Invertebrate Biology x(x): 1–11. © 2013, The American Microscopical Society, Inc. DOI: 10.1111/ivb.12012 1 2 3 Androdioecy and hermaphroditism in five species of clam shrimps 4 (Crustacea: : Spinicaudata) from India and Thailand 5 6 Justin S. Brantner,1 Donald W. Ott,1 R. Joel Duff,1 La-orsri Sanoamuang,2 7 3 4 1,a 8 Gulli Palli Simhachalam, K. K. Subhash Babu, and Stephen C. Weeks 9 1 Integrated Bioscience Program, Department of Biology, The University of Akron, 10 Akron, Ohio 44325-3908, USA 11 2 Applied Taxonomic Research Center, Department of Biology, Khon Kaen University, 12 Khon Kaen 40002, Thailand 13 3 Department of Zoology, Acharya Nagarjuna University, Guntur, Andhra Pradesh, India 14 4 Department of Marine Sciences, Biochemistry and Microbiology, Cochin University of Science and Technology, 15 Lakeside Campus, Cochin, Kerala, 16, India 16 17 18 Abstract. in the order Spinicaudata display a broad range of reproductive 19 types, ranging from pure hermaphroditism to pure dioecy (separate males and females), and 20 mixes in between. One particularly interesting genus of these “clam shrimps” is Eulimnadia. 21 Based on offspring sex ratios, it has been suggested that all members of the genus are 22 androdioecious: populations consist of mixtures of males and hermaphrodites. However, 23 only two of the ~40 species in this genus have been examined histologically to confirm the 24 presence of ovotestes in the purported hermaphrodites of this group. Here, we report both 25 sex ratio and histological evidence that populations of five additional Eulimnadia species 26 from India and Thailand are indeed mixes of males and hermaphrodites (four species) or 27 hermaphrodite only (one species). Sex ratios of adults and offspring from isolated hermaph- 28 rodites are in accordance with those previously reported for 15 Eulimnadia species, and 29 histological assays of four of the five species show the presence of both testicular and ovar- 30 ian tissue in these hermaphrodites. As has been previously reported, the testicular tissue in 31 members of these Eulimnadia spp. is located in a small section at the distal end of the 32 gonad. In addition, the sperm produced in these hermaphrodites forms distinct plaques of 33 compacted chromatin. Overall, these data are consistent with a single origin of hermaphro- 34 ditism in the Eulimnadia, and support the notion that members of the entire genus are either 35 androdioecious or all-hermaphroditic. 36 Additional key words: Eulimnadia, Conchostraca, breeding systems, ovotestes 37 38 39 40 41 The Branchiopoda (Arthropoda; Crustacea) repre- bers of the (which includes clam 42 sents one of the most ancient lineages shrimps and water fleas) contain at least five differ- 43 that still includes extant representatives (Walossek ent breeding systems: dioecy (true males and true 44 1993; Martin & Davis 2001; Olesen 2007). The class females), hermaphroditism (individuals capable of 45 Branchiopoda is subdivided into the orders Anostra- self-fertilization due to testicular and ovarian tissue 46 ca, , and Diplostraca (Martin & Davis occurring concomitantly in the same reproductive 47 2001; Olesen 2007). These taxa have been exten- tract), androdioecy (hermaphrodites and males), 48 sively studied by evolutionary biologists interested parthenogenesis (asexual reproduction), and cyclic 49 in, among other things, the diversity of breeding sys- parthenogenesis (multiple episodes of asexual repro- 50 tems that exist within and among branchiopod duction followed by marked periods of dioecy) 51 groups (Dumont & Negrea 2002). Specifically, mem- (Hebert & Finston 1993; Sassaman 1995; Martin & 52 Davis 2001; Dumont & Negrea 2002; Olesen 2007; 53 aAuthor for correspondence. Weeks et al. 2008). Of these five breeding systems, 54 E-mail: [email protected] four are found specifically in one suborder of the

IVB 12012 Dispatch: 2.1.13 Journal: IVB CE: Tushara Journal Name Manuscript No. B Author Received: No. of pages: 11 PE: Hari Prakash 2 Brantner, Ott, Duff, Sanoamuang, Simhachalam, Babu, & Weeks

1 Diplostraca: the Spinicaudata. Such reproductive One taxon for which a detailed analysis of repro- 2 diversity makes these branchiopods ideal candidates ductive systems is needed is the genus Eulimnadia. 3 for studies of the evolution of breeding systems. Members of Eulimnadia inhabit temporary playas, 4 The suborder Spinicaudata comprises three fami- ditches, and many other ephemeral freshwater habi- 5 lies (, Leptestheriidae, and ) tats throughout the world, from the tropics to the 6 that are collectively known as “clam shrimps” deserts (Dumont & Negrea 2002). Hermaphrodites 7 (Martin & Davis 2001; Olesen 2007). Historically, produce desiccation-resistant cysts, which they bury 8 sex ratio studies on the Spinicaudata have been the within the top several millimeters of the soil. These 9 method used to identify breeding systems (Sassaman cysts hatch rapidly following hydration under spring 10 1995). Sex ratio determination in popu- and summer conditions (at water temperatures 11 lations is based solely on external morphological above 18°C), releasing a nauplius larva. Larval and 12 characters (i.e., claspers in males, and eggs in juvenile growth is extraordinarily rapid: most 13 females/hermaphrodites). In many species of clam shrimp reach reproductive size in 4–7 d in the labo- 14 shrimps, sex determination occurs 5–8 d following ratory at 27–30°C (Sassaman & Weeks 1993; Weeks 15 hatching from desiccation-resistant eggs (Sassaman et al. 1997), and in as little as 4–6 d in the field 16 & Weeks 1993; Brendonck 1996; Weeks et al. 1997, (Vidrine et al. 1987). The hermaphrodites produce 17 2002). Using these morphological indicators to iden- thousands of eggs in their lifetime, generating 18 tify the sex of individuals in populations of clam clutches ranging from 100 to 300 eggs, one to two 19 shrimps, four breeding systems have been identified times a day (Knoll 1995; Weeks et al. 1997). Sexual 20 in the suborder Spinicaudata: dioecy, androdioecy, dimorphism is pronounced. The thoracic append- 21 hermaphroditism, and parthenogenesis. ages of hermaphrodites are unmodified, but the first 22 Before evolutionary hypotheses regarding the evo- two pairs of thoracic appendages in males undergo 23 lution of breeding systems can be tested within the differentiation into claw-like claspers, which are 24 Spinicaudata, a more accurate assessment of the used to hold on to the margins of a hermaphro- 25 reproductive status of species is necessary. Such dite’s carapace during mating. Natural populations 26 assessments require examinations that extend of Eulimnadia are typically hermaphrodite-biased 27 beyond mere sex ratio determinations. Aside from (Mattox 1954), with some populations completely 28 Wingstrand’s (1978) descriptive account of bran- lacking males (Zinn & Dexter 1962; Stern & Stern 29 chiopod spermatology, which included examination 1971). Weeks et al. (2008, 2009a) have inferred that 30 of some representatives of Spinicaudata, few studies the entire genus Eulimnadia comprises either 31 have looked at the reproductive biology of the Spin- androdioecious or all-hermaphroditic species. 32 icaudata from a cellular and histological perspec- Androdioecious species typically are mixtures of all- 33 tive, despite its importance in understanding hermaphrodite and mixed male/hermaphrodite pop- 34 phylogenetic relationships (Martin & Davis 2001). ulations (Sassaman & Weeks 1993; Weeks et al. 35 A few cellular and histological studies have exam- 2008). Sex is determined in these clam shrimp by a 36 ined clam shrimps in the family Leptestheridae, e.g., Z-W chromosomal system (Weeks et al. 2010) in 37 Leptestheria dahalacensis RUPPELL€ 1837 and Eolep- which there are three chromosomal types: ZZ 38 testheria ticinensis BALSAMO-CRIVELLI 1859 (Tomma- males, ZW “amphigenic” hermaphrodites, and WW 39 sini & Scanabissi Sabelli 1992; Scanabissi Sabelli & “monogenic” hermaphrodites; both hermaphroditic 40 Tommasini 1994; Scanabissi & Mondini 2000). In types can self-fertilize or mate with males (Sass- 41 addition, a few ultrastructural and histological stud- aman & Weeks 1993). When selfing, the monogenic 42 ies have been conducted to assess reproductive hermaphrodites “breed true”, producing 100% 43 diversity in the family Limnadiidae; these have monogenic hermaphroditic offspring, whereas the 44 focused on Eulimnadia texana PACKARD 1871 (Zuc- amphigenic hermaphrodites produce 25% male, 45 ker et al. 1997; Scanabissi et al. 2006), Eulimnadia 50% amphigenic hermaphrodite, and 25% mono- 46 agassizii PACKARD 1874 (Weeks et al. 2005), and genic hermaphrodite offspring when selfed (Sass- 47 Limnadia lenticularis LINNAEUS 1761 (Zaffagnini aman & Weeks 1993). Weeks et al. (2008) used 48 1969; Tommasini & Scanabissi Sabelli 1992; Scan- these sex ratio predictions to assess offspring rea- 49 abissi & Mondini 2002a). Clearly, to better under- rings from isolated hermaphrodites and found 15 50 stand the evolution and diversity of breeding Eulimnadia species to be either androdioecious or 51 systems found within the Spinicaudata, a more all-hermaphroditic. Hermaphrodites from only two 52 extensive examination of the histological and cellu- of these 15 species (E. texana and E. agassizii) have 53 lar structure of clam shrimp reproductive systems been examined histologically (Zucker et al. 1997; 54 must be undertaken. Weeks et al. 2005). Because the genus Eulimnadia

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1 contains over 40 species (Brtek 1997), an assessment NN1 from Nagarjuna Nagar, Guntur district, And- 2 of reproduction in more members of the genus must hra Pradesh), E. azisi from India (two populations, 3 be made before we can confidently assert that this Ghat 1 and Ghat 2, from the Western Ghats, Vetti- 4 genus contains only androdioecious and all-her- lapara area), Eulimnadia sp. from India (two popu- 5 maphroditic species. lations: Veli and Karuvatta), and E. michaeli from 6 Toward that goal, this study combines micros- Thailand (six populations, KK2A, KK4A, KK6A, 7 copy with offspring rearings to assess reproductive KK7A, KK8A, and KK9A, from Khon Kaen). The 8 mode in five species of Eulimnadia from India and soil samples were kept in a dry, dark cabinet for 9 Thailand: Eulimnadia gibba SARS 1900, E. gunturen- 6 months–2 years before hydration. 10 sis RADHAKRISHNA &DURGA PRASAD 1976, E. micha- In cases where ample soil was available, approxi- 11 eli NAYAR &NAIR 1968, E. azisi SUBHASH BABU & mately 500 mL of field site soil was added to a 27-L 12 BIJOY NANDAN 2011, and an undescribed Eulimnadia aquarium, and then hydrated using deionized (DI) 13 sp. We show that these five species are indeed a mix- water. In cases where only sparse amounts of soil 14 ture of androdioecious and all-hermaphroditic were available, approximately 250 mL of field site 15 reproductive modes, and note that the anatomical soil was added to a 10-L aquarium, and then 16 layout of the ovotestis in E. gibba, E. gunturensis, E. hydrated using DI water. “Standard conditions” 17 michaeli, and E. azisi has the same structural design were maintained for all aquaria (see Sassaman & 18 as E. texana and E. agassizii. These findings are Weeks 1993; Weeks et al. 1997, 2000, 2001, 2008, 19 consistent with a single origin of hermaphroditism 2009a). These included a room temperature of 25– 20 in this genus, as asserted previously (Weeks et al. 28°C, continuous light, minimal tank aeration (using 21 2009a). air stones), and daily feeding of a mixture of ground 22 baker’s yeast and TetraminTM fish food (Tetra Wer- 23 Methods ke, Melle, Germany) (2.5 g of each in 500 mL of DI 24 water). On day five after hydration, up to 100 25 immature clam shrimps were individually isolated in Rearing of samples from soil 26 500-mL plastic cups. Approximately, 6–10 d follow- 27 The cysts of clam shrimps were collected in soil ing the appearance of nauplii, the shrimps in both 28 samples from dry temporary pools (i.e., populations) the isolated and larger aquaria were sexually 29 as summarized in Table 1. Cysts of Eulimnadia gib- matured. Sexual maturity was noted when males 30 ba were collected from India (one population, developed claspers and hermaphrodites produced 31 PED1, from Pedakakani, Guntur district, Andhra eggs within “brood chambers” on their dorsal sur- 32 Pradesh), E. gunturensis from India (one population, face. Total population sex ratios were documented 33 34 Table 1. Population sex ratios of five species of Eulimnadia reared from soil samples. Herm., hermaphroditic; SE, stan- 35 dard error. Bolded numbers in the “% male” column indicate male percentages that were significantly different from 36 zero (see Weeks et al. 2008). Breeding systems are hermaphroditic (Herm.) or androdioecious (Andro.); see text for 37 determinations of breeding system. 38 39 Species Location Population # Male # Herm. Total % Male SE (%) Breeding System 40 41 E. azisi Ghat, India Ghat 1 0 112 112 0.0 0.0 42 E. azisi Ghat, India Ghat 2 0 120 120 0.0 0.0 Total 0 232 232 0.0 0.0 Herm. 43 E. gunturensis Nagarjuna Nagar, India NN1 1 30 31 3.2 3.2 Herm. 44 E. michaeli Khon Kaen, Thailand KK2A 50 56 106 47.2 4.8 45 E. michaeli Khon Kaen, Thailand KK4A 0 51 51 0.0 0.0 46 E. michaeli Khon Kaen, Thailand KK6A 0 32 32 0.0 0.0 47 E. michaeli Khon Kaen, Thailand KK7A 3 18 21 14.3 7.6 48 E. michaeli Khon Kaen, Thailand KK8A 0 39 39 0.0 0.0 49 E. michaeli Khon Kaen, Thailand KK9A 1 7 8 12.5 11.7 50 Total 54 203 257 21.0 2.5 Andro. 51 E. gibba Pedakakani, India PED1 11 40 51 21.6 5.8 Andro. 52 Eulimnadia sp. Veli, India Veli 27 159 186 14.5 2.6 53 Eulimnadia sp. Karuvatta, India Karuvatta 0 41 41 0.0 0.0 Total 27 200 227 11.9 2.4 Andro. 54

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1 for both isolated and aquarium-reared shrimp. uals were female (no viable offspring expected) 2 Males from the isolated cups and all shrimp in the versus asexual/hermaphroditic (viable offspring pro- 3 aquaria were frozen upon sexual maturity. Females/ duced in isolation; see Sassaman & Weeks 1993). If 4 hermaphrodites in the isolated cups were allowed to isolated individuals produced viable offspring, sex 5 produce eggs for egg banks (see below) for ~7d, ratios of their offspring can be used to distinguish 6 and then were frozen for potential future genetic hermaphroditic type (Sassaman & Weeks 1993). 7 analyses. For all-hermaphroditic populations, all isolated 8 hermaphrodites were expected to produce 100% 9 hermaphroditic offspring, termed “monogenic” her- Rearing from egg banks 10 maphrodites (Sassaman & Weeks 1993). For andro- 11 Egg banks generated above were used to assess dioecious populations, expectations were that some 12 the sex ratios of offspring produced by isolated indi- isolates would produce 0% males (monogenics) 13 viduals. Eggs and DI water were added to 500-mL while others would produce ~25% males (amphi- 14 cups along with 2 mL of baker’s yeast/TetraminTM genics) among their offspring (Sassaman & Weeks 15 food. Cups were maintained under continuous light 1993). To test for a 3:1 ratio of hermaphrodites to 16 at temperatures of 25–28°C. In addition, 10-L rear- males in amphigenic clutches, a Chi-squared analy- 17 ing tanks were prepared with water from “condi- sis was used (Sassaman & Weeks 1993). 18 tioning” tanks that had 27 L DI water and 500 mL 19 of soil collected from either Arizona or Indiana Microscopy 20 (United States). This water was screened through a 21 63-lm sieve to ensure no cysts or nauplii were trans- Sexually mature clam shrimp hermaphrodites 22 ferred to the rearing tanks. These rearing tanks from populations of four clam shrimp species (E. 23 allowed growth of the nauplii once they hatched. gibba, E. gunturensis, E. michaeli, and E. azisi) were 24 The 500-mL cups were periodically checked for nau- removed from rearing tanks and prepared for fixa- 25 plii over a period of 2 days. Once observed, nauplii tion. A minimum of three hermaphrodites were sur- 26 were transferred from the 500-mL cups to the 10-L veyed from each of these four species. All fixation 27 rearing aquaria, which were then kept under the and embedding procedures were carried out at room 28 standard conditions noted above. Isolated sex ratios temperature unless otherwise noted. Samples were 29 were determined after sexual maturity, as noted placed into small glass vials and pre-fixed in a 2% 30 above. glutaraldehyde solution buffered with 0.1 mol LÀ1 31 sodium cacodylate (pH of 7.2) for 2 h. At this 32 point, the carapace of each individual was removed. Testing sex ratios 33 The samples were then placed into small glass vials 34 To assess whether the five species of Eulimnadia that contained fresh 2% glutaraldehyde buffered 35 studied here are mixtures of all-hermaphrodite and with 0.1 mol LÀ1 sodium cacodylate (pH of 7.2) for 36 androdioecious populations, as noted in other Eu- 1 h. The samples were then washed in three changes 37 limnadia species (Weeks et al. 2008), we compared of 0.1 mol LÀ1 sodium cacodylate buffer for 1 h. 38 both population and isolated sex ratios as deter- Next, the samples were post-fixed in 2% osmium À1 39 mined above to expectations derived from previ- tetroxide (OsO4) with 0.1 mol L sodium cacody- 40 ously studied species. Expected population sex ratios late buffer (pH of 7.2) for 1.5 h, following which 41 for all-hermaphroditic populations were 0% males: samples were washed with three changes of deion- 42 100% hermaphrodite (Weeks et al. 2006b), whereas ized (DI) water for 30 min. Samples were en bloc 43 there were no specific population sex ratio expecta- stained for 30 min using an aqueous 2% uranyl ace- 44 tions for androdioecious populations because the tate solution, then washed with three changes of DI 45 ratio of monogenics to amphigenics in natural popu- water at 10-min intervals. After washing the samples 46 lations determines overall (“population”) sex ratios thoroughly, the specimens were dehydrated over- 47 (Otto et al. 1993). Deviations of population sex night (approximately 13 h) in an acetone desiccator 48 ratios from expectations of all-hermaphrodite spe- (Ott & Brown 1974). To ensure complete dehydra- 49 cies were examined by constructing 95% confidence tion, 100% acetone was added and then removed 50 intervals around the sex ratios measured, and noting seven times at 10-min intervals the following day. 51 whether the CI included 0% males (see Weeks et al. Next, samples were covered in a 90% acetone-10% 52 2008). plastic (Embed-812; Electron Microscopy Science, 53 Sex ratios from isolated females/hermaphrodites Hatfield, PA, USA) solution, covered with alumi- 54 were used to test both whether the isolated individ- num foil with several holes punched in the foil, and

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1 placed under a fume hood overnight (~13 h) to were all significantly different from 0% male 2 allow slow evaporation of the acetone and total (Table 1). These male percentages are consistent 3 infiltration of the plastic. To ensure complete infil- with an androdioecious breeding system. Two of 4 tration, 100% plastic was added, and then partially these three species (E. michaeli and Eulimnadia sp.) 5 removed three times from the samples at 30-min had one or more populations that had no males 6 intervals the following day. Samples were placed in (Table 1), which is also consistent with most andro- 7 a mold, covered in 100% plastic, and placed in a dioecious species (Weeks et al. 2008). 8 60°C oven for 48 h. The embedded specimens were Although population sex ratios are generally 9 removed from the oven and allowed to cool. The indicative of breeding system type in clam shrimps 10 specimens were then sectioned with a Reichert (Weeks et al. 2008), sex ratios from egg banks col- 11 OMU-3 ultramicrotome using a diamond knife. lected from isolated females/hermaphrodites 12 Thick sections (1.5 lm) were gently placed on a (Table 2) are better indicators of breeding systems 13 slide, heat-fixed, and stained with a 1% toluidine because these sex ratios can be tested against specific 14 blue-1% sodium borate solution for light micros- predictions (Sassaman & Weeks 1993). All four spe- 15 copy. Thick sections were examined using an Olym- cies tested produced viable offspring from eggs col- 16 pus BX60 digital light microscope (Olympus lected from females/hermaphrodites reared in 17 1 America Inc) equipped with an Olympus DP71 digi- isolation, indicating that they were either self-com- 18 tal camera. patible hermaphrodites or asexual females (Weeks 19 et al. 2008). Isolated females/hermaphrodites pro- 20 Results duced broods without males, or broods that con- 21 tained 13–30% males. Although the 0% male 22 isolates could have been produced by asexual Sex ratios 23 females, the most parsimonious inference is that 24 The population samples of Eulimnadia azisi pro- these isolates comprised monogenic and amphigenic 25 duced all hermaphrodites (i.e., 0% males), suggest- hermaphrodites, as has been found in all other spe- 26 ing the species has an “all-hermaphroditic” breeding cies of Eulimnadia examined to date (Sassaman & 27 system (Table 1). One other species, E. gunturensis, Weeks 1993; Weeks et al. 2008). 28 had a low enough male sex ratio (3.2%) to not be Isolated sex ratios averaged by population are 29 significantly different than 0% males (Table 1), shown in Table 2A. Populations Ghat 1, Karuvatta, 30 which is consistent with an all-hermaphroditic and KK4A comprised only monogenic hermaphro- 31 breeding system (but see below). The remaining dites, while the remaining populations had 23 to 32 three species had average male percentages ranging 52% (mean 34.1%) monogenic hermaphrodites 33 from 12 to 22% in the population samples, which (Table 2A). As was found for the population sex 34 35 Table 2. Sex ratios of offspring produced by isolated hermaphrodites of Eulimnadia. A. Overall sex ratios combined 36 across hermaphroditic parents (each parent produced one clutch). % Monogenic indicates the percentage of the iso- 37 lated hermaphroditic parents that were monogenic (i.e., had only hermaphrodite offspring). B. Sex ratios of offspring 38 from isolated amphigenic hermaphrodites only. Chi-square is the calculated deviation from a 3:1 sex ratio expected for 39 hermaphrodites:males among the offspring of a selfing amphigenic hermaphrodite. Other columns are as in Table 1. 40 41 A. Species Population N parents # Male # Herm. Total % Male SE (%) % Monogenic 42 43 E. azisi Ghat1 14 0 861 861 0.0 0.0 100 E. gibba PED1 6 36 274 310 11.6 1.8 33.3 44 Eulimnadia sp. Veli 44 150 984 1134 13.2 1.0 22.7 45 Eulimnadia sp. Karuvatta 3 0 82 82 0.0 0.0 100 46 E. michaeli KK2A 20 232 2116 2348 9.9 0.6 40.0 47 E. michaeli KK4A 22 0 1416 1416 0.0 0.0 100 48 E. michaeli KK8A 21 83 1023 1106 7.5 0.8 52.4 49 B. Species Population # Male # Herm. Total % Male SE (%) Chi-square p-value 50 51 E. gibba PED1 38 129 167 22.8% 3.2% 0.45 0.5023 52 Eulimnadia sp. Veli 150 588 738 20.3% 1.5% 8.6 0.0033 < 53 E. michaeli KK2A 232 1130 1362 17.0% 1.0% 46.1 0.0001 < 54 E. michaeli KK8A 81 537 618 13.1% 1.4% 46.6 0.0001

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1 ratios, Ghat1, Karuvatta, and KK4A all had aver- sex ratio data were correct, we longitudinally sec- 2 age isolate sex ratios not significantly different from tioned the gonads of hermaphrodites of four of the 3 0% males, while PED1, Veli, and KK2A were all purported hermaphroditic/androdioecious species 4 significantly different from 0% males. KK8A was (E. gibba, E. gunturensis, E. michaeli, and E. azisi) 5 significantly different from 0% males in the isolated to test for the presence of ovotestes. Positioned 6 rearings (Table 2A), which differed from the 0% adjacent to the digestive tract within the hemocoel 7 males seen in the population rearings (Table 1). was a bi-lobed, tubular gonad. Beginning at the 8 Average sex ratios for isolates producing males anterior tip and constituting the majority of the 9 (i.e., amphigenics) ranged from 13 to 23% males gonad was a distinct female wall. This thick female 10 (Table 2B), with only one of the four populations wall (which surrounded a central luminal space) 11 (PED1) having a sex ratio not significantly different consisted mostly of closely compacted epithelial 12 from the 3:1 hermaphrodite to male ratio predicted cells, sporadically interrupted by developing oocytes. 13 by the ZW chromosomal system of other species of The female wall continued down the length of the 14 Eulimnadia. gonad until it was interrupted by a thin-walled 15 region (Fig. 1). This thin wall represented the male 16 wall, and the amoeboid cells strewn throughout the Histology 17 wall (as well as located in the lumen of the gonad) 18 To determine whether the conclusions of her- were clearly defined male gametes (Figs. 1, 2). 19 maphroditism and androdioecy inferred from the Often, male gametes toward the center of the lumen 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 Fig. 1. Longitudinal thick sections of the posterior region of the hermaphroditic gonads of Eulimnadia spp. A. Eulimn- adia gibba. B. Eulimnadia gunturensis. C. Eulimnadia michaeli. D. Eulimnadia azisi. In each of these species, the gonad 51 is surrounded by muscle (M). Internally, the gonad includes a female wall (FW) that is interrupted at the posterior- 52 most tip by a male wall (MW). From the male wall, male gametes (MG) are released into the gonad lumen (L) and 53 are often found surrounded by eggshell secretory matter (EM). DT, digestive tract; H, hemocoel; O, oocyte. Scale 54 bars=200 lm.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Fig. 2. Male gametes (MG) released from the male wall (MW) into the lumen (L) of the hermaphroditic gonads of Eu- 29 limnadia spp. A. Eulimnadia gibba. B. Eulimnadia gunturensis. C. Eulimnadia michaeli. D. Eulimnadia azisi. Many of the * 30 luminal male gametes possess sporadically arranged chromatin blocks ( ) against the nuclear membrane. Intercellular 31 bridges (arrows) are evident between the male gametes in E. azisi (D). EM, eggshell secretory matter; FW, female wall. Scale bars=50 lm. 32 33 34 35 could be seen in clusters associated with the eggshell 36 matter secreted by cells of the female wall of the Discussion 37 gonad (Figs. 1, 2). Phylogenetic evidence suggests that the common 38 Nuclear arrangement appeared to differ depend- ancestor of the genus Eulimnadia was androdioe- 39 ing on the location of the male gametes. Male cious (Weeks et al. 2006b, 2009a). To date, all spe- 40 gametes that were closely associated with the cies of Eulimnadia that have been surveyed have 41 male wall typically had well-defined nuclei with either been androdioecious or all-hermaphroditic 42 less compacted nuclear material. As male gametes (Weeks et al. 2008). Herein, we have evidence of an 43 were liberated from the male wall and began to additional five species of Eulimnadia that are also a 44 mature, nuclei were still readily identifiable, mix of androdioecious and all-hermaphroditic popu- 45 although many began to show pronounced nuclear lations. 46 condensation. These condensed chromatin blocks Weeks et al. (2006b, 2008, 2009a) used sex ratio 47 formed along the nuclear membrane were not pre- data combined with offspring-rearing studies and 48 valent in male gametes still attached to the male genetic assays to determine hermaphroditism in 15 49 wall, and appeared to increase in frequency within species of Eulimnadia from all over the world. In 50 male gametes toward the center of the gonad this study, population sex ratios ranged from 0% to 51 lumen (Fig. 2). Interestingly, many of the luminal 47% males (Table 1). These ratios fall within the 52 male gametes of E. azisi were connected to range previously reported for Eulimnadia species: 0– 53 each other via pronounced intercellular bridges 50% males with a mode of 15–18% males (Weeks 54 (Fig. 2D). et al. 2008). These ratios suggested that E. azisi is

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1 all-hermaphroditic (sex ratios not significantly differ- from isolated hermaphrodites in four of these six 2 ent from 0% males) and that E. gibba, E. michaeli, populations (Table 2A), and three of these four 3 and Eulimnadia sp. are androdioecious (sex ratios populations (Ghat 1, KK4A, and Karuvatta) indeed 4 significantly different than 0% males, and a mix of had no males among a total of 2359 offspring 5 androdioecious and all-hermaphroditic populations reared, suggesting that these populations were quite 6 for the latter two species). The one population of E. likely all-monogenic. In the one remaining popula- 7 gunturensis provided ambiguous results: even though tion (KK8A), 83 males were found of a total of 8 one male was found, the population’s sex ratio was 1106 offspring, a sex ratio of 7.5% males, with 11 9 not significantly different from 0% males (Table 1). of the 21 isolates producing no males (i.e., 52% of 10 The smaller sample size (31 total shrimp) and single the isolates were monogenic). Thus, although the 11 population studied makes the assignment of all-her- population sex ratio suggested all-hermaphroditism 12 maphroditism to this species quite tentative. for this population, it instead turned out to be 13 A second method for assessing androdioecy and androdioecious, with ~50% of the isolated hermaph- 14 hermaphroditism is to assess sex ratios from isolated rodites being amphigenic (see below). 15 hermaphrodites that are forced to self-fertilize Of the four populations containing males (PED1, 16 (Sassaman & Weeks 1993; Weeks et al. 2006b, Veli, KK2A, and KK8A), the percentage of mono- 17 2008). We were able to do this in four of the five genics ranged from 23% to 52% (mean 37.1%). 18 species studied (Table 2), indicating that these iso- This is very similar to the average percentage of 19 lates were capable of producing viable offspring monogenics in other androdioecious Eulimnadia spe- 20 without being fertilized by males, which is consistent cies (mean 41.1%; Weeks et al. 2008), indicating 21 with the self-fertilizing hermaphroditism noted in all that these populations were also likely androdioe- 22 other species of Eulimnadia studied to date (Weeks cious. 23 et al. 2008). When these offspring were reared to To further test for androdioecy consistent with 24 sexual maturity, overall offspring sex ratios ranged that previously found in other Eulimnadia species, 25 from 0% to 13% males (Table 2A). These male per- sex ratios of clutches produced by isolated amphi- 26 centages were low compared with those of other genic hermaphrodites were compared with the 3:1 27 species of Eulimnadia, which ranged from 0% to hermaphrodite:male sex ratio predicted by the ZW 28 43% males (Weeks et al. 2008). Offspring sex ratios sex determining system of related clam shrimp spe- 29 from isolated hermaphrodites can include all- cies (Sassaman & Weeks 1993; Weeks et al. 2010). 30 hermaphrodite clutches (i.e., from isolated mono- Sex ratios for amphigenics only (Table 2B) ranged 31 genic hermaphrodites) and those that have ~25% from 13% to 23% males (mean 18.3%), which is 32 males (i.e., from isolated amphigenic hermaphro- somewhat lower than reported for E. texana (mean 33 dites). When considering sex ratios from isolates 23.1%; Sassaman & Weeks 1993). Only one popu- 34 from the populations with males, the current range lation, PED1, had sex ratios that strictly con- 35 was 7.5–13.2% males (mean 10.5%). This is half of formed to the 3:1 prediction. The remaining three 36 that found in previous studies of Eulimnadia (21.8% populations had male sex ratios significantly lower 37 male; Weeks et al. 2008). Previous studies have than the predicted 25% (Table 2B). Finding fewer 38 shown that male mortality is partly due to expres- males than the predicted 25% is common in an- 39 sion of deleterious recessive alleles on the Z sex drodioecious Eulimnadia species (Sassaman & 40 chromosome (Weeks et al. 2011). It is possible that Weeks 1993; Weeks et al. 2006b, 2008). Males have 41 the Z chromosomes in the current species have a poorer survival than hermaphrodites (Strenth 1977; 42 greater number of deleterious alleles, which could Zucker et al. 2001) due to both expression of dele- 43 then cause lower male frequency due to higher mor- terious alleles on the Z-chromosome (as noted 44 tality rates because of expression of these alleles above), as well as increased energy expenditures of 45 (Weeks et al. 2010). To test this, very specific crosses males relative to hermaphrodites (Weeks et al. 46 would need to be made to assess the possibility of 2011). The lower percentage of males was mainly 47 the expression of these deleterious recessive alleles in the two Thai populations, which may suggest 48 (Weeks et al. 2010). that males of E. michaeli have a lower relative sur- 49 In populations that had no males (i.e., Ghat 1, vival than other Eulimnadia males (as suggested 50 Ghat 2, KK4A, KK6A, KK8A, and Karuvatta), we above). 51 expected all monogenic hermaphrodites, and thus Considering population and isolate sex ratios 52 overall offspring sex ratios should have been 0% together, E. azisi appears to be all-hermaphroditic, 53 male among egg banks reared from isolated her- E. michaeli, E. gibba, and Eulimnadia sp. appear to 54 maphrodites. Offspring were successfully reared be androdioecious, and we await further evidence

Invertebrate Biology vol. x, no. x, xxx 2013 Hermaphroditism in clam shrimp 9

1 for E. gunturensis, which we had difficulty rearing in Combining the sex ratio with the anatomical evi- 2 the laboratory. dence, we propose that three (E. gibba, E. michaeli, 3 Although parsimonious interpretation of sex and Eulimnadia sp.) of the five species studied herein 4 ratios to infer androdioecy and all-hermaphroditism are androdioecious. Eulimnadia gunturensis is defini- 5 is reasonable, a stronger conclusion for these two tively hermaphroditic, but sex ratio data are not suf- 6 breeding systems would be to find ovotestes in the ficient to determine whether it is all-hermaphroditic 7 purported hermaphrodites of these species. Our ana- or androdioecious. The fifth species, E. azisi, 8 tomical examinations demonstrate that E. gibba, E. appears to be solely hermaphroditic, as noted in E. 9 gunturensis, E. michaeli, and E. azisi definitively agassizii. However, it is possible that E. azisi is also 10 contain hermaphrodites (Fig. 1). As in the previous androdioecious, but that we have only sampled two 11 species of Eulimnadia assayed, E. gibba, E. gunturen- all-hermaphroditic populations. As noted here and 12 sis, E. michaeli, and E. azisi possess only a small elsewhere (Sassaman 1989; Weeks et al. 2008), an- 13 section of the ovotestis devoted to sperm produc- drodioecious species of Eulimnadia are a mix of all- 14 tion, and this section is located at the posterior end hermaphroditic and androdioecious populations. 15 of the gonad. Such a bias toward egg production in The all-hermaphrodite populations are likely the 16 hermaphrodites is expected in species that can only products of colonization by monogenic hermaphro- 17 self-fertilize, because hermaphrodites only need to dites, which cannot produce males (Pannell 2008). 18 produce a small number of sperm to fertilize their Thus, it would be helpful to study more populations 19 own eggs; any extra sperm production would be of both E. gunturensis and E. azisi to determine 20 wasted energy (Charnov 1982). Thus, to date, all six whether they are fully hermaphroditic or androdioe- 21 species examined for the presence of testicular tissue cious. 22 in the hermaphroditic gonad (E. texana, E. agassizii, All of the findings reported above are consistent 23 E. gibba, E. gunturensis, E. michaeli, and E. azisi) with the prediction that the genus Eulimnadia com- 24 have exhibited the same pattern: a small portion of prises either androdioecious or all-hermaphroditic 25 the gonad at the distal end of the ovotestis devoted species (Weeks et al. 2006b, 2009a). These data 26 to sperm production (Zucker et al. 1997; Weeks strengthen the hypothesis that androdioecy is the 27 et al. 2005). ancestral breeding system for these shrimps. As a 28 Among the Eulimnadia analyzed histologically in result, we can be confident that this breeding system 29 this study, male gametes liberated into the lumen has been in place for at least 25 million and possibly 30 of the ovotestis appear to contain distinct plaques up to 180 million years (Weeks et al. 2006b). Hence, 31 formed against the nuclear membrane (Fig. 2). this crustacean taxon represents the longest lived an- 32 These plaques represent highly compacted chroma- drodioecious clade in the or plant kingdoms 33 tin (chromatin blocks), and are similar to those (Pannell 2002; Weeks et al. 2006a). 34 found in male gametes reported in other male 35 branchiopods (Scanabissi & Mondini 2002b; Scan- Acknowledgments. The authors thank Dr. Matthew 36 abissi et al. 2005, 2006). Although these plaques Shawkey for the use of his laboratory during parts of this 37 have been previously associated with degenerating project. JSB would also like to thank Chris Kolaczewski- 38 sperm in E. texana (Scanabissi et al. 2006), the Ferris from the Office of Student Academic Success (The 39 hermaphrodites surveyed here (E. gibba, E. guntur- University of Akron) for providing graduate assistantship 40 ensis, E. michaeli, and E. asizi) did not display any funding. 41 appreciable signs of male gamete degeneration, 42 such as the widespread cytoplasmic voiding found References 43 in males of E. texana. However, to completely rule 44 out all features suggestive of complete male gamete Brendonck L 1996. Diapause, quiescence, hatching 45 degeneration in the hermaphrodites surveyed, requirements: what we can learn from large freshwa- 46 future studies using transmission electron micros- ter branchiopods (Crustacea:Branchiopoda:, Notostraca, Conchostraca). Hydrobiologia 320: 47 copy would be helpful. In addition, it would be – 48 85 97. beneficial in future studies to examine male speci- Brtek J 1997. Checklist of the valid and invalid names of 49 mens from the present androdioecious populations the “large branchiopods” (Anostraca, Notostraca, Spin- 50 to determine if, in fact, there are any signs of male icaudata and Laevicaudata), with a survey of the tax- 51 gamete degeneration, as have been reported in var- onomy of all Branchiopoda. Zbornik Slovenskeho 52 ious other branchiopods (Wingstrand 1978; Scan- Narodneho Muzea Prirodne Vedy. 43: 2–65. 53 abissi & Mondini 2002b; Scanabissi et al. 2006; Charnov EL 1982. The theory of sex allocation. Princeton 54 Weeks et al. 2009b). University Press, Princeton, NJ. 355 pp.

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1 Dumont HJ & Negrea SV 2002. Introduction to the class Scanabissi F, Cesari M, Reed SK & Weeks SC 2006. Ultra- 2 Branchiopoda. Backhuys, Leiden. 398 pp. structure of the male gonad and male gametogenesis in 3 Hebert PDN & Finston TL 1993. A taxonomic reevalua- the clam shrimp Eulimnadia texana (Crustacea, Bran- 4 tion of North-American Daphnia (Crustacea, ) chiopoda, Spinicaudata). Invertebr. Biol. 125: 117–124. 5 .1. The D-similis complex. Can. J. Zool. 71: 908–925. Stern DH & Stern MS 1971. Morphology and culture of 6 Knoll L 1995. Mating-behavior and time budget of an an- Eulimnadia diversa (Crustacea, Conchostraca) found in drodioecious crustacean, Eulimnadia texana (Crustacea, Louisiana. Trans. Am. Microsc. Soc. 90: 483–486. 7 – 8 Conchostraca). Hydrobiologia 298: 73 81. Strenth NE 1977. Successional variation of sex ratios in Martin JW & Davis GE 2001. An updated classification Eulimnadia texana Packard (Crustacea, Conchostraca). 9 of the recent Crustacea. Natural History Museum of Southwest. Nat. 22: 205–212. 10 Los Angeles County, Los Angeles. Tommasini S & Scanabissi Sabelli FS 1992. Morphologi- 11 Mattox NT 1954. A new Eulimnadia from the rice fields cal and functional aspects of the female gonad of the 12 of Arkansas with a key to the American species of the conchostracan Leptestheria dahalacensis Ruppel, 1837 13 genus. Tulane Stud. Zool. 2: 3–10. (Crustacea, Branchiopoda), and a comparison with the 14 Olesen J 2007. Monophyly and phylogeny of Branchiopoda, gonads of other branchiopoda. Can. J. Zool. 70: 15 with focus on morphology and homologies of 511–517. 16 branchiopod phyllopodus limbs. J. Crust. Biol. 27: 165– Vidrine MF, Sissom SL & McLaughlin RE 1987. Eulimn- 17 183. adia texana Packard (Conchostraca, Limnadiidae) in Ott DW & Brown RM 1974. Developmental cytology of rice fields in southwestern Louisiana. Southwest. Nat. 18 – 19 the genus Vaucheria I Organization of the vegetative fil- 32: 1 4. ament.. Brit. Phycol. 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Inbreeding and sex-ratio variation in Weeks SC, Crosser BR & Gray MM 2001. Relative fit- ness of two hermaphroditic mating types in the andro- 30 female-biased populations of a clam shrimp, Eulimn- adia texana. Bull. Mar. Sci. 45: 425–432. dioecious clam shrimp, Eulimnadia texana. J. Evol. 31 ————1995. Sex determination and evolution of unisexu- Biol. 14: 83–94. 32 ality in the Conchostraca. Hydrobiologia 298: 45–65. Weeks SC, Marcus V, Salisbury RL & Ott DW 2002. 33 Sassaman C & Weeks SC 1993. The genetic mechanism Cyst development in the conchostracan shrimp, Eulimn- 34 of sex determination in the conchostracan shrimp adia texana (Crustacea: Spinicaudata). Hydrobiologia 35 Eulimnadia texana. Am. Nat. 141: 314–328. 486: 289–294. 36 Scanabissi F & Mondini C 2000. Sperm transfer and Weeks SC, Posgai RT, Cesari M & Scanabissi F 2005. 37 occurrence of spermatophore in the Conchostraca Androdioecy inferred in the clam shrimp Eulimnadia a- Leptestheriidae (Crustacea, Branchiopoda). 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1 Weeks SC, Reed SK, Ott DW & Scanabissi F 2009b. (Phyllopoda, Conchostraca). Experientia 25: 650– 2 Inbreeding effects on sperm production in clam shrimp 651. 3 (Eulimnadia texana). Evol. Ecol. Res. 11: 125–134. Zinn DJ & Dexter RW 1962. Reappearance of Eulimn- 4 Weeks SC, Benvenuto C, Sanderson TF & Duff RJ 2010. adia agassizii with notes on its biology and life history. 5 Sex chromosome evolution in the clam shrimp, Eulimn- Science 137: 676–677. – 6 adia texana. J. Evol. Biol. 23: 1100 1106. Zucker N, Cunningham M & Adams HP 1997. Anatomi- Weeks SC, Sanderson TF, Wallace BF & Bagatto B 2011. cal evidence for androdioecy in the clam shrimp Eu- 7 – 8 Behavioral cost of reproduction in a freshwater crusta- limnadia texana. Hydrobiologia 359: 171 175. cean (Eulimnadia texana). Ethology 117: 880–886. Zucker N, Stafki B & Weeks SC 2001. Maintenance of 9 Wingstrand KG 1978. Comparative spermatology of the androdioecy in the freshwater clam shrimp Eulimnadia 10 Crustacea Entomostraca. 1 Subclass Branchiopoda. texana: longevity of males relative to hermaphrodites. 11 2 Biologiske Skrifter. 22: 1–66. Can. J. Zool. 79: 393–401. 12 Zaffagnini F 1969. Rudimentary hermaphroditism and 13 automictic parthenogenesis in Limnadia lenticularis 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

Invertebrate Biology vol. x, no. x, xxx 2013 Author Query Form

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O n c e y o u h a v e A c r o b a t R e a d e r o p e n o n y o u r c o m p u t e r , c l i c k o n t h e C o m m e n t t a b a t t h e r i g h t o f t h e t o o l b a r :

h i s w i l l o p e n u p a p a n e l d o w n t h e r i g h t s i d e o f t h e d o c u m e n t . h e m a j o r i t o f

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t o o l s o u w i l l u s e f o r a n n o t a t i n g o u r p r o o f w i l l e i n t h e A n n o t a t i o n s s e c t i o n ,

b

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b

o x w h e r e r e p l a c e m e n t t e x t c a n e e n t e r e d . d e l e t e d .

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i g h l i g h t a w o r d o r s e n t e n c e . i g h l i g h t a w o r d o r s e n t e n c e . H

‚ H ‚

R e p l a c e ( I n s ) i c o n i n t h e A n n o t a t i o n s S t r i k e t h r o u g h ( D e l ) i c o n i n t h e l i c k o n t h e l i c k o n t h e C

‚ C ‚

s e c t i o n . A n n o t a t i o n s s e c t i o n .

p e t h e r e p l a c e m e n t t e x t i n t o t h e l u e o x t h a t

b b y

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a p p e a r s .

i g h l i g h t s t e x t i n e l l o w a n d o p e n s u p a t e x t

a r k s a p o i n t i n t h e p r o o f w h e r e a c o m m e n t

M

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o x w h e r e c o m m e n t s c a n e e n t e r e d .

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n e e d s t o e h i g h l i g h t e d .

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l i c k o n t h e A d d s t i c k n o t e i c o n i n t h e

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‚ ‚ C

A n n o t a t i o n s s e c t i o n .

l i c k o n t h e A d d n o t e t o t e x t i c o n i n t h e

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l i c k a t t h e p o i n t i n t h e p r o o f w h e r e t h e c o m m e n t

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a p p r o p r i a t e p a c e i n t h e t e x t . p l a c e i n t h e p r o o f .

l i c k o n t h e A t t a c h F i l e i c o n i n t h e A n n o t a t i o n s l i c k o n t h e A d d s t a m p i c o n i n t h e A n n o t a t i o n s C

‚ C ‚

s e c t i o n . s e c t i o n .

l i c k o n t h e p r o o f t o w h e r e y o u ’ d l i k e t h e a t t a c h e d e l e c t t h e s t a m p y o u w a n t t o u s e . ( T h e A p p r o v e d S

‚ C ‚

f i l e t o b e l i n k e d . s t a m p i s u s u a l l y a v a i l a b l e d i r e c t l y i n t h e m e n u t h a t

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t h i s w o u l d n o r m a l l y b e o n t h e f i r s t p a g e ) .

i n t h e p r o o f . l i c k O K .

C

A l l o w s s h a p e s , l i n e s a n d f r e e f o r m a n n o t a t i o n s t o b e d r a w n o n p r o o f s a n d f o r

c o m m e n t t o b e m a d e o n t h e s e m a r k s . .

l i c k o n o n e o f t h e s h a p e s i n t h e D r a w i n g

‚ C

s e c t i o n . M a r k u p s

l i c k o n t h e p r o o f a t t h e r e l e v a n t p o i n t a n d

‚ C

d r a w t h e s e l e c t e d s h a p e w i t h t h e c u r s o r .

o a d d a c o m m e n t t o t h e d r a w n s h a p e ,

‚ T

m o v e t h e c u r s o r o v e r t h e s h a p e u n t i l a n

a r r o w h e a d a p p e a r s .

o u b l e c l i c k o n t h e s h a p e a n d t y p e a n y

‚ D

t e x t i n t h e r e d b o x t h a t a p p e a r s .