The colubrid radiation in Africa (Serpentes: Colubridae): phylogenetic relationships and evolutionary patterns based on immunological data

JOHN E. CADLE

Museum of Comparative

Received December 1992, revised and accepted May 1993

Phylogenetic relationships among genera of African colubrids were evaluated using estimates of divergence among serum albumins compared by microcomplement fixation. Representatives of about half of the extant genera of African colubrids, as well as the Elapidae, Atractaspis and the Madagascan colubrid Leioheterodon, were analysed. The tree of best fit to the data has an unresolved basal polychotomy comprising at least five lineages of colubrids, as well as Elapidae and Atractaspis; thus, colubrids were not demonstrably monophyletic with these data. Two cosmopolitan clades, colubrines and natricines, are represented in Africa by series of closely related genera, but divergence among other genera is relatively great. Rate tests show that this is apparently not due to higher rates of albumin evolution in these, relative to other colubrids. Among the other associations supported by the immunological data are: (I) Psammophis-(Rhamphiophis-Dipsina)-Malpolon- Psarnmophylax; (2) Ambhodipsas-Macrelaps; (3) (Lycodonomorphus-Lamprophis)-Meheha; and (4) Colubrinae-Natricinae. Grayia is questionably associated with the colubrine-natricine lineage. Prosymna and Lycodon are clearly members of the colubrine clade, and Amplorhinus possibly associates with Leioheferodon. Gonionotophis, Duberria, Lycophidion and Pseudaspis show no strong association with any other genera, and represent other basal or near-basal clades within the colubridlelapid radiation. The immunological data do not support a clade comprising the Elapidae, Atractaspic and some ‘aparallactines’ relative to Viperidae and other colubrids. The basal colubrid-elapid-Afractaspis divergence occurred more than 30 Myr ago, and the fossil record of colubrids in Africa greatly underestimates both the age and clade diversity of this group. In contrast to the pattern of radiation in the neotropics, where most colubrids belong to one of three major clades, in Africa only the colubrine lineage comprises a substantial portion of the extant generic diversity; most other genera stem from relatively ancient cladogenetic events and have few living representatives.

ADDITIONAL KEY WORDS:-Africa - snakes - systematics - biogeography - phylogeny molecular systematics - albumin.

CONTENTS

Introduction ...... 104 Bogert’s arrangement of African colubrids and the scope of the present work. . . . 105

This paper is dedicated to the memory of Charles M. Bogert (1908-1992), who not only pioneered the modern study of African colubrid systematics but also showed us the way in many other areas of reptile biology. 103 0024-4082/94/002 103 + 38 $08.00/0 0 1994 The Linnean Society of London 104 J. E. CADLE

Materials and methods ...... 107 Antigens and antisera ...... 107 Basic taxa, outgroups, and phylogenetic trees ...... 107 Results ...... 111 Reciprocal comparisons and phylogenetic trees ...... 111 Rate tests for albumin evolution in African colubrids ...... 1 I3 Unidirectional comparisons (Bogert’s Groups 1, 2, 5-7, 12, 16, 17) . . . . . 114 Lycodon and the status of the Lycodontinae ...... 118 Psammophis-Rhamphiophis clade (Bogert’s Group 16) ...... 119 Comments on McDowell’s (1986) Homoroselaps Group (Bogert’s Groups 7 and 17) . 121 Problems in colubrid phylogeny ...... 123 Discussion ...... 124 Other immunological data bearing on higher relationships among colubrids . . . 124 Bogert’s arrangement of African colubrids in retrospect ...... 127 The fossil record of snakes in Africa and the age of its colubrid lineages . . . . 128 Biogeographic considerations ...... 130 A comparison of African and Neotropical colubrid radiations ...... 132 A note on nomenclature of higher taxa within colubrids ...... 133 Acknowledgements ...... 136 References ...... 137 .4ppendix ...... 140

INTRODUCTION The central importance of Africa for understanding the evolutionary and biogeographic history of Gondwanan and Laurasian taxa has been widely acknowledged (Maglio & Cooke, 1978; Storch, 1990, 1992; Gheerbrant, 1990; Rage, 1988a; Ciochon & Chiarelli, 1980). Recent fossil discoveries from Africa and elsewhere have sharply focused attention on our poor understanding of the relationship between Africa and its early Tertiary neighbours. These discoveries provide new insights into the composition of early African faunas (e.g. marsupials in the Eocene and Oligocene [Crochet, 1984, 1986; Bown & Simons, 19841; tarsiiform and possible anthropoid primates from the early Tertiary [Simons & Bown, 1985; Godinot & Mahboubi, 1992; Simons, 19921, and faunal exchanges with other regions (Bernor, 1983; Bernor et al., 1987; Rage, 1988a). These discoveries and improvements in understanding the phylogenetic relationships of Africa’s many endemic groups, have resulted in more complete understanding of faunal evolution on that continent and its bearing on faunal evolution on other continents throughout the Cenozoic (Maglio & Cooke, 1978; Bernor et al., 1987; Storch, 1992). Long-standing hypotheses about the biogeographic histories of some groups have been questioned or revised to reflect these findings (Martin, 1985; Benton, 1985; Bernor et al., 1987; Janis, 1988; Culotta, 1992; Gheerbrant, 1990; Ciochon & Chiarelli, 1980). Nevertheless, much of the early history of Africa remains shrouded in mystery as a result of the paucity of Tertiary terrestrial fossil-bearing deposits in Africa (Savage, 1967; Cooke, 1978; Winkler, 1992). Hence, studies of the phylogenetic relationships of the extant fauna continue to play major roles in formulating hypotheses for the evolutionary history of the African fauna (Luckett & Hartenberger, 1985). This is especially true for groups such as snakes, which have a very poor fossil record in Africa, as in all tropical regions (Rage, 1973; Meylan, 1987; Cadle, 1987). Of special concern for understanding the Tertiary history of Africa are relationships between some African and South American lineages. The most PHYLOGENY OF AFRICAN COLUBRIDS 105 extensively-studied examples are the endemic platyrrhine primates of the Neotropics, and hystricognath rodents, found in both Africa and South America (both groups reviewed in Ciochon & Chiarelli, 1980; see also Luckett & Hartenberger, 1985). The potential relevance of African lineages to understanding the evolutionary history of several clades of endemic neotropical snakes of the family Colubridae was recognized early in a broad-scale phylogenetic study using estimates of albumin divergence measured immunologically (Cadle, 1984a-c, 1985, 1988). An initial series of molecular comparisons revealed, however, that the situation in Africa was much more complicated than that in the neotropics, and exploration of African-Neotropical relationships was postponed until additional data concerning the phylogenetic structure of the colubrid radiation in Africa could be obtained. This report is a first exposition of the African situation. My goals in this paper are (1) to present a hypothesis of relationships among major lineages of African colubrids and other taxa of advanced (caenophidian) snakes (Elapidae, Atractaspis); (2) to evaluate the significance of the hypothesis for African colubrid systematics; (3) to use this hypothesis as a framework for evaluating some other hypotheses and scenarios for the evolution of advanced snakes; and (4) to examine some general characteristics of the radiation of African colubrids as compared to Neotropical radiations. The relevance of the phylogenetic hypotheses to aspects of the biogeographic and fossil history of African snakes is discussed, and some nomenclatural issues within Colubridae are addressed.

BOGERT’S ARRANGEMENT OF AFRICAN COLUBRIDS AND THE SCOPE OF THE PRESENT WORK Perhaps the most influential modern work on the systematics of African colubrids is Bogert’s (1940) account of several collections made during the 1920s and 1930s. Bogert developed a comprehensive classification of African genera using features that Dunn (1928) found useful in assessing relationships among New World colubrids (vertebral hypapophyses, hemipenes and dentition), although the relative importance he ascribed to these features differed from Dunn’s. Bogert’s paper has formed the point of departure for much subsequent systematic work on African colubrids, and some of his groups have been given formal systematic status (see, for example, Bourgeois, 1968; Dowling, 1967, 1969; Dowling and Duellman, 1978). Using other sets of characters, particularly additional osteological and soft anatomical information, other workers have in some cases reached different conclusions from Bogert’s (1940) concerning the relationships of particular genera. Some of these differences are discussed below with reference to the immunological data. Bogert’s (1940) groups are summarized in Table 1 and are referred to in the text. However, discussion of immunological data on some of Bogert’s groups are deferred to future papers. These include Groups 8-10, 13-15 and 18, which collectively comprise a clade (along with many non-African representatives) relative to all other groups in Bogert’s scheme (Cadle, unpublished data). These are the Colubrinae, in the sense of Dowling et al. (1983) and McDowell (1987); the African members of these groups have been discussed by Rasmussen (1979, 1985). Likewise, Group 4 comprises genera usually referred to the Natricinae (sensu Malnate, 1960; Rossman & Eberle, 1977; Dowling et al., 1983), whose 106 J. E. CADLE TABLE1. Classification of African genera of colubrids according to Bogert ( 1940). Generic names and synonymies have been updated. Genera examined in the present study are marked with asterisks; those for which antisera were available are marked with double asterisks. Groups 3, 8-10, 13-15 and 18 are not discussed in detail in this paper

Group 1 Group 6 Group 13 Lycodonomorphus* Amplorhinus* Boiga* Lamprophis* * Group 7 Crotaphopeltis* Bo~rophtha~m~s Aparal[actus* Dipsadoboa Pseudoboodon Miodon Group 14 Bothrolycus AmblyodipsaP * Telescopus* * Group 2 Group 8 Macroprotodon * Hormonotus Coronella Group 15 Gonionoiophis* Meizodon Dispholidus* * Mehelya** Cohber* Thelotomis* * Lycophidion* Aeluroglena Group 16 Chamaeocus Spalerosophis Hemirhagerrhis Group 3 Lytorhynchus Malpolon* Geodipsas Group 9 Dromophis Group 4 Philothamnus* * Psammophis* * Afronatrir* Hapsidophrys* RhamphiophiP * NatricitereP Gastropyxis Dipsina* Hydraethiops Group 10 Group 17 Limnophis Rhamnophis Xenocalamus Group 5 Thrasops* * Chilorhinophis Grayia* * Group 11 Marrelaps* Duberria* Scaphiophis Micrelaps PseudaspiP Group 12 Group 18 Prosymna* DasypeltiP *

Eurasian and American representatives have been the focus of several biochemical studies (reviewed by Dessauer et al., 1987). Natricines and Colubrines are the two most universally recognized clades within colubrids, although with somewhat varying compositions among classifications. Considerably more controversy exists concerning the monophyletic status, relationships and/or composition of the remainder of Bogert’s groups. Of the remainder, Group 3 comprises a single genus, Geodipsas, shared between Africa and Madagascar, but no material for biochemical analysis is presently available for African Geodipsas. Although Bogert ( 1940) did not explicitly consider the colubrids of Madagascar in his study, the 16 or so colubrid genera present there have been discussed in the context of African colubrids by subsequent workers (e.g. McDowell, 1987). Molecular evolutionary studies of the Madagascan colubrids are presently underway, and their relationships will be considered at a later time. However, one Madagascan colubrid, Leioheterodon, is represented by an antiserum in the present study. No material for biochemical study has been available for the single genus of Group 11 (Scaphiophzs);with the exception of this group, all of Bogert’s groups are represented in this study by one or more taxa. Thus, this study will focus on relationships within and among Bogert’s Groups 1, 2, 5-7, 12, 16 and 17, to the extent that material is available. Colubrines (Groups 8- 1 1, 13- 15, 18), Natricines (Group 4) and other non-colubrid lineages of advanced snakes are included as OTUs for analysis (see sampling below) but will be discussed elsewhere in greater detail. I have found it necessary to use names for some higher categories within colubrids, but the history of many of these names is bewilderingly complex, and there often seems to be little consistency between classifications as to the generic PHYLOGENY OF AFRICAN COLUBRIDS 107 composition for many named higher categories (e.g. compare Underwood, 1967; Bourgeois, 1968; Dowling & Duellman, 1978; Dowling et al., 1983; McDowell, 1987). I do not add to that confusion here, and eschew erecting new categories or altering previous usages. However, as a necessary shorthand, I follow McDowell (1987) as modified by Cadle (1987; see also Dowling & Duellman, 1978; Dowling et al., 1983) for the following named categories: (1) Colubrinae (colubrines), a cosmopolitan clade characterized by derived hemipenial features and some osteological features (McDowell, 1987); (2) Natricinae (natricines), a primarily Eurasian and North American clade (a few African representatives), used here in its more traditional sense (Malnate, 1960; Rossman & Eberle, 1977) and characterized by derived hemipenial features; (3) ‘Xenodontines’, comprising two major neotropical clades and several North American genera which, collectively, have not been demonstrated to be monophyletic (hence the use of quotation marks around the name; see Cadle, 1984a-c, 1987, and further discussion of nomenclature herein); and (4)Psammophiini (psammophiines) for Bogert’s Group 16 (see Dowling, 1967, and Bourgeois, 1968).

MATERIALS AND METHODS Antigens and antisera Cadle (1988) detailed the immunological methods used in the present study. Briefly, plasma samples were collected from snakes in the field and frozen in liquid nitrogen, or whole blood samples were preserved in 2-phenoxyethanol phosphate sucrose (PPS; Nakanishi et al., 1969). Albumins were isolated by precipitation in a pH 8.4 Tris-EDTA-Borate buffer with 6,9-diamino-2-ethoxy- acridine lactate (‘rivanol’), regenerated in 0.5 M Tris hydrochloride (Sigma T-3253), concentrated by vacuum dialysis, and further purified by polyacrylamide gel electrophoresis (see Cadle, 1988, for details). Antisera to each albumin were prepared in three rabbits, titred using the microcomplement fixation (MC’F) method (Champion et al., 1974), and pooled in inverse proportion to their titres. Antigens used in cross reactions consisted of plasma, whole blood, or tissue extracts, either frozen or preserved in PPS. Albumin immunological distances (AIDS) were calculated as described (Champion et al., 1974). Non-reciprocity of reciprocal AID measurements was assessed and corrected using Sarich & Cronin’s (1976) method (see Cadle, 1988, for further discussion); the original data matrix was subjected to this procedure until all matrix row/column ratios were within 1 f0.1.

Basic taxa, outgroups and phylogenetic trees Because the phylogenetic structure of the African colubrid radiation is so poorly understood (the work of Bogert, Bourgeois, McDowell and others notwithstanding), I attempted to make few assumptions in analysing this radiation from the standpoint of the immunological data. To make the data analytically tractable, I used colubrid genera as the basic units for analysis. However, I have not made comprehensive investigations of the monophyly of the genera, and with few exceptions have not made extensive intragenetic comparisons; hence, the interpretation of my statements concerning the 108 J. E. CADLE relationships of particular genera should take due consideration of the sampling. Nevertheless: I have made some comparisons, not reported here, to additional species of genera discussed herein; none of these comparisons contradicts general conclusions based on the data given here. Because of controversies about higher relationships among advanced snakes, in which African colubrids continue to play a pivotal role (McDowell, 1986, and references therein), I also included as basic taxa for analysis the Elapidae and the enigmatic genus Atractaspis, which has sometimes been considered a colubrid (see reviews in Cadle, 1983; McDowell, 1986; Underwood & Kochva, 1993). Selection of an appropriate outgroup for this study poses additional complications. I make the assumption that viperids do not share a closer common ancestry with any of the basic ingroup taxa than the latter do among themselves, and that viperids therefore can be used as an outgroup for assessing amounts of albumin change among ingroup OTUs, and for rooting the phylogenetic tree of the ingroup taxa. This is contradictory to McDowell’s (1986) hypothesis that viperids and a majority of extant colubrid lineages are monophyletic relative to elapids, Atractaspis and a few colubrid genera. My assumption that viperids are a justifiable outgroup taxon is supported by previous immunological evidence showing that elapids, Atractaspis and some colubrid lineages are monophyletic relative to vipers, when Boa and chicken albumins are used to root the advanced snake tree (Cadle, 1988). Additional data bearing on this point are presented herein. Fourteen genera, treated as nominal OTUs for analysis, were included in a matrix of reciprocal immunological comparisons (see Table 2). In addition, the Elapidae (cobras and their relatives) and Viperidae (vipers), were added to this matrix. In these two cases, immunological distances to the other 14 genera in the matrix were determined by using pooled antisera to albumins of several species of each of these taxa. For the Elapidae, the antiserum pool included individual antisera to the following species: Bungarus fasciatus, Dendroaspis poblepis, Elapsoidea semiannulata, Hydrophis melanosoma, Laticauda semifasciata, Micrurus spixii and Naja haje. The series for the Viperidae included Bothrops atrox, Crotalus enyo and Bitis nasicornis. This approach permits an assessment of immunological differences between a higher taxon or lineage (e.g. Elapidae or Viperidae), represented by a pooled antiserum, and a suite of other lineages. Such antisera give a broad representation of antigenic determinants present among taxa contributing to the pool and make higher order comparisons among groups tractable without resorting to the use of monospecific antisera to represent the groups (see Sarich, 1985, who used this approach to examine mammalian interordinal relationships). However, these pooled antisera cannot be used to assess intra- group relationships. The assumption is made that species contributing to the antiserum pool are monophyletic relative to other OTUs in the data matrix. This assumption is justified in this study, in that Elapidae and Viperidae, as used herein (i.e. as represented by the respective antiserum pools), are widely accepted to be monophyletic relative to the other clades discussed (see Cadle, 1988, for discussion). Hence, the matrix for generating a phylogenetic framework within which to evaluate the African colubrid radiation comprises 16 OTUs, of which 14 were represented by reciprocal comparisons. In addition to the reciprocal matrix, representatives of 13 additional African colubrid genera and one Asian genus TABLE2. Albumin immunological distances among African snakes. Reciprocal comparisons are given for 14 taxa (Lamprophis-Dasypeltis). Antiserum abbreviations (columns) correspond to the species listed at the left. The Elapidae and Viperidae are each represented by antisera to multiple species of those taxa (see text). The row sums/column sums use the corresponding values for reciprocal comparisons only, and are the correction factors for individual antisera (see text) cd 2 ANTISERA s Antigens Lam Rha Psa Meh Lei Gra Amb Atr Thr Dis The Tel Phi Das Elapidae Viperidae @ .e Lamprophisfuliginosus 0 84 83 70 76 82 106 86 60 60 64 81 83 67 74 89 Rhamphiophisoxyrhynchus 85 0 56 91 73 98 105 77 84 84 89 76 104 74 93 109 % Psammophis subtaaniatus 92 45 0 83 90 64 101 84 54 77 67 76 109 64 77 89 % Mehelyacrossi 53 88 96 0 81 105 97 84 72 88 85 78 101 79 85 92 z Leioheterodonmadagascariensis 73 82 98 87 0 69 85 64 57 57 69 70 103 69 75 90 n Grayiasmythii 75 117 71 92 79 0 105 81 32 53 53 60 91 49 69 100 > Amblyod$sas polylpis 88 112 127 88 85 88 0 93 84 80 87 103 130 85 90 105 z Atractaspisbibronii 85 75 89 87 64 85 80 0 71 78 86 89 100 77 85 94 n Thrasops jacksoni 52 86 67 100 62 47 99 79 0 13 13 33 67 26 64 94 C DispholidusQpus 66 90 74 84 80 52 102 81 13 0 15 30 65 25 69 90 m Thelotomiscapensis 60 87 75 88 71 47 108 73 12 7 0 30 59 25 67 90 ?Fj b Telescopus semiannulatus 82 89 67 82 73 67 108 90 35 36 36 0 72 16 80 109 rA Philothamnus angolensis 55 88 102 76 64 77 95 82 54 49 50 57 0 37 79 92 Dasypeltisscabra 91 103 87 94 92 75 109 96 40 42 47 40 81 0 88 98

Row sum/column sum: 1.05 0.96 0.92 0.99 0.99 1.00 0.96 1.00 1.11 1.07 0.95 1.04 0.76 1.44 110 J. E. CADLE (whose inclusion is explained below), were compared to all major clades identified from the analysis of the reciprocal immunological distance matrix. For these unidirectional comparisons, a colubrine antiserum analogous to the viperid and elapid pools described above was prepared from six African colubrine antisera ( Thrasops jacksoni, Dispholidus &pus, Thelotornis capensis, Telescopus semiannulatus, Philothamnus angolensis and Dasypeltis scabra) . Hence, of the somewhat more than 50 colubrid genera in Africa, representatives of about half (27) have been examined during this study, including representatives of all Bogert’s groups except Group 11 (Table 1). Specimens used are listed in Appendix 1. Note that no assumption is made here about the monophyly of Bogert’s groups-those are considered questions to be investigated, though clearly more sampling is needed before this question can be addressed for many of the groups (Table 1). The reciprocal matrix of AIDS was corrected as described above, and reciprocal measurements were averaged and used for phylogenetic analysis. Phylogenetic trees were constructed by the method of Fitch & Margoliash (1967), using the program EVOLVE provided by W. M. Fitch and using the FITCH program of PHYLIP (version 3.41; Felsenstein, 1991). With the latter program, several OTU input orders were used, including random order entries, as were the global optimization routine and several user-defined trees. Global optimization is a branch-swapping routine which successively removes all possible subtrees, reinserting each at all possible positions, and evaluating the resulting trees; this option was repeated 10 times using a different random number seed to determine taxon input order. Thus, the trees discovered are considered the best under the evaluation criteria. Fitch-Margoliash trees are evaluated using the percent standard deviation criterion (YoSD),a measure of differences between output distances on a constructed phylogenetic tree and the input values (Fitch & Margoliash, 1967). Lower values of %SD reflect better agreement between input and output data matrices. In addition to the Fitch- Margoliash trees, PHYLIP was also used to construct trees with the neighbour- joining method of Saitou and Nei (1987). The conceptual basis for the tree- building methods used herein is discussed by Swofford & Olsen (1990). Taxa for which only unidirectional comparisons to several antisera were available were attached to the Fitch-Margoliash trees by the method of Beverley & Wilson (1982), which allows the attachment of an OTU to any branch or node of a given phylogenetic tree; in some cases several different attachment sites were evaluated using the YoSD criterion. The Beverley and Wilson method only permits the assessment of the relationship of species to an already-constructed tree, and not to other OTUs for which only unidirectional comparisons are available. Moreover, estimations of branch lengths using this method become more accurate as more outgroups are used. Because of some peculiarities of this particular data set (see following), only a single outgroup could be confidently used herein, so placement of taxa using this method should be considered approximate. Variability in the rate of albumin differentiation was assessed using methods previously described (Beverley & Wilson, 1984, and references therein; Cadle, 1988). Briefly, rate tests using the pooled antisera to the viperid outgroup were performed for all ingroup taxa. Secondly, constructed phylogenetic trees were used to estimate and compare the amounts of albumin differentiation along all PHYLOGENY OF AFRICAN COLUBRIDS 111 lineages stemming from the basal ingroup node of the tree. Estimates of rate variability followed methods outlined by Nei (1977) and Beverley & Wilson (1984).

RESULTS Reciprocal comparisons and phylogenetic trees Titres for pooled antisera ranged from 2600 to 6800 (mean 4300). Non- reciprocity of the 14 x 14 distance matrix (Table 2) is 8.82y0, a small figure for a matrix of that size. Most of the non-reciprocity is due to comparisons involving Philothamnus and Daypeltis; the average non-reciprocity for comparisons not involving these OTUs is 5.81 yo. Correction of the matrix for non-reciprocity using Sarich & Cronin’s (1976) procedure required only three passes through the procedure (the Philothamnus, Daypeltis and Thrasops columns), and the non- reciprocity for the corrected matrix was 5.77%. The Fitch-Margoliash tree having the lowest standard deviation (8.03y0), with the restriction that branch lengths be non-negative, was found using the FITCH program of PHYLIP with global optimization (1060 trees examined; Fig. 1). Seventy-five trees were evaluated using EVOLVE. A significant feature of the optimal tree (Fig. 1) is the very short length of many of the internal branches. With the exception of internal branches 8-19 AID units long joining the pairs Lamprophis-Meheba and Psammophis-Rhamphiophis, and the series of clades including Grayia and the colubrines (Philothamnus-Daypeltis-Telescopus- Thrasops-Dispholidus- Thelotornis), all other internal branches are about 5% or less

53 Amblyodipsas 4 36 Atractaspis 28 I 2 Leioheterodon 25 Lamprophis 37 Mehelya 2 29 L

Figure 1. A phylogenetic tree produced from the average corrected reciprocal data of Table 2 using the FITCH algorithm of PHYLIP, global optimization, and disallowance of negative branches. Branch lengths have been rounded to the nearest integer. The standard deviation of this tree is 8.03%. 112 J. E. CADLE ( I4AID units) of the average distance between taxa stemming from the basal series of dichotomous branches. None of these short branches is considered significant, given the large immunological distances involved and the experimental error of the MC’F approach. Alternative trees with slightly higher percent standard deviations differed from the tree in Fig. 1 in the relative order of branching of the major clades at the base of the tree, another indication that these very short internal branches are non-significant. In contrast to clades defined by short internal branches, OTUs united by internal branches of length 2 8 were not disrupted in any of the alternative trees with low %SD, indicating the robustness of these clades in trees of increasingly poorer fit to the data matrix. Of these latter clades, the association of Grayia with the colubrines was the least stable, and this genus was pulled out of the colubrine clade and attached to other basal lineages in the EVOLVE trees of SD 2 approximately 9.4%. None of the 75 EVOLVE trees disrupted any of the other clades joined by internal branches 18. Moreover, Philothamnus was not dissociated from the other colubrines in any of the trees examined, despite its position outside a Grayia- (other colubrines) clade in the globally optimized tree (Fig. 1). A tree identical to that in Fig. 1, with the reversal of the positions of Grayia and Philothamnus, has nearly the same SD (8.1% versus 8.03%) and introduces no negative branches (see Fig. 2). Thus, I view this series of relationships as an unresolved three-way split (Philothamnus-Grayia-other colubrines) . Given the unstable position of Grayia within alternative trees, future data may suggest a more secure linkage for this genus to a lineage other than to the colubrine clade, as shown in Fig. 1. Within the colubrines (in the strict sense), all trees examined preserved the branching order shown in Figs 1 and 2: (Philothamnus ( (Dugpeltis, Telescopus) ( Thrasops (Dispholidus, Thelotornis)))) . Several Fitch-Margoliash trees were produced with relaxation of the requirement for non-negative branches, resulting in some with lower YoSD than the tree in Fig. 1. However, these invariably introduced rather large negative branches and differed largely from Fig. 1 in the arrangement of basal dichotomous branches (the tree with lowest SD, 7.4y0, was found using global optimization and allowing negative branches, but had three negative branches of length 9- 10). The tree produced by Saitou and Nei’s neighbour-joining method had a topology identical to several of the Fitch-Margoliash trees with low SDs; it had no negative branches. As with all of the Fitch-Margoliash trees, the neighbour- joining tree was very unresolved at the base (internal branches 1-5 units in length) and had long terminal branches. The clusters Lamprophis-Mehelya, Psammophis-Rhamphiophis and the series of clades including Grayia and the colu brines (Philothamnus-Dugpeltis-Telescopus- Thrasops-Dispholidus- Thelotornis) all appeared in the neighbour-joining tree; within the latter clade, the branching order of the genera was identical to that in Fig. 1. Although not here considered significant, it is worth noting that the association of Atractaspis, Leioheterodon and Amblyodipsas by a short internal branch (as in Fig. 1) appeared also in the neighbour-joining tree, where the common branch had length = 5. Although a more detailed discussion of colubrine relationships is deferred, one issue brought out by Fig. 1 should be addressed here. The colubrines (represented by Philothamnus-Dugpeltis-Telescopus- Thrasops-Dispholidus- Thelotornis in Figs 1 and 2) are, from the standpoint of molecular comparisons, one of the PHYLOGENY OF AFRICAN COLUBRIDS 113

Leiohelemdon

Lycodonomorphus

Figure 2. A phylogenetic tree produced using the tree in Fig. 1 as a framework and applying Beverley & Wilson’s (1984) method to the data of Table 4. Taxa were added only when clear associations with a reference OTU (antisera in Tables 4 and 5) were apparent considering the rate test data. Basal branches of the tree have been collapsed into a polychotomy to reflect non-resolution at this level, and the positions of Gfayia and Philothamnus have been reversed from their positions in Fig. 1 (see text). Lycodon, not shown in the figure, is within the colubrine lineage, and Prosymna, shown as a clade basal to Colubrinae, is probably within this lineage (see text). most cohesive clades of colubrids, with albumin immunological distances generally Iabout 55 among genera worldwide (Cadle, 1984c, 1987; Dowling et al., 1983; Dessauer et al., 1987). This cohesiveness is reflected in Figs 1 and 2, where a branch of length 8 or 9 roots the colubrine clade to the rest of the tree. However, the placement of Grayia within this clade (Fig. 1) is somewhat surprising since this genus lacks the derived hemipenial features of the colubrines, and no special relationship between these taxa has been proposed previously. The placement of Grayia within the colubrine radiation (Fig. 1) would require homoplasy in the derived hemipenial features between Philothamnus and the other colubrines (or reversal to a primitive state in Grayia). Clearly, other data bearing on the phylogenetic position of Grayia are needed. Hence, Fig. 1 is taken to be the ‘best’ tree (minimizing %SD) with the constraint of non-negative branches, and suggests that within the limits of the approach, the radiation of these taxa can be viewed as a polychotomy with seven basal branches (Figs 1 and 2): ( 1 ) Arnblyodipsas; (2) Atractaspis; (3) Leioheterodon; (4)Lamprophis-Meheyla; (5) Rhamphiophis-Psammophis; (6) Elapidae; (7) Grayia plus the colubrines. Notably, the Elapidae and Atractaspis appear as derivatives of this basal radiation, and not as clades separate from the colubrids (although there is a marginal association of Atractaspzs with Arnblyodipsas and Leioheterodon, as noted above).

Rate tests for albumin evolution in African colubrids Rate tests performed by measuring the immunological distances between the viperid outgroup and all ingroup taxa show that the standard deviation of measured distances averages about 12% of the mean AID (Table 3). This is 114 J. E. CADLE TABLE3. Rate tests and variation in albumin evolutionary rate for African colubrids. Numbers in parentheses after each taxon are sample sizes. Values are meanfone SD

1. Rate tests using viperid outgroup' Colubrines (Africa only (6) 95.5k 7.3 Psammophines (5) 96.2+ 13.6 ~UNatricilereslAfronnhix (Natricines) (2) 107k 12.7 All other colubrids (Tables 1 and 2) (1 1) 90.1 13.0 Overall colubrid mean (24) 94.1 f 12.2 Variance/mean* 3.16 2. Tree method3 Distance from root (overall mean) ( 15) 42.3+ 7.07 Variance/mean 1.18

' Values given are average immunological distances measured between the viperid outgroup and each ingroup taxon (Tables 1 and 2). Calculated as in Nei (1977). See also Beverley and Wilson (1984). Value given is the average of the summed branch lengths between the basal colubrid/elapid node and each ingroup OTU (Fig. 1). comparable to other vertebrate albumin data sets (Sarich & Cronin, 1976; Beverley & Wilson, 1984). The average distance between viperids and the colubrids in this study is slightly greater than a comparable average for other colubrids calculated from data in Cadle (1988). This very probably reflects the inclusion herein of the viperine Bitis, which is known to have a higher albumin evolutionary rate than the viperids used in the previous study (Crotalus and Bothrops; Cadle, 1992). The rate tests (Tables 2-4) and estimates of branch lengths from the basal colubrid/elapid node in Fig. 1 identify several OTUs whose albumins have changed relatively more (Amblyodipsas, Rhamphiophis, Daypeltis, Telescopus, Natriciteres) or less (Amplorhinus, Psammophylax, Lycodonomorphus) than one standard deviation from the mean rate. Relative rate tests were also performed by comparing the calculated amounts of albumin change along branches of the phylogenetic tree (Fig. 1) from the basal colubrid/elapid node to each OTU, which gives results comparable to the method using simultaneous equations outlined by Beverley & Wilson (1984). Hence, all pairwise ratios of estimated branch lengths from the tree in Fig. 1 were calculated. For these data the average difference for all pairwise comparisons is 22%, again comparable to other albumin data sets (Beverley & Wilson, 1984). Two methods of calculating the variance of evolutionary rate for the snake albumins (Nei, 1977) yield estimates of that variance as about 1-3 times the mean (Table 3); similar estimates for other albumin data sets are 2-9 times the mean (Nei, 1977; Beverley & Wilson, 1984). Combined, the outgroup rate tests and the relative rate tests performed using the tree method, as well as variance estimates, suggest that these snake taxa are comparable to other vertebrates in terms of variation in albumin evolutionary rate.

Unidirectional comparisons (Bogert's Groups 1, 2, 5-7, 12, 16, 17) Unidirectional comparisons between the genera representing each basal clade in Fig. 1 and species of 13 additional African colubrid genera are presented in Table 4. Highlighted in that table (values in bold italics) are those PHYLOGENY OF AFRICAN COLUBRIDS 115 TABLE4. Albumin immunological distances among African and Asian snakes. Antiserum abbreviations (columns) correspond to those in Table 1, except for COL (pool of colubrine antisera) and VIP (pool of viperid antisera), as explained in the text. Distances given are the raw data values mutiplied by the respective antiserum correction factors given in Table 2. Numbers in parentheses under each antiserum and adjacent to antigens used in cross-reactions refer to Bogert's groups (Table 1). Immunological distances in bold denote significant associations, as discussed in the text. Lycodon, an Asian colubrid, is included here for reasons explained in the text

ANTISERA

Psa Rha Meh Lorn Lk Gra Amb Air COL VIP Antigens (16) (16) (2) (1) (5) (7)

Lycodonomorphus rufulus (1) 77 85 69 39 64 84 99 69 63 75 Lycophidion capensis (2) 74 81 69 65 51 60 76 92 57 - Gonionofophis granfi (2) 78 81 73 71 80 66 91 86 74 97 Natriciteres oliuacea (4) 97 101 132 90 84 86 91 103 60 116 Afronatrix anoscopus (4) 95 91 101 90 89 82 121 113 56 98 Duberria lufrix (5) 71 79 72 72 67 70 93 78 67 89 Pseudaspis cana (5) 80 84 92 110 73 86 102 69 81 107 Amplorhinus mulfimaculafus (6) 73 74 72 56 44 70 72 58 63 62 Prosymna sundeuallii ( 12) 68 71 97 71 60 72 95 62 48 85 Psammoplcylax fn~aeniafur(16) 51 54 105 98 63 79 104 122 73 79 Dipsina multimaculala (16) 46 32 81 80 83 87 102 62 76 93 Malpolon monspessulanus ( 16) 38 36 104 93 99 76 94 73 80 111 Macrelaps microlepidofus ( 17) 102 77 76 82 71 75 41 67 73 - Lycodon laoem's (Asia) 60 99 86 87 53 55 92 78 32 102 immunological distances which represent significant associations; that is, the distances so denoted can be apportioned along the branches in Fig. 1 with unambiguous association of these taxa with the clades indicated at the head of each column (note that these apportionments take account of the relative rates of change indicated by comparisons to the viperid outgroup). Strikingly, of the 1 17 ingroup comparisons in Table 4, only 13 values indicate strong associations with one or more of the basal clades in Fig. 1. By inference, then, the remaining taxa in Table 4 represent additional basal or near-basal clades, with albumin immunological distances averaging 78 from all other ingroup taxa. Although reciprocal comparisons may eventually permit the association of some of these additional genera with those already represented by antisera,- the unidirectional comparisons in Table 4 suggest that those associations will' not be very strong (especially given the good reciprocity of the origina1,Ziiatrix). Hence, present sampling would suggest several additional lineagts involved in the basal colubrid/elapid/Atractaspis polychotomy of Fig. 1. A summary of these unidirectional associations, using Beverley & Wilson's ( 1982) method of branch length estimation, is given in Fig. 2. This tree indicates the uncertainty inherent in the basal series of branching events, and also reverses the positions of Grayia and Philothamnus shown in Fig. 1 (see above). Because more detailed comparisons are available for the Psammophis- Rhamphiophis group, and because of substantive implications of the Amblyodipsas- Macrelaps association noted in Table 4 and Fig. 2, discussion of these taxa is deferred until following sections. Similarly, the nomenclatural implications of the comparisons involving the Asian genus Lycodon are treated separately below. Among the other significant associations denoted in Table 4 and Fig. 2, a few other comments are warranted. 116 J. E. CADLE The Lamprophis-L~codonomorphus association was recognized in Bogert’s ( 1940) classification (Group 1), and has generally been recognized since (e.g. Dowling, 1969); hence, there is nothing especially surprising in the immunological results, although the branch length estimated for their period of common ancestry is short relative to the length of the lineage. The relationships of the other African genera showing significant associations in Table 4, Amplorhinus, Prosymna and Afatriciteres-Afronatrix, have not been as clear. Both Amplorhinus and Prosymna were placed in their own groups by Bogert (1940; Groups 6 and 12, respectively, Table l), implying some uncertainty concerning their relationships. Of these, the peculiar skull (Bourgeois, 1968) and hemipenis (Broadley, 1980, 1983) of Prosymna have hampered understanding of its relationships to other colubrids; the immunological data clearly ally Prosymna with the colubrine/Grayia lineage. However, because comparisons of Prosymna with antisera to albumins of individual species of colubrines have not been completed, and therefore its more precise placement within this radiation cannot be estimated, Fig. 2 shows its arbitrary attachment at the base of the colubrine clade. The association of Prosymna with the colubrines perhaps facilitates understanding some aspects of the peculiar hemipenial morphology of Prosymna, despite its other autapomorphies: all Prosymna hemipenes are simple (non- bilobed) and have an undivided sulcus spermaticus (Broadley, 1980)- synapomorphies shared with virtually all colubrines. Thus, there seem to be good grounds for considering Prosymna as simply a colubrine modified for a burrowing mode of life, at least superficially much as seen in the North American colubrines, Chionactis and Chilomeniscus (Stickel, 1943; Savitzky, 1983). Less definitively, the immunological data suggest that a possible relationship between Amplorhinus (restricted to southeast Africa) and Leioheterodon (Madagascar) merits further evaluation. The difficulty here is that Amplorhinus has a very conservative albumin, in fact, the most conservative albumin examined in this study (AID = 62 to viperids, whereas all other viperid- colubrid distances are 2 75; Tables 2 and 4).Consistent with this demonstrated conservatism, and as seen in Table 4, Amplorhinus shows relatively low immunological distances to several taxa other than Leioheterodon (Lamprophis, Atractaspis and the colubrines). Nevertheless, the Leioheterodon-Amplorhinus distance is substantially lower (44) than any other distance measured to Amplorhinus (53-79; Table 4), and the Lamprophis, Atractaspis and colubrine antisera tend to give relatively low distances to some other taxa as well. Clearly, reciprocal immunological distances in this case would be extremely helpful, as the placement of Amplorhinus vis- 8-vis Leioheterodon using the unidirectional comparisons (Table 4, Fig. 2) shows an unresolved trichotomy involving these two genera and Atractaspis (that is, a ‘branch’ of length 0 attaches Amplorhinus to Leioheterodon, using the Beverley and Wilson method). Hence, notwithstanding some hesitation in making a definitive statement concerning the A~plorh~nus- Leioheterodon association, that relationship warrants further exploration, particularly in light of the geographic contiguity of these taxa. Two other African colubrids of Table 4, JVatriciteres and Afronatrix, are members of Bogert’s Group 4 and seem clearly to ally with the colubrine/Grayiu clade. These genera are members of a much larger worldwide radiation of colubrids, the natricines (E. V. Malnate, personal communication; Gartside & Dessauer, 1977; Rossman & Eberle, 1977; Dowling et al., 1983; see also PHYLOGENY OF AFRICAN COLUBRIDS 117 McDowell, 1987). Dowling et al. (1983), also using MC’F comparisons of albumins, concluded that natricines and colubrines comprised a clade relative to several African and New World clades; however, as that study used no outgroups or rate tests, those conclusions were subject to confirmation (see following). The present results confirm that association for Afronatrix and Xutriciteres, which have, in comparison to the viperid outgroup, among the most-changed albumins examined (Table 4), and yet still show similar very low distances to the colubrines. No comparisons with other antisera (Table 4) approach these values. As in the case of Prosymna, and because reciprocal comparisons among individual taxa of these two diverse clades are presently unavailable, AfronatrixlXutriciteres are arbitrarily attached at the colubrinelGrayia node (Fig. 2); more precise assessment of the relationship between these taxa will be possible once reciprocal comparisons are completed. Connecting Afronatrix/.Natriciteres to the common Grayiu-colubrine lineage (i.e. to the branch below the Grayia-colubrine node; Fig. 2) results in negative branches. Using an antiserum to the albumin of the North American natricine, Tharnnophis sirtalis, Dowling et al. (1983) reported an AID of 46 to Xutriciteres, the largest intra-natricine value in their study, and probably close to the maximum intra-lineage AIDs to be expected within natricines or colubrines (see Dessauer et al., 1987). The average colubrine- natricine distances given by Dowling et al. (1983), using antisera to several colubrines and a natricine from North America, was 65. This is consistent with the African colubrine-Afronatrix/Xatriciteres AID reported here (mean = 58), and lower than all other inter-lineage AIDs obtained. Hence, these immunological data provide the first indication that two major colubrid lineages (often considered subfamilies or families) form a clade relative to a large number of others (Fig. 2). Many other lineages remain to be included in these broad-scale colubrid comparisons, however, but it is nonetheless interesting to note that virtually all colubrines and most natricines (E. V. Malnate, personal communication) share a simple sulcus spermaticus, a potential synapomorphy for a combined colubrine-natricine clade (and one found only occasionally outside these groups; see McDowell, 1987). Moreover, these two clades are the dominant colubrid lineages of the Holarctic region and have low diversity in tropical Africa and America. In view of the likely natricine-colubrine association, and the ambiguous position of Grayia discussed above, the possible association of Grayia with the natricines (a view not endorsed by McDowell, 1987) should be explored. The immunological data for the four remaining African genera in Table 4 do not suggest strong associations with any of the basal clades of Fig. 1. However, without reciprocal measures the possibility of weak associations with those basal clades cannot be ruled out. Two members of Bogert’s Group 2, Gonionotophis and, especially, Lycophidion (whose albumin appears markedly conservative using the viperid outgroup; Table 4), show somewhat low distances to several other conserative taxa but no strong association with Mehelya, the member of Group 2 represented by an antiserum. That Gonionotophis in particular shows no association with Mehelya is puzzling in view of strong similarities between the two genera in venom gland structure (McDowell, 1986) and in the presence of peculiar expansions on the pre- and post-zygapophyses (Bogert, 1964; McDowell, 1987). Given that only a single sample of Gonionotophis has been available for study, in this case I am tempted to suspect the immunological I I8 J. E. CADLE result, which should be confirmed with additional samples. In contrast to Bogert (1940), McDowell (1987) did not specifically ally Lycophidion with Mehelya but, instead, with Chamaelycus, Bothrolycus and Hormonotus; none of these latter genera has been available for immunological study. Duberria and Pseudaspis (Bogert’s Group 5) likewise show no strong association with Grayia, also a member of Group 5, nor with any other genera represented by an antiserum. Bourgeois (1968) discussed many skull pecularities of Duberria and Pseudaspis and disagreed with Bogert’s implied relationship among these genera and Grayia. However, her data on skull morphology suggested no special relationship between Duberria and Pseudaspis, nor between either of these genera and other African colubrids. McDowell (1987) placed both of these genera within a large African colubrid assemblage (‘Boodontinae’), and considered them related to one another and to Pythonodipsas, based on the presence of musculature associated with the hypapophyses, live-bearing (Duberria and Pseudaspis only), and unusual chromosome number (2n = 42; known only for Pseudaspis and Pythonodipsas). This hypothesis cannot be evaluated in detail, as no antiserum to Duberria or Pseudaspis, nor any material of Pythonodipsas, have been available for study. However, as discussed below, the existence of a clade of African colubrids equivalent to the ‘Boodontinae’ of McDowell (1987) and others is questionable from the standpoint of the immunological data.

Lycodon and the status of the Lycondontinae The remaining genus which, from the unidirectional comparisons (Table 4), shows a strong association with a major clade of Fig. 1, is Lycodon. This genus is not African, but its relationships bear on an important nomenclatural issue for many African and Asian colubrid genera. Lycodon, represented here by L. laoensis from Thailand, is distributed across southern Asia from the Caspian Sea into Australasia. It is the type genus of a higher category, the Lycodontinae (Bonaparte, 1845), to which most non-colubrine/natricine African colubrid genera have been assigned in much of the recent literature (e.g. Dowling & Duellman, 1978; Dowling et al., 1983; Cadle, 1987). McDowell’s (1987) results on the morphology of several species of Lycodon suggested that, far from representing a distinct clade of colubrids, Lycodon fits comfortably into the colubrine radiation, the most cosmopolitan of extant colubrid lineages as that clade has usually been conceived. This result has clear nomenclatural implications with direct bearing on the classification of African colubrids. Therefore, investigation of the relationships of Lycodon in this study appeared warranted. The immunological results confirm McDowell’s ( 1987) assessment, placing this genus well within the colubrine clade (Table 4). Therefore, the use of this genus as a basis for a higher category within Colubridae in traditional and more recent senses (Underwood, 1967; Dowling & Duellman, 1978; Dowling et al., 1983) should be discontinued. Dowling et al. (1983) resurrected the name Boodontinae (Cope, 1893) for those ‘lycodontines’ having hypapophyses on the posterior vertebrae, but retained Lycodontinae for those lacking posterior hypapophyses. McDowell ( 1987) used Boodontinae for most African/ Madagascan colubrids of the former Lycodontinae. He did not use the latter term, a choice supported by his conclusions on the relationships of Lycodon, which PHYLOGENY OF AFRICAN COLUBRIDS 119 are bolstered by the immunological data presented herein. But equally significant, the immunological results do not support the existence of clades for either the Boodontinae or the Lycodontinae, in the broad sense that these terms have recently been used (Dowling & Duellman, 1978; Dowling et al., 1983; McDowell, 1987). Further nomenclatural implications of these results are discussed below.

Psammophis-Rhamphiophis clade (Bogert’s Group 16) The largest non-colubrine African assemblage for which the immunological data are capable of providing some phylogenetic resolution comprises genera of Bogert’s (1940) Group 16 (Hemirhagerrhis and Dromophis not examined). Even here, however, the albumin differentiation between the two reference species (Psammophis subtaeniatus and Rhamphiophis oxyrhynchus) , 48 AID units using the corrected values (Table 5), is substantial. Additional comparisons (Table 5) show that, with the exception of the Rhamphiophis-Dipsina distance and the low distance to Malpolon shown by both reference antisera, albumin differentiation among genera within this clade is relatively great. The relationships suggested by these data are summarized as a tree in Fig. 3. The data in Table 5 suggest that Dipsina and Rhamphiophis are sister taxa relative to the other genera, but the branching order among the other genera is unresolved. Furthermore, within Psammophis, the Asian species condanarus is the earliest diverging species. Among the other species of Psammophis, the immunological data (Table 5 and Fig. 3) suggest especially close relationships between P. subtaeniatus, P. phillzjuii, and P. rukwae. Note that, although the P. subtaeniatus-P. phillipsii distance is relatively high (30) and comparable to the distances involving P. biseriatus and P. elegans, the comparisons of these species with Rhamphiophis suggest that the albumin of P. phillipsii is relatively much more changed than that of these other species; hence, this species associates much more strongly with P. subtaeniatus and has an

TABLE5. Albumin immunological distances among snakes of Bogert’s Group 16 (Psammophiini). Distances are the raw data values multiplied by the respective antiserum correction factors given in Table 2. All snakes are from Africa, except for Psarnmophis condanarus, which is from Thailand

ANTISERA

Psammophis Rhamphiophis subtaeniatus oxyrhynchus

Psammophis subtaeniatus 0 43 P. rukwae 6 47 P. punctulatus 17 41 P. biseriatus 23 47 P. elegans (Togo) 24 51 P. elegans (Ghana) 29 52 P. phiU;Psii 30 71 P. condanarus 40 59 Psammophylax tritaeniatus 51 54 Rhamphiophis oxyrhynchus 52 0 Dipsina multimaculata 46 32 Malpolon monspessulanus 38 36 120 J. E. CADLE

Psammophis condanarus

P. subtaeniatus Figure 3. A phylogenetic tree for psammophiines (Bogert’s Group 16) using Beverley & Wilson’s (1984) method on data in Table 5. As this tree used only the comparisons in Table 5, calculated branch length are somewhat different from those of Fig. 2, which were based on the entire matrix of Table 4. accelerated rate of albumin change (Fig. 3). Conversely, the calculated rate of change for P. punctulatus (Fig. 3) is very conservative relative to other species of Psammophis. Kate tests, then, prove critical for proper phylogenetic interpretation of the immunological data for these taxa. In summary, the immunological data on differentiation within Psammophis suggest early divergence for the Asian species P. condanarus, later divergence of a series of northern and eastern African species (biseriatus, elegans, punctulatus) , and finally three closely related species (phillipsii, subtaeniatus and rukwae) belonging to a widespread African complex. The phylogenetic relationships suggested by the immunological data on Group 16 (Fig. 3) conform to most hypotheses concerning this group. Virtually all workers since Bogert (1940) have recognized his Group 16 as a clade (the Psammophiini, Psammophinae or psammophiines; see e.g. Bourgeois, 1968; Dowling & Duellman, 1978), characterized by hemipenial synapomorphies (Bogert, 1940) and, perhaps (observed in Malpolon and several Psammophis spp.), by a peculiar ‘polishing’ behaviour and associated glands (Steehouder, 1984). The association of the monotypic genus Dipsina with Rhamphiophis is consistent with its former status as a species of Rhamphiophis (Broadley, 1983). Within Psammophis, the strong association of subtaeniatus-rukwae-phill$sii was recognized by Broadley (1977, 1983) in erecting the ‘sibilans complex’ (P. rukwae is a sibling species of P. sibilans; Broadley, 1977). The relatively primitive status of elegans- biseriatus-punctulatus among African species of Psammophis (Broadley, 1977) is consistent with the placement of these species as early diverging members of the African complex (Fig. 3), and these species may prove to form part of a monophyletic group, as suggested by Broadley (1977), once molecular differentiation among them is assessed. Additional questions raised by the immunological results concern the relationship between the Asian and African species of Psammophis. Clearly it would be of interest to determine if the Asian species are monophyletic relative to the African ones. Furthermore, given the PHYLOGENY OF AFRICAN COLUBRIDS 121 placement of P. condanarus at the base of the Psammophis lineage (Fig. 3), the precise relationship between African and Asian Psammophis vis-a-vis other psammophiines should be investigated more fully.

Comments on McDowell’s (1986) Homoroselaps Group (Bogert’s Groups 7 and 17) Bogert’s Groups 7 and 17 (Table 1) have formed the focal point for many of the more controversial aspects of the phylogeny of advanced snakes. Bogert himself (1940 : 10) recognized that these two groups were close relatives despite their wide separation in his system (a result of variation in the simpleldivided nature of the sulcus spermaticus among these genera). In the late 1960s there was growing consensus that the genera of these two groups formed a natural (monophyletic) colubrid group, the Aparallactinae. Moreover, two genera previously assigned to other families, Atractaspis and Homoroselaps, were suggested to be related to this group, although interpretations on this point are not in total agreement (Bourgeois, 1968; Kochva et al., 1967; McDowell, 1968; Cadle, 1983; McCarthy, 1985; Underwood & Kochva, 1993). Because Atractaspis and Homoroselaps were not considered to be colubrids, Bogert (1940) did not consider them in his groupings. Although the relationships of Atractaspis and Homoroselaps continue to be controversial (Cadle, 1983; McCarthy, 1985; Underwood & Kochva, 1993), the Aparallactinae gained general acceptance in its original formulation including Bogert’s Groups 7 and 17 (references cited above; Table 1) . Nevertheless, a phylogenetic association between Amblyodipsas and Aparallactus was not confirmed by preliminary immunological data on aparallactines (Cadle, 1983). Moreover, McDowell ( 1986), in a broad reclassification of advanced snakes, divided the Aparallactinae into a core of genera (the Homoroselaps group, including Miodon, Amb~ud~sas,Xenocalamus, C~ilor~inophis,Micrelaps and Homoroselaps), which he considered possibly related to the E1.apidae and Atractaspis, and a clade comprising Aparallactus, Macrelaps, all other colubrids and Viperidae. Thus, both immunological data (Cadle, 1983) and McDowell’s ( 1986, 1987) morphological comparisons suggested the artificiality of the Aparallactinae in its original formulation (Bourgeois, 1968; McDowell, 1968). The controversies concerning the relationships of ‘aparallactines’ and Atractaspis are not directly addressed in this paper because of the continuing absence of a broad array of comparative material of aparallactines for biochemical analysis. Inferences drawn here must be viewed in the light of this limitation. The available immunological data are relevant primarify to three points concerning this group, as follows: (1) In all comparisons involving a broad spectrum of antisera to advanced snake albumins, Atractaspis shows no significant association with other aparallactines (sensu lato; McDowell, 1968) or with any other lineages (Tables 2 and 6; Figs 1 and 2; see also Cadle, 1983, 1988). (2) Aparallactus is, at best, very distantly related to either Amblyodipsas or Atractaspis (Table 6; Cadle, 1983; see also McDowell, 1986). The immunological data currently available place Aparallactus at the common colubrid/elapid node (Figs 1 and 2). However, it should be stressed that Aparallactus needs to be thoroughly re-evaluated with respect to the numerous lineages discovered here which stem from the earliest diversification of the colubrid/elapid clade (Figs 1 and 2), and in view of McDowell’s (1987) hypothesized relationship between I22 J. E. CADLE

r- 1 ABLE 6. Albumin immunological distances among ‘aparallactines’, using antisera to Atractaspis bibronii and Amblyodipsas polylepis. Distances given are the raw data values multiplied by the respective antiserum correction factors given in Table 2. Some of these data were reported and discussed by Cadle (1983)

ANTISERA TO ALBUMINS

Atractaspis Ambbodipsas bibroni poblepis

~ Aparallactus capensis 104 87 Aparallactus lunulatus 105 86 Amblyodipsas poblepis 93 0 Ambbodipsas unicolor 83 17 hlacrelaps microlepidotus 67 41

Aparallactus and a diverse assemblage of other African and Madagascan genera. Given the degree of resolution possible at this level with the immunological data, placement at the common colubrid/elapid node is consistent with McDoweli’s ( 1986) conclusions concerning the relationship of Aparallactus to Ambiyodipsas and Atractaspis, but, in contrast to McDowell, the immunological data do not ally Aparallactus with any of the other colubrids examined, including (by inference) Macrelaps, which the MC’F data ally with Amblyodipsas (see following). (3) In contrast to McDowell’s conclusions concerning the relationships of Amblyodipsas and Macrelaps, these two genera do appear to be relatively closely related-in fact, this is one of the strongest associations among all the African genera surveyed (Table 4 and Fig. 2). In this respect the immunological data agree with Bogert’s ( 1940 : 10) conclusion that Amblyodipsas and Macrelaps are closely related. In summary, the major point of similarity between the hypotheses developed here and those of McDowell (1986, 1987) is the great antiquity hypothesized for the divergences between some aparallactines and other colubrid genera. Our hypotheses differ with respect to the relationships postulated for Atractaspis vis-a-vis the Elapidae and Amblyodipsas, the Amblyodipsas-Macrelaps association relative to other caenophidians and, most significantly, the relationship of viperids to elapids and colubrid lineages (see Cadle, 1988). Clearly, the unresolved basal node of the colubrid/elapid clade (Fig. 2) may eventually be resolved in a manner consistent with McDowell’s (1986, 1987) hypotheses concerning Atractaspis and ‘aparallactines’; however, I remain sceptical until a well-resolved series of clades is presented with their defining synapomorphies-a result not yet realized. Of the main points of disagreement, that concerning the relationship of viperids to other caenophidians appears to be the most trenchant, and additional evidence relative to this point should be avidly sought. The latest contribution to the controversy concerning the relationships of Atractaspis and ‘aparallactines’ is that of Underwood & Kochva ( 1993). They provided much new information on the morphology of these snakes, but some aspects of their analysis are troubling with respect to the present immunological data and McDowell’s recent interpretations concerning ‘aparallactines’. In particular, Underwood & Kochva (1993) accepted the phylogenetic unity of the ‘Aparallactinae’ (sensu Bourgeois, 1968, and McDowell, 1968), despite both PHYLOGENY OF AFRICAN COLUBRIDS 123 immunological data (Cadle, 1983, and herein) and morphological data (McDowell, 1986) suggesting otherwise. They then analysed the relationships of Atractaspis to this group, concluding that Atractaspis stems from within the ‘Aparallactinae’, and that Macrelaps is the sister taxon to all other genera of the group (sensu lato). These hypotheses conflict with those developed herein, which show Atractaspis as an early-diverging lineage of the advanced snakes, and a strong association and much later divergence between Macrelaps and Ambly odipsas. Given the basal divergences within caenophidians here postulated for Atractaspis and some ‘aparallactine’ genera (Aparallactus, Amblyodipsas-Macrelaps) , resolving these relationships will be a complex undertaking with any character systems, since the problem encompasses most extant caenophidian lineages. That is, the problem concerns the relative branching order of basal clades within advanced snakes (see the following section ‘Problems in Colubrid Phylogeny’). Future analyses of the relationships of Atractaspis and ‘aparallactines’ should confront this complexity by developing comparative data for all of these basal lineages without a priori and undue restriction of the OTUs for analysis, or assumptions about their monophyly. As the monophyly of Aparallactinae has been questioned, this assemblage in particular should be subjected to phylogenetic re-analysis.

Problems in colubrid phylogeny The issues raised through continuing controversy over the relationships of ‘aparallactines’ to other advanced snakes encapsulate some of the problems faced by each of the many studies that has attempted to examine broad patterns of colubrid phylogeny. Part of the problem is surely one of scope, as one is talking about an enormously diverse assemblage. But equally important is conscientious attention to underlying assumptions and the effects of sampling when designing a study to examine broad patterns of colubrid phylogeny. These are rarely discussed in the literature and, not surprisingly, many studies have arrived at conflicting conclusions (e.g. compare Underwood, 1967; Dowling & Duellman, 1978; Dowling et al., 1983; McDowell, 1986, 1987). Certainly, immunological data are fraught with problems of their own (Farris, 1987, and references therein; Felsenstein, 1986, and references therein), but they have shown consistent strength in elucidating broad patterns of phylogeny for many groups (see Sarich & Cronin, 1976; Cadle, 1988, and references therein). Results presented in this paper show that resolving the relationships among major lineages of African colubrids will be a very complex undertaking. The reciprocal and unidirectional albumin comparisons (Fig. 1) show a lack of support for the monophyly of colubrids relative to Atractaspis and elapids, snakes usually placed in other families. This remains one of the major outstanding problems of colubrid evolution (Cadle, 1987, 1988). This perceived lack of support for colubrid monophyly from the immunological data should not be construed as conclusive on the non-monophyly of Colubridae-as noted above, the data simply are not sufficient to resolve the branching order of lineages at this level. But this in itself may reflect a pattern of significance for interpreting aspects of the evolutionary history of this radiation. The phylogenetic hypotheses developed here suggest a very rapid and early diversification of the colubrid/ 124 J. E. CADLE elapidlAtractaspis clade into many lineages, none of which shared a lengthy common ancestry relative to the age of the lineages. Thus, any derived features which might have been shared between two or more of the basal lineages in Fig. 1 would have had ample time for subsequent evolutionary modification. Hence, interpreting character homologies among advanced snakes has proven difficult (McDowell, 1986, 1987). A pessimistic view of the inferences to be drawn from Figs 1 and 2 would suggest that very little has been solved. That is, is a tree with seven (or more; Table 4) lineages stemming from a node telling us very much about the evolutionary history of this group? Clearly, if the answer is evaluated only in terms of whether we have produced a tree with a rigously hierarchical series of dichotomous branches, then the answer is ‘No’. But this result in itself may tell us something important about the pattern of diversification of this group, and about the means we must use to recover more about the details of that diversification. It may tell us one reason (though by no means the only one) why there have been conflicting hypotheses for the relationships among some of the major lineages of advanced snakes, and why even associating many genera into higher monophyletic groups has proven problematic. As indicated by the short internal branches and long terminal branches in Fig. 1, many extant African colubrid genera have been separate lineages for much of the history of advanced snakes. In other words, the time period over which these genera shared a common ancestor was short relative to the age of the lineages, resulting in few shared molecular changes between genera over those periods of common ancestry, and still fewer of those retained to the present. Similar problems have arisen in the examination of mammalian relationships, in which immunological (Sarich, 1985) and genomic sequence data sets (Baker et al., 1991; Ammerman & Hillis, 1992) have failed to provide a definitive series of dichotomous branches for many interordinal divergences. Comparable problems have also plagued attempts to resolve mammalian relationships at this level using morphological characters (Novacek, 1992), and may yet be seen when similar comprehensive character sets are available for advanced snakes. Extensive character sets and/or substantial work at the sequence level may be required to sort these lineages into a series of well-corroborated dichotomous branches. In the meantime we should accept less resolved phylogenies for such groups to reflect this reality.

DISCUSSION Other immunological data bearing on higher relationships among colubrids Although there have been other immunological studies of colubrids, few have included a significant representation of the taxa which were the focus of this study, i.e. the African Colubridae. Previous molecular work was reviewed by Dessauer et al. (1987) and will not be repeated here. One other recent study has, however, attempted to resolve some relationships among major lineages of colubrids using the immunological approach (Dowling et a!., 1983). My conclusions differ somewhat from those of the Dowling et al. study and, since that study also used MC’F comparisons of albumins to infer phylogeny, it is worthwhile to examine why our conclusions differ. Dowling et al. (1983) used antisera to several colubrines (Elaphe, Lampropeltis PHYLOGENY OF AFRICAN COLUBRIDS 125 and Masticophis), a natricine ( Thamnophis), a North American ‘xenodontine’ (Diadophis) and a Madagascan colubrid (Muduguscarophis, referred to the ‘Lycodontinae’), to infer relationships. Curiously, they presented two phylogenetic trees which differ in the branching order of major clades, and discussed a third with yet another branching order. These three trees are (using major clades only): (1, Dowling et al., 1983: Fig. 1): (Muduguscarophis ((natricines, ‘xenodontines’) colubrines)); (2, Dowling et al., 1983: Fig. 2): (Madugascarophis ((natricines, colubrines) ‘xenodontines’)); and (3, the ‘Swofford’ tree, Dowling et ul., 1983 : 3 14): ( (natricines, ‘xenodontines’) (Mudagascurophis, colubrines)). Trees 1 and 3 had equally good fit to their immunological distance matrix, but Dowling et ul. presented no tree statistics for Tree 2, even though this seemed to be their preferred tree. My analysis of their data using the FITCH algorithm of PHYLIP showed that Trees 1 and 3 had equivalent standard deviations (SD = 8.9%). (Using the FITCH algorithm these two trees are equivalent, as the basal node is forced to be a trichotomy. This topology results from global optimization of trees produced from the Dowling et al. reciprocal matrix.) Tree 2 had a SD = 9.1% when evaluated by FITCH and negative branches were disallowed, but the branch connecting Diadophis (‘xenodontine’) to the colubrine-natricine clade had length = 0. When Tree 2 is evaluated with negative branches permitted, the Diudophis-(colubrine- natricine) branch has a large negative value (-9), and the SD of the tree is 8.5%. Since Tree 2 has a higher SD than the other trees, or a large negative branch, it is not clear why Dowling et al. (1983:322) appear to prefer this topology in their summary. With respect to the data presented by Dowling et al. (1983)’ topologies for Trees 1 and 3 fit their data matrix better and do not introduce negative branches, and are to be preferred on these grounds. Note that the differences between the three trees presented by Dowling et al. ( 1983) are not minor rearrangements of non-significant branches. For example, the natricinelxenodontine association of Tree 1 is by a common lineage of length 9 in Dowling et al. (1983: Fig. 1) (this branch has a length of 5 in the FITCH trees produced by PHYLIP) . Although some evidence has been presented herein for the association of natricines and colubrines (consistent with Dowling et al., 1983; Tree 2), there is no corroboration from this study for the highly resolved phylogeny of major colubrid lineages presented by Dowling et al. (no matter which of their three trees is examined), and it is unclear from their study as to how we are to interpret the substantive differences between their three trees. Why, then, do we arrive at different conclusions despite very similar technical approaches? I believe our differences relate to two major concerns, both having much to do with underlying assumptions of our respective analyses, and both important concerns for any phylogenetic study using the immunological approach: (1) Assumption about regularity of rates of albumin evolution. This assumption is implicit in the Dowling et al. (1983) study in their failure to include an outgroup for evaluating rates of albumin change among ingroup OTUs (all of the OTUs in the Dowling et al. study are ingroup OTUs, unless one wishes to make additional assumptions about the pattern of relationships, as in Fig. 2 of Dowling et al., 1983). Hence, rates of albumin change in various lineages can be neither evaluated nor accounted for in constructing phylogenetic hypotheses from the Dowling et al. data. 126 J. E. CADLE The critical importance of rate test information as a prerequisite for drawing phylogenetic conclusions was evident from the earliest modern studies of phylogeny from immunological data (Sarich & Wilson, 1967; Sarich, 1969, 1973; reviewed by Cadle, 1988). In essence, as with character data, without rate tests or outgroups it becomes impossible to attach any special significance (phylogenetic or otherwise) to any measure of immunological difference between two taxa (are two very similar taxa similar because they are related or because their protein sequence differences, as measured immunologically, evolved slowly?). Rate tests and outgroups help disentangle us from the often-cited criticism (e.g. Kluge, 1983) that synapomorphies and symplesiomorphies are inseparable with distance data. Rate tests allow us to apportion those data in a phylogenetically meaningful fashion and should accompany any phylogenetic study based on molecular distances. (2) Assumptions about the monophyly of basic taxa (OTUs) for analysis. My introductory comments outlined some of the reasons for making as few assumptions as possible concerning basic taxa in phylogenetic analyses of colubrids, and it is clear from the results of this and previous studies (Cadle, 1984a-c, 1988), that that concern has some justification. Dowling et al. (1983) made, in my opinion, unwarranted assumptions about the monophyly of basic taxa in developing their phylogenetic hypotheses. At the most general level, this includes even the family Colubridae, for which corroborative evidence of monophyly, as just discussed, is lacking (which is why outgroups and rate tests are crucial in analysing relationships among ‘colubrids’). Within colubrids, Dowling et ill. (1983) used two other large groups, ‘lycodontines’ and ‘xenodontines’, as basic taxa (though represented by a single antiserum each). This study has shown for ‘lycodontines’, as previous immunological studies showed for ‘xenodontines’ (Cadle, 1984a-c, 1988), no conclusive evidence of monophyly for these groups, and their use in the traditional broader senses should be discontinued for phylogenetic analyses (see Cadle, 1987). (These inferences have substantive nomenclatural implications, which are discussed below.) Rather, constituent clades within these assemblages for which evidence suggests monophyly should be used for phylogenetic analyses at higher levels. For ‘xenodontines’ these clades comprise the large ‘Central American’ and ‘South American’ clades (sensu Cadle, 1984a-c, 1985), and several other smaller clades. For ‘lycodontines’ the results of this study show that many additional phyletic lines will have to be treated separately for phylogenetic analyses until their interrelationships are more fully understood. (Although Dowling et al. [ 1983 : 3261 recognized the phyletic heterogeneity of ‘xenodontines’ and ‘lycodontines’, rather than take full account of the implications of that heterogeneity for their taxonomic conclusions or phylogenetic analyses, their persistence in “. . . adjust[ing] the current classification . . .” [Dowling et al., 1983 : 3261, while still recognizing large assemblages of these taxa, amounts to ignoring their own data concerning the complexity of resolving relationships at this level.) .4 result of making assumptions concerning rates of evolution and the monophyly of basic OTUs is that Dowling et al. (1983) have overstated the confidence with which major lineages are resolved within colubrids using the immunological approach (compare Figs 1 and 2 of this study with Dowling et al., 1983: Figs 1 and 2). As shown herein, once rates of albumin evolution are PHYLOGENY OF AFRICAN COLUBRIDS 127 properly assessed using outgroups, there is no support for a strongly hierarchical arrangement, nor, for that matter, for most of the clades in the three alternative trees presented by Dowling et al. (1983) (the natricine/colubrine association is the one clade also supported by this study, but without rate tests that association in one of the trees given by Dowling et al. is considered spurious, and it is not supported by the trees of lowest SD from their data matrix). Of the two assumptions, the failure to include definitive outgroups and to perform rate tests are the major analytical problems with the Dowling et al. (1983) study. A major insight of the earliest modern immunological approaches to phylogenetics (Sarich & Wilson, 1967) was that outgroups are as crucial in this endeavour as they are for cladistic analyses of character data; all systematic studies should incorporate this practice as a matter of course.

Bogert’s arrangement of African colubrids in retrospect Bogert (1940) did not intend his system to be a purely phylogenetic one, recognizing the artificiality of separating the genera of Groups 7 and 17, for example. In fact, it seems to have been a largely intuitive system, for Bogert did not explicitly state the characters upon which most of the groups were based. However, he clearly intended them to represent assemblages of related genera within a larger, somewhat arbitrary, framework imposed by the nature of the sulcus spermaticus and presencelabsence of hypapophyses (this higher framework is omitted from Table 1). Of the groups recognized by Bogert, most have gained some acceptance as natural groups, and some have been corroborated by further study using additional characters. The colubrines (Groups 8-15, 18 of Bogert’s system) are a strongly-supported clade from the standpoint of immunological data (Dessauer et al., 1987). They, and Bogert’s Group 3 (Geodipsas), will be considered in future works. Here, I briefly summarize the taxonomic status of the remaining groups, and summarize the support for these groups from the standpoint of the immunological data. Except for Natricinae, which has had a long and reasonably stable usage, names of formal taxa have been homogenized as ‘tribes’ (-ini ending) regardless of original usage (usually at subfamilial level). Groups 1 (Boaedontini; Dowling, 1967), 4 (Natricinae; Malnate, 1960; Rossman & Eberle, 1977; E. V. Malnate, personal communication), 7 + 17 (Aparallactini; Bourgeois, 1968; McDowell, 1968; but see McDowell, 1986), and 16 (Psammophiini; Bourgeois, 1968; Broadley, 1983) have each been characterized by further study, with some characters of scalation, dentition, hemipenes and/or osteology interpretable as derived features for each. To the extent that material has been available for study, the Boaedontini, Psammophiini and Natricinae are supported by the immunological data presented herein and elsewhere (reviewed by Dessauer et al., 1987). With the exception of the Natricinae, however, samples for biochemical analysis have not been adequate to fully evaluate Bogert’s system, and many genera remain untested (Table 1). For the Aparallactini, the immunological data do not support a close relationship between Aparallactus and Amblyodipsas, the only member of that group to which an antiserum has been produced, but the existence of a smaller clade comprising some subset of ‘aparallactines’ (including at least AmbEyodipsas and Macrelaps, as documented herein) needs to be evaluated. 128 J. E. CADLE With regard to this group, it should be noted that the unresolved nature of the basal colubrid radiation (Figs 1 and 2) allows the possibility of a very ancient diversification of ‘aparallactines’ in the broader sense, but documentation of their monophyly from the standpoint of either molecular or morphological features has not been forthcoming (see discussion in McDowell, 1986). The remaining groups of Bogert’s system (2, 5, 6, 11 and 12) do not receive support from the current immunological data. Group 2 (Hormonotus, Gonionotophis, Mehelya, Lycophidion, Chamaelycus) has been recognized formally (Lycophidini of Bourgeois [ 19681; Dowling & Duellman [ 1978]), and the included genera share a number of morphological features (form of the maxilla, hypapophyses on posterior vertebrae, venom gland structure; Bourgeois, 1968; McDowell, 1986). Clearly, the immunological results conflict with these observations if the morphological similarities are interpreted to reflect phylogenetic affinity. More efforts should be directed toward putting the morphological observations in a more secure phylogenetic context, as well as verifying and extending the molecular data on this group (see Results). Dowling & Duellman (1978) united the genera of Bogert’s Groups 5 (Duberria, Grayia, Pseudaspis), 6 (Amplorhinus), 11 (Scaphiophis) and 12 (Prosymna) as a tribe (Pseudaspini). Bourgeois (1968) found no support from skull morphology for Group 5, in agreement with the MC’F results reported here, and she considered the relationships of all of these genera ambiguous (she did not discuss Amplorhinus). Broadley ( 1983) concurred, concluding that Scaphiophis is a colubrine, and pointing out that Amplorhinus and Prosymna are not closely related to the genera of Group 5. These hypotheses are confirmed for Amplorhinus and Prosymna in this study, with the immunological results also placing Prosymna within the colubrine clade. As no synapomorphies for the Pseudaspini appear to be available, this taxon should neither be formally recognized nor used as an OTU for phylogenetic analyses (see comments on nomenclature below).

The-fossil record of snakes in Africa and the age of its colubrid lineages The spotty temporal and geographic distribution of Tertiary fossil-bearing localities for terrestrial vertebrates in Africa is well known (e.g. Cooke, 1972, 1978; Bernor, 1983), resulting in a generally poor understanding of faunal evolution during this period of African history. The mammalian record suggests a rapid turnover in the composition of faunas from early to late Miocene (Cooke, 1978; Winkler, 1992), and comparisons among contemporaneous faunas indicate exchanges with neighbouring peri-Tethyan land masses during various periods (Bernor, 1983; Bernor et al., 1987; Rage, 1988a; Gheerbrant, 1990). For most groups, however, the available African deposits present an extremely biased record of actual diversity through most of the Tertiary, even for groups that usually fossilize well (see, for example, Meylan & Auffenberg, 1986, on African tortoises; and Simons, 1992, on primates). The fossil record of advanced snakes in Africa is much more sparse than for terrestrial mammals, and inferences about their history must rely heavily on present distributions and phylogenetic hypotheses. Colubrids, poorly preserved and of unknown relationships, appear in Lower Miocene deposits of Uganda (Rage, 1979), with an age estimated from radiometric dating of 17-20 Myr (Bishop et al., 1969; Cooke, 1978). A long interval separates these from the next PHYLOGENY OF AFRICAN COLUBRIDS I29 colubrid fossil (a single vertebra) in Pliocene deposits at Laetoli, Tanzania (Meylan, 1987), with an age of 3.5-4 Myr. Although tentatively assigned to the extant genus Rhamphiophis, this specimen is not diagnosable from a variety of terrestrial colubrid genera of diverse lineages. Colubrids of at least four genera (tentatively Lamprophis, Coluber, cf. Grayia and Dagpe2ti.r) are known from Pleistocene deposits ( < 2 Myr old) at Olduvai, Tanzania (Rage, 1973). Several Pliocene and Pleistocene sites in east Africa have yielded a few fossils belonging to extant genera or species of pythons, cobras and vipers (Rage, 1979; Meylan, 1987). Outside Africa, only two colubrid genera with clear African affinities are known from fossils. Mulpolon, known from the Pliocene of Spain (Szyndlar, 1988, 1991a), is extant in Europe and north Africa but belongs to the primarily African psammophiine lineage of snakes (Table 5 and Fig. 3). Telescopus, a colubrine with several extant species in Africa and Europe, is known from Pleistocene deposits of Bulgaria (Szyndlar, 1991a) within the present range of the European species. A fossil cobra (Elapidae) from the Upper Miocene of Spain is related to an extant African lineage (Szyndlar & Rage, 1990). Clearly, we must look to modes of inference other than fossils to piece together some of the broad outlines of colubrid evolution in Africa-though the fossils must guide us as to minimum ages for particular points in that history. This study has shown that many of the extant clades of African colubrids (as well as elapids and Atractaspis) stem from an explosive diversification early in the history of the group, and the question to be addressed is ‘How early did this diversification occur?’. As the Lower Miocene African colubrid fossils (and, indeed, all of the African pre-Pleistocene fossils) cannot be assigned with confidence to any extant lineage, we consider the fossil record of colubrids elsewhere as it potentially bears on the African radiations. This leaves us with only the European and North American records, as no other areas provide the requisite documentation (Cadle, 1987), and for these records we assume that potentially relevant fossils have been correctly placed as to lineage. The earliest colubrid (of indeterminate systematic relationships) comes from late Eocene deposits of Thailand (Rage et al., 1992). By the Lower Oligocene in Europe (age approximately 33 Myr; Rage, 1988b), two colubrid lineages, natricines and colubrines, are present (Rage, 1974, 1988b), a fact significant for interpreting the age of the African colubrid radiation. These fossils allow us to infer that the basal colubrid/elapid radiation (Figs 1 and 2) had occurred at the latest by about 33 Myr ago. If colubrines and natricines share a significant period of ancestry subsequent to their separation from the basal colubrid/elapid node, as suggested by some data in this paper (Fig. 2), then the age of the basal node would be older than suggested here. Szyndlar ( 1991 b) arrived at similar conclusions based on consideration of the fossil record of colubroid diversity in Europe and North America. Thus, the radiation of colubrids in Africa is, minimally, older by a factor of 1.5-2 than is indicated by the African fossil record of the group. A similar finding for the temporal dimensions of the South American colubrid radiation (2.5-3 times older than the oldest South American colubrid fossils; Cadle, 1984c) suggests that we should not be surprised if future fossil discoveries require us considerably to revise these age estimates upward. Furthermore, the phylogeny developed herein emphasizes not only a potentially strong temporal bias of the fossil record as we know it on southern continents, but also strong taxic and 130 J. E. CADLE geographic biases as well. A majority of colubrid fossils belong to the two major extant Holarctic clades, colubrines and natricines, and essentially all of those fossils are from Europe and North America (Rage, 1987; Cadle, 1987). If colubrids had very early in their history diversified into as many major clades as implied by results of this study (Figs 1 and 2), then the diversity of extant clades entirely missing from the fossil record is sobering as regards interpreting details of colubrid evolution from fossils. (Significantly, Figs 1 and 2 include only African lineages, so they undoubtedly underestimate the true diversity of early colubrid clades; several New World clades are probably at least as old [see Cadle, 1984c, 1985, 19881.) Inferences derived primarily from the paleontological record about broad patterns of phylogeny or rates of diversification in snakes (e.g. Stanley, 1979) risk missing major components of lineage diversity and temporal scale.

Biogeographic considerations A striking aspect of the contemporary African colubrid fauna is its high endemicity, and the fossil record of surrounding areas (mostly European: Rage, 1974; Szyndlar, 1991a, 1991b) gives few indications of past exchanges with Africa. With reference to the phylogenetic hypothesis developed herein (Fig. 2), only the Elapidae and two colubrid lineages, colubrines and natricines, are known to have a substantial representation outside Africa (natricine diversity in Africa is low compared to other areas). Colubrines and natricines are each diverse throughout the distribution of the Colubridae, except for Australia, where both clades are depauperate, and the neotropics, where natricines are poorly represented (see Cadle, 1987). At the generic level, virtually all of the approximately 55 colubrid genera of Africa are endemic (see details following). Similarly, genera of the other caenophidian lineages in Africa (Elapidae, Viperidae, Atractaspis) are largely endemic, and only the elapid genus Naja is substantially represented outside Africa. Its distribution is similar to that of Psammophis, but fossil species of Naja are relatively common in Miocene- Pleistocene deposits of Europe (Szyndlar & Rage, 1990; Szyndlar & Zerova, 1990). Even within Naja, however, phylogenetic studies indicate that all of the European fossils, except those of the Iberian Peninsula, belong to a putatively monophyletic complex comprising the contemporary Asiatic species, whereas the contemporary African species are more primitive, perhaps paraphyletic, and largely endemic to Africa (Szyndlar & Rage, 1990; Szyndlar & Zerova, 1990). High endemicity, then, characterizes all contemporary African caenophidian clades, just as it does present neotropical clades (see Cadle, 1984c, 1987). Having made the case for endemicity, however, I close with a strong caveat-no extensive comparisons (either the biochemical ones here reported, or previous morphological studies) have been made between African snakes and those of Asia, which remain largely unknown in terms of lineage composition. In addition, considerably more work needs to be directed toward comparisons between African and neotropical lineages. Future phylogenetic studies will specifically focus on these issues. The highly endemic nature of all extant African caenophidian lineages, and the antiquity of most of the lineages hypothesized here, suggests substantial isolation of this fauna from other land masses through much of its history. Similar patterns are seen in the present-day mammal fauna of Africa (Maglio & Cooke, 1978; Luckett & Hartenberger, 1985). Yet, increasingly, improved PHYLOGENY OF AFRICAN COLUBRIDS 131 understanding of critical fossil deposits in Africa and elsewhere strongly suggest contributions from Africa to the faunas of peri-Tethyan regions during several periods of the Tertiary (Bernor, 1983; Bernor et al., 1987; Rage, 1988a; Gheerbrant, 1990; Storch & Schaarschmidt, 1992). The earliest exchanges possibly occurred by the Cretaceous-Tertiary boundary and the Paleocene- Eocene boundary (Gheerbrant, 1990; Storch & Scharrschmidt, 1992); later significant exchanges occurred by the early Miocene (Bernor et al., 1987). The possibility of exchanges between Africa and the Neotropics during the early Cenozoic (most likely pre-Oligocene, if at all) are more controversial, but have been invoked to explain the deployment of neotropical primates, African marsupials and some rodents of both areas (see papers in Ciochon & Chiarelli, 1980; Rage, 1988a). Moreover, the presence of ‘South American’ groups (anteaters, ziphodont mesosuchian crocodiles) in early Eocene deposits at Messel, Germany, possibly reflects African/Neotropical exchanges prior to implacement of these groups in Europe via trans-Tethyan migration (Storch & Schaarschmidt, 1992). Although critical fossil discoveries have played major roles in interpreting the relationships between African and other areas at discrete times in the past, phylogenetic studies are crucial for certain groups lacking informative fossils (see, for example, Ciochon & Chiarelli, 1980, on primates). It seems unlikely that the fossil record for colubrids in either African or surrounding areas will significantly augment our understanding of their historical biogeography in the near future. Hence, it is worthwhile to explore the implications of the present molecular studies insofar as they may direct attention toward fruitful areas of further research. Contemporary African colubrid genera presently found in extra-African regions are: (1) three present in Europe and the Middle East (Coluber, Telescopus and Malpolon); (2) two present throughout southern Asia and/or Australasia (Psammophis, Boiga); and (3) one (Geodipsas) shared with Madagascar. These represent three of the basal colubrid lineages (Figs 1 and 2), including colubrines (Coluber, Telescopus, Boiga) and psammophiines (Psammophis, Malpolon) . (The status of African species of Geodipsas has received careful phylogenetic evaluation with respect neither to the Madagascan species of Geodipsas nor to major clades identified during this study (Fig. 2) and, therefore, is considered no further in this discussion. Its relationships, along with those of Mimophis, another Madagascan endemic possibly belonging to the psammophiine clade [see Mocquard, 1895; Domergue, 1962; McDowell, 19871, will be considered in a future comprehensive study of the Madagascan colubrids.) Although all African natricine genera are endemic to Africa, their precise relationships to other natricine clades is at present unclear (E. V. Malnate, personal communication; see also Dessauer et al., 1987), and no information is available concerning molecular divergence among them or their relationships to one another. Thus, this commentary focuses on the psammophiines and colubrines. Although the rate tests (see Results) do not preclude use of the MC’F data presented here in a molecular clock context, the lack of specific calibration points means that only general indications of temporal range are presently possible (see Cadle, 1988, for discussion). More specifically, this discussion focuses on the minimum estimated age for separation among basal colubrid clades, 33 Myr (Fig. 2). For the clades here considered (psammophiines and colubrines), albumin divergence among the psammophiine genera is the greatest (Tables 2, 4 132 J. E. CADLE and 5), suggesting that the oldest divergences occur among these. Indeed, the only Asian species of Psammophis examined (P. condanarus) stems from the base of the Psammophzs lineage (Fig. 3), implying very early speciation events giving rise to extant members of this diverse and widespread assemblage. An estimate for the minimum age of the extant psammophiine genera would be 15-20 Myr, based on the estimated 33 Myr divergence among the basal colubrid lineages and the AIDs among genera at the common mode of the extant psammophiine genera (about 45, somewhat more than half the mean AIDs among lineages stemming from the basal polychotomy of Fig. 2). By the same reasoning, most of the colubrine genera are much more recent than this, but more detailed information on the phylogenetic structure of this worldwide lineage will be required before their ages can be estimated. The antiquity of the colubrid radiation in Africa hypothesized here (see also Cadle, 1984c, 1988, concerning similar age estimates for the neotropical radiation), requires consideration of the early Tertiary relationship of Africa to neighbouring areas in developing hypotheses for the biogeographic deployment of these snakes. In particular, if the radiation of extant members of clades such as the psammophiines began minimally by early Miocene, then early Tertiary trans- Tethyan exchanges with neighbouring areas, as indicated by some mammalian fossils (Bernor, 1983; Bernor et al., 1987), become more likely possibilities for these clades. Furthermore, investigation of relationships between African clades (basal lineages of Fig. 2) and those of the neotropics should be a focus of future phylogenetic studies of the relatively primitive clades found in both areas (see McDowell, 1987). Given the number of lineages involved in the basal colubrid radiation (Figs 1 and 2), resolving some aspects of their early biogeographic history may prove quite complex. Nevertheless, now that some of the structure of the African radiation is apparent (Figs 1-3), studies of the phylogenetic relationships of African colubrids to those of other areas should be facilitated, as we now have an estimate of how many independent lineages are potentially involved. Obviously, the next steps in this analysis should focus on detailed testing of possible relationships between these African lineages and those of the neotropics (whose phylogenetic structure is relatively well known; Cadle, 1984.a-c; see also McDowell, 1987), and those of Asia (whose phylogenetic structure is essentially unknown at present).

A comparison of African and Neotropical colubrid radiations The final evolutionary pattern to be considered concerns the differing phylogenetic structures of the radiation of colubrids in Africa as compared to the Neotropics. A major inference from phylogenetic studies of Neotropical colubrids is that most extant species (>go% of the genera) belong to one of three monophyletic clades (Cadle, 1984a-c, 1985) whose divergence had occurred by about 30 Myr ago (Cadle, 1988). One of these clades, the colubrines, occurs worldwide (Cadle, 1987) and includes many African representatives, as shown herein (Figs 1 and 2); its radiation is inferred to have been more recent than the divergence among other much more geographically restricted clades (for example, all of the colubrid clades resulting from the basal polychotomy in Fig. 2). The other two Neotropical lineages (‘xenodontines’) are endemic to the New World ((Cadle, 1985). PHYLOGENY OF AFRICAN COLUBRIDS 133 The phylogenetic hypothesis developed herein for African colubrids suggests a pattern of radiation for these snakes that contrasts sharply with that for the Neotropical clades. Aside from the worldwide colubrine lineage, no other basal clade (Figs 1 and 2) comprises a substantial portion of the extant genera or species of African colubrids. Instead, the hypothesis suggested herein (Fig. 2) implies an early ( > 30 Myr ago) and rapid caenophidian diversification, giving rise to elapids, Atractaspis and many colubrid lineages. No extant African colubrid lineage has a present diversity equal to that seen in either of the endemic Neotropical clades (collectively comprising > 500 species), even though many African and Neotropical lineages are possibly about the same age (see above; Cadle, 1988). Why should the African and Neotropical colubrid radiations show such contrasting patterns? Resurgence in interest in the origins of such macro- evolutionary patterns has focused on ecological and evolutionary factors that promote differences in clade diversification (Vrba, 1980, 1984, 1987). Clearly, study of historical differences between the colubrid radiations of Africa and the Neotropics might provide some clues. Superficially, at least, the phylogenetic hypothesis for African colubrids (Figs 1 and 2) suggests that, in comparison to the Neotropical radiation, the African lineages stemming from the basal diversi- fication of the colubrid/elapid clade either have undergone little speciation throughout their history or else their high speciation rates have been matched by rather high extinction rates. Although these alternatives cannot be evaluated without greatly improved knowledge of past clade diversity through additional fossil discoveries, several neotropical ‘xenodontine’ clades have very high species diversities despite relatively recent origins, as indicated by molecular data (Cadle, 1984a-c; Dessauer et al., 1987). This suggests high speciation rates for these lineages. Examples are several clades (Geophis, Sibon-Dipsas, Liophis), each with > 35 species and relatively recent origins within their respective ‘xenodontine’ clades (Cadle, 1984a, b). In contrast, the extent of albumin divergence within Psammophis, the African colubrid genus with highest species diversity (about 17 species) and, with the exception of P. phillipsii, no indication of extremely divergent albumins within the genus (Tables 1 and 5; Fig. 3), suggests an age two to three times that of the neotropical clades. Consequently, speciation rates within Psammophis are potentially much lower, or long-term extinction rates are much higher, than in the neotropical clades. Examination of the behavioural and ecological characteristics of snakes with such contrasting speciational modes, then, might help explain differences in the macro- evolutionary pattern of radiation of these lineages (see, for example, Vrba, 1984, 1987). Cadle & Greene (1993) showed that, even within the Neotropics, two major clades of ‘xenodontines’ had substantive differences in ecology and feeding behaviour as a result of contrasting patterns of body size evolution within each lineage. Such inter-lineage evolutionary comparisons may help us understand the evolutionary forces operating to promote differential speciational and macro- evolutionary diversification patterns.

A note on nomenclature of higher taxa within colubrids Finally, what implications does the phylogenetic hypothesis developed here (Figs 1 and 2) have for the nomenclature of higher taxa within colubrids? A 134 J. E. CADLE significant aspect of the phylogenetic hypothesis (Figs 1 and 2) is the large number of unresolved basal lineages for the colubrid/elapid radiation. As this study has examined only some African members of that radiation in detail, it is useful to pursue, in closing, the implications of the hypothesis for the nomenclature of higher taxa within colubrids. As noted in the Introduction, considerable variation in the application of names for higher categories within Colubridae already exists. Much of that variation is due to emphasis on the formulation of classifications and erection of categories, without equal consideration being given to establishing phylogenetic hypotheses for taxa under study and the clear elucidation of derived characters to provide evidence of group membership. For many classifications, there has been almost total devotion to a few character systems and little attention to exploration of other characters. The result too often has been a system of higher categorical names that communicate little phylogenetic (or other) information, and one is usually faced with the added burden of needing lists of included taxa to attach significance to the names. Perusal of several recent classifications that use many of the same higher categories, but which differ considerably in their content, dramatically illustrates the ensuing confusion (compare, for example, the content of higher categories in Underwood, 1967; Bourgeois, 1968; Dowling, 1975; Dowling & Duellman, 1978; Dowling et al., 1983; Rasmussen, 1985; McDowell, 1987). Clearly, such a system facilitates neither communication nor understanding. The case of the Lycodontinae vis-a-vis the phylogenetic relationships of Lycodon discussed herein (see also McDowell, 1987) is perhaps unusual in the extent of disparity between a taxonomic category and the relationships of its name-forming taxon. But the phylogenetic basis for many named categories within Colubridae is just as poorly understood, and this should cause us to reflect on a general consensus that classifications should convey information about evolutionary history (notwithstanding some disagreements concerning which aspects of evolutionary history they should convey). For the African colubrid radiation, the immunological data suggest that many extant genera stem from very early divergences in the history of the Colubridae, which itself is not clearly monophyletic relative to elapids and Atractaspis; there are few demonstrably monophyletic clades which include a substantial number of the genera surveyed (Fig. 2). Many of these genera (non-colubrine/natricine colubrids) are commonly united as the ‘Lycodontinae’ (e.g. Atractaspis, the psammophiines, Amblyodipsas-Macrelaps, Grayia, Prosymna) or ‘Boodontinae’ (Lamprophis- Lycodonomorphus, Leioheterodon) (see, e.g. Dowling & Duellman, 1978; Dowling et al., 1983; McDowell, 1987). However, no synapomorphies for these enormously diverse ‘taxa’ have been discovered, and the results of immunological (herein) and morphological (McDowell, 1987) studies on Lycodon (see Results) raise questions concerning the utility of continuing formally to recognize such taxa. Should we recognize these categories in the broad sense that they have generally been used? I suggest, given the confusing usage of these terms in the past, that a more fruitful (and informative) approach would be to use a system of names for smaller clusters of genera for which it is possible to provide diagnoses in the form of derived characters, or for which data such as the immunological comparisons presented herein suggest phyletic unity. These would essentially be PHYLOGENY OF AFRICAN COLUBRIDS 135 equivalent to the basal lineages of Fig. 2. Some such categories already exist (e.g. Psammophiini for Bogert’s Group 16, whose morphological basis was established by Bogert and extended through the work of Bourgeois [ 1968]), but most named taxa for African colubrids have not received such treatment. Once the phylogenetic relationships among such clusters are more fully elucidated, higher categories should directly follow to reflect the improved understanding. In the meantime, we may need a system of names for discussing larger assemblages of taxa that are not clearly monophyletic, but with full recognition that these named assemblages cannot constitute basic taxa for phylogenetic analyses. Gauthier el al. (1988 : 15-16) recognized the same problem for lizard taxa, and suggested using quotation marks around known para- or polyphyletic taxa, and the asterisk (*) to denote metataxa, i.e. taxa for which evidence supporting either monophyly or paraphyly is lacking (see also De Queiroz & Gauthier, 1992). Using this system, groups such as the Xenodontinae* are metataxa, whereas Colubrinae and Natricinae, used in their more restricted senses (McDowell, 1987, for Colubrinae; Malnate, 1960, and Rossman & Eberle, 1977, for Natricinae) are monophyletic groups. Note that although Xenodontinae* is a metataxon, there is support from immunological comparisons for the monophyly of two clades within that taxon, Central American Xenodontines and South American Xenodontines (sensu Cadle, 1984a-c, 1985). Dowling et al. [1983] used the formal names ‘Dipsadidae’ and ‘Xenodontinae’, respectively, for rough equivalents of these two clades as recognized by Cadle (1984a-c, 1985). However, they included taxa within each (e.g. Alsophis and Farancia in the Dipsadidae; Diadophis in the Xenodontinae) whose inclusion in those clades is not corroborated by immunological data (Cadle, 1984a, b; see also Dowling & Dueilman, 1978; Dowling, 1975). Because of contradictory immunological evidence, and since Dowling et al. (1983) provided no synapomorphies for these taxa as they conceived them, I consider the composition of Xenodontinae and Dipsadidae as given in that paper suspect. This illustrates precisely the problem outlined at the beginning of this section. Of the two xenodontine clades identified by the immunological data, only the Central American clade is as yet supported by morphological synapomorphies (Cadle, 1984c, and unpublished data). Although the use of the metataxon concept and the denotation of known paraphyletic groups with quotation marks aids in the recognition of such groups when those terms are used, for phylogenetic analyses I advocate using only the largest assemblages for which there is evidence of monophyly. Thus, for example, the Central American and South American clades (sensu Cadle, 1984a-c) of Xenodontinae* could be used in phylogenetic analyses, whereas the Xenodontinae* in toto could not. For the African colubrid radiation, only the following taxa have enough support to be used in phylogenetic analyses of higher taxa: (1) Psammophiini for Bogert’s Group 16 (Table 1; see also Bourgeois, 1968); (2) Boodontini for Bogert’s Group 1 (see also Dowling, 1969); (3) Colubrinae (see McDowell, 1987) for a diverse assemblage of colubrids distributed worldwide, and including Bogert’s Groups 8-15 + 18; (4) Natricinae for Bogert’s Group 4 plus other Eurasian and American taxa (see Malnate, 1960; Rossman & Eberle, 1977); (5) possibly the Aparallactini (Bogert’s Groups 7 + 17; Bourgeois, 1968; but see McDowell, 1986, and the results concerning Macrelaps and Aparallactus discussed herein); and (6) other isolated genera 136 J. E. CADLE showing no association with other lineages, or of ambiguous relationships as suggested herein (e.g. Duberria, Pseudaspis, Lycophidion or any of the basal clades of Fig. 2; note that the inclusive nature of clades represented by these genera could be modified as required by examination of all those genera not included in this study [see Materials and Methods]). Other higher taxa for African colubrids have been proposed (Dowling et al., 1983, and references therein; McDowell, 1987), but there has been no clear delineation of synapomorphies for these (but see discussion of Bogert’s Group 2, including Mehelya and Gonionotophis, above), and none of these receives support from the immunological studies presented herein. I specifically recommend that larger metataxa such as Boodontinae* and many of the tribes included within Boodontinae* and ‘Lycodontinae’ (see Dowling et al., 1983 : 323), not be used in phylogenetic analyses until further study demonstrates that these are clades. Based on phylogenetic studies of Lycodon (Results), use of ‘Lycodontinae’ for a higher taxon within Colubridae should be discontinued. Clearly, immunological data such as those presented herein can provide some insights into evolutionary relationships. Indeed, I believe that they can aid in the recognition of monophyletic groups, such as the Central American and South American xenodontine clades, a usage that would not be consistent with the view that only discrete character data can support clades. However, although approaches such as comparative immunology can be very powerful analytical tools for analysing colubrid phylogeny, they are no panacea for either phylogenetic reconstruction or classification. We need a much richer understanding of morphological, behavioural and biochemical variation of colubrids and other snakes. Full understanding of the evolutionary history of this diverse clade will require the integration and synthesis of different perspectives, with concerted efforts toward understanding evolutionary patterns at all levels, and with properly conceived scepticism when different perspectives disagree.

ACKNOWLEDGEMENTS With pleasure I acknowledge the generosity of colleagues in providing some of the samples used in this study: D. G. Broadley, S. D. Busack, H. C. Dessauer, R. C. Drewes, R. Fisher, H. W. Greene, L. G. Hoevers, A. Lambiris, B. Hughes, S. Reilly, S. Spawls and V. Wilson. During fieldwork in Africa, I was honoured to be the guest of B. Hughes (Legon), S. Spawls (Wa),the family of D. Leona.rd (Nairobi), L. Frank (Mara) and D. G. Broadley (Bulawayo); authorities and countless acquaintances and friends in Ghana, Kenya and Zimbabwe kindly facilitated the fieldwork. The field and laboratory work were supported by grants from the National Science Foundation (DEB 80-14101, BSR 84-00166 and BSR 89-18558). H. C. Dessauer provided advice, laboratory facilities and encouragement during the long gestation of the work, and J. Felsenstein and W. M. Fitch generously supplied copies of their phylogenetics programs. I have been helped immensely in my understanding of African snakes through discussions with B. Hughes, D. G. Broadley, E. V. Malnate and H. W. Greene. For comments on the manuscript I thank D. G. Broadley, D. Cundall, H. C. Dessauer, H. W. Greene, E. V. Malnate and S. B. McDowell. PHYLOGENY OF AFRICAN COLUBRIDS 137

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APPENDIX

Specimens used as sources of antigens and antisera (* = antiserum) in this work. Abbreviations for respositories of specimens are: CAS (California Academy of Sciences, San Francisco); LSUMZ (Louisiana State University Museum of Natural Science, Baton Rouge); MVZ (Museum of Vertebrate Zoology, Berkeley); NMZB (National Museums of Zimbabwe, Bulawayo); UGZD (University of Ghana Zoology Department, Legon j. Afronatrix anoscopus, Liberia: Harbel: Firestone Plantation (LSUMZ 29575); Amblyodipsaspulyfepis, Zimbabwe: Midlands Prov.: Gwelo District, Guelo (MVZ 176471*); Amblyodipsas unicolor, Ghana: Upper West Region, Wa secondary school, Wa (MVZ 176436); Amplorhinus multimaculatus, Zimbabwe: Manicaland Prov.: Inyanga District, Inyanga National Park (NMZB 6583); Aparallactus capensis, Zimbabwe: Matabeleland South Prov.: Umzinguane District, Inyankuni Dam, approx. 15 km NE Balla Balla (MVZ 176472); Aparallactus lunulafus, Somalia: Lower Juba Region: Juba Sugar Project near Mareri (CAS I531 78); Atractaspis bibroni, Locality unknown (MVZ 175868*); Dasylpeltis scabra, Zimbabwe: Matabeleland South Prov.: Beitbridge District, Mazunga River Bridge (NMZB 6013*); Dipsina multimaculata, Namibia: Luderitz Bay (LSUMZ 55389); Dispholidus !@us, Zimbabwe: Manicaland Prov.: Umtali District, Umtali (MVZ 176475*); Duberria lutrix, Zimbabwe: Mashonaland Prov., Atlantica Ecological Research Station, 26 km W Salisbury [Harare] (MVZ 176474); Gonionotophis grantii, Ghana: Upper East Region, Navrongo (MVZ 176439); GrLsyia smythii, Togo: Locality unknown (LSUMZ 44409*); Lamprophisfuliginosus, Locality unknown (MVZ 172384*); Leioheterodon rnadagascariensis, Madagascar: Locality unknown (LSUMZ 30404*); Lycodon laoensis, Thailand: Locality unknown (LSUMZ 37500); Lycodonomorphus rufulus, Zimbabwe: Manicaland Prov.: Umtali District (MVZ 176477); Lycophidion cupense, Zimbabwe: Matabeleland North Prov.: Bulawayo District, Bulawayo (MVZ 176478); Macrelaps microlejidotus, Location unknown (LSUMZ 55387); Malpolon monspessulanus, Spain: Cadiz Prov., 22.6 km SW Facinas on CA-221 (S.D. Busack 1544); Mehelya crossi, Ghana: Eastern Region, University of Ghana campus, Legon (MVZ I76441 *); Natriciteres olivacea, Zimbabwe: Mashonaland South Prov.: Harare District, Salisbury [Harare] (MVZ 176480); Philothamnus angolensis, Zimbabwe: Manicaland Prov.: Melsetter District, Martin Forest Reserve (MVZ 176481 *); Prosymna sundeualli, South Africa: Northern Transvaal, Tshipise (NMZB 6014); Psammophis biseriatus, Kenya: Locality unknown (CAS); Psummophis condanarus, Thailand, Locality unknown (LSUMZ 32727); Psammophis elegans, Ghana: Eastern Region, vicinity of Legon (MVZ 176445); Psammophis elegans, Togo, Locality unknown (LSUMZ 44665); Psammophzs phillipsii, Ghana: Upper West Region, Wa secondary school, Wa (UGZD); Psammophis punctulatus, Kenya, Locality unknown (CAS); PsammophiJ rukwae, Ghana: Upper West Region, Wa secondary school, Wa (MVZ 176447); Psammophis subtaeniatus, Zimbabwe: Matabeleland South Prov.: Gwanda District, Gwanda (MVZ 176482*); Psammuphylax kitaeniatus, Zimbabwe: Matabeleland North Prov.: Bulawayo District (MVZ 176479); Pseudapis cana, Locality unknown (LSUMZ 42678); Rhamphiophis oTrhynchus, Ghana: Upper West Region, Wa secondary school, Wa (MVZ 176504*); Telescopus semiannulatus, Zimbabwe: Manicaland Prov.: Umtali District, Umtali (MVZ 176490*); Thelotornis capensis, Zimbabwe: Matabeleland South Prov.: Gwanda District, Gwanda (MVZ 176491*); Thrasopsjacksoni, Kenya: Kakamega District, Kakamega forest (CAS 152795*).