FREDRIC J. JANZEN AND JAMES G,KRENZ

Phylogenetics

Which was First, TSD or GSD?

The basic challenge of evolutionary biology is to explain ever, that this temperature-dependent sex determination variation or the lack thereof, be it phenotypic, genetic, phy- (TSD) does not imply a lack of genetic involvement in sex logenetic, spatial, temporal, andso on. To illustrate, onegross determination, just an absence of sex chromosomes and generalization is that phenotypic traits we think of as being temperature-insensitive sex-determining genes (Valenzuela very important to organisms tend to be highly conserved et al. 2003). TSD can be contrasted with the more familiar (e.g., binocular vision in vertebrates), probably because genotypic sex determination (GSD), sometimes referred to the genomic and developmentalunderpinnings are essentially as chromosomal sex determination (Deeming and Fergu- fixed. Thus. one striking feature about sex-determining son 1988), wherein sex is fixed permanently by generic fac- mechanisms (SDMs), a fundamental aspect of sexual or- tors at conception. Exceptions exist (e.g., sex-changing ganisms, is the enormous variety (Bull 1983). fish), but these two categories serve to organize, identiiy, This great diversity of SDMs (in vertebrates in particu- and elucidate the key evolutionary issues involved (Valen- lar) has long puzzled biologists. Given the existence of sex- zuela et al. 2003). ual reproduction and its demonstrable adaptive significance In the course of cataloguingthis diversity, a controversy of (e.g., West et al. 1999), one might assume that the means by sorts has ensued over the origins of various sex-determining which it is accomplished might be highly conserved. More- systems in vertebrates. How many times has each mecha- over, SDMs and the primary sex ratio are inexnicably linked nism evolved and, as exemplified in the chapter title, which (Bd 1983), and selection for a 1:l primary sex ratio is mechanism is ancestral? The answers to these and related strong (Fisher 1930); thus, one might also assume that any questions strike to the heart of at least two critical and con- SDM producing a skewed primary sex ratio would be non- troversial issues involving SDMs: (1) adaptive significance existent. But these two assumptions are, remarkably, incor- and (2) molecular genetic and physiological underpinnings. rect. In fact, sex is determined in various vertebrate taxa Traits as fundamental as SDMs are typically not amenable in an extraordinary variety of ways (Bull 1983;Janzen and to experimental evolution, necessarily leaving informed PaMs199la). speculation and theoretical exploration to fill the void. In- SDMs in vertebrates nonetheless can be collected roughly deed, such has been the case regarding evolutionary transi- into two major categories. One is environmental sex deter- tions among SDMs in vertebrates (Ohno 1979; Bull 1983; mination (BSD), wherein sex is fixed permanently by envi- Karlin and kssard 1986; Ewert and Nelson 1991; Janzen ronmental cues (primarily temperature in vertebrates) during and Paukstis 1991a; Solari 1994; but see MUk and Green a discrete period after fertilization (Bull 1983). Note, how- 1990; Janzen and Paukstis l99lb; Kraak and Pen 2002). However, the development of rigorous, phylogeneticdy fish primarily have TSD Ib, crocodilians TSD 11, sphen- based comparative methods over the past two decades pro- odontians TSD Ib (or possibly TSD 11), lizards TSD Ib and vides another valuable approach: the retrodicrion of the TSD 11, and TSD la and TSD II. evolutionary history of SDMs.This method is not perfect of course (e.g., it is correlative in nature), but it does pro- Hypotheses on Evolutionary Transitions vide a strong framework for much modern evolutionary re- search on the nature of variation (e-g., Harvey et al. 1996; Scenarios regarding evolutionary transitions between SDMs Martins 1996; Avise 2000; Page and Holmes 1998). in vertebrates have historically been qualitative. For es- In this chapter, the evolutionary history of SDMs in ver- ample, Witschi (1959) stated that fish had morphologically tebrates will be explored. First previous thinking on this indistinguishable (i-e., homomorphic) sex chromosomes, if topic will be discussed to illustrate the issues involved. Then any at all, representing "a primitive and ancient condition" comparative methods will be applied to rigorous (mainly (see also Ohno 1979), with genetically based sex determha- molecular) phylogeneuc hypotheses 111 vertebrates and par- uon thus arising in tetrapods in the Jurassic around 150 ticular vertebrate lineages, with special emphasis on lepi- million years ago. Similarly, Ohno (1967) proposed inde- dosaurs (lizards, snakes, andsphenodontians) and turtles, to pendent evolutionary transitions from ancestral homomor- evaluate evolutionary transitions between SDMs. These phic to derived heteromorphic sex chromosomes in lizards, comparative analyses will provide explicit tests of hypothe- snakes, and birds and a comparable more recent event in ses concerning the evolutionary diversity of SDMs in verte- mammals. These kinds of scenarios are understandable in brates. After the hypothesized ancestral SDM in vertebrates the absence of (1) information on the true diversity of is identified and the approximate phylogenetic loci of em- SDMs in vertebrates, (2) rigorous phylogenetic hypotheses, lurionary transitions between SDMs noted, the implica- and (3) acceptable formal comparative or molecular meth- tions of these results for important biological issues involv- ods. Furthermore, these propositions have no doubt per- ing TSD will be discussed. In particular, this discussion will sisted and become established dogma owing to support focus on the ramifications of these findings for research into from some population genetic models of the evolution of the adaptive significance and mechanistic underpinnings of sex chromosomes (e.g.,Chariesworth 1978; reviewed in Bull TSD in vertebrates. 1983; Charlesworth 1991) and a few indirect observations of sex chromosome degradation after autosoma1 conversion Taxonomic Distribution (e.g., reviewed in White 1973). To the authors' knowledge, however, these verbal scenarios remain untested explicitly SDMs are diverse and nonrandomly distributed in verte- using modern comparative methods. brates. Few fish (see Conover, Chapter 2)and no amphibians The qualitative nature of hypotheses concerning the (Hayes 1998; Chardard et aL, Chapter 7), snakes (Janzen and evolution of SDMs in vertebrates has characterized most Paukstis 199la), birds, or mammals (Bull 1983) are known scenarios involving TSD as well. Most authors have weighed to have TSD. Instead, male and female heterogamety are in cautiously on. this subject, which is summed up nicely as, "widely distributed in fish and amphibians, whereas snakes "There is no dear empirical evidence suggesting the evolu- and birds only have female heterogamety, and mammals tionary order of [TSD] compared to GSD .. ." (Karlin and have only male heterogamety Crocodilians and sphenodon- Lessard 1986; see also Bull 1983;Janzen and Pauksris 19Pla). tians have exclusively TSD (Deeming, Chapter 4; Nelson et Kraak and Pen (2002) qualitatively interpreted their phylo- al., Chapter 6), but lizards and turtles exhibit a variety of genetic representation of SDMs in vertebrates to indicate genotypic and environmental SDMs (Bull 1983;Janzen and that both GSD and TSD have evolved multiple times (see Paukstis 1991a; Harlow, Chapter 5; Ewert et al., Chapter 3). also Janzen and Paukstis I991a) but staked no position re- GSD is much more common in lizards than in turtles; the garding their polarity. Even so, an underlying sentiment converse is true for TSD (Janzen and Paukstis 1991 a). More- regarding the ancestral nature of TSD (at least in ) over, TSD is not monotypic; in fact, three types have been is evident (Karlin and Lessard 1986; Webb and Cooper- recognized, based on laboratory incubation of eggs (Bull Preston 1989; Janzen and Paukstis 199la; Solan 1994). On 1983; Conowr 1984; Ewert and Nelson 1991; Ewert et al. the other hand, some workers have cautioned against too 1994; Viets et d. 1994; Deeming, Chapter 4; Nelson et al., quickly rejecting an ancestd GSD scenario for reptiles Chapter 6; Conover, Chapter 2). The distribution of these (Bull 1983;Janzen and Paukstis 1991a). types of TSD, like that of SDMs in general, is nonrandom: To the authors' knowledge, only two studies have at- PHTLOGBNETICS: WHICH WAS FIRST, TSD OR GSDt 123

tempted quantitative tests of these verbal hypotheses, and several major nodes, particularly where sauropsids (reptiles at that only for turtles. Ewert and Nelson (1991), dung Gaff- and birds) split from mammals. Even with this poor resolu- ney's (1984) morphology-based phylogeny, inferred tion at deep branches in the tree, several firm conclusions that TSD must have been lost independently four to six times can be drawn. First and foremost, GSD is an ancient con- in this group. Janzen and Paukstis (1991a), using phyloge- dition in vertebrates, and there is at least one dearly docu- netic information accumulated for another study (Janzen mented origin of TSD in fishes (Conover,Chapter 2). How- and Paukstis 199lb) and employing a parsimony-based ever, how early TSD arose 'within sauropsids cannot be comparative analysis, found that TSD was the ancestral determined unambiguously by this particular analysis. None- condition for turtles . The authors now explore these issues theless, TSW is nearly ubiquitous in turtles, crocodilians, further in a larger context, using phylogenetic hypotheses and sphenodontians and occurs in a number of squamates for vertebrates, with a particular focus on the SDM-diverse i.e.,the clade containing lizards and snakes) as well (Figure lepidosaurs and turtles. 13.1). Within squamates, this comparative analysis indicates a minimum of five independent origins of TSD: (1) euble- Comparative Analyses pharids, (2) gekkonids, (3) lacertids, (4) agamids, and (5) vara- nids. Alternatively, within turtles, this analysis identifies The authors employ a parsimony-based statistical frame- at least three independent origins of GSD: (I)staurotypids, work implemented in MacClade 4.0 (Maddison and Maddi- (2)emydids, and (3) batagurids. Overall then, the crirerion son 2000) to test hypotheses concerning the evolutionary of parsimony applied to this phylogenetic hypothesis for history of SDMs in vertebrates and thereby provide guid- vertebrates confirms multiple independent origins of both ance for future "work. Information on SDMs was gathered GSD and TSD. from the literature (Table 13.1). General relationships among The previous analysis treats GSD as a homogeneous vertebrate groups were based on the traditional view (e-g., category. A more informative approach may be to explore Benton 2000). Modem phylogenetic hypotheses for major evolutionary transitions among SDMs after breaking out vertebrate lineages (mainly family level and above) con- the different types of GSD. Labeling a species as having socucted using DNA sequence data from the nudear gene GSD without indicating the heterogametic sex (i.e., homo- Rag1 were extracted primarily from Krenz et al. (impubl. morphic) may simply reveal a lack of sufficiently detailed data) and T. Townsend (unpubl. data). Relationships within genetic information. However, male and female heterogam- emydine, deirochelyine, and batagurid turtles and within ety are quire dissimilar and likely not subject to rapid, di- lacertid and gekkotan (eublepharids, diplodactylids, pygopo- rect intertransitions due to the time required for the com- dids, and gekkonids) lizards were derived from additional plex evolutionary and genetic mechanisms (e.g., Muiler's sources (respectively: Feldman and Parham 2002; Bickham ratchet) to operate (Bull 1983; Charlemrth 1991; but see, et al. 1996; Gaffney and Meylan 1988; Fu 2000; Kluge 1987 e.g., Schmid and Steinlein 2001). Thus, equating all such and Grismer 1988). The numbers of lepidosaur and turde SDMs under the umbrella term of GSD may inadvertenfly species have been targeted to 35 each, with broad within- obscure legitimate patterns of SDM evolution. In contrast, group sampling to minimize the complexity of the analyses distinguishing between the different patterns of TSD, which yet adequately capture the trends in SDM evolution. This probably share closely homologous genetic pathways (e.g., decision also limits controversies regarding supportable Deeming and Ferguson 1988), is less justifiable in the com- phylogenetic relationships among the focal taxa. In aB com- parative analyses. parative analyses in MacClade, the authors further adopted Analyzing SDMs with respect to different types of GSD the conservative tactic of ascribing no ordering between versus TSD provides considerable insight into the evotu- SDMs: gain or loss of any particular SDM is thus assumed tionary history of vertebrates (Figure 13.2). Indeed, parsi- to be equally likely. This parsimony approach seems pru- mony reconstruction of ancestral states in MacClade in this dent in the absence of information on the molecular under- context resolves several major issues revealed in the initial pinnings of SDMs in most vertebrates. analysis. In addition to the fish case, TSD is now identified The authors first examined evolutionary transitions be- as having originated early in sauropsid history, apparently tween the two major categories of SDMs without distin- from ancestors possessing male heterogamety. indepen- guishing among the different types of SDMs within each dent origins of TSD in eublepharids and gekkonids are less category (Figure 13.1). A parsimony analysis in MacClade certain in this analysis, but the other three instances within under these conditions cannot pinpoint the likely SDM at squamates (lacerrids, varanids, and agarnids) are solid. In Table 13.1 Information on Sex-Determining Mechanisms (SDMs) in Vertebrate Taxa Analyzed in This Chapter

Taxon SDM Source

FISH H,XY, ZW, TSD Conover, Chapter 2 AMPHIBIA H,XY, ZW Hayes 1998 MAMMALLA XY Bull 1983 Podomemis () TSD Valenzuela 2001b Pelomedusu (Pelomedusidae) TSD Ewert et al. 1994 (Pelomedusidae) TSD Ewert and Nelson 1991 () H Janzen and Paukstis 199la Chelodiw (Chelidae) H Janzen and Paukstis 1991a Acan thwhelys (CheIidae) XY Janzen and Pauksds 1991a Carettochelys (Carrertochelyidae) TSD Janzen and Paukstis 1991 a Pelmiisc~s(Trianychidae) H Choo and Chou 1992 Apdione (TMonychidae) H Janzen and Paukstis 19913 Staura~w(Staurotypidae) XY Janzen and Paukstis 1991a Chwdiiu (Staurotypidae) H Vogt and Flores-Villela 1992 () TSD Ewert and Nelson 1991 Kimstm(Kinosternidae) TSD Ewert and Nelson 1991 Dermatemys () TSD Vogt and ora as-Villela 1992 () TSD Ewert and Nelson 1991 Dermochelys () TSD Ewert and Nelson 1991 CMonid () TSD Ewert and Nelson 1991 Corrtta (Cheloniidae) TSW Ewert and Nelson 1991 Ca1emys (Emydinae) H Janzen and Paukstis 1991 a Terrapme (Emydinae) TSD Ewert and Nelson 1991 Clemmys (Emydinae) TSD Ewert and Nelson 1991 (Emydinae) TSD Ewert and Nelson 1991 Deirochelys (Deirochelyinae) TSD Bwert and Nelson 1991 (Deirochelyinae) TSD Ewert and Nelson 1991 Malademys (Deirochelyinae) TSD Ewert and Nelson 1991 Chrysemys (Deirochelyinae) TSD Ewert and Nelson 1991 Psmdemys (Deirochelyinae) TSD Ewert and Ndson 1991 Sichrockieh (Bataguridae) XY Janzen and Pauksris 1991a Chmemys (Bataguridae) TSD Janzen and Pauksris 1991a Kachnga (Bataguridae) zw Janzen and Paukstfs 1991a Maurewys ('Bataguridae) TSD Ewert and Nelson 1991 Melanpchelys (Bataguridae) TSD Ewert and Nelson 199 1 Rhmoclemmys (Bataguridae) TSD Ewert and Nelson 1991 Gophems (Testudinidae) TSD Spotila et al. 1994 Testwio (Testudinidae) TSD Jmzen and Paukstis 1991a SPHENODONTIA TSD Nelson et al., Chapter 6 Coleonyx (Eublepharidae) H Viexs et a1. 1994 Hemitht-conyx (Eublepharidae) TSD Vets et a1. 1994 EubIcpfiari5 (Eublepharidae) TSD Viers et al. 1994 Lialu (Pyeopodidae) XY Janzen and Paukstis 1991b Gkko gecko (Gekkonidae) XY Ewert and Nelson 1991 Gekko japunicus (Gekkonidae) TSD Viets et d. 1994 Gehyra (Gekkonidae) zw Janzen and Paukstis 1991b Phehma (Gekkonidae) TSD Viets et d. 1994 PhylloSacty!~~~(Gekkonidae) zw Oh01986 Tarentola (Gekkonidae) TSD Viets et al. 1994 Gonatodes (Gekkonidae) XY McBee et d.1987 Scincelld (Sdncidae) XY Janzen and Paukstis 1991b PHYLOGENETICS: WHICH WAS FIRST, TSD OR GSD? 125

Taxon SWM Source

Bumeces (Sdnadae) H Mets et d. 1994 Eremias (Lacertidae) zw Gorrnan 1973 Marcis pityusensis (Lacerridae) TSD Viets et d. 1994 Podarcis erhardii (Lacertidae) zw Oimo et d.1990 hum vivipam (Lacertidae) zw Janzen and PaiAstis 1991b Gallotfa (Lacerridae) 2W oh-1101986 Rip(Arnphisbaenidae) zw Cole and Cans 1987 GymnophthaImw (Gymnophthalinidae) XY Cole et al. 1990 Cnemidophm (Teiidae) XY Janzen and Paukstis 199 1b Ameiva (Teiidae) XY Peccinini-Seak and de Ameida 1986 Chamaeieo (Chamaelemidae) H Vie= et al. 1994 Agam (Agamidae) TSD et a]. 1994 Fogom (Agamidae) H nets et a]. 1994 Sasiliscw (Iguanidae) H Viets et al. 1994 Sceloporus (Iguanidae) XY Janzen and Paukatis 199lh Amlis (Iguanidae) XY Janzen and Paukstis 1991b Crotaphytw (Iguanidae) H Wets et a]. 1994 Dipsosauw (Iguanjdae) H Janzen and Paukstis 1991b Trof"duw (Ignanidae) XY Janzen and Paukstis 1991b Vamnus ~lwtor(Varanidae) TSD Wets et al. 1994 Varanw acanthurw (Varanidae) 2W lanzen and Pauksds I 991b SERPENTBS 2W Bull 1983, Olmo 1986 AVES zw Bull 1983 CROCODIU A TSD Deeming, Chapter 4

Note: Reviews that contain the relevant information are ated in many cases in the table to minimize the length of the literature ated section Please consult those reviews for citations m the original solutes OF the research. 1-1 = homornorphic sex chromosorne~ XT = male hctcrogamcty, ZW = female httcrogamtty, and TSD = ternpcratuitdepcnctent sex dettminadon. For the purpose of this analysis hcterogamecy implies hctcromorphicsea chroinosomes as well.

the former two instances, TSD appears to have been derived The majority of scenarios regarding evolutionary transitions from ancestors possessing female heterogamety; no firm between SDMs, including within the vertebrate lineage, conclusion can be drawn for agamids. Different types of have been qualitative, although clearly based on an apprecia- GSD have also arisen independently within squamates, al- tion of the historical origins of different taxonomic groups. chough the polarity and specific nodes of the transitions are Widespread adoption of such "tree thinking," accompanied often unclear (e.g., Is male heterogamety, female heterogam- by dramatic improvements in phylogenetic tree reconstruc- ety or TSD the basal SDM in gekkonids?).These transitions tion and comparative analysis, has aided rigorous quantita- are greatly clarified within turtles, however. Different types tive evaluation of many analogous evolutionary questions of GSD have originated in turtles a minimum of six times: (e.g., Geffeney et al. 2002). The formal modem compara- (I) trionychds, (2) chelids, (3) staurotypids, (4) emydids, tive analyses of SDM evolution in vertebrates herein, based and (5 and 6) twice in batagurids. Once again, both major on current understanding of phylogenetic relationships and categories of SDMs are identified as having originated SDM distribution, are instructive about the phylogenetic many times within vertebrates. loci where SDM transitions probably occurred and identify crucial taxa to target for further empirical work. These Implications analyses also provide a launching point for further discus- sion of the implications of this striking phenotypic diversity Biologists have long struggled with intriguing and impor- for the adaptive significance of TSD and its molecular ge- tant questions surrounding the origins of different SDMs. netic and physiological underpinnings in vertebrates. E3 FISI ^A- AMF 1 Pelomedusa -1 Pelusios a Ernydura 1=1

ci 1 a Claudius JB Sternotherus 1 1Dermatemys 1 Chelydra 1 Dermochelys IChelonia 1 Caretta /in Calemys

1 Emys m Deirochelys 1Trac hernys A 1 Malaclemys m Chrysemys

1 1Gopherus lTestudo SPHENODONTIA IColeonvx

-.-..- J,4 J,4 1 Gekko gecko / Â Gekko japonicus a Gehyra 1 Phelsuma

-- ~ -- \m ~~natodes 0 Scincella A n Eurneces

~FDLacerta vivipara dallntia

GSD vs. TSD .-.am-.. - /inChamaeleo unordered Agama -a Pogona GSD 1 Basiliscus TSD A 1 Scelo~orus - polymorphic KOIOIll equivocal

Figure 13.1 Parsimony analysis of SDM evolution on a vertebrate phylogeny, emphasizing lepidosaurs and turtles. This comparative analysis of GSD vs. TSD was conducted in MacClade 4.0 (Maddison and Maddison 2000) using broadly recognizedrelationships among major amniote lineages. Information on systematics and SDMs (black = TSD,white = GSD) is provided in Table 13.1. 4-a E: Lizards Turtles But first the authors present a cautionary note concern- node distances are short (P. Lewis, pen. comm.). The au- ing some of the limitations of comparative phylogenetic thors also assumed that different patterns of TSD are varia- approaches. Like any analysis, garbage in equals garbage tions on a homologous theme and thus could be treated as our. If the phylogenetic relationships or SDMs assigned to a single trait (sensu Deeming and Ferguson 1988). To as- particular tasa are incorrect, the resulting conclusions may sume otherwise would alter some of the conclusions (results fail as well (e.g., Losos 1994). The authors have attempted not shown). In the end, the reader must thus recognize the to minimize this potential problem by working at higher potential limitations of the comparative analyses and ac- taxonomic levels with well-supported phylogeneuc hypothe- cept the authors' interpretations with appropriate caution. ses. Even so, variation exists in the hypothesized phylo- Regardless of any methodological concerns, the au- genetic relationships among major vertebrate groups (e.g., thors believe that the comparative phylogenetic approaches Hedges and Pohng 1999). Fortunately this variation does adopted in this chapter have provided dear tests of several not alter the main conclusions of this chapter (results not hypotheses and have thereby generated a few robust con- shown). However, more importantly, lepidosaurian rela- elusions about SDM evolution in vertebrates. Above all, the tionships are not resolved with confidence for all branches ancestral state of sex determination in vertebrates is almost on the tree. A well-supported phylogenetic hypothesis for certainly GSD (Figure 13.2) (contra Ohno 1979). TSD has gekkotan lizard genera, in particular, would likely lead to a been clearly documented in fishes, but most cases occur dramatic improvement in resolving the polarity of SDM within a single ; there are many more recognized in- evolution in sauropsids. stances of GSD in those basal vertebrate lineages (Conover, The authors are less assured of the validity of all desig- Chapter 2). Moreover, no amphibians (Hayes 1998; Chardard nations of SDMs, particularly for many fish species and for et al., Chapter 7) or mammals (Bull 1983) can be said to the less common and /or Afro-Asian sauropsids (Valenzuela have TSD, properly defined (Valenzuela et al. 2003). Even et al. 2003; Harlow, Chapter 5;Conover, Chapter 2). Informa- so, there is some circumstantial evidence for TSD (or more tion on SDMs for taxa exposed to more detailed, replicated likely a thermally modified GSD system) in lampreys (Bea- work is simply more robust. hi the authors' opinion, follow- mish 1993), which is the sister taxon to the rest of verte- up investigation of SDMs in , Kachug~,Tam- brates, and next to nothing is known about SDMs in the tola, PoddrrU* Bipes. Cftflmaeleo,and Varawwould strengthen phylogenetically important chondrichthyaii, coelacanth, our understanding greatly (see Figures 13.1 and 13.2). More- and lungfish lineages (Maddock and Schwartz 1996; Devlin over,a number of key lineages, especially (1) numerous fish and Nagahama 2002; Conover, Chapter 2). Although much species, (2) trionychid, platysternid, and batagurid turtles, crucial work on SDMs in basal vertebrates is necessary, the and (3) eublepharid, gekkonid, scincid, lacertid, amphis- authors nonetheless conclude from their comparative ex- baenid, agamid, varanid, xantusid, xenosaurid, heloderma- animation that some form of GSD exemplifies the ancestral tid, anniellid, dibarnid, cordylid, and anguid lizards, have condition of sex determination in vertebrates: GSD was first! not been sufficiently explored for SDMs. Basic research on The authors' parsimony-basedcomparative analyses (esp. SDMs in a11 of these groups would again considerably im- Figure 13.2) also lend credence to other key hypotheses prove our view of SDM evolution in vertebrates. regarding the evolution of TSD in vertebrates. In particular, Another important issue involves the statistical assump- the authors found that TSD appears to be the ancestral con- tions underlying the comparative method employed. Some dition of sex determination in sauropsids. The results fur- or all of the actual evolutionary processes involved in the ther support contentions (Ewert and Nelson 1991; Janzen transitions between SDMs in vertebrates may have fol- and Paukstis 1991a) that TSD has been lost at least six times lowed different "rules" (Valenzuela, Chapter 14). Although in turtles and has originated at least three times in lizards. the authors were explicit in describing the assumptions be- Thus, SDMs exhibit remarkable phylogenetic lability: both hind their analyses, it nonetheless remains the case that, for major categories of SDMs have been "lost" and "found" example, evolutionary transitions of character states be- numerous times in vertebrates. It is curious, however, that tween lineages may not be best or always reflected by the temperature is the cue in all known independent origins of criterion of parsimony (e.g., Collins et al. 1994). Alternative ESD in vertebrates (although Conover, Chapter 2, men- methods such as maximum likelihood Markov-transition tions the possibility of pH-dependent sex determination in approaches (Pagel 1999) may be better suited in such dr- fish), when a variety of factors are involved in other groups cumstances, particularly where branch lengths or inter- (reviewed in Korpelainen 1990, 1998). Why this should be so is unclear, but it might reflect the widespread importance microevolutionary analyses directed toward the derived of temperature in affecting sex-specific fitness attributes of cases of TSW in lacerrids, agamids, and varanids (and of vertebrates (reviewed by Deeming and Ferguson 1991b; GSD in turtles; Janzen and Paukstis 1988) will prove to be Janzen and Paukstis 1991a; Shine 1999; but see Rhen and the most fruitful and enlightening research projects. Alter- Lang,Chapter 10; Valenzuela, Chapter 14) or perhaps insuffi- natively, an explanation for the adaptive significance of TSD dent exploration by biologists of alternative environmental in turtles, crocodilians, sphenodontians, and perhaps gekko- signals that might influence sex determination in this group. tans may lie outside the framework of the Charnov-Bull What does the pattern of transitions between SDMs re- model (e.g.,in sex ratio evolution). veal about the evolutionary forces and molecular mecha- The authors' phylogenetic historical analyses also shed nisrns involved? Most researchers have proceeded on [he light on, and provide guidance for research into, the mech- expectation that TSD in vertebrates is adaptive. However, anistic underpinnings of TSD in vertebrates, a hot (no pun despite considerable effort devoted to detailed study of a intended) topic these days. One general prediction from the variety of sauropsid ma (especially turtles, crocodilians, parsimony-based comparative assessments is that the basic and eublepharids), the adaptive significance of TSD has molecular and physiological pathways involved in TSD in only been clearly demonstrated for one species of fish turtles, crocodilians, sphenodontians, and perhaps gekko- (Conaver 1984 and Chapter 2; see alsoVaIemela, Chapter tans should be broadly conserved. Different patterns of 14). This outcome is all the more surprising because one TSD, for example, occur in various species in these lineages elegant theoretical framework (Charnov and Bull 1977) has (Ewert et a]. 1994; Viets et al. 1994; Deeming, Chapter 4; robustly explained the adaptive significance of other forms Nelson et al., Chapter 61, yet the underlying major develop- of BSD in numerous taxonomically diverse invertebrates(re- mental pathways should be similar (e.g., Smith et al. 1999a) viewed in Bull 1983; Korpelainen 1990, 1998; Shine 1999). if they share a common ancestry. The authors eagerly await The lack of success of the Chamov-Bullmodel in explaining empirical tests of this conjecture. the existence of TSD in sauropsids has led some researchers On the other hand, the mechanics involved in the newer to question the current adaptive significance of this unusual independent origins of TSD in vertebrates might very well SDM (e.g., Janzen and Paukstis 1988; Girondot and Pieau be different in each case. Still, it is puzzling to note that the 1999; Valenzuela, Chapter 14). type of TSD appears to be identical in all four instances Our comparative analyses provide a fresh perspective (probably TSD Ib)! Why this should be so, other than by on this overarching issue. With the exception of the origin chance, remains to be explained.The pattern (in addition to of TSD at the sauropsid/marnrnal node, the remaining the mechanism itself) may be adaptive in the same way in clearly independent cases of TSD (fish, lacertids, agamids, each case, but the phenotypic similarity might instead re- and varanids;Figure 13.2) have most likely arisen relatively fleet constraints on the molecular and physiological path- recently. If so, and if these latter four derived instances of ways involved in evolving TSD from GSD. Empirical work TSD were in fact driven by the forces of natural selection, on fish, lacertids, agamids, and varanids is greatly needed to then the signature of adaptation may be more likely to be distinguish between these competing explanations. detected therein than in the ancient origin of TSD at the Fascinating to many, the remarkable diversity of SDMs sauropsid/rnamrnal split. In the authors' view then, effort in vertebrates holds an elevated position in the pantheon of expended on evaluating the adaptive significance of TSD in longstanding evolutionary enigmas. In particular, how the turtles, crocodilians, sphenodontians, and perhaps gekko- unusual mechanism of TSD arose and withstood untold tans is potentially misdirected. TSD in these taxa may in- environmental upheavals over geological eons has long cap- stead be quasi neutral (sensu Janzen and Paukstis 1988; tivated the attention of scientists and interested laypersons Girondot and Pieau 1999; Valenzuela, Chapter 141, and the al&e (e.g., Deeming and Ferguson 1989~).How taxa with signature of adaptation, if it ever existed in this case, has TSD will respond to current rapid climatic changes and simply attenuated over the 300+ million years since the ar- swift habitat modifications are especially timely and unset- dent transition event occurred. Indeed, is it any wonder tling questions, particularly since many of these species are that none of the considerable experimental effort devoted already imperiled. Whether the prediction is more dire (e.g., to turtles, crocodilians, and eublepharids has produced a Janzen 1994a; Morjan 2002) or more hopeful (e.g., Rhen broadly convincing explanation for the adaptive significance and Lang 1998; Girondot and Pieau 1999), research on TSD of TSD across these taxa? If the authors are correct, then in vertebrates promises to continue to challenge us practi- cally and scientifically.The authors hope that their compara- sity) for his generosity in sharing his unpublished Rag1 lepidosaur tive analyses lend guidance to researchers intending to phylogeny;Aaron Bauer, Lee Grismer, and Arnold Huge for help- tackle these and related evolutionary challenges. ingclarify gekkotan syscernatics;and Mike Ewert,John Wiens, and an anonymous reviewer for their constructive criticisms on the manuscript. Funding of Janzen's most recent research in this area Acknowledgments~Manythanks to Nicole Valenzuela for invit- has been provided by U.S. National Science Foundation grants ing us to write this chapter; Ted Townsend (Washington Univer- DEB-9629529and DEE-0089680,