiCK et al. 1997). Experiments in D. melanogaster revealed no detectable incorporation of ejaculate-derived material into female somatic or ovarian tissues (PITNICK et al. 1997). While ejaculate donations are often perceived to be of nutritive value, a cost to remating has been observed in D. mojavensis females, suggesting the possibility of sexual conflict (ETGES and HEED 1992). Another major difference in the reproductive biology of repleta group vs. melanogasier subgroup flies is that repleta group males require significantly more time to reach sexual maturity. For example, D. arizonae and D. mojavensis require 4-5 days posteclosion to reach maturity, compared to 1-2 days for D. melanogaster males (PITNICK et al. 1995).
Data on natural variation in reproductive traits suggest a more dynamic postmating interaction between the sexes in desert Drosophila compared to melanogaster subgroup flies. Immediately after mating, a pronounced insemination reaction occurs in the female reproductive tract of D. arizonae and D. mojavensis (PATTERSON 1947; PATTERSON and STONE 1952) but is absent in D. melanogaster (WHEELER 1947; MARKOW and ANKNEY 1988). The reaction manifests itself as a large mass within the vaginal pouch and acts as a barrier that prevents remating for the several hours that it persists (PATTERSON 1947; KNOWLES and MARKOW 2001). Seminal fluid proteins may be the primary male contributor to this phenotype, as it is triggered in the absence of live spermatozoa (PATTERSON 1947). Comparisons between desert Drosophila species, as well as between different populations within species, show that postcopulatory male-female interactions change across short evolutionary time periods. For example, heterospecific matings between D. arizonae and D. mojavensis trigger an exaggerated insemination reaction that is both harder and longer lasting than that of the respective conspecific matings of either species (PATTERSON 1947). Moreover, both D. arizonae and D. mojavensis show larger and longer insemination reactions in interpopulation vs. intrapopulation crosses (KNOWLES and MARKOW 2001) within species. Further evidence of rapid evolution of reproductive traits comes from the observation that D. mojavensis shows significant among-population variation in the correlated traits of male sperm size and female sperm-storage organ length (PITNICK et al. 2003).
These data support the idea that properties of ejaculates or ejaculate-female interactions evolve very quickly in desert Drosophila, possibly as a result of antagonistic coevolution between the sexes (RICE 1996, 1998) and/ or cryptic female choice (EBERHARD 1996). We should expect such elaboration of ejaculate characteristics to extend to the molecular level. The purpose of this study is to add a molecular framework to investigation of desert Drosophila reproduction. First, we report the composition of D. mojavensis male reproductive tract cDNA libraries relative to the gene annotations of D. melanogaster. Many of these data are presented as supplementary online material (http://www.genetics.org/ supplemental). second, we report results from molecular and evolutionary analyses of genes expressed in male reproductive tracts in D. mojavensis and D. arizonae and compare these results to those previously reported from D. melanogaster/D. simulons.
MATERIALS AND METHODS
D. mojavensis reproductive tract library: Poly (A)-enriched mRNA was prepared with the MicroPoly(A)-Pure kit (Ambion, Austin, TX) from 50 whole reproductive tracts of adult male D. mojavensis flies. First-strand cDNAwas reverse transcribed with the SMART PCRcDNA synthesis system reagents and protocol (CLONTECH, PaIo Alto, CA). second-strand product was produced with the Expand high-fidelity polymerase system (Roche Molecular Biochemicals, Indianapolis). Cycling parameters were programmed as instructed by the manufacturer, including a 4-min extension step for 10 total cycles. The secondstrand product was cloned into the TOPO vector (Invitrogen, San Diego) and used for bacterial transformations according to the manufacturer's instructions. Colony PCR was carried out using cloning-vector-derived primers (Ml 3 reverse and T7) on 480 colonies (i.e., five 96-well plates). The resulting PCR products were purified prior to sequencing with M13R and T7 primers on an Applied Biosystems (Foster City, CA) 377 automated sequencer (ABI, Columbia, MD). These sequences included 54 unique transcripts. Expressed sequence tags (ESTs) from this library can be found under accession nos. DR033184DR033386 and DR033894-DR033895.
Preliminary expression analysis and D. mojavensis testis cDNA library production: Dot blots prepared from PCR products of the 54 unique clones were hybridized separately to 32P-labeled cDNAs derived from D. mojavensis accessory glands and testes. Hybridizations were carried out at 65° in a buffer consisting of 0.5 M NaPi (pH 7.2), 7% SDS, 1 ITLM EDTA. Filters were washed at 60° with buffer at 40 ITLM NaPi, 1% SDS, and 1 ITLM EDTA. Comparison of signal intensities from hybridization of labeled accessory gland vs. testis cDNA suggested that the majority of the clones represented accessory gland transcripts.
To increase the sample size of testis-enriched transcripts we made a testis cDNA library. This library was produced as described above for whole reproductive tracts, but with 50 D. mojavensis dissected testes as the source tissue. This library was sequenced to the point of producing 118 unique ESTs. ESTs from the testis library can be found under accession nos. DR033387-DR033542.
BLAST methodology and characterization of amino acid sequences: All unique ESTs were compared to D. melanogaster through a pipeline of BLAST analyses to one or more FlyBase Release 3.1 databases (ALTSCHUL et al. 1997). Default BLAST parameters were used except that the cutoff value for significance was set to E - 0.01. The pipeline started with BLASTp (protein to predicted D. melanogaster proteins) queries of all ESTs for which an open reading frame (ORF) was well established (as described below). ESTs that returned significant (E < le-8) D. melanogaster sequences were not queried further. The remaining ESTs were BLASTx (nucleotide to protein) queried to the same D. melanogaster database. Once again, ESTs that returned small ?-values were not queried further. This pipeline continued through tBLASTx (nucleotide to nucleotide query, using all six possible protein translations of the sequences) and BLASTn (nucleotide to nucleotide) queries of predicted D. melanogaster genes and chromosome arms. For the ESTs that returned no D. melanogaster sequences at E< 0.0001, the NCBI whole-genome shotgun (wgs) database was tBLASTx queried with the same default parameters (ALTSCHUL et al. 1997). The NCBI wgs database includes many complete genomes, including D. pseudoobscura and the mosquito , Anopheles gambiae. All D. mojavensis ESTs were also tBLASTx or BLASTn queried (BLASTn was used only if tBLASTx failed to return sequences of E < 0.0001) to the D. melanogaster dbEST database using default BLAST parameters and an ?-score cutoff of 0.01. Finally, we queried the SignalP 3.0 (NIELSEN and KROGH 1998; BENDTSEN etal. 2004) and NCBI CDD (MARCHLER-BAUER et al. 2003) servers with amino acid sequences corresponding to ESTs with identifiable ORFs to identify the presence of signal peptides and conserved domains, respectively.
A subset of genes isolated from both libraries was scrutinized in greater detail to winnow candidates for population genetic analysis. Each clone sequence was subjected to an ORF analysis by the Genejockey software program (Biosoft, Ferguson, MO). If a putative initiation codon followed by an ORF covering at least 70% of the EST could not be identified, we used RACE to gather additional cDNA sequence data.
Reproductively mature D. mojavensis adults of both sexes served as the tissue source for RACE-ready template. mRNA was isolated using the MicroPoly(A)-Pure kit (Ambion, Austin, TX). RACE-ready cDNA was prepared and target molecules were PCR amplified and isolated using the GeneRacer (Invitrogen) protocol, which preferentially selects full-length transcripts for first-strand cDNA synthesis. RACE products derived from such a library should provide high-quality information on the 5' ends of transcripts. Several criteria were used to identify the set of ORFs ultimately used in molecular evolutionary analysis: (i) size and position of candidate ORFs within an EST, (ii) presence of a predicted signal peptide sequence for putative Acp's (WoLFNER et al. 1997; SWANSON et al. 2001), (iii) tBLASTx homology to genes in public databases (e.g., D. melanogaster genome release 3.1), and (iv) presence/ absence of INDEL mutations and/or premature termination codons in polymorphism data from genomic DNA. Only strongly supported ORFs were used in evolutionary analysis.
Quantitative PCR evaluation of ESTs: Genes targeted for population genetic analyses as accessory gland vs. testisenriched in expression on the basis of dot blots were subjected to more rigorous quantification of transcript distribution and abundance by real-time quantitative PCR. For the subset of genes in which a related D. melanogaster gene was identified, quantitative PCR was also carried out in D. melanogaster to provide comparisons of expression between lineages. The purpose of this analysis was to assign genes to three expression classes: Acp, testis enriched, and other tissues. A total of 58 and 33 genes were investigated in D. mojavensis and D. melanogaster, respectively.
Tissue dissections consisted of 80 D. mojavensis and 40 D. melanogastermaie flies. All flies were reproductiveIy mature and were dissected in RNAfofer (Ambion) into three tissue categories: accessory glands, testes, and carcasses without the reproductive tracts. Each collection of dissected tissues was divided equally into two replicate samples for RNA isolation. Likewise, whole, reproductively mature female flies from each species (n = 40) were evenly split into two replicate RNA preps. Total RNA was extracted using Trizol Reagent (Invitrogen), purified through RNeasy (QIAGEN) columns, and treated with DNase according to manufacturer's instructions (QIAGEN). RNAs were then reverse transcribed at a concentration of 20 ng/μι using TaqMan reverse transcription (RT) reagents (Applied Biosystems). These first-strand cDNAs served as the templates for quantitative PCR analysis.
Quantitative PCR was performed using an ABI Prism 7700 sequence detector and SYBR green PCR core reagents (Applied Biosystems). Amplification primers were designed with Primer Express (Applied Biosystems). For every 200 -μ? PCR reaction, 0.5 μι of first-strand cDNA was used. Quantitative PCR conditions were 94° for 10 min followed by 40 cycles of 94° for 20 sec, 59° for 30 sec, and 72° for 30 sec. A dissociation step was added to the end of the run to ensure that only a single amplicon was produced in each reaction. All primer pairs produced a single product. A total of 13 quantitative PCRs were processed for each gene. Three reactions were run for each of the four tissues: one for each of the two replicate RT reactions as well as a single minus-RT reaction derived by drawing equally from the minus-RT templates of paired replicates. The 13th reaction was a no-template control. We found no evidence of genomic contamination or primer-by-reagent interactions.
Quantitative PCR quantification: Quantification followed the g-AACr methods of LIVAK and SCHMITTGEN (2001). Quantitative PCR provides an estimate of CT, the cycle at which the quantity of amplified product exceeds a predetermined threshold. Therefore, more abundant transcripts should yield lower Cr scores. To control for different first-strand cDNA concentrations across templates, as well as run and reagent effects, our Δ Cp scores were calculated by subtracting experimental gene CT scores from housekeeping gene CT scores derived from the same tissue and experimental microtiter plate. The housekeeping control for both species was the ribosomal protein gene CG7808, which was identified in the D. mojavensis reproductive tract cDNA library (moj!2) and is highly conserved between D. mojavensis and D. melanogaster (96% protein similarity).
Our calculation of 2~ΔΔ?ΙΓ reflects fold change in gene expression of the most abundant tissue template (lowest ACr score) relative to the second most abundant tissue template for any given gene. There were two justifications for this approach. First, we observed several instances in which quantitative PCR product was detected in only two of the four templates. second, compared to approaches estimating fold differences across all tissues, our approach minimizes fold difference values, thereby providing conservative lower-bound estimates for actual differences between tissue transcriptome profiles. The two replicate 2~ΔΔ?ΙΓ scores for each gene were always independently calculated and then averaged for the reported values.
Quantitative PCR statistics: Replicate 2~ΔΔ<3τ scores for every gene and for each of the four templates can be used to determine the amount of experimental error. A scatter plot of replicate ACp scores for the most abundant tissue of each surveyed gene (n - 91, Figure 1) reveals a high degree of similarity between replicate pairs (-R2 = 0.979). The slope of this line (m = 0.985) is very close to BZ= 1, showing that the high repeatability of our assays holds across alarge range of expression estimates.
We used our replicate 2~AACr scores to determine threshold fold differences that are sufficiently disparate to represent significant differences. To approximate a gamma distribution, we calculated ratios of replicate pairs by dividing the higher g-AACr score by its counterpart and then subtracting one. A total of 91 replicate reaction pairs generated a distribution ranging from 0.0 to 18.24. We then used the XQ value at which the area under the frequency distribution (O ^ χ ^ X0) is equal to 0.95 to establish a critical threshold for significant differences between 2~ΔΔ& scores. For the complete data set, 2~ΔΔ& scores >7.84 represent significant differences between tissues (P < 0.05). This is a conservative critical threshold estimate because genes that are highly tissue specific (those with high g-AACr SCOres) are susceptible to larger error in terms of relative expression differences. This is a consequence of fold differences being derived by comparing the most abundant tissue (lowest ACp) to the second most abundant tissue. Thus, fold difference for a gene that is highly tissue specific in expression is estimated by comparison to a tissue showing very low transcript abundance. In such cases, experimental error associated with the less abundant tissue expression will affect 2~AACr scores of highly tissue-specific genes. Many of our genes have large 2~ΔΔ?ΙΓ scores (see supplementary Table S2; http://www. genetics.org/supplemental), which indicate high tissue specificity. Restricting our statistical analysis to genes with 2~ΔΔ& < 50 (η - 28), the critical threshold for significance is reduced to 3.25 (P < 0.05). Further narrowing the analysis to genes with 2~AACr < 15 (η = 24) reduces the critical threshold to 2.10 (P < 0.05).
The different critical values for different subsets of the data support the idea that error variance of relative expression levels is greater for genes with the highest 2 ΔΔ?ΙΓ scores. Therefore, we view the critical threshold of 2.10 as most informative because it is derived from the very data whose relative expression patterns are most in doubt. Even so, we choose a conservative critical threshold of 2~ΔΔ?ΙΓ = 5.0 (fivefold difference in relative expression for the most abundant vs. next most abundant tissue) for the purpose of categorizing genes as either Acp's or testis enriched. Though somewhat arbitrary, we note that categorization of genes would not be substantially altered by choosing a more conservative threshold. For example, a critical threshold of 18 would only recategorize three testis-enriched genes as genes showing no strong pattern of tissue enrichment.
D. mojavensis genomic library: A genomic library was constructed to provide flanking data around gene sequences to help identify regions of homology between D. melanogaster and D. mojavensis (e.g., WAGSTAFF and BEGUN 2005; see supplementary material, http://www.genetics.org/supplemental). D. mojavensis genomic DNA was partially digested with SaaSA and size fractionated by electrophoresis through a 0.6% agarose gel. DNA fragments between 9 and 23 kb were selected via gel extraction (QIAGEN), ligated to λ-DASH ll/BamHl vector (Stratagene, LaJoIIa, CA), and packaged using the Lambda DASH II/Gigapack II cloning kit (Stratagene). The resultant library consisted of ~2.3 X 106 plaque-forming units. Plaques were screened with 32P-labeled D. mojavensis target DNA. Lambda DNA was purified from selected plaques and D. mojavensis genomic inserts were amplified using T3/T7 vector primers and LA-Taq long PCR polymerase (TaKaRa, Shiga, Japan). The resulting PCR products were sheared by sonication and the fragments were blunt ended using Klenow fragment of DNA polymerase and T4 DNA polymerase. Fragments of 1-2 kb were isolated from a low-melting agarose electrophoresis gel and cloned into the pUC18/SmaI/BAP vector with a Ready-to-Go kit (Amerisham Biosciences, Piscataway, NJ). Sequencing of the phage through ~7X coverage was performed on an ABI Prism 3700 sequencer. Consensus sequences were assembled using the SeqMan program of the DNASTAR software package (Lasergene, Madison, WI).
Nomenclature: Unique ESTs were assigned numbers (1-54 for reproductive tract library ESTs, 100-217 for testis library ESTs). Genes from the quantitative PCR analysis showing at least fivefold greater expression (2~ΔΔ?ΙΓ>5) in either accessory glands or testes were categorized as Acp's and testis enriched in expression (hereafter referred to as testis-enriched genes), respectively. Prefixes for numbered EST names were added according to these expression patterns, with Acp preceding accessory gland genes and Tes preceding testis-enriched genes. Those genes that did not exceed this threshold (moj9, moj29, moj30, moj32, moj!37, and moj!52) were given the moj prefix to avoid a connotation of tissue specificity. Four Acp's (Acp5, Acpl6, Acp21, and Acp27) are members of recently duplicated gene families (B. J. WAGSTAFF, unpublished data) and are given an additional -a or -b suffix to differentiate between members. Five genes were named as Acp's (Acp4, Acpl5, Acpl 7, Acp23, and Acp36) on the basis of very strong evidence from our dot blot data rather than from quantitative PCR experiments. The remaining ESTs were given the mo/prefix, as no relative expression data were gathered for the associated genes.
Stocks and DNA sequencing: A total of 15 fly stocks from the Drosophila Species Stock Center (Tucson, AZ) were used for collection of most population genetic data. D. arizonae (150811271.00,15081-1271.04,15081-1271.05,15081-1271.08,150811271.12, 15081-1271.13, and 15081-1271.14; various locations, mainland Mexico) and D. mojavensis were represented by seven lines each, while a single D. mulleristock (15081-1371.00; Lake Travis, TX) provided outgroup data. Of the seven D. mojavemis stocks, four were D. mojavensisbaja (15081-1351.03, 15081-1351.09, 15081-1351.12, and 15081-1351.14; various locations, Baja, Mexico) and three were D. mojavensis mojavenMS (15081-1352.00,15081-1352.01, and 15081-1352.02; various locations, southern California). Primers used for amplification of genomic DNA were designed from ESTs or from extended sequences identified by RACE analysis. Expand High-Fidelity polymerase (Roche Molecular Biochemicals) was used for PCR amplification. Single alleles for sequencing were isolated by cloning PCR products into the TOPO vector (Invitrogen) and selecting one bacterial colony for PCR amplification for each allele. Amplified colony-PCR products and their associated sequences were obtained using Ml 3 reverse and T7 primers. A second collection ?ι D. mojavensis mainland and Baja strains (kindly provided by W. Etges, University of Arkansas) was used for additional population sequencing of Acp 7. PCR products from the Etges strains were directly sequenced. All sequencing was done on an Applied Biosystems 377 automated sequencer (ABI). Sequences were aligned and edited using the DNASTAR software package (Lasergene). Generally, the small, predicted size of most Acp's resulted in survey data for most codons. Compared to Acp's, testis-enriched genes, on average, provided lower coverage of codons on a per gene basis (see Table 1).
Statistical analysis of aligned sequences: The DnaSP program (RozAS and ROZAS 1999) was used for most of the population genetic analyses. Average levels of polymorphism or divergence for different groups of genes (e.g., Acp vs. testis enriched) refer to means weighted according to sequence length. For genes sampled for multiple alleles, replacement and synonymous divergence represent the average pairwise difference. Fixations for polarized McDonald-Kreitman tests were assigned using parsimony. Only codons with single mutations that could be clearly assigned to either the D. arizonae or D. mojavensis lineage were considered.
Lineage-specific synonymous and replacement divergences were estimated using the free-ratio maximum-likelihood model of the PAML computer program (YANG 1997). For most of these analyses we used one randomly selected allele from each of three species: D. arizonae, D. mojavensis, and D. mulleri. In some cases for which D. mulleri data were unavailable, we used a duplicated gene that predated the D. arizonae/D. mojavensis speciation event (B.J. WAGSTAFF, unpublished data). We used only duplicated genes showing synonymous divergence that was comparable to or less than the average D. mulleri synonymous divergence (see Table 3). Hypothesis testing was carried out using likelihood-ratio tests (GoLDMAN and YANG 1994; YANG 1998). To determine whether or not ?Α significantly exceeds -K8 in a particular lineage, the likelihood value for the null hypothesis (?Α - K5; i.e., the one-ratio model) was also calculated. Twice the log-likelihood difference between the two models is then compared to a ^-distribution with one d.f. to determine the level of significance.
Content and characterization of D. mojavensis male reproductive tract libraries
The content and basic characteristics of the D. moja