phylogenetic analysis of sperm storage in female squamates

In: Animal Reproduction: New Research Developments ISBN 978-1-60692-595-9 Editor: Lucas T. Dahnof © 2009 Nova Science Publishers, Inc.

Chapter 5

PHYLOGENETIC ANALYSIS OF SPERM STORAGE IN FEMALE SQUAMATES

Mallory E. Eckstut∗1,2, David M. Sever1, Mary E. White1 and

Brian I. Crother1 1Department of Biological Sciences, Southeastern Louisiana University,

Hammond, LA 70402, USA 2Present Address: School of Life Sciences, University of Nevada, Las Vegas, 4505 Maryland Parkway, Las Vegas, NV 89154, USA

ABSTRACT

The storage of sperm by females is a common but variable phenomenon observed in a number of vertebrate lineages. The utility of female sperm storage has been widely debated, but has been suggested to benefit species by lengthening breeding seasons as well as enhancing colonization abilities. Additionally, the variation in sperm storage traits has been suggested to have value for assessing phylogenetic hypotheses. To date, little is known regarding the evolutionary and ecological implications of sperm storage in female squamates (lizards and snakes). Utilizing previous studies of reproductive morphology and ultrastructural studies and three competing squamate phylogenies (one morphological and two molecular, including a new hypothesis of 611 taxa), we address character state evolution of sperm storage characters and overlay a variety of ecological factors in order to assess ecological function. We also test the hypothesis that sperm storage may offer reciprocal illumination in choosing among hypotheses of squamate phylogeny. At present there is minimal value for phylogenetic inference from these reproductive characters. We found that several sperm storage characters are relatively conserved across the sampled squamates (including presence of sperm storage and sperm storage tubules (Ssts), embedding of sperm, and sperm storage with eggs in utero). Alternatively, location of Ssts, secretions within the Ssts, and length of sperm storage are highly variable traits. We suggest that these differences are correlated with ecological differences in the reproductive tactics of each species. Because of limited data available for many of the taxa, however, statements regarding the ecological utility of the traits are highly

∗ Corresponding Author: Email: [email protected]

Mallory E. Eckstut, David M. Sever, Mary E. White et al. 186

speculative. Additional studies will likely find that these traits are more variable than currently recognized. Despite the limited amount of information currently available, our preliminary results indicate that further comparative work on sperm storage traits may be useful in phylogenetic analyses.

INTRODUCTION Female sperm storage has frequently been observed in many animals, including

arthropods, mollusks, and all classes of jawed vertebrates (Sivinski 1980; Todd et al. 1997; Uhl 2000; Sever and Hamlett 2004). Sperm storage in females allows: (1) the decoupling of oogenesis, ovulation and fertilization from mating activity; (2) delay of fertilization until environmental conditions are most favorable; (3) extension of the mating period; facilitates multiple matings; (4) enhances colonization abilities; and (5) provides the conditions for sperm competition (Fox 1963; Conner and Crews 1980; Sever and Brizzi 1998). Functional sperm storage, which we define as the ability to utilize sperm to fertilize multiple clutches, is especially important in colonization. If a female can functionally store sperm, she would be the only one necessary to found a new population, whereas without sperm storage, a mating pair would need to be present and have the capacity to locate one another (Conner and Crews 1980).

Variations in reproductive traits of organisms, including the ability to store sperm, often exist among related taxa, and thus present an important area of research in evolutionary biology, given their direct impact on fitness and survival. The nature of sperm storage is variable among even the most closely related species, including variation within four species of gekkonids (geckos), a group which is noted for conservatism in reproductive traits (Girling et al. 1998). Geckos comprise a squamate lineage that is often thought to be reproductively conservative, in part because of a fixed clutch size of two calcareously-shelled eggs throughout all but a few species in the entire family, which includes more than 75 genera and 1000 species (Girling et al. 1998; Pianka and Vitt 2003; Rose and Barbour 1968). Sperm storage characters were shown to have important family-level phylogenetic trends in other organisms such as salamanders (Sever and Brizzi 1998), and potential phylogenetic implications are also seen across the subphylum Vertebrata as well as within the sub-order Squamata (Sever and Hamlett 2002).

Sever and Hamlett (2002) addressed the evolution of lizard sperm storage, summarizing the variability found within the group. They compiled previous studies of lizard sperm storage to propose a phylogenetic hypothesis for the evolution of sperm storage tubule (Sst) locations in lizards, and they separately compared Sst characters based on transmission electron microscopy (TEM) studies. More recently, Sever and Hopkins (2004) compared many aspects of lizard sperm storage and reported a large amount of variation both between and within families.

Complicating issues of assessing sperm storage in a phylogenetic context, there are currently several competing phylogenetic hypotheses proposed for the squamates (Townsend et al. 2004; Lee 2005; Vidal and Hedges 2005). Sperm storage characters have previously been hypothesized to be valuable for phylogenetic inference in squamates, because of the variety of conserved and variable traits (Sever and Hamlett 2002). However, the lack of a robust phylogenetic hypothesis for the group as a whole makes it difficult to assess the

Evolution of Squamate Sperm Storage 187

phylogenetic utility of sperm storage characters and to test hypotheses regarding development of these characters.

This chapter expands on the Sever and Hopkins (2004) study on the evolution of lizard sperm storage by addressing two hypotheses: i) that sperm storage characters will offer reciprocal illumination for squamate phylogeny (congruence between sperm storage evolution and the squamate phylogeny), and ii) that character state evolution of sperm storage characters will vary in relation to utility of the characters. Some characters that were examined are: presence of Ssts, location of Ssts in the oviduct, characteristics of Sst linings, and length of sperm storage. Several of these characteristics are depicted using light microscopy in Figure 1.

The evolutionary histories of traits are not always tied to functional adaptation (Gould and Lewontin 1979). However, because of the hypothesized functional importance of sperm storage, we assumed adaptive utility of the sperm storage character states unless otherwise stated in the respective sections.

Figure 1. Representation of several sperm storage characters using light microscopy. A. Nactus multicarinatus (Squamata: Gekkonidae) oviduct stained with hemotoxilin-eosin (H&E) for basic cytological analysis, emphasizing three basic regions associated with all squamate lineages: infundibulum, uterus, and vagina. B. Hemidactylus turcicus (Squamata: Gekkonidae) sperm storage tubules stained using H&E, depicting both Ssts and sperm embedded within the Ssts. C. Nactus multicarinatus Ssts stained with periodic-acid sciff and alcian blue (PAS/AB), depicting AB+ (carboxylated glycosaminoglycans present) secretory activity throughout the Ssts of the gekkonid species during a reproductive season.


Furthermore, Sever and Brizzi (1998) noted that the phylogenetic value of sperm storage characters varies greatly, and many studies concerning squamate sperm storage vary regarding which issues they address. For that reason, some of the characters analyzed overlap between studies, and others were not used for all studies. Each trait was analyzed separately in this chapter because of both the hypothesis of variable phylogenetic value between characters as well as the differing amounts of data available for each character.

MATERIALS AND METHODS

Phylogenetic Inference In addition to mapping the reproductive characters on published squamate phylogenies,

we have included a new phylogenetic hypothesis for squamates. This new hypothesis is based on a singular nuclear gene, c-mos, but to date is the most densely sampled phylogenetic study of squamates with 611 species included. The original sequences and the data set can be obtained upon request. The c-mos sequences were data-mined from Genbank and originally included multiple duplicates as well as sequences that could not be aligned and were considered either mislabeled or contaminants and removed from the study. The sequences varied in length from 192 – 953 positions. The sequences were aligned by eye in Sequencher 3.1.1 (GeneCodes, Ann Arbor, MI). For the subsequent analyses the sequences were trimmed to position 178 to 754 to reduce the number of missing characters and to maximize the aligned sequence. The aligned data were analyzed within parsimony (MP) and likelihood (ML) frameworks. Sphenodon punctatus (the tuatara) was the outgroup for all analyses.

The MP analyses were conducted with the software package TNT (Tree Analysis Using New Technology, Goloboff et al. 2000). All heuristic searches employed the parsimony ratchet, sectorial searches, tree fusing, and drifting method (Goloboff 1999; Nixon 1999). Specific parameters for these searches can be obtained by request. Because of the immense number of possible trees there is a real concern that tree space cannot be adequately searched with a single run so the analyses were run 25 times with 100 random addition iterations for every run. The ML analyses were conducted with the software package RAxML-III (Stamatakis et al. 2005), which is a fast ML program developed specifically for the analysis of large data sets. The equivalent of the GTR + G + I model (MIX) and a non-parametric bootstrap (CAT) were used to infer and evaluate the ML inference. As an indirect measure of character support, nonparametric bootstrap proportions were obtained via 2000 fast heuristic search iterations (Felsenstein 1985).

Reproductive Data Collection Sperm storage characters and character states were obtained from several squamate taxa

(Table 1). Analyzed sperm storage characters [proposed by Sever and Hamlett (2002) and Sever and Hopkins (2004)] included (Table 2): Presence of sperm storage, length of sperm storage, sperm presence with eggs in utero, embedding of sperm, multiple clutches, sperm storage tubule (Sst) presence, Sst location, Sst structure, and Sst linings.


Table 1. References for recorded sperm storage data

Order Suborder Family Species Reference

Aves

Phasianidae Gallus domesticus Bakst (1987), King et al. (2002)

Crocodilia Alligatoridae Alligator mississipienssis Gist et al. (2008)

Testudines Widespread;

6 families Widespread;14 species Gist and Jones (1989) Squamata Sauria Agamidae Psammophilus dorsalis Srinivas et al. (1995) Agamidae Calotes versicolor Kumari et al. (1990) Chameleonidae Chameleo sp. St. Girons (1962) Crotaphytidae Crotaphytus collaris Cuellar (1966) Gekkonidae Coleonyx variegatus Cuellar (1966)

Gekkonidae Phyllodactylus homolepidurus Cuellar (1966)

Gekkonidae Hemidactylus frenatus Murphy-Walker and Haley (1996)

Gekkonidae Hemidactylus turcicus Eckstut et al. (2009a) Gekkonidae Nactus multicarinatus Eckstut et al. (2009b) Lacertidae Acanthodactylus scutellatus Bou-Resli et al. (1981) Phyrnosomatidae Urosaurus microsctatus Cuellar (1966) Phyrnosomatidae Uta stansburiana Cuellar (1966) Phyrnosomatidae Uta squamata Cuellar (1966) Phyrnosomatidae Uta palmeri Cuellar (1966) Phyrnosomatidae Callisaurus dragonoides Cuellar (1966) Phyrnosomatidae Holbrookia propinqua Adams and Cooper (1988) Phyrnosomatidae Holbrookia elegans Cuellar (1966) Phyrnosomatidae Phyrnosoma cornutum Cuellar (1966) Phyrnosomatidae Sceloporous undulatus Cuellar (1966) Phyrnosomatidae Sator grandaevus Cuellar (1966) Polychrotidae Anolis carolinensis Conner and Crews (1980) Polychrotidae Anolis sagrei Sever and Hamlett (2002) Polychrotidae Anolis pulchellus Ortiz and Morales (1974) Scincidae Hemiergis peronii Smyth and Smith (1968) Scincidae Eumeces egregius Schafer and Roeding (1973)

Scincidae Mabuya scincoides Sarkar and Shivanandappa (1989)

Scincidae Scincella lateralis Sever and Hopkins (2004)

Serpentes Colubridae Thamnophis sirtalis Hoffman and Wimsatt (1972)

Colubridae Seminatrix pygaea Sever and Ryan (1999) Viperidae Agkistrodon piscivorous Siegel and Sever (2008)

To analyze possible ecological influences of sperm storage evolution, the following

characters were also recorded for analysis with sperm storage characters (Table 3): Reproductively active female snout-vent length (RAF SVL), number of eggs/clutch, season of vitellogenesis, season of mating, season of clutch deposition, habitat location (aquatic, terrestrial, semi-arboreal, or arboreal), daily activity (diurnal or nocturnal), and diet (carnivorous, herbivorous, or omnivorous).


Table 2. Character state analysis and coding. For Sst Location, PV indicates posterior vagina, AV = anterior vagina, Inf = infundibulum, MAV = mid and anterior vagina, AV

+ Inf = anterior vagina and infundibulum, V = entire vagina, and V + Inf = entire vagina and infundibulum

Character State Code Represented Taxa Sst Location (A) PV 1 Polychrotidae (1), Scincidae AV 2 Agamidae, Aves, Chameleonidae, Polychrotidae (2) Inf 3 Colubridae (1), Gekkonidae, Lacertidae, Testudines,

Viperidae MAV 4 Crotaphytidae, Phrynosomatidae (1) AV + Inf 5 Phrynosomatidae (2), Crocodilia V + Inf 6 Colubridae (2) Sst Location (B) V 1 Agamidae, Aves, Chameleonidae, Crotaphytidae,

Phrynosomatidae (1), Polychrotidae, Scincidae Inf 2 Colubridae (1), Gekkonidae, Lacertidae, Viperidae V + Inf 3 Colubridae (2), Phrynosomatidae (2), A Sst Secretions Proximal 1 Aves, Polychrotidae Distal 2 Colubridae (1), Phrynosomatidae, Scincidae,

Viperidae Throughout 3 Agamidae, Colubridae (2), Lacertidae, Gekkonidae Storage Length <1 month 1 Aves 2.5 months 2 Phrynosomatidae 4 months 3 Agamidae, Gekkonidae (1) 6 months 4 Lacertidae > 6 months 5 Gekkonidae (2), Testudines

Table 3. References for recorded ecological data Order Suborder Family Species Reference Aves Phasianidae Gallus domesticus Caceci (2008)

Squamata Sauria

Agamidae Psammophilus dorsalis

Radder et al. (2005), Radder et al. (2006)

Agamidae Calotes versicolor Enge and Krysko (2004), Kumari et al. (1990), Zug et al. (2006)

Chameleonidae Chameleo sp. Herrmann and Herrmann (2005)

Crotaphytidae Crotaphytus collaris

McAllister (1985), Trauth (1978)

Gekkonidae Coleonyx variegatus Parker (1972)

Gekkonidae Hemidactylus frenatus Rose and Barbour (1968)

Lacertidae Acanthodactylus scutellatus Perez-Mellado (1992)

Phyrnosomatidae Callisaurus draconoides Pianka and Parker (1972)


Order Suborder Family Species Reference

Phyrnosomatidae Holbrookia propinqua Judd (1976)

Phyrnosomatidae Sceloporous undulatus

Gillis and Ballinger (1992), Lemos-Espinal et al. (2003)

Polychrotidae Anolis carolinensis Conner and Crews (1980),

Lovern et al. (2004) Polychrotidae Anolis sagrei Lee et al. (1989)

Polychrotidae Anolis pulchellus Gorman and Licht (1974), Goto and Osbourne (1989), Ortiz and Morales (1974)

Scincidae Hemiergis peronii Smyth and Smith (1968) Scincidae Eumeces egregius Mount (1963) Scincidae Scincella lateralis Brooks (1967), Preest (1991)

Serpentes Colubridae

Thamnophis sirtalis

Larsen et al. (1993), Shine et al. (2000)

Colubridae Seminatrix pygaea Siegel et al. (1995)

Viperidae Agkistrodon piscivorous

Aldridge and Duvall (2002), Beyer (1893), Blem (1977), Burkett (1966)

Sperm Storage Data Analysis Several squamate species from several studies were utilized in this chapter (Table 1).

Each character was analyzed individually to account for the fact that not all studies contributed the same amount of information nor are all sperm storage characters hypothesized to have equal phylogenetic value (Sever and Brizzi 1998).

Each character state was coded numerically and there was no prior assumption of a character state being ancestral or derived. Three competing squamate phylogenies were utilized for this project, including the recent nuclear molecular phylogenies of Vidal and Hedges (2005), which utilized 19 taxa and 6192 basepairs of nuclear DNA, and a new phylogeny described in this chapter, utilizing 611 taxa and 953 basepairs of nuclear DNA, as well as the morphological phylogeny proposed by Lee (2005) (Table 4). The Townsend et al. (2004) tree was discarded from these analyses because it is entirely consistent with the Vidal and Hedges (2005) tree, but there is substantially less resolution.

The outgroup for these analyses was the order Aves (birds), specifically utilizing the domestic fowl (Gallus domesticus), as this is the closest outgroup which has had all relevant sperm storage characters analysed. Tuatara (Sphenodon punctatus) would be an ideal outgroup, but little is known; however, it has been proposed that they lack specialized Ssts (Gabe and Saint Girons 1964). These procedures follow protocols of Sever and Brizzi (1998) and Sever and Hamlett (2002). Crocodilians (crocodiles and alligators) and testudines (turtles) were also considered as possible ancestral states in analysis, but birds were the primary outgroup because of the extensive amount of literature on avian sperm storage. The placement of turtles in amniote phylogenetics is widely debated, with hypotheses ranging from turtles as the basal amniote; sister to archesaurs (crocodilians and birds); sister to lepidosaurs (tuatara and squamates; nested within Archesauria and sister to crocodilians; and sister to Sauria (both archesaurs and lepidosaurs) (Meyer and Zardoya 2003, and references within).


Table 4. Phylogenetic hypotheses analyzed and associated references

Phylogenetic Hypothesis

Characters Analyzed Inference Method Study Reference

Morphological Osteological, Soft Anatomical, and Ecological

Maximum Parsimony Lee (2005) p. 39, Figure 8A

Molecular 953 bp: C-mos Maximum Parsimony This chapter This chapter Molecular 6192 bp: C-mos,

RAG1, RAG2, R35, HOXA12, Jun, α-enolase, amelogenin, MAFB

Maximum Likelihood / Bayesian

Vidal and Hedges (2005)

p. 1003, Figure 1

Furthermore, turtles and crocodilians have Ssts, but the structure differs from birds and

squamates, because their Ssts are tubal-albumin glands. For this reason, in analysis of Sst structure, these two groups were discarded. However, for Sst location, they were still considered. Each character was optimized using parsimony on each of the squamate phylogenies using MacClade version 4.08 (Maddison and Maddison 2000), and character states were subsequently optimized by hand using delayed transformation (DELTRAN) and accelerated transformation (ACCTRAN).

Tree length (TL), consistency index (CI), retention index (RI), and rescaled consistency index (RCI) were calculated for each character in MacClade version 4.08 (Maddison and Maddison 2000).

Evolution of character states was qualitatively assessed by frequency of each character state on the trees and evaluated by the number of convergences, and reversals and quantitatively assessed using descriptive statistics (TL, CI, RI, and RCI). Ecological characters were subsequently analyzed in conjunction with the sperm storage characters and qualitatively assessed to identify potential relationships.

To assess potential reciprocal illumination of squamate phylogenies, the resulting trees were both qualitatively analyzed for presence of monophyly of traits and quantitatively assessed using descriptive statistics (TL, CI, RI, and RCI) for number of steps per tree (TL), amount of homoplasy (CI) (Kluge and Farris 1969), amount of synapomorphic value (RI) (Farris 1989a, 1989b), and amount of noise for each character (RCI), which is the product of CI and RI and is predominantly used for character weighting in phylogenetic analysis (Farris 1989b).

RESULTS

Phylogenetic Inference The most parsimonious solution found from MP analyses was 4634 steps. In the 25

separate runs with 100 random addition iterations, a total of 128 most parsimonious trees (mpts) were found on multiple islands.

Almost certainly more islands of the same tree length exist in the tree space, but it should be noted that the hypotheses vary mostly at higher levels in the tree and not at the levels of


interest for this chapter. The ML showed significantly less resolved structure at higher level relationships.

As such, the MP tree (Appendix 1) will be used in the subsequent analyses of the reproductive traits.

In general, snakes are monophyletic, sister to an iguanid-anguimorph clade, with acrodonts (agamids and chamaeleonids) a monophyletic clade sister to snakes-iguanids-anguimorphs (the ML tree inferred acrodonts as sister to the iguanids). A large resolved scincoid clade containing lacertids, rhineurids, amphisbaenians, cordylids, dibamids, xantusiids and scincids is sister to the aforementioned large group. Gekkonids were inferred to be the penultimate basal lineage while teiids and gymnophthalmids were inferred to be the sister group to all other squamates.

Bootstrap proportions for squamate families were all relatively high (>70%), indicating strong synapomorphic signal for those clades. However, for most of the deeper nodes inferring relationship among families, bootstrap proportions were low (<70%). Nonetheless, the density of sampling makes this an important contribution to our knowledge of squamate phylogeny.

Evolution of Sperm Storage Characters The majority of analyzed sperm storage characters depicted little, if any, variation among

character states (Figures 2-5). Sperm storage and associated Ssts were present in all species with the exception of the skink Mabuya scincoides (Sarker and Shivanadappa 1989). Additionally, all species except for Scincella lateralis (Scincidae) (Sever and Hopkins 2004) and Nactus multicarinatus (Gekkonidae) (Eckstut et al. 2009b) retained sperm while eggs were in utero.

Figure 2. Evolution of sperm storage length using parsimony optimization with Aves as the designated outgroup. Colors indicate variant character states, while lined patterning indicates unresolved character states. A) Morphological phylogeny (Lee 2005) depicting gradual increase and then decrease in length of sperm storage. B) Nuclear molecular phylogeny (Vidal and Hedges 2005) indicating an initial increase in length from 0.5 months to 9 months, followed by a gradual decrease in sperm storage length. C) Nuclear molecular phylogeny (this chapter) also indicating a large initial increase and subsequent decrease in sperm storage length.


Figure 3. Evolution of Sst secretion location using parsimony optimization with Aves as the designated outgroup. Colors indicate variant character states, while lined patterning indicates unresolved character states. A) Morphological phylogeny (Lee 2005) showing that either secretions through as well as distal secretions evolved twice or secretions throughout evolved once, distal secretions evolved twice, and proximal secretions revolved once. B) Nuclear molecular phylogeny (Vidal and Hedges 2005) showing secretions throughout evolving once, distal secretions evolving twice, and proximal secretions revolving once. C) Nuclear molecular phylogeny (this chapter) also indicating the same pattern as the solely nuclear analysis. Analysis of these patterns indicates that the molecular phylogenies – B and C - depict the most parsimonious evolution of the Sst secretion locations.

Figure 4. Evolution of Sst location (A) using parsimony optimization with Aves as the designated outgroup. Colors indicate variant character states, while lined patterning indicates unresolved character states. A) Morphological phylogeny (Lee 2005) indicating that there is a single evolution of solely infundibular Ssts, and multiple evolutions of variant vaginal Ssts within a vaginal Sst clade, as well as two evolutions of infundibular and vaginal Ssts occurring in unison. B) Nuclear molecular phylogeny (Vidal and Hedges 2005) showing the most likely scenario of infundibular Ssts evolving once, with a reversal back to vaginal Ssts on three independent occasions. C) Nuclear molecular phylogeny (this chapter) indicates the least amount of Sst location resolution, and it is unclear if the number of times vaginal and infundibular Ssts evolved.

Sst Location

The location of Ssts was the most highly variable character analyzed, with six recorded character states; Ssts in the anterior vagina is the ancestral state if birds represents the ancestral state, and Ssts in the posterior infundibulum is the ancestral state if turtles represent the ancestral state. The morphological phylogeny suggests that the basal squamate clade


Figure 5. Evolution of Sst location (B) using parsimony analysis with Aves as the designated outgroup. Colors indicate various character states, while lined patterning indicates unresolved character states. A) Morphological phylogeny (Lee 2005) indicating vaginal Ssts as the ancestral state, and infundibular Ssts as the derived state, with one independent evolution of vaginal Ssts (Scincidae), one evolution of vaginal in addition to infundibular (Colubridae 2), and one evolution of infundibular in addition to vaginal in the vaginal Sst clade (Phrynosomatidae 2). B) Nuclear molecular phylogeny (Vidal and Hedges 2005) indicates the least amount of Sst location resolution, and the number of times vaginal and infundibular Ssts evolved is unclear. C) Nuclear molecular phylogeny (this chapter) depict a most parsimonious case where infundibular Ssts arose independently three times, with one evolution of infundibular in addition to vaginal Ssts (Phrynosomatidae).

retained vaginal Ssts, with one evolution of Ssts located both in the vagina and infundibulum, and no occurrences of solely infundibular Ssts in this clade.

Additionally, the vaginal Sst clade showed either one evolution of mid and anterior vaginal Ssts and two reversals to anterior vaginal Ssts, or two independent evolutions of mid and anterior vaginal Ssts. In the morphological phylogeny, there was one evolution of an infundibular clade, and within the infundibular clade there was one evolution of Ssts in the posterior vagina, as well as one evolution of Ssts located in both the infundibulum and the vagina (Figure 4a). Each clade independently gave rise to an evolution of posterior vaginal Ssts (Figure 4a).

The analysis of character state evolution lacks a substantial amount of resolution in both molecular phylogenies as a result of the lack of data on several taxa. However, the most parsimonious resolution of the nuclear phylogeny (Vidal and Hedges 2005) depicted the vaginal Sst clade (which is consistent with the morphological vaginal Sst clade) as derived from the infundibular Ssts (Figure 4b). Alternatively, a lack of clarity for the character state reconstruction in this nuclear phylogeny suggests two equally parsimonious situations: either the infundibular clade gave rise to the vaginal Sst clade, or infundibular Ssts evolved twice, and posterior vaginal Ssts gave rise to the vaginal Sst clade as well as two subsequent independent evolutions of infundibular Ssts (this is the result of both lack of resolution in the character state evolution on this phylogeny as well as the lack of sampled taxa). Furthermore, if it is the case that posterior vaginal Ssts gave rise to both clades, either two independent evolutions of infundibular Ssts occurred, or one reversal to vaginal Ssts in colubrids.

These characters were subsequently analyzed grouping all vaginal, infundibular, and both vaginal and infundibular Sst clades (Figure 5). With Aves as the ancestral state, the morphological phylogeny (Lee 2005; Figure 5a) indicates vaginal Ssts as the ancestral state, and infundibular Ssts as the derived state, with one independent evolution of vaginal Ssts


(Scincidae), one evolution of vaginal in addition to infundibular (Colubridae 2), as well as one evolution of infundibular in addition to vaginal in the vaginal Sst clade (Phrynosomatidae 2). The nuclear DNA phylogeny (Vidal and Hedges 2005; Figure 5b), indicates the least amount of Sst location resolution, and the number of times vaginal and infundibular Ssts evolved is unclear. The nuclear phylogeny generated here (Figure 5c) depicts a most parsimonious case where infundibular Ssts arose independently three times, with one evolution of infundibular in addition to vaginal Ssts (Phrynosomatidae). However, if infundibular Ssts are the ancestral state as is seen in turtles, then all three phylogenies have infundibular Ssts retained in squamates, but in the morphological phylogeny (Lee, 2005) the evolution of the vaginal clade occurred in a basal-most squamate clade.

Despite the more parsimonious results of the second Sst location analysis (Sst location B analysis) (Sst location A, TL = 7-8+; Sst location B, TL = 4-5+), the descriptive statistics (Tables 6 and 7) indicate less homoplasy for Sst location A analysis (Sst location A, CI = 0.71 – 0.50; B, CI = 0.50 – 0.40), and a higher amount of synapmorphic value for Sst location A analysis (A, RI = 0.60 – 0.40; B, RI = 0.50 – 0.25). Additionally, the data for Sst location A analysis, while still considerably noisy (RCI = 0.43 – 0.25), were relatively less noisy than the data for Sst location B analysis (RCI = 0.25 – 0.10).

Figure 6. Scatter plot depicting potential relationship between length of sperm storage and Sst secretion location. While there appears to be a preliminary relationship between Sst secretion location and length of sperm storage, more data should be collected to properly assess this relationship.

Sst Linings

All Ssts had ciliated as well as secretory epithelium. However, the location of the secretions varies among taxa, including epithelial secretions that occur distally, proximally,


and throughout the Ssts. For all phylogenies, the ancestral state is proximal secretions. The morphological phylogeny showed two equally parsimonious states: first, secretions throughout the Ssts likely evolved twice, as did distal secretions. Alternatively, secretions throughout evolved once, whereas distal secretions evolved twice, and reversal to proximal secretions once (Figure 3a). Additionally, the molecular phylogenies showed that secretions throughout were first derived from the hypothesized ancestral state of proximal secretions, followed by at least two independent evolutions of distal secretions and one reversal to proximal secretions (Figure 4b-c).

Table 5. Ecological character states associated with taxa. * = Produces one egg at a time, but the oviducts alternate and are continually reproducing, ** = Reproduces every other

year. *** = Anolis sagrei only deposits clutches year round in the tropics. All species examined were carnivorous/insectivorous, except for Gallus domesticus (Aves) and

Callisaurus draconoides (Phrynosomatidae), which were omnivorous Character State Represented Taxa RAF SVL < 50mm Gekkonidae, Lacertidae, Polychrotidae, Scincidae 50 – 100mm Agamidae, Crotaphytidae, Phrynosomatidae 100 – 150mm Chameleonidae > 150mm Aves, Colubridae, Viperidae # Eggs/Clutch 1 Polychrotidae*, Aves 2 Gekkonidae 3 – 12 Chameleonidae, Colubridae (2), Phrynosomatidae,

Scincidae, Viperidae > 12 Agamidae, Colubridae (1) Vitellogenesis Early Spring Agamidae, Crotaphytidae, Phrynosomatidae (1),

Scincidae (1) Late Spring Phrynosomatidae (2) Spring Colubridae, Viperidae Spring – Summer Gekkonidae Summer Scincidae (2) Year Round Aves, Chameleonidae Mating Spring Crotaphytidae, Gekkonidae, Scincidae (1) Spring – Summer Agamidae, Phrynosomatidae Summer Colubridae Fall Scincidae (2) Spring, Summer, Fall Viperidae Year Round Aves Clutch Deposition

Spring – Summer Crotaphytidae, Gekkonidae (1), Polychrotidae (1), Scincidae

Summer Colubridae (2), Phrynosomatidae Summer – Fall Agamidae, Colubridae (1)**, Gekkonidae (2),

Polychrotidae (2), Polychrotidae (3)***, Viperidae** Location Aquatic Colubridae (2), Viperidae Terrestrial Agamidae (1), Aves, Colubridae (1), Gekkonidae (2),

Lacertidae, Phrynosomatidae (1), Polychrotidae (1), Scincidae

Semi-arboreal Phrynosomatidae (2), Polychrotidae (2), Crotaphytidae Arboreal Agamidae (2), Chameleonidae, Gekkonidae (1) Activity Diurnal Agamidae, Aves, Chameleonidae, Colubridae,

Crotaphytidae, Phrynosomatidae, Polychrotidae, Scincidae (1), Viperidae

Nocturnal Gekkonidae, Scincidae (2)


Table 6. Summary statistics of sperm storage character mapping. TL = Tree length, CI = Consistency Index, RI = Retention Index, and RCI = Rescaled Consistency Index

Character Phylogeny TL CI RI RCI Sst Location (A) Lee (2005) 7 0.71 0.60 0.43 Vidal and Hedges (2005) 8 0.63 0.40 0.25 This chapter >8 0.63 0.40 0.25 Sst Location (B) Lee (2005) 4 0.50 0.50 0.25 Vidal and Hedges (2005) 5 0.40 0.25 0.10 This chapter >5 0.40 0.25 0.10 Sst Secretion Location Lee (2005) 5 0.60 0.33 0.20 Vidal and Hedges (2005) 6 0.40 0.00 0.00 This chapter >5 0.50 0.33 0.17 Sperm Storage Length Lee (2005) 4 0.80 0.00 0.00 Vidal and Hedges (2005) 4 1.00 0.00 0.00 This chapter >4 1.00 0.00 0.00

Table 7. Number of homoplastic events for each character as delineated by Parsimony, Accelerated (ACCTRAN), and Delayed (DELTRAN) Transformation Optimization. “-”

indicates ambiguity

Character Phylogeny

Homoplastic Events

Parsimony ACCTRAN DELTRAN

Sst Location (A)

Lee (2005) # Convergences 1 (PV) 1 (PV) 1 (PV); 1 (MAV)

# Reversals - 1 (AV) 0

Vidal and Hedges (2005) # Convergences 1 (PV) 1 (PV)

1 (PV); 1 (MAV)

# Reversals 1 (AV) 2 (AV) 1 (AV) This chapter # Convergences 1 (PV) 1 (PV) 1 (PV) # Reversals - 1 (AV); 1 (Inf) 1 (AV); 1 (Inf) Sst Location (B) Lee (2005) # Convergences 1 (V+Inf) 1 (V+Inf) 1 (V+Inf) # Reversals 1 (V) 1 (V) 1 (V)

Vidal and Hedges (2005) # Convergences - 1 (V+Inf) 1 (V+Inf)

# Reversals - 2 (V); 1 (Inf) 2 (V)

This chapter # Convergences 3 (V); 1 (V+Inf) 3 (V); 1 (V+Inf) 3 (V); 1 (V+Inf)

# Reversals 0 0 0 Sst Secretion Location Lee (2005) # Convergences 1 (D) 1 (D) 1 (TH); 1 (D) # Reversals 1 (TH) 1 (TH); 1 (P) 1 (TH)


Character Phylogeny

Homoplastic Events

Parsimony ACCTRAN DELTRAN

Vidal and Hedges (2005) # Convergences - 0 4 (TH); 3 (D)

# Reversals - 2 (TH); 1 (D); 1 (P) 1 (P)

This chapter # Convergences 1 (D) 1 (D) 1 (D) # Reversals 1 (TH); 1(P) 1 (TH); 1(P) 1 (TH); 1(P) Storage Length Lee (2005) # Convergences 0 0 0 # Reversals 0 0 0

Vidal and Hedges (2005) # Convergences 0 0 0

# Reversals 0 0 0 This chapter # Convergences 0 0 0 # Reversals 0 0 0 The descriptive statistics of the Sst secretion location showed moderate homoplasy in all

phylogenies (Table 6, CI = 0.33-0.40; Table 7). Alternatively, RI indicated no synapomorphic quality of Sst secretion location in the Vidal and Hedges (2005) phylogeny (RI = 0.00), whereas the Lee (2005) and this chapter’s phylogenies indicated low synapomorphic quality of Sst secretion location (RI = 0.25). The data for both the Lee (2005) and phylogeny and the new phylogeny presented here were both substantially noisy (RCI = 0.10).

Sperm Storage Length

Only five taxa had well-defined literature regarding length of sperm storage. The length of sperm storage exhibited significant variation amongst the five studied species, and no two taxa had the same length of sperm storage. No definitive statements can be made regarding the evolution of the length of sperm storage at present due to the lack of available data and highly variable nature of this character, though one may speculate regarding general trends.

With birds as the ancestral state, the morphological phylogeny (Lee 2005) showed a general increase in length of sperm storage, with a progression of 0.5 months to 2.5 and 4 months, and more recent evolution of 9 months. However, a backward progression then occurs in length of time from 9 months to 6 months (Figure 2a). Alternatively, both molecular phylogenies (Vidal and Hedges 2005; this chapter) depicted an initial extreme increase followed by a progressive decline in the length of sperm storage (Figures 2b-c). Alternatively, with turtles as the ancestral state, all phylogenies depict an initial decrease in sperm storage length.

The descriptive statistics of the sperm storage length character (Tables 6 and 7) indicated that all four phylogenies had high consistency indices (CI), indicating little to no homoplasy of the characters. However, the retention indices (RI) for all phylogenies were 0.00, demonstrating no synapomorphic qualities of these traits. These results are likely the result of all of the variations of sperm storage length being uniquely derived.


Relationships within and between Reproductive Characters and Ecological Traits

Sst Location

Analysis of Sst Location showed no relationship with respect to Sst secretion location, RAF SVL, eggs/clutch, time of vitellogenesis, time of mating, time of clutch deposition, location of habitat, and activity.

However, Sst location did show possible implications for relationship to sperm storage length, as the three of the known taxa storing sperm vaginally had sperm storage lengths of four months or less, whereas the three of the known taxa storing sperm in the infundibulum store sperm for greater than 6 months (Table 5).

Sst Secretory Cell Location

A possible relationship between Sst secretion location and sperm storage length may exist, because the four known taxa with both sets of data exhibit a relationship between longer sperm storage and secretions throughout the Ssts (Figure 6). Additionally, a relationship exists between Sst secretion location and the number of eggs per clutch. The taxa with proximal secretions in the sperm storage tubules carry one egg per clutch, whereas those taxa with distal secretions or secretions throughout carry two or more eggs per clutch.

No apparent relationships occur between Sst secretion location and RAF SVL, time of vitellogenesis, time of mating, and time of clutch deposition, and location of habitat. Furthermore, no data were collected on the Sst secretion location of nocturnal squamates, so it is unknown if a relationship exists between Sst secretion location and time of daily activity.

Sperm Storage Length

The preliminary analysis of sperm storage length shows relationships with both Sst location and Sst secretion location. No apparent relationship, however, occurs between sperm storage length and RAF SVL or location of habitat. Eggs/clutch, time of vitellogenesis, time of mating, and time of clutch deposition show no relationship except possibly a relationship between exceptionally short lengths of sperm storage (<2.5 months) and year round reproduction (Table 5).

CONCLUSION

Variability of Sperm Storage Characters The majority of sperm storage characters exhibited very little variation. These particular

characters (including presence of sperm storage and sperm storage tubules, embedding of sperm, multiple clutches, and Sst structure) may be less variable and unchanged because of overall utility of each character.

In order to produce as many offspring as possible, efficient sperm storage characteristics are conserved in many squamate lineages, including: producing multiple clutches, storing sperm, and having specialized structures with which to store sperm.


Alternatively, some sperm storage characteristics are highly variable, as is seen by the amount of variation in length of sperm storage, Sst linings, and Sst location. All of these characters have a significant impact in reproduction, and how these have evolved could imply a substantial amount about what derived characters may be beneficial given the life history and lifestyle of each organism.

Sperm Storage Length The length of sperm storage is only known for six squamate families, is unknown for the

other eleven families with known Sst locations, and is likely more variable than observed in this chapter. If, however, these data show a representative trend, then the following may be considered with bids as the ancestral state: All three analyses exhibited an initial increase in length of sperm storage (Figure 2). However, they also all show a decrease in length of sperm storage as well, although the timing varies between the molecular and morphological phylogenies. The molecular phylogenies showed an extreme increase in sperm storage length, followed by a progressive decline (Figure 2b-c), whereas the morphological phylogeny showed a general progression of increase in sperm storage length followed by a decrease (from nine months to six months) (Figure 2a). However, if turtles represent the ancestral state, all phylogenies depict an initial decrease in sperm storage length.

Both scenerios suggest that, while there may be benefit to storing sperm for longer than two weeks (the proposed ancestral state) for some lineages, the length of sperm storage may also be related to events occurring during sperm storage. Some lineages may utilize sperm for fertilization of clutches, whereas others may simply just have residual sperm from a previous mating. Eckstut et al. (2009a) and Yamamoto and Ota (2006) found evidence for Hemidactyline geckos fertilizing follicles with stored sperm throughout their reproductive seasons. Alternatively, Sever and Brizzi (1998) proposed that residual sperm in plethodontid salamanders are not used for additional fertilizations occurring in subsequent breeding seasons. Thus, length of sperm storage may be related to purpose of sperm storage in each lineage. This character is especially variable, and the observed progression of character states is likely inaccurate. No two analyzed taxa shared the same length of sperm storage, and only five taxa have available sperm storage length data, so increasing study of sperm storage length would likely show that even more variation exists than currently noted.

Further analysis of this trait would be greatly enhanced by examining it in conjunction with seasonality of sperm storage and use by both sexes. Aldridge (pers. comm.) found that North American colubrid species with bimodal mating (in the spring and late summer/fall) have females storing sperm over the winter, whereas species with unimodal mating (spring mating) have males producing sperm over the winter. However, it is currently unknown if similar seasonality of sperm storage differentiates between the sexes in other squamate lineages.

Sst Secretory Cell Locations Combined analysis of all three hypotheses indicate that the likely evolution of these

character states follows the molecular evolution patterns. The two competing hypotheses


(throughout and distal secretory cells evolving twice each, or one evolution of secretory cells throughout, two evolutions of distally secretory cells, and one reversal to proximal secretory cells) for the morphological phylogeny are equally parsimonious. One of the hypotheses (one evolution of secretory cells throughout, two evolutions of distally secretory cells, and one reversal to proximal secretory cells) agrees with the implications of all molecular phylogenies (Figure 3).

Hypotheses regarding function of secretions in Ssts include spermiophagy, sperm nourishment, and sperm attraction (Dent 1970). The skink Scincella lateralis was shown to have distally secretory cells that were associated with primary lysosomes, which may be used for spermiophagy (Sever and Hopkins 2004), and degradation of sperm may be related to the concept that certain vertebrate lineages do not use embedded sperm for fertilization (Sever and Brizzi 1998). Alternatively, the gecko Hemidactylus turcicus has secretory cells throughout the Ssts. The sperm in H. turcicus are hypothesized to fertilize follicles during the reproductive season, when the species has multiple clutches (Eckstut et al. 2009a), and thus are unlikely to be phagocytized during the reproductive season. However, secretions are present throughout the sperm storage duration, and thus may be hypothesized to nourish sperm during that time period. Alternatively, Siegel and Sever (2008) found that sperm are stored in female Agkistrodon piscivorous for longer than secretions are present (see Figure 11). Although not addressed in their study, one hypothesis proposed by Siegel (pers. comm.) is that, in this case, secretions are produced to attract the sperm to the tubules.

Sst Location There are two competing hypotheses present with these data on the evolution of Sst

location when Aves are considered the appropriate outgroup. First, the morphological phylogeny suggests that infundibular Ssts were entirely derived from vaginal Ssts. Second, the nuclear molecular phylogenies suggest that infundibular Ssts were derived from vaginal Ssts, but then vaginal Ssts were subsequently derived from infundibular Ssts. Alternatively, if turtles are indeed sister to squamates, then infundibular Ssts are the ancestral state, and vaginal Ssts independently evolved both within squamates, and within birds and crocodilians.

Currently, we may say that the most parsimonious states would be indicated by the morphological phylogeny. However, the results indicated by this phylogeny are still somewhat ambiguous. Resolving the evolution of these character states and determining whether the variant states (all instances of vaginal Ssts and infundibular Ssts) are homologous or analogous would require a more refined analysis of structure and placement within the oviduct that would be consistent throughout studies.

If the infundibular Ssts are derived in squamates, as indicated by the morphological phylogeny with the Avian outgroup, it may imply that infundibular Ssts are more advantageous for storing sperm for long periods of time, because lineages with vaginal Ssts may have their sperm destroyed by eggs passing through the vagina during oviposition. This may be a frequent occurrence in squamates that utilize sperm to fertilize multiple clutches. This hypothesis agrees with the length of time that squamates with infundibular Ssts can store sperm. Lacertidae and Gekkonidae have six and nine month sperm storage lengths, respectively, and turtles have been seen to store sperm for over one year. These are the two longest presently observed sperm storage lengths in squamates. Additionally, they are the


only two families with information on both sperm storage length and Sst location, and they both have infundibular Ssts. Derived infundibular Ssts may prevent the embedded sperm from being destroyed as oviposition occurs.

Relationships between Characters Relationships exist between Sst location and sperm storage length as well as Sst secretion

location and sperm storage length. For Sst location and sperm storage length, those lineages with storing sperm four months or less had vaginal Ssts, whereas those with sperm storage greater than four months had infundibular sperm storage, indicating that infundibular sperm storage may be more beneficial for long term sperm storage.

The relationship between Sst secretion location and sperm storage length indicates correlations between long term sperm storage (four months or greater) and secretory cells throughout, moderate sperm storage lengths (two and a half months) and distally secretory cells, and short term sperm storage (two weeks) and proximal secretory cells. These relationships may be related to the function of sperm storage and thus the function of the secretions. Are the short-term lineages merely going through spermiophagy, which could have either proximal or distal secretory cells and thus secretions produced that may be associated with spermiophagy? Are those lineages with long-term sperm storage fertilizing their follicles with the stored sperm and thus need to nourish the sperm, or are the secretions present merely there to attract sperm?

Ancestral State of Squamates Crocodilians have intermediary sperm storage traits between birds and turtles. Sst

location is both posterior infundibular (seen in turtles) as well as anterior vaginal (seen in birds). However, their Sst structure is shared with turtles. Length of sperm storage is also unknown for crocodilians, but is hypothesized to be less than a month (Bagwill, pers. comm.), which is consistent with birds. These issues confound rather than illuminate the debates over both amniote phylogenetics as well as the ancestral state of squamate sperm storage characters.

We were unable to hypothesize the squamate ancestral state based on the existing literature. Birds, turtles, and crocodilians have differing sperm storage traits, and whether these traits are derived or retained from ancestors is unknown. Furthermore, very few taxa within Aves, Crocodilia, and Testudines have had sperm storage analyzed, and it is possible that sperm storage traits in these classes are equally as variable as observed in squamates.

Phylogenetic Implications Minimal phylogenetic inference can be drawn for the analyzed sperm storage characters,

even though the molecular phylogenies are similar (Vidal and Hedges 2005). Additionally, for Sst location, the morphological phylogeny (Lee 2005) showed the most amount of resolution, but that could be due, in part, to the fact that the analyzed traits are


morphologically based. Although these histological characters may show different rates and trends of evolution from other anatomically based characters, they still may show similar trends, which would confound the use of these characters.

Sperm storage characters appear to be highly plastic and variable, with no strong phylogenetic signal for any of the characters, especially given the high amount of homoplasy and lack of synapomorphic value for each trait (Table 6). We suggest that, while the evolution of these characters may have strong implications with respect to the lifestyles of the analyzed organisms, the analyzed characters do not contribute to any debates over squamate phylogeny.

However, these results are all preliminary and limited information was available on sperm storage for each taxon. For instance, with sperm storage length, only one species was sampled per family (except Gekkonidae with two species sampled), and only four families were represented. With future studies, these traits will likely prove substantially more variable than currently recognized. Despite the limited amount of information, these preliminary results may hold strong implications for the ecological utility of these variant sperm storage traits, such as function of secretions in either maintaining or destroying stored sperm.

ACKNOWLEDGMENTS This chapter is part of a Master’s thesis submitted by the senior author to Southeastern

Louisiana University Department of Biological Sciences. We would also like to thank R.A. Pyron and F.T. Burbrink for assistance in building the molecular dataset, as well as for providing comments on the manuscript. This manuscript was also improved by comments and suggestions from R. Valverde and C.D. McMahan. Funding for this project was provided by a grant from Southeastern Louisiana University Center for Faculty Excellence.

APPENDICES Appendix 1. The detailed squamate phylogeny inferred for 611 species. This is a strict

consensus tree of 128 most parsimonious trees. Values represent bootstrap proportions and are only those >50 are shown. The two taxa with asterisks are questionable in terms of names matching the sequences given the highly unusual location of them in the phylogeny. Taxa labeled with A, B, C, etc. represent different sequences of the same species.


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Reviewed by Roldán Valverde, Southeastern Louisiana University, Hammond, Louisiana 70402 (Email: [email protected]).

phylogenetic analysis of sperm storage in female squamates

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