Abstract Descent of testes from a position near the kidneys into the lower abdomen or into the scrotum is an important developmental process that occurs in all placental mammals, with the exception of five afrotherian lineages. Since soft-tissue structures like testes are not preserved in the fossil record and since key parts of the placental mammal phylogeny remain controversial, it has been debated whether testicular descent is the ancestral or derived condition in placental mammals. To resolve this debate, we used genomic data of 71 mammalian species and analyzed the evolution of two key genes (relaxin/insulin-like family peptide receptor 2 [RXFP2] and insulin-like 3 [INSL3]) that induce the development of the gubernaculum, the ligament that is crucial for testicular descent. We show that both RXFP2 and INSL3 are lost or nonfunctional exclusively in four afrotherians (tenrec, cape elephant shrew, cape golden mole, and manatee) that completely lack testicular descent. The presence of remnants of once functional orthologs of both genes in these afrotherian species shows that these gene losses happened after the split from the placental mammal ancestor. These “molecular vestiges” provide strong evidence that testicular descent is the ancestral condition, irrespective of persisting phylogenetic discrepancies. Furthermore, the absence of shared gene-inactivating mutations and our estimates that the loss of RXFP2 happened at different time points strongly suggest that testicular descent was lost independently in Afrotheria. Our results provide a molecular mechanism that explains the loss of testicular descent in afrotherians and, more generally, highlight how molecular vestiges can provide insights into the evolution of soft-tissue characters.

Author summary While fossils of whales with legs demonstrate that these species evolved from legged ancestors, the ancestral state of nonfossilizing soft-tissue structures can only be indirectly inferred. This difficulty is also confounded by uncertainties in the phylogenetic relationships between the animals concerned. A prime example is the case of testicular descent, a developmental process that determines the final position of testes, which occurs in most placental mammals but is absent from several afrotherian lineages. Here, we discovered that afrotherians possess remnants of genes known to be required for testicular descent. These “molecular vestiges” show that testicular descent was already present in the placental ancestor and was subsequently lost in Afrotheria. Our study highlights the potential of molecular vestiges in resolving contradictory ancestral states of soft-tissue characters.

Citation: Sharma V, Lehmann T, Stuckas H, Funke L, Hiller M (2018) Loss of RXFP2 and INSL3 genes in Afrotheria shows that testicular descent is the ancestral condition in placental mammals. PLoS Biol 16(6): e2005293. https://doi.org/10.1371/journal.pbio.2005293 Academic Editor: Laurence Hurst, University of Bath, United Kingdom of Great Britain and Northern Ireland Received: January 1, 2018; Accepted: May 24, 2018; Published: June 28, 2018 Copyright: © 2018 Sharma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All genome alignments are available at https://bds.mpi-cbg.de/hillerlab/testicondy/. Sanger sequences are available from GenBank (accession numbers MH347268-MH347272). All data are also available on request. Funding: Max Planck Society. Received by MH. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. German Research Foundation (grant number HI 1423/3-1). Received by MH. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Leibniz Association (grant number SAW-2016-SGN-2). Received by TL, HS, MH. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: CESAR, Coding Exon-Structure Aware Realigner; INSL3, insulin-like 3; Mya, million years ago; RXFP2, relaxin/insulin-like family peptide receptor 2; SRA, Sequence Read Archive

Introduction In placental mammals—the eutherian crown group consisting of the clades Afrotheria, Xenarthra, and Boreoeutheria [1]—optimal testicular function requires a temperature that is lower than the body temperature. To achieve this, the testes are located outside of the abdominal cavity in a scrotum in many species such as primates, most rodents, lagomorphs, most carnivores, and most terrestrial artiodactyls [2, 3]. Alternatively, testes are located in the lower abdomen in dolphins, true seals, pangolins, and other mammals. In these species, testicular cooling is achieved by vascular countercurrent heat exchanger systems, as observed in dolphin [4]; direct cooling with blood from the hind limbs, as observed in seals [5]; or testicular cooling may not be necessary, as these species have lower body temperatures [2, 6, 7]. The position of the testes in the lower abdomen or in the scrotum is the result of a developmental descent process (S1 Fig). During mammalian development, testes initially form at a position near the kidneys in the embryo. Testicular descent into the scrotum occurs in two phases: first from the abdomen to the inguinal canal and second through the inguinal canal into the scrotum [8, 9]. The first transabdominal phase is governed by the growth and reorganization of the gubernaculum, a ligament that connects the lower pole of the testes and inner ring of the future inguinal canal [8–10]. Migration of the testes is caused by the swelling of the distal gubernaculum, which anchors the testis to the inguinal canal, while the abdominal cavity enlarges. The second inguinoscrotal phase is dependent on androgen signaling and requires the elongation of the gubernaculum, which migrates into the scrotum [8–10]. The involved signaling and mechanics make testicular descent a difficult and complex developmental process. Failure in any of the descent phases results in a pathological condition called cryptorchidism (absence of testes from the scrotum), which is a congenital birth defect observed at an appreciable frequency in human males (2%–4% at birth [11]) and other animals (up to 10% in male dogs [12], 2% in male cats [13], 2%–8% in male horses [14]). Almost all placental mammals exhibit either partial descent (only the transabdominal phase), which results in ascrotal testes located in the lower abdomen, or complete descent (transabdominal and inguinoscrotal phase), which results in scrotal testes [2, 3]. A notable exception is Afrotheria, in which five of the six main lineages (represented here by the lesser hedgehog tenrec, cape golden mole, cape elephant shrew, manatee, elephant, and rock hyrax) do not show any testicular descent and have testes positioned at their initial abdominal position near the kidneys [2, 3, 15–17]. This lack of any testicular descent is termed testicondy. The aardvark is the only afrotherian exhibiting descended but ascrotal testes [2, 3, 18]. A schematic illustration of the different position of testes in mammals is shown in S1 Fig. Since Afrotheria represent one of the three main clades of placental mammals (together with Xenarthra and Boreoeutheria), two different evolutionary scenarios could explain testicondy in several afrotherian lineages. First, if testicondy is the ancestral condition in placental mammals, then testicular descent was gained two or three times (depending on the phylogeny) in Xenarthra, Boreoeutheria, and the aardvark lineage. Second, if testicular descent is the ancestral condition in placental mammals, then testicular descent was lost once or more often (again depending on the phylogeny) in five of the six afrotherian lineages. Since soft-tissue structures like testes and the transient gubernaculum ligament are typically not preserved in the fossil record, the evolution of such soft-tissue structures can only be inferred by analytical methods such as parsimony, extant phylogenetic bracketing, or maximum likelihood [19–22], all of which rely on the given phylogenetic tree. Consequently, resolving whether testicondy or testicular descent is the ancestral condition in placental mammals requires accurate knowledge of the underlying phylogeny. Unfortunately, although integrative approaches using both morphological and molecular characters have brought major advances in our understanding of mammalian phylogeny [23–26], there is still no final consensus on the relationships between (and sometimes within) the main clades of placental mammals. In particular, the placental root and branching pattern of the clades Afrotheria, Xenarthra, and Boreoeutheria are still debated [26–28] (S2 Fig), and an analysis of rare genomic events raised the concrete possibility of a near-simultaneous split [29]. Furthermore, the phylogeny within Afrotheria is not well resolved, because of conflicting evidence for the position of the aardvark (the only nontesticond afrotherian lineage) and the relationships between manatees, elephants, and hyraxes [30–34] (S3 Fig). Given these phylogenetic uncertainties, it is probably not surprising that two different studies reached opposite conclusions about whether testicondy is the ancestral or derived state for placental mammals and for Afrotheria. Werdelin and Nilsonne [2] inferred that testicular descent in placental mammals and Afrotheria is the ancestral condition (testicular descent was subsequently lost). However, their results were based on a phylogeny in which Afrotheria were nested within Boreoeutheria, which is not supported by current phylogenies. More recently, Kleisner and colleagues [3] reexamined the evolution of testicular descent in the context of current phylogenies and came to the opposite conclusion that testicondy in placental mammals and Afrotheria is the ancestral phenotypic character. Here, we sought to resolve this debate whether testicondy or testicular descent is the ancestral condition in placental mammals and in Afrotheria by using molecular evidence. First, we reasoned that if testicular descent is ancestral, then testicond afrotherian lineages may have lost key genetic information that is necessary for testicular descent. Such a loss of genetic information may be detectable by comparative genomics analysis. Second, we reasoned that if testicular descent is ancestral and if aardvarks are nested within Afrotheria, then testicondy would have evolved independently several times. This is expected to leave a signature of independent loss of the genetic information that is necessary for testicular descent. By analyzing the evolution of two key genes (relaxin/insulin-like family peptide receptor 2 [RXFP2] and insulin-like 3 [INSL3]) that are required for gubernaculum development and function in 71 placental mammals, we found that both genes have loss-of-function mutations only in several testicond afrotherian species. The absence of shared inactivating mutations and our age estimates for the loss of RXFP2 further suggest that testicondy evolved independently in afrotherian lineages at different time points. Together, these results provide not only a molecular mechanism that explains the loss of testicular descent in afrotherian lineages but also shows that testicular descent is the ancestral state for placental mammals and Afrotheria.

Discussion The evolution of testicondy and whether testicular descent is the ancestral [2] or derived state [3] for placental mammals and for Afrotheria has been controversial, despite agreement in the phenotypic character assignment. The different conclusions are mainly due to persisting differences in the phylogeny, which affect ancestral character reconstruction. To resolve this debate, we investigated the evolution of RXFP2 and INSL3, two genes encoding a hormone receptor pair that is required for the development of the testes-descending gubernaculum ligament. We found remnants of once functional orthologs of RXFP2 and INSL3 as molecular vestiges in four testicond afrotherian lineages. Together with the presence of orthologs of both genes in other Afrotheria and other placental mammals, this shows that these genes were lost after the testicond lineages split from the afrotherian ancestor. This allows us to conclude that testicular descent is the ancestral condition in placental mammals and was subsequently lost in different afrotherian lineages. Importantly, our conclusion holds regardless of persisting phylogenetic discrepancies that involve the branching pattern of Afrotheria, Xenarthra, and Boreoeutheria at the placental root, and the phylogeny within Afrotheria (S2 and S3 Figs). Our study also provides three lines of evidence that testicondy evolved independently in Afrotheria. First, if testicondy evolved in the common ancestor of any two testicond afrotherians, we would expect inactivating mutations in RXFP2 and INSL3 that are shared among species. However, both genes do not exhibit any shared inactivating mutations. Second, by estimating how long these genes have been evolving neutrally, we found different time intervals in which gene loss and likely testicondy evolved. These estimated time intervals may be helpful to better understand the ecological conditions under which testicondy evolved repeatedly. Third, independent evolution of testicondy is further supported by our finding that the testicond elephant and rock hyrax have intact RXFP2 and INSL3 genes that still appear to evolve under purifying selection. Since recent shifts from purifying selection to neutral evolution will not leave a detectable signature of significantly increased Ka/Ks ratios, our results suggest that elephant and rock hyrax RXFP2 and INSL3 may have come under relaxed or no selection in recent evolutionary time. In agreement with a more recent evolution of testicondy, the rock hyrax still exhibits rudiments of gubernacular structures [15]. It is possible that amino acid mutations or cis-regulatory mutations that affect the expression of RXFP2 or INSL3 are responsible for testicondy in elephant and rock hyrax. Indeed, the RXFP2 promoter region exhibits more sequence divergence in the rock hyrax than in all other nontesticond species, and the elephant sequence also shows elevated divergence compared to most but not all mammals (S12 Fig). However, the precise genomic basis of testicondy in elephant and rock hyrax remains to be elucidated. Spermatogenesis and sperm storage require temperatures below 37 °C. Body temperatures below 35 °C, as observed in the heterothermic tenrecs and golden moles [7], may abolish the need for testicular cooling, which would explain why testicondy is tolerated in these species. However, other afrotherian species have body temperatures similar to many scrotal mammals. For example, the body temperature of elephants and elephant shrews is 36.8 °C and 37.2 °C, respectively [7, 58]. This raises the questions of how these testicond afrotherian lineages maintain normal testicular function at a body temperature of approximately 37 °C and whether these species have evolved novel cooling mechanisms. Previous anatomical studies did not reveal conclusive evidence of such cooling systems. In particular, testicond afrotherians do not possess a pampiniform plexus or a vascular countercurrent heat exchanger that could act as a comparable cooling system [17, 59, 60]. In the dugong, the epididymis has a large, folded surface and is surrounded by highly vascularized tissue [60]; however, whether this arrangement acts as a cooling system is not clear. In rock hyrax and elephant, it was observed that the part of the testicular duct that stores spermatozoa lies close to the body surface [59], but it remains unknown whether this anatomical feature has a cooling function during sperm storage. Hence, detailed comparative and functional studies of the testes, epididymis, and their associated anatomical structures are required to understand whether Afrotheria evolved novel mechanisms for sperm cooling during spermatogenesis and storage. Furthermore, it would be of great interest to learn why several afrotherian but not any other placental lineages have completely lost testicular descent. For example, it is unknown whether the loss of testicular descent is beneficial for these species, whether anatomical constraints associated with body plan or life history explain the loss of descent, or whether this phenotypic reversal is an evolutionary tradeoff for another advantageous trait. More anatomical studies that in particular compare the development of Afrotheria are required to address these questions. More generally, our results highlight how molecular evolution can shed light on the evolution of phenotypes. While the fossil record has contributed substantially to our understanding of how hard-tissue characters such as bones, teeth, or shells evolved, the evolution of soft-tissue structures that do not fossilize can often only be inferred with analytical methods. Moreover, conclusions about the ancestry of such characters are dependent on the underlying phylogeny. Molecular vestiges offer an alternative strategy to investigate character ancestry. This strategy may be broadly applicable, since molecular vestiges are also known for other phenotypes whose ancestry is not controversial based on fossil evidence and accurate knowledge of the underlying phylogeny. For example, remnants of enamel-related genes in toothless mammals [61–63], remnants of hair development genes in hairless cetaceans [63], remnants of gastric genes in vertebrates without a stomach [64], or remnants of eye-related genes in subterranean mammals with degenerated eyes [65–68] show that these species descended from ancestors with teeth, hair, a stomach, and functional eyes, respectively. Thus, instead of investigating a soft-tissue structure directly, one can trace the evolution of genes that are crucial for the development of this structure. Molecular vestiges of such genes can then provide insights into the ancestry of soft-tissue structures, even if the phylogenetic positions of the respective species remain controversial.

Materials and methods Genome alignments To investigate the coding region of RXFP2 and INSL3 in placental mammals, we used an alignment between the human hg38 genome assembly and the genomes of 68 nonhuman placental mammals [45]. Since this alignment does not provide the rock hyrax (Procavia capensis) and Hoffmann’s two-toed sloth (Choloepus hoffmanni), two mammals for which improved genome assemblies have recently become available, we used the pipeline of lastz (version 1.03.54, parameters K = 2,400, L = 3,000, Y = 9,400, H = 2,000, and the lastz default scoring matrix) [69], chaining (parameters chainMinScore 1,000, chainLinearGap loose), and netting (default parameters) [70] to compute new genome alignments. Since several species have assembly gaps overlapping INSL3 exon 1, likely because this GC-rich region gets poor coverage in Illumina sequencing, we further computed genome alignments using updated assemblies of bonobo, domestic goat, camel, and dolphin. We also inspected the genome alignment to the recent gorGor5 assembly of the gorilla, provided by UCSC [71]. For gorilla, goat, and dolphin, the new assemblies closed these assembly gaps and revealed an intact INSL3 exon 1. For bonobo and camel, the new assemblies still have assembly gaps overlapping INSL3 exon 1. S3 Table lists all species and their assemblies that were analyzed. We examined the colinear alignment chains (loaded in the UCSC genome browser) in order to assess if gene order around the RXFP2 and the INSL3 genes is conserved in Afrotheria. This showed that both genes and several neighboring genes occur in a conserved order in afrotherian genomes. Furthermore, existing gene annotations (Ensembl) and reciprocal-best BLAST of the human proteins confirmed that the aligning loci contain orthologous RXFP2 and INSL3 genes or their remnants. To perform ultra-sensitive genome alignments that could reveal any hitherto undetected functional copies of RXFP2 or INSL3, we first computed genome alignments between the human genome (hg38 assembly) and each of the seven afrotherians, using highly-sensitive lastz parameters by setting K = 2,000 and L = 2,500. Subsequently, we used even more sensitive parameters to find additional local alignments by running lastz with parameters K = 1,500, L = 2,200, and W = 5 on all chain gaps (nonaligning regions flanked by aligning blocks) that are at least 30 bp and at most 500 Kb long, as described in [45, 72]. We built alignment chains from all local alignments [70] and visualized them in the UCSC genome browser [71]. Investigating RXFP2 and INSL3 in 70 nonhuman mammals We used the genome alignments to systematically search each exon for frameshifting insertions and deletions, mutations that create in-frame stop codons, and mutations that disrupt the conserved splice site dinucleotides (donor GT/GC, acceptor AG). We validated each putative mutation by the following steps: First, we realigned the exonic sequence of each exon with an inactivating mutation, using CESAR [49, 50] with default parameters. CESAR is a Hidden-Markov-Model based aligner that takes the reading frame and the splice sites into account and tries to produce an intact exon alignment (no inactivating mutations, consensus splice sites) between human and a query species, wherever possible. All exons for which CESAR confirmed the existence of at least one inactivating mutation are shown in Fig 3. S10 Fig shows an example for which CESAR found an alternative alignment that lacks inactivating mutations. Second, we validated the remaining inactivating mutations by searching SRA for unassembled sequencing reads that have a 100% match to the genomic context comprising at least 50 bp around the mutation, as described in [73]. Furthermore, we searched SRA for a putative sequence, in which the inactivating mutation was reversed to its ancestral state. Every mutation that is supported by at least 10 sequencing reads without any hit to the putative “ancestralized” sequence was considered as real. Third, we examined all exons that do not align in the query genome, since these are either truly deleted or an artifact arising from incomplete genome assemblies [68, 74]. We used the nearest up- and downstream aligning blocks in the alignment chain to determine the genomic locus that corresponds to this exon in the query genome. If this locus does not overlap an assembly gap, we concluded that the exon is lost (either deleted or accumulated so many mutations that it does not align anymore). If this locus overlaps an assembly gap, we conservatively considered this exon as missing sequence, as described in [63]. Indeed, as shown in S9 Fig, exons that overlapped assembly gaps in a previous assembly readily aligned with an intact reading frame in an improved genome assembly, showing that assembly gaps should not be mistaken for exon loss. Determining if the genes are under relaxation in different species and the associated Ka/Ks values To determine if the RXFP2 coding sequence evolves neutrally in different species, we used RELAX version 2 [52]. Briefly, given an alignment, a tree topology, and branches labeled as either test or reference, RELAX determines if the gene evolves under relaxed or intensified selection in the test branches relative to the reference branches. We used RELAX’s partition descriptive method that fits three Ka/Ks classes to the test and the reference branches and also estimates an overall Ka/Ks value. To determine if RXFP2 evolves under relaxed selection in any mammal, we iteratively applied RELAX, labeling each of the 71 species as the test branch. The resulting P values were corrected for multiple testing using the Benjamini and Hochberg method (S1 Table). To exclude any bias caused by relaxed selection in the RXFP2-loss species, which might inflate the reference Ka/Ks values, we further applied RELAX, labeling only one afrotherian species as test and excluding all other afrotherian species from the reference branches. These tests confirmed that RXFP2 evolves under relaxed selection only in the four afrotherians with an inactivated RXFP2 gene but not in the three other afrotherian species (S1 Table). Dating gene loss events To date the loss of RXFP2, we followed the procedure described in [55, 56]. For a branch along which the gene was inactivated, this method assumes that a gene evolves under a selective pressure similar to that in other species until it is inactivated. Afterward, the gene is assumed to accumulate both synonymous and nonsynonymous mutations at a neutral rate. The Ka/Ks value (K) estimated for this entire branch is then the average of the Ka/Ks value for the part of the branch where the gene was under selection (K s ) and the Ka/Ks value for the part of the branch where the gene evolved neutrally (K n = 1), weighted by the proportion of time for which the gene was evolving under selection (T s / T) and neutrally (T n / T): where T represents the time since the split from the last common ancestor. Using the lower and upper bound of the confidence interval for the species divergence time T obtained from TimeTree [57] (S4 Table) and using the Ka/Ks value for mammals with a functional RXFP2 (K s = 0.2491), one can estimate a lower and upper bound for T n as which provides an estimate of how long RXFP2 has been evolving neutrally (S2 Table). PCR experiments DNA of the greater hedgehog tenrec (Setifer setosus) and the lesser hedgehog tenrec (Echinops telfairi) was kindly provided by Athanasia Tzika (University of Geneva). Dugong tissue was kindly provided by Joerns Fickel (Leibniz-Institute of Zoo and Wildlife Research, Berlin). DNA was extracted using the innuPrep DNA Mini Kit (Analytik Jena, Jena, Germany) following the manufacturer’s instructions (protocol for DNA isolation from tissue samples or rodent tails) but extending tissue lysis overnight and including RNA digestion. Standard PCR reactions were performed in a total volume of 10 μl containing 5–20 ng DNA, 5 μl Phusion Flash High-Fidelity PCR Master Mix (Thermo Scientific, Waltham, United States of America; #F-548S), 0.25% DMSO (New England Biolabs, Frankfurt Main, Germany), and 0.5 μM of each primer (S5 Table). Cycling conditions were as follows: 35 cycles were used with denaturation at 98 °C (15 s but 2 min for the first cycle), annealing at a primer pair–specific temperature (S5 Table, 20s), and extension at 72 °C (10 s–1 min but 5 min after the last cycle). PCR products were Sanger-sequenced after enzymatic cleanup with CleanDTR (GC biotech, Waddinxveen, Netherlands) followed by cycle sequencing with the PCR primers and a mix of the BigDye Terminator v3.1 Cycle Sequencing Kit and dGTP BigDye Terminator v 3.0 Kit (Applied Biosystems, Foster City, CA, USA). Analyses were performed on an ABI 3730xl DNA Analyser (Applied Biosystems). Promoter sequence divergence We analyzed per-species sequence divergence of the promoter region of RXFP2 and INSL3. Using the human hg38 assembly coordinates chr13:31739464–31739544 and chr19:17821524–17821716, we extracted the sequence of all placental mammals, used PRANK [75] with parameters “-keep -showtree -showanc -prunetree” to align these sequences and to reconstruct the most likely sequence of the placental mammal ancestor and measured the percent sequence identity between the ancestral sequence and the sequence of every extant mammal, as described in [68, 74]. The result is visualized in S12 Fig.

Acknowledgments We thank the genomics community for sequencing and assembling the genomes, the UCSC genome browser group for providing software and genome annotations, Nikolai Hecker for computing hyrax and sloth genome alignments, and the Computer Service Facilities of the MPI-CBG and MPI-PKS for their support. We also thank Athanasia Tzika and the LANE animal facility at the University of Geneva for tenrec DNA, and Joerns Fickel for dugong tissue.