DIRECT EVIDENCE OF SEX IN SYMBIODINIACEAE AND A HYPOTHESIS ABOUT MEIOSIS

Dinoflagellates in the family Symbiodiniaceae are obligate endosymbionts of diverse marine invertebrates, including corals, and impact the capacity of their hosts to respond to climate change-driven ocean warming. Understanding the conditions under which increased genetic variation in Symbiodiniaceae arises via sexual recombination can support efforts to evolve thermal tolerance in these symbionts and ultimately mitigate coral bleaching, the breakdown of the coral-Symbiodiniaceae partnership under stress. However, direct observations of meiosis in Symbiodiniaceae have not been reported, despite various lines of indirect evidence that it occurs. We present the first cytological evidence of sex in Symbiodiniaceae based on nuclear DNA content and morphology using Image Flow Cytometry, Cell Sorting and Confocal Microscopy. We show Symbiodiniaceae cells undergo gamete conjugation, zygote formation and meiosis within a dominant reef-building coral in situ . On average, sex was detected in 1.5% of the cells analyzed (N=10.000-40.000 cells observed per sample in a total of 20 samples obtained from 3 coral colonies). We show that meiosis follows a two-step process described in other dinoflagellates, in which diploid zygotes form dyads during meiosis I and triads and tetrads as final products of meiosis II. This study sets the stage for investigating environmental triggers of Symbiodiniaceae sexuality and can accelerate the assisted evolution of a key coral symbiont in order to combat reef degradation. and to identify Symbiodiniaceae with and that can definitively of complete compatible the identification of zygotes the of and aligns a two-step 29,45,46 ). Below, we highlight key DNA content and cell morphology observations that allow us to establish differences between mitosis and meiosis in Symbiodiniaceae compare our hypothesis for sex in to the sexual in other and highlight outstanding questions regarding the conditions that promote Symbiodiniaceae sex.


INTRODUCTION
Reef-building corals and other marine invertebrates establish obligate symbioses with a diverse group of dinoflagellates in the family Symbiodiniaceae (reviewed in 1,2 ). This symbiosis can be disrupted by environmental stressors including elevated sea surface temperatures (SSTs) and increased UV radiation, resulting in bleaching-the mass loss of Symbiodiniaceae cells and/or chlorophyll from the hostand frequently, host mortality 3,4 . Thermal stress due to anthropogenic climate change is recognized as the leading cause of coral reef degradation [5][6][7] . Despite this, the onset of coral bleaching from 2007 to 2017 occurred at significantly higher SSTs (+~0.5°C) than over the decade preceding that period 8 . This suggests that thermally susceptible genotypes may have adapted and/or declined such that the thermal threshold for bleaching has increased. As reefs continue to experience thermal stress under committed (and likely additional) warming due to climate change, supporting the assisted evolution of thermal tolerance in Symbiodiniaceae 9 is critical to increasing reef resilience 10 and contributing to the restoration of ecologically and economically valuable ecosystems 11 .
The most direct mechanism for adaptation to environmental challenges is sex 12,13 . Sexual recombination of parental genotypes during meiosis promotes new (and potentially beneficial) genetic combinations in offspring, the basic prerequisite for evolution via natural selection. Indeed, various field observations and experimental evolution studies across diverse taxa have documented that stressful or novel environments can select for higher levels of sexuality 14 , and microorganisms, including Symbiodiniaceae, are predicted to have a high adaptive capacity in selective environments 15,16 . Meiosis is the hallmark of sex, consisting of two nuclear divisions (karyokinesis) and one simultaneous or two successive cytoplasmic divisions (cytokinesis). Recombination in meiosis 'remixes' genetic material from both parents to increase genetic variation in the progeny, in contrast to mitosis-the division typical of ordinary cell growth-where daughter cells have the same number and kind of chromosomes as the parent cell 17 .
More than 10% of the approximately 2.000 known marine dinoflagellate species produce cysts and are thought to undergo facultative sex as part of their life cycle 18 . In these dinoflagellates, reproduction is primarily asexual (through mitosis, Fig.  1A), but sex can be induced within a subset of cells in a population under certain environmental conditions. Foundational studies, dating back to the 1970s, linked dinoflagellate sexuality to the formation of highly resistant, benthic stages ('resting cysts'), considered a mechanism for surviving harsh environmental conditions 19 . When resting cysts germinate, meiosis results in the release of novel genotypes that are potentially better adapted to local conditions. Although dinoflagellate sex was first proposed to be rare in nature 20 , research over the last decade revealed that sex in these microeukaryotes is a relatively frequently and flexibly utilized reproductive mechanism. The capacity for sex in dinoflagellates is also now recognized as independent of a species' ability to form resting cysts (see reviews by 21,22 ). Initial studies of Crypthecodinium cohnii suggested that dinoflagellates could undergo only a one-step meiosis 23 (Fig. 1B.1), but later works on different species consistently reported the existence of a two-step meiotic process ( 24 and references therein). In two-step meiosis, there is a delay in meiosis II: a single division occurs in the zygote, whereas the second division takes place at postzygotic stages ( Fig. 1B.2). Despite these advances, sexuality remains difficult to identify in most dinoflagellate species due to (i) morphological similarities between sexual and vegetative stages; and (ii) the potential for co-occurrence of 2C DNA content stages derived from both mitosis (haploid) and gamete fusion (diploid) within the same population of cells. Given this, a general consensus has emerged that the detection of a 4-fold DNA content stage, which is formed during meiosis (but not mitosis), is key to identifying sex in dinoflagellates [25][26][27][28][29] .
A growing body of molecular evidence shows that Symbiodiniaceae possess functional sexual machinery, and thus suggests that these key coral reef symbionts can reproduce sexually. Indirect evidence for sexual reproduction in this group includes: i) the existence of a sufficient inventory of essential Symbiodiniaceae meiotic genes 30,31 , as well as genes related to gamete formation 32 ; and ii) population-level genetic signatures [33][34][35][36] and codon usage trends 32 most parsimoniously interpreted as arising from meiotic recombination. Upregulation of meiosis-related genes has also been documented to occur under thermal stress 37,38 . This temperature-associated regulation suggests that sexual reproduction may be key for the adaptation of Symbiodiniaceae under current warming trajectories, driven by climate change.
In contrast to this growing body of evidence, genomic evidence for the absence of canonical synaptonemal complex (SC), as well as for a reduced set of cohesin complex genes 32 , have been reported from this dinoflagellate family. The synaptonemal complex (SC) mediates the pairing of homologous chromosomes during the early stages of meiotic prophase I and cohesin proteins play a role in sister chromatid cohesion. However, the absence of the SC and reduction of cohesin complex genes does not preclude meiotic capability in Symbiodiniaceae; similar patterns have been reported in other dinoflagellates known to be sexual 32 .
Despite strong molecular evidence of sexual reproduction in Symbiodiniaceae, no direct cytological proof for fertilization and meiosis in this group have been available. The first cytological descriptions of Symbiodiniaceae (which was previously recognized as a single genus, Symbiodinium 2 ) life cycle stages 39 indicated the existence of motile gymnodinoid zoospores and vegetative cells (the dominant, non-motile stage). Freudenthal 39 observed that vegetative cells (haploid) either divide by binary fission or form cysts, which are characterized by a thicker wall ( Fig. 2A). Cysts could divide or turn into a zoosporangium, which could either release a swimming gymnodinoid zoospore or remain as a non-motile spore (aplanospores, Fig. 2A). In cultures described as "old", which could indicate nutritional deficiencies, cysts were observed to contain dividing autospores (according to their external morphology, typically two and rarely four). Under certain conditions (not clarified, although the cultures used were clonal), cysts could even give rise to multiple cells resembling a process of gametogenesis. However, morphologies related to gamete conjugation were not detected 39 . Fitt and Trench 40 subsequently argued that the term "coccoid stage" should be used to describe the non-motile form of Symbiodiniaceae (as opposed to "cyst"). This is because "cyst" in dinoflagellates is usually related to a dormant (non-active), resistant (thick wall) stage, whereas "coccoid stage" can be used independently of a cell's metabolic activity or cell wall thickness (a highly variable character). Instead, the haploidy of the coccoid (vegetative) stage was considered key to sexuality by Fitt and Trench 40   Progression of mitosis was evident in two-nuclei "2C" cells (e.g., Fig. 5C-F) based on the presence of an equatorial constriction which is formed after nuclear division. As division progresses, the pyrenoid is shared (Fig. 5D, arrow), to later appear clearly in each cell ( Fig. 5E-F, arrows), whereas the accumulation body remains single and unshared (Fig. 5E, arrowhead). At the final mitotic stage, nuclei were positioned either opposite or adjacent to each other (Figs. 5E and F, respectively). In the other group of "2C" cells with a single nucleus, classified as non-mitotic cells (Fig. 6), two pyrenoids and accumulation bodies were observed, and the outer morphologies suggested the existence of two newborn cells (splitting apart) or two fusing cells (i.e., mating). Two accumulation bodies (e.g., Fig. 6A-C arrowheads) and two pyrenoids (e.g., Fig. 6D, arrows) were observed in these cells.

DISCUSSION
Foundational studies previously generated evidence delineating much of the Symbiodiniaceae life cycle, and strong molecular evidence indirectly supported the existence of a sexual phase in this group of dinoflagellates. However, cytological proof of sexual reproduction in Symbiodiniaceae was still needed to advance our understanding of the basic biology of the ecologically and economically valuable Symbiodiniaceae-coral mutualism, and to catalyze subsequent research into when, where, and how sex occurs in this dinoflagellate group. This study is the first to apply cutting edge approaches (IFC, sorting and confocal analyses) to identify Symbiodiniaceae cells with DNA content and nuclear processes that can definitively be interpreted as sexual activity. This first description of a complete nuclear progression compatible with sex in Symbiodiniaceae, ranging from the identification of putative zygotes to the formation of dyads, triads and tetrads, aligns with a meiotic two-step process already described in other dinoflagellates (e.g. 29,45,46 ). Below, we highlight key DNA content and cell morphology observations that allow us to establish differences between mitosis and meiosis in Symbiodiniaceae cells, compare our hypothesis for sex in Symbiodiniaceae to the sexual stages reported in other dinoflagellate species, and highlight outstanding questions regarding the conditions that promote Symbiodiniaceae sex.

Identifying sexual stages: Key differences between mitotic and meiotic cells
Image flow cytometry (IFC) indicated that most of the Symbiodiniaceae cells processed in this study fell into a single group representing the vegetative stage, characterized by low DNA content ("1C", haploid) and a single, oval to round-ish nucleus. As recently shown in other dinoflagellate species 47,48 , close examination of the haploid cells with confocal microscopy showed that chromosomes were not all identical as previously thought 49 , but in fact, highly variable in size (e.g., Fig. 4C').
Other single nucleus cells fell between "1C" and "2C" DNA contents and were interpreted to be replicating their DNA as part of the mitotic cycle ("S" phase).
However, some cells with a single nucleus had a DNA content higher than 2C; these cells were consistent with a replicating zygote in meiosis I during a two-step meiosis ( Fig. 1B.2).
Following a similar dichotomy to the cells with one nucleus, cells with two nuclei could have either 2C DNA content and be in a mitotic cycle, or have a DNA content >2C and be in a non-mitotic cycle. The two nuclei of cells in this latter group varied in size, shape and chromatin condensation state; such cells were interpreted as precursors to a triad stage (i.e., cell with three nuclei). To distinguish cells that were part of this non-mitotic sequence, cells with two nuclei and DNA content between "2C" and "4C" were considered dyads, as opposed to what we will simply call "mitotic coccoid stages". Cells with three and four nuclei (triads and tetrads, respectively) were also detected. Although the morphologies of these stages were difficult to analyze in the IFC images (Fig. 3D-E), such cells could be examined at higher magnification using sorting and confocal microscopy. The variability observed in tetrad morphology (Fig. 9) indicates these cells were dividing stages derived from triads (Fig. 8), in what we interpret to be a delayed Meiosis II. Taken together, we infer that the observed Symbiodiniaceae tetrad cells were undergoing two-step meiosis (Fig. 1B.2); this reproductive strategy has been observed in most studied dinoflagellates. For example, Prorocentrum micans and Prorocentrum minimum form tetrads as a final meiotic product 29 . Additionally, asynchronous divisions of the zygote in these Prorocentrum species also lead to the formation of triads 29 . It should be noted that some free-living dinoflagellates (e.g., members of the genus Alexandrium) produce chains of cells during two consecutive mitotic divisions. In these Alexandrium species, cell chains are formed frequently and are readily detectable in any growing culture. Since  5). Some of the 2C-single nucleus cells in this study were therefore identifiable as zygotes because they contained two accumulation bodies (Fig. 6A-C); such cells could not have been undergoing mitotic division.
The nuclear morphology and pyrenoid count of some cells also allowed zygotes to be distinguished from mitotically dividing cells. For example, 2C-two nuclei cells with duplicated pyrenoids would only be observed at advanced stages of mitotic division; such cells were frequently detected in our study (e.g., Fig. 5C-D). However, we also observed cells that contained only one "2C" DNA content nucleus but had two well-developed pyrenoids (Fig. 6D, Fig. 7A); such cells could not have been undergoing mitosis and were interpreted as fusing gamete products or early stage zygotes (these differences in accumulation bodies and pyrenoids are summarized in Fig. 2B.2 mitotic stage versus 2B.3 zygote).

Sexual stages of Symbiodiniaceae are similar to those reported in free-living dinoflagellates
In free-living dinoflagellates, the identification of zygotes in non-resting stage cells (planozygotes) has been impeded due to the high morphological similarity between planozygotes and mitotic cells. In past studies, the number of flagella was considered the hallmark of a zygote (although this characteristic is unreliable due to the weak nature of flagella under fixation). Number of flagella is inapplicable to Symbiodiniaceae, however, since this family alternates between mobile and coccoid stages ( Fig. 2A), the latter of which lacks flagella. Therefore, the key to morphological discrimination of zygotes in Symbiodiniaceae (and other dinoflagellates in non-resting stages) lies in differentiating features of sex from mitosis, either during nuclear fusion of gametes or the meiotic process. The process of zygote formation we posit for Symbiodiniaceae here is very similar to that observed in other dinoflagellates. For example, during gamete fusion in the naked dinoflagellates Gymnodinium catenatum and Gymnodinium nolleri, karyokinesis occurs first, and, during the process, one gamete nucleus migrates to the position of the other, and they fuse. This occurs while the cell wall is in early stages of fusion, allowing two fusing cytoplasms to be distinguished 45,46 (Fig. 10, first row). In other species, such as Prorocentrum micans, the process looks similar at the nuclear level, but the cytoplasms never fuse, and instead, one of them degenerates 26 . Early stages of meiosis in Gymnodiniaceae are characterized by a big and round-ish zygotic nucleus that changes into a bi-lobed form with a central 'cytoplasmic channel', and a DNA-decondensed state in which chromosomes appear thinner (Fig. 10, second row). In dinoflagellates, chromosome segregation occurrs via binding to the nuclear envelope surrounding the cytoplasmic channels and microtubule bundles 53 . Although the formation of cytoplasmic channels was first described during mitosis 54,55 , it was later also confirmed to occur during meiosis. Specifically, a main channel centrally positioned during meiosis I is often visible via conventional fluorescence microscopy 45,46 . Given this, some of the one- These researchers indicated that during Symbiodiniaceae cell division, karyokinesis (nuclear division) occurs first, and later an equatorial zone of constriction in the cytoplasm separates the two daughter cells, which split pyrenoids but not accumulation bodies. As occurs in other dinoflagellate species (e.g. 45,46 ), we propose that nuclear fusion is faster than cytoplasmic fusion during the process of gamete conjugation, given the existence of cells with elongated external shapes and duplicated pyrenoids and accumulation bodies but a single "2C" DNA content nucleus; such cells further corroborate ongoing gametogenesis and zygote formation. Thus, the hypothesized Symbiodiniaceae life cycle (summarized in Fig. 2A) put forward by initial, foundational works 39,40 constitutes the foundation of our updated life cycle (Fig. 2B-C, Supp. Fig. 1). This study documents the entire meiotic process in Symbiodiniaceae, and includes: i) the novel observation of (2C-4C) DNA content cells with a single nucleus (i.e., direct proof of meiosis); ii) the identification of previously unpredicted intermediate stages, including dyads and triads; and iii) the first images of tetrads, which had a relatively linear morphology, compared to the previously described coccoid morphology (Fig. 2B4 and Fig. 9 vs Fig. 2A4, respectively). Integrating our observations with foundational works, we provide a revised proposed life cycle for Symbiodiniaceae in supplementary figure S1.

Outstanding questions regarding Symbiodiniaceae sex
Both techniques (IFC and conventional flow cytometry) applied in this study indicated that sex occurs rarely in Symbiodiniaceae in hospite (typically occurring in less than 1% of cells observed in a sample and at a maximum of ~5% of cells); sex was only detectable using high resolution imaging following cell sorting. Conventional flow cytometry has a lower capacity to discriminate cells from other fluorescent particles or aggregates; this is likely why the technique reported a slightly higher percentage of cells with (2C-4C) DNA content than IFC analyses (Table 1). Regardless, findings from both techniques agree with previous molecular analyses that indicated Symbiodiniaceae display a mixed reproductive strategy, which is mainly asexual with occasional to frequent sex 35 . In free-living dinoflagellates, sex also originally appeared to occur rarely and as a last resort in case of severe nutrient deficiency 20 . However, later studies concluded that sexual reproduction in these dinoflagellates is probably more common and flexible in nature than previously thought, although induced under species-specific environmental conditions 21,22 . Now that sexual stages are confirmed for Symbiodiniaceae, we can investigate the environmental factors promoting sex and the physiological details of the sexual stages documented in this study. Although no relationships between sexuality and time of day or temperature stress were detected in the present study, more comprehensive analyses are needed to investigate their possible role as triggers of sexuality (Fig. 11). For example, certain experimental designs have previously missed meiosis in other dinoflagellates, since this process can be strongly entrained with specific points in the diel cycle 28 . The abiotic triggers of sexuality and other research directions that are likely to advance our understanding of the role and constraints on Symbiodiniaceae sex in nature are summarized as outstanding questions in Figure 11. Pursuing such lines of inquiry will shed light on the evolutionary implications that may accompany the induction of new genetic diversity in Symbiodiniaceae via enhanced sexuality, including the potential for rapid symbiont adaptation to thermal stress 58 .

Conclusion
This study is the first to categorically demonstrate sexual reproduction in Symbiodiniaceae, establishing a foundation from which to explore the potential role of symbiont evolution in coral resilience to global change. Based on DNA content and morphological evidence, we propose that Symbiodiniaceae species may follow the same two-step meiotic process described for other dinoflagellates, in which the first meiotic division produces a dyad of cells, whereas the second division produces an intermediate triad state, with meiosis II ultimately resulting in a tetrad stage of haplontic cells. Beyond basic biology, understanding sexuality in Symbiodiniaceae can advance experimental evolution work on this group, with the goal of enhancing the capacity of coral holobionts to cope with warming ocean temperatures and other stressors under rapid global change. Restriction enzyme digests of LSU rDNA PCR amplicon products were conducted with Taq1 and Hha1 (New England BioLabs) following 60 . Colonies were then split in half and maintained in flow-through seawater aquaria; half of each colony was subjected to an elevated temperature treatment (~3°C above ambient), while the other half was maintained at ambient temperatures. To maximize the chance of detecting meiotic events, samples were collected and preserved from both the heated and ambient tank intermittently throughout the day and night over a three-day period (Table 1). For each sample, one branch (~5cm 2 ) was removed from a given coral colony, airbrushed using 0.22µm filtered seawater, and homogenized using a Fisherbrand 150 handheld homogenizer (Waltham, Massachusetts). The homogenate was filtered through 70µm mesh and fixed to a final concentration of 3% formalin. Fixed cells were concentrated by centrifugation and stored at 4°C until further processing. Details of the samples analyzed in this study (n=6 time points and 20 replicates total from 3 coral colonies) are provided in Table 1.

Flow cytometry analyses
Imaging Flow Cytometry (IFC). Samples were centrifuged at 7000 × g for 5 min and the pellet resuspended in 2.5 mL of cold methanol, where they were stored for at samples and was named "1C" (Fig. 3A). This peak indicates the basic DNA content of Cladocopium cells examined. A region with the same width as "1C" was centered at 2x the Geometric mean of the 1C population (based on PI fluorescence, channel 4) and named "2C" (Fig. 3A). Cells detected in the "2C" region could be ready to undergo mitosis, or be zygotes (Fig. 1). The region in between "1C" and "2C" was termed "S" phase ( Fig. 3A); cells in this region were interpreted to be in the DNA synthesis phase.
A final region was established that ranged from the end of the "2C" region to the cells with the highest detected DNA fluorescence, which had approximately "4C" mean positions. This region was called "2C-4C" (Fig. 3A) and could include cells ready to undergo two-step meiosis (Fig. 1B.2). Additionally, regardless of the number of nuclei they contained, cells were classified as "individual" (i.e., one cell observed) or as "in cell chains" (i.e., more than one cell observed) according to the nuclear-aspect ratio (width vs. height of the mask used to more precisely adjust the area to the U-shaped nucleus) and the cell area. The precision of the aspect ratio adjustment was made manually by studying the acquired images. During data collection, between 40.000-70.000 events were acquired in the gating region selected for the Symbiodiniaceae population.

Cell sorting
Cells were prepared for sorting using the same fixation and staining protocol described above for IFC analyses. Cells were sorted at low speed and in high purity mode on a SH800Z cell sorter (Sony Biotechnology Inc.) equipped with a 488-nm diode laser. Peaks were analyzed using the SH800Z software, and three regions of DNA content were established as described above in the IFC analyses. The entire sample was sorted to increase the number of rare events following two sorting rounds: First, "C" and "2C" cells were sorted until approximately 5.000 events were sorted into the "1C" population (control). The rest of the sample was sorted in a second round separating "2C" from ">2C-4C"cells. For statistical analyses, populations with different DNA content regions were analyzed using histograms of PI fluorescence in linear scale using FlowJo TM 10.7.1

Confocal Microscopy
Sorted cells were observed using a confocal LEICA SP8 microscope equipped with 3 laser lines (405, 488 and 552 nm) after mounting the cells on slides using ProLong TM Gold medium antifade reagent (Invitrogen). The preparations were allowed to rest 1-2hrs before observations were performed. To improve spatial analyses and decrease the probability of misinterpretation during visual scoring, all images were extracted from 3D videos; all videos are available as Supplementary Files S2-S7. Imaging was performed at 63x or 100x magnification using the super-resolution mode LIGHTNING. Images were optimized for best contrast and brightness, and then analyzed, using LASX software (LEICA Microsystems).

Statistical analyses
Basic statistics (mean, standard deviation (SD)) and tests for equal means (t-tests) comparing treatment conditions were performed using Past 4.02 software 62 . All tests were performed with a significance level of p-value = 0.05.  Previously published direct observations include the production of two mobile haploid cells (mastigotes, referred to as 'zoospores' by Freudenthal 39 ) from mitosis within the coccoid stage (termed 'cysts' or 'aplanospores' by Freudenthal 39 ), which could behave as isogametes or transform into coccoid stages. The formation of zygotes through gamete fusion, as well as the formation of tetrads (called 'autospores' by Freudenthal 39 ) via meiosis were hypothesized but not documented. B. Schematic view of the results of the present study in relation to the previously proposed Symbiodiniaceae life cycle (in Fig. 2A). Discriminating morphological features (nuclei, pyrenoids and accumulation bodies) are shown in the sexual stages unless in dyads, triads and tetrads, as these stages are transitory and were found in different evolving grades. C. Confocal images corresponding to the proposed sexual stages depicted in Fig. 2B.     Fig. 7. Confocal images of cells sorted in the (>2C-4C) DNA content gate that have one nucleus or two nuclei. Chromatin was less condensed in these cells, in which two pyrenoids were detected (A, arrows). A central cytoplasmic channel (cc, bent arrow) was observed in one of these cells (B). Cells with two nuclei (dyads) were considered meiotic and are distinct from two nuclei mitotic cells due to their higher DNA content and the different morphology and shape displayed between both nuclei (C-F). Dividing furrows in dyads are indicated by an arrow.     Table 1. Details of the Symbiodiniaceae samples analyzed in this study and the % of DNA content classifications made from each sample. All samples analyzed (Sample ID) originated from three colonies of Pocillopora species complex (Colony ID) collected from the north shore forereef in Mo'orea, French Polynesia during the dry season (July 2019). Pocillopora colonies contained Symbiodiniaceae in the genus Cladocopium. Treatment Condition: C: Control, ambient temperature, ~27°C; H: Heat stress, ~30°C. 'Hours after T0' indicates the cumulative hours that corals had been exposed to the treatment condition at the time of sampling. Analysis Type: IFC = Image Flow Cytometry; Sorting= Sorting Flow Cytometry. A minimum of 10.000 cell nuclei were analyzed for DNA content in each sample.