Mitochondrial DNA sequence relationships of the ...

crystallin (60 to 110 mU per mg to total protein [11]). These latter values are due to normal, anaerobic glycolysis in the lens. The high values in Phelsuma indicate that e-crystallin is expressed as an active enzyme, similar to the situation in birds. It has been proposed that the recruitment as crystallins of NAD(P)H-binding enzymes, which causes increased levels of reduced pyridine nucleotides, could protect the lens against oxidative stress and/or serve to reduce glare at wavelengths in the near UV [4, 7, 12]. eCrystallin has - with the exception of the nocturnal caiman - indeed been found only in lenses of diurnal species. However, the possession of e-crystallin or other NAD(P)H-binding enzyme crystallins cannot be a decisive requirement for effective functioning of lenses in diurnal animals, as there are many diurnal birds and reptiles without such crystallins, e.g., the fully diurnal geckonid genera Lygodactylus and Gonatodes [13]. It may be noted that the Phelsuma lens is remarkable for the complexity of its composition: it contains, in addition to the ubiquitous a-, fl-, and y-crystallins, no less than four enzyme crystallins: 6/argininosuccinate lyase, r/ct-enolase,

~z/glyceraldehyde 3-phosphate dehydrogenase, and e / L D H [4, 11] (Fig. 1). The presented data demonstrate that the occurrence of e-crystallin is not restricted to the archosaurian lineage. This could mean that the potential of the Ldh-B gene to become highly expressed in the lens developed before the divergence of archosaurs and lepidosaurs, culminating in certain birds and crocodiles. However, considering that ecrystallin has hitherto not been detected in any lepidosaurian reptile other than Phelsuma [ 5 - 7 , 11], it seems more likely that the evolutionary recruitment of e-crystallin occurred independently in different reptilian lineages by distinct regulatory mechanisms. Such an independent recruitment has recently been shown for (-crystallin in camels and hystricomorph rodents [14]. The capacity for increased lens expression may, in fact, be an innate property of certain housekeeping genes, because also in mammalian lenses various enzymes, present as genuine crystallins in other vertebrates, tend to show elevated levels of expression [15].

Received October 23, 1995 and January 11, 1996

Naturwissenschaften 83, 178-182 (1996) © Springer-Verlag 1996

Mitochondrial DNA Sequence Relationships of the Extinct Blue Antelope

Hippotragus leucophaeus T.J. Robinson, A.D. Bastos Department of Zoology and Entomology, University of Pretoria, Pretoria 0002, South Africa K.M. Halanych* Department of Zoology, University of Texas, Austin, Texas 78712, USA B. Herzig Naturhistorisches Museum Wien, 1. Zoologische Abteilung, A-1014 Vienna, Austria

*Present address: Department of Zoology and Entomology, University of Pretoria, Pretoria 0002, South Africa 178

1. Wistow, G.J., Piatigorsky, J.: Science 236, 1554 (1987) 2. De Jong, W.W., Lubsen, N.H., Kraft, H.J., in: Progress in Retinal and Eye Research, Vol. 13, p. 391. Amsterdam: Elsevier 3. Wistow, G.L.: Trends Biochem. Sci. 18, 301 (1993) 4. Jimenez-Asensio, J., Gonzalez, E, Zigler, Jr., J.S., Garland, D.L.: Biochem. Biophys. Res. Comm. 209, 796 (1995) 5. ROll, B., van Boekel, M. A. M., Amons, R., de Jong, W.W.: ibid. 217, 452 (1995) 6. Stapel, S.O., Zweers, A., Dodemont, H.J., Kan, J.H., de Jong, W.W.: Eur. J. Biochem. 147, 129 (1985) 7. Wistow, G.J., Mulders, J.W.M., de Jong, W.W.: Nature 326, 622 (1987) 8. Hendriks, W., Mulders, J.W.M., Bibby, M.A., Slingsby, C., Bloemendal, H., de Jong, W.W.: Proc. Nat. Acad. Sci. USA 85, 7114 (1988) 9. Wistow, G.J., Anderson, A., Piatigorsky, J.: ibid. 87, 6277 (1990) 10. Hedges, S.B.: ibid. 91, 2681 (1994) 11. R011, B.: Herpetol. J. 5, 298 (1995) 12. Rao, C.M., Ziglel, Jr., J.S.: Photochem. Photobiol. 56, 523 (1992) 13. R011, B.: Verh. Dt. Zool. Ges. 88.1, 172 (1995) 14. Gonzales, R, Rao, P.V., Nufiez, S.B., Zigler, Jr., J.S.: Mol. Biol. Evol. 12, 773 (1995) 15. Wistow, G., Kim, H.: J. mol. Evol. 32, 262 (1991)

The blue antelope (Hippotragus leucophaeus) is the first African mammal to have become extinct in historic times (ca. 1799-1800). However, the processes which may have precipitated its initial decline are thought to have been in effect prior to European settlement of southern Africa, with habitat degradation being a probable cause [1]. Nonetheless, its brief recorded history (first reported in 1719, formally described in 1766, and extirpated by 1800) makes it one of the most obscure of Africa's large mammals [2], with the only evidence of its existence limited to a skull of dubious provenance (the Hunterian Museum, University of Glasgow; but see [2]), two sets of horns, and four mounted museum specimens. The species' early extinction precluded even the most cursory investigations of its life history, and has confounded

Naturwissenschaften 83 (1996) © Springer-Verlag 1996

studies of its taxonomic status and phylogenetic relationships. While more recent works of Mohr [3] and, in particular, Klein [11 have removed much speculation, these authors hold conflicting views on the taxonomic status and relationships of this endemic and little-unknown antelope. Mohr [3], who relied extensively on the mounted H. leucophaeus specimens in her morphological analysis, concluded that the blue antelope was merely a subspecies of roan. In contrast, Klein's examination, principally of fossil horn cores and teeth, showed that the blue antelope and roan once occurred sympatrically in the southwestern Cape Province, supporting recognition of the former as a distinct species. Until fairly recently, molecular phylogenetic studies relied almost exclusively on the extrapolation of information from living organisms to reconstruct evolutionary history; however, the discovery that DNA can be extracted from the remains of extinct species has contributed an added dimension to these investigations. Given successes in obtaining ancient DNA from museum skins ([41 and references therein), we report on efforts to resolve the taxonomic status and evolutionary relationships of the blue antelope by sequencing DNA from a mounted museum specimen of this enigmatic species, and comparing these to orthologous sequences derived from its extant congeners, the roan and sable antelope. Total genomic DNA was extracted ([51, with minor modification) from a mounted blue antelope museum skin (H. leucophaeus NMW St 7/5; Natural History Museum, Vienna) and from cultured fibroblasts [6] of roan (H. e. cottoni, Namibia, N = 1, Malawi, N = 1; H. e. langheldi, southwest Kenya, N = 1); and sable antelope (H. n. kirkii, Malawi, N = 1). Additionally, DNA was extracted from heart tissue from a bontebok D. p y g a r g u s (subfamily Alcelaphinae) and its geographic variant the blesbok, which were obtained under permit from the West Cape Department of Nature and Environmental Conservation; the alcelaphine sequences were use to root the phylogeny and polarize the characters. Primers were initially chosen to amplify a single 652-bp stretch of cytochrome b (defined by L14841 and H15494 - the

complement of L15513 [7]). However, given the difficulties experienced in amplifying this region from the blue antelope museum skin, we subsequently chose to use two primer pairs L14841 - H15149 (308 bp) and L15162-H15494 (322bp). Although the amplified products overlap, we were unable to sequence through this region, and the data are therefore derived from two almost contiguous portions of cytochrome b. As is convention stringent precautions were implemented to avoid contamination [8]. Among others, these included deriving our blue antelope sequence data from four independent DNA extractions, spread over a 6-month interval. In three of four extractions the blue antelope was the only sample worked on and, at the time of study, no other antelope (or ungulate) DNA or cytochrome b products, other than those contained herein, were present in the laboratory. Sequences were aligned [9] and proofread by visual inspection; these are available from the authors on request, and have been deposited in GenBank under accession numbers U18274 and U18275 (H. leucophaeus), U18276, U18277, U/8278, and U/8279 (D. pygargus, bontebok, and blesbok, respectively), U18280 and Ul8281 (H. e. cottoni - Malawi), U18282 and U18283 (H.e. cottoni - Namibia), U18284 and U18285 (H. e. langheldi Kenya), U18286 and U18287 (H. niger).

Analyses were conducted on 502 bp of cytochrome b corresponding to positions 14631 - / 4 8 9 7 and 14933-15167 in the cow cytochrome b sequence [10]. A range of transversion (tv) to transition (ts) weightings were used in the analyses (1:1, 2: 1, 3:1, 10:1), including transversions weighted at 1.6 x transitions; the latter was empirically determined using maximum likelihood to find the ts/tv ratio with the highest likelihood score. Finally, we included an assessment of phylogenetic relationships, using only the transversions contained in our data set. Parsimony analyses of the aligned sequences were conducted using PAUP version 3.1.2d5 (D. L. Swofford, Illinois Natural History Survey). We estimated the reliability of nodes on the shortest trees with 1000 bootstrap replicates (using the Branch-and-Bound option of

Naturwissenschaften 83 (1996) © Springer-Veriag 1996

PAUP), which resulted in 50°7o majority rule consensus trees. Additionally we examined the skewness of the treelength distributions that resulted from an exhaustive analysis of all possible trees [11 ]. Maximum likelihood (dnaml) and neighbor-joining (neighbor) analyses were performed using PHYLIP version 3.5c (J. Felsenstein, University of Washington, Seattle). There was only one instance of negative control amplification in the PCR, determined by sequencing to be contaminated by blesbok DNA, and this was discarded. The single instance (of four extractions) where blue antelope DNA was extracted in conjunction with other species resulted in identical blue antelope sequences. Additionally, factors consistent with working with ancient DNA included failure to amplify blue antelope DNA under standard conditions (without the inclusion of Bmercaptoethanol and bovine serum albumin, which were required to overcome inhibitory substances in the ancient DNA extracts [5]), and our failure to amplify the larger mtDNA fragment (652-bp fragment defined by primers L14841 and H15494) despite success with the same primers in the DNA of the extant species. Finally, the blue antelope sequences conformed to a phylogentic placement closely related to the extant congeners, the roan and sable antelope (see below). The exhaustive search option of PAUP resulted in a single most parsimonious tree of 112 steps (Fig. l a). The tree topology was invariant over a range of weighting parameters (transversions weighted 1:1, 1.6:1, 2:1, 3:1, 10:1). The measure of skewness (gl = 1.209638), associated with the actual distribution of all possible tree lengths (characters unweighted), showed that the data set was significantly more structured than random [11], and therefore contains significant phylogenetic signal. Of the 502 aligned base pairs, 98 are variable and 44 phylogenetically informative (i.e., parsimony sites). Of the variable sites, 11 were at the first position (11.2%), 10 at the second (10.2%), and 76 at the third codon position (77.6%). One site (number 268), which was on the boundary between the two stretches of sequence, was out of frame, and not phylogenetically informative. There were only 22 -

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instances o f n o n s y n o m o u s mutations. O n l y 2 5 % o f the variable n u c l e o t i d e positions in our data set were characterized by transversions c o n f i r m i n g the transition bias generally observed in vertebrate m t D N A [7].

a.

B o o t s t r a p values indicate strong support for all the relationships p r o p o s e d by p a r s i m o n y analysis (Fig. 1 a). T h e n o d e s u p p o r t i n g the r o a n and sable as sister taxa to the exclusion o f the blue a n t e l o p e is s u p p o r t e d in 76.7% o f 1000

Blue antelope

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b o o t s t r a p replications (and 83.7% w h e n using a t s / t v ratio o f 1.6: 1). Since b o o t s t r a p p r o p o r t i o n s o f >_ 70°70 usually c o r r e s p o n d to a > 9 5 % probability that the c o r r e s p o n d i n g clade is m e a n i n g f u l [13], these values are clear-

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Fig. 1. Phylogenetic relationships of blue antelope (14. leucophaeus) and its extant congeners, the roan (H. e. eottoni, Malawi; H. e. cottoni, Namibia; H. e. langheldi, Kenya) and sable antelope (H. niger), based on cytochrome b sequences. Trees were rooted using the Alcelaphinae representatives, the bontebok and bleshbok (D. pygargus), as the outgroup, a) Single most parsimonious tree. The tree had a length of 112 with the CI = 0.818 (when excluding uninformative sites), and RI = 0.831; numbers are bootstrap indices based on 1000 iterations showing level of support for individual branches with characters weighted (1.6 : 1) and unweighted (in parenthesis), b) Neighbor-joining tree based on pairwise sequence divergence values corrected for multiple hits [12] and transitional bias using the empirically derived 1.6:1 ts/tv ratio; values along branches (not drawn to proportion) show bootstrap support based on 1000 iterations, c) Maximum likelihood tree; bootstrap values from 100 replicates are given on each branch Naturwissenschaften 83 (1996)

© Springer-Verlag 1996

ly significant. The topologies contained in the suite of next most parsimonious trees were also examined. These were three steps longer ( N = 2; CI = 0.776 when excluding uninformative characters; RI = 0.780) and four steps longer ( N = 1; C I = 0 . 7 6 3 when excluding uninformative characters; RI = 0.763) than the most parsimonious solutions; the blue antelope was represented as a distinct clade in one of the trees (116 steps) and in the remaining two as sister taxon to the roan and sable, respectively (115 steps). Finally, transversion parsimony similarly supported an independent blue antelope lineage, placing the blue antelope outside an unresolved roan and sable polytomy, an association supported in 63% of 1000 bootstrap cycles (not shown). The analysis of the cytochrome b data by neighbor-joining (Fig. 1 b) revealed a phylogenetic affinity for the blue antelope that differed from the single most parsimonious solution. Here, the blue antelope was clustered with the sable to the exclusion of the roan, irrespective of whether the data were unweighted or transversions weighted at 1.6: 1, 2: l, 3:1, or 10: 1. However, bootstrap support for this association (blue antelope and sable as sister taxa) was less strong (69.6% of 1000 bootstrap iterations compared to the 83.7% for recognition as a distinct clade by parsimony analysis). In contrast to neighbor-joining, but concordant with the parsimony result, maximum likelihood analysis placed the blue antelope outside the roan/sable clade (Fig. 1c), an association which was strongly supported in 78.0°70 of 100 bootstrap iterations. Our results, based on cytochrome b sequence data, contradict the hypothesis that the blue antelope was merely a subspecies of the roan, H. equinus. Support for the placement of this antelope as a distinct lineage outside the roan/ sable assemblage is reflected in the outcomes from two independent methods of phylogeny construction, parsimony analysis and maximum likelihood. Moreover, both parsimony and maximum likelihood (Fig. I a, c) resulted in bootstrap proportions that gave strong support for an independent blue antelope lineage (83.7% in parsimony using all character evidence and 63% with transversion parsimony; 78% in maximum likelihood). This conclusion,

while at variance with the close but novel sable: blue antelope association depicted in the neighbor-joining result (Fig. lb), is congruent with that advanced for the fossil data [1]. When taken together, these two unrelated data sets provide robust support for the recognition of the blue antelope as a distinct species, the prior name for which is H. leucophaeus. While this study has applied molecular techniques to infer evolutionary relationships between the blue antelope and the contemporary Hippotragus species, the causes for the decline and subsequent extinction of the blue antelope remain subject to speculation. Although the blue antelope's distribution was reported to be extremely limited by the time of settlement at the Cape Province [14], fossil data suggest that it was once both more widely distributed and more numerous. Clearly, the area in which the species occurred at the

time of historic contact (Fig. 2), estimated at approximately 100 km (eastwest) by 60kin (north-south), would have been far too restricted to maintain a viable population of this relatively large herbivore for an extended period [2]. It seems likely, therefore, that the species was in decline prior to European settlement. Fossil evidence in support of a wider Holocene (10000 years B.p.) distribution suggests that the blue antelope occurred throughout the southern Cape [1] and at least as far inland as the eastern Orange Free State Province [15]. Even if its distribution was patchy and discontinuous within this region, it would nonetheless have formed an area greatly in excess of the historical record. This wider distribution begs the question as to what precipitated the extinction spiral in the blue antelope. It would seem from the fossil data that the blue antelope was the only species

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181

of Hippotragus in the southern Cape 7 0 0 0 0 - 3 5 0 0 0 years B.p. [1, 16]; at approximately 11000 years B.p., the roan became predominant in this region. This ecological change is thought to coincide with diminution of the extensive grasslands (regarded as the species' preferred habitat) through its progressive replacement by bush, forest, and the unique "fynbos" of the southwestern Cape [16], and therefore climatic change and accompanying habitat deterioration and fragmentation may have been responsible for the initial reduction in the species' range. Irrespective of the cause, however, habitat loss would inevitably be followed by a concomitant decline in population numbers. In turn, this may have progressed to the point where genetic impoverishment through inbreeding and drift, coupled with stochastic demographic factors and increasing human pressures, combined to depress the species' viability to a point from which it could not recover.

The analysis of the sequence data was largely completed while T JR was a visiting researcher in David Hillis' Laboratory at the University of Texas at Austin. Drs. D.M. Hillis and C.W. Cunningham are thanked for sharing their expertise in phylogeny reconstruction. This research was supported by a sabbatical leave grant and core program funding from the South African Foundation for Research Development. Received December 1, 1995 and January 19, 1996

1. Klein, R.G.: Ann. S. Afr. Mus. 65, 99 (1974) 2. Gould, S.J.: Nat. Hist. 5, 16 (1993) 3. Mohr, E.: Der Blaubock Hippotragus leueophaeus (Pallas, 1766). Eine Dokumentation. Hamburg: Parey 1967 4. Thomas, R.H., Schaffner, W., Wilson, A.C., P~tbo, S.: Nature 340, 465 (1989) 5. Cooper, A., in: Ancient DNA, p. 149 (B. Hermann, S. Hummel, eds.). New York: Springer 1994

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Fate of Ant Foundress Associations Containing "Cheaters" S.W. Rissing, G.B. Pollock, M.R. Higgins Department of Zoology, Arizona State University, Tempe, Arizona 85287-1501, USA The "cheater" dilemma often frames evolutionary analyses of cooperation [ 1 - 3 ] : what prevents a behavioral mutation to exploit the cooperation of others from spreading in a population of cooperators? Kin selection provides one answer, noting that cheaters suffer indirect fitness costs when victims are close relatives [2, 4]. Game theory provides another method for eliminating cheaters through punishment [3, 5 - 7 ] . A m o n g Hymenoptera, the latter alternative is difficult to isolate experimentally from the former, since hymenopteran cooperators are often closely related [8, 9]. Ant cofoundresses, however, are often unrelated [10-15], providing a natural opportunity to exclude 182

kin selection analytically from studies of cooperation. Cofoundresses of the leaf-cutter ant A c r o m y r m e x versicolor, while associating regularly with nonrelatives [13, 15], nonetheless parallel several species of kin-assorting wasp foundresses by exhibiting differential risk allocation ("altruism") during colony foundation in the form of a forager specialist prior to development of workers [14]. A m o n g kin-assorting wasps, differential risk is attributed to kin selection [8, 16]; the behavior of A. versicolor foundresses suggests some other, non-kin-selected mechanism can produce altruistic risk assumption as well. As a first step in identifying such a mechanism, we have employed a

6. Maniatis, T., Fritsch, E.E, Sambrook, J.: Molecular Cloning: a Laboratory Manual: New York: Cold Spring Harbor Laboratory 1982 7. Irwin, D.M., Kocher, T.D., Wilson, A.C.: J. Mol. Evol. 32 128 (199l) 8. Janczewski, D.N., Yuhki, N., Gilbert, D., Jefferson, G.T., O'Brien, S.J.: Proc. Nat. Acad. Sci. USA 89, 9769 (1992) 9. Higgins, D.G., Bleasby, A.J., Fuchs, R.: CABIOS 8, 189 (1992) 10. Anderson, S.M., De Bruin, M.H.L., Coulson, A.R., Eperon, E.C., Sanger, R., Young, I.B.: J. Mol. Biol. 165, 683 (1982) t 1. Hillis, D.M., Huelsenbeck, J.P.: J. Here& 83, 189 (1992) 12. Kimura, M.: J. Mol. Evol. 16, 111 (1980) 13. Hillis, D.M., Bull, J.J.: Syst. Biol. 42, 182 (1993) 14. Skead, C.J.: Historical Mammal Incidence in the Cape Province. Vol. 1. Cape Town: Department of Nature and Environmental Conservation 1980 15. Plug, I., Engela, R.: S. Aft. Arch. Bull. 47, 16 (1992) 16. Klein, R.G., in: Fynbos Palaeoecology: a Preliminary Synthesis, p. 116 (H. J. Dekan, Q.B. Hendey, J.J.N. Lambrechts, eds.). Pretoria: South African National Scientific Programme Report No. 75, 1983

"pseudomutant" design [17] to create A. versicolor foundress associations that contain an apparent "cheater", i.e., a foundress made to appear to cheat by precluding her from continuing her role as foraging specialist. Our design reveals behavior among the "cheater's" nonforaging cofoundresses consistent with game-theoretic punishment. We suggest that such punishment, when coupled with other aspects of A. versicolor natural history, provides a mechanism precluding evolutionarily viable cheating (avoiding foraging task assignment) among A. versicolor foundresses. Any behavior performed outside the nest, especially foraging, is a high-risk task among social insects [18-20]. A single individual within A. versicolor foundress associations becomes a foraging specialist, searching for fungus garden substrate outside the nest while her colony mates perform within-nest tasks. The cofoundresses are not closely related [13, 15]. Further, the degree of ovarian development of the foraging specialist is indistinguishable from that
