COMMENTARY AND PERSPECTIVE

Evolution of Viviparity in Squamate Reptiles: Reversibility Reconsidered DANIEL G. BLACKBURN* Department of Biology, Electron Microscopy Center, Trinity College, Hartford, Connecticut

ABSTRACT

J. Exp. Zool. (Mol. Dev. Evol.) 324B:473–486, 2015

Viviparity in squamate reptiles is widely recognized as having evolved convergently from oviparity more than 100 times. However, questions persist as to whether reversals from viviparity back to oviparity have ever occurred. Based on a theoretical model, a recent paper (Pyron and Burbrink, 2014) has proposed that viviparity is ancestral for squamates and that viviparity—oviparity reversals have far outnumbered origins of viviparity in reproductive history. Close examination of this analysis reveals features that cast doubt on its plausibility, notably the requirement of repeated, sequential transformations back and forth between these reproductive modes, as well as numerous, uncounted evolutionary transformations that have produced inaccurate estimates of parsimony. Evidence derived from studies of anatomy, physiology, and developmental biology strongly supports the inference that oviparity is ancestral for squamates and has given rise to viviparity on numerous occasions. Biological data provide important insights into the likelihood of evolutionary transformations, and deserve to be incorporated fully into future analyses of the evolution of reproductive modes. J. Exp. Zool. (Mol. Dev. Evol.) 324B:473–486, 2015. © 2015 Wiley Periodicals, Inc. How to cite this article: Blackburn DG. 2015. Evolution of viviparity in squamate reptiles: Reversibility reconsidered. J. Exp. Zool. (Mol. Dev. Evol.) 324B:473–486.

Viviparity in squamate reptiles (lizards and snakes) has been a focus of biological investigation for more than a century. Squamates are considered to be ideal for studies of viviparity on the grounds that this reproductive pattern has evolved on more than 100 separate occasions, often at low taxonomic levels (Blackburn, '99,2006). Although squamate viviparity is widely recognized to have evolved from oviparity (Packard et al., '77; Tinkle and Gibbons, '77; Shine, '85), the question occasionally arises as to whether the reverse transformation has ever occurred (e.g., Fenwick et al., 2012; Lynch and Wagner, 2010). A recent analysis in Ecology Letters has proposed that viviparity actually is ancestral for squamates and that viviparity ! oviparity “reversals” have predominated in their reproductive history (Pyron and Burbrink, 2014). If correct, these assertions would require us to reinterpret the results of numerous studies and to reject widely held assumptions and conclusions. The present paper contributes to a dialogue in the Journal of Experimental Zoology B on the potential evolutionary reversibility of squamate viviparity. First, this paper summarizes the historical background of the issues. Second, it highlights previously unexplored aspects of the recent Ecology Letters analysis (Pyron and Burbrink, 2014) that bear upon its implications and plausibility. Third, this paper considers what

squamate reproductive biology reveals about the directionality of transformations between oviparity and viviparity. An implicit issue underlying the present controversy is the role that diverse biological data should play in assessments of evolutionary transformations. Historical Perspective In scientific work that date back to the mid 1800s, viviparity is inferred to have evolved convergently from oviparity in multiple squamate lineages. More than a thousand studies have documented anatomical, physiological, biochemical, and ecological specializations for viviparity (see Shine, '85; Yaron, '85; Blackburn, 2000; Blackburn and Stewart, 2011; Murphy and Thompson, 2011; Stewart and Blackburn, 2014; van Dyke et al.,



Correspondence to: Daniel G. Blackburn, Deptartment of Biology, Electron Microscopy Center, Trinity College, Hartford, CT 06106. E-mail: [email protected] Received 30 January 2015; Accepted 20 March 2015 DOI: 10.1002/jez.b.22625 Published online 13 May 2015 in Wiley Online Library (wileyonlinelibrary.com).

© 2015 WILEY PERIODICALS, INC.

474 2014), many of which have analyzed individual clades to document details of its evolution (e.g., Qualls et al., '95; Qualls and Shine, '96, '98; Andrews, 2000; Andrews and Mathies, 2000; Swain and Jones, 2000; Smith et al., 2001; Stewart and Thompson, 2003; 2009a). The first serious attempts to quantify the origins of reptile viviparity were analyses in the 1980s that drew on phylogenies and phylogenetic classifications, under the assumption that viviparity evolves irreversibly from oviparity (Blackburn, '82, '85; Shine, '85). A subsequent compilation of these analyses defined a minimum of 102 such origins (Blackburn, '99). Over the years, a few of these potential origins were eliminated and several new origins were uncovered through refined understandings of phylogenetic relationships.1 From these changes, a minimum of 115 origins of viviparity were defined in squamate reptiles, more than in all other vertebrates combined (Blackburn, 2014). Despite its magnitude, this total is a conservative figure that underestimates the origins of viviparity in such speciose squamate groups as the Lygosominae and Colubroidea. This figure also does not include evolutionary origins defined in extinct reptiles of the Mesozoic and late Paleozoic (Blackburn, 2014; Blackburn and Sidor, 2015). While most published studies have assumed that squamate viviparity evolves irreversibly from oviparity, a few have suggested a viviparity ! oviparity reversal in a particular group, based on the phylogenetic distribution of reproductive modes. The rationale was that allowing for such reversals yields interpretations that require fewer character state changes than otherwise (Lynch and Wagner, 2010; Surget-Groba et al., 2006). However, nearly all of the alleged reversals have not withstood scrutiny. In many cases, subsequent study has shown that interpretations involving a putative viviparity ! oviparity reversal are no more parsimonious than those that lack such reversals (Mink and Sites, '96; Lee and Doughty, '97; Blackburn, '99; Stanley et al., 2011; also see Odierna et al., 2004; Kupriyanova et al., 2006). For example, assessment of several alleged cases of reversal (including anguids, gekkos, and cylindrophine snakes) found that all had weak support (Lee and Shine, '98). One review of a limited number of taxa proposed that viviparity ! oviparity reversals have occurred almost as frequently as oviparity ! viviparity transitions (de Fraipont

BLACKBURN et al., '96). However, this analysis has been refuted based on its faulty methodology and erroneous data (Blackburn, '99; Shine and Lee, '99). Among other vertebrates, viviparity has arisen many times, but alleged reversions to oviparity are rare. No good reason exists to suspect reversals among lissamphibians or (non-tetrapod) osteichthyans, which account for at least 9 and 13 origins of viviparity, respectively (Blackburn, 2014).2 In chondrichthyans, among which 9 origins have been defined, oviparous rajoids (traditionally thought to lie nested within a viviparous group) represent a potential reversal (Blackburn, 2005; Dulvy and Reynolds, '97). However, a recent phylogenetic analysis has revised the position of rajoids (Naylor et al., 2012) and diminished reasons to infer a reversion (Blackburn, 2014; cf. Musick, 2010). Despite more than 150 recognized origins of vertebrate viviparity (Blackburn, 2014; Blackburn and Sidor, 2015), little evidence supports the idea that reversals to oviparity have played a significant role in vertebrate evolutionary history. The Ecology Letters Analysis The recent analysis by Pyron and Burbrink (2014) in Ecology Letters has constructively stimulated debate by challenging widely accepted views about reptile viviparity. In the process, the paper has beneficially highlighted a type of evolutionary question that seldom draws public attention.3 The paper’s publication provides an opportunity for a diversity of biologists to engage with significant issues that are fundamental to our understanding of reptile reproduction and evolution (Griffith et al., 2015; Pyron and Burbrink, 2015; Shine, 2015). It also provides a useful way to evaluate the efficacy of its theoretical models, thereby contributing to ongoing attempts to evaluate and refine these models (King and Lee, 2015; Rabosky and Goldberg, 2015). The Ecology Letters paper presented results of a model-based BiSSE (Binary State Speciation and Extinction) procedure and a MP (Maximum Parsimony) analysis, each of which was designed to allow bidirectional transformations between oviparity and viviparity. They were compared to a third analysis, which assumed that the transformation from oviparity to viviparity is irreversible. Two additional procedures (state-independent Mk2 analyses) reportedly yielded results similar to the BiSSE analysis. Results of the procedures were presented as large tree diagrams in

1

Three origins eliminated from the 1999 analysis are Chamaesaura (Stanley et al., 2011), Typhlops diardi (Vitt and Caldwell, 2009) and one in Sceloporus (Blackburn, 2000). Several new origins of viviparity have been uncovered via phylogenetic modifications, including four in Liolaemus (Schulte et al., 2000), two in amphisbaenians (the genera Monopeltis and Loveridgea: Andrade et al., 2006), a second origin in the reproductively bimodal Zootoca vivipara (Odierna et al., 2004; Surget et al., 2006), plus one each in a Cretaceous lizard (Wang and Evans, 2011), in Phrynosoma (Hodges, 2004; Zamudio et al., 2000), in Trioceros affinis (Tilbury and Tolley, 2009), and in Shinisaurus (Vidal and Hedges, 2009), and a few others through revisions of skinks, elapids, and colubrids (Pyron et al., 2013; Wiens et al., 2012).

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2 A ninth origin of viviparity in amphibians was documented in a recent paper (Iskander et al., 2014).

“The old snake-and-egg question,” New York Times, 23 December 2013; “Ancestor of snakes, lizards likely gave birth to live young,” Science News, 17 December 2013. The latter quoted one of the authors: “This is a very unusual and controversial finding, and a major overturn of an accepted school of thought. Before, researchers long assumed that the ancestor of snakes and lizards laid eggs, and that if a species switched to live birth, it never reverted back. We found this wasn't the case.” 3

VIVIPARITY AND REVERSIBILITY online appendices and were summarized in an online Supporting Information (SI) section. Comparison of Results. The BiSSE analysis produced three heterodox conclusions: (a) that viviparity (not oviparity) is ancestral for squamate reptiles and higher squamate taxa, having first originated more than 174 million years ago; (b) that viviparity (V) ! oviparity (O) reversals have occurred much more frequently than origins of viviparity; and (c) that warm climates have driven the evolution of oviparity in numerous viviparous clades (Pyron and Burbrink, 2014). These views conflicted with work that viewed squamates as ancestrally oviparous and studies that traced origins of viviparity to Cretaceous and Cenozoic time frames (Lynch, 2009; Schulte and Moreno-Roark, 2010), with some having occurred as recently as the Pliocene and Pleistocene (Heulin and Guillaume, '89; Blackburn, '95; Calder
on-Espinosa et al., 2010). They also conflicted with numerous experimental and analytical studies that implicated cold (rather than warm) climates in evolutionary shifts in reproductive modes (Shine and Bull, '79; Shine, '85; Lynch, 2009; Lambert and Wiens, 2013; Watson et al., 2014; see Shine, 2014 for a historical review). The three analytical methods produced very different quantitative results. The BiSSE analysis was said to yield 34 origins of viviparity4 and 59 reversals back to oviparity (Ecology Letters text, p. 4). (These figures appear to exclude two origins of viviparity that are indicated in the data tree of Appendix S15 as well as a reversal.6) In marked contrast, the MP analysis produced 73 unambiguous origins of viviparity and 12 reversals (Supporting Information lines 452–459). Most of these putative reversals were in groups with poor resolution at the species level (e.g., Liolaemus and Lygosominae) (see SI, line 429–431). The model with no reversions revealed 121 origins of viviparity (SI lines

4 In a minor discrepancy, the 34 nominal origins of viviparity are said to include (EL page 4, paragraph 4) as well as to exclude the root node (Supporting Information line 424). Tabulation of changes diagrammed in Appendix S1 supports the latter statement; thus 35 origins are actually indicated. 5 If viviparity is ancestral for squamates, dibamids (as the sister group to all other squamates) must represent a reversal to oviparity. Dibamids are indicated as such in the diagram of Appendix S1 but are not included in the list of reversals (Supporting Information lines 417–424). Likewise, the MRCA of Alethinophidia must be viviparous (as shown in Appendix S1, and as required for subsequent unambiguous reversions to oviparity), representing an origin not included in the quantitative summary (SI lines 424–428). Minor discrepancies of this nature do not materially affect conclusions of the Ecology Letters paper, but are worth noting for the sake of quantitative precision. 6 Assuming that Gerrhosauridae and Platysaurinae reflect independent transformations (as implied by SI line 420 and the corresponding tree of Appendix S1), a total of 60 reversals are listed.

475 439–440).7 The irreversibility analysis yielded origins of viviparity that are broadly consistent with those defined previously (Blackburn, '99, 2014), despite its sophisticated methodology and use of updated phylogenetic information (Pyron et al., 2013). However, the Ecology Letters paper omitted three previously defined origins in snakes (represented by natricine and lamprophiid species not included in the phylogeny). In addition, it suggested new origins in Viperidae, Elapidae, Colubridae, Scincidae, and Liolaemidae. The three procedures also differed fundamentally in inferences about ancestral reproductive modes of key taxa (Table 1). The most recent common ancestor (MRCA) of squamates was reconstructed as viviparous in the BiSSE model but as oviparous in the MP analysis. In addition, the two procedures differed in the ancestral parity modes inferred for Colubroidea, Viperidae, nondibamid squamates, and the large genus Liolaemus. In an unorthodox area of agreement between the BiSSE and MP models, viviparity was inferred to be ancestral in such major taxa as Scincidae, Lygosominae, Anguimorpha, Booidea, and Crotalinae. In the irreversibility model, each of the above taxa was reconstructed ancestrally as oviparous (Table 1), as in previously analyses (Blackburn, '99). Sequential Transformations. An important (and potentially surprising) feature of the BiSSE model is that it requires most species to have resulted from sequential transformations back and forth between oviparity and viviparity (see King and Lee, 2015). This issue is not mentioned in the Ecology Letters paper and is easily overlooked without detailed examination of the diagram of Appendix S1. Under its provisions, many viviparous lizard clades must eventually have resulted from three successive transformations as follows: O (the MRCA of Lepidosauria) ! V (MRCA of Squamata) ! O ! V. Examples include live-bearing iguanians, gekkotans, lacertids, scincids, and amphisbaenians. Some oviparous Iguania (certain Sceloporus and seven clades of Liolaemus: see SI line 422) would have resulted from a sequence of four transformations: O (Lepidosauria) ! V (Squamata) ! O (MRCA of Iguania) ! V ! O. The great majority of oviparous snakes (i.e., alethinophidians) emerge as a product of transitions between five reproductive modes, a sequence of O (Lepidosauria) ! V (Squamata) ! O (MRCA of snakes) ! V ! O. Many viviparous snake clades reflect successive transformations between no fewer than six reproductive modes, an O ! V ! O ! V ! O ! V sequence. Examples include viviparous Natricinae (thamnophiines, Sinonatrix annularis), Colubrinae (Coronella

7

In the irreversibility analysis, five origins of viviparity are scored for Phrynosomatidae (SI line 443). However, the corresponding tree (Appendix S4) indicates a total of seven origins, two for Phrynosoma and five for Sceloporus (assuming that S. lundelli is correctly scored). The latter set of numbers is consistent with previous analyses of these genera, although for Sceloporus, phylogenetic details differ.

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Table 1. Ancestral reproductive modes of squamate taxa as inferred from analyses in the Ecology Letters paper (Pyron and Burbrink, 2014). MRCA of taxon Squamata Non-dibamid squamates Scincoidea Cordylidae Scincidae Lacertoidea Anguimorpha Iguania Liolaemidae Liolaemus Serpentes Alethinophidia Booidea Boidae Colubroidea Viperidae

BiSSE model

MP analysis

Irreversibility analysis

V V V ? V Oa V Oa ? V Oa Vb V V V V

O O Vc ? V O Vc O O O O O Vc ? O O

O O O O O O O O O O O O O O O O

Information is taken from the appendices, and where possible, the text. O, oviparity; V, viviparity; “?,” ambiguous (undefined); MRCA, most recent common ancestor. a Listed as oviparous in the Supporting Information for this model, but represented as ambiguous in the corresponding data tree (Appendix S1). Serpentes is represented as viviparous in the text figure (Fig. 1). b See text footnote 5. c Listed as viviparous in the Supporting Information, but represented as ambiguous in Appendix S5.

austriaca, Elaphe rufodorsata, Conopsis, Ahaetulla), and Dipsadinae, as well numerous clades of the families Lamprophiidae and Elapidae. In the MP analysis, in contrast, zero to two reproductive mode transformations lie in the ancestry of each taxon. The great majority of oviparous species are shown to have retained their reproductive modes from the oviparous MRCA of squamates, and most viviparous species result from a single, orthodox O ! V transformation. Very few egg-laying forms are indicated as the result of an O ! V ! O sequence (e.g., Plestiodon [Scincinae], Trachylepis [Lygosominae], Lachesis [Crotalinae], and Eryx jayakari [Boidae]). In the analysis that assumes viviparity to be irreversible, the evolution of reproductive modes involves (by definition) only O ! V transformations. Uncounted Transformations. Another notable, unrecognized feature of the Ecology Letters analyses is the degree to which they underestimate the number of transformations in reproductive patterns. The issue is significant if the quantity of transformations is used as a criterion for plausibility (see below). In addition to the well-defined (unambiguous) origins and reversals, many other transformations between oviparity and viviparity are necessary to account for the phylogenetic distribution of reproductive modes. These transformations understandably were not tabulated in the Ecology Letters paper J. Exp. Zool. (Mol. Dev. Evol.)

because their polarity (and in several cases, their precise positions) are ambiguous. Nevertheless, minimal quantitative figures can be generated from the diagrammatic data trees (Appendices S1–S5). The issue is best illustrated with examples. Phrynosoma (which is ancestrally oviparous) contains two viviparous clades, each of which is a sister group to an oviparous clade (Appendix S1). In the BiSSE analysis, the clades are simply scored as one origin of viviparity, due to a character state ambiguity in their MRCA. However, given the relationship of each viviparous clade to an oviparous group, at least one more transformation is required to account for the distribution of reproductive patterns. (These include either a second origin of viviparity [as in Hodges, 2004], or two reversals, or a second origin plus one reversal.) In a similar example, the genus Chamaeleo also contains two viviparous clades, each of which is separately linked to multiple oviparous forms (Appendix S1). Under the inference that oviparity is ancestral, this genus is scored as one origin of viviparity.8 However, the phylogenetic distribution of patterns in Chamaeleo either requires a second origin of viviparity or a larger number of origins þ reversals. In another case from the BiSSE model, the snake families Homalopsidae (viviparous) and Lamprophiidae 8

See SI line 426, which also includes a distant origin in the confamilial Bradypodion.

VIVIPARITY AND REVERSIBILITY (ancestrally oviparous) are inferred to be sister groups derived from an MRCA of ambiguous parity mode. One of these two clades must represent a transformation in reproductive mode from their common ancestor, reflecting either one origin of viviparity or one reversal. A more complex situation is presented by Scincinae, which is inferred to be derived from a viviparous ancestry, and within which three reversals to oviparity are quantified (SI line 420). Assuming the tree to be correct, the distribution of oviparous species in the subfamily actually requires a minimum of three (and as many as five or more) additional, uncounted transformations in reproductive modes. Analysis of the BiSSE results (Appendix S1) reveals a minimum of 22 undefined (yet unavoidable) transformations in lizards and at least seven in snakes (Table 2).9 The Maximum Parsimony analysis results (Appendix S5) requires at least 15 additional transformations in lizards and 12 in snakes (Table 2).10 Although conservative, these numbers assume validity of the phylogenetic relationships represented. Thus, they could shift with modifications to the trees, especially in groups with poor species level resolution. However, the same is true of the defined origins and reversions in each of the analytical procedures (SI lines 429–431). Another type of undefined transformation is represented by species that contain both oviparous and viviparous populations. Reproductively bimodal species were omitted from the Ecology Letters analyses because they are not readily handled by the BiSSE procedure (SI line 342). Nevertheless, of ten species in which reproductive bimodality has been well-established,11 each 9

In the BiSSE analysis, uncounted transformations in lizards involve (by my analysis) one in Chamaeleo, one in Phrynosoma, two in Liolaemus, three in Sceloporus, three in Scincinae, and 12 in Lygosominae. In snakes, they include two in Lamprophiidae, four in Elapidae, and one in the above-mentioned homalopsid/ lamprophiid dichotomy.

10 In the MP procedure, unscored transformations in lizards entail (again by my count) one in Liolaemidae, one in Scincinae, three in Phrynosomatidae, two in Cordylidae þ Xantusiidae, four in Anguidae and four in Lygosominae. Those in snakes involve two in Lamprophiidae, two in Viperinae, three in Booideae, and five in Crotalinae. 11

Ten of the 12 reproductively bimodal species listed in the Ecology Letters paper (SI lines 323–328) (most of which came from previous analyses: Blackburn, '85, '99; Shine, '85) are considered here as reliable: Zootoca vivipara, Glaphyromorphus nigricaudis, Lerista bougainvilli, Saiphos equalis, Trachylepis capensis, Madascincus igneocaudatus, Helicops angulatus, Psammophylax variabilis, Protobothrops jerdonii, and Echis carinatus. The other two species listed are excluded herein: Trachylepis sulcata (for which I find no corroborative evidence) and “Sceloporus aeneus.” The latter nominal taxon was split several years ago along the lines of reproductive mode (Mink and Sites, '96; Smith et al., '93) and the viviparous member of the pair (S. bicanthalis) is already included elsewhere in the Ecology Letters analysis. However, S. bicanthalis itself may be bimodal (Benabib et al., '97; Mink and Sites, '96). Two subspecific origins (rather than just one) may have occurred in Z. vivipara (Kupriyanova et al., 2006; Odierna et al., 2004).

477 arguably represents a potential transformation in reproductive mode. Although to infer the polarity of the associated transformations may be premature (other than under the assumption of irreversibility), seven of the ten bimodal species are wellembedded within oviparous groups and therefore probably contain origins of viviparity rather than reversions. Other likely origins of viviparity (or alternative transformations) are represented by viviparous species that were not included in the Ecology Letters analyses.12 Implications. As outlined above, the BiSSE and MP analyses have yielded diverse, mutually incompatible interpretations with regard to inferred, ancestral reproductive modes of key taxa (Table 1) as well as to numbers of definable evolutionary origins and reversions (Table 2). That developing methodologies yield inconsistent results is to be expected, and tests and discussions of their reliability continue (Goldberg and Igi
c, 2008; O'Meara, 2012; Garamszegi, 2014; King and Lee 2015; Wright and Hillis, 2015); in fact recent analyses have shown that BiSSE results in particular should be reconsidered (Rabosky and Goldberg, 2015). The discrepant interpretations of reptile reproduction raise the issue of which (if any) set of conclusions research biologists should trust, or whether an agnostic stance must be maintained as long as theoretical methodological issues remain unresolved. The latter approach raises logistical problems for ongoing research, because whether ancestral squamates and other higher order taxa were oviparous or viviparous is fundamental to basic interpretations of reproduction and evolution. Under the circumstances, reproductive and other biological data become all the more important in reconstructions of squamate evolutionary history. Results of the analytical procedures offer internal evidence of their relative plausibility. First, as outlined above, the BiSSE model yields repeated sequential transformations to and from viviparity. To suggest an occasional viviparity ! oviparity reversal (Lynch and Wagner, 2010; Fenwick et al., 2012) is very different than to postulate a protracted string of sequential transformations in both directions that lies in the deep history of most squamate species. Results of the BiSSE model indicate that many oviparous snakes and lizards would be the ultimate product of successive transformations between five reproductive patterns, and most viviparous snakes would be a product of sequential transformations between six patterns (an O ! V ! O ! V ! O ! V sequence). Notably, most of the alleged transformations reflect the inference that viviparity is ancestral for squamates and other higher—order taxa (Table 1), as reflected

12

Among them are the amphisbaenian Loveridgea ionidesii (Andrade et al., 2006), the natricine snake Amphiesma ishigakiense (Ota and Iwanaga, '97), and the lamprophiids Psammophylax variabilis (Branch, '82), and Aparallactus jacksoni (Blackburn, '85).

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Table 2. Transformations in reproductive modes under each of three analytical models in the Ecology Letters paper (Pyron and Burbrink, 2014). Transformations Origins of viviparity Lizards Snakes Total Reversions to oviparity Lizards Snakes Total Total defined transformations Undefined transformations Lizardsf Snakesf Total undefined Total transformationsg

BiSSE model

MP analysis

Irreversibility analysis

21 b,d 13 a,b,d 34

47 26 73

76c 45 121c

36e 23 59e 93

10 2 12 85

0 0 0 121c

23 8 31 124

15 12 27 112

0 0 0 121

a,b

Defined transformations (origins and reversions) are taken from the paper’s Supporting Information. Other than those manifested at subspecific levels, transformations of undefined polarity are assessed herein from the diagrammatic trees (see text). “Lizards” represent a paraphyletic group consisting of squamates exclusive of Serpentes. a Not counting an origin of viviparity at the root node (text footnote d). b These “origins” all represent “re-reversals” from oviparity (O ! V ! O ! V transformations) c Not including two potential origins in Sceloporus (text footnote g). d Not including an origin leading to the MRCA of Alethinophidia (text footnote e). e Not counting the reversal in dibamids (footnote d) and separate reversals in Gerrhosauridae and Platysaurinae (text footnote f). f As determined from the corresponding diagrams (Appendix S1, S4, and S5), and excluding 10–12 subspecific transformations (text footnotes 9– 11). g Excluding 10–12 subspecific transformations, as well as four additional transformations in the BiSSE analysis (Table footnotes a, d, & e above) and two origins in the irreversibility analysis (Table 2 footnote c above).

in the much smaller number of putative reversions required by the MP procedure.13 Given the impact of root state assumptions (Goldberg and Igi
c, 2008), this point raises questions as to robustness of conclusions of the BiSSE analysis. Second, a disproportionate number of alleged reversals and ambiguous transformations lies within groups whose phylogenetic relationships have long been problematic and which exhibit poor resolution at lower taxonomic levels. For example, the scincid subfamily Lygosominae accounts for 29 transformations in the BiSSE analysis (with three origins of viviparity, 14 reversions, and 12 ambiguous transitions) and for 26 in the MP procedure (with 16 origins, 6 reversals, and 4 undefined 13

The inference that viviparity is ancestral for squamates as well as alethinophidians under the BiSSE procedure contributes to a total of four of the sequential transformations and two reversions. If the inferences as to ancestry are wrong (King and Lee, 2015), the need for multiple transformations and reversions disappears. Similarly, if ancestral squamates are coded as oviparous, the succession of three transformations with two reversions (O ! V ! O ! V) that putatively lies in the ancestry of many viviparous lizards evaporates to leave orthodox O ! V transformations.

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transitions). A single lizard genus—Liolaemus—accounts for 13 transformations in the BiSSE analysis (including four origins of viviparity, seven reversals, and two ambiguous transformations), as well as 12 in the MP analysis (including ten origins of viviparity, one reversal, and one undefined transition). In the MP analysis, all ten of the viviparity ! oviparity reversals alleged to have occurred in lizards lie within the Scincidae and Liolaemidae. In many instances, minor shifts in position of a species or small clade eliminate multiple alleged transformations in reproductive modes. Thus, several of the potential transformations in reproductive modes rest on phylogenetic hypotheses of questionable robustness. As King and Lee (2015) point out, the BiSSE model tends incorrectly to recognize multiple reversals in groups with high rates of change. This issue raises additional reason to question the large number of recognized reversals in these particular groups. Third, the postulate that the MRCA of squamates was viviparous (as supported by the BiSSE model) has significant implications. Since oviparity in extant squamates would be homoplastic, uniquely squamate features associated with that pattern must also be independently derived, an aspect that is not

VIVIPARITY AND REVERSIBILITY evident in extant species (see below). A related point is that no viviparous squamate can simply be the product of an orthodox squamate O ! V transformation. Rather, viviparity in particular squamate clades either must have been retained from the MRCA of squamates or must have arisen convergently from a form of oviparity that was also convergently evolved. These issues offer powerful means by which falsifiable predictions from the BiSSE model can be tested. A related point that is worth noting (albeit a semantic one) is that if viviparity is ancestral, O ! V transformations (not V ! O transformations) would constitute actual “reversions” to the ancestral squamate state. Fourth, inclusion of transformations of undefined polarity changes the relative plausibility of the analyses as judged on quantitative grounds. Under the principle of parsimony, conservative interpretations are preferable. On first consideration, the BiSSE model with its 93 defined transformations (origins and reversals) appears much more conservative than the irreversibility analysis (with its 121 origins). However, with inclusion of omitted, undefined transformations, numbers for the BiSSE analysis are elevated to 124 transitions, more character state changes than either of the other two approaches (Table 2). Insights From the Biology of Lizards and Snakes The foregoing discussion yielded three distinct scenarios as expanded from the Ecology Letters analysis (Pyron and Burbrink, 2014). The first is that viviparity is ancestral for squamates; has reverted to oviparity on numerous (59–61) occasions; has re-evolved back into viviparity many (34–35) times; and has vacillated back and forth between viviparity and oviparity as many as four and five times in the history of many lineages. The second is that an ancestral pattern of oviparity has evolved into viviparity on many (73) occasions, and has re-evolved into oviparity in relatively few (12) clades. These two scenarios leave many transformations unclassified, such that total transformations (origins and reversals) are on the order of 124 and 112, respectively, plus  10 transformations at subspecific levels. The third scenario is that ancestral squamates were oviparous and that viviparity has originated many (121) times, but has not reverted to oviparity. From these scenarios, we can derive falsifiable predictions that may be tested against empirical information from the literature. If oviparity is ancestral for squamates, we would predict commonalities in structural and functional features associated with oviparity that are broadly shared among lizards and snakes. Likewise, specializations for viviparity should be diverse, reflecting the independent origins of that pattern. Alternatively, if viviparity is ancestral, we could predict the opposite: widespread commonalities in how viviparity is achieved that cut across taxonomic boundaries, particularly in groups that allegedly have retained viviparity from the MRCA of squamates (see Appendix S1). Likewise, if oviparity is derived, we would also expect diversity between clades in how oviparity is accomplished.

479 Accordingly, derived features associated with oviparity that postdate the MRCA of squamates such as “the squamate eggshell” would refer to non- homologous features. Homogeneity of Oviparous Features. Empirical tests of these predictions strongly support the view that oviparity is ancestral for squamates. For example, squamates have eggshells that (excluding specialized Gekkota: Pike et al., 2012) show only modest variation in structure and mode of formation but that differ from eggshells of other reptiles including the tuatara Sphenodon (Packard et al., '82, '88; Schleich and K€astle, '88; Packard and DeMarco, '91; Palmer et al., '93; Thompson and Speake, 2004). The shared derived features of squamate eggshells are indicative of their homology and inconsistent with the postulate that eggshells have originated independently many times. Likewise, oviductal glands that synthesize and secrete the eggshell fibers show strong evidence of a single evolutionary origin. Throughout oviparous squamates, the glands are very similar in structure, function, and mode of endocrinological control (Fox, '77; Blackburn, '98; Siegel et al., 2011; Stewart and Blackburn, 2014), and their commonalities leave no doubt as to their homology. In addition, oviparous squamates typically lay eggs that lie within a very restricted range of developmental stages (Blackburn, '95; Shine, '83), corresponding to the limb bud stage. In contrast, other extant Reptilia (chelonians, crocodilians, sphenodontids, and birds) lay eggs in very early stages of development (Romanoff, '60; Magnusson and Taylor, 1980; Ewert, '85). In populations of a very few bimodal lizard species, oviparous females retain eggs until later stages of development (Smith and Shine, '97; Garc
ıaCollazo et al., 2012). However, these lizards are widely thought to reflect transitional stages in the evolution of viviparity. The oviposition of developing eggs at the limb bud stage is a shared derived feature of squamates, one that parsimoniously is explained as an ancestral feature of squamate oviparity. To view squamate oviparity as derived implies that this uniquely squamate pattern has evolved (or has been “re-expressed”: see below) independently in >60 oviparous lineages. Diverse Specializations for Viviparity. Empirical evidence also is consistent with multiple evolutionary origins of viviparity from oviparity. Because an eggshell would inhibit maternal-fetal gas exchange during pregnancy (Thompson et al., 2004), under natural selection it has been reduced to a minuscule vestige in viviparous squamates. However, shell membranes differ between viviparous species in overall structure, vary in thickness by more than an order of magnitude, and vary in fate between species (disappearing very early in development, or later in development, or being retained throughout gestation) (Qualls, '96; Blackburn, '98; Heulin et al., 2002; Stewart et al., 2004; Stewart and Blackburn, 2014). Among species in which the shell membrane is shed, it disappears entirely in some species (Blackburn and J. Exp. Zool. (Mol. Dev. Evol.)

480 Flemming, 2012; Jerez and Ram
ırez-Pinilla, 2003) but in others, it is shed and accumulates as a pad of degenerating material at the abembryonic pole (Weekes, '30; Blackburn, '93a; Villagr
an et al., 2005; Anderson et al., 2011). Furthermore, the mechanisms by which shell membranes are reduced during gestation differ between species. They include digestion by the chorioallantois, by the omphalopleure, and by the uterine epithelium, as well as expansion of the chorionic vesicle and mechanical splitting of the membrane (Weekes, '27; Jacobi, '36; Blackburn, '93b, Blackburn and Lorenz, 2003a,b). Homologues of the uterine glands that normally deposit the eggshell also are reduced in viviparous forms (Giersberg, '22; Jacobi, '36; Guillette and Jones, '85; Picariello et al., '89; Qualls, '96; Blackburn and Callard, '97; Girling et al., '97). However, the means by which this reduction is accomplished differ between lineages. In some viviparous species, the glands are reduced in size, but in others, the glands are reduced in quantity or in density (Blackburn, '98; Heulin et al., 2005; Stewart et al., 2010). Such diversity is what one would predict from the independent losses of uterine shell glands. Placentas also show variation that is entirely consistent with the inference of multiple origins of viviparity. All viviparous squamates have placentas which are responsible (at minimum) for gas exchange and water provision. However, placentas differ between clades in terms of patterns of development, fetal membrane contributions, cellular composition, and types and quantities of nutrients provided (Blackburn, '93b; Stewart, '93; Blackburn and Flemming, 2009; Stewart and Blackburn, 2014). For example, in most viviparous squamates, placentas provide limited quantities of nutrients, whereas in others, placental transfer is responsible for virtually all nutrients for development (Yaron, '85; Thompson and Speake, 2006; Ram
ırez-Pinilla, 2006; Fleming and Blackburn, 2012; Stewart and Blackburn, 2014). In squamates of the former category, clades differ in the type of nutrients supplied and whether such provision is facultative or obligatory (Stewart, 1989, 1992; Stewart and Thompson, 2000; Thompson et al., 2000; Stewart, 2013). Variation in placental morphology also is consistent with independent origins of viviparity. Detailed interspecific comparisons have been made in five distinct lineages: thamnophine snakes (Stewart, '90; Blackburn et al., 2002, 2009; Blackburn and Lorenz, 2003a,b; Stewart and Brasch, 2003); South American Mabuya (Scincidae) (Blackburn et al., 2002; Blackburn and Vitt, '92, 2002; Jerez and Ram
ırez-Pinilla, 2001; Ram
ırez-Pinilla, 2006, 2014; Leal and Ram
ırez-Pinilla, 2008; Ram
ırez-Pinilla et al., 2006, 2011); a clade of North American Sceloporus (Phrynosomatidae) (Villagr
an et al., 2005; Blackburn et al., 2010; Anderson et al., 2011); and two distantly related clades of Australian skinks (Stewart et al., 2000; Stewart and Thompson, '98, 2000, 2009a,b; Thompson et al., 2002; Adams et al., 2005). In each case, placental features are similar or identical in species within a lineage but vary extensively between clades. Furthermore, placentas of lizards J. Exp. Zool. (Mol. Dev. Evol.)

BLACKBURN derived from three other potential origins of viviparity differ strikingly from those of other clades (Blackburn, '93a; Flemming and Branch, 2001; Flemming and Blackburn, 2003; Blackburn and Flemming, 2012). Other means by which viviparity is accomplished also vary extensively among live bearing squamates, just as one would predict from the independent origins of this pattern. For example, numerous specializations enhance maternal-fetal gas exchange in the intrauterine environment (summarized by Blackburn, 2000, 2006). One set of specializations is the increased vascularity of the pregnant uterus and of the chorioallantois (Murphy et al., 2010; Parker et al., 2010a, b; Ram
ırez-Pinilla et al., 2012). Another is represented by the attenuated chorionic and/or uterine epithelia that reduce the interhemal distance (Blackburn and Lorenz, 2003a; Stewart and Brasch, 2003; Blackburn, '93b; Adams et al., 2005). Yet another is the progressive replacement of the avascular omphalopleure with vascularized chorioallantois, a pattern that is accomplished by any of three different developmental patterns, depending on the species (Stewart and Blackburn, '88, 2014; Stewart, '93). In yet another feature that enhances gas exchange, fetal blood has higher affinity for oxygen than maternal blood (Grigg and Harlow, '81; Birchard et al., '84; Ingermann, '92). Species vary in how this differential oxygen affinity is achieved. In some, a specialized fetal hemoglobin with high oxygen affinity has evolved (Ragsdale and Ingermann, '93). In others, the oxygen affinity of maternal blood is lowered during pregnancy through manipulation of nucleoside triphosphate levels, a process regulated by maternal progesterone (Ingermann et al., '91; Ragsdale et al., '93). Even the mechanisms by which gestation is accomplished vary between viviparous species. Progesterone from the corpus luteum has been implicated in the maintenance of pregnancy in many species. However, species show great variation in lifespan of the corpus luteum and in whether or not it is necessary to maintain pregnancy, as well as in sources of progesterone, steroid hormone profiles during gestation, and effects of progesterone on parturition (summarized in Yaron, '85; Xavier, '87; Jones and Baxter, '91; Blackburn, 2006). Implications. The homogeneity of oviparous mechanisms and the diversity of specializations for viviparity are easily explained if oviparity is ancestral and viviparity is derived. In contrast, to interpret the wealth of biological data in a way consistent with the BiSSE scenario requires the adoption of elaborate ad hoc hypotheses and highly implausible scenarios for which biological evidence is scarce or absent. One must presume that each time oviparity has evolved that it involves the same mechanisms— the opposite of what one would predict. Likewise, one must presume that viviparous mechanisms are highly diverse, despite the shared ancestries of this pattern in clades that purportedly have retained this pattern from the MRCA of squamates (i.e., viviparous xantusiids, cordylids, anguids, xenosaurids, and

VIVIPARITY AND REVERSIBILITY various scincines and lygosomines). Such arguments also apply to the Maximum Parsimony analysis (though to a smaller degree), given the inference of 12 V ! O reversals (plus a subset of 27 unclassified transformations). Speculation that viviparity has frequently and easily reverted to oviparity may be based on misconceptions about differences between the two patterns. Pyron and Burbrink (2014, p. 6) state: “Squamates with simple placentae… are essentially retaining shell-less eggs in utero (Thompson and Speake, 2006), and the reversion to oviparity apparently only involves the re-introduction of shell deposition during embryonic development.” On the contrary, as documented above (as well as in the 2006 paper by Thompson and Speake and in other work by these authors), viviparity entails a wide range of specializations necessary for sustenance of embryos and maintenance of pregnancy. The differences between oviparous and viviparous species extend far beyond presence versus absence of an eggshell, and include anatomical, physiological, developmental, endocrinological, biochemical, behavioral, and ecological specializations. If viviparity ! oviparity transformations have occurred frequently, the unanswered question is why no apparent sign of ancestral specializations for viviparity have been retained in derivative oviparous forms. For example, in the BiSSE analysis, the scincid lizard Bassiana duperreyi is shown as having reverted to oviparity from within Pseudemoia, a viviparous clade with morphological and physiological specializations for placentotrophy (Pyron and Burbrink, 2014: Appendix S1). However, this oviparous species shows no trace of a viviparous ancestry (Griffith et al., 2015), in (for example) its fetal membranes, uterus, or yolk nutrient supply. Instead its biological features are entirely consistent with their being ancestral to the viviparity of Pseudemoia (Stewart et al., 2003; Stewart and Thompson, '96, 2003, 2009a; Thompson et al., 2000; Stewart and Blackburn, 2014). The MP analysis holds open another theoretical possibility given ambiguities in parity modes of the MRCA of these taxa (Appendix S5): independent evolution of viviparity and placentation in P. entrecasteauxii and P. pagenstecheri. However, the close similarities of unique structural and functional features of the placentas of these two species and their congeners virtually preclude their independent origins. As an alternative to the hypothesis that oviparous features evolved convergently during reversions from viviparity, one could postulate the reappearance of long lost oviparous features through suppression and eventual re-expression of genes in derivative forms (Pyron and Burbrink, 2015). In accord with results of the BiSSE analysis, one must postulate that such genes arose in unidentified hypothetical lepidosaurian ancestors, were suppressed more than 174 million years ago in the MRCA of squamates, yet were retained in the history of numerous viviparous clades. Accordingly, throughout repeated vacillations between viviparity and oviparity, such genes would have been re-

481 expressed each time oviparity re-evolved, and suppressed with each re-evolution of viviparity. Likewise, in the MP analysis, oviparity (putatively) would have been lost 166.6 mya in the line leading to Scincoidea and 118 mya in ancestral Anguimorpha, yet its genetic basis was retained for many millions of years in viviparous clades that subsequently reverted to oviparity. One must question the likelihood that such genes were conserved over such extended time frames (see Griffith et al., 2015), while those for viviparous reproduction (a pattern supposedly present in the MRCAs of squamates and of Scincoidea) apparently were not. Furthermore, the idea that flip-flops between reproductive patterns have occurred through successive suppression and re-expression of homologous genes is incompatible with the diversity of mechanisms by which viviparity is accomplished. An overarching issue to recognize is that biological features associated with oviparity and viviparity offer invaluable information for reconstruction of the evolutionary history of those patterns. This is particularly the case, given recent challenges to the use of BiSSE analysis (King and Lee, 2015; Rabosky and Goldberg, 2015). For example, one beneficial feature of the gene suppression hypothesis is that it is testable by genetic analyses on extant species (Pyron and Burbrink, 2015), particularly those that allegedly have resulted from successive transformations back and forth between oviparity and viviparity. Likewise, careful examination of oviparity in some of the better supported inferences of reversal (Lynch and Wagner, 2010; Fenwick et al., 2012; King and Lee, 2015) may well yield evidence relevant to the reversibility hypothesis. As noted by Pyron and Burbrink (2015): “It is unlikely that phylogenetic analyses alone will suffice to understand the evolution of parity mode in squamates, particularly with respect to the likelihood, mechanism, and frequency of transitions to and from oviparity.” Indeed, evolutionary transformations in general are best reconstructed through analysis of phenotypic and genetic features (Cristin et al., 2010; Galis et al., 2010; also see Kohlsdorf et al., 2010). Consequently, notwithstanding the utility of theoretical models in generating evolutionary hypotheses and the attractions of their usage, empirical data warrant being fully incorporated into evolutionary reconstructions. Multiple lines of biological evidence not only provide a valuable reality check on modelbased and other theoretical approaches, but also offer both the means and the rationale for achievement of a full, integrative understanding of reproductive diversity and its evolutionary history.

ACKNOWLEDGEMENTS I thank Gunter Wagner, editor of the Journal of Experimental Zoology B, for inviting me to contribute to the journal’s special issue on squamate viviparity, and the anonymous reviewers for their comments on the paper. Laurie Bonneau reviewed and formatted the manuscript. J. Exp. Zool. (Mol. Dev. Evol.)

482

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