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This is the first comparative study of commercial scallop species in the Pacific coast of the MP combining morphological and molecular characters. Our phylogenetic analyses highlight the association between A. natans and Ad. colbecki; two members of monospecific tribes and last extant representatives of their Southern Ocean-restricted genera.These results confirm the presence of both Magallanes scallops in the MP, as well as the so-far unsuspected presence of mixed “banks” where both species occur in sympatry. The BND/VH ratio helps discriminate between two distinct entities that belong to the genetic lineage of Z. patagonica and to a different lineage, highly divergent from the former, which corresponds to A. natans. A. natans is the only species of a whole lineage with a particular phylogenetic value, therefore having developed and tested an accurate identification criterion for both scallops will allow efficient fishery management in the future.Here we discuss the phylogenetic position and the taxonomic status of both Magallanes scallops, as well as the implications of these results for the future management and conservation of Z. patagonica and A. natans in the Magallanes Region. Despite the numerous classifications built on morphological, ecological or molecular data, the relationships among pectinids are still under constant modification depending on the number of taxa, loci, length of the sequence and the selected outgroups1,4. The work of Alejandrino et al.7 is the most inclusive so far in terms of taxon sampling, with 81 species. Although Scherrat et al.25 included 143 species, the node supports of the phylogenetic trees are not provided, making it difficult to assess the robustness of this large phylogeny. In order to define the phylogenetic position of Zygochlamys patagonica and Austrochlamys natans, we included 93 pectinid taxa (43 genera) representative of tribes Chlamydini, Crassadomini, Fortipectini, Palliolini, Aequipectinini, Pectinini and Amussini. Comparing to Waller’s5 and Dijkstra’s15 classifications, only the subfamily Camptonectinae and the tribe Mesoplepini are missing. We used three ribosomal regions (one nuclear and two mitochondrial). Compared to Alejandrino et al.7, histone H3 is missing here, however this locus is among the least informative4. The family Pectinidae appears to be monophyletic with high support values (Fig. 5, S2), as previously demonstrated4,7,26,27,28. According to Dijkstra15 there are currently five subfamilies of Pectinidae, two of which are absent from our analysis: Camptonectinae and Pedinae. This topology supports the classifications of Waller5 and Dijkstra15, except for the position of the tribe Austrochlamydini.Our Magallanes scallops separated into two very divergent clades: Z. patagonica is associated with its conspecifics and congenerics in a single lineage (Fig. 5), which also contains species of Veprichlamys and Talochlamys. This lineage already appeared well supported as the sister clade to Palliolinae and Pectininae in Alejandrino7. For the first time, Talochlamys dichroa and T. gemmulata are nested with high support values into the Zygochlamys clade, making this latter genus paraphyletic (Fig. 5). These taxa are all restricted to high latitudes of the Southern Ocean. Due to phylogenetic and geographic affinities, we suggest that these three genera may constitute a tribe separate from Chlamydini. Since Dijkstra15 moved the two Atlantic ‘Crassadoma’ into the genus Talochlamys, the affinities among Talochlamys spp. had not been explored until now. Talochlamys species rather associate according to geographic affinities, splitting the genus into two highly divergent entities corresponding to European and New Zealand Talochlamys. A systematic revision of these four species would be useful.Austrochlamys natans associated with the Palliolinae, which was elevated to a subfamily rank by Waller5. Of the three extant tribes that compose this group, Mesopleplini are missing from our phylogenetic analyses. We included 4 genera (8 species) of the remaining two tribes: Adamussium (Adamussini) and Palliolum, Pseudamussium, Placopecten (Palliolini). The present sampling of Palliolini is the most inclusive to date and led to the monophyly and full support of the tribe Palliolini. Our phylogenetic results do not support any of the previous classifications of the tribe Austrochlamydini1,5,9,13,15, and introduce this monospecific tribe as a new member of the subfamily Palliolinae. Indeed, Austrochlamys natans clusters together with Adamussium colbecki, both in a sister clade to Palliolini. The first molecular characterization of Ad. colbecki did not lead to a clear classification due to the low polymorphism of the 18S26. Later, Ad. colbecki appears either as sister species to Chlamydinae or to Palliolini, depending on tribe sampling and the choice of outgroup and loci4,10,11. However, in the most recent and inclusive studies of taxon sampling7 (present study) or genomic cover29, Ad. colbecki is the sister group of the tribe Palliolini, as in the present phylogeny.The subfamily Palliolinae originated from a Chlamydinine ancestor in the Cretaceous and subsequently underwent diversification in the Northern Hemisphere1 and in the Southern Hemisphere, where the extinct genus Lentipecten spread in the Paleocene–Eocene Thermal Maximum30. The genus Adamussium derived from Lentipecten and appeared in the early Oligocene; it comprises 5 endemic Antarctic species; Ad. colbecki is the only one extant13,31,32. The genus Austrochlamys also appeared in the Oligocene and was first restricted to King George Island (South Shetlands), then spread around the north of the Antarctic Peninsula and achieved a circum-Antarctic distribution until the Pliocene13,33,34. Austrochlamys persisted during the progressive cooling of the Antarctic Continent from the Paleocene to the Pliocene, dominating the coastal areas, while Adamussium occupied the deep seas and continental platform33. The opening and deepening of the Drake Passage and the intensification of the Antarctic Circumpolar Current during the Pliocene provoked a drastic cooling and the extension of sea ice over the coastal habitat, which caused the northward movement of Austrochlamys and its subsequent disappearance from Antarctica, along with the circumpolar expansion of Ad. colbecki in Antarctic shallow waters33. The colonization of the coastal habitat has been related to the sea ice extent that provided a more stable environment and low-energy fine-grained sediment with which Adamussium was associated in the deep waters. Austrochlamys fossils appear in the Subantarctic Heard Island in late Pliocene layers (3.62–2.5 Ma35). Today Ad. colbecki is a circum-Antarctic and eurybathic species that reaches high local density in protected locations13,36, while all Austrochlamys became extinct except for A. natans, which is restricted to southern South America33. The phylogenetic affinity highlighted here between A. natans and Ad. colbecki has its origins in the Southern Ocean; the deep divergence between the lineages of these monospecific tribes attests to the long time since their common origin in the Paleogene. These results point out both species as relevant biogeographic models to address longstanding questions regarding the origin of marine biota from Southern Ocean.The nomenclature, taxonomy and ecology of both A. natans and Z. patagonica have been problematic for almost 200 years. Since its original description37, Z. patagonica, a.k.a. the “Ostión Patagónico” has been named with more than 10 synonyms, probably due to the great intra-specific morphological variability throughout its distribution19,38 (see the nomenclatural history in Supplementary Table S1). In contrast, there are very few records in the scientific literature and no genetic data on A. natans, a.k.a. the “Ostión del Sur”13,14,17,19, and some problems of nomenclature and establishing diagnostic characters persist since its description13,39. Many of the current junior synonyms of both species were described from small and juvenile specimens (under 52 mm VH39,40,41). Indeed, all deposited type material of A. natans ranges from 23.5 to 52 mm VH; the latter is half of the maximum size39. The criteria most commonly used for the identification of both scallops were number of radial primary ribs, maximum size, shell colour and presence of laminated concentric lines (Supplementary Table S1). Specimens with marked primary and secondary radial ribs alternated regularly and more whitish colouring of the right shell were attributed to Z. patagonica, while those with weaker and less markedly coloured radial ribs and the maximum size were considered as A. natans42. However, the number of radial ribs overlaps between Z. patagonica (26–4212,43) and A. natans (22–5017,19). These characters also have high variability across different environments and during ontogeny13,17. Thus the use of a taxonomy based on environment-sensitive and allometric characters has led to confusion in the morphological identification of these species13,38. The criterion used in the present study, the BND/VH ratio established by Jonkers13, discriminates the species efficiently. As attested by the narrow dispersal cluster in Fig. 3, this character has low intra-population variability13. In some cases a level of intraspecific variation can be detected, and this is mainly due to the environments where the scallop populations inhabit19 (e.g. exposed, protected, substrate type, fjord, oceanic). However, although there may be some intraspecific variability between populations, this variability does not generate problems for the identification of the two species. Individuals of A. natans generally presented a significantly greater BND/VH ratio than those of Z. patagonica. However, it is important to consider that, given that this character varies during ontogeny, it is more accurate in individuals over 25 mm VH13. Only the molecular identification was able to discriminate juvenile scallops of both species accurately.According to the literature, A. natans is restricted to interior waters of channels and is associated with kelp forests of M. pyrifera (Supplementary Table S1). Z. patagonica inhabits a wider range of environments such as bottoms of shells, sand, mud and gravel in protected and exposed areas, between 2 and 300 m depth (Supplementary Table S1), but is also associated with kelp forests in fjords with different degrees of glacial retreat12,16,44. The juveniles of both scallops recruit in kelp forests44,45. According to the local artisanal fishermen, adults of “Ostión del Sur” (A. natans) occur in fjords with glaciers (orange circles in Fig. 123). We included two sampling locations near glaciers (in Pia and Montañas fjords), where large individuals (between 46 and 86 mm) of A. natans and Z. patagonica occur in sympatry. This sympatry was previously reported in Silva Palma Fjord between 5 and 25 m depth16. In conclusion, scallop banks are not monospecific but rather mixed and Z. patagonica occurs in the interior waters of the channels and fjords. Consequently, these two species have overlapping ecology (recruiting zone and glacial affinity) in the channels and fjords, overturning a long-held view that these scallops have marked habitat segregation.The fishery for both species was established in the 1990s in the political-administrative Region of Magallanes16, despite the complexity of the morphological recognition of scallops. The distinction between species was based on shell colour and radial ribs42, two characters that, given the results of this study, do not have this diagnostic capacity. Consequently, the scallop fisheries in the Magallanes Region are currently based on inaccurately discriminative characters. Scallop banks in MP have always been considered as monospecific16,47. A great part of scallop landing has always been attributed to A. natans47, about which the scientific literature is scarce (Supplementary Table S1). Conversely, Z. patagonica, which was erroneously considered as the commercial species of southern Chile, has more scientific research (Supplementary Table S1).The difficulty to discriminate A. natans and Z. patagonica morphologically may lead to incorrect fishery statistics and uncertain conservation status of A. natans. Incorrect fishery statistics could overestimate the abundance of banks of A. natans compared to Z. patagonica. If the minimum catch size is reduced23 in the context of the fishing overuse of the last decade, A. natans may suffer a reduction of its maximum size48. Therefore, an identification criterion between species is a need to improve fishery management. We showcased a quantified criterion that is useful to identify both species. In the short-term, this method can be used, but it is difficult to enforce in practical ways. We suggest to train fishing inspectors, following three guidelines. First, the identification should consider only the right valve (RV) for species identification, since the left valve is not taxonomically informative. Second, for visual classification, check the outline of the BN, mainly because the individuals of Z. patagonica have a more arcute BN. Third, a reliable identification has to measure the depth of the byssal notch (BND) and shell height (VH) ratio. Lastly, future research and fishery monitoring should follow these criteria to carry out a correct identification and subsequently better landings statistics.Molecular tools allowed evaluating the phylogenetic relationships of scallops globally or regionally and incorporating parameters that can be used for the management and conservation of species of commercial interest49. For example, in the last few decades metrics have been developed to address conservation problems that give us a measure of the current state of particular taxa. These conservation priorities are often seen as measures for threatened species categorized by the IUCN Red List (World Conservation Union, 1980), one of the most widely and recognized systems. Although this prioritization metric incorporates phylogenetic distinctiveness (PD), this factor has been updated due to the importance of quantifying the loss of evolutionary diversity that would be implied by the extinction of a species50. The magnitude of the PD loss from any species will depend (but not exclusively) on the fate of its close relatives51. The “Ostión del Sur”, Austrochlamys natans is the last representative of its tribe (Austrochlamydini) in the Southern Ocean. Its phylogenetic position and the long branch length (i.e. the length of the branch from the tip to where it joins the tree), which represents an important amount of evolutionary change, highlights the degree of isolation of A. natans and calls attention to the possible loss of a unique genetic lineage. There is currently no conservation value for this relict species; we sought to alert the current fishery management that the “Ostión del Sur” is a distinct taxon and provide integrative evidence for further conservation studies.Finally, regarding the overlapping niche of these scallops and the conservation importance of the clade of A. natans, we propose three key recommendations for the future scallop fishery policies in the sub-Antarctic channels. First, it is necessary to assess the proportion of both species per bank and landing to generate a distribution map through the sub-Antarctic channels. For this assessment, the byssal notch depth is the most appropriate morphological character. Second, we recommend reassessments of biological and ecological parameters (e.g. size at first maturity) for A. natans across the glacial fjords, which are the most relevant fishing sites. As a final point, today there is a complete lack of knowledge of the genetic connectivity along the Subantarctic Channels. Thus we should generate more research about spatial population genetics at different temporal scales. The integration of genomic approaches (e.g. SNPs) with macro- and micro-environmental modelling approaches provide enormous opportunity to establish a new regional zoning for fishery management and conservation scallop strategy. More

Soil propertiesStone contentThe stone content ranged from (15,pm ,8%) (w/w) at V.18 to (44,pm ,13%) (w/w) at F.3 (Fig. 1a, Table 1). These differences between sites may partly be due to different sources of the raw material used to create the SIS. Further, the stone content increased with depth within the first 15 cm (V.18) and 10 cm (W.10), but remained approximately constant over depth at F.3. Hence, the stone content in the upper layers of the older SIS (W.10 and V.18) was lower than in the lower layers. These depth-related differences at each site may be related to time-dependent developments within the SIS. In the uppermost layers of V.18 and W.10, stone content was comparatively low probably due to input of fine mineral and organic particles by storm water. For the oldest SIS (V.18), this assumption is supported by the field observation of soil material lying on a bricked stone border near the inflow within the SIS.Figure 1Depth-dependent soil properties: (a) stone content [% (w/w)], (b) bulk density ((hbox {g},hbox {cm}^{-3})), (c) pH (0.01 M (hbox {CaCl}_{2})) and (d) organic carbon content (OC) [% (w/w)] of the three sites F3, W.10, V18. The error bars are the standard deviation ((hbox {n}=4)).Full size imageTable 1 Soil properties of SIS.Full size tableBulk densityThe bulk density in the upper layers of the different SIS increased in the following order: V.18 < W.10 < F.3 (Fig. 1b, Table 1). At V.18, we observed the strongest change with depth from (1.0,pm ,0.1,hbox {g},hbox {cm}^{-3}) (0–5 cm) to (1.5,pm ,0.1,hbox {g},hbox {cm}^{-3}) (15–20 cm). In contrast, we observed almost no depth-dependent change of bulk density at the youngest site of F.3 ((1.6,pm ,0.2,hbox {g},hbox {cm}^{-3})).In samples of the older sites of V.18 and W.10, low bulk densities in the uppermost layers compared to deeper layers were probably caused by the activity of macrofauna, an intensive rooting, a higher organic carbon (OC) content and the input of strongly sorted fine material. The older the SIS, the stronger the effect of these factors.At F.3 the bulk density was relatively high. Here, we supposed an uniform compaction of the soil layer under the topsoil during construction. This assumption was supported by the observation of redox characteristics (iron-red stains next to grey iron-depleted areas) in the soil at approximately 25 cm depth caused by the lack of oxygen due to accumulating water45 in compacted soil.TextureThe mean texture of fine soil at all SIS was very similar: 57–80% (w/w) sand, 16–34% (w/w) silt and 5–9% (w/w) clay, since similar textured materials were used for construction to guarantee solute retention and sufficient hydraulic conductivity31. Average clay contents of all SIS were within acceptable ranges of Best Management Practice (BMP) claimed by ATV-DVWK A-138 (( More

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Design of the novel Zinc-Finger TaqMan assayIn order to establish the level of sequence conservation of the Zinc-Finger gene within the elephantine taxa, we aligned the known ZFX/Y regions for both alleles and each genus using Geneious R9. For the Asian elephants (Elephas maximus), we used the previously published Sanger sequences24 (Supplementary Table S1). For the woolly mammoths (Mammuthus primigenius) and the African elephants (Loxodonta africana and Loxodonta cyclotis), due to the lack of actual Zinc-Finger sequences deposited in sequence databanks, we recovered the corresponding sequences via the mapping of published whole-genome NGS reads from known male specimens20,29 (Supplementary Table S2). This alignment shows the complete conservation of the signatures discriminating the ZFX and ZFY alleles in Asian elephants at the scale of the elephantine subfamily. Low coverage data of this region (4X) are also available for the American mastodon (Mammut americanum; Supplementary Table S2), an extinct proboscidean species, which is a quite distant relative to the elephantine taxa: their most recent common ancestor dates back to 25–30 Mya (clade Elephantimorpha30). The comparison with the elephantine sequences strongly suggests the antiquity of these allelic signatures within the proboscideans (Supplementary Fig. S1). Conversely, when added to our comparison, the overlapping ZFX/Y sequences of modern humans show several fixed divergent positions from the elephantids (Fig. 1).Figure 1Alignment of the Zinc-Finger amplicon of interest for the ZFX and ZFY alleles from humans and elephantine taxa: Loxodonta (African elephants), Elephas (Asian elephants) and Mammuthus (mammoths). The top sequence represents the elephant ZFX allele; identities are indicated by dots. Primers and MGB probes are displayed in annealing position.Full size imageWe designed one pair of primers: ZF_Forward (5′-ACAAAATGGTGCATAAGGAAAAG-3′; Tm = 58.9 °C) and ZF_Reverse (5′-CTCAGCTGTCTCGTATTCACA-3′; Tm = 60.3 °C), which promote the amplification of a 74 bp long amplicon surrounding two sex-specific polymorphic sites. We chose priming sites exhibiting fixed divergent positions with human ZFX/Y sequences—specifically the final 3′ position of the forward primer—to reduce the risk of amplification of human contaminants. Based on the melting temperatures of the chosen primers, we designed two sex-specific Minor Groove Binding (MGB) fluorescent probes diverging from each other by two of their 13 nucleotides (Fig. 1): ZFX 5′-VIC/AGCCAACAAAATG/NFQ/MGB-3′ (Tm = 69.0 °C) and ZFY 5′-FAM/ATCCAGCAAAATG/NFQ/MGB-3′ (Tm = 68.8 °C), labelled with the two fluorescent dyes used by default in bi-allelic discrimination31, and manufactured by Applied Biosystems (Foster City, CA).In vitro sensitivity experimentsTo address the sensitivity of our assay, we first generated sex-specific quantitative standards: we diluted a male mammoth DNA extract (Lyakhov mammoth; Supplementary Table S4) until the point when real-time PCR reactions using this dilution as a template would only yield the amplification of one or the other sex-specific allele (or no product at all). We pooled three reactions for which only the X allele was detected in one microtube, and three other Y-positive reactions in another microtube. Each pool was purified using the minelute PCR purification kit (Qiagen, Venlo, NL) and concentrated separately in 10 µl of EBT buffer (Qiagen EB buffer supplemented with 0.05% Tween-20). We quantitated each sex-specific standard using the Qubit High Sensitivity assay kit (Invitrogen, Waltham, MA) and prepared a tenfold dilution series ranging from 1010 copies down to 10−1 copy per µl. Standard series were stored in frozen aliquots and thawed only before use.We analyzed the sensitivity of the assay in two dimensions: (I) the sensitivity of the PCR amplification in absolute copy numbers and (II) the relative sensitivity of both X- and Y-specific allele diagnostics. We first tested the general sensitivity of the assay using a SYBR Green I approach, with 1X Sso-Advanced Supermix (Bio-Rad, Ipswich, MA) and a standard series of each allele (105 down to 10−1 each), using 6 replicates of the standards at the low end (2 × 100 and 2 × 10−1). We then evaluated the reciprocal sensitivity of each MGB probe via a TaqMan reaction using a standard series ranging from 105 down to 100 each, with three replicates for the latest. For probe-based PCR reactions, we used the dedicated TaqMan Fast Advanced Master Mix (Applied Biosystems, Foster City, CA) which contains dUTP and Uracyl-N-glycosylase (UNG) pre-treatment steps to avoid PCR contamination from carryover PCR products.Quantitative PCR optimization and genotype analysesWe compared the behaviour of TaqMan reactions with various combinations of primer concentrations between 400 and 900 nM, final probe concentrations ranging between 200 and 600 nM, and an annealing/extension temperature gradient (55–65 °C). The best sensitivity was obtained around 60.5 °C regardless of the reagent concentrations: the Cq of the standards were retarded by up to 0.8 or 1.2 cycles when lower or higher temperatures were picked, respectively. Balanced MGB probe concentrations systematically yielded a higher response of the FAM probe over the VIC one (up to 150%), and sometimes caused a shallow crosstalk-signal artifact within the VIC detection range. Implementing uneven probe concentrations—increasing VIC by one-third and lowering FAM by as much—addressed both issues. We thus adopted the following conditions for all subsequent experiments: final reaction volumes of 15 µl with 1X of TaqMan Supermix, 800 nM of each primer, 375 nM of Y-FAM probe, 525 nM of X-VIC probe, and 1–2 µl of DNA extract.We performed all PCR reactions on a CFX-96 real-time thermocycler (Bio-Rad, Ipswich, MA) using the following 2-step conditions: after a first denaturation of 2′ at 95 °C, we performed 40 cycles of 95.0 °C 10 s and 60.5 °C 35 s. We conducted the allelic discrimination from the qPCR output with the CFX-Manager software v3.1 (Bio-Rad, Ipswich, MA) using the following set of parameters: baseline subtracted curve fit, quantification cycle (Cq) determined via a single threshold set to 10% of average plateau fluorescence (measured in Rescaled Fluorescence Units, RFU), call of alleles on the last PCR cycle.Specificity analysesWe investigated the level of specificity of our assay against human contaminants via straight qPCR attempts with various concentrations of control human genomic DNA (Thermofisher, cat. number 4312660): 1, 5, and 25 ng per reaction. We complemented this analysis with an in silico assessment of our assay: we used BLASTn32 to analyze the ‘nr’ collection database in GenBank, and identify which taxa shared sequence identity with at least one of our primers. Among those hits, we focused on the putative sympatric taxa of elephantids (modern and extinct) for which we aligned the available ZFX/Y fragments.Although the risk of non-specific detection is extremely low with an MGB-TaqMan methodology31, we chose to monitor the specificity of PCR design in our case study experiments. We prepared two pools—one per case study—from all positive PCR reactions from actual specimens across an entire replicate series. We transformed these pools in double-indexed Illumina libraries33 and performed a shallow sequencing of each (in paired-end 2 * 75 bp).Case study on elephant fecal extractsWe conducted the fecal sampling of wild elephants from November 2016 to January 2019 in Sebitoli area in the vicinity of Kibale National Park (south-western Uganda). The wildlife of this forest area, located at the north of the protected area, is studied by the Sebitoli Chimpanzee Project/Great Apes Conservation Project and the Muséum national d’Histoire naturelle (MNHN, Paris, France). Commercially logged in the 1970s, the Sebitoli forest is now composed of 70% of regenerating forests and only 14% of old-growth forest34. In areas adjacent to Kibale, human population density is high35 (circa 300 inhabitants/km2). They grow monocultures such as tea fields, eucalyptus, and banana plantations as well as crops like maize, which attract elephants and primates out of the forest. This survey is part of a project aiming at mitigating the human-wildlife conflict at the edge of the protected area in the framework of the Memorandum of Understanding SJ 445-12 between MNHN, Uganda Wildlife Authority, and Makerere University in Uganda and the MoU between UWA and GACP.To avoid the repeated sampling of the same individuals, we collected only once when we encountered several dung boli of similar size on the same day and location. Since female elephants live in close family groups36,37—while the adult males are mostly solitary—this strategy made the sampling of male dung more likely than female ones. A quantity of 10 to 15 g of feces was stored in 70% ethanol for 24 h. After removing the supernatant, feces were placed in gauze on silica gel beads and stored at ambient temperature until processed in the laboratory. After removing the largest vegetal compounds, between 150 and 200 mg of dried feces were extracted with the Power fecal DNA Isolation Kit (MoBio, Carlsbad, CA). The DNA extraction was performed in France, at the modern lab of the ‘Plateau de Paléogénomique et Génétique Moléculaire’ (P2GM platform) from the MNHN. Total DNA yields from the extracts, as measured with a NanoDrop 2000 (ThermoFisher Scientific, Waltham, MA), ranged from 2.9 to 186.7 ng/μl (Supplementary Table S3).To validate the assay, we used a set of 12 elephant extracts for which sex was known a priori: six male and six female specimens. We then implemented our assay in a case study that involved 91 specimens of unknown sex. Two PCR replicates per individual extract were performed, in parallel with a total of 7 PCR negative controls (NTC for ‘No Template Control’ reactions).Case study on mammoth ancient DNAOver the last 20 years, we have gathered several dozens of woolly mammoth samples that have been used in various paleogenetic analyses38,39. They are part of a broad comparative genomics project of diachronic specimens from Beringia which objective is to address the diversity and gene flow throughout the Late Pleistocene populations of woolly mammoths. Here we attempted to derive the genetic sex for a subset of 29 specimens using the novel assay. These samples all come from the Late Pleistocene in Siberia, and the radiocarbon-dated specimens range from 4420 up to beyond 50 ky BP (Supplementary Table S4).DNA extractions and PCR setup of mammoth samples took place in the dedicated ‘ancient DNA cleanroom’ at the P2GM platform, which is physically isolated from the modern lab. We used a protocol previously published for DNA extraction from bone39 and extracted the specimens in 5 different series—each along with one extraction blank. We first tested six specimens of known sex (thanks to a morphological diagnosis): Lyakhov, Jarkov and Oymiakon (all males), 2001/174, Lyuba and Khroma (all females). We then implemented the assay on 23 extracts of unknown sex together with each extraction blank, several NTCs, and one absolute standard series to establish the number of template molecules for each X and Y allele available from our mammoth extracts.Our sexing assay relies on the identification of one homozygous genotype (female) and one heterozygous genotype (male) via a bi-allelic target. In such a design, the risk of false assignation of a male to the female genotype due to allelic dropout of the Y allele is a limitation, particularly when working with templates of low DNA content40,41. We carried out all mammoth PCR reactions in triplicates, to comply with the multi-tube strategy developed to control for that risk. The implementation of a quantitative PCR framework in our sexing assay provided us with the ability to refine the estimate of the accuracy of the genotypes. Taberlet et al.41 showed that (i) the allele amplification of a bi-allelic marker behaves stochastically for very dilute samples, and (ii) for a known amount (U) of diploid genome copies in a reaction, the probability of allelic dropout can be precisely modeled (Supplementary Fig. S4). We posited that the sum of ZFX and ZFY allele copy numbers per reaction inferred via qPCR is a relevant proxy of this amount among our samples—a reasonable assumption when one considers that Zinc-Finger is a single copy nuclear gene. We derived the absolute copy number (CN) based on the Cq calculations for both Y-FAM and X-VIC between the positive specimens and the corresponding standard series. We then used this metric to estimate the probability PXX of allele dropout per reaction for a true heterozygote, based on Taberlet et al.’s model. For each specimen, the theoretical risk of wrongly being deemed a female due to allelic dropout thus translates as (PXX)n from the binomial distribution of parameters n and PXX, where n refers to the number of PCR attempts that yielded a genotype (Supplementary Table S6). More

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