Strict Sex-Specific mtdna Segregation in the Germ line of the DUI Species Venerupis philippinarum (Bivalvia: Veneridae) Research article.

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1 Strict Sex-Specific mtdna Segregation in the Germ line of the DUI Species Venerupis philippinarum (Bivalvia: Veneridae) Fabrizio Ghiselli,*,1 Liliana Milani, 1 and Marco Passamonti 1 1 Dipartimento di Biologia Evoluzionistica Sperimentale, Università di Bologna, Bologna, Italy *Corresponding author: Associate editor: Helen Piontkivska Abstract Doubly Uniparental Inheritance (DUI) is one of the most striking exceptions to the common rule of standard maternal inheritance of metazoan mitochondria. In DUI, two mitochondrial genomes are present, showing different transmission routes, one through eggs (F-type) and the other through sperm (M-type). In this paper, we report results from a multiplex real-time quantitative polymerase chain reaction analysis on the Manila clam Venerupis philippinarum (formerly Tapes philippinarum). We quantified M- and F-types in somatic tissues, gonads, and gametes. Nuclear and external reference sequences were used, and the whole experimental process was designed to avoid any possible cross-contamination. In most male somatic tissues, the M-type is largely predominant: This suggests that the processes separating sex-linked mitochondrial DNAs (mtdnas) in somatic tissues are less precise than in other DUI species. In the germ line, we evidenced a strict sex-specific mtdna segregation because both sperm and eggs do carry exclusively M- and F-types, respectively, an observation that is in contrast with a previous analysis on Mytilus galloprovincialis. More precisely, whereas two mtdnas are present in the whole gonad, only the sex-specific one is detected in gametes. Because of this, we propose that the mtdna transmission is achieved through a three-checkpoint process in V. philippinarum. The cytological mechanisms of male mitochondria segregation in males and degradation in females during the embryo development (here named Checkpoint #1 and Checkpoint #2) are already well known for DUI species; a Checkpoint #3 would act when primordial germ cells (PGCs) are first formed and would work in both males and females. We believe that Checkpoint #3 is a mere variation of the mitochondrial bottleneck in species with standard maternal inheritance, established when their PGCs separate during embryo cleavage. Key words: mitochondrial inheritance, Doubly Uniparental Inheritance, Venerupis philippinarum, real-time qpcr, multiplex. Research article Introduction Mitochondria are commonly inherited maternally in animals, a feature known as standard maternal inheritance; however, its most striking variation is doubly uniparental inheritance (DUI: Skibinski et al. 1994a, 1994b; Zouros et al. 1994a, 1994b). DUI has been found, so far, in bivalve mollusks only: In those species, two mitochondrial lineages are present, one transmitted through eggs (henceforth called F) and the other transmitted through sperm (henceforth called M). Because of this, two different mitochondrial genomes are detectable in DUI bivalves with a peculiar distribution pattern: F is found in eggs, ovaries, and somatic tissues of both sexes, whereas M is found in sperm and testes and is sometimes detected in traces in male soma (Garrido-Ramos et al. 1998; Dalziel and Stewart 2002). These mitochondrial DNA (mtdna) variants show an unexpected high level of sequence divergence: up to 52% (Doucet-Beaupré et al. 2010). DUI is not a case of biparental inheritance of organelles because both male and female mitochondria are transmitted uniparentally. Phylogenetic analyses demonstrated that in some DUI animals, F-type mtdna can invade the male line and be transmitted through sperm, an event known as masculinization or role reversal : This resets to zero the divergence between the genomes. Masculinization was observed in Mytilus mussels but not, until now, in unionid freshwater bivalves (Breton et al and references therein) and venerids (Passamonti and Scali 2001). DUI has been found in species belonging to seven different bivalve families: Donacidae, Hyriidae, Margaritiferidae, Mytilidae, Solenidae, Unionidae, and Veneridae (Breton et al and references therein; Theologidis et al. 2008). Because of its wide taxonomic distribution and its scattered occurrence, there has been contention about whether DUI evolved once or many times among Bivalvia. However, because of the evident DUI analogies in all analyzed species, the soundest hypothesis is that DUI originated once, at the evolutionary radiation of Bivalvia, at least in the early Ordovician (leading to all Autobranchia bivalves) (Theologidis et al and references therein). Consequently, if DUI was the ancestral condition, reversions to standard maternal inheritance should have occurred many times, thus producing the scattered distribution of DUI, with some bivalve species not showing any trace of sex-related mtdna heteroplasmy. However, DUI may be difficult to detect, and its absence cannot be certain (as discussed in Theologidis et al. 2008). In Mytilus, evidence that sperm-derived mitochondria are actively segregated to germ cells in male embryos has been found: The sperm mitochondria distribution shows two different patterns depending on the sex of The Author Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please Mol. Biol. Evol. 28(2): doi: /molbev/msq271 Advance Access publication October 15,

2 Ghiselli et al. doi: /molbev/msq271 the embryos. If the embryo is a male, sperm mitochondria aggregate in a single blastomere, likely the precursor of the male germ line, whereas in female embryos, they are dispersed and degraded (Cao et al. 2004; Obata and Komaru 2005; Cogswell et al. 2006). Fluorescence and confocal microscopy observation of sperm mitochondria stained with MitoTracker Green in V. philippinarum embryos (Milani L et al., in preparation) showed the same two patterns observed in Mytilus, whereas in Crassostrea gigas, a standard maternal inheritance bivalve species, sperm mitochondria do not aggregate during development (Obata et al. 2008). Traces of M genome were also found in the somatic tissues of females (Garrido-Ramos et al. 1998; Dalziel and Stewart 2002) as well as in unfertilized eggs of Mytilus galloprovincialis (Obata et al. 2006), although some kind of sperm contamination cannot be excluded in this case; in contrast, the male germ line excludes F genomes and retains the M ones only (Venetis et al. 2006). This implies that DUI is achieved through a series of molecular signals between nucleus and mitochondria because sperm mitochondria appear to be recognized and actively transferred to the male germ line. Molecular details of such mechanisms are largely unknown, but, analogous to what has been observed regarding standard maternal inheritance (Sutovsky et al. 1999), spermderived mitochondria might be labeled with a molecule similar to ubiquitin (Kenchington et al. 2009). Moreover, active segregation of male mitochondria would imply some kind of microtubule-related transport (Yaffe 1999; Obata and Komaru 2005). To better understand the distribution of M- and F-type mtdna in somatic tissues, gonads, and gametes of V. philippinarum, we set up a fluorescence-based real-time quantitative polymerase chain reaction (qpcr) (Higuchi et al. 1992, 1993; Wittwer et al. 1997) because of its capacity to detect and measure small amounts of nucleic acids in a wide range of samples. Materials and Methods Sample Preparation All clams were collected in Goro (Italy). Single clams were placed in beakers with artificial seawater (e.g., reverse osmosis water added with RedSea Coral Pro aquariology sea salt) that was changed every 12 h to remove any external contamination (i.e., sperm from possible former spawnings). After h, the clams were analyzed. We obtained samples from tissues (adductor muscle, mantle, and gonads), eggs, and sperm. Every possible precautions to avoid contaminations between male and female tissues were taken: 1) different laboratory glassware and plastic ware were used for males and females; the same for pestles and scalpels; 2) males and females were processed on different days or by different people on different benches; 3) two different sets of micropipettes were used, one for males and the other for females; 4) presterilized filter tips were used; and 5) to avoid cross-contamination between tissues, a different scalpel was used to dissect each tissue. Moreover, particular care was taken in tissue excision by avoiding contiguous regions between different tissues and by keeping samples well frozen (thus allowing more precise cuts). Spawning was induced by cyclic thermal manipulation. Again, each clam was kept separate from the others. Sperm was purified using Percoll (GE Healthcare) density gradient as described by Venetis et al. (2006). Finally, eggs and sperm were briefly centrifuged, the seawater was removed and replaced with absolute ethanol, and they were stored at 20 C. After spawning, clams were sacrificed and frozen: Females were used for qpcr analyses, whereas males used for spawning were excluded because spawned sperm could have contaminated the tissues. Therefore, only males sacrificed before spawning were used for qpcr analyses of tissues. A total of 440 DNA samples was extracted from tissues, eggs, and sperm with the DNeasy Blood and Tissue Kit (Qiagen) and the MasterPur Complete DNA and RNA Purification Kit (Epicentre). Genomic DNA was then run on 1% agarose gel to check its integrity, and degraded samples were discarded from the analysis. The remaining was quantified using a Nanodrop 1000: Of those, 220 had the proper quantity and quality. After quantification, samples were diluted to a concentration of ;12.5 ng/ll with TE buffer (Ambion). A second quantification was performed, then the samples were again diluted to reach a concentration of ;2.5 ng/ll. These samples were used to generate the Stock I solution (Grubor et al. 2004; Gallup and Ackermann 2008; see below) and as templates for qpcr experiments. Real-Time qpcr Experimental Design We chose TaqMan hydrolysis probe-based qpcr (see, e.g., Bustin 2000; Kutyavin et al. 2000), instead of the SYBR green (Morrison et al. 1998), because of its higher specificity and sensitivity. This method also allows for multiplexing, which is the simultaneous detection of multiple targets in the same reaction. A nuclear reference gene was used for normalization, calibration, and as internal control. Mitochondrial and nuclear sequences of V. philippinarum were used as targets for probe design. Whole sequences of both male and female mitochondrial genomes are already available (Okazaki M and Ueshima R, unpublished data, GenBank accession numbers: AB and AB065375, respectively), but very few nuclear genes have been sequenced so far. To find a nuclear target, we amplified, cloned, and sequenced several genes: histones (H1, H2A, H2B, H3, and H4), metallothionein (mt), b-actin (actb), and hsp70. Primers for such amplifications were designed with the Primer3 software (Rozen and Skaletsky 2000). The metallothionein-obtained sequence was too short (;200 bp), and no reliable primer/probe set was found, whereas the b-actin fragment (;1,000 bp) showed excessive variability (we got six different alleles in ten clones). In the case of histone sequences, the design of an appropriate combination of primers/probes failed at the multiplexing stage (see below). The only sequence that was suitable and that turned out to be compatible with the 950

3 mtdna Inheritance in Venerupis philippinarum doi: /molbev/msq271 two mtdnas when multiplexed was hsp70. We are confident that this is a good target because it is extremely conserved (so mutations cannot affect its reliability), and it is also a low copy number gene (e.g., one to six copies per haploid genomes in Leishmania, Drosophila, and Trypanosoma: Requena et al. 1988; Bettencourt and Feder 2001; Brochu et al. 2004). For qpcr primer and probe design, we used the Beacon Designer 7.02 Software (Premier Biosoft International; see: for details [last accessed October 29, 2010]). This software automates the design of real-time qpcr experiments: a BLAST search of sequences and a search for template structures are performed, and the regions that exhibit significant cross-homologies and template structures are avoided automatically. Avoidance of cross-homology gives specificity to the primers/probes, so they are highly targeted and only amplify the intended sequence and no other target. More precisely, the three primer pairs and the three TaqMan probes were checked for 1) self-priming/cleavage, 2) cross-homologies/amplifications, 3) secondary structures, and 4) multiplex compatibility. The first and most obvious choice for primer/probe design was to find mtdna fragments that are exclusive to M or F genomes (i.e., present in one genome and absent in the other). The majority of such sequences are located in untranslated regions and are characterized by the presence of several repeats and secondary structures (Okazaki M and Ueshima R, unpublished data; GenBank accession numbers: AB and AB065375; Ghiselli F et al., in preparation), a feature that is not suitable for the design of appropriate primers and probes. Other exclusive regions (i.e., inside female COII duplicated gene) did not give any functional sets. The F primer/probe set was then designed within the NADH dehydrogenase subunit 1 (nd1) gene, whereas the M one within the rrns gene. The set quality was rated good by the software, with a score of To assess the variability in the amplicon regions, we aligned all the available M- type sequences of rrns. There was a perfect sequence identity in the target region of samples from Italy, Japan, and USA, so we are confident that possible detection failures of M due to mutation are very unlikely events. On the female side, only one nd1 sequence was available in Gen- Bank, so an alignment was not possible. Then, we checked the available sequences among all bivalves, and we found a very low sequence variability in the target region (data not shown). Moreover, F-type mtdna in V. philippinarum is known to be much less variable than M (Passamonti et al. 2003), so possible detection failures should be very unlikely for the F-type too. The program output (including primer/probe sequences) is reported in supplementary material 1, Supplementary Material online. Probes were purchased from Sigma, and we chose FAM, HEX and ROX reporters (for, respectively, nuclear target, M mtdna, and F mtdna) coupled with Black Hole Quenchers for better background noise suppression. Primer/Probe Specificity Tests Several tests were performed to assess the specificity of the set generated by Beacon Designer. The three primer pairs were initially tested with end-point PCR (i.e., traditional PCR) using genomic DNA extracted from eggs and purified sperm as targets. The PCR cycle was set as follows: 94 C for 5 min, then 35 cycles (94 C, 51 C, and 72 Cfor 30 s), and a final extension at 72 C for 7 min. The malespecific primer pair amplified sperm DNA only, femalespecific primers amplified egg DNA only, whereas the hsp70 specific primer pair amplified both targets as expected. The obtained amplicons were cloned and sequenced (ten sequences per amplicon), and there was a perfect match between the obtained sequences and those available on GenBank. The cloned amplicons were then used as templates in a second cross-amplification experiment with end-point PCR. Each primer pair was tested using all the three cloned amplicons as targets, and no unspecific amplification was detected once more corroborating the high specificity of the designed primers. Moreover, end-point PCR being far less sensitive than realtime qpcr, it may overlook small amounts of unspecific amplification. For this reason, we ran several real-time qpcr plates to further check the specificity of the designed set: Each TaqMan probe and primer pair was tested against all the three cloned amplicons. The cycle was programmed as follows: initial denaturation at 95 C for 3 min, then 40 cycles (95 C for 10 s and 58 C for30s). The fluorescence data acquisition was set at the 58 C step. Because the end-point test PCRs had an annealing temperature of 51 C and they did not show any crossamplification, the increase in that temperature to 58 C made the primer binding even more specific. All the tests confirmed the effectiveness of the set generated by Beacon Designer. In the same experiments, each primer/probe combination was checked both in singleplex and multiplex, and the concentration of primers and probes was optimized in order to achieve the same reaction efficiency in both conditions. A TaqMan probe gives a fluorescent signal only once it is degraded by a DNA polymerase. Therefore, to have nonspecific target amplification, nonspecific annealing of both primers and probe must occur; given all the tests we ran, this is improbable in our experimental conditions. Further evidence against unspecific amplification comes from the results: In most female tissues as well as in some male ones and in all gametes, we did not detect one of the two mtdna variants (either M or F), whereas the nuclear target and the other variant were always present (see Results). If cross-amplification had happened, such observed distribution patterns could not be possible. Quantification The use of a reference nuclear gene allows a relative quantification of the mitochondrial targets, but for an absolute quantification, an external standard curve is needed (Bustin 2000). A standard dilution series with a known concentration of initial target copy number is used to generate 951

4 Ghiselli et al. doi: /molbev/msq271 a standard curve by plotting the Cq (i.e., the quantification cycle, formerly the threshold cycle, see Bustin et al. 2009) values against the logarithm of the initial copy numbers (Ke et al. 2000). The range of the standard dilution series must include the Cq values expected for the experimental RNA/ DNA samples. The copy number of unknown samples can be calculated from the linear regression of that standard curve, with the slope providing the amplification efficiency. Standard curves can be constructed from cloned PCR fragments, in vitro T7-transcribed RNA, single-stranded sense-strand oligodeoxyribonucleotides, or commercially available universal reference RNAs (Pfaffl and Hageleit 2001; Mohammadi and Day 2004; Dhanasekaran et al. 2010). The accuracy of absolute quantification depends entirely on the accuracy of standards; however, external standards cannot detect or compensate for inhibitors that may be present in the samples (Bustin 2005). To overcome this and many other problems related to efficiency and inhibition of qpcr, we used the PREXCEL-Q (P-Q) software (Gallup and Ackermann 2008). Among its many features, it identifies and avoids inhibition of qpcr reactions, identifies the valid log-linear-amplificationcapable ranges for all target standard curves, calculates the valid dilution series for each nucleic acid sample on a per target basis, and is able to achieve nearly 100% reaction efficiency, assuming an appropriate primer probe design (Gallup and Ackermann 2008). P- Q also establishes/suggests the use of a Stock I solution (Grubor et al. 2004; Gallup and Ackermann 2008), which ensures the uniformity of all qpcrs: It is obtained by pooling small amounts of all samples. Stock I serves as the serially diluted sample for preliminary test plate, which allows for the assessment of the qpcr dynamics of each sample and target to identify and avoid working within the inhibitory range of the assay. Moreover, it is used as the standard curve material on all final qpcr plates for individual sample/qpcr target assessments. A common problem with qpcr is selecting a sample that is representative of all samples in a study and a source of material to use for qpcr target standard curves. Stock I will behave identically to actual samples because it is made of the samples themselves. To get a very accurate quantification, we wanted to correlate the standards obtained with the Stock I serial dilutions with more precisely quantified standards. Therefore, we decided to use synthesized oligonucleotides (IDT Ultramers) also to avoid contaminations that may easily occur during plasmid preparation. The use of Ultramers has two main advantages: 1) purity of standards (because they do not have a biological derivation and because their preparation does not require the handling of genomic DNA) and 2) quantification precision (the exact molecular weight is known, so the copy number calculation is more precise). Three sense and three antisense Ultramers were ordered (sequences reported in supplementary material 1, Supplementary Material online). The double-strand standards were prepared resuspending the oligos in an RNAse-free duplex buffer, heating them at 94 C for 2 min, and cooling them down slowly to room temperature. The Ultramers were then checked by agarose electrophoresis and stored at 20 C. The copy number calculation (with associated errors) and the correlation between the two standard series ( Stock I and Ultramers derived) were obtained by means of the P-Q software, using the equations shown in supplementary material 2, Supplementary Material online. All the Cq values were corrected by the efficiency-corrected DDCq method (known as the Pfaffl method, see Pfaffl 2001). Propagation of errors was calculated as in Karlen et al. (2007) (see supplementary material 2, Supplementary Material online, for details). In this work, we considered the samples showing not detectable (N/D) signal as having no target DNAs, even if N/D might also mean the targets were so scarce that our experimental procedures could not detect them. However, given the experimental design, quantities of ;10 copies/sample gave clear signals between 25 and 35 cycles. Actually, the Cq values for approximately ten target copies of synthetic Ultramers were (hsp70), (M-type mtdna), and (F-type mtdna); similar Cqs were recovered when using genomic DNA. As all samples were run up to the 40th cycle, we are confident that whenever no signal was detected, the real copy number was zero or very close to it. According to Wittwer and Kusakawa (2004), the most sensitive limit of detection (LOD) theoretically possible for qpcr is three copies per sample. Our qpcrs could identify approximately ten copies within the 35th cycle at the latest, so that the theoretical LOD would have been reached well before the 40th cycle. Increasing the number of cycles above the 40th would have had the unwanted effect of increasing background noise, preventing more fine detection. Finally, we also want to point out that the N/D values were not due to reaction failures because in the very same reaction, the other two targets always gave good amplification (see tables 1 and 2). In the light of all this, we are treating the N/D data in a qualitative way as the mere detection of a nucleic acid template rather than an accurate quantification (Bustin et al. 2009). Test Plate and Standard Series The Stock I solution was generated mixing 3 ll of each sample (diluted to ;2.5 ng/ll). To test the dilution range in which the reaction worked best, 11 serial dilutions of Stock I were prepared (test plate). The chosen range enabled testing of the samples from ;2,500 cells out to ;1cellworthof material (assuming that there are ;4 pg/cell because the haploid genome of V. philippinarum is ;2pg; see: [last accessed October 29, 2010]). The log-linear range within which to generate the standard curve stretched from 1:8 to 1:150 for the two mtdna targets. This range was subdivided into five standards: 0.31, 0.25, 0.12, 0.04, and 0.01 ng/ll, that were used to quantify the two mtdna targets. The hsp70 had a narrower range from 1:8 to 1:21, so it was interpreted by three standards at 0.31, 0.25, and 0.12 ng/ll because its log-linear range did not extend beyond that. The P-Q software also calculated the most suitable sample dilution to use in order to amplify correctly within the 952

5 mtdna Inheritance in Venerupis philippinarum doi: /molbev/msq271 Table 1. Quantification of M- and F-type mtdna in Female Tissues and Eggs. Sample Adductor Muscle M/Nu SD Sample M-TYPE IN FEMALES Mantle M/Nu SD Sample f09-6a N/D f09-6m N/D f09-6g N/D f09-7a N/D f09-7m N/D f09-7g N/D f09-8a N/D f09-8m N/D f09-8g N/D f09-14a N/D f09-14m N/D f09-14g N/D f09-16a N/D f09-16m N/D f09-16g N/D f09-17a N/D f09-17m N/D f09-17g N/D f09-18a N/D f09-18m f09-18g f09-20a N/D f09-20m N/D f09-20g N/D f09-23a N/D f09-23m N/D f09-23g N/D f09-28a f09-28m N/D f09-28g N/D f09-29a N/D f09-29m N/D f09-29g N/D f09-30a N/D f09-30m N/D f09-30g N/D f09-31a N/D f09-31m N/D f09-31g N/D f09-32a N/D f09-32m N/D f09-32g N/D f07-9a N/D f07-9m N/D f07-9g N/D f07-10a N/D f07-10m N/D f07-10g N/D f07-17a N/D f07-17m N/D f07-17g N/D f07-24a N/D f07-24m N/D f07-24g N/D f07-36a N/D f07-36m N/D f07-36g N/D f07-39a N/D f07-39m N/D f07-39g N/D f07-42a N/D f07-42m N/D f07-42g N/D f07-45a N/D f07-45m N/D f07-45g N/D f07-46a N/D f07-46m N/D f07-46g N/D f07-47a N/D f07-47m N/D f07-47g N/D f07-48a N/D f07-48m N/D f07-48g N/D f07-49a N/D f07-49m N/D f07-49g N/D f07-59a N/D f07-59m N/D f07-59g N/D f07-61a N/D f07-61m N/D f07-61g N/D f07-62a N/D f07-62m N/D f07-62g N/D f07-63a N/D f07-63m N/D f07-63g N/D f07-64a N/D f07-64m N/D f07-64g N/D f07-70a N/D f07-70m N/D f07-70g N/D f07-72a N/D f07-72m N/D f07-72g N/D Gonad M/Nu SD Sample Adductor Muscle F\Nu SD Sample F-TYPE IN FEMALES Mantle F\Nu SD Sample f09-6a f09-6m f09-6g f09-7a f09-7m f09-7g f09-8a f09-8m f09-8g f09-14a f09-14m f09-14g f09-16a f09-16m f09-16g f09-17a f09-17m f09-17g f09-18a f09-18m f09-18g f09-20a f09-20m f09-20g f09-23a f09-23m f09-23g f09-28a f09-28m f09-28g f09-29a f09-29m f09-29g f09-30a f09-30m f09-30g f09-31a f09-31m f09-31g f09-32a f09-32m f09-32g f07-9a f07-9m f07-9g f07-10a f07-10m f07-10g f07-17a f07-17m f07-17g f07-24a f07-24m f07-24g f07-36a f07-36m f07-36g f07-39a f07-39m f07-39g f07-42a f07-42m f07-42g f07-45a f07-45m f07-45g f07-46a f07-46m f07-46g f07-47a f07-47m f07-47g f07-48a f07-48m f07-48g f07-49a f07-49m f07-49g Gonad F\Nu SD 953

6 Ghiselli et al. doi: /molbev/msq271 Table 1 Continued Sample Adductor Muscle F\Nu SD Sample F-TYPE IN FEMALES Mantle F\Nu SD Sample f07-59a f07-59m f07-59g f07-61a f07-61m f07-61g f07-62a f07-62m f07-62g f07-63a f07-63m f07-63g f07-64a f07-64m f07-64g f07-70a f07-70m f07-70g f07-72a f07-72m f07-72g Gonad F\Nu SD Sample M\Nu SD EGGS Sample F\Nu SD f09-16e N/D f09-16e f09-18e N/D f09-18e f07-24e N/D f07-24e f07-26e N/D f07-26e f07-35e N/D f07-35e f07-36e N/D f07-36e f07-37e N/D f07-37e f07-39e N/D f07-39e f07-40e N/D f07-40e f07-45e N/D f07-45e f07-46e N/D f07-46e NOTE. The copy numbers are normalized to the nuclear reference gene (M/Nu and F/Nu). N/D, not detectable; SD, standard deviation. noninhibited log-linear range ( Tier 1 as in Gallup and Ackermann 2008). The Tier 1 was 0.25 ng/ll; for this reason, all the samples were further diluted to ;1.25 ng/ll. Using 4 ll of this DNA dilution in the final qpcr volume of 20 ll entails an additional 1:5 dilution that makes the in-well concentration of the samples ;0.25 ng/ll. For the Ultramers standards, we prepared a dilution series, from to copies, that was loaded onto a 96-well plate in triplicates. The qpcr reaction (for the test plate, the Ultramers standards, and all the following experiments) was set up as indicated in the iq Multiplex Powermix (Bio-Rad) instruction manual. The final concentration of each probe and primer was 100 and 150 nm, respectively, and the cycle was programmed as already indicated in the Primer/Probe Specificity Tests paragraph. The quantification experiments were performed in duplicates. All the qpcr setup and optimization experiments were performed under the guidance of the Iowa State University qpcr Consultation Service (see isuqpcrconsultationservices for information [last accessed October 29, 2010]). Statistical Analyses To test whether the M mtdna distribution is different among the adductor and mantle tissues of the 32 males, we calculated the normalized M/(F þ M) values, made the arcsine transformation, and tested the results for fit into the normal distribution with the Wilk Shapiro and Kolmogorov Smirnov tests. The Mann Whitney U test is a nonparametric test for assessing whether two samples of observations have equally large value, and its alternative hypothesis is that one distribution is stochastically greater than the other. It was used to evaluate whether the total mtdna amount in males is greater than that in females (see table 3). Mann Whitney U test, Wilk Shapiro normality test, and Kolmogorov Smirnov test were performed online at VassarStats: Website of Statistical Computation (http:// faculty.vassar.edu/lowry/vassarstats.html [last accessed October 29, 2010]). Results Real-time qpcr analysis enabled quantification of mtdna variants in somatic tissues, gonads, and gametes of V. philippinarum. Altogether, 33 females and 32 males were analyzed for adductor muscle, mantle, and mature gonad. Eleven egg and 14 purified sperm samples were tested. Four hundred and forty multiplex (three targets each reaction) reactions were performed for this study, including experimental replicas. mtdna copy numbers are normalized to the nuclear reference gene. M- and F-type mtdna quantifications in female tissues are shown in table 1. Among the 33 individuals analyzed, M-type mtdna has been detected only in two: f09-18 has a predominance of M-type in both mantle and gonad (M/F ratio: 5.7 ± and ± , respectively), whereas in f09-28, it is present only in the adductor muscle where, however, F-type is about three times more abundant. The average total mtdna/ hsp70 ratio in female tissues is 1.57 (±0.095) (adductor), 0.93 (±0.465) (mantle), and 3.71 (±1.857) (gonad) (table 3). 954

7 mtdna Inheritance in Venerupis philippinarum doi: /molbev/msq271 Table 2. Quantification of M- and F-type mtdna in Male Tissues and Sperm. Sample Adductor Muscle M/Nu SD Sample M-TYPE IN MALES Mantle M/Nu SD Sample m09-1a m09-1m m09-1g m09-3a m09-3m m09-3g m09-4a m09-4m m09-4g m09-9a m09-9m m09-9g m09-10a m09-10m m09-10g m09-11a N/D m09-11m m09-11g m09-13a m09-13m m09-13g m09-15a m09-15m m09-15g m09-19a N/D m09-19m N/D m09-19g m09-24a m09-24m m09-24g m09-26a m09-26m m09-26g m09-34a m09-34m m09-34g m09-35a m09-35m m09-35g m09-36a m09-36m m09-36g m07-4a m07-4m m07-4g m07-7a m07-7m m07-7g m07-8a m07-8m m07-8g m07-12a m07-12m m07-12g m07-13a m07-13m m07-13g m07-21a m07-21m m07-21g m07-27a N/D m07-27m N/D m07-27g m07-34a N/D m07-34m N/D m07-34g m07-38a m07-38m N/D m07-38g m07-53a m07-53m m07-53g m07-54a m07-54m m07-54g m07-58a m07-58m m07-58g m07-65a m07-65m m07-65g m07-66a m07-66m m07-66g m07-67a m07-67m m07-67g m07-68a m07-68m m07-68g m07-69a m07-69m m07-69g m07-71a m07-71m m07-71g Gonad M/Nu SD F-TYPE IN MALES Sample Adductor Muscle F\Nu SD Sample F\Nu SD Sample F\Nu SD m09-1a m09-1m m09-1g m09-3a m09-3m m09-3g m09-4a m09-4m m09-4g m09-9a m09-9m m09-9g m09-10a m09-10m m09-10g m09-11a m09-11m m09-11g m09-13a m09-13m m09-13g m09-15a m09-15m m09-15g m09-19a m09-19m m09-19g m09-24a m09-24m m09-24g m09-26a m09-26m m09-26g m09-34a m09-34m m09-34g m09-35a m09-35m m09-35g m09-36a m09-36m m09-36g m07-4a m07-4m m07-4g m07-7a m07-7m m07-7g m07-8a m07-8m m07-8g m07-12a m07-12m m07-12g m07-13a m07-13m m07-13g m07-21a m07-21m m07-21g m07-27a m07-27m m07-27g m07-34a m07-34m m07-34g m07-38a m07-38m m07-38g m07-53a m07-53m m07-53g m07-54a m07-54m m07-54g m07-58a m07-58m m07-58g

8 Ghiselli et al. doi: /molbev/msq271 Table 2 Continued F-TYPE IN MALES Sample Adductor Muscle F\Nu SD Sample F\Nu SD Sample F\Nu SD m07-65a m07-65m m07-65g m07-66a m07-66m m07-66g m07-67a m07-67m m07-67g m07-68a m07-68m m07-68g m07-69a m07-69m m07-69g m07-71a m07-71m m07-71g Sample F\Nu SD SPERM Sample F\Nu SD m09-1s m09-1s N/D m09-2s m09-2s N/D m09-3s m09-3s N/D m09-4s m09-4s N/D m09-5s m09-5s N/D m09-33s m09-33s N/D m09-34s m09-34s N/D m09-35s m09-35s N/D m09-36s m09-36s N/D m07-43s m07-43s N/D m07-65s m07-65s N/D m07-66s m07-66s N/D m07-67s m07-67s N/D m07-68s m07-68s N/D NOTE. The copy numbers are normalized to the nuclear reference gene (M/Nu and F/Nu). N/D, not detectable; SD; standard deviation. Our analyses show that no M-type mtdna is present in the eggs of V. philippinarum. The results are reported in table 1. Another interesting finding is that the eggs spawned by f09-18 (having a strong predominance of M-type in the gonad, see above) do contain only F-type mtdna, meaning that the M-type is restricted to the somatic part of the ovary in this unusual specimen. The quantity of mtdna in V. philippinarum eggs is variable, ranging from about (±11.682) to (±40.949) copies per hsp70 (average ± ), and quite interestingly, the eggs with the highest ratio are those spawned by f09-18 (see table 1). Both M- and F-type mtdna were always detected in male tissues, and their quantification is shown in table 2. Most of the specimens have a predominance of M-type in somatic tissues, but it is absent in four adductor muscle samples (m09-11, m09-19, m07-27, and m07-34). The normalized M/F ratio is 3.24, and the average total mtdna/ hsp70 ratio in male adductor muscle is 5.35 (±0.234) (table 3). The arcsine-transformed M/(F þ M) values fitted into a normal distribution, suggesting no statistical differences of M/F ratio among the 32 males. In male, mantle M-type is absent in four samples (m09-19, m07-27, m07-34, and m07-38), three of which lack it in the adductor muscle too (m09-19, m07-27, and m07-34). As in the adductor muscle, the M-type is dominant in most of the samples but at a lower percentage (table 2). The normalized M/F ratio is 2.23, and the average total mtdna/hsp70 ratio in male mantle is 2.56 (±1.280) (table 3). In contrast with adductor muscle data, arcsine transformation for mantle did not fit into a normal distribution. In male gonads, the M-type is always strongly dominant, and the M/F normalized ratio ranges from a minimum of (±10.163) (m07-7) to a maximum of 3,439 (±1, ) (m09-10) (see table 2). The normalized M/F ratio is , and the average total mtdna/hsp70 ratio in male gonads is (±16.627) (table 3). Although it was present in the gonad, no F-type was detected in sperm, meaning that the purification through Percoll gradient was successful in eliminating potential somatic cells contamination and that in V. philippinarum, sperm does not carryf-typemtdna.theaveragemtdnacontentperhsp70in sperm is (±2.522) (table 3) ranging from a minimum of (±0.812) to a maximum of (±8.936) (table 2). The average total mtdna amount (M þ F) normalized to the reference nuclear gene is shown in table 3. In both males and females, it follows the trend: mantle, adductor, gonad, gametes. The highest concentration is, not surprisingly, in eggs. The ratio in male gonads is very close to the ratio in sperm, whereas there is a consistent difference between the amount of mtdna in the ovary and in the eggs. An interesting finding is that males have more Tabel 3. Average Total mtdna Amount in Females ($) and Males (#). Total mtdna $ # Significance Adductor 1.57 (60.095) 5.35 (60.234) P < Mantle 0.93 (60.465) 2.56 (61.280) P < Gonad 3.71 (61.857) ( ) P < Gametes ( ) (62.522) NOTE. Normalization to the nuclear reference gene has been calculated. The significance of the Mann Whitney U test is reported. The test has not been calculated in gametes for obvious reasons. 956

9 mtdna Inheritance in Venerupis philippinarum doi: /molbev/msq271 FIG. 1.The proposed model for germ line mitochondria selection under DUI. The arrows indicate the three DUI checkpoints. The curves represent a schematic visualization of the mtdna copy number variation during development. mtdna than females in all their tissues. The Mann Whitney U test showed that the differences in mtdna content between tissues of males and females are highly significant (P, 0.001) (table 3). The test was not performed in gametes for obvious reasons (eggs and spermatozoa are different in structure, function, and dimension, so that a comparison is not meaningful). Discussion Tissue and Gamete Distribution of the Sex-Linked mtdnas in V. philippinarum This study represents the first analysis of DUI sex-linked mtdna haplotypes with multiplex TaqMan real-time qpcr, performed according to the most recent experimental prescriptions (Bustin et al. 2009). The data we obtained enabled us toreconstruct the distribution of M and F mtdnas, andthe results are here interpreted in the light of the DUI system of V. philippinarum. The use of TaqMan chemistry allowed us to get high sensitivity and specificity and also to obtain consistent quantifications, thanks to multiplexing: As a matter of fact, the three targets (M and F mtdnas and nuclear DNA) were detected simultaneously in the same reaction for every sample, achieving a high uniformity of reaction conditions (sample, primer,probeandmastermixconcentrations,reactionvolume and efficiency, etc.), which strengthened the reliability of results. For the above-mentioned reasons, and thanks to the use of a nuclear gene (hsp70) as internal control and for data normalization, this experimental design greatly improved on the first-attempted qpcr analysis performed on a DUI species (M. galloprovincialis; Sano et al. 2007). In the Mytilus species complex (Garrido-Ramos et al. 1998; Dalziel and Stewart 2002) as well as in Musculista senhousia (Passamonti 2007), mature males contain different ratios of F and M genomes in their tissues, with gonads containing predominantly M-type and somatic tissues containing mainly F-type. Compared with mytilids, the first most striking difference is the strong dominance of M-type mtdna in somatic tissues of V. philippinarum males. This was already observed by Passamonti and Scali (2001) using PCR and cloning, but our qpcr data confirm this finding using a more sensitive and reliable technique. Although the normalized M/(F þ M) ratio in the adductor fits the normal distribution, it does not in the mantle. This different pattern may be tentatively related to the specific segregation mechanism of sperm mitochondria during male embryo development in DUI species (Cao et al. 2004; Obata and Komaru 2005; Cogswell et al. 2006; Milani et al., in preparation), which drives sperm-derived mitochondria into the primordial mesodermic blastomeres (from which the adductor derives), whereas it allows only stochastic leakage in ectodermic blastomeres (from which the mantle originates). In male gonads, both mtdnas are present, with a strong predominance of M, as expected for a DUI system. In our analysis of V. philippinarum, despite the presence of a small quantity of F-type in testes, sperm never carries F-type mtdnas, which is in contrast to what was observed by Sano et al. (2007). Actually, as stated by Venetis et al. (2006), the dependability of gamete-specific transmission of the two mitochondrial genomes is a basic requirement for the stability of DUI. To obtain their samples, Venetis et al. (2006) purified sperm through a solution of Percoll (a protocol widely used to select viable sperm for artificial insemination) to get rid of somatic cells/debris contaminations. After that, they did not detect any F-type mtdna in sperm, showing that the male germ line is protected against invasion by the maternal genome. In our analysis, we always purified sperm samples with the same protocol, and no F-type was found, thus confirming for V. philippinarum what Venetis et al. (2006) found in Mytilus. Another controversial topic regarding DUI is the transmission of M-type through eggs. Obata et al. (2006, 2007) and Sano et al. (2007) stated that M mtdna was present in all female tissues and eggs analyzed. Our results confirm its presence (although rare) in somatic tissues that could be 957

10 Ghiselli et al. doi: /molbev/msq271 explained by a malfunctioning of the elimination mechanism of sperm mitochondria (which is also not perfect in species with standard maternal inheritance, see paternal leakage in: Kondo et al. 1990; Shitara et al. 1998; Kvist et al. 2003; Xu 2005), but we observed a very different situation in female gametes, them being homoplasmic for the F-type. Thus, we find the regular and repeated cotransmission of two mitochondrial variants through the germ line of the same lineage to be very unlikely because of the genomic conflicts rationale (see Passamonti and Ghiselli 2009 and references therein). In fact, we never found M-type mtdnas in eggs of V. philippinarum. Despite the very strict homoplasmy of gametes, some females of V. philippinarum showed somatic heteroplasmy: Among 99 female tissue samples analyzed, 3 showed the presence of M mtdna (i.e., the adductor muscle of f09-28, in which the F-type was predominant, plus the mantle and the gonad of f09-18, in which M was dominant). These findings show, once again, that the elimination mechanism of sperm mitochondria is not unfailing for somatic tissues. However, the most striking piece of data here is that f09-18 spawned eggs contained only F-type mtdnas, even if they had a strong predominance of M-type in the ovary (normalized M/F ratio ± ), meaning that M-type is restricted to somatic cells only. Therefore, our qpcr observations suggest that a strict mechanism controlling the transmission of the mtdna through the germ line is present in both males and females of V. philippinarum. The M-type detected in f09-18 gonad, restricted to the somatic part of the ovary, escaped degradation during embryonic development but failed to enter the germ line, thanks to the above-mentioned selection mechanism. Mitochondrial Copy Number The mtdna copy number (roughly linked to the number of mitochondria) is under nuclear control and is related to the metabolic requirements of the cell (Marin-Garcia et al. 1994; Medeiros 2008; Clay Montier et al. 2009), so it is quite obvious that, in both males and females, the quantity of mtdna follows the pattern: gonad. adductor. mantle. The intriguing observation is that V. philippinarum males have significantly more mtdna copies than females: about 9, 3.5, and 3 times more in gonad, adductor, and mantle, respectively (P, 0.001). This finding leads to two nonmutually exclusive considerations: 1) More copies of M mtdna are required to fulfill the metabolic requirements of male somatic cells, meaning that the gene products of the M-type may possibly have a lower enzymatic activity than those of the F-type, as suggested by Breton et al. (2008), probably because of the higher mutational rate observed in M-type mtdna (Hoeh et al. 1996; Liu et al. 1996; Stewart et al. 1996; Quesada et al. 1998; Skibinski et al. 1999; Passamonti et al. 2003; Smietanka et al. 2009) and 2) M mtdnas may have a higher replication rate that may help them to overcome the F-types, when both are present in the same tissue. Following the genomic conflicts rationale, a mutation that boosts the replication rate of an organelle is positively selected, even in the presence of slightly deleterious mutations. The capacity for quicker replication is not important in somatic tissues because they are an evolutionary dead end for mitochondria, but it becomes fundamental when M mtdna has to invade the germ line and replace the F- type. The mechanisms that allow M-type mitochondria to displace F-type ones in the male germ line are unknown. Oocytes of many organisms (amphibians, fishes, insects, planarians, chaetognaths, nematodes, and some mammals) possess a cytoplasmic region called germplasm or pole plasm that segregates with the germ line and is necessary for its specification (Kloc et al. 2004). It usually contains the Balbiani body, which consists of an electrondense material rich in ribonucleoproteins (nuage) and mitochondria. Some of these mitochondria during oogenesis aggregate with germ line determinants (reviewed in: Kloc et al. 2004), and it is pretty obvious that in DUI species, the amplification of M mitochondria in primordial germ cells (PGCs) of males has to overcome or prevent the replication of F mitochondria coming from the Balbiani body, so that they can replace them and enter the germ line. An M- type mitochondria replicative advantage was proposed by Cogswell et al. (2006), and it might account for the differences between M- and F-type control regions (CRs). Above all, it could explain why segments of an M-type CR are always present in the CRs of recently masculinized mtdnas (i.e., Burzynski et al. 2003, 2006). As a matter of fact, mtdna CR contains the signals that control RNA and DNA synthesis, and M-type CR could contain enhancers or other structures that favor a faster replication. Another hypothesis could be a complete prevention of F- type mtdna replication and/or its degradation. Both hypotheses still have to be investigated. Finally, the strong (most times predominant) presence of M mtdna in male somatic tissues of V. philippinarum raises some considerations concerning the mechanism of active segregation of sperm mitochondria in male embryos. Although in mytilids, this system seems to be quite precise because the presence of M mtdna outside the gonad is rare, a leakage of M-type is evidently very frequent in V. philippinarum. The Mitochondrial Bottleneck Mature mammal oocytes contain at least 100,000 copies of mtdna (one to two copies per organelle). Despite this high copy number, mtdna sequence variants segregate rapidly between generations, and this fact led to the concept of a developmental bottleneck for the transmission of mtdna (Olivo et al. 1983; Laipis et al. 1988; Ashley et al. 1989). Embryonic mitochondria originate from a restricted founder population present in the PGCs and undergo amplification during oogenesis. The bottleneck provides a homoplasmic population of mitochondria in the embryo despite the high mutation rate of mtdna, and the survival of the cleaving embryo depends on its 958

11 mtdna Inheritance in Venerupis philippinarum doi: /molbev/msq271 inheriting a functional complement of mitochondria from the egg (Dumollard et al. 2007). In V. philippinarum, the mtdna quantity in eggs (on average ;300 mtdnas per hsp70; see table 3) is significantly lower when compared with mammals, which is not surprising given the different complexity and metabolic requirements. One may think that in DUI species, M and F mitochondria experience two very different bottlenecks because the spermatozoon only contains few mitochondria (usually four to five), whereas in V. philippinarum, the egg contains hundreds of them. Nonetheless, the bottlenecks are not so different because sperm mitochondria probably derive from the fusion of many small ones, so they carry many copies of mtdna. Actually, our data indicate that about 34 mtdna molecules per hsp70 copy are present in V. philippinarum spermatozoon. The mechanisms for mitochondrial bottleneck are largely unknown: Is it just a random subsampling (i.e., the mitochondria that, by chance, end up in a germ cell/gamete), or is there any active selection? According to Shoubridge and Wai (2007), there is genetic evidence that segregation of mtdna sequence variants is a stochastic process that occurs during the mitotic divisions of the PGCs because in their experiments, no strong selection against oocytes carrying pathogenic mutations was observed. On the other hand, according to Dumollard et al. (2007), the localization of mitochondria in the egg during maturation and their segregation to blastomeres in the cleaving embryo are strictly regulated. Gradients in the distribution of mitochondria in the egg entail different numbers of mitochondria, first in blastomeres and then in tissues (differences that we observed in the present work, see Results). Such differences in mitochondrial distribution are thought to play roles in defining the long-term viability of blastomeres and embryonic axes and patterns. In ascidians, a mitochondria-rich domain spanning the egg vegetal subcortex has been found to be maintained during development and to segregate exclusively with the muscle lineage (Roegiers et al. 1999). Sea urchin eggs have a higher density of mitochondria in a region that is segregated to the oral pole of the embryo, and artificial manipulations of this mitochondria gradient can reorientate the oral aboral axis of the embryo (Coffman et al. 2004). All these studies showed that the location of mitochondria during embryonic development is somehow regulated rather than purely stochastic. Can we say the same about the segregation of germ line mitochondria? What we observed in our analyses is that there must be a very efficient mechanism for selection of such mitochondria in V. philippinarum because, despite the leakage during M-type active segregation in males and the sporadic malfunctioning of M-type selective elimination in females, both sperm and eggs are strictly homoplasmic for their sex-specific mtdnas. It is not clear whether the PGCs are formed only once at the beginning of clam life or if they arise every time from somatic cells during seasonal development of gonads (i.e., during the reproductive season). However, this would not change the selective process: In the first case, there would be a single selection event in germ line mitochondria and in the second case, there would be many. It is a matter of conjecture whether this mechanism is restricted to DUI species only or whether it is rather a variation of a more common mechanism that usually segregates mitochondria to the germ line. A Model for DUI mtdna Segregation: A Working Hypothesis Two main selection processes operate on sperm-derived mitochondria to grant (in males) or prevent (in females) their invasion of the germ line and therefore their transmission to the next generation. In chronological order, the first process (which we refer to here as DUI Checkpoint #1; see fig. 1) is the active segregation of sperm mitochondria, as observed in male embryos of M. edulis (Cao et al. 2004; Cogswell et al. 2006) and M. galloprovincialis (Obata and Komaru 2005). This mechanism starts from the very first embryo division, and the aggregate pattern of M mitochondria is observed in male embryos until the D-larvae stage. Beyond that stage (and very often even before), mitochondrial staining is no longer visible, but the most acknowledged hypothesis is that the aggregate ends up first in the mesoderm, then in the gonad. The massive presence of M mtdna we observed in the somatic tissues of V. philippinarum males suggests that male mitochondrial leakage does happen frequently during embryo cleavage; as a consequence, M mtdnas are commonly present in other tissues of mesodermic derivation, like the adductor muscle, and even in tissues of ectodermic derivation, like the mantle. Checkpoint #1 appears to be more relaxed in V. philippinarum than in mytilids, and more analyses are needed to understand exactly how this happens. A second mechanism (DUI Checkpoint #2 in fig. 1) operates in females, and it works exactly as it does in standard maternal species. The sperm-derived mitochondria are selectively degraded, and this process has been related to ubiquitination (Sutovsky et al. 1999): The sperm mitochondria are labeled with ubiquitin during spermatogenesis and are degraded shortly after fertilization (between third and fourth division) (Shitara et al. 1998). In DUI species, this process is blocked in males and occurs later in females (not before the D-larvae stage: Cao et al. 2004; Cogswell et al. 2006). The finding of M-type mtdna in females shows that this mechanism is not always error free. Our analysis showed that both DUI Checkpoint #1 and Checkpoint #2 are not perfect in V. philippinarum. As a consequence, the strict mtdna homoplasmy we observed in sperm and eggs may require the presence of a third selection mechanism, which we refer to as Checkpoint #3 (fig. 1). This mechanism would act as a filter for germ line mitochondria, and it may operate when PGCs establish themselves (i.e., whenever PGCs do separate from somatic cells). This Checkpoint #3 should be considered as the mitochondrial bottleneck for DUI species. Given the supposed origin of DUI from standard maternal inheritance, Checkpoint #3 is probably established with a similar molecular machinery for both. 959

12 Ghiselli et al. doi: /molbev/msq271 Supplementary Material Supplementary materials are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/). Acknowledgments We gratefully thank: Prof. Giovanni Perini and Prof. Giuliano Della Valle (Functional Genomics R.U. University of Bologna) for letting us to use the Bio-Rad iq5 Optical System in their laboratory; Dr. Jack Gallup (qpcr Consultation Service Iowa State University) for precious help and guidance with the qpcr setup and optimization; Dr. Silvia Ghesini and Dr. Federico Plazzi for their help in statistical analyses; and the anonymous reviewers for their valuable comments that improved the manuscript. This work was supported by the Italian Ministero dell Università e della Ricerca Scientifica funding (PRIN07) and by the Donazione Canziani bequest. References Ashley MV, Laipis PJ, Hauswirth WW Rapid segregation of heteroplasmic bovine mitochondria. 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