Chromosome-level genome assembly of the Common tenrec, Tenrec ecaudatus (Schreber, 1778), a new model for early placental mammal evolution

Chromosome-level genome assembly of the Common tenrec, Tenrec ecaudatus (Schreber, 1778), a new model for early placental mammal evolution | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Chromosome-level genome assembly of the Common tenrec, Tenrec ecaudatus (Schreber, 1778), a new model for early placental mammal evolution Damián Hernández-Roco, Evgeny Leushkin, Jaccqueline Galeas, Katharine Grabek, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7991728/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Mar, 2026 Read the published version in BMC Genomics → Version 1 posted 11 You are reading this latest preprint version Abstract Background Our understanding of many biological aspects of early placental mammals is still very limited. Due to the paucity of the fossil record, attention is often turned to those extant organisms that share plesiomorphic characters in order to gain insights into the evolutionary history of mammals. Afrotheria is one of the four major clades of placental mammals, accounting for around a third of all mammalian orders, and encompasses a wide array of differently adapted species. Within Afrotheria, tenrecs are a more species-rich group of small mammals native to the island of Madagascar that display several special traits resembling those hypothesized on early placentals regarding reproductive strategies, thermoregulation and growth metabolism. Despite this, tenrecs remain heavily understudied in many aspects. Genomic information for this group of mammals is scarce and not up to modern quality standards. Results We present here the complete, chromosome-scale reference genome and annotation of the common tenrec, Tenrec ecaudatus . To put this new resource to use, we conducted a phylogenetic reconstruction and divergence time estimation for Afrotheria using all the available genomic resources for afrotherian mammals. This analysis recovered the phylogenetic order containing hyraxes as a sister group to elephants and a younger molecular divergence of tenrecs than previously estimated. Added to this, our comparative chromosome-synteny analyses showed significant rearrangements within afrotherians, especially on the clade shared by tenrecs, elephant-shews and the aardvark (Afroinsectiphilia). Conclusion This newly produced high quality genome assembly proves to be a valuable resource to complement our genomic understanding of Afrotheria, allowing for insights into chromosome evolution, time of molecular divergence and phylogenetic reconstruction. This establishes a basis for further studies to utilize this resource to further pursue evolutionary questions regarding tenrecs adaptations and comparative analyses within Afrotheria. Tenrec Genomics Afrotheria Afroinsectiphilia Madagascar Phylogenetics Synteny Chromosome-level Genome Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Afrotheria is one of the four major placental mammal clades, comprising around one third of all eutherian orders ( 1 ). This taxon unites ungulate-like (Paenungulata: proboscideans, sea cows, hyraxes) and insect-feeding lineages (Afroinsectiphilia: aardvarks, sengis, tenrecs, golden moles). Despite displaying this diverse array of morphological features and ecological specializations that have sparked debate about the validity of the clade ( 2 – 4 ), this group has been categorized as monophyletic by numerous taxonomic assessments employing modern molecular methods ( 5 – 8 ). Afrotherians, as their name suggests, are hypothesized to have originated on the African continent around 100 and 65 million years ago (Mya) and later highly diversified during the late Cretaceous period when the South American landmass fully separated from the rest of Africa, leaving it isolated ( 9 – 11 ). However, not all afrotherian species remained confined within continental Africa. Such is the case of the afrosoricidan (tenrecs and golden moles) family Tenrecidae, an insectivoran-grade lineage that colonized the island of Madagascar in the Indian ocean between 30 to 56 Mya ( 12 ) and subsequently radiated into the species-rich yet understudied group of mammals ( 13 , 14 ). Commonly known as ‘Malagasy tenrecs’, this group of small mammals includes 31 currently accepted extant species ( 15 ), with four species classified as Vulnerable (VU) and two species classified as Endangered (EN) by the International Union for Conservation of Nature (IUCN). Yet, the number of threatened species is expected to increase in the following decade ( 16 ). Though, alpha taxonomy of tenrecs is still not fully understood and the cryptic diversity might be higher ( 17 , 18 ). This relatively unknown group of mammals displays several unique characteristics that makes them worthy of broader scientific interest ( 19 ). For example, tenrecs retain reproductive flexibility that likely mirrors early placental mammals ( 20 ). Unlike most modern placentals, they exhibit superfetation, polyovulation, large litter sizes, and a lack of traditional antrum formation, suggesting a reproductive strategy that prioritized offspring quantity over metabolic efficiency ( 21 – 24 ). Tenrecs also undergo intrafollicular fertilization, a system thought to optimize fertilization success before the development of more complex implantation strategies ( 20 , 22 ). This reproductive flexibility may reflect strategies employed by early placental mammals, which likely relied on high reproductive output rather than sustained metabolic investment in individual offspring ( 25 ). Indeterminate growth is also considered the ancestral trait of amniotes ( 26 ) and it’s rarely documented in mammals, with known cases limited to marsupials (e.g. red kangaroos, wallabies). Tenrecs vary widely in size at birth and exhibit striking developmental variability. Some juveniles grow only ~ 20% in their first month, while others increase in size by ~ 400% ( 24 ). Such extreme growth plasticity suggests a disconnect between chronological and developmental age, allowing for profound variation in growth trajectories over a lifetime. Tenrec ecaudatus (Schreber, 1778), also known by the common name ‘tailless tenrec’ or ‘common tenrec’, is the only species of tenrec to have been introduced through human activity to several islands along the Mascarenhas archipelago, the Comoros and the Seychelles ( 19 , 27 ). Tailless tenrecs being such a widespread species in between islands has resulted in the existence of multiple reports of individuals acting as a reservoir of Leptospira and causing several zoonosis outbreaks ( 28 – 30 ). This added to the fact that tenrecs are an important part of the bushmeat diet of Malagasy households ( 31 – 33 ), makes T. ecaudatus a species of special human interest. Among other particularities of this species, T. ecaudatus has remarkable thermal plasticity. Active season tenrecs housed at 12°C have variable core body temperatures ranging from ~ 13°C to ~ 28°C and can maintain full locomotor abilities across that range ( 24 ). Alternatively, hibernating tenrecs successfully hibernate at ambient temperatures of 28°C. Further, this thermoregulatory plasticity is accompanied by tremendous metabolic plasticity wherein torpid tenrecs at 12°C have metabolic rates that are nominally lower than that of a torpid Arctic ground squirrel hibernating at 4°C. Taking into account all the peculiarities of Tenrecidae in general and the human relevance of the species, T. ecaudatus makes a suitable candidate to be a model organism for physiological, developmental and evolutionary research of tenrecs, afrotherians and placental mammals as a whole ( 34 – 36 ). Increased availability of chromosome-scale genome assemblies and high throughput sequencing technology has made it both possible and almost a requirement to generate high quality genomic data for non-model species ( 37 – 40 ). In this study, we generated a haplotype-resolved, chromosome-level genome assembly for the tailless tenrec, T. ecaudatus . Furthermore, we supplemented this genomic resource with orthology based annotation and comparative genomic analyses of chromosome synteny in order to delve into the phylogenetic history of Afrotheria and their chromosome evolution. As a result, this genomic resource makes possible further research into Tenrecidae from a genomic standpoint, allowing for insight into previously unknown aspects of this group's biology and evolutionary history. Materials and Methods Sample collection: Samples were taken from lab-reared common tenrec individuals from the breeding colony maintained at the University of Nevada, Las Vegas (UNLV), USA (Fig. 1 ). The breeding colony originated from 40 wild-caught individuals imported from Mauritius in June 2014 under appropriate U.S. federal and state permits. Laboratory-reared tenrecs of male and female sex (Table S2) were euthanized via cervical dislocation followed by pneumothorax, and immediately dissected on ice. Heart, brain, kidney, testis, liver and embryotic tissue was extracted, rinsed in diethyl pyrocarbonate (DEPC)-treated water, and snap-frozen in liquid nitrogen. Samples were stored at − 80°C until sent to FaunaBio (Emeryville, CA, U.S.) for further processing. In addition, liver, kidney, uterus, and placenta were collected as above from a pregnant female reported in Stadtmauer et al. (2025) ( 20 ), preserved in RNAlater (AM7021, Thermo Fisher) and shipped to Yale University (New Haven, CT, USA) for processing. DNA/RNA extraction and PacBio Sequencing: Heart, brain, kidney and liver samples underwent liquid nitrogen pulverization and were then extracted using the Monarch HMW DNA extraction kit for tissue (New England Biolabs) in accordance with protocol instructions. To process the liver sample, NaCl was utilized to remove polysaccharides and avoid glycogen contamination. Final quality measuring of DNA and concentration was performed with NanoDrop, Qubit Fluorometer and Femto quantification. Library preparation was carried out using the SMRTbell prep kit 3.0 (Pacific Biosciences, Menlo Park, CA). The library was sequenced on a PacBio Sequel II using the Sequel II binding kit 3.2 at QB3 Genomics, (UC Berkeley, Berkeley, CA). While aiming for 50x coverage, a total of 9 flow cells were run. Additionally, at this same facility PacBio Iso-Seq data was generated for liver, kidney, brain, testes, heart and embryonic tissue using 1,250 ng/sample. RNAlater-preserved specimens from the pregnant female were extracted using a Qiagen RNEasy Plus Micro Kit (Q74034) and sequenced on a PacBio Sequel II at the Yale Center for Genome Analysis. Hi-C chromatin conformation capture: Chromatin conformation capture was carried out in the facilities of Arima Genomics, Carlsbad, USA using the ARIMA-HiC kit for animal tissue (Document Part Number: A160132 v01) and following protocol’s guidelines. Illumina library preparation was performed following the ARIMA Library Prep Module user guide (Document Part Number: A160432 v02). The barcoded HiC library was then sequenced on an Illumina NovaSeq6000 platform with 150 bp paired-end reads. Genome assembly & annotation: Prior to the assembly, we called HiFi reads from PacBio subreads using pbccs v.6.4.0 ( https://github.com/nlhepler/pbccs ) and deepconsensus v.1.1.0 ( 41 ). HiFi reads were assembled using hifiasm v.0.18.7 ( 42 ), with Hi-C reads used for phasing of the two haplotypes. We then mapped HiFi reads back to each assembly with minimap2 v.2.24 ( 43 ) to remove unambiguous heterozygous sites, removed duplicates using Picard MarkDuplicates tool v.3.1.0 ( http://broadinstitute.github.io/picard ), and called variants using DeepVariant v1.5.0 ( 44 ). Finally, we corrected corresponding nucleotide sites in the assembly using BCFtools consensus v.1.13 ( 45 ). To scaffold the polished haplotype assemblies, we mapped the Hi-C reads to each of the two assemblies using Chromap v.0.2.5 ( 46 ) and then processed mapped reads with yahs v1.1a ( 47 ). We then concatenated the two yahs-scaffolded assemblies and remapped the Hi-C reads but now allowing for multimapping reads (-q 0) to avoid losing reads, mapped to regions identical between two haplotypes. This allowed us to cross-validate two haplotypes by manually curating them in parallel in PretextView v.0.2.5 ( https://github.com/wtsi-hpag/PretextView ). Additionally, telomeric sequences were identified with tidk v.0.2.31 ( https://github.com/tolkit/telomeric-identifier ) and where necessary were used to correct wrong contig orientations to have telomeres in the ends of resulting scaffolds. To finalize changes made via manual curation, as recently proposed by the Vertebrate Genomes and Darwin Tree of Life Projects, we used the rapid curation framework ( https://gitlab.com/wtsi-grit/rapid-curation/-/tree/main ) from the Genome Reference Informatics Team ( 48 ). To estimate the assembly base accuracy, we used Merqury ( 49 ) with the HiFi reads. To assess gene completeness, we used compleasm 0.2.6 ( 50 ) with the BUSCO (Benchmarking universal single-copy orthologues) odb10 set of 9226 near-universally conserved mammalian genes. We used a combination of orthology inference and transcriptome sequencing to produce whole-genome annotations. For each of the two haplotype assemblies we generated alignment chains to human (hg38) as described previously ( 51 , 52 ) and then used TOGA v.1.1.4 ( 53 ) to annotate coding genes with human GENCODE 38 annotation serving as reference. We then processed previously produced Iso-Seq reads to obtain transcriptomic evidence for each tissue separately. Namely, circular consensus was generated from the subreads using ccs v.6.4.0 ( https://github.com/PacificBiosciences/pbbioconda ). Adapter trimming and base quality filtering were performed with lima v2.2.0 ( https://github.com/PacificBiosciences/pbbioconda ). Processed reads were cleaned from polyA tails and polymerase switching artifacts using “refine” command from the Iso-Seq package v.4.0.0 ( https://github.com/PacificBiosciences/IsoSeq/blob/master/isoseq-clustering.md ) and then clustered together and aligned to generate consensus reads using the “cluster” command from the Iso-Seq package. Additionally, reads were filtered for alignment quality using a custom Perl script (Supplemental Code). Finally, StringTie v.2.1.2 ( 54 ) was used to produce assembled transcripts. Individual tissue annotations were then merged together. Finally, in cases where TOGA prediction was missing, transcriptome evidence was used as a primary annotation source, incorporating for the most part lineage-specific and noncoding transcripts. Comparative quality metrics with the additional genome assemblies included in the study were latter assessed with Quast v5.2 ( 55 ), BBMap v37.62 ( 56 ) and Compleasm v0.2.6 ( 50 ) Phylogenetic reconstruction and date estimation: For the phylogenetic reconstruction of afrotheria, we first downloaded full genome annotation data already available at the time, focusing on Tenrecidae and adding representatives from all remaining afrotherian clades. We assembled a dataset obtaining human reference-annotated data from the TOGA annotations for mammals repository on the Senckenberg Genome Browser ( https://genome.senckenberg.de/TOGA.mammals.html ). For species of interest without an available annotation on this database, we supplemented it by producing our own annotation for available afrotherian genome assemblies also using the make_lastz_chains ( https://github.com/hillerlab/make_lastz_chains ) and TOGA v1.1.4 ( 53 ) pipeline. This resulted in the creation of a dataset comprising 14 afrotherian species (Table S1 ), including our own Tenrec ecaudatus annotation data. This dataset encompassed sea cows (Sirenia: Dugong dugon , Trichechus manatus ), elephants (Proboscidea: Loxodonta africana , Elephas maximus ), hyraxes (Hyracoidea: Heterohyrax brucei , Procavia capensis ), the aardvark (Tubulidentata: Orycteropus afer ), a golden mole (Chrysochloridae: Chrysochloris asiatica ), sengis (Macroscelidea: Rhynchocyon cirnei , Elephantulus edwardii , Petrodromus tetradactylus ) and tenrecs (Tenrecidae: Nesogale talazaci , Echinops telfairi , Tenrec ecaudatus ). To generate amino acid alignments as the basis for phylogenetic reconstruction, we filtered the human reference-aligned amino acid sequences produced by TOGA. We used the generated orthology classification to filter the transcripts based on genes that were classified as one to one (1:1) orthologs between the reference human genome and the query species genome. Given that even one to one orthologs can have multiple ortholog chains, we filtered for duplicated transcripts based on sequence length and prevalence of gaps. Next, we filtered for genes that occurred at least once per species, obtaining a list of 12062 genes. This list of genes was then used to filter transcripts in order to create one alignment per gene for the 14 chosen species using MAFFT v7.5 ( 57 ). These alignments were then trimmed using ClipKit v2.3 ( 58 ), in kpi-smart-gap mode to dynamically determine the threshold of gaps to remove and keep parsimony informative sites only. Subsequently, the resulting alignments were filtered by sequence length keeping only those that were > 50 amino acids in length, thus obtaining a new filtered dataset corresponding to 7119 genes that are all over 50 parsimony informative sites in length. Individual, unrooted maximum-likelihood gene trees for each of the 7119 amino acid alignments were generated using IQ-TREE v2.1.4 ( 59 ). A Q.mammal + F + R9 substitution model was used for calculating each gene tree, with independently calculated log likelihood parameters. To account for anomalous long branches due to the estimation of an unrooted topology, we used TreeShrink v1.3.9 ( 60 ) to filter out outlier tip branches. A coalescence-based species tree was generated using ASTRAL v5.7.8 ( 61 ) in order to use the resulting topology as the fixed input tree to use with the MCMCTree program in the PAML suite ( 62 ) for divergence time calculation. Consequently, the R package MCMCTreeR ( 63 ) was used to further prepare the input files and node age priors for the MCMCTree divergence time inference analysis. Fossil calibration estimates were designated using the oldest known fossil proving the existence of the branch and multiplying the hard minimum age by 1.25 to estimate a soft maximum age ( 64 , 65 ) (Table 1 ). In line with Heritage et al., 2021 ( 66 ), age constraint intervals were placed on eight nodes representing crucial clade divergences within Afrotheria as follows: We used the Ocepeia daouiensis fossil (Node A; hard minimum at 60.5 Mya) to constrain the Paenungulata-Afroinsectiphilia split and thus as the oldest calibration for this dataset, this being the earliest known afrotherian fossil. Eritherium azzouzorum (Node B; 59 Mya) was chosen to calibrate the Paenungulata node; Priscosiren atlantica (Node D; 28 Mya) for the split between Dugongidae and Trichechidae within Sirenia; Daouitherium rebouli (Node E; 56 Mya) for the split between Proboscidea and Hyracoidea; Heterohyrax auricampensis (Node F; 10.4 Mya) for the divergence within Hyracoidea; and the earliest Loxodonta sp. (Node G; 6.5 Mya) for the split between extant proboscideans. Finally, within Afroinsectiphilia, Todralestes variabilis (Node C; 56.8 Mya) was used to constrain the split between Afrosoricida and Macroscelidea; Oligorhynchocyon songwensis (Node H; 25.2 Mya) for the split between Rhynchocyonidae and Macroscelididae within Macroscelidea. Table 1 Fossil calibration ages used for divergence time inference within afrotheria. All ages are displayed in the Mya (Million years ago) unit. Species Hard minimum age (Mya) Soft maximum age (Mya) Node Ocepeia 60.5 75.625 (A) Eritherium 59 73.75 (B) Todralestes 56.8 71 (C) Priscosiren 28 35 (D) Daouitherium 56 70 (E) Heterohyrax auricampensis 10.4 13 (F) Loxodonta sp. 6.5 8.125 (G) Oligorhynchocyon 25.2 31.5 (H) The approximate likelihood calculation for protein data function of MCMCTree v4.10.0 ( 67 ) was used in order to calculate divergence times within Afrotheria. Two datasets were created for this; a concatenated set of filtered gene trees that share the proposed coalescence-based topology of the species tree produced with ASTRAL and the concatenated set of all gene trees. IQtree’s QMaker function ( 68 ) was used to calculate amino acid substitution models for each dataset. Having specified the amino acid models, MCMCTree then uses CODEML ( 69 ) to calculate the Hessian matrix for protein data. Later, a Markov Chain Monte Carlo (MCMC) approach was used to calculate divergence times estimations using 1 million runs with 200 thousand burnin for both datasets. Multiple MCMCTree runs were performed and multiple dating scenarios were tested to check for convergence and effective sample sizes with Tracer v1.7.2 ( 70 ). Estimates for both the filtered and the complete dataset were then visualized with TreeViewer v2.2.0 ( 71 ). Synteny analysis: In order to gain insights into chromosome evolution among afrotherian mammals, we obtained all high quality chromosome-level genome assemblies produced for Afrotheria (Table S1 ). Chromosomes synteny of two elephants ( Loxodonta africana , Elephas maximus ), two sea cows ( Dugong dugon , Trichechus manatus ), one hyrax ( Heterohyrax brucei ), one aardvark ( Orycteropus afer ) and one elephant shrew ( Rhynchocyon petersi ) was compared to our assembly of Tenrec ecaudatus chromosomes. Genome assemblies were manually curated to extract only autosomal chromosome scaffolds and the X sex chromosome scaffold, due to not having a male representative for all species. We used ntSynt v1.0.2 ( 72 ) to infer multi-genome synteny between all the available chromosome-level genome assemblies of afrotherian species and our new Tenrec ecaudatus genome. This program utilizes a minimizer graph-based approach, allowing for the calculation of multiple genome homologous synteny blocks with over 15% overall genome divergence using minimal resources. Furthermore, we used Mash v.2.3 ( 73 ) to calculate pairwise mutation distance and therefore, genome divergence between assemblies. For visualizing the synteny plots we used ntSynt-viz v1.0.0 ( 74 ). Results Genome assembly and annotation: We obtained a highly complete and contiguous haplotype-resolved genome assembly for the common tenrec, T. ecaudatus . (Table 2 ). Our primary haplotype was assembled to a total length of 4.06 Gb, whereas our secondary haplotype only had 3.44 Gb in length. Both reconstructed haplotypes showed remarkable BUSCO completion scores based on the set of conserved mammalian genes (Haplotype 1: mammalia odb10: C: 99.3% [S:96.92%, D:2.43%], F: 0.07%, M: 0.59%, n:9226; Haplotye 2: mammalia odb10: C: 97.16% [S:95.09%, D:2.07%], F: 0.11%, M: 2.73% n:9226). Added to this, the estimated k-mer based consensus quality (QV) portrays a very high base accuracy of 64.9 and 64.7 for both haplotypes respectively, indicating roughly one error per ~ 3.1 million base pairs. Table 2 Primary and secondary haplotype assembly statistics of the Tenrec ecaudatus genome assembly. Assembly Primary haplotype Secondary haplotype # contigs 5663 3611 Largest contig (mb) 294 295 Total length (Gb) 4.06 3.44 GC (%) 44.97 44.35 scaffold N50 (mb) 133.2 156.4 scaffold L50 (mb) 12 9 BUSCO completion 99.3% 95.09% QV (Merqury) 64.9 64.7 Along this orthology based quality measure, the annotation based quality measure calculated with TOGA yielded a total count of 17420 genes with intact open reading frames, 1720 genes with a type of inactivating mutation and 324 missing genes. Both these measures of quality positively highlight our Tenrec ecaudatus genome assembly and annotation compared to the available set of afrotherian mammal genome resources (Fig. 2 ). In this assemblage of genomes, the previous representation of Tenrecidae consisted only of the scaffold-level genome assemblies of Nesogale talazaci (BUSCO C: 66.7%; TOGA Intact ORF genes: 61.8%) and Echinops telfairi (BUSCO C: 95.9%; TOGA Intact ORF genes: 66.1%) (Table S3). Phylogenomic reconstruction and dating: We reconstructed the phylogenetic relationships of Afrotheria (Fig. 3 ) using a protein alignment dataset for both species with available annotation data and those with annotations generated in this study. We rooted our resulting topology on the basal split within Afrotheria between Afroinsectiphilia and Paenungulata. The recovered topology coincides with that of recent studies employing multiple molecular evidence ( 6 , 7 , 12 , 75 ). Notably, our coalescent-based topology recovered Hyracoidea as a sister group to Proboscidea, thus rejecting the Tethytheria hypothesis (elephants + sea cows) ( 76 , 77 ). The uncalibrated nodes within Afroinsectiphilia estimate the split of tenrecs from golden moles at around 49.4 Mya. Consequently, the following split between Tenrecinae and Oryzorictinae is estimated around 27.06 Mya. and the divergence time between T. ecaudatus and E. telfairi is estimated at 14.37 Mya. Synteny analysis: We inferred multi-genome synteny for eight chromosome-level genome assemblies of afrotherian species including our novel Tenrec ecaudatus genome assembly, representing all six orders within the clade (Fig. 4 ). All inferred chromosomes were aligned based on synteny with T. ecaudatus . The first synteny blocks corresponding with Chr. 1–2 in T. ecaudatus show to be highly conserved between clades. Though Afroinsectiphilia in particular showed remarkably high chromosome divergence. Between the common tenrec ( T. ecaudatus ) and the giant sengi ( R. petersi ) there is a complex display of chromosome rearrangements including fission/fusion events on all chromosomes as well as multiple instances of inversions. Similarly, several inversions and fission/fusion events can be found between the chromosomes of R. petersi and O. afer , most notably, the event that resulted in Chr. 4 of the latter with one half corresponding to the inverted Chr. 10 of R. petersi and the other half corresponding to the inverted Chr. 4 of the same species. In the case of Paenungulata, the compared representatives show a much more conserved chromosome synteny than their sister clade. Both sea cow species show the same chromosome number, yet with a different chromosome configuration. Moreover, both representatives of extant Proboscidea show full microsynteny, while differing in overall chromosome scaffold length after chromosome 13. Discussion Two high quality haplotypes were assembled representing the genome of T. ecaudatus , adding to this the annotation of said genome. Though, the discordance in quality observed between the assembled haplotype one and haplotype two is more likely due to this last assembly containing only autosomal chromosomes. For comparative analyses, only haplotype one was considered on the quality metrics. Although commonly used as an indicator of genome quality, the scaffold N50 metric does not directly link to better quality of genome assembly ( 78 ) in this particular phylogenetic order-wide comparison, due to the highly variable chromosome number and genome sizes across Afrotheria ( 79 ). Such is the case of the aardvark assembly, showing a highly superior N50 metric (644 mb) due to its massive chromosomes, and therefore, scaffold sizes. In cases like this where the compared assemblies are of outstanding quality and mostly at a chromosome-scale, BUSCO ( 80 ) completion can be a more appropriate metric of assembly quality. Currently, Afrotheria is not well genomically represented. Despite being a diverse superorder, only a fraction of afrotherian species have an available high quality genome assembly. Out of the now 16 species that have an available genome assembly, our Tenrec ecaudatus assembly ranks as the highest quality assembled and annotated one for Tenrecidae and Afrosoricida, while also qualifying as one of the top five highest quality genomes for Afrotheria in terms of annotation and BUSCO completion (Fig. 2 ; Table S3). High quality annotation can be attributed to the use of multiple-tissue transcriptome data used to complement the TOGA produced annotation ( 53 , 81 ). Using eight fossil calibration points, our results are consistent with modern phylogenetic inference approaches that recover Afroinsectiphilia as a monophyletic clade ( 12 , 75 , 82 ). These calibrations corresponded to the oldest available fossil for a determined group and estimated soft maximum bounds. Since including multiple calibrations, the determination of the upper bound is not as key of a factor as when using a single calibration point ( 65 ). Accordingly, calibrated nodes showed small confidence intervals around the estimated hard minimum and soft maximum ages. Though, nodes that remained uncalibrated, such as the ones within Afrosoricida showed broad intervals of confidence. The afrosoricidan fossil record is very limited and fragmented, and the phylogenetic position of most extinct species remains unclear ( 15 ). Thus, these nodes currently lack any fossil-informed calibration that could help increase the precision of temporal estimates ( 66 ). In our estimates, the nodes comprising Afrosoricida (95%HPD=[45.105, 53.5972] Mya), Tenrecidae (95%HPD=[21.5568, 31.0638] Mya) and Tenrecinae (95%HPD=[9.55502, 18.9804] Mya), show younger mean divergence times than in Everson et al. (2016), which estimates a mean divergence of 58.4 Mya for Afrosoricida, 35.5 Mya for Tenrecidae and 25.7 Mya for Tenrecinae. Our results further show that even with a genome-wide dataset of phylogenetic data, divergence dating can be challenging for some nodes if there is no uniform temporal signal among genes. The choice of the clock model, too, has recently been shown to affect divergence dating within afrotherians when ingroup fossils for calibration are lacking ( 64 ). We recommend future work to focus on this issue of divergence dating within afrosoricidans as particularly the split within Tenrecidae can be indicative of the minimum colonization date of Madagascar. Tenrecidae is also currently the family among Afrotheria with the least amount of cytogenetic studies ( 83 ). In terms of currently known chromosome numbers, afrosoricidans range from 2n = 26 to 2n = 34 in golden moles ( 84 – 86 ), keeping highly conserved chromosome numbers, whereas in Tenrecidae chromosome numbers are more variable. This variation ranges from 2n = 30 (or even from 2n = 14 according to an unsupported claim of Gilbert (2008)( 86 )) to 2n = 56 ( 83 , 87 ). Therefore, our chromosome number estimation ranges within this reported variation. Although some studies involving chromosome painting proposed the aardvark’s karyotype to share a strong resemblance with the eutherian ancestral state ( 88 ), more recent studies on mammal chromosome evolution hypothesize that the last common ancestor (LCA) of all placental mammals possessed 19 autosomal pairs (2n = 40) and showed great chromosomal conservation with the LCA of Amniota. Similarly, the hypothesized ancestral karyotype for Atlantogenata (Xenarthra + Afrotheria) would have shown the same number of chromosomes, differing only by four chromosome inversions accumulated over 5 million years ( 89 ). Thus, chromosome number in T. ecaudatus is closer to the ancestral state in placental mammals and potentially afrotherians as well. Several million years of evolution have resulted in Afrotheria displaying great chromosome variation between orders, meanwhile maintaining a high degree of synteny for the sex chromosomes, which has been very well documented in mammals ( 88 , 90 – 92 ). This can be seen in the chromosome rearrangements observed between the common tenrec ( T. ecaudatus ), giant sengi ( R. petersi ) and aardvark ( O. afer ), which despite varying a great amount in chromosome number, size and arrangement, still maintain conserved X chromosome synteny. In contrast to this highly variable group, elephant species E. maximus and L. africana , having had their divergence event much closer to present times, show virtually no chromosome rearrangements. The west indian manatee ( T. manatus ) and dugong ( D. dugon ) do not show the same level of conserved chromosome synteny as the compared elephant species, showing multiple chromosome reorganization events converging on the same chromosome number (2n = 48) ( 93 ). Interestingly, Trichechus inunguis , a particular manatee species not included in this study, displays a karyotype of 2n = 56, eight more autosomes than its sister species T. manatus . These species of manatee have been reported to have diverged via chromosomal fusion events, a pericentric inversion and six Robertsonian translocations ( 94 ). In other words, D. dugong and T. manatus exhibit the derived karyotypic state, whereas T. inunguis retains the ancestral state. This is further supported by Hyracoidea being hypothesized as having 2n = 54 as their ancestral state, which H. brucei and other hyrax species maintain until today ( 95 ). This same species also shows very few chromosome rearrangements that separate them from proboscideans, mainly an inversion on Chr. 19 and fission/fusion of Chr. 9 and 10 into three and two separate chromosomes respectively. Overall, the high quality genomic resource generated in this study for T. ecaudatus is compatible with genomic analyses such as phylogenomic reconstruction, multi-genome chromosome synteny inference and although challenging, molecular divergence dating. These insights gained into the evolutionary history of T. ecaudatus and Afrosoricida are just a first example of the potential that genomics offers to bridge the gap in the knowledge of tenrec evolutionary history. Further analyses should focus more on delving into the physiological plesiomorphic characters present in T. ecaudatus and their genomic elements in order to explore how these make tenrecs unique creatures and how mammalian genomes may have evolved through the ages. Abbreviations Mya: Million years ago IUCN: International Union for Conservation of Nature DEPC: Diethyl pyrocarbonate ng: Nanograms PacBio: Pacific Biosciences bp: Base pairs BUSCO: Benchmarking universal single-copy orthologues Gb: Giga base-pairs MCMC: Markov Chain Monte Carlo ORF: Open reading frame Chr: Chromosome HPD: Highest posterior density LCA: Last common ancestor Declarations Data availability The genome annotation data newly generated with TOGA for Tenrec ecaudatus and the annotation data generated previously are available at: https://genome.senckenberg.de/download/TOGA/human_hg38_reference/Afrotheria/ Raw data and genome assembly data have been deposited in NCBI under BioProject accession number PRJNA1359382. Funding D.H.R. and P.A. acknowledge support from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, HO3492/25-1 & AR1307/5-1). Funding for F.vB. was provided by the National Science Foundation grants IOB 0448396 and IOS 1655091. Additional sequencing funding was provided by the Yale Institute for Biospheric Studies (to D.J.S.), the John Templeton Foundation (61329, to G.P.W.), and a SMRT cell donated for novel species pilots by the Yale Center for Genomic Analysis. Acknowledgements D.J.S. and G.P.W. thank the Yale Center for Genome Analysis and Keck Microarray Shared Resource at Yale University for PacBio sequencing services, funded in part by the National Institutes of Health instrument grant 1S10OD028669-01. Author contributions F.vB. and G.R.S. provided tissue samples. D.J.S., J.G. and C.L. performed wet lab procedures. L. G., D. J. S. and G.P.W. produced/provided sequencing data. E.L. and M.H. handled T. ecaudatus genome assembly and annotation. D.H.R. handled bioinformatic analyses and data interpretation. D.H.R. drafted the manuscript and crafted the figures. P.A., F.vB., E.L. and D.J.S. contributed to writing/reviewing the manuscript. F.vB., L.G., K.R.G., M.H. and P.A. supervised the study. All authors have read and approved the final version of this manuscript. Ethics statement Sampling protocols for this study were approved by the University of Nevada, Las Vegas Institutional Animal Care and Use Committee (IACUC) under project number [IACUC-01176]: 1397752 from 17/01/2025. Consent for publication Not applicable. Competing interests L.G., K.G., J.G. and C.L. are employed by FaunaBio. The rest of the authors have declared no competing interests. References Springer MS. Afrotheria. Curr Biol. 2022;32(5):R205–10. Averianov AO, Lopatin AV. Fossils and monophyly of Afrotheria: a review of the current data. Archives of the Zoological Museum of Lomonosov. 2016;54:146–60. Seiffert ER, Simons EL, Ryan TM, Bown TM, Attia Y. 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14:01:37","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":66204,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7991728/v1/0963046cc2b48b0fda2f7648.png"},{"id":96914185,"identity":"bd7bf502-8d97-498e-af3c-aae96b9c5b32","added_by":"auto","created_at":"2025-11-27 14:05:33","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":141071,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7991728/v1/5352c8adc6a1784bc161860a.png"},{"id":96914118,"identity":"7f15cbf0-4669-4f56-9b83-5caa5d8fa0aa","added_by":"auto","created_at":"2025-11-27 14:05:28","extension":"xml","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":181589,"visible":true,"origin":"","legend":"","description":"","filename":"54cda20b4bac41a4ad2fb42fc312b6fb1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7991728/v1/cdcd8f7d82c43c69ced98a58.xml"},{"id":96914032,"identity":"a240a8ed-f0d6-4fcf-9686-c35c32266e3e","added_by":"auto","created_at":"2025-11-27 14:05:13","extension":"html","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":197026,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7991728/v1/780033f98e3b06c9834132b1.html"},{"id":96726500,"identity":"6842896f-3097-4e4d-a17b-e00a91723fe2","added_by":"auto","created_at":"2025-11-25 12:34:35","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":15582915,"visible":true,"origin":"","legend":"\u003cp\u003eIndividual of \u003cem\u003eT. ecaudatus\u003c/em\u003e from the breeding colony at the University of Nevada, Las Vegas, USA. Photograph by Sophia Lorenzana.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7991728/v1/c1e52b031ec523c181fe2a7c.jpg"},{"id":96726490,"identity":"da676de5-69c9-4092-9202-da843178b274","added_by":"auto","created_at":"2025-11-25 12:34:35","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1124277,"visible":true,"origin":"","legend":"\u003cp\u003eQuality metrics of all available genome assemblies of afrotherian mammals. A) Shows the quality of the genome assembly on the percentage of BUSCO orthologous genes (%) that were classified as Complete, Fragmented and Missing. B) Shows quality of the genome assembly and annotation in the percentage (%) of genes catalogued by TOGA as Intact, Inactivated or Missing.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7991728/v1/a63d65cf243d27c442789906.jpg"},{"id":96726488,"identity":"65064cac-3420-4251-9345-8c83d09693d6","added_by":"auto","created_at":"2025-11-25 12:34:35","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":549623,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic reconstruction and divergence time estimation using amino acid alignments. Fossil calibrated nodes are indicated alphabetically. Relevant clades mentioned are highlighted and colored by group. Branching points with 95% confidence intervals are indicated in blue rectangles on each node, along with the inferred age of divergence in a scale of Million Years Ago (Mya).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7991728/v1/8ceb9e05b991891d9f8b6cec.jpg"},{"id":96726516,"identity":"1cf7288b-fb77-4c87-8b87-3dee51cc2f4e","added_by":"auto","created_at":"2025-11-25 12:34:36","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":43305930,"visible":true,"origin":"","legend":"\u003cp\u003eRibbon plot representing chromosome synteny between chromosome-scale genome assemblies available for afrotherian mammals and aligned to the karyotype of \u003cem\u003eT. ecaudatus\u003c/em\u003e. Chromosomes are represented by bars on a colour gradient, numbered at the top from longer to smaller autosomal scaffold for \u003cem\u003eT. ecaudatus\u003c/em\u003e. X chromosomes are represented at the rightmost end.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7991728/v1/b19a2df2cff385d97feb77c8.jpg"},{"id":106343793,"identity":"356e4de3-22ac-4ac2-ba04-4a853b49ba25","added_by":"auto","created_at":"2026-04-07 16:09:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":61435718,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7991728/v1/046806e6-792d-4d0f-947d-8005b99d7db2.pdf"},{"id":96913564,"identity":"a90bf8b0-b549-42e0-8daa-d8c0b9449d1a","added_by":"auto","created_at":"2025-11-27 14:02:46","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":19667,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7991728/v1/5cadb8a4e6d1b34315f18bf6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chromosome-level genome assembly of the Common tenrec, Tenrec ecaudatus (Schreber, 1778), a new model for early placental mammal evolution","fulltext":[{"header":"Background","content":"\u003cp\u003eAfrotheria is one of the four major placental mammal clades, comprising around one third of all eutherian orders (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). This taxon unites ungulate-like (Paenungulata: proboscideans, sea cows, hyraxes) and insect-feeding lineages (Afroinsectiphilia: aardvarks, sengis, tenrecs, golden moles). Despite displaying this diverse array of morphological features and ecological specializations that have sparked debate about the validity of the clade (\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), this group has been categorized as monophyletic by numerous taxonomic assessments employing modern molecular methods (\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Afrotherians, as their name suggests, are hypothesized to have originated on the African continent around 100 and 65\u0026nbsp;million years ago (Mya) and later highly diversified during the late Cretaceous period when the South American landmass fully separated from the rest of Africa, leaving it isolated (\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHowever, not all afrotherian species remained confined within continental Africa. Such is the case of the afrosoricidan (tenrecs and golden moles) family Tenrecidae, an insectivoran-grade lineage that colonized the island of Madagascar in the Indian ocean between 30 to 56 Mya (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) and subsequently radiated into the species-rich yet understudied group of mammals (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Commonly known as \u0026lsquo;Malagasy tenrecs\u0026rsquo;, this group of small mammals includes 31 currently accepted extant species (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), with four species classified as Vulnerable (VU) and two species classified as Endangered (EN) by the International Union for Conservation of Nature (IUCN). Yet, the number of threatened species is expected to increase in the following decade (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Though, alpha taxonomy of tenrecs is still not fully understood and the cryptic diversity might be higher (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis relatively unknown group of mammals displays several unique characteristics that makes them worthy of broader scientific interest (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). For example, tenrecs retain reproductive flexibility that likely mirrors early placental mammals (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Unlike most modern placentals, they exhibit superfetation, polyovulation, large litter sizes, and a lack of traditional antrum formation, suggesting a reproductive strategy that prioritized offspring quantity over metabolic efficiency (\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Tenrecs also undergo intrafollicular fertilization, a system thought to optimize fertilization success before the development of more complex implantation strategies (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). This reproductive flexibility may reflect strategies employed by early placental mammals, which likely relied on high reproductive output rather than sustained metabolic investment in individual offspring (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIndeterminate growth is also considered the ancestral trait of amniotes (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) and it\u0026rsquo;s rarely documented in mammals, with known cases limited to marsupials (e.g. red kangaroos, wallabies). Tenrecs vary widely in size at birth and exhibit striking developmental variability. Some juveniles grow only\u0026thinsp;~\u0026thinsp;20% in their first month, while others increase in size by ~\u0026thinsp;400% (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Such extreme growth plasticity suggests a disconnect between chronological and developmental age, allowing for profound variation in growth trajectories over a lifetime.\u003c/p\u003e\u003cp\u003e\u003cem\u003eTenrec ecaudatus\u003c/em\u003e (Schreber, 1778), also known by the common name \u0026lsquo;tailless tenrec\u0026rsquo; or \u0026lsquo;common tenrec\u0026rsquo;, is the only species of tenrec to have been introduced through human activity to several islands along the Mascarenhas archipelago, the Comoros and the Seychelles (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Tailless tenrecs being such a widespread species in between islands has resulted in the existence of multiple reports of individuals acting as a reservoir of \u003cem\u003eLeptospira\u003c/em\u003e and causing several zoonosis outbreaks (\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). This added to the fact that tenrecs are an important part of the bushmeat diet of Malagasy households (\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), makes \u003cem\u003eT. ecaudatus\u003c/em\u003e a species of special human interest. Among other particularities of this species, \u003cem\u003eT. ecaudatus\u003c/em\u003e has remarkable thermal plasticity. Active season tenrecs housed at 12\u0026deg;C have variable core body temperatures ranging from ~\u0026thinsp;13\u0026deg;C to ~\u0026thinsp;28\u0026deg;C and can maintain full locomotor abilities across that range (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Alternatively, hibernating tenrecs successfully hibernate at ambient temperatures of 28\u0026deg;C. Further, this thermoregulatory plasticity is accompanied by tremendous metabolic plasticity wherein torpid tenrecs at 12\u0026deg;C have metabolic rates that are nominally lower than that of a torpid Arctic ground squirrel hibernating at 4\u0026deg;C.\u003c/p\u003e\u003cp\u003eTaking into account all the peculiarities of Tenrecidae in general and the human relevance of the species, \u003cem\u003eT. ecaudatus\u003c/em\u003e makes a suitable candidate to be a model organism for physiological, developmental and evolutionary research of tenrecs, afrotherians and placental mammals as a whole (\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Increased availability of chromosome-scale genome assemblies and high throughput sequencing technology has made it both possible and almost a requirement to generate high quality genomic data for non-model species (\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). In this study, we generated a haplotype-resolved, chromosome-level genome assembly for the tailless tenrec, \u003cem\u003eT. ecaudatus\u003c/em\u003e. Furthermore, we supplemented this genomic resource with orthology based annotation and comparative genomic analyses of chromosome synteny in order to delve into the phylogenetic history of Afrotheria and their chromosome evolution. As a result, this genomic resource makes possible further research into Tenrecidae from a genomic standpoint, allowing for insight into previously unknown aspects of this group's biology and evolutionary history.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSample collection:\u003c/h2\u003e\u003cp\u003eSamples were taken from lab-reared common tenrec individuals from the breeding colony maintained at the University of Nevada, Las Vegas (UNLV), USA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The breeding colony originated from 40 wild-caught individuals imported from Mauritius in June 2014 under appropriate U.S. federal and state permits. Laboratory-reared tenrecs of male and female sex (Table S2) were euthanized via cervical dislocation followed by pneumothorax, and immediately dissected on ice. Heart, brain, kidney, testis, liver and embryotic tissue was extracted, rinsed in diethyl pyrocarbonate (DEPC)-treated water, and snap-frozen in liquid nitrogen. Samples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until sent to FaunaBio (Emeryville, CA, U.S.) for further processing. In addition, liver, kidney, uterus, and placenta were collected as above from a pregnant female reported in Stadtmauer et al. (2025) (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), preserved in RNAlater (AM7021, Thermo Fisher) and shipped to Yale University (New Haven, CT, USA) for processing.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDNA/RNA extraction and PacBio Sequencing:\u003c/h3\u003e\n\u003cp\u003eHeart, brain, kidney and liver samples underwent liquid nitrogen pulverization and were then extracted using the Monarch HMW DNA extraction kit for tissue (New England Biolabs) in accordance with protocol instructions. To process the liver sample, NaCl was utilized to remove polysaccharides and avoid glycogen contamination. Final quality measuring of DNA and concentration was performed with NanoDrop, Qubit Fluorometer and Femto quantification. Library preparation was carried out using the SMRTbell prep kit 3.0 (Pacific Biosciences, Menlo Park, CA). The library was sequenced on a PacBio Sequel II using the Sequel II binding kit 3.2 at QB3 Genomics, (UC Berkeley, Berkeley, CA). While aiming for 50x coverage, a total of 9 flow cells were run. Additionally, at this same facility PacBio Iso-Seq data was generated for liver, kidney, brain, testes, heart and embryonic tissue using 1,250 ng/sample. RNAlater-preserved specimens from the pregnant female were extracted using a Qiagen RNEasy Plus Micro Kit (Q74034) and sequenced on a PacBio Sequel II at the Yale Center for Genome Analysis.\u003c/p\u003e\n\u003ch3\u003eHi-C chromatin conformation capture:\u003c/h3\u003e\n\u003cp\u003eChromatin conformation capture was carried out in the facilities of Arima Genomics, Carlsbad, USA using the ARIMA-HiC kit for animal tissue (Document Part Number: A160132 v01) and following protocol\u0026rsquo;s guidelines. Illumina library preparation was performed following the ARIMA Library Prep Module user guide (Document Part Number: A160432 v02). The barcoded HiC library was then sequenced on an Illumina NovaSeq6000 platform with 150 bp paired-end reads.\u003c/p\u003e\n\u003ch3\u003eGenome assembly \u0026 annotation:\u003c/h3\u003e\n\u003cp\u003ePrior to the assembly, we called HiFi reads from PacBio subreads using pbccs v.6.4.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/nlhepler/pbccs\u003c/span\u003e\u003cspan address=\"https://github.com/nlhepler/pbccs\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and deepconsensus v.1.1.0 (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). HiFi reads were assembled using hifiasm v.0.18.7 (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e), with Hi-C reads used for phasing of the two haplotypes. We then mapped HiFi reads back to each assembly with minimap2 v.2.24 (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e) to remove unambiguous heterozygous sites, removed duplicates using Picard MarkDuplicates tool v.3.1.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://broadinstitute.github.io/picard\u003c/span\u003e\u003cspan address=\"http://broadinstitute.github.io/picard\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and called variants using DeepVariant v1.5.0 (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Finally, we corrected corresponding nucleotide sites in the assembly using BCFtools consensus v.1.13 (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo scaffold the polished haplotype assemblies, we mapped the Hi-C reads to each of the two assemblies using Chromap v.0.2.5 (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e) and then processed mapped reads with yahs v1.1a (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). We then concatenated the two yahs-scaffolded assemblies and remapped the Hi-C reads but now allowing for multimapping reads (-q 0) to avoid losing reads, mapped to regions identical between two haplotypes. This allowed us to cross-validate two haplotypes by manually curating them in parallel in PretextView v.0.2.5 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/wtsi-hpag/PretextView\u003c/span\u003e\u003cspan address=\"https://github.com/wtsi-hpag/PretextView\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Additionally, telomeric sequences were identified with tidk v.0.2.31 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/tolkit/telomeric-identifier\u003c/span\u003e\u003cspan address=\"https://github.com/tolkit/telomeric-identifier\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and where necessary were used to correct wrong contig orientations to have telomeres in the ends of resulting scaffolds. To finalize changes made via manual curation, as recently proposed by the Vertebrate Genomes and Darwin Tree of Life Projects, we used the rapid curation framework (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gitlab.com/wtsi-grit/rapid-curation/-/tree/main\u003c/span\u003e\u003cspan address=\"https://gitlab.com/wtsi-grit/rapid-curation/-/tree/main\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) from the Genome Reference Informatics Team (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). To estimate the assembly base accuracy, we used Merqury (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e) with the HiFi reads. To assess gene completeness, we used compleasm 0.2.6 (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e) with the BUSCO (Benchmarking universal single-copy orthologues) odb10 set of 9226 near-universally conserved mammalian genes.\u003c/p\u003e\u003cp\u003eWe used a combination of orthology inference and transcriptome sequencing to produce whole-genome annotations. For each of the two haplotype assemblies we generated alignment chains to human (hg38) as described previously (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e) and then used TOGA v.1.1.4 (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e) to annotate coding genes with human GENCODE 38 annotation serving as reference. We then processed previously produced Iso-Seq reads to obtain transcriptomic evidence for each tissue separately. Namely, circular consensus was generated from the subreads using ccs v.6.4.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/PacificBiosciences/pbbioconda\u003c/span\u003e\u003cspan address=\"https://github.com/PacificBiosciences/pbbioconda\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Adapter trimming and base quality filtering were performed with lima v2.2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/PacificBiosciences/pbbioconda\u003c/span\u003e\u003cspan address=\"https://github.com/PacificBiosciences/pbbioconda\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Processed reads were cleaned from polyA tails and polymerase switching artifacts using \u0026ldquo;refine\u0026rdquo; command from the Iso-Seq package v.4.0.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/PacificBiosciences/IsoSeq/blob/master/isoseq-clustering.md\u003c/span\u003e\u003cspan address=\"https://github.com/PacificBiosciences/IsoSeq/blob/master/isoseq-clustering.md\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and then clustered together and aligned to generate consensus reads using the \u0026ldquo;cluster\u0026rdquo; command from the Iso-Seq package. Additionally, reads were filtered for alignment quality using a custom Perl script (Supplemental Code). Finally, StringTie v.2.1.2 (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e) was used to produce assembled transcripts. Individual tissue annotations were then merged together. Finally, in cases where TOGA prediction was missing, transcriptome evidence was used as a primary annotation source, incorporating for the most part lineage-specific and noncoding transcripts.\u003c/p\u003e\u003cp\u003eComparative quality metrics with the additional genome assemblies included in the study were latter assessed with Quast v5.2 (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e), BBMap v37.62 (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e) and Compleasm v0.2.6 (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e)\u003c/p\u003e\n\u003ch3\u003ePhylogenetic reconstruction and date estimation:\u003c/h3\u003e\n\u003cp\u003eFor the phylogenetic reconstruction of afrotheria, we first downloaded full genome annotation data already available at the time, focusing on Tenrecidae and adding representatives from all remaining afrotherian clades. We assembled a dataset obtaining human reference-annotated data from the TOGA annotations for mammals repository on the Senckenberg Genome Browser (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://genome.senckenberg.de/TOGA.mammals.html\u003c/span\u003e\u003cspan address=\"https://genome.senckenberg.de/TOGA.mammals.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). For species of interest without an available annotation on this database, we supplemented it by producing our own annotation for available afrotherian genome assemblies also using the make_lastz_chains (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/hillerlab/make_lastz_chains\u003c/span\u003e\u003cspan address=\"https://github.com/hillerlab/make_lastz_chains\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e and TOGA v1.1.4 (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e) pipeline. This resulted in the creation of a dataset comprising 14 afrotherian species (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), including our own \u003cem\u003eTenrec ecaudatus\u003c/em\u003e annotation data. This dataset encompassed sea cows (Sirenia: \u003cem\u003eDugong dugon\u003c/em\u003e, \u003cem\u003eTrichechus manatus\u003c/em\u003e), elephants (Proboscidea: \u003cem\u003eLoxodonta africana\u003c/em\u003e, \u003cem\u003eElephas maximus\u003c/em\u003e), hyraxes (Hyracoidea: \u003cem\u003eHeterohyrax brucei\u003c/em\u003e, \u003cem\u003eProcavia capensis\u003c/em\u003e), the aardvark (Tubulidentata: \u003cem\u003eOrycteropus afer\u003c/em\u003e), a golden mole (Chrysochloridae: \u003cem\u003eChrysochloris asiatica\u003c/em\u003e), sengis (Macroscelidea: \u003cem\u003eRhynchocyon cirnei\u003c/em\u003e, \u003cem\u003eElephantulus edwardii\u003c/em\u003e, \u003cem\u003ePetrodromus tetradactylus\u003c/em\u003e) and tenrecs (Tenrecidae: \u003cem\u003eNesogale talazaci\u003c/em\u003e, \u003cem\u003eEchinops telfairi\u003c/em\u003e, \u003cem\u003eTenrec ecaudatus\u003c/em\u003e).\u003c/p\u003e\u003cp\u003eTo generate amino acid alignments as the basis for phylogenetic reconstruction, we filtered the human reference-aligned amino acid sequences produced by TOGA. We used the generated orthology classification to filter the transcripts based on genes that were classified as one to one (1:1) orthologs between the reference human genome and the query species genome. Given that even one to one orthologs can have multiple ortholog chains, we filtered for duplicated transcripts based on sequence length and prevalence of gaps. Next, we filtered for genes that occurred at least once per species, obtaining a list of 12062 genes. This list of genes was then used to filter transcripts in order to create one alignment per gene for the 14 chosen species using MAFFT v7.5 (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). These alignments were then trimmed using ClipKit v2.3 (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e), in kpi-smart-gap mode to dynamically determine the threshold of gaps to remove and keep parsimony informative sites only. Subsequently, the resulting alignments were filtered by sequence length keeping only those that were \u0026gt;\u0026thinsp;50 amino acids in length, thus obtaining a new filtered dataset corresponding to 7119 genes that are all over 50 parsimony informative sites in length.\u003c/p\u003e\u003cp\u003eIndividual, unrooted maximum-likelihood gene trees for each of the 7119 amino acid alignments were generated using IQ-TREE v2.1.4 (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). A Q.mammal\u0026thinsp;+\u0026thinsp;F\u0026thinsp;+\u0026thinsp;R9 substitution model was used for calculating each gene tree, with independently calculated log likelihood parameters. To account for anomalous long branches due to the estimation of an unrooted topology, we used TreeShrink v1.3.9 (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e) to filter out outlier tip branches. A coalescence-based species tree was generated using ASTRAL v5.7.8 (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e) in order to use the resulting topology as the fixed input tree to use with the MCMCTree program in the PAML suite (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e) for divergence time calculation. Consequently, the R package MCMCTreeR (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e) was used to further prepare the input files and node age priors for the MCMCTree divergence time inference analysis. Fossil calibration estimates were designated using the oldest known fossil proving the existence of the branch and multiplying the hard minimum age by 1.25 to estimate a soft maximum age (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In line with Heritage et al., 2021 (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e), age constraint intervals were placed on eight nodes representing crucial clade divergences within Afrotheria as follows: We used the \u003cem\u003eOcepeia daouiensis\u003c/em\u003e fossil (Node A; hard minimum at 60.5 Mya) to constrain the Paenungulata-Afroinsectiphilia split and thus as the oldest calibration for this dataset, this being the earliest known afrotherian fossil. \u003cem\u003eEritherium azzouzorum\u003c/em\u003e (Node B; 59 Mya) was chosen to calibrate the Paenungulata node; \u003cem\u003ePriscosiren atlantica\u003c/em\u003e (Node D; 28 Mya) for the split between Dugongidae and Trichechidae within Sirenia; \u003cem\u003eDaouitherium rebouli\u003c/em\u003e (Node E; 56 Mya) for the split between Proboscidea and Hyracoidea; \u003cem\u003eHeterohyrax auricampensis\u003c/em\u003e (Node F; 10.4 Mya) for the divergence within Hyracoidea; and the earliest \u003cem\u003eLoxodonta sp.\u003c/em\u003e (Node G; 6.5 Mya) for the split between extant proboscideans. Finally, within Afroinsectiphilia, \u003cem\u003eTodralestes variabilis\u003c/em\u003e (Node C; 56.8 Mya) was used to constrain the split between Afrosoricida and Macroscelidea; \u003cem\u003eOligorhynchocyon songwensis\u003c/em\u003e (Node H; 25.2 Mya) for the split between Rhynchocyonidae and Macroscelididae within Macroscelidea.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eFossil calibration ages used for divergence time inference within afrotheria. All ages are displayed in the Mya (Million years ago) unit.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHard minimum age (Mya)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoft maximum age (Mya)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNode\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eOcepeia\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e60.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e75.625\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(A)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eEritherium\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e73.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(B)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eTodralestes\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e56.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(C)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePriscosiren\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(D)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eDaouitherium\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(E)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eHeterohyrax auricampensis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(F)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eLoxodonta sp.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(G)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eOligorhynchocyon\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(H)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe approximate likelihood calculation for protein data function of MCMCTree v4.10.0 (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e) was used in order to calculate divergence times within Afrotheria. Two datasets were created for this; a concatenated set of filtered gene trees that share the proposed coalescence-based topology of the species tree produced with ASTRAL and the concatenated set of all gene trees. IQtree\u0026rsquo;s QMaker function (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e) was used to calculate amino acid substitution models for each dataset. Having specified the amino acid models, MCMCTree then uses CODEML (\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e) to calculate the Hessian matrix for protein data. Later, a Markov Chain Monte Carlo (MCMC) approach was used to calculate divergence times estimations using 1\u0026nbsp;million runs with 200 thousand burnin for both datasets. Multiple MCMCTree runs were performed and multiple dating scenarios were tested to check for convergence and effective sample sizes with Tracer v1.7.2 (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e). Estimates for both the filtered and the complete dataset were then visualized with TreeViewer v2.2.0 (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eSynteny analysis:\u003c/h2\u003e\u003cp\u003eIn order to gain insights into chromosome evolution among afrotherian mammals, we obtained all high quality chromosome-level genome assemblies produced for Afrotheria (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Chromosomes synteny of two elephants (\u003cem\u003eLoxodonta africana\u003c/em\u003e, \u003cem\u003eElephas maximus\u003c/em\u003e), two sea cows (\u003cem\u003eDugong dugon\u003c/em\u003e, \u003cem\u003eTrichechus manatus\u003c/em\u003e), one hyrax (\u003cem\u003eHeterohyrax brucei\u003c/em\u003e), one aardvark (\u003cem\u003eOrycteropus afer\u003c/em\u003e) and one elephant shrew (\u003cem\u003eRhynchocyon petersi\u003c/em\u003e) was compared to our assembly of \u003cem\u003eTenrec ecaudatus\u003c/em\u003e chromosomes. Genome assemblies were manually curated to extract only autosomal chromosome scaffolds and the X sex chromosome scaffold, due to not having a male representative for all species. We used ntSynt v1.0.2 (\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e) to infer multi-genome synteny between all the available chromosome-level genome assemblies of afrotherian species and our new \u003cem\u003eTenrec ecaudatus\u003c/em\u003e genome. This program utilizes a minimizer graph-based approach, allowing for the calculation of multiple genome homologous synteny blocks with over 15% overall genome divergence using minimal resources. Furthermore, we used Mash v.2.3 (\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e) to calculate pairwise mutation distance and therefore, genome divergence between assemblies. For visualizing the synteny plots we used ntSynt-viz v1.0.0 (\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eGenome assembly and annotation:\u003c/h2\u003e\u003cp\u003eWe obtained a highly complete and contiguous haplotype-resolved genome assembly for the common tenrec, \u003cem\u003eT. ecaudatus\u003c/em\u003e. (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Our primary haplotype was assembled to a total length of 4.06 Gb, whereas our secondary haplotype only had 3.44 Gb in length. Both reconstructed haplotypes showed remarkable BUSCO completion scores based on the set of conserved mammalian genes (Haplotype 1: mammalia odb10: C: 99.3% [S:96.92%, D:2.43%], F: 0.07%, M: 0.59%, n:9226; Haplotye 2: mammalia odb10: C: 97.16% [S:95.09%, D:2.07%], F: 0.11%, M: 2.73% n:9226). Added to this, the estimated k-mer based consensus quality (QV) portrays a very high base accuracy of 64.9 and 64.7 for both haplotypes respectively, indicating roughly one error per ~\u0026thinsp;3.1\u0026nbsp;million base pairs.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimary and secondary haplotype assembly statistics of the \u003cem\u003eTenrec ecaudatus\u003c/em\u003e genome assembly.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAssembly\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimary haplotype\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSecondary haplotype\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e# contigs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5663\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3611\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLargest contig (mb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e294\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e295\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal length (Gb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.44\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGC (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e44.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e44.35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003escaffold N50 (mb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e133.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e156.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003escaffold L50 (mb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBUSCO completion\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e99.3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e95.09%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eQV (Merqury)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e64.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e64.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAlong this orthology based quality measure, the annotation based quality measure calculated with TOGA yielded a total count of 17420 genes with intact open reading frames, 1720 genes with a type of inactivating mutation and 324 missing genes. Both these measures of quality positively highlight our \u003cem\u003eTenrec ecaudatus\u003c/em\u003e genome assembly and annotation compared to the available set of afrotherian mammal genome resources (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In this assemblage of genomes, the previous representation of Tenrecidae consisted only of the scaffold-level genome assemblies of \u003cem\u003eNesogale talazaci\u003c/em\u003e (BUSCO C: 66.7%; TOGA Intact ORF genes: 61.8%) and \u003cem\u003eEchinops telfairi\u003c/em\u003e (BUSCO C: 95.9%; TOGA Intact ORF genes: 66.1%) (Table S3).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003ePhylogenomic reconstruction and dating:\u003c/h2\u003e\u003cp\u003eWe reconstructed the phylogenetic relationships of Afrotheria (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) using a protein alignment dataset for both species with available annotation data and those with annotations generated in this study. We rooted our resulting topology on the basal split within Afrotheria between Afroinsectiphilia and Paenungulata. The recovered topology coincides with that of recent studies employing multiple molecular evidence (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e). Notably, our coalescent-based topology recovered Hyracoidea as a sister group to Proboscidea, thus rejecting the Tethytheria hypothesis (elephants\u0026thinsp;+\u0026thinsp;sea cows) (\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e). The uncalibrated nodes within Afroinsectiphilia estimate the split of tenrecs from golden moles at around 49.4 Mya. Consequently, the following split between Tenrecinae and Oryzorictinae is estimated around 27.06 Mya. and the divergence time between \u003cem\u003eT. ecaudatus\u003c/em\u003e and \u003cem\u003eE. telfairi\u003c/em\u003e is estimated at 14.37 Mya.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eSynteny analysis:\u003c/h2\u003e\u003cp\u003eWe inferred multi-genome synteny for eight chromosome-level genome assemblies of afrotherian species including our novel \u003cem\u003eTenrec ecaudatus\u003c/em\u003e genome assembly, representing all six orders within the clade (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). All inferred chromosomes were aligned based on synteny with \u003cem\u003eT. ecaudatus\u003c/em\u003e. The first synteny blocks corresponding with Chr. 1\u0026ndash;2 in \u003cem\u003eT. ecaudatus\u003c/em\u003e show to be highly conserved between clades. Though Afroinsectiphilia in particular showed remarkably high chromosome divergence.\u003c/p\u003e\u003cp\u003eBetween the common tenrec (\u003cem\u003eT. ecaudatus\u003c/em\u003e) and the giant sengi (\u003cem\u003eR. petersi\u003c/em\u003e) there is a complex display of chromosome rearrangements including fission/fusion events on all chromosomes as well as multiple instances of inversions. Similarly, several inversions and fission/fusion events can be found between the chromosomes of \u003cem\u003eR. petersi\u003c/em\u003e and \u003cem\u003eO. afer\u003c/em\u003e, most notably, the event that resulted in Chr. 4 of the latter with one half corresponding to the inverted Chr. 10 of \u003cem\u003eR. petersi\u003c/em\u003e and the other half corresponding to the inverted Chr. 4 of the same species. In the case of Paenungulata, the compared representatives show a much more conserved chromosome synteny than their sister clade. Both sea cow species show the same chromosome number, yet with a different chromosome configuration. Moreover, both representatives of extant Proboscidea show full microsynteny, while differing in overall chromosome scaffold length after chromosome 13.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTwo high quality haplotypes were assembled representing the genome of \u003cem\u003eT. ecaudatus\u003c/em\u003e, adding to this the annotation of said genome. Though, the discordance in quality observed between the assembled haplotype one and haplotype two is more likely due to this last assembly containing only autosomal chromosomes. For comparative analyses, only haplotype one was considered on the quality metrics.\u003c/p\u003e\u003cp\u003eAlthough commonly used as an indicator of genome quality, the scaffold N50 metric does not directly link to better quality of genome assembly (\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e) in this particular phylogenetic order-wide comparison, due to the highly variable chromosome number and genome sizes across Afrotheria (\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e). Such is the case of the aardvark assembly, showing a highly superior N50 metric (644 mb) due to its massive chromosomes, and therefore, scaffold sizes. In cases like this where the compared assemblies are of outstanding quality and mostly at a chromosome-scale, BUSCO (\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e) completion can be a more appropriate metric of assembly quality. Currently, Afrotheria is not well genomically represented. Despite being a diverse superorder, only a fraction of afrotherian species have an available high quality genome assembly. Out of the now 16 species that have an available genome assembly, our \u003cem\u003eTenrec ecaudatus\u003c/em\u003e assembly ranks as the highest quality assembled and annotated one for Tenrecidae and Afrosoricida, while also qualifying as one of the top five highest quality genomes for Afrotheria in terms of annotation and BUSCO completion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Table S3). High quality annotation can be attributed to the use of multiple-tissue transcriptome data used to complement the TOGA produced annotation (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eUsing eight fossil calibration points, our results are consistent with modern phylogenetic inference approaches that recover Afroinsectiphilia as a monophyletic clade (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e). These calibrations corresponded to the oldest available fossil for a determined group and estimated soft maximum bounds. Since including multiple calibrations, the determination of the upper bound is not as key of a factor as when using a single calibration point (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). Accordingly, calibrated nodes showed small confidence intervals around the estimated hard minimum and soft maximum ages. Though, nodes that remained uncalibrated, such as the ones within Afrosoricida showed broad intervals of confidence. The afrosoricidan fossil record is very limited and fragmented, and the phylogenetic position of most extinct species remains unclear (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Thus, these nodes currently lack any fossil-informed calibration that could help increase the precision of temporal estimates (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e). In our estimates, the nodes comprising Afrosoricida (95%HPD=[45.105, 53.5972] Mya), Tenrecidae (95%HPD=[21.5568, 31.0638] Mya) and Tenrecinae (95%HPD=[9.55502, 18.9804] Mya), show younger mean divergence times than in Everson et al. (2016), which estimates a mean divergence of 58.4 Mya for Afrosoricida, 35.5 Mya for Tenrecidae and 25.7 Mya for Tenrecinae. Our results further show that even with a genome-wide dataset of phylogenetic data, divergence dating can be challenging for some nodes if there is no uniform temporal signal among genes. The choice of the clock model, too, has recently been shown to affect divergence dating within afrotherians when ingroup fossils for calibration are lacking (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e). We recommend future work to focus on this issue of divergence dating within afrosoricidans as particularly the split within Tenrecidae can be indicative of the minimum colonization date of Madagascar.\u003c/p\u003e\u003cp\u003eTenrecidae is also currently the family among Afrotheria with the least amount of cytogenetic studies (\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e). In terms of currently known chromosome numbers, afrosoricidans range from 2n\u0026thinsp;=\u0026thinsp;26 to 2n\u0026thinsp;=\u0026thinsp;34 in golden moles (\u003cspan additionalcitationids=\"CR85\" citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e), keeping highly conserved chromosome numbers, whereas in Tenrecidae chromosome numbers are more variable. This variation ranges from 2n\u0026thinsp;=\u0026thinsp;30 (or even from 2n\u0026thinsp;=\u0026thinsp;14 according to an unsupported claim of Gilbert (2008)(\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e)) to 2n\u0026thinsp;=\u0026thinsp;56 (\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e, \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e). Therefore, our chromosome number estimation ranges within this reported variation.\u003c/p\u003e\u003cp\u003eAlthough some studies involving chromosome painting proposed the aardvark\u0026rsquo;s karyotype to share a strong resemblance with the eutherian ancestral state (\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e), more recent studies on mammal chromosome evolution hypothesize that the last common ancestor (LCA) of all placental mammals possessed 19 autosomal pairs (2n\u0026thinsp;=\u0026thinsp;40) and showed great chromosomal conservation with the LCA of Amniota. Similarly, the hypothesized ancestral karyotype for Atlantogenata (Xenarthra\u0026thinsp;+\u0026thinsp;Afrotheria) would have shown the same number of chromosomes, differing only by four chromosome inversions accumulated over 5\u0026nbsp;million years (\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e). Thus, chromosome number in \u003cem\u003eT. ecaudatus\u003c/em\u003e is closer to the ancestral state in placental mammals and potentially afrotherians as well.\u003c/p\u003e\u003cp\u003eSeveral million years of evolution have resulted in Afrotheria displaying great chromosome variation between orders, meanwhile maintaining a high degree of synteny for the sex chromosomes, which has been very well documented in mammals (\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e, \u003cspan additionalcitationids=\"CR91\" citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e). This can be seen in the chromosome rearrangements observed between the common tenrec (\u003cem\u003eT. ecaudatus\u003c/em\u003e), giant sengi (\u003cem\u003eR. petersi\u003c/em\u003e) and aardvark (\u003cem\u003eO. afer\u003c/em\u003e), which despite varying a great amount in chromosome number, size and arrangement, still maintain conserved X chromosome synteny. In contrast to this highly variable group, elephant species \u003cem\u003eE. maximus\u003c/em\u003e and \u003cem\u003eL. africana\u003c/em\u003e, having had their divergence event much closer to present times, show virtually no chromosome rearrangements. The west indian manatee (\u003cem\u003eT. manatus\u003c/em\u003e) and dugong (\u003cem\u003eD. dugon\u003c/em\u003e) do not show the same level of conserved chromosome synteny as the compared elephant species, showing multiple chromosome reorganization events converging on the same chromosome number (2n\u0026thinsp;=\u0026thinsp;48) (\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e). Interestingly, \u003cem\u003eTrichechus inunguis\u003c/em\u003e, a particular manatee species not included in this study, displays a karyotype of 2n\u0026thinsp;=\u0026thinsp;56, eight more autosomes than its sister species \u003cem\u003eT. manatus\u003c/em\u003e. These species of manatee have been reported to have diverged via chromosomal fusion events, a pericentric inversion and six Robertsonian translocations (\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e). In other words, \u003cem\u003eD. dugong\u003c/em\u003e and \u003cem\u003eT. manatus\u003c/em\u003e exhibit the derived karyotypic state, whereas \u003cem\u003eT. inunguis\u003c/em\u003e retains the ancestral state. This is further supported by Hyracoidea being hypothesized as having 2n\u0026thinsp;=\u0026thinsp;54 as their ancestral state, which \u003cem\u003eH. brucei\u003c/em\u003e and other hyrax species maintain until today (\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e). This same species also shows very few chromosome rearrangements that separate them from proboscideans, mainly an inversion on Chr. 19 and fission/fusion of Chr. 9 and 10 into three and two separate chromosomes respectively.\u003c/p\u003e\u003cp\u003eOverall, the high quality genomic resource generated in this study for \u003cem\u003eT. ecaudatus\u003c/em\u003e is compatible with genomic analyses such as phylogenomic reconstruction, multi-genome chromosome synteny inference and although challenging, molecular divergence dating. These insights gained into the evolutionary history of \u003cem\u003eT. ecaudatus\u003c/em\u003e and Afrosoricida are just a first example of the potential that genomics offers to bridge the gap in the knowledge of tenrec evolutionary history. Further analyses should focus more on delving into the physiological plesiomorphic characters present in \u003cem\u003eT. ecaudatus\u003c/em\u003e and their genomic elements in order to explore how these make tenrecs unique creatures and how mammalian genomes may have evolved through the ages.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eMya: Million years ago\u003c/p\u003e\n\u003cp\u003eIUCN: International Union for Conservation of Nature\u003c/p\u003e\n\u003cp\u003eDEPC: Diethyl pyrocarbonate\u003c/p\u003e\n\u003cp\u003eng: Nanograms\u003c/p\u003e\n\u003cp\u003ePacBio: Pacific Biosciences\u003c/p\u003e\n\u003cp\u003ebp: Base pairs\u003c/p\u003e\n\u003cp\u003eBUSCO: Benchmarking universal single-copy orthologues\u003c/p\u003e\n\u003cp\u003eGb: Giga base-pairs\u003c/p\u003e\n\u003cp\u003eMCMC: Markov Chain Monte Carlo\u003c/p\u003e\n\u003cp\u003eORF: Open reading frame\u003c/p\u003e\n\u003cp\u003eChr: Chromosome\u003c/p\u003e\n\u003cp\u003eHPD: Highest posterior density\u003c/p\u003e\n\u003cp\u003eLCA: Last common ancestor\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003e\u0026nbsp;\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genome annotation data newly generated with TOGA for \u003cem\u003eTenrec ecaudatus\u003c/em\u003e and the annotation data generated previously are available at: https://genome.senckenberg.de/download/TOGA/human_hg38_reference/Afrotheria/\u003c/p\u003e\n\u003cp\u003eRaw data and genome assembly data have been deposited in NCBI under BioProject accession number PRJNA1359382.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD.H.R. and P.A. acknowledge support from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, HO3492/25-1 \u0026amp; AR1307/5-1). Funding for F.vB. was provided by the National Science Foundation grants IOB 0448396 and IOS 1655091. Additional sequencing funding was provided by the Yale Institute for Biospheric Studies (to D.J.S.), the John Templeton Foundation (61329, to G.P.W.), and a SMRT cell donated for novel species pilots by the Yale Center for Genomic Analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD.J.S. and G.P.W. thank the Yale Center for Genome Analysis and Keck Microarray Shared Resource at Yale University for PacBio sequencing services, funded in part by the National Institutes of Health instrument grant 1S10OD028669-01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eF.vB. and G.R.S. provided tissue samples. D.J.S., J.G. and C.L. performed wet lab procedures. L. G., D. J. S. and G.P.W. produced/provided sequencing data. E.L. and M.H. handled \u003cem\u003eT. ecaudatus\u003c/em\u003e genome assembly and annotation. D.H.R. handled bioinformatic analyses and data interpretation. D.H.R. drafted the manuscript and crafted the figures. P.A., F.vB., E.L. and D.J.S. contributed to writing/reviewing the manuscript. F.vB., L.G., K.R.G., M.H. and P.A. supervised the study. All authors have read and approved the final version of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSampling protocols for this study were approved by the University of Nevada, Las Vegas Institutional Animal Care and Use Committee (IACUC) under project number [IACUC-01176]: 1397752 from 17/01/2025.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.G., K.G., J.G. and C.L. are employed by FaunaBio. The rest of the authors have declared no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSpringer MS. Afrotheria. Curr Biol. 2022;32(5):R205\u0026ndash;10.\u003c/li\u003e\n\u003cli\u003eAverianov AO, Lopatin AV. 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John Wiley \u0026amp; Sons; 2006.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Tenrec, Genomics, Afrotheria, Afroinsectiphilia, Madagascar, Phylogenetics, Synteny, Chromosome-level, Genome","lastPublishedDoi":"10.21203/rs.3.rs-7991728/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7991728/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eOur understanding of many biological aspects of early placental mammals is still very limited. Due to the paucity of the fossil record, attention is often turned to those extant organisms that share plesiomorphic characters in order to gain insights into the evolutionary history of mammals. Afrotheria is one of the four major clades of placental mammals, accounting for around a third of all mammalian orders, and encompasses a wide array of differently adapted species. Within Afrotheria, tenrecs are a more species-rich group of small mammals native to the island of Madagascar that display several special traits resembling those hypothesized on early placentals regarding reproductive strategies, thermoregulation and growth metabolism. Despite this, tenrecs remain heavily understudied in many aspects. Genomic information for this group of mammals is scarce and not up to modern quality standards.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eWe present here the complete, chromosome-scale reference genome and annotation of the common tenrec, \u003cem\u003eTenrec ecaudatus\u003c/em\u003e. To put this new resource to use, we conducted a phylogenetic reconstruction and divergence time estimation for Afrotheria using all the available genomic resources for afrotherian mammals. This analysis recovered the phylogenetic order containing hyraxes as a sister group to elephants and a younger molecular divergence of tenrecs than previously estimated. Added to this, our comparative chromosome-synteny analyses showed significant rearrangements within afrotherians, especially on the clade shared by tenrecs, elephant-shews and the aardvark (Afroinsectiphilia).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThis newly produced high quality genome assembly proves to be a valuable resource to complement our genomic understanding of Afrotheria, allowing for insights into chromosome evolution, time of molecular divergence and phylogenetic reconstruction. This establishes a basis for further studies to utilize this resource to further pursue evolutionary questions regarding tenrecs adaptations and comparative analyses within Afrotheria.\u003c/p\u003e","manuscriptTitle":"Chromosome-level genome assembly of the Common tenrec, Tenrec ecaudatus (Schreber, 1778), a new model for early placental mammal evolution","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-25 12:34:30","doi":"10.21203/rs.3.rs-7991728/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-29T10:56:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-27T21:18:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"143383345487035256423676327974339878592","date":"2026-01-13T16:53:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-19T13:24:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"302420686188520967302726673638647063944","date":"2025-11-18T09:23:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"334087878621082382862391202224756359743","date":"2025-11-17T09:41:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-13T09:03:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-13T08:57:18+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-12T15:47:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-12T14:37:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2025-11-12T14:32:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c915689c-12bb-4341-8f6e-9e994ec99b88","owner":[],"postedDate":"November 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-07T16:05:02+00:00","versionOfRecord":{"articleIdentity":"rs-7991728","link":"https://doi.org/10.1186/s12864-026-12794-9","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2026-03-31 15:59:49","publishedOnDateReadable":"March 31st, 2026"},"versionCreatedAt":"2025-11-25 12:34:30","video":"","vorDoi":"10.1186/s12864-026-12794-9","vorDoiUrl":"https://doi.org/10.1186/s12864-026-12794-9","workflowStages":[]},"version":"v1","identity":"rs-7991728","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7991728","identity":"rs-7991728","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}