Proc. Natl. Acad. Sci. USA Vol. 87, pp. 2496-2500, April 1990 Evolution DNA "fingerprinting" reveals high levels of inbreeding in colonies of the eusocial naked mole-rat (cooperative breeding/eusoality/kin selection/hypervariable minisateilite DNA) HUDSON K. REEVE*, DAVID F. WESTNEATt, WILLIAM A. NOONt, PAUL W. SHERMAN*, AND CHARLES F. AQUADROt *Section of Neurobiology and Behavior, Seeley G. Mudd Hall, and tSection of Genetics and Development, Biotechnology Building, Cornell University, Ithaca, NY 14853 Communicated by Richard D. Alexander, January 2, 1990 ABSTRACT Using the technique of DNA fingerprinting, MATERIALS AND METHODS we investigated the genetic structure within and among four wild-caught colonies (n = 50 individuals) of a eusocial mam- Samples. Naked mole-rats from four distinct colonies were mal, the naked mole-rat (Heterocephalus glaber, Rodentia: captured between 1977 and 1980 in southeast Kenya. Three Bathyergidae). We found that DNA fingerprints of colony- ofthe four colonies (A, B, and TT) were collected within 5 km mates were strikingly similar and that between colonies they of each other near Mtito Andei (30 S, 380 E); the fourth (K) were much more alike than fingerprints of non-kin in other was collected near Ithumba, approximately 40 kmn northwest free-living vertebrates. Extreme genetic similarity within col- and across the Athi River. Mole-rats caught from the same onies is due to close genetic relationship (mean relatedness colony were housed together in a Plexiglas tunnel system; estimate ± SE, i = 0.81 ± 0.10), which apparently results from each colony was housed separately. Pups were born in three consanguineous mating. The inbreeding coefficient (F = 0.45 of our captive colonies (A, K, and TT); all of them were ± 0.18) is the highest yet recorded among wild mammals. The conceived in intracolony matings. genetic structure of naked mole-rat colonies lends support to The DNA of 50 individual mole-rats was sampled: 23 kin and ecological constraints models for the evolution field-caught adults, 25 lab-born offspring from intracolony selection matings among a different subset of field-caught adults, and of cooperative breeding and eusociality. 2 offspring of a mating between an additional field-caught adult and a lab-born offspring of wild-caught parents. All the Naked mole-rats (Heterocephalus glaber) are virtually hair- individuals in our sample had died ofnatural causes; we were less and sightless mammals that live in large subterranean reluctant to sacrifice live animals or to risk injury or disease colonies [mean size, 74 individuals; range, 25 to >295 (1, 2)] by sampling tissues because of the likelihood ofjeopardizing in northeastern Africa. H. glaber are particularly interesting long-term behavioral studies (e.g., ref. 6). because they are eusocial: within a colony, only-one female DNA Extraction, Digestion, and Hybridization. Nuclear and her 1-3 mates reproduce, and the young from previous DNA was isolated from individual liver, muscle, or brain litters maintain and defend the colony and assist in rearing samples as described by Saghai-Maroof et al. (21). Approx- newborns as do workers in colonies of the social insects imately 6-8 jFg of DNA was digested with Hae III (a (1-6). Despite intense interest in this remarkable mammal, four-base restriction enzyme), electrophoresed in a 1% aga- the genetic structure of naked mole-rat colonies is unknown. rose gel in 40 mM Tris acetate/1 mM EDTA (pH 8.0), blotted A knowledge of intracolony genetic relatedness, in particu- onto a nylon membrane, and hybridized to radiolabeled lar, is essential for understanding whether or not close wild-type M13 DNA or to Jeffreys's 33.15 or 33.6 probes kinship (7-10) underlies the extreme reproductive selfless- (which are cloned in M13; ref. 11) as described by Westneat ness (altruism) displayed by subordinate mole-rats. et al. (22). M13 hybridizations were carried out with both We investigated the genetic structure of naked mole-rat dATP-labeled and dCTP-labeled M13 probes. Each of the colonies by using DNA probes derived from tandem repeti- three DNA probes we used detects a distinct family of tive sequences in humans (11-16) and the M13 phage (17, 18) minisatellites. to detect repeat-sequence length variants (minisatellites). Scoring and Interpretation of Bands. For each probe, we These minisatellites typically are so polymorphic that they scored only prominent bands that were clearly distinct from can generate DNA "fingerprints" (individual-specific re- those obtained with the other probes. The M13 bands were striction fragment patterns), which have proven useful for scored first, followed by the 33.15 and 33.6 bands (the order assigning paternity and maternity in humans (11, 12) and of scoring does not affect our conclusions). A mean of 12.1, birds (15, 16, 19). We used three such probes (wild-type M13 9.9, and 7.3 bands were scored per individual for each of the phage DNA and A. J. Jeffreys's human-derived probes num- respective probes (Fig. 1); scored bands represented DNA bered 33.15 and 33.6) to generate DNA fingerprints of naked fragments ranging between 1.8 and 16 kilobases in size. DNA mole-rats from four field-caught colonies. We then inferred samples from a set of animals representing each colony (i.e., mean intracolony relatedness and degree of inbreeding by (i) controls) were run in multiple gels to facilitate comparisons directly estimating mean relatedness, using Pamilo and Cro- among fingerprints obtained from different gels. Samples zier's (20) regression technique, and (ii) comparing DNA from all four colonies were run side by side in each gel, and fingerprint similarity among field-caught colony members samples from a given colony were divided between gels; with that among littermate siblings conceived in within- these procedures ensured that a relatively high within-colony colony matings in the laboratory. similarity would not arise spuriously from between-gel dif- ferences in band migration rates. No variation was evident in The publication costs of this article were defrayed in part by page charge fingerprints of the same animal on the same or different gels payment. This article must therefore be hereby marked "advertisement" (regardless of the tissue from which the DNA had been in accordance with 18 U.S.C. §1734 solely to indicate this fact. isolated). 2496 Downloaded by guest on September 30, 2021 Evolution: Reeve et al. Proc. Natl. Acad. Sci. USA 87 (1990) 2497 PROBE FIG. 1. Examples of DNA finger- print patterns for naked mole-rats. M13 33.15 33 6 The bands represent DNA fragments of different size (kilobases, kb) that Size (kb) were obtained by cutting mole-rat nu- clear DNA with Hae III (a four-base - 7.8 restriction enzyme) and probing with _ *- r66 __ - _w _w wild-type M13 DNA (Left), Jeffreys's 4w, 33.15 probe (Center), and Jeffreys's 33.6 probe (Right). Fingerprints of _ 9 the same eight mole-rats are shown in the three panels; K, A, B, and IT 4 1 0 4 _39w^ refer to each animal's colony oforigin (colony B individuals were field- 40, ... s l, s.sI. caught animals; the others are off- 40a~~ spring of within-colony laboratory matings). In the M13 patterns, the 7.8- .4#$E~~~~~~l-"j and 6.6-kb fragments were identified as alleles at a variable bi-allelic locus i .f :: ao;3; I and the 4.1- and 3.9-kb fragments 181 were identified as alleles at a variable ;t 211~~~~40 4 bi-allelic locus II by the multiple re- -1I8 J striction enzyme method of Jeffreys et al. (ref. 23; see Materials and T TT B B A K II TiTT B B B A K TT T. TT B B B A K Methods). In the M13 patterns, two fragments were identified as - F) for AA x Aa matings, 2p2(1 - p)2(1 - F) for AA X aa alleles at a variable bi-allelic locus I and two other fragments matings, 4p2(1 - p)2(1 - F) for Aa x Aa matings, and were identified as alleles at a variable bi-allelic locus II, using 4p(1-p)3(1 - F) forAa x aa matings. The expected value for Jeffreys et aL.'s multiple restriction enzyme method (23). In the modified regression estimate is equal to W - Z, where W both cases, the putative allelic fragments were never both = [(1 - F)(p3 - 6p2 + Sp + 4) + 4F]/[4(1- F)(2 - p) + 4F] absent, and they exhibited the same size difference regardless and Z = (1 - F)p(1 - p)[1 - (p/4)]/[1 - p(l - F)]. When of which of three restriction enzymes (Hae III, Alu I, or F = 0, the regression estimate reduces to [(p3 - 6p2 + 5p + HinfI) was used to cut the DNA. The latter finding suggests 4)/4(2 - p)] - p(l - p14), which is <0.50 for p > 0 (the that the two fragments were allelic by indicating that their amount of underestimation approaches zero as p becomes embedded tandem repeats had similar flanking sequences smaller). This analysis assumes single paternity; if it is (23). Sequence similarity, hence allelism, of the two putative assumed that two or three breeding males share paternity locus II bands was further suggested by the observation that equally, the expected regression estimates for F = 0 are also these two bands, unlike all the others, appeared only after always less than the true values of r = 0.375 and r = 0.333, hybridization to dATP-labeled M13 probe (all the other bands respectively, and again the amount of underestimation de- in the M13 fingerprints appeared after hybridization to both creases as the allele frequency decreases.