A SYSTEMATIC STUDY of the GENUS EULIMNADIA Sadie K. Reed, R. Joel Duff, and Stephen C. Weeks

JOURNAL OF CRUSTACEAN BIOLOGY, 35(3), 379-391, 2015

A SYSTEMATIC STUDY OF THE GENUS EULIMNADIA

Sadie K. Reed, R. Joel Duff, and Stephen C. Weeks ∗

Program in Integrated Biosciences, Department of Biology, The University of Akron, Akron, OH 44325-3908, USA

ABSTRACT The clam shrimp genus Eulimnadia Packard, 1874 is the most speciose and widely distributed in the Spinicaudata. Taxonomic determinations based on morphology have been controversial because of intraspecific variability in many of the characters used to date. Most recently, egg shell morphology has been the preferred source of species specific characters. We explore the phylogenetic relationships of 19 Eulimnadia species and assess previously proposed synonymies of E. diversa based on egg shell morphology. Phylogenetic studies were based on cytochrome b and elongation factor 1α, and were analyzed using Maximum Likelihood and Bayesian Inference approaches. Phylogenetic analyses support the monophyly of Eulimnadia, yet a large amount of polyphyly exists for species identified via morphology. Specimens of Eulimnadia diversa s.l. were highly unresolved and polyphyletic. Overall, species level phylogenetic resolution was low, emphasizing the great need for a systematic revision of Eulimnadia. KEY WORDS: Androdioecy, clam shrimp, hermaphrodites, Spinicaudata DOI: 10.1163/1937240X-00002345

INTRODUCTION presented a discussion of limnadiid generic relationships based on morphological characters. She chose to recognize the similarities of Eulimnadia and Limnadia by including Eulimnadia Packard, 1874 is classified within Limnadi- them in the subfamily Limnadiinae Burmeister, 1843. This idae along with Limnadia Brongniart, 1820, Limnadopsis taxonomic classification was based on a comparison of 10 Spencer and Hall, 1896, Imnadia Hertzog, 1935, Metalim- morphological characters (Straškraba, 1964). Rogers et al. nadia Mattox, 1952, Paralimnadia Sars, 1896, Afrolimnadia (2012) revised the extant genera of Limnadiidae based on Rogers et al., 2012, Calalimnadia Rogers et al., 2012, and the molecular analyses of Weeks et al. (2009) and provided Austrolimnadia Timms and Schwentner, 2012. Of these Eu- morphological characters specific to each genus. Yet species limnadia is the most speciose and widely distributed (Brtek, distinctions are still in need of study. 1997). The next most speciose genus is Limnadopsis, which Most molecular phylogenies to date have focused on contains 12 species (Schwentner et al., 2012). Brtek (1997) higher level analyses of the branchiopod families (Hanner compiled a checklist of the valid and invalid species names and Fugate, 1997; Spears and Abele, 2000; Braband et al., 2002). Braband et al. (2002) did a combined analysis of of the large Branchiopods, within which he listed 52 de- 12S rDNA and elongation factor 1α (EF1α) and found sup- scribed species of Eulimnadia, but only recognized 43 valid port for a sister relationship between (Imnadia and Limna- species (Table 1). Additionally, Brtek (1997) did not men- dia) and (Eulimnadia and Limnadopsis) that were both sup- tion three species that had been described prior to his check- ported as monophyletic clades. Hoeh et al. (2006), using list (Martin and Belk, 1988; Roessler, 1990), and since his 28S rDNA, 12S rDNA, and cytochrome b (cytb) sequences, list, an additional seven species have been described (Pereira found support for the monophyly of Eulimnadia but the sis- and Garcia, 2001; Durga-Prasad and Simhachalam, 2004; ter group remained ambiguous. However, Limnadopsis was Timms and McLay, 2005; Babu and Nandan, 2010; Rogers never found to be sister to Eulimnadia. Schwentner et al. et al., 2010) yielding a total of 53 species (Table 1). (2009) analyzed 41 species, including 15 of Eulimnadia, Webb and Bell (1979) synonymized Eulimnadia with with data from three genes: 28S, 16S, and cytochrome c oxidase I (COI). This study reached the same conclusions Limnadia, stating that within Spinicaudata, genus diagnos- as Hoeh et al. (2006) regarding the monophyly of Eulim- tic characters can be highly variable in different stages of nadia and lack of sister relationship with Limnadia.Inthe ontogeny, in different sexes, and in different species within same year, Weeks et al. (2009) reported strong support for the same genus. Martin and Belk (1988) and Belk (1989) the monophyly of Limnadiidae including the genera Eulim- disputed this reasoning, suggesting it was based on a sur- nadia, Metalimnadia, Imnadia, and Limnadopsis. Two unde- vey of poor taxonomic drawings in the literature, but never- scribed eulimnadoid species included in the analysis fall into theless recognized the morphological similarities of the ge- distinct clades, the first from the island Republic of Mauri- nera and assumed them to be sister taxa. In 1964 Straškraba tius and the second from South Africa. These two new taxa

∗ Corresponding author; e-mail: [email protected]

Table 1. (Continued.) Table 1. List of valid Eulimnadia species names along with generalized locality information obtained from the literature or from our own collec- Species Locality tions. Synonymies are indented underneath the valid taxon. Eulimnadia ovilunata Argentina Species Locality Martin and Belk, 1989 Eulimnadia ovisimilis Paraguay Eulimnadia acutirostris Daday, 1913 West Africa Martin and Belk, 1988 Eulimnadia aethiopica Daday, 1913 Sudan Eulimnadia packardiana Ishikawa, 1895 Japan Eulimnadia agassizii Packard, 1874 Massachusetts, Eulimnadia pulchra Mohammad, 1986 Iraq USA Eulimnadia similis Sars, 1900 India Eulimnadia stoningtonensis Berry, 1926 Eulimnadia subtropica Daday, 1913 Madagascar Eulimnadia alluaudi Daday, 1913 Madagascar Eulimnadia taoluoensis Hu, 1986 China Eulimnadia antillarum (Baird, 1852) Mexico, West Eulimnadia texana Packard, 1871 USA, Mexico Indies, Brazil Eulimnadia tropica Rammner, 1933 Caribbean Eulimnadia antlei Mackin, 1940 USA Islands Eulimnadia astraova BeIk, 1989 USA Eulimnadia azerbaidshanica Azerbaijan Smirnov, 1936 Eulimnadia azisi Babu and Nandan, 2010 India Eulimnadia behningi Smirnov, 1949 Uzbekistan were assigned by Rogers et al. (2012) the two generic names Eulimnadia belki Martin, 1989 Mexico, Calalimnadia and Afrolimnadia, respectively. Venezuela Rogers et al. (2012) resurrected the genus Paralimnadia Eulimnadia brasiliensis Sars, 1902 Brazil Sars, 1896 to encompass the Australian representatives of Eulimnadia braueriana Ishikawa, 1895 Japan Limnadia. They further noted the monophyly of (Eulimna- Eulimnadia chacoensis Gurney, 1931 Paraguay dia + Metalimnadia) and (Paralimnadia + Limnadopsis). Eulimnadia colombiensis Roessler, 1990 Colombia Weeks et al. (2009) analyzed a total of 71 specimens repre- Eulimnadia compressa (Baird, 1860) India senting 15 species of Eulimnadia. The analysis of multiple Eulimnadia chaperi (Simon, 1886) Eulimnadia curvirostris Roen, 1952 China specimens per species highlighted problematic species de- Eulimnadia cylindrova Belk, 1989 USA, Mexico, terminations for specimens of E. diversa, E. follisimilis, and Galapagos E. cylindrova which were not monophyletic in the phyloge- Eulimnadia dahli Sars, 1896 Australia netic analyses (Weeks et al., 2009). Eulimnadia diversa Mattox, 1937 USA While Brtek (1997) did recognize 43 valid species of Eulimnadia alineata Mattox, 1953 Eulimnadia (disregarding the inclusion in Limnadia), he also Eulimnadia francesae Mattox, 1953 suggested that many of these would be synonymized in the Eulimnadia inflecta Mattox, 1939 future, after careful examination of additional material. This Eulimnadia oryzae Mattox, 1954 Eulimnadia thompsoni Mattox, 1939 underscores the taxonomic difficulties that have faced this Eulimnadia ventricosa Mattox, 1953 group. Many of the early species descriptions were based on Eulimnadia dubia Daday, 1913 New Guinea few specimens, often from one locality. Frequently details Eulimnadia feriensis Dakin, 1914 Australia of characters were vague or missing and drawings have Eulimnadia follisimilis Venezuela been misleading (Belk, 1989). An excellent example of these Pereira and Garcia, 2001 issues is that of E. diversa. Mattox (1954) recognized 12 Eulimnadia garretti (Richters, 1882) Tahiti North American species of Eulimnadia. He found these to be Eulimnadia geayi Daday, 1913 Venezuela, distinct through the use of several morphological characters Colombia, Mexico including: carapace growth lines, carapace size and shape, Eulimnadia gibba Sars, 1900 India telson spines, head shape, male claspers, and antennae length Eulimnadia graniticola Rogers et al., 2010 Georgia, USA (Belk, 1989). However, Belk (1989) cites these characters as Eulimnadia gunturensis India unreliable due to high levels of intraspecific variability. He Radhakrishna and Durga-Prasad, 1976 also found that in some cases, such as that of E. alineata, Eulimnadia indocylindrova India the type specimens were immature. Like many since, he Durga-Prasad and Simhachalam, 2004 found egg morphology to be the most important in species Eulimnadia kobai Ueno, 1940 China diagnoses and subsequently synonymized six (E. inflecta, E. Eulimnadia magdalensis Roessler, 1990 Colombia thompsoni, E. ventricosa, E. francesae, E. oryzae, and E. Eulimnadia margaretae Bond, 1934 South Arabica Eulimnadia marplesi New Zealand alineata) of the North American species into E. diversa due Timms and McLay, 2005 to similarities in egg structure. However, while Belk (1989) Eulimnadia mauritiana (Guérin, 1837) Ilse de France, did find egg morphology to be the most useful character, Mauritania he recognized that the full extent of its value, as well as its Eulimnadia michaeli Nayar and Nair, 1968 India limitations, needs to be explored further. Eulimnadia minuta Daday, 1913 West Africa The current study presents the most comprehensive mo- Eulimnadia ovata Nayar, 1965 India lecular analysis of the genus Eulimnadia to date with 19 Eulimnadia ovata ssp. inversa India species across 5 continents using a portion of the nuclear- Battish, 1981 encoded EF1α and a portion of the mitochondrion-encoded cytb. The resulting phylogenetic analyses are used to test the REED ET AL.: A SYSTEMATIC STUDY OF THE GENUS EULIMNADIA 381 proposal by Belk (1989) to broaden the species delineation of 27 million generations and resulted in 54,240 saved trees. The cytb criteria for Eulimnadia diversa. analysis consisted of 8 million generations and resulted in 17,014 saved trees. The EF1α analysis consisted of 5 million generations and resulted in 100,002 saved trees. In all analyses, the first 50% of trees were considered MATERIALS AND METHODS as the burn in and were discarded. Majority rule consensus trees were Study Organisms created incorporating posterior probabilities (PP) which are percentage values representing the frequency at which each clade was recovered in the Accessions included in this study are listed in Table 2. The phylogenetic sampled trees. analysis included all available species of Eulimnadia as the ingroup. Calal- imnadia (Rogers et al., 2012) and Limnadia lenticularis (Linnaeus, 1761) served as out group taxa in all analyses. Limnadopsis parvispinus Henry, RESULTS 1924 was included as an out group in the cytb analysis. Limnadopsis birchii (Baird, 1860), Imnadia yeyetta Hertzog, 1935, Leptestheria dahalacensis Sequence data of the mitochondrial gene cytb and nuclear (Rüppel, 1837), and Caenestheria lutraria (Brady, 1886), were included as gene EF1α from a total of 112 specimens representing 28 out group taxa in the EF1α analyses. species were newly generated or obtained from GenBank in Specimen collection, identification, and rearing this study (Table 2). Adult clam shrimp collected in wet field conditions were preserved in 95% ethanol for DNA extraction. In dry field conditions, sediment was collected Combined Analyses from dry pools and placed in plastic bags for subsequent lab hydrations. Both genes were analyzed using BI and ML for 44 speci- Collections were obtained by the Weeks’ lab team or obtained through Eulimnadia colleagues. Those samples reared from sediment collections were kept in mens. forms a monphyum in this analysis but a temperature controlled animal facility at the University of Akron at 26- not all limnadiid genera were included. Both trees had a sim- 28°C and under 24 hour light conditions (Durotest Sunlight Simulating ilar topology and so only the ML tree is presented (Fig. 1). fluorescent bulbs). A small sample of sediment (about 500 ml) was placed Species, identified primarily on egg morphology, of Eulim- in the bottom of a 38-l glass aquarium and hydrated with deionized water. nadia were found to be either well supported, weakly sup- Each aquarium received approximately 10 ml of food each day. Food was composed of 2.5 g of ground Tetramin® flake food for algae eating fish ported or, in many cases, polyphyletic. Generally Eulimna- and 2.5 g of baker’s yeast suspended in 500 ml of deionized water. This dia constitutes a trichotomy with E. brasiliensis and two protocol has been shown to be the most successful in raising a variety of clades of limited support (Fig. 1). Within the two clades, species of clam shrimp (Weeks et al., 1997, 2006, 2009). When clam shrimp individual samples of E. diversa, E. texana, E. follisimilis, − reached sexual maturity, they were frozen at 70°C for DNA extraction. E. thompsoni, E. inflecta, and E. oryzae do not cluster by All samples were split into two portions: one portion was used for DNA analyses (see below) and the other was sent to C. Sassaman or D. C. Rogers species designation, nor do the species synonymized with where they were identified to species. E. diversa constitute a single monophylum (this is also not the case in the single gene analyses). Some well-supported DNA Sequencing clades of specimens represent groups of species which have Total DNA was extracted from individual clam shrimp using the Qiagen historically been identified as species based on morpholo- DNeasy Animal Tissue Kit (Qiagen, Germantown, MD, USA). Polymerase gical characters (E. michaeli,PP= 100, BS = 100; E. braue- chain reactions (PCR) were used to amplify fragments of EF1α and cytb = = = = using the primer pairs presented in Table 3. Each reaction consisted of riana,PP 100, BS 100; E. feriensis,PP 100, BS 1.5 μl of template DNA, 0.75 μl of each primer (125 mg/ml), 18.0 μlQ- 87; E. graniticola,PP= 100, BS = 98). master mix, and 10.5 μlofH2O for a total volume of 30 μl. The reactions were run in an Eppendorf personal thermocycler (Eppendorf, Hamburg, EF1α Analyses Germany) for 40 cycles. Each cycle consisted of a 1-min denature step at 94°C, a 1-min annealing step at 45°C for cytb and 53°C for EF1α,anda Trees inferred from BI and ML (Fig. 2) for the 66 specimens 1.5-min extension step at 72°C. using only EF1α sequences differed only in the placement PCR products were cleaned with the Qiagen MinElute PCR Purifica- of one taxon (E. brasiliensis) from each other. In these tion kit (Qiagen). Sequencing was carried out with an ABI 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Sequences were analyses Eulimnadia does not form a monophylum, but evaluated and initially aligned using Sequencher 4.7 (Genecodes, Ann Ar- instead forms a paraphyletic clade (PP = 100, BS = 17) bor, MI, USA). Subsequent editing and alignment was conducted with Se- with Calalimnadia mahei and L. lenticularis. In the ML tree, qApp (Gilbert, 1992). These were combined with sequences acquired from = ® E. brasiliensis is sister, with little support (BS 17), to the GenBank genetic sequence database (http://www.ncbi.nlm.nih.gov/) Eulimnadia + Calalimnadia + Limnadia (Benson et al., 1997). Further sequence evaluation was performed in MEGA the entire clade (Molecular Evolutionary Genetics Analysis) v. 5.1 (Tamura et al., 2011). while in the BI tree E. brasiliensis is included in a clade with E. michaeli, E. asizi, E. africana, E. braueriana,the Phylogenetic Analyses unidentified species from India, E. dahli and E. feriensis Analyses were conducted using concatenated data sets with all genes (PP = 62). As expected for a single gene tree, resolution partitioned by codon position. Maximum Likelihood analyses were per- is limited. Only four species were found to be supported as formed using Randomized Accelerated Maximum Likelihood version 7.0.3 = = (RAxML) (Stamatakis, 2006). Two methods of analysis were used: 1. a two monophyletic: E. braueriana (PP 56, BS 86), E. sp. 2 gene concatenation approach, in which the gene sequences were concate- from India (PP = 100, BS = 92), E. graniticola (PP = 99, nated head-to-tail to form a two gene alignment; and 2. a single gene con- BS = 86), and E. cylindrova (PP = 100, BS = 100). The rest sensus approach, where each gene was analyzed individually. The General of the species had individuals falling in different clades, or Time Reversible (GTR) model (Taver, 1986) of nucleotide substitution with were part of polytomies with other species. I model (Yang, 1996) of rate heterogeneity was implemented for all analyses. Support for the ML tree was obtained by with 1000 bootstrap iterations. Bayesian Inference (BI) analyses were computed with MrBayes v. 3.1.2 cytb Analyses (Ronquist and Huelsenbeck, 2003). The GTR + I + G substitution model (Ronquist et al., 2012) was implemented with two simultaneous runs in The analyses of 94 individuals using only cytb sequences each analysis and terminated when the average standard deviation of the showed somewhat different topologies when inferred with split frequencies reached less than 0.01. The two-gene analysis consisted ML (Fig. 3) and BI (Fig. 4). In both the BI and ML 382 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 35, NO. 3, 2015

Table 2. List of accessions included in this study. Newly obtained sequences are represented by GenBank accession Nos KM923800-KM923890, KP330266-KP330298, and KP705255. All other published sequences obtained from GenBank are noted by accession numbers. General locality information is listed. AZ, Arizona; FL, Florida; GA, Georgia; IL, Illinois; IN, Indiana; LA, Louisiana; MA, Massachusetts; MS, Mississippi; NE, Nebraska; NM, New Mexico.

Species Speci- cytb EF1α Collection locality men no. Calalimnadia mahei (Rogers et al., 2012) 124 KM923844 KM923888 Republic of Mauritius 125 – KP330283 Republic of Mauritius 126 KM923843 KM923888 Republic of Mauritius 127 KM923842 KM923887 Republic of Mauritius N47 AY779719 – Republic of Mauritius Eulimnadia africana (Brauer, 1877) 42 KM923803 KM923848 South Africa 59 KP330286 – South Africa 60 KP330287 – South Africa Eulimnadia agassizii Packard, 1874 111 KP330298 – United States, MA 113 – KP330276 United States, MA 74 KM923830 KM923875 United States, MA 75 KM923829 KM923874 United States, MA N59 AY779709 – United States, MA Eulimnadia brasiliensis Sars, 1896 132 KM923812 KM923857 Brazil 133 KP330290 – Brazil 134 KP330291 – Brazil 135 KP330292 – Brazil Eulimnadia braueriana Ishikawa, 1895 141 – KP330268 Japan 142 KM923808 KM923853 Japan 143 KM923807 KM923852 Japan 146 KP330288 – Taiwan 593 – KP330267 Japan N40 AY779724 – Japan N41 AY779726 – Japan Eulimnadia colombiensis Roessler, 1990 136 KM923819 KM923864 Venezuela 137 KP330294 – Venezuela N106 AY779715 – Venezuela Eulimnadia cylindrova Belk, 1989 102 KM923840 KM923885 Mexico 103 KM923841 KM923886 Mexico N103 AY779715 – Venezuela N104 AY779716 – Venezuela N11 AY779697 – Mexico N16 AY779699 – Mexico N17 AY779698 – Mexico N64 AY779701 – Ecuador, Galapagos Islands N65 AY779700 – Ecuador, Galapagos Islands Eulimnadia dahli Sars, 1896 87 KP330284 – Australia 88 KM923802 KM923847 Australia 89 KP330285 – Australia Eulimnadia diversa Mattox, 1937 21 KM923831 KM923876 United States, IN 22 – KP330277 United States, IN 23 – KP330278 United States, NE 24 – KP330279 United States, NE 37 KM923835 KM923880 United States, IN 38 KP330293 – United States, IN 46 KM923832 KM923877 United States, NE 47 KM923833 KM923878 United States, NE 48 KM923834 KM923879 United States, NE N4 AY779720 – United States, AZ N8 AY779721 – United States, AZ Eulimnadia feriensis Dakin, 1914 90 KP705255 Australia 91 KM923801 KM923846 Australia 92 KM923800 KM923845 Australia Eulimnadia follisimilis Pereira and Garcia, 2001 35 KM923811 KM923856 United States, NM 36 KP330289 – United States, NM 76 KM923810 KM923855 United States, NM Eulimnadia graniticola Rogers et al., 2010 100 KM923827 KM923872 United States, GA 101 KM923828 KM923873 United States, GA 31 KM923826 KM923871 United States, GA REED ET AL.: A SYSTEMATIC STUDY OF THE GENUS EULIMNADIA 383

Table 2. (Continued.)

Species Speci- cytb EF1α Collection locality men no. 32 – KP330275 United States, GA 72 – KP330274 United States, GA Eulimnadia gunterensis 4 KM923806 KM923851 India Radhakrishna and Durga-Prasad, 1976 Eulimnadia inﬂecta Mattox, 1939 62 KM923815 KM923860 United States, LA 63 KM923814 KM923859 United States, LA 64 KM923813 KM923858 United States, LA Eulimnadia magdalensis Roessler, 1990 N100 AY779728 – Venezuela N107 AY779729 – Venezuela N108 AY779730 – Venezuela N99 AY779731 – Venezuela Eulimnadia michaeli Nayar and Nair, 1968 68 KM923825 KM923870 Thailand 69 KM923824 KM923869 Thailand 85 KM923823 KM923868 Thailand Eulimnadia oryzae Mattox, 1954 108 – KP330271 United States, MS 109 KM923818 KM923863 United States, MS 110 KM923817 KM923862 United States, MS 77 – KP330270 United States, MS 78 KM923816 KM923861 United States, MS 79 – KP330272 United States, MS Eulimnadia packardiana Ishikawa, 1894 28 – KP330281 Japan 29 – KP330282 Japan 44 KM923837 KM923882 Japan 93 KM923836 KM923881 Japan N85 AY779726 – Japan Eulimnadia sp. A6 KM923805 – India Eulimnadia sp. 2 128 KM923809 KM923854 India 129 – KP330269 India Eulimnadia azisi Babu and Nandan, 2010 I1 KM923804 KM923849 India Eulimnadia texana Packard, 1871 11 – KP330282 United States, NM 12 KM923838 KM923883 United States, NM 21 KM923839 KM923884 United States, NM 5 KP330295 – United States, NM N35 AY779712 – United States, NM N50 AY779702 – Mexico, Baja California N51 AY779703 – Mexico, Baja California N5 AY779714 – United States, NM N70 AY770707 – United States, NM N71 AY779706 – United States, NM N72 AY779717 – United States, NM N73 AY779704 – United States, NM N9 AY778708 – United States, NM W65 AY779704 – United States, NM Eulimnadia thompsoni Mattox, 1939 49 KM923821 KM923866 United States, IL 50 KM923822 KM923867 United States, IL 51 – KP330273 United States, IL 80 KP330296 – United States, IL 81 KP330297 – United States, IL 97 KM923820 KM923865 United States, IL Imnadia yeyetta Hertzog, 1935 289 – AF526290 Austria Leptestheria dahalacensis Ruppel, 1837 291 – AF562691 Europe Limnadopsis birchii Baird, 1860 290 – AF562690 Australia Limnadopsis parvispinus Henry, 1924 N44 AY779734 – Australia N45 AY779665 – Limnadia lenticularis (Linnaeus, 1761) W66 AY779732 – United States, FL NS25 AY779733 KM923890 United States, FL analyses, Eulimnadia is a monophyletic group (PP = 100, Arizona, USA (N8 and N4) are sisters to all remaining BS = 77). These trees show better resolution than the Eulimnadia lineages (PP = 73, BS = 39). However, six other EF1α trees. In both trees, two specimens of E. diversa from specimens of E. diversa are scattered among specimens of 384 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 35, NO. 3, 2015

Table 3. Primer pairs used for DNA ampliﬁcation in this study: mitochondrion-encoded cytochrome b (cytb) and nuclear-encoded elongation factor 1-α (EF1α).

Gene Primer Sequence (5 → 3) Amplicon size/citation cytb UcytB151F TGTGGAGCNACYGTWATYACTAA Approx. 400 bp UcytB270R AANAGGAARTAYCAYTCNGGYTG Merrit et al. (1998) EF1α M44-1 GCTGACCGYGARCGTGGTATCAC Approx. 1300 bp 3EF1 GGAAGTCAGAGAAGGACTC Braband et al. (2002) other species. Of particular note, in the ML tree, a large be found in each locality. Multiple specimens were included clade, denoted by an arrow in Fig. 3, includes E. cylindrova in each analysis to test the consistency of these characters for from Mexico and Venezuela, E. packardiana from Japan, E. species identification. Only one North American species was colombiensis from Venezuela, E. follisimilis and E. texana monophyletic: E. graniticola. Eulimnadia cylindrova from from Mexico and the USA, E. graniticola from the USA, E. Mexico is monophyletic in the combined gene analysis, but agassizi from the USA, and E. diversa and its synonyms (E. when more specimens are added in the cytb and EF1α sin- thompsoni, E. oryzae, and E. inflecta) from the USA. This gle gene analyses, the species becomes polyphyletic, though clade corresponds to one found in the BI tree (PP = 100), lack of resolution makes such a result preliminary. Eulimna- also denoted by an arrow in Fig. 4. The topology between dia agassizi is monophyletic in the combined gene analyses the ML and BI trees for the arrow-denoted clade in Figs. 3 and the cytb analyses, but is polyphyletic in the EF1α anal- and 4 is very different and often poorly supported. However, yses, again likely due to lack of resolution. In all analyses, it is clear that in the species for which multiple samples were specimens identified as E. texana are polyphyletic, dispersed included, very few form monophyla according to species in the tree with specimens of E. diversa, E. follisimilis, and identification. Only E. graniticola (PP = 64, BS = 74) and E. colombiensis. Eulimnadia diversa specimens, and those E. agassizi (PP = 94, BS = 61) form monophyla, while all identified as its synonyms, also do not form a monophylum other specimens fall in polyphyletic groups. and thus is not supported as a valid taxon. These results highlight the troubles in identification of the DISCUSSION species of Eulimnadia. In this study, we assume that all spec- The taxonomic status of Eulimnadia has been thoroughly imens were correctly identified based on the diagnostic char- debated in the literature (Sars, 1895; Sayce, 1903; Daday, acters of the species descriptions. Under this assumption, the 1925; Barnard, 1929; Brehm, 1933; Ueno, 1940; Mattox, molecular analyses suggest that many of the current charac- 1954; Straškraba, 1964; Webb and Bell, 1979; Martin and ter states used to distinguish species are not taxonomically Belk, 1988; Belk, 1989; Martin, 1989; Brtek, 1997; Pereira useful. Alternatively, we could assume that some of the spec- and Garcia, 2001). Recently, several molecular phylogenetic imens were incorrectly identified and thus led to such poly- studies have supported its monophyly (Hoeh et al., 2006; phyly as seen in the molecular analyses. If this were the case, Schwentner et al., 2009; Weeks et al., 2009). it would likely be due to the variability in morphological The two gene analyses, as well as the single gene cytb characters. We believe that these species all best fit the cur- analyses presented here, appear to support Eulimnadia as rent species delineations and thus represent our current un- a monophlum, but a lack of important members of the derstanding. However, either scenario suggests that the char- Limnadiidae makes a definitive conclusion unwarranted acters used to identify species, including those of the egg as at this time. The single gene EF1α analysis does not proposed by Belk (1989), are not reliable. support Eulimnadia monophyly, as Calalimnadia mahei Despite the uncertainty of the species designations, given and Limnadia lenticularis render Eulimnadia paraphyletic. the polyphyly of the three Mattox (1954) species included However, much of this single gene tree is ambiguous, with in this study (E. inflecta Mattox, 1939; E. oryzae Mattox, many polytomies, and low nodal support for many of the 1954; and E. thomsoni Mattox, 1939) that Belk (1989) branches. synonymized within E. diversa, we can conclude that Belk The current molecular phylogenetic analyses allow for was correct in his assessment that these are not valid species. independent testing of some of the taxonomic conclu- However, in all analyses E. diversa is also polyphyletic. sions based on morphological observations. Currently, eight Eulimnadia texana has been extensively studied as a model species of Eulimnadia are recognized from North America for the androdioecious mating system (Chasnov, 2010). Belk (Table 4). This study includes five of these, as well as three (1989) found the eggs of E. texana to be morphologically of the species that Belk (1989) synonymized into E. diversa conservative between two wide ranging populations, one (Table 4). The specimens representing these species were in Texas and one in California. In the current study, we identified by D. C. Rogers and C. Sassaman, both leading were only able to obtain both genes for two specimens clam shrimp taxonomists, based on the original type char- and these do not form a monophylum, falling in a clade acters used to diagnose the species prior to Belk’s (1989) with E. follisimilis, E. colombiensis, and the E. diversa synonymization. It must be noted, however, that the strategy specimens from Nebraska. In the EF1α tree, three specimens of separating specimens based on locality and sending some are included, but the tree is poorly resolved at this level. for identification while retaining others for the DNA analysis The cytb trees offer the most information on this species may have been problematic if more than one species could as we were able to include 13 specimens, 11 of which are REED ET AL.: A SYSTEMATIC STUDY OF THE GENUS EULIMNADIA 385

Fig. 1. Phylogenetic tree resulting from maximum likelihood analysis of 44 taxa with both cytb and EF1α sequences. Calalimnadia mahei and Limnadia lenticularis comprise the outgroups. Values along branches represent Bayesian posterior probabilities (PP) ﬁrst and maximum likelihood bootstrap support values (BS) second, both expressed as percentages. Eulimnadia diversa and its synonyms are all noted with asterisks. from populations in New Mexico and two from a population specimens of E. follisimilis (also from New Mexico), E. in Mexico (Baja of California). In both the BI and ML colombiensis from Venezuela, and the E. diversa specimens analyses, these specimens form a polyphyletic group with from Nebraska. Also, in both analyses, the specimens 386 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 35, NO. 3, 2015

Fig. 2. Phylogenetic tree resulting from maximum likelihood analysis of 66 taxa with partial EF1α sequences. Values along branches represent Bayesian posterior probabilities (PP) first and maximum likelihood bootstrap support values (BS), second, expressed as percentages. Eulimnadia diversa and its synonyms are all noted with asterisks. from the population in Mexico were significantly grouped populations. Thus it seems specimens from populations into a clade. It is also interesting to note that the two form monophyletic groups, while specimens from different specimens of E. colombiensis and the two specimens of populations identified as the same species do not. This E. follisimilis do represent monophyletic groups within the highlights that it is the morphological species diagnosis that E. texana polytomy. Yet, these specimens are from single is in question. REED ET AL.: A SYSTEMATIC STUDY OF THE GENUS EULIMNADIA 387

Fig. 3. Phylogenetic tree resulting from maximum likelihood analysis of 94 taxa with partial cytb sequences. Values above branches maximum likelihood bootstrap support values (BS) in percentages. The clade denoted with the arrow does not include BS values for spatial constraints. Eulimnadia diversa and its synonyms are all noted with asterisks.

Eulimnadia agassizii is a North American species with Zinn and Dexter, 1962). In the two gene analysis and in the a very small range in Massachusetts, USA (Packard, 1874; single cytb analysis, E. agassizii specimens ﬁnd support as 388 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 35, NO. 3, 2015

Fig. 4. Phylogenetic tree resulting from Bayesian inference analysis of 94 taxa with cytb sequences. Values above branches are posterior support values in percentages (PP). The clade denoted with the arrow does not include all PP values because of spatial constraints. Eulimnadia diversa and its synonyms are all noted with asterisks. monophyletic (PP = 65, BS = 46 and PP = 94, BS = 61, Eulimnadia cylindrova is a widely distributed species, oc- respectively) and we consider them a valid species. curring throughout North and South America. Here, we have REED ET AL.: A SYSTEMATIC STUDY OF THE GENUS EULIMNADIA 389

Table 4. The currently recognized taxonomic status of the North Ameri- presented herein further support the validity of E. graniticola can species of Eulimnadia. Note the synonyms of E. diversa proposed by as a species supported by both morphological and molecular Belk (1989). Synonymies are indented underneath the valid taxon. Those characters. included in the present study are noted. Overall, from these data, it is evident that Belk (1989) is North American species Included correct in his assessment that the North American species in study designated by Mattox (1954) are not valid. However, our data do not support that these taxa should be synonymized Eulimnadia agassizii Packard, 1874 Yes into E. diversa (Fig. 1). Eulimnadia diversa, E. texana and Eulimnadia stoningtonensis Berry, 1926 E. cylindrova are all wide ranging species and seem to Eulimnadia antlei Mackin, 1940 Eulimnadia astraova Belk, 1989 form species groups with other North and South Ameri- Eulimnadia belki Martin, 1989 can species. Eulimnadia graniticola and E. agassizii are Eulimnadia cylindrova Belk, 1989 Yes narrowly distributed and they are well supported as valid Eulimnadia diversa Mattox, 1937 Yes species. Eulimnadia alineata Mattox, 1953 The current molecular study highlights the need to fur- Eulimnadia francesae Mattox, 1953 ther investigate inter-population variability within species Eulimnadia inﬂecta Mattox, 1939 Yes to better understand what constitutes a species in Eulimna- Eulimnadia oryzae Mattox, 1954 Yes dia. Species based on morphological characters are not sup- Eulimnadia thompsoni Mattox, 1939 Yes ported but species delimitation based on molecular charac- Eulimnadia ventricosa Mattox, 1953 Yes ters will require more data before a consistent and practical Eulimnadia graniticola Rogers et al., 2010 Yes Eulimnadia texana Packard, 1871 Yes species deﬁnition can be applied.

ACKNOWLEDGEMENTS included specimens from Mexico, Venezuela, and Ecuador We thank M. Hamer, N. Rabet, M. Grygier, B. Timms, A. Ohtaka, B. Lang, G. Pereira, L. Sanoamuang, A. Ooyagi, A. Ferreira, S. Wu, J. Garcia, D. (the Galapagos Islands). Although all of these specimens do Smith and A. Maeda-Martinez for soil samples, eggs and/or preserved clam not form a monophyletic group, some do occur in mono- shrimp. Many thanks are extended to Clay Sassaman and D. Christopher phyletic groups at the population level. Brendonck et al. Rogers for sample identification. We thank Walter R. Hoeh for running the (1990) conducted a large survey of the Branchiopod fauna of final phylogenetic analyses. Francisco Moore was kind enough to review an early draft of this paper. This project was supported by the National Science the Galapagos Islands, including E. cylindrova. In this study, Foundation Doctoral Dissertation Improvement Grant (No. DEB-0709618) Brendonck et al. (1990) noted that there were slight differ- to SKR. ences in the morphology of the eggs from those described by Belk (1989), but not enough to warrant a new species desig- REFERENCES nation. However, these differences led the authors to observe Babu, K. K. S., and S. B. Nandan. 2010. Two new clam shrimp species egg morphology across populations of E. cylindrova from (Crustacea: Branchiopoda: Spinicaudata) from Kerala, India. Zootaxa one population in the USA (Arizona), two populations in 2713: 55-64. Mexico, as well as the Galapagos Islands populations. Bren- Baird, W. 1852. Monograph of the family Branchipodidae, a family of donck et al. (1990) noted variability in morphology and size crustaceans belonging to the division Entomostraca, with a description of a new genus and species of the family, and two new species belonging characteristics of the eggs from these different populations. to the family Limnadiidae. Proceedings of the Zoological Society of This is in contrast to the suggestions by Belk (1989) that London 20: 18-37. egg morphology is a conservative character. They concluded . 1860. Description of a new entomostracous crustacean, belonging that further study is needed to clarify the taxonomic level at to the order Phyllopoda, from South Australia. Proceedings of the Zoological Society of London 28: 392-393. which these populations should be considered and resorted Barnard, K. H. 1929. Contributions to the crustacean fauna of South Africa. to classifying all populations into an extended species group: A revision of the South African Branchipoda (Phyllopoda). Annals of the E. cylindrova sensu lato. The molecular data in the current South African Museum 29: 181-270. study, particularly the cytb analysis (which included three Battish, S. K. 1981. On some conchostracans from Punjab with the different populations of E. cylindrova), supports the conclu- description of three new species and a new subspecies. Crustaceana 40: 178-196. sions of Brendonck et al. (1990) that populations of Eulim- Belk, D. 1989. Identification of species in the conchostracan genus nadia can be quite different and might warrant species level Eulimnadia by egg-shell morphology. Journal of Crustacean Biology 9: distinction. 115-125. The support for monophyly of E. graniticola in our Benson, D. A., M. S. Boguski, D. J. Lipman, and J. Ostell. 1997. GenBank. Nucleic Acids Research 25: 1-6. analyses reaffirms this as a good species. Interestingly, this Berry, E. W. 1926. Description and notes on the life history of a new species is the most recently described species of Eulimnadia from of Eulimnadia. American Journal of Science 11: 429-433. North America (Rogers et al., 2010) and the designation of Bond, R. M. 1934. Report on Phyllopod Crustacea (Anostraca, Notostraca, it as a new species is based primarily on egg morphology. It and Conchostraca), including a revision of the Anostraca of the Indian Empire. Memoirs Connecticut Academy of Arts and Sciences 10: 29-62. is noted that eggs of E. graniticola are most similar to eggs Braband, A., S. Richter, R. Hiesel, and G. Scholtz. 2002. Phylogenetic of E. follisimilis and these two species might form a species relationships within the Phyllopoda (Crustacea, Branchiopoda) based group. Brendonck et al. (1990) suggested that these species on mitochondrial and nuclear markers. Molecular Phylogenetics and groups may indicate single species. However, Rogers et al. Evolution 25: 229-244. Brady, G. S. 1886. Notes on freshwater entomostraca from South Australia. (2010) used molecular analysis as support for E. graniticola Proceedings of the Zoological Society of London 54: 82-93. as a unique species: they found three apomorphies in the Brehm, V. 1933. Phyllopoden. Mitteilungen von der Wallacea – Expedition sequences of 28S of four specimens. The molecular data Wolterek, 5. Zoologischer Anzeiger 104: 31-40. 390 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 35, NO. 3, 2015

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