Clam Shrimp’ (Crustacea: Branchiopoda) As a Model Organism for the Study of Sexual System Evolution

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Sexual discrimination at work: Spinicaudatan ‘Clam Shrimp’ (Crustacea: Branchiopoda) as a model organism for the study of sexual system evolution

T.I. Astrop, L.E. Park, B. Brown, and S.C. Weeks

ABSTRACT

Biological interactions are rarely preserved in the fossil record and where they do occur, are often difficult to discern. Therefore, the evolution of sexual systems over geologic time in animals has been difficult to investigate. The reproductively labile Spinicaudata ('clam shrimp') are a model clade for the study of sexual systems, containing dioecious (males and females), androdioecious (males and hermaphrodites) and self-fertilizing hermaphrodites. Herein we present a methodology in which mating systems can be inferred by the quantification of carapace shape differences attributable to sexual dimorphism in fossil specimens. We develop our methodology by comparing the carapaces of six species from two families of extant Spinicaudatans using eigenshape analyses. Sexual dimorphism was successfully quantified using morphometric techniques combined with discriminant analyses, correctly identifying males and females/hermaphrodites 92% of the time in extant taxa. Thirty-four specimens of the Jurassic clam shrimp Carapacetheria disgragaris were ana- lyzed utilizing the methods developed with extant species. From these fossil data, we were able to detect two distinct carapace shapes and assign 100% of individuals to either shape. The mean carapace shapes of the fossil specimens fit well with the average outlines for males and females in the extant species, enabling us to calculate a sex ratio of 51:49 males:females and thereby assign the sexual system of dioecy. This study begins to successfully utilize the fossil record of the Spinicaudata to elucidate ancient sexual systems, which will likely have far reaching implications for our understanding of the evolutionary dynamics of sexual systems over geologic timescales.

T.I. Astrop. Program in Integrated Bioscience, Department of Biology, The University of Akron, Akron, Ohio, USA. [email protected] L.E. Park. Department of Geology and Environmental Sciences, Environmental Scanning Electron Microscope Laboratory, University of Akron, Akron, Ohio 44325-410, USA [email protected] B. Brown. Department of Biology, The University of Akron, Akron, Ohio 44325-3908, USA. [email protected] S.C. Weeks. Program in Integrated Biosciences, Department of Biology, The University of Akron, Akron, Ohio 44325-3908, USA. [email protected]

Keywords: Eigenshape; morphometrics; carapace; Conchostraca; Ostracod

PE Article Number: 15.2.20A Copyright: Palaeontological Association June 2012 Submission: 11 October 2011. Acceptance: 27 April 2012

Astrop, T.I. , Park, L.E., Brown, B., and Weeks, S.C. 2012. Sexual discrimination at work: Spinicaudatan ‘Clam Shrimp’ (Crustacea: Branchiopoda) as a model organism for the study of sexual system evolution. Palaeontologia Electronica Vol. 15, Issue 2;20A,15p; palaeo-electronica.org/content/2012-issue-2-articles/262-sexual-systems-in-fossilsr ASTROP ET AL.: SEXUAL SYSTEMS IN FOSSILS

INTRODUCTION fossil record, they almost exclusively are restricted to examples of epibiosis (Brande, 1982; Fernan- Within evolutionary biology, one of the most dez-Leborans, 2010), predator-prey interactions perplexing questions is: why are there so many (Kelley and Hansen, 2003) or plant-insect relation- methods of reproduction? (Williams, 1975; Bell, ships (Labandeira and Sepkoski, 1993; Pott et al., 1982; Barrett, 2002; Kondrashov, 1993; Charles- 2008). Although the importance to evolutionary worth, 2006; Jarne and Auld, 2006; Vallejo-Marin biology of understanding the long-term evolution of et al., 2010). The more common reproductive reproductive systems cannot be overstated, infor- forms include asexuality (clonal females only), her- mation concerning the mating systems of extinct maphroditism (all individuals are both male and taxa remain almost completely unknown (Sassa- female) and dioecy (separate males and females). man, 1995) except for rare instances (Klimov and The list of less common forms is enormous, includ- Sidorchuk, 2011). ing cyclic parthenogenesis (asexuality punctuated “Clam shrimp” (Spinicaudata) are a group of by periods of dioecy), sequential hermaphroditism primitive crustaceans within the Class Branchio- (one sex first then switching to the other sex), gyn- poda; their fossil record is very good and extant odioecy (females and hermaphrodites) and taxa exhibit a diverse array of reproductive sys- androdioecy (males and hermaphrodites), to name tems. They are identifiable as adults due to their a few. distinctive bivalved carapace from which they One of the most frustrating aspects of study- derive their common name (Figure 1). Extant clam ing the evolution of reproductive systems is that we shrimp are geographically widespread, occurring have been unable, to date, to utilize information on every continent except Antarctica (although locked within the fossil record to assess breeding there is an excellent Antarctic fossil record for system evolution in deep time. While the fossil these crustaceans; Tasch, 1987) with a particularly record provides us with information on an organ- good freshwater fossil record extending back 400 ism’s living environment, as well as some aspects Ma to the Devonian period (Novojilov, 1961; Tasch, of its ecology, the preservation of biological interac- 1969; Shen, 1978). Restricted to freshwater, the tions (sex, feeding, symbiosis) in the fossil record majority of modern taxa inhabit lakes, ponds and is an exceedingly rare phenomenon. When evi- ephemeral water bodies (Weeks et al., 2009; dence of these interactions is present within the

FIGURE 1. Hermaphrodite Eulimnadia dahli. 1) Antennae. 2) Brood chamber with egg clutch. 3) Head. 4) Phyllopod appendages. 5) Telson. 6) Trunk.

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Dumont and Negrea, 2002). Like other branchio- A likely candidate for assessing fossil repro- pods, they exhibit naupliar larval stages and have a ductive system is the sex ratio, which has been variable number of body segments (Frank, 1988). described as the ‘only information concerning The clam shrimp are a problematic group, tax- reproductive systems able to enter the fossil onomically. Traditionally referred to as the ‘Con- record’ in fossil ostracodes (Abe, 1990). Sex ratio chostraca’ containing the orders Spinicaudata, information has been successfully used to deter- Laevicaudata and Cyclestheridae, the group is now mine reproductive systems in extant clam shrimp deemed paraphyletic with the Cyclestheridae (Weeks et al., 2008). Ongoing taphonomic studies showing affiliations with the closely related Clado- also indicate no bias in preservation potential cera (proposed Cladoceramorpha; Olesen, 2007). between sexes; meaning extension of this method The comprehensive work on crustacean taxonomy into the fossil record should yield reliable informa- by Martin and Davis (2001) suggests that the Con- tion (Astrop and Hegna, unpublished data). Sex chostraca should be discarded and that the group, ratio is thus a promising trait for assessing fossil including the clam shrimp, Notostraca (tadpole reproductive modes. Many palaeontological works shrimp) and Anostraca (fairy shrimp) be referred to have suggested that discrete variation in clam as Phyllopoda (Phyllopoda + Cladoceramorpha shrimp carapace shape is indeed evidence of sex- being the Branchiopoda). ual dimorphism (Table 1). The bimodal distribution The Spinicaudata are of particular interest as of these data in many cases, and the similar num- they are diverse, geographically widespread and ber of growth rings in the two morphs, supports the have an excellent fossil record, as noted above. idea that these two categories represent sexual The Order Spinicaudata comprises three extant dimorphism rather than variation attributable to dif- families: the Limnadiidae, Cyzidae and Leptes- ferences in ontogenetic stages of development theriidae. The Leptestheriidae are not as diverse or (Brown and Astrop, unpublished data). abundant as either the Cyzidae or Limnadiidae and The carapace of the Spinicaudata has not are definitely not as well represented in the fossil been subjected to a contemporary, comprehensive record. study and observations that have been made are The Spinicaudata contain species that exhibit often incomplete or conflicting (Martens, 1983, dioecious, androdioecious and hermaphroditic 1985; Rieder et al., 1984; Wang, 1989; Chen and mating systems (Sassaman, 1995; Weeks et al., Hudson, 1991; Olempska, 2004). This neglect has 2009; Brantner, 2011) spread across the three con- resulted in a poor understanding of a unique ana- stituent families, providing an excellent opportunity tomical feature. What is known is that the carapace to study the evolution of sexual systems over geo- is constructed via multiple lamellae from succes- logic time. Many authors have proposed that sive growth stages, and that these lamellae are androdioecy is a relatively unstable system that found in three discrete layers: a thin epicuticle on should not persist over long periods of time (Lloyd, top of a thicker exocuticle and protocuticle (Martin, 1975; Charlesworth, 1984; Pannell, 1997; Wolf and 1992). The carapace itself is weakly biomineralized Takebayashi, 2004) and is transitional between with a chitin and calcium-phosphate complex (Sti- dioecy and hermaphroditism. The widespread gall and Hartman, 2008), the structural details of occurrence of androdioecy within the family Limna- which are poorly known (Tasch, 1969). The Spini- diidae (Weeks et al., 2006, 2009), along with a caudatan carapace is unique in that partial molting hypothesized age of the androdioecious genus during ecdysis produces discrete growth lines that Eulimnadia at between 180 (Weeks et al., 2006) – record the ontogenetic history of each individual. 45 mya (Tasch, 1969), would seem to be evidence Although the growth lines represent discrete onto- to the contrary. It is also important to note that the genetic stages, the formation of the lines appears closely related Notostraca also exhibit dioecy, to be influenced by their growth environment rather androdioecy and hermaphroditism (Sassaman, than a consistent set of lines set down in a specific 1989, 1991; Zierold et al., 2007), and that the time sequence (Bishop, 1967). The exact mecha- divergence of these two groups is estimated to nisms involved in the partial molting in clam shrimp have occurred approximately 330 Ma (Regier et are not understood but are thought to differ from al., 2005). Thus, these branchiopod crustaceans the molt retention seen in some ostracodes, such provide an interesting set of taxa with clearly labile as the Cytherellidae (Jones, 2003) and Eridostraca breeding systems (Korpolainen, 2007) and a good (Olempska, 2011). A dedicated contemporary fossil record, which could prove exceptionally use- investigation of the Spinicaudatan carapace is ful for the study of breeding system evolution.

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TABLE 1. Suggested instances of dimorphism in fossil clam shrimp inferred via carapace outline or dimensions.

Species Superfamily Age Locality Source

Cyzicus (Lioestheria) Cyzoidea U.Carb- ANTARCTICA - Shackleton Glacier Babcock et al.et al.2002 shackletonensis L.Perm Estheriina astartoides Limnadioidea U.Cret BRAZIL - Potiguar Basin Lana andCarvalho 2002 Endolimnadiopsis rusconii Lioestherioidea U.Tria ARGENTINA - Mendoza Province Gallego 2005 Shizhuestheria truncata Eosestheriteoidea M.Jura CHINA - Sichuan Basin Li ,Hiranoet al.et al.2009 Hemicycloleaia grantrangicus/ Leaioidea E.Carb AUSTRALIA Jones and Chen - 2000 Hemicycloleaia andersonae Cratostracus? Cheni Estheriteoidea L.Cret - Aptian CHINA - western Liaoning Li and Batten 2004 Yanjiestheria dabeigouensis Eosestherioidea E.Cret CHINA - eastern Jilin Li et al.et al.2007 Dictyestheriae longata Estheriteoidea Cret? CHINA - Yaojia Formation Li, Wanet al.et al. 2009 Plectestheria songhuaensis Estheriteoidea U.Cret CHINA - Jilin Province Li et al.et al.2009b Halysestheria yui Estheriteoidea Cret? CHINA - Jilin Province Li,Wan et al.et al. 2009 Isaura (Euestheria) harveyi Cyzoidea Perm USA - Kansas Tasch 1958 Pemphicyclus-Gabonestheria Cyzoidea L.Carb Tasch 1961 Menucoestheria wichmanni Eosestherioidea Tria ARGENTINA - Vera Formation Gallego 2010 Abrestheria rotunda Eosestherioidea L.Cret CHINA - NorthenHebei Li et al.et al. 2006 Carapacestheria disgregaris L.Jura ANTARCTICA - Shackleton Glacier Shen 1994

needed in order to understand the structure and Lohmann and Schweitzer, 1990). It has been effec- function of this important diagnostic feature. tively applied in previous biological, paleontological The quantification of carapace shape using and paleoenvironmental analyses (MacLeod, contemporary morphometric techniques, as imple- 2002; Krieger et al., 2007; Astrop, 2011) and has mented in this study, builds on historical sugges- also been successful in detecting sexual dimor- tions of preserved dimorphism in clam shrimp in phism in fossil Ostracoda (Elewa, 2003). Standard order to diagnose both sex ratio and by extension eigenshape analysis is suitable for the exploration the reproductive system. A more detailed examina- of variation in the Spinicaudatan carapace because tion of carapaces in relation to sexual dimorphism multiple, discrete, homologous features (i.e., land- allows a new approach to fossil “Conchostracan” marks in geometric morphometric analyses; Book- studies and the interpretation of mating systems in stein, 1986) are absent. The changes in carapace fossil species (via sex ratio comparisons and/or shape (associated with the maintenance of a brood generic identities – see below). This new approach chamber, development of claspers and other ana- is of great importance for studies of breeding sys- tomical differences between the sexes) are not tem evolution. only suspected to be significant, but in light of previous applications, likely to be detected through METHODS analysis of outlines. Changes in the shape of carapace outlines are described as changes to individ- The Morphometric ‘revolution’ (Rohlf and Mar- ual margins. The location of margins and major cus, 1993) and the subsequent development of anatomical features of a clam shrimp can be seen modern, computer-based techniques (see Adams in Figure 1. et al., 2004; Laffont et al., 2011 for reviews) has Eigenshape analyses (sensu MacLeod, 1999) enabled the investigation of morphological variabil- operate via the conversion of the digitized outline ity by quantifying features traditionally assigned of an individual specimen into equidistant, Carte- qualitative values based on primary homology sian (x-y) coordinates. The digitized co-ordinates assessments between and within taxonomic are then transformed into a shape function as groups (de Pinna, 1991). angular deviations (phi function: φ; Zahn and Eigenshape analysis (MacLeod, 1999) is a Roskies, 1972) from the previous step (coordinate) powerful contemporary technique employed in the in order to describe the shape of the curve ordination of curved outlines (Lohmann, 1983;

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(sequestering size from the analysis). This descrip- models v0.7 Mathematica notebooks available via tion is derived from a set of empirical, orthogonal the morphotools site (http://www.morpho- shape functions via an eigenfunction analysis of a tools.net). The analysis interpolates and standard- matrix of correlations between shapes. Eigen- izes the raw Cartesian data before performing a shape ‘scores’ can be then used to project individ- singular value decomposition to produce eigenval- ual specimens into a multi-dimensional ues, eigenscores and eigenshapes that describe morphospace that allows the visualization of indi- variation of shape within the dataset. The eigen- vidual vectors of shape change and highlight shapes produced by the analysis describe two- whether particular vectors of deviation from the dimensional axes of shape change that can be ‘mean shape’ are characteristic of a particular used to construct morphospaces that specimens group. may be projected into, allowing trends in shape This study utilizes three independent data- variation to be observed. All specimens used in this sets. The first consists of four species from two study are adult, exhibiting multiple growth lines, genera within the family Limnadiidae: the dioecious claspers (in males) and egg clutches in the brood species Limnadia badia and Limnadia stanleyana, chamber (in females/hermaphrodites) (Figure 2). along with the androdioecious species Eulimnadia A total of 120 adult specimens were imaged dali, and Eulimnadia texana. Fifteen males and 15 (left valve only) to create the first dataset. Popula- females/hermaphrodites were selected from popu- tions of L.badia, L.stanleyana, E.texana and lations reared in the laboratory, producing a data- E.dahli were reared in the laboratory following the set of n=120. This data set was designed to protocols outlined in Weeks and Zucker (1999). Of investigate the strength and extent of any dimor- these taxa, 15 of each sex (male/female for Limna- phic ‘signal’ in carapace shape. A second, smaller dia, male/hermaphrodite for Eulimnadia) were pro- dataset was assembled to assess the phylogenetic cessed. Preserved specimens of L. occidentals consistency of shape dimorphism within the group and C. mexicanus were used to create the smaller, by using the Australian endemic species Limna- second dataset, while 34 well preserved speci- dopsis occidentalis and a representative of the mens of C. disgragaris from palaeontological col- family Cyzidae, Cyzicus mexicanus. lections at Ohio University and the Natural History In order to apply the above protocol to extinct Museum, London (Figure 2.7, 2.8) were identified taxa, it was important to develop a methodology to to create the third, fossil dataset. All specimens validate the existence of ‘morphotypes’ within a were imaged facing left and outlines digitized to fossil sample. Therefore a third dataset of the 500 equidistant points beginning from the junction Lower Jurassic (Ferrar Group, Toarcian Stage), of the anterior and dorsal margins of the carapace. Estheriid Carapacestheria disgragaris (Tasch A composite .tps file of outline data for all 120 1987), was assembled. Fossil specimens were specimens was constructed using the free tpsUtil originally collected from the Shackleton Glacier v1.44 (Rohlf, 2009) software. These data were region, Carapace Nunatak, South Victoria Land, then subjected to a standard eigenshape analysis. Antarctica (Stigall and Hartman, 2008). The sedi- The outlines were treated as closed curves and mentary interbeds of Kirkpatrick Basalt deposits were mean centered and standardized to remove from which these fossils are derived represent high size, scale and rotation from the analysis (see palaeolatitude lakes where algal biofilms enabled Slice, 2005). All statistical analyses (ANOVA, Clus- the preservation of weakly biomineralized lake flora tering, Discriminant) where performed using PAST and fauna (Stigall and Hartman, 2008) (For v2.14 (Hammer et al., 2001). detailed geologic, stratigraphic and palaeontological information see Stigall and Hartman, 2008; Sti- RESULTS gall-Rode et al., 2005; Tasch, 1969; Tasch. 1987). Standard eigenshape analysis of all 120 out- All specimens were imaged using a Jenoptik Pro- lines produced 119 eigenshapes, the first four of gres C5 digital camera attached to a binocular which account for ~30% of the observed variance microscope and desktop computer with Jenoptik captured by the analysis (Figure 3). Capture Pro software. The outlines of individual By observing the trends in shape change cap- carapaces were digitized using tpsDig v2.10 tured by each eigenshape axis, it is possible to (Rohlf, 2008) and then subjected to an eigenshape describe corresponding biological change seen in analysis. Digitized outline data were saved as .tps the extant clam shrimp datasets. The first eigen- files which were then processed using modified shape axis (Figure 3) exhibits considerable change versions of the Eigenshape v2.6 and Guide to in the anterior portion of the dorsal margin which

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FIGURE 2. Limnadia badia female (1) and male (2). Carapace dimorphism prominent on dorsal margin associated with presence/absence of brood chamber. Limnadopsis occidentalis female (3) and male (4) displaying similar carapace dimorphism to that seen in L. badia, despite gross morphologic difference. Female (5) and male (6) Cyzicus mexicanus. The dimorphism in Cyzicus is more subtle than that seen within the Limnadiidae, the female (5) being slightly more elongate with a flattened postero-ventral margin. ‘Female’ morphotype (7) and ‘male’ morphotype (8) of Carapacestheria disgragaris. All scale bars equal 1 mm. corresponds to the presence/absence of the brood posterior and ventral margins while eigenshape chamber in egg-bearing individuals and males, four represents the ‘roundedness’ of the dorsal respectively. This axis also captures change in margin. angularity of the junction between the posterior and A plot of the first two eigenshapes clearly dorsal margins. The second eigenshape axis is shows a divergent trend between the two sexes related to the relative position of the brood cham- along either axis (Figure 4). Despite some clear ber. Eigenshape axis three captures change in the overlap, the two groups are well defined when this

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FIGURE 3. Modes of shape change represented by eigenshape (ES) axes 1-4. Eigenshapes produced via standard eigenshape analysis (sensu MacLeod 1999) of digitized outlines of 120 total specimens of E. dahli, E. texana, L. badia and L. stanleyana. Dark-light shading indicates representative shapes with low to high scores on each of the four axes.

dataset is assessed using a hierarchical clustering described here, it is possible to correctly identify approach (Ward’s method) (Figure 5). The distinc- species for 92.3% (112 of 120 properly classified) tion between the two sexes was highly significant of the measured individuals. (Table 2). The fact that the overlap on the first two Using these same techniques to analyze eigenshape axes did not translate to higher levels smaller datasets (n= 20-25) of the phylogenetically of mis-scoring (Figure 5) and exceptionally high disparate clam shrimp Cyzicus mexicanus (Figure levels of distinction between the two sexes (Table 2.5, 2.6) and morphologically disparate Limnadop- 2) underscores the importance of the second two sis occidentalis (Figure 2.3, 2.4), sexes were also eigenshapes at further distinguishing the two successfully discriminated 76% and 73% of the sexes. A MANOVA of this dataset using the first time, respectively (Table 2). MANOVAs performed four eigenshapes reports a significant difference on the first four eigenshape scores of either data between scores for both groups. sets (Table 2) revealed that the between-group dif- The raw eigenscores for the first four eigen- ference in carapace shape is not significant for shapes produced were utilized in discriminant anal- Cyzicus (p=0.151; Figure 6) and weakly significant yses that successfully identified 96% of all for Limnadopsis (p=0.0497876; Figure 7), in these specimens based on sex (Table 2). The five mis- samples. Although these data exhibit a less clear classified individuals are likely outliers in terms of distinction between sexes (Figures 6 and 7), than erroneous shape capture, mis-identifications or the first data set (Figures 4 and 5), it is likely that possibly juveniles. Mis-classified individuals the smaller sample size and more subtle dimor- occurred in both genera. phism influenced the ability of the analyses to both Carapace shape can also be used to discrimi- capture the shape of and discern the sexes pres- nate between taxa. This has been shown in previ- ent, highlighting an important consideration for ous studies using combinations of linear future work. measurements and area-estimations (e.g., Zier- Initial observations of C. disgragaris revealed old, 2007; Hethke al., 2010) as well as landmark- two distinct carapace shapes: one elongate (Figure based methods (Stoyan et al., 1994). However, 2.8) and one more rounded (Figure 2.7). These these studies did not take into account carapace shapes are reflected in both the outline of the cara- shape dimorphism. Using the eigenshape method pace (Figure 8) and the growth lines preserved.

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FIGURE 4. A plot of 120 extant specimens (E. dahli, E. texana, L. badia and L. stanleyana) by sex (red = female/hermaphrodite, blue = male) on the first and second eigenshape axes. Mean shapes for males (blue) and females/hermaphrodites (red) shown both separate and overlapping to highlight dimorphism.

Eigenshape analysis of the fossil dataset produced ure 8 shows branch lengths based on distance, four major vectors of shape change that accounted clearly illustrating the existence of two distinct for ~30% of the observed variance. Hierarchical groups. Discriminant analysis of these two groups cluster analysis of C. disgragaris using the first four (proposed by the hierarchical cluster analysis eigenshapes produces two distinct groups (Figure approach) using the first four eigenshapes, 8, which were highly significantly different from one resulted in 97% success of classifying individual another (Table 2). These groups correspond to the fossils into these two groups (Table 2). morphotypes suspected as being different sexes. In order to test the effectiveness of the hierar- The branch lengths supporting these groups are chical clustering approach, extant androdioecious slightly shorter than those for the extant dataset. E. texana (Figure 9) and dioecious L. badia (Figure This length difference is likely due to increased 10), where the sex of individuals is known a priori, noise in the fossil dataset attributable to tapho- were tested using the same protocol described nomic sources. above. Hierarchical clustering produced two-group In order to test the initial observations of two assignments that successfully assigned sex 97 or discreet morphotypes within C. disgragaris, a hier- 100% of the time, respectively. MANOVAs using archical cluster analysis was performed (using the the first four eigenshapes of either data set reveal first four eigenshapes and applying Ward’s cluster- that between group differences are highly signifi- ing method). The hierarchical cluster seen in Fig- cant (Table 2).

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FIGURE 5. Hierachical cluster analysis (Ward’s method) of all 120 extant specimens using eigenshape axes 1-4. Note distinct clustering of two groups with little error. Terminal nodes labeled with species/sex abbreviations.

DISCUSSION been previously suggested but not rigorously tested (Bock, 1953; Oleynikov, 1969; Tasch, 1969; The long-term evolutionary forces driving the Tintori and Brambilla, 1991; Gallego, 2005, 2010; diversity and maintenance of breeding systems Li et al., 2007, 2008, 2009a, 2009b, 2010; Zierold, have traditionally been addressed by theory alone 2007). Traditional attempts at utilizing morphomet- (Williams, 1975; Kondrashov, 1993; Charnov, ric measurements to quantify carapace shape via 1982; Charlesworth, 1984, 2006; Nunney, 1989; linear measurements (Oleynikov, 1969; Tintori and Pannell, 2009) due to the lack of detailed historical Brambilla, 1991; Machado et al., 1999; Jones and information and absence of breeding system Chen, 2000; Babcock et al., 2002; Zierold, 2007) assignments in the fossil record. This study suc- and, in one case, landmark-based methods cessfully quantifies and interprets sex-specific car- (Stoyan et al. 1994), have produced mixed results apace morphology for the first time in fossil that likely do not best represent the gross dimor- Spinicaudata and allows informative biological phic carapace morphology. Although dimorphism inferences to be made from fossil taxa. had been previously noted in C. disgragaris by We have shown that carapace shape does Tasch (1987), Shen (1994) deemed dimorphism indeed carry a strong, sex-based signal, as had “more difficult to determine” due to the distorting

TABLE 2. Species used in this study with associated MANOVA significance values (p*) of between group differences and discriminant scores (DS) as a percentage classified correctly. The first four eigenshapes produced by analysis of the separate datasets were used in the MANOVA and discriminate analyses.

Dataset M F MANOVA (p*) DS (%)

L. badia, L. stanleyana, E. texana, E. dhali 60 60 2.03E-41 97

L. occidentalis 11 11 0.0497876 73

L. badia 15 15 6.92E-14 100

E. texana 15 15 1.17E-12 100

C. mexicanus 9 15 0.151 76

C. disgragaris † 18 16 1.74E-10 97

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FIGURE 6. Plot of C. mexicanus on eigenshape axes 1 and 2 with dendrogram of hierarchical cluster analysis (Ward’s method) using the first four eigenshapes. Mean shapes for males (blue) and females (red) shown separate and overlapping to highlight dimorphism.

FIGURE 7. Plot of L. occidentalis on eigenshape axes 1 and 2 with dendrogram of hierarchical cluster analysis (Ward’s method) using the first four eigenshapes. Mean shapes for males (blue) and females (red) shown separate and overlapping to highlight dimorphism. effects associated with fossilization despite being imens of C. disgragaris were collected (Stigall and able to provide some distinction between proposed Hartman, 2008), the specimens selected for this morphotypes (similar to those described via eigen- study showed no signs of warping or distortion of shape analysis in this study) using linear height/ any kind. Ongoing taphonomic studies (Astrop and length measurements of the valves. Although Hegna, unpublished data) have revealed that the some degree of tectonism in the form of folding is Spinicaudatan carapace is resistant to extensive reported in the Kirkpatrick Basalt from where spec- aqueous post-mortem transport. Also of import is

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FIGURE 8. Plot of C. disgragaris on eigenshape axes 1 and 2. Mean shapes for morphotype 1 ‘male’ (blue) and morphotype 2 ‘female’ (red) highlight possible dimorphism with associated Hierarchical cluster analysis (Ward’s method) using eigenshape axes 1-4. The designation of specimens to either two morphotypes is based on the distinct two- group clustering.

FIGURE 9. Plot of E. texana males and self-fertilizing hermaphrodites on eigenshape axes 1 and 2 with dendrogram of hierarchical cluster analysis (Ward’s method) using the first four eigenshapes. that after deposition, remains of Spinicaudatans the detailed eigenshape methodology to correctly are surrounded by sediment and subsequently pre- distinguish sexes in species where sex is known a vented from laterally expanding during compaction, priori. The observations that these methods clearly as found by Briggs and Williams (1981). extract sexual type in extant species, the clear sim- The usefulness of the current approach is our ilarity of shape between the fossil and extant male/ ability to use these methods on both extant and female (hermaphrodite) carapaces, as well as the fossil clam shrimp and underscores the ability of strong signal of two morphotypes in the fossil data

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FIGURE 10. Plot of L. badia males and females on eigenshape axes 1 and 2 with dendrogram of hierarchical cluster analysis (Ward’s method) using the first four eigenshapes. show these methods can be successfully used to occurring species: most modern clam shrimp popu- assess individual sex in fossil clam shrimp cara- lations are monospecific, but where clam shrimp paces. Using individual gender determination we species do occur together they are morphologically can further distinguish population sex ratios in fos- and phylogenetically disparate (Orr and Briggs, silized death assemblages. 1999; Dumont and Negrea, 2002). Given that The results from the extant taxa highlight that these two morphotypes are consistent with shape morphological dimorphism in the form of carapace differences between the sexes observed in extant shape occurs throughout the Spinicaudata. Differ- clam shrimp taxa (Figure 2), the morphotypes may ences in the mode of dimorphism between dispa- be considered male and female (Figure 8; compare rate taxa is likely the result of differing anatomical also with average shapes in Figures 9 and 10). locations of the egg-clutch/brood chamber (dorsal Thus, it is likely that C. disgragaris was a dioecious in the Limnadiidae, lateral in the Cyzidae and Lep- clam shrimp species. testheriidae), and possibly the size and position of In addition, it is also important to note that due claspers in males of all taxa. Upon application of to their thin, chitinous carapace and weakly sclero- the eigenshape method to a fossil taxon, it is clear tized soft integument, clam shrimp may be suscep- that it is indeed possible to detect dimorphism in tible to pre-depositional (transport, predation, extinct clam shrimp and subsequently, it is reported desiccation) and post-depositional (decay, com- here that the sex ratio between the two morpho- paction) processes, and as such, these sources of types of C. disgragarisis 51:49 male:female. variation may distort carapace shape in particular Weeks et al. (2008, 2009) have noted that sex ratio ways. In order to account for these sources of vari- can be used to assign breeding system in extant ation, a dedicated taphonomic study is currently clam shrimp species: 0% male samples are self- underway to allow us to characterize how such fertilizing hermaphrodites, 20-30% males are variables influence the entry of clam shrimp into androdioecious and 50+% males are dioecious. It the fossil record and elucidate how to recognize is unlikely that the bimodal variance observed in and account for these sources of taphonomic vari- both the clustering and scatter plot (Figure 8) is the ation. result of ontogenetic variation as all specimens The successful elucidation of sex-specific car- exhibit several growth lines, which do not occur in apace shape, and by extension sex ratios, in this juveniles. It is also unlikely that the two ‘morpho- study shows that contemporary morphometric types’ are the result of the preservation of co- analyses of fossil clam shrimp are capable of pro-

12 PALAEO-ELECTRONICA.ORG viding the quantitative information required to Brande, S. 1982. Epibiont analysis of the fossil interac- assign breeding system to extinct clam shrimp. tions among a benthic infaunal bivalve, a barnacle The extraction of breeding system from fossils can and a drilling gastropod. Journal of Paleontology, now allow tests of hypotheses concerning the long- 56(5):1230-1234. term evolution and maintenance of breeding sys- Brantner, J. 2011.Mating system inferences in represen- tatives from two clam shrimp families (Limnadiidae tems in these interesting, and reproductively labile, and Cyzcidae) using histological and cellular obser- crustaceans. vations. Thesis: Master of Science, University of Akron. ACKNOWLEDGMENT Briggs, D.E.G. and Williams, S.H. 1981.The restoration We thank A. Stigall, T. Hegna, J. Brantner, A. of flattened fossils. Lethaia 14, 157-164. Charlesworth, D. 1984. Androdioecy and the evolution of Calabrese, L. Vaz Tassi and L. Roketenetz for their dioecy. Biological Journal of the Linnean Society, helpful advice, correspondence and material, the 22:333-348. Natural History Museum, London for access to Charlesworth, D. 2006. Evolution of plant breeding sys- their collections, D. Polly, our handling editor, and tems. Current Biology, 16(17):R726-R735. two anonymous reviewers for their helpful sugges- Charnov, E.L. 1982. The Theory of Sex Allocation. Princ- tions. This research was funded in part by a Den- eton University Press, 363 p. ton Belk Memorial Scholarship from the Chen, P., and Hudson, J.D. 1991.The Conchostracan Crustacean Society to T.I. Astrop. L.E. Park fauna of the great estuarine group, Middle Jurassic, (Boush) was supported by the National Science Scotland. Palaeontology, 34(3):515-545. Foundation as part of the author’s Individual de Pinna, M.C.C. 1991. Concepts and tests of homology Research Plan. She is currently a Program Officer in the cladistic paradigm. Cladistics. 7: 367-394. Dumont, H.J. and Negrea, S.V. 2002. Introduction to the within the Earth Science Division of the Geosci- class Branchiopoda. In H.J. Dumont (ed.), Backhuys ence Directorate. Publishers, Leiden. Elewa, A. 2003. Morphometric studies on three ostracod REFERENCES species of the genus Digmocythere mandelstam Abe, K. 1990. What the sex ratio tells us: A case from from the middle Eocene of Egypt. Palaeontologia marine ostracods, p.175-185. In Ostracoda and Electronica, 6(2):1-11. Global Events, Whatley, C.R. and Maybury, C. (ed.), Fernandez-Leborans, G. 2010.Epibiosis in Crustacea: an Chapman and Hall, Cambridge. overview. Crustaceana, Volume 83(Number 5):549- Adams, D.C., J.F. Rohlf, and Slice, D.E. 2004. Geometric 640(92). morphometrics: Ten years of progress following the Frank, P.W. 1988. Conchostraca. Palaeogeography, ‘revolution’. Italian Journal of Zoology, 71:5-16. Palaeoclimatology, Palaeoecology. 62(1-4), 399-403. Astrop, T.I. 2011. Phylogeny and evolution of Mecochiri- Gallego, O. F. 2005. First record of the family Palaeolim- dae (Decapoda: Reptantia: Glypheoidea): an inte- nadiopseidae Defretin-le Franc, 1965 (Crustacea– grated morphometric and cladistic approach. Journal Conchostraca) in the Triassic of Argentina. Journal of of Crustacean Biology, 31(1):114-125. South American Earth Sciences, 18(2), 223-231 Babcock, L.E., Isbell, J.L., Miller, M.F. and Hasiotis, S.T. Gallego, O.F. 2010. A new crustacean clam shrimp 2002. New Late Paleozoic Conchostracan (Crusta- (Spinicaudata: Eosestheriidae) from the Upper Trias- cea: Branchiopoda) from the shackleton glacier area, sic of Argentina and its importance for ‘Conchostra- Antarctica: age and paleoenvironmental implications. can’ taxonomy. Alcheringa: An Australasian Journal Journal of Paleontology, 76(1):70-75. of Palaeontology, 34(2):179-195. Barrett, S.C.H. 2002. The evolution of plant sexual diver- Hammer, Ø., Harper, D.A.T., and Ryan, P.D. 2001. PAST: sity. Nature Reviews Genetics, 3:274-284. Palaeontological statistics software package for edu- Bell, G. 1982. The Masterpiece of Nature. University of cation and data analysis. Palaeontologica Electron- California Press, Berkeley, CA. ica 4(1):9p Bishop, J.A. 1967. Seasonal occurrence of a branchio- Hethke, M., Fursich, F.T. and Jiang, B. 2010. Determin- pod crustacean, Limnadia stanleyana King (Concho- ing morphotypes of fossil clam shrimps by fourier straca) in Eastern Australia. Journal of Animal analysis: Possible implications and limitations tested Ecology, 36(1):77-95 on Middle Jurassic specimens from Gansu, China. Bock, W. 1953. American Triassic Estherids. Journal of Earth Science Frontiers 17:217-218 Paleontology, 27(1):62-76. Jarne, P. and Auld, J.R. 2006. Animals mix it up too: The Bookstein, F. L. 1986. Size and shape spaces for land- distribution of self-fertilization among hermaphroditic mark data in two dimensions. Statistical Science, animals. Evolution 60:1816-1824. 1(2):181-222.

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