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Home , Female sperm storage, Fisherian runaway

Does Drive the of the Female Storage in ?

by Tiffini Smith

B.A. in Biological Sciences, December 2012, Virginia Commonwealth University

A Thesis submitted to

The Faculty of The Columbian College of Arts and Sciences of The George Washington University in partial fulfillment of the requirements for the degree of Master of Science

May 20, 2018

Thesis directed by

Mollie Manier Assistant Professor of

ã Copyright 2018 by Tiffini Smith All rights reserved

Dedication

I want to dedicate this thesis to the family and friends that have supported me throughout

this journey. I would also like to dedicate this thesis to all the first, only, and different people in the world that are breaking barriers, following their dreams, and paving the way

for those like them.

iii Acknowledgements

I wish to acknowledge my fellow graduate students specifically the students in Dr.

Patricia Hernandez and Dr. Arnaud Martin lab for answering questions, providing support, and being sounding boards for my projects.

iv Abstract of Thesis

Does Sexual Selection Drive the Evolution of the Female Sperm Storage Organ in Drosophila?

In polyandrous species, the sperm of multiple males may coexist within the female reproductive tract, generating postcopulatory sexual selection (PCSS) that can lead to rapid diversification of male and female traits. In Drosophila, PCSS drives the evolution of extremely long sperm, which are also coevolving with the female sperm storage organ, the seminal receptacle (SR). This male-female coevolutionary dynamic may be mediated by Fisherian sexual selection, fueled by a genetic correlation between SR length and sperm length. SR length is also genetically correlated with remating rate within D. melanogaster, suggesting that the intensity of PCSS may be driving the evolution of SR length in this species. Here, I ask if this microevolutionary process can explain the macroevolutionary pattern of male-female across the Drosophila lineage. I investigate the association between PCSS and SR length across 17 Drosophila species and find that they are not significantly associated. I additionally test alternative hypotheses that SR evolution is mediated by on fecundity and again fail to find support for this hypothesis. Although it is assumed that Fisherian sexual selection drives the evolution of extraordinarily long sperm through postcopulatory female choice mediated by SR length, it is still unclear what is driving SR evolution

v Table of Contents

Dedication ...... iii Acknowledgements ...... iv Abstract of Thesis ...... v List of Figures ...... vii List of Tables ...... viii List of Symbols / Nomenclature ...... ix Does Sexual Selection Drive the Evolution of the Female Sperm Storage Organ in Drosophila?...... 1 Methods...... 9 Results ...... 12 Discussion ...... 13 Conclusions ...... 15 References ...... 16 Figure Legend ...... 26 Figures...... 27 Table Legend ...... 31 Tables ...... 32

List of Figures

Figure 1 ...... 27

Figure 2 ...... 27

Figure 3 ...... 28

Figure 4 ...... 28

Figure 5 ...... 29

Figure 6 ...... 29

Figure 7 ...... 30

vii List of Tables

Table 1 ...... 32

Table 2 ...... 33

Table 3 ...... 34

viii List of Symbols / Nomenclature

1. PCSS: Postcopulatory Sexual Selection

2. CFC: Cryptic Female Choice

3. FRT: Female reproductive tract

4. SR: Seminal Receptacle

5. SSO/SSOs: Sperm storage organ/s

6. PIC: Phylogenetic Independent Contrast

ix Does Sexual Selection Drive the Evolution of the Female Sperm Storage Organ in Drosophila?

In polyandrous species, the sperm of multiple males may coexist within the female reproductive tract, generating postcopulatory sexual selection (PCSS) in the form of (Parker 1970) or cryptic female choice (Eberhard 1996) that may bias sperm use for fertilization. Male to PCSS in sperm include increased swimming speed and energetics, as well as modified sperm morphology to compete for position, storage, and ultimately fertilization (Parker 1970; Birkhead et al., 1999; Hunter & Birkhead 2002;

Higginson et al., 2012; Arnqvist 2014; Firman et al., 2017). It is not enough for sperm to be fertilization competent; sperm must successfully fertilize eggs in sperm competition to have reproductive success.

Sperm competition as a Mechanism of PCSS

Sperm competition is male-male competition that in internal fertilizers occurs within the female reproductive tract (FRT), where successful sperm have a greater fertilization rate and increase the reproductive success of the respective male. This competition causes male traits to experience a high degree of diversification and evolution; this rapid evolution provides an avenue for males to adapt to better compete in sperm competition to enhance paternity success (Miller & Pitnick 2002; Manier et al., 2013c).

Sperm has been shown to develop adaptations in swimming speed, sperm energetics

( due to ATP content which enhances sperm swimming), as well as sperm morphology to compete for position, storage and ultimately egg fertilization (Miller &

Pitnick 2002; Lüpold et al., 2009; Higginson et al., 2012; Manier et al., 2013c; Rowe et al.,

1 2013; Fitzpatrick & Lüpold 2014; Rowe et al., 2015). Rowe et al., (2015), showed in passerine that sperm competition drives the evolution of midpiece and flagellum length which is positively correlated with swimming speed (Rowe et al., 2015) and is hypothesized to increase thrust and aid drag resistance during sperm competition (Lüpold et al., 2009; Rowe et al., 2015). Postcopulatory sexual selection can also drive the evolution of sperm length. Miller and Pitnick (2002) used to demonstrate that long sperm outcompete short sperm during sperm competition. The caveat being that long sperm only outcompete short sperm in long seminal receptacles (sperm storage organ;

Miller & Pitnick 2002). This indicates reproductive success is not solely mediated by the male.

Cryptic Female Choice as a Mechanism of PCSS

Females can also alter reproductive success of males by using cryptic female choice

(CFC) to control and bias sperm. Cryptic female choice (Thornhill 1983), the second major mechanism of PCSS, is generally defined as the use of morphological, biological, and behavioral mechanisms by polyandrous females to create nonrandom paternity biases

(Pitnick & Brown 2000). Females can bias paternity at any stage of the reproductive process, including through control of duration, sperm ejection, sperm storage, and sperm use for fertilization (Birkhead & Møller 1993; Pitnick et al., 1999; Manier et al.,

2013a; Firman et al., 2017). Female control of copulation duration can alter male ejaculate size and sperm transfer (Pilastro et al., 2006; Herberstein et al., 2011; Manier et al., 2013b;

Manier et al., 2013c). In the sexually cannibalistic spider Argiope keyserlingi, researchers demonstrated that copulation duration increased the relative paternity of males

2 (Herberstein et al., 2011). When females end copulation early, sperm transfer is inhibited, reducing the relative number of sperm in the female reproductive tract (FRT) (Mandelli et al., 2006). Another method of cryptic female choice is sperm ejection or the elimination of sperm from the FRT prior to storage or use. Sperm ejection has been recorded in many different including multiple species (Birkhead & Møller 1993; Birkhead et al., 1983; Dean et al., 2011), mammals, and (Manier et al., 2013b & c). In some birds, females eject sperm from the cloaca between 30-60 minutes after insemination. If sperm transfer or storage is not complete, ejection results in a loss of paternity success

(Moller 1997; Sasanami et al., 2013).

Cryptic Female Choice Generated by Reproductive Tract Morphology

In addition to the control of copulation duration and ejection females also generate

CFC by using reproductive tract morphology. The female reproductive tract is a complex environment composed of ducts, glands, secretory cells and, in many polyandrous species, sperm storage organs (SSOs) (Eberhard 1996; Bloch et al., 2003; Pitnick 1999; Orr &

Brennan 2015). Sperm storage organs are female reproductive organs that temporally separate mating from fertilization. This separation allows females to maintain and store more sperm from multiple males within the reproductive tract over time (Birkhead et al.,

1993; Birkhead 1998; Pitnick et al., 1999; Schnakenberg et al., 2012; Bloch et al., 2003;

Manier et al., 2013a; Manier et al., 2013b; Orr & Brennan 2015; Ala-Hankola & Manier

2016), promoting sperm competition, and generating opportunities for CFC. It also provides fertility insurance, giving females the opportunity to potentially develop a broader range of genotypes (Bloch et al., 2003).

3 Sperm storage organs have also been shown to secrete proteases that interact with male seminal promoting sperm motility and egg laying (Schnakenberg et al.,

2011). SSOs can be seen across many taxa, and can vary in number, size, and shape (Parker

1970; Pitnick 1999; Simmons et al., 1999; Bloch et al., 2003; Miller & Pitnick 2003;

Higginson et al., 2012; Orr & Brennan 2015; Arnqnist 2014). For example, yellow dung have three chitinous spermathecal SSOs, (Simmons et al., 1999; Ward 2000) whereas the Australian only has a pair spermathecae. In the Australian redback spider males transfer sperm into both spermathecae through two separate matings. If females, however, can prevent the second mating through premature cannibalism or aggression, paternity success decreases (Andrade 1996; Andrade 1998; Snow & Andrade

2005).

Drosophila mechanisms of CFC and SSO’s

Sperm storage organs and FRT morphology also play a role in fertilization bias.

Fertilization bias includes the preferential use of sperm in different sperm storage organs to fertilize an egg (Manier et al., 2013b). In Drosophila, females have 2 types of SSOs,

(Pitnick et al., 1999) a pair of spermathecae (Parker 1970; Snow & Andrade 2005;

Presgraves et al., 1993) and the seminal receptacle (SR) (Parker 1970; Pitnick 1999;

Schnakenberg et al., 2012; Lüpold et al., 2016). The SR, the primary SSO (Bloch et al.,

2003; Manier et al., 2010; Manier et al., 2013a) is thought to have evolved as a more efficient SSO (Bloch et al., 2003) and has been shown to play a major role in CFC as well as PCSS (Orr & Brennan 2015; Lüpold et al., 2016). The SR has been shown to have an evolutionary relationship with sperm, (Pitnick et al., 1999; Pitnick et al., 2001; Bloch et

4 al., 2003; Miller & Pitnick 2003; Schnakenberg et al., 2011; Higginson et al., 2012) with high degrees of inter- and intraspecific variation which has been correlated to the variation seen in sperm length (Pitnick et al., 1999; Miller & Pitnick 2003). These sperm storage organs are responsible for amplifying PCSS through cryptic female choice. Furthermore,

SSOs allow females that mate with multiple males to store sperm for extended periods of time.

In Drosophila, when females remate, sperm are first deposited in the bursa before entering one of the two types of sperm storage organs. Drosophila exhibit last-male precedence, where sperm from the last male to mate displaces the previous male’s sperm in the SR (but not the spermathecae, where a “topping off” mechanism occurs), resulting in a higher proportion of progeny sired by the second male (P2) (Parker 1970; Manier et al., 2010; Manier et al., 2013a). Larger ejaculates and longer sperm are both associated with competitive fertilization success (Manier et al., 2010; Lüpold et al., 2013; Manier et al., 2013c). Furthermore, larger ejaculates (in terms of more sperm) displace a greater proportion of resident sperm from sperm storage. There is also a correlation between ejaculate size and mating status, males alter ejaculate size when mating with non-virgin females (Lüpold et al., 2011; Manier et al., 2013a). These results suggest that males transfer fewer sperm when mating with virgin females due to the lack of resident sperm to displace, whereas in matings with non-virgin females, males increase ejaculate size to better displace resident sperm. Postcopulatory sexual selection can therefore drive the evolution of traits associated with sperm morphology and ejaculate size that increase male success in sperm competition.

5 Drosophila females can exhibit three different mechanisms of CFC: timing of ejection, SR length, and differential use of sperm storage organs. After insemination, sperm enters storage in the SR or spermathecae. Within the SR, sperm entering storage physically displace resident sperm from previous matings, leading to last-male precedence, in which the last male to mate with a female sires the majority of the progeny. Drosophila females control sperm ejection times, with early sperm ejection terminating the sperm storage process and having a negative relationship with second male paternity (P2) (Birkhead &

Møller 1993; Lupold et al., 2013; Manier et al., 2013a; Manier et al., 2013b). Sperm ejection also terminates sperm storage by removing the source of second-male sperm from the bursa and preventing further displacement of resident sperm. Ejection also establishes the fertilization set, the of sperm in storage that are available for fertilization

(Manier et al., 2010). In Drosophila, ejection can take up to 5 hours and can also be a mechanism of conspecific sperm precedence, in which females mating with a conspecific and heterospecific male favor conspecific sperm (Manier et al., 2010; Manier et al., 2013a;

Manier et al., 2013b). Copulation termination and sperm ejection are important, because second-male sperm that can enter storage have an increased likelihood of displacing resident sperm and escaping ejection from the bursa. Drosophila females can also bias fertilization success through the differential selection of sperm from different sperm storage organs. Drosophila simulans females bias sperm storage use regardless of mating order in favor of conspecific males when mated to both conspecific and heterospecific males (Manier et al., 2013b). In intraspecific matings, however, females use ejection to favor sperm from larger males but adjust sperm use during fertilization to select for male population identity (Ala-Honkola & Manier 2016).

6

The Evolution of SSOs and SR coevolution with sperm

In general, females can use one or more mechanisms of cryptic female choice to bias paternity success and impose postcopulatory sexual selection. In Drosophila, PCSS can be driven by evolution of the female reproductive tract when males adapt to CFC, resulting in the rapid evolution and diversification of male and female reproductive traits that increase competitive fertilization success (Manier et al., 2010; Arnqvist 2014) and may lead to reproductive isolation (Manier et al., 2013a, b, & c). Sperm length can determine male fertilization success, with long sperm outcompeting short sperm but primarily in long

SRs (Miller & Pitnick 2002). Drosophila species have also been shown to have highly variable SR lengths with SR length driving the evolution of sperm length and coevolving with sperm length both among and within species (Pitnick et al., 1999, Miller & Pitnick

2002; Miller & Pitnick 2003; Pitnick et al., 2003). The coevolution of SR and sperm length provides evidence for a Fisherian runaway scenario which explains extreme male ornamentation due to female preferences (Fisher 1930). However, for a Fisherian runaway scenario to exist there must be an advantage to the extreme male ornamentations such as reproductive success and the two traits male ornamentation/female preference must be genetically correlated (Fisher 1930).

In 2016 Lüpold et al., showed that when male and females of Drosophila melanogaster are genetically identical there is still a correlation between sperm and SR length. It was also shown that when remating rate is genetically correlated with SR length

(Lüpold et al., 2016) females which remate more frequently have longer SRs. In addition, in 2013, using Drosophila melanogaster, Lüpold et al., identified that females with longer

7 SRs also store more sperm and lay more eggs. The genetic correlation between SR and sperm length as well as SR length and remating rate provides evidence that a Fisherian runaway scenario may be occurring across the Drosophila lineage. Where female SR length is driving the evolution of sperm length amplifying PCSS and altering competitive fertilization success, PCSS may be driving the evolution of the SR. While the evidence of longer SRs having greater sperm storage and egg laying capacities provides evidence fecundity may also be involved in SR evolution.

This evidence suggests that variation in PCSS and fecundity across the Drosophila lineage may play a role in sperm-SR coevolution and species divergence. Using remating rate which is genetically correlated with SR length and is an indicator of the strength of

PCSS, where species that remate frequently having an enhanced degree of sexual selection,

I will investigate the relationship between SR length and fecundity as well as PCSS and

SR length in 17 Drosophila species (Lüpold et al., 2016).

8 Methods

Stock Maintenance

Flies used in this experiment were obtained from the Drosophila Species Stock Center in

San Diego, CA and then in Ithaca, NY. Species were maintained according to the husbandry given by the stock center. Flies were collected in the morning and the evening immediately after eclosion under CO2 anesthesia and transferred into sex-specific vials at densities of 10 females or 20 males per vial. Flies were maintained in 10 mL plastic vials until the start of each experiment when they were 2-5 days old.

Remating trials

Remating rate was calculated as the number of matings in a week with a four-hour opportunity to mate each day for approximately 20-50 virgin females per species. Females were aspirated without anesthesia into individual vials the day before the experiment to allow females to acclimate and access fresh food. For each four-hour mating trial, two males were aspirated into a female’s vial. At the start of copulation, the non-copulating male was removed. Upon copulation completion, the mated male was removed, and two more males were transferred to the vial. Copulation durations were recorded. At the end of the 4-hour time period all males were removed from vials until the next day. During the experiment, females were transferred to new vials once or twice (depending on the species).

After the experiment, females were allowed to oviposit in fresh vials for one week, and progeny were counted from all vials.

SR Length

For each species, five additional females were collected for measuring SR length and etherized or frozen in food vials at -20°C. Females were dissected under a Nikon dissecting scope in phosphate-buffered saline (PBS) on a glass slide using fine forceps. The ovipositor

9 was gently pulled, removing the reproductive tract, and the SR was carefully uncoiled using a fine pin. Reproductive tracts were then covered with a cover slip, sealed with nail polish, and imaged and measured under phase contrast or DIC microscopy on a Nikon Ni-

U upright microscope with an Andor Zyla (SMZ745) monochrome stereoscope and NIS- elements software (Figure 1).

Phylogeny

A molecular phylogeny was generated using a subset of 5 coding genes from a 13 gene Drosophila supermatrix (Figure 4) (van der Linde et al., 2010) that were well represented across all 17 species. FASTA files of the amino acid sequences were compiled from GenBank (Table 1; NCBI Research Coordinators 2017) and prepared for phylogenetic analysis.

Amino acid sequences were aligned using Saté (v.2.27) to ensure an accurate coestimation of both sequence alignments and phylogenetic trees (Liu et al., 2012), uing MAFFT as the aligner, MUSCLE as the merger, FASTTREE was the tree estimator, and GTR + G20 as the model. Gene alignments were manually inspected in Aliview (v.1.18.1; Larson 2014) to detect and correct potential regions of uncertainty and disagreement among species and concatenated with Geneious (v.11.0.5; Kearse et al., 2012). Phylogenetic analyses were conducted under Bayesian (v.3.2.6) and maximum likelihood approaches on XSEDE and

–HPC2 XSEDE (respectively) version 8.2.8 using the Cipres Portal 3.3 (Miller et al.,

2015). For Bayesian analysis, two runs were conducted with 2 chains for 5000 generations each, and trees and parameters were recorded every 1000 generations. For maximum likelihood, bootstrap analyses were executed (100 replications) with joint branch length optimization. All other parameters and conditions were kept at the default settings. The

10 best tree from the Bayesian analyses with posterior probabilities and the ML analysis with bootstap values and bipartitions were visualized using FigTree version 1.4.3 (Rambaut,

2007).

Statistical Analysis

All statistical analyses were conducted in R (v.3.3.2). The following packages were used in the R environment to complete statistical analysis: ape (Pradis 2012), ggplot2

(Wickham, 2009), caper (Orme et al., 2013), geiger (Harmon et al., 2008), psych (Revelle,

2018), calibrate (Graffleman & van Eeuwijk, 2005), and picante (Kembel et al., 2010). A correlation analysis between SR length and sperm length was performed. A correlation analysis was conducted between SR length and remating rate as well as SR length and fecundity. To confirm the results of both correlations phylogenetic methods were used.

Prior to conducting phylogenetic comparative methods, Blomberg (K) (Blomberg et al.,

2003), Pagel (�) (Pagel 1992) and phylogenetic generalized least squares (PGLS) (Grafen

1989) phylogenetic tests were performed using the caper (Orme et al., 2013) and geiger

(Harmon et al., 2008) package to identify the importance of phylogenetic relationships in trait evolution. Then the ape (Pradis 2012) package was used for the phylogenetic independent contrasts (Felsenstein 1985) analysis which correlated SR length and remating rate as well as SR length and fecundity.

11 Results

SR and Sperm Length

Consistent with the results of Pitnick et al., (1999), seminal receptacle (SR) and sperm length were positively correlated across all 17 species (slope = 0.714, p < 0.0001, R2 =

0.8614) (Figure 2).

SR Length and Remating Rate Correlation

There was no significant correlation between SR length and remating rate. The linear regression for SR length ~ remating rate resulted in a slope = -0.861, R2 = -0.028 and p =

0.464 (Figure 3).

Upon testing for phylogenetic signal using Blomberg (K) (Blomberg et al., 2003), Pagel

(�) (Pagel 1992) and phylogenetic generalized least squares (PGLS) (Grafen 1989) remating rate displayed a weak phylogenetic signal for all three methods (Table 3). SR length and SR length ~ remating rate (SR length as a of remating rate) however received strong phylogenetic signal values, where for SR length K = 0.9, � = 1, and PGLS

= 0.92 (Table 3). With such strong phylogenetic signal, a phylogenetic independent contrast was conducted. The phylogenetic independent contrast (PIC) confirmed the results of the correlation. There was no significant relationship found between SR length and remating rate, for the PIC slope = 0.62, R2 = -0.038, p = 0.51 (Figure 5).

SR Length and Fecundity Correlation

There was no significant correlation between SR length and fecundity. The linear regression for fecundity ~ SR length resulted in a slope = -3.58, R2 = -0.004 and p = 0.317

(Figure 6). The phylogenetic independent contrast (PIC) confirmed the results of the correlation, for the PIC, slope = 3.63, R2 = -0.054, p = 0.63 (Figure 7)

12 Discussion

Previous work showed that within D. melanogaster, longer sperm have an advantage in sperm competition (Miller & Pitnick 2002; Lüpold et al. 2013), primarily in longer SRs

(Miller & Pitnick 2002). Coevolution of SR length and sperm length in Drosophila (Pitnick et al., 1999) may be mediated by Fisherian sexual selection, fueled by a genetic correlation between SR length and sperm length (Lüpold et al., 2016). SR length in turn, is genetically correlated with remating rate and females with longer SRs also store more sperm and lay more eggs within D. melanogaster (Lüpold et al., 2013; Lüpold et al., 2016). This suggests that the intensity of postcopulatory sexual selection (predicted by female remating rate) or fecundity may be driving the evolution of SR length among species. This evolutionary scenario assumes that sexual selection acting within species can be invoked to explain patterns of divergence among species and that microevolutionary processes can predict macroevolutionary patterns. My research tested these relationships by examining the correlation between SR length and remating rate as well as fecundity across species.

My results fail to find a correlation between SR length and PCSS or fecundity. Although

Fisherian sexual selection explains the relationship between sperm and SR length, with the idea that SRs drive sperm evolution it is still unclear what is driving SR evolution. The lack of correlation between SR length and fecundity indicate that SR evolution may not be a result of the need to increase progeny production. The lack of support for a macroevolutionary relationship between PCSS and SR length could imply that this relationship, while true for Drosophila melanogaster, is not necessarily generalizable to other Drosophila species. It could also imply that Fisherian sexual selection only acts at the microevolutionary scale, and macroevolutionary patterns are not experiencing

Fisherian selection. Furthermore, because of the strong phylogenetic signal found in SR

13 length the relationship between PCSS and SR length may also be seen in Drosophila melanogaster sister species Drosophila mauritiana and Drosophila simulans. If phylogeny is truly important in the evolution of SR length then closely related species are likely to experience similar evolutionary patterns.

Another possible driver of SR evolution is body size. Pitnick et al., (1999) found a significant correlation between female body size and sperm length and a marginally insignificant correlation in female body size with SR length. Furthermore, when female body size and phylogeny are controlled, there is a stronger relationship between sperm and

SR length (Pitnick et al., 1999). Thus, body size may be imposing constraints on SR development.

Another possibility is that PCSS is a stronger driver of SR evolution in species that use the

SR as the primary SSO and not seen in species that use the spermathecae as the primary

SSO or both equally. Out of 17 species represented in this data set 12 species have known

SSO preferences while 5 species SSO preferences are unknown, the 12 species with known preferences use the SSOs equally (Pitnick et al., 1999). In order to determine the factor causing the evolution of the SR and initiating the Fisherian runaway event seen between sperm and SR length more research must be done investigating SSO use, body size and independent PCSS evolution across the Drosophilidae family.

14 Conclusions

Although previous research in Drosophila melanogaster between remating rate and SR length indicated that PCSS or fecundity may drive the evolution of SR length, our interspecific comparative data provides evidence against these relationships on a macroevolutionary scale. Furthermore, the phylogenetic signal data indicates that phylogenetic relationships are important and should be accounted for when investigating the evolution of SR length but not when investigating remating rate. Further research should be conducted on body size as a possible initiator or restrictor of SR length, as well as the effect of sperm storage use on the PCSS-SR relationship and the possibility of independent PCSS evolution across the Drosophilidae family.

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25 Figure Legend

Figure 1: Dissected, uncoiled, and measured SRs of Drosophila albomicans (1A),

Drosophila simulans (1B), and Drosophila pseudoobscura (1C).

Figure 2: Correlation of SR length and sperm length showing similar results to Pitnick et al., 1999.

Figure 3: Correlation of SR length and remating rate with bidirectional standard error bars.

Figure 4: constructed using 5 protein coding genes represented across all 17 species. Branch values are the RaxML bootstrap supports and the lowest 80% posterior is listed for the Drosophil busckii, Drosophila willistoni relationship.

Figure 5: Phylogenetic independent contrast correlation between SR length and remating rate.

Figure 6: Correlation of SR length and fecundity with bidirectional standard error bars.

Figure 7: Phylogenetic independent contrast correlation between SR length and fecundity.

26 Figures

Figure 1

Figure 2

27

Figure 3

Figure 4

28

Figure 5

Figure 6

29

Figure 7

30 Table Legend

Table 1: Data collected during remating experiments and from dissections. * sperm lengths unpublished data from Scott Pitnick at Syracuse University.

Table 2: Genes used to construct phylogeny (Van der linde et al., 2016).

Table 3: Data from phylogenetic signal tests.

31 Tables

Species Copulation Remating SR Length *Sperm Fecundity Duration Rate (mm) Length (mm) (avg, min) D. persimilis 4 1.51 0.33 0.32 63.3 D. miranda 4 1.12 0.37 0.31 28 D. pseudoobscura 2 1.46 0.41 0.31 36.14 D. mauritiana 25 2.5 1.23 0.98 13.9 D. erecta 19 1.11 1.49 1.15 25.29 D. busckii 1.25 1.8 1.65 1.18 43.63 D. melanogaster 26 2 2.07 1.85 103.57 D. simulans 23 3 2.13 1.1 161.83 D. yakuba 29 1.7 2.2 1.75 10.73 D. takahashii 19 1.2 2.47 1.87 48.89 D. kikkiwai 4 3 2.87 2.87 18.16 D. bipectinata 8 1.5 5.24 1.75 35.15 D. albomicans 21 1.31 5.33 5.35 32 D. americana 2 1.33 5.98 5.22 46.1 D. virilis 2 2.11 7.38 4.7 48.21 D. willistoni 17 1.18 7.9 6.62 7.25 D. subpalustris 7 1.25 8.92 5.96 7.71 Table 1

32 Adh AmyRel COI COII per Drosophila M60997 - AF451101.1 + M95143 L81264.1 persimilis U51609.1 Drosophila Y00602 U82556 AF519412.1 + AF519348.1 L81280 pseudoobscura U51606.1 + AF451073.1 Drosophila AF459771 U96156 AF050746.1 AY737608. - kikkawai 1 Drosophila X54116 AF039562 AF050744.1 - AF251239.1 erecta Drosophila X54120 AF039561 NC_001322.1 X03240 X61127 yakuba Drosophila Z00033 U96157 NC_005779.1 AF474081 AF251240.1 mauritiana Drosophila M36581 U96159 NC_005781.1 AF200834 U11801+AF simulans 251250 Drosophila X98338 AF022713 M57910.1 U37541 X03636 melanogaster Drosophila U95251 AF039560 - AY335206. U51056 willistoni 1 Drosophila - - GU597451 GU597481 - busckii Drosophila U26846 AF136603 DQ471577.1 DQ426813. X13877 virilis 1 + AY646749. 1 Drosophila AF136650 AY736529 DQ471597.1 AY646727. L81302 americana +U26837. * 1 1 Drosophila AB03364 AF462595 - - AF102153 albomicans 2+AY044 126 Drosophila AB93267 AY736539. - - - subpalustris 5.1 1 Drosophila AF459749 U96161 AB027264.1 AF474089 - takahashii Drosophila - - U51608.1 M95148.1 AY238807. miranda 1 Drosophila - AF136936 AY757287.1 AY757275. - bipectinata + AJ844778.1 1 Drosophila EU877944 - AB032132.1 KC183710. - ananassae .1 1 Drosophila DQ36323 AF250055. - AF474079.1 - eugracilis 1.1 1 Table 2

33

Blombergs K Pagels � PGLS SR Length 0.9 1 0.92 Remating 0.2 0.22 0 Rate Table 3

34