Contributions to Zoology, 71 (4) 123-129 (2002) SPB AcademicPublishing bv. The Hague A note on the systematic position of the Bathynellacea (Crustacea, Malacostraca) using molecular evidence Ana+Isabel Camacho Isabel Rey²,Beatriz+A. Dorda Annie ³ ², Machordom Antonio+G. Valdecasas ¹ ³, 'Museo Naturales Nacional de Ciencias (CSIC). Dpto. Biodiversidady Biologia Evolutiva. C/ Jose Gutierrez Ahascal 28006- 2 2, Madrid. Spain, [email protected]; Museo Nacional de Ciencias Naturales (CSIC), de Colecciones. Jose Gutierrez 3 Dpto. C/ Ahascal 2, 28006- Madrid; Museo Nacional de Ciencias Naturales (CSIC), Dpto. Biodiversidady Biologia Evolutiva. C/ Jose Gutierrez Ahascal 2, 28006- Madrid. Keywords: Systematics, Crustacea, Malacostraca, Bathynellacea, 16S rDNA. lar, is the Abstract usually homogeneous amongst special- ists. The traditional of way working is to use the latest information or the latest Molecular data for the 16S of ba- hypothesis mt rDNA gene fragment a published in order to thynellacean is here presented for the first time and used to select those views that are going to be the of within the analyze relationship the group crustacean class compared. However, it must be universal that where Malacostraca Two (Arthropoda, Bathynellacea). contrasting there is more than one taxonomist working on a views have classified the bathynelids as being cither within the there will be group not a single hypothesis or in- order Syncarida or in a separate super-order Podophallocarida of the How terpretation descent. or small based group’s big belonging to the infra-class Eonomostraca, a disagreement are these irrelevant on debates internal The discrepancies and does it not mainly over external and morphology. preliminary analyses offered here in question the placement of invalidate this universal law. this Bathynellacea within the Syncarida, and suggest the need The group known as “Bathynellacea” (Serban, for further of the malacostracan a study relationships among 1972) is a good example of the above. First dis- groups. covered by Vejdovsky (1882), their systematic (po- has been sition) a problem since then. They were considered an “aberrant” member of Malacostraca Contents (Caiman, 1909). Although nobody knows what “aberrant” members in evolution the arc, term Abstract 123 gen- that do not fit Introduction 123 erally implies organisms well into our limited reconstructed Brief natural history of the Bathynellacea 124 ground patterns. Other Material and methods 124 authors have designated them as simplified syncarids Results 125 (see Schminke, 1981). However, the morphologi- Discussion 126 cal taxonomist that most deeply studied the inter- On the phylogenetic relationships of Bathynellacea 126 nal and external morphology of the Bathynellacea, Acknowledgements 128 References 128 Eugene Serban, never joined the Bathynellacea with the Syncarida, but proposed a more radical model of relationships (Serban, 1970, 1972): the order in Introduction Bathynellacea his scheme would belong to the superorder Podophallocarida Serban, 1970 under The the infraclass Eonomostraca relative merit of the systematic position given Serban, 1972. True to a certain of animals Syncarida, on the other hand, would be placed group or plants, depends by Serban more often under a different infraclass, the than not on how thoroughly the group Anomostraca. has In this we the first been studied rather than the technique used. paper present nucleotide data for a and Contrasting morphological studies with molecular bathynellacean species apply these to- wards ones has often of an of the of this had the undesirable effect con- analysis relationship group with other malacostracan crustaceans. cealing that neither view, morphological or molecu- 124 Camacho al. — A.I. et Systematics ofBathynellacea (Crustacea) using molecular evidence Brief natural history of the Bathynellacea Material and methods 0.5 Bathynellacea are small crustaceans, between are rare and difficult Bathynellids creatures, very 3 in and mm size, with an almost to colect. The cylindrical body sample used for this study was com- and vermiform without shape, eyes or pigmenta- posed of 11 specimens of Iberobathynella (Espano- tion. All known live in the interstitial envi- species bathynella) magna Camacho& Serban, 1998, found ronment either in in (stygobionts), cave groundwater a pool in the Cave CO.246 (Sierra de la Collada, or phreatic water close to rivers and epigean aqui- Cantabria, Spain) collected at -30 m by C. Puch & fers. 2 that live Only species free in Lake Baikal F. Molinero (13/04/2001). Two female specimens seem to be the but it the exception, is to were used for molecular and eleven interesting analysis up to note that they live on the dark beds of deep the for the morphological identification. All the speci- lake. lack do They free swimming larvae and not mens were frozen at -20°C after sorting from the swim well as adults, to crawl preferring amongst unfixed sample. the of sand. grains Extraction was carried out with Chelex following Bathyncllaceans females at time. lay one egg a Walsh et al. (1991). Specimens were crushed with The lasts for 9 months wall of ml embryonic development a pipette tip against the a 1.5 microcen- stammeri (Antrobathiynella ) and the newly emerg- trifuge tube. Each tube contained 100 pi Chelex form the ing is similar to adult, except that it has 100 (Bio-Rad; 5% in distilled with 2 water) pi pro- fewer legs (1 to 4) than the adult. The forms teinase K The young (20 mg/ml; Promega). specimens were successively acquire legs, up to 8, in the following incubated overnight at 56°C, followed by 10 minutes moults. The leads adults development to which look at 100°C and centrifuged at 16, OOOg for 10 minutes. like larvae lok, and this has been related with their A ofmitochondrial 16S rDNA fragment (mt) was colonization of the interstitial environment (Coineau, amplified using primers 16Sar (5'-CGCCTGTTT- 2000). ATCAAAAACAT-3') and 16Sbr(5'-CCGGTCTG- arc distributed They widely in all the subterra- AACTCAGATCACTG-3') (Palumbi et al., 1991) nean waters of the world, except at the Poles. At at 0.4 pM. Five pi of the Chelex-extracted DNA present we know of 2 60 and al- solution used families, genera was as a template. Other components most 200 of 25 species. the pi PCR reaction were 75 mM Tris HCI There is no known fossil but the species, geo- (pH 9.0), 2 mM MgCl 50 mM KCI, 20 mM 2, distribution graphic suggest an origin not later than (NH ) S0 10 mM dNTPs, 0.02% BSA, and 0.625 4 2 4, the Paleozoic upper (Schram, 1977). units Taq DNA polymerase (Biotools). The of the adult morphology bathynelids can be PCR was carried out for an initial 10 min dena- summarized the characters; by following turation at 95°C initial incubation, followed by 40 • absence of cephalic caparace; cycles at 95°C for 30s 51 °C for 45s , and 72°C for • of 8 thoracic and free and presence 5 abdominal 45s, a final extension of 10 min at 72°C. The PCR segments; end products were purified and concentrated • and eyes statocysts absent; with Kit Bioclean columns (Biotools) and stored • 1 to 7 4°C until strand thoracopods biramous; at sequencing. Each was sequenced • 8 male transformed thoracopod in copulatory using “Big Dye Terminator” (Applied Biosystems, and female organ reduced; Inc; AB1) sequencing reactions for each primer. • absent; end petasma Sequencing products were analysed on an ABI • furca and always present, Capillary 3700 Genetic Analyzer. • with not tail-fan. DNA uropod pleotelson forming The sequences were truncated at the primer ends. The CLUSTAL W program (Thompson et al. 1994) was employed to align the mt 16S rDNA Find made sequences. adjustements were by eye. Additionalmt 16S rDNA sequences for 23 crus- tacean species were downloaded from GenBank Contributions to 71 - 2002 Zoology, (4) 125 Table I. Crustacean species used in the phylogenetic analysis (Table 1). were Phyllopoda used as out-groups for and GenBank accession numbers for mt 16S rDNA. Classification class Malacostraca. The in-group, the class Malaco- according to Schram 1986. is straca, represented by the subclases, superorders Class Remipcdia:Reinipedia: and orders, following the Lange & Schram (1999) Order Nectiopoda: Speleonectes gironensis classification In (Table 1). some cases, more than (AF370874) one species per family or order was included in the Class Phyllopoda: analysis. Nucleotide saturation was evaluated Subclass Cephalocarida: by Order transition and transversion Brachypoda: Hutchinsoniella macracantha plotting changes against (AF370875) uncorrected (“p”) divergence values. Preliminary Subclass Branchiopoda treatments based on neighbor-joining (NJ), maxi- Order Anomostraca: Branchinectapaludosa mum parsimony (MP), and maximum likelihood (AF209055) principles were carried out the PAUP* 4.10 Class Malacostraca: using best Subclass Hoplocarida: package (Swofford, 2002). The model of evo- Order lution that Stomatopoda:Stomatopoda : Gonodactylus chiragra fitted our data was obtained using the (AF107614) program Model test 3.06 (Posada & Crandall, 1998). Squilla empusaempusa (AF 107617) Thus, the GTR (General Time Reversible) model Subclass Eumalacostraca et al. (Lavane 1984; Rodriguez et al. 1990) was Superorder Syncarida Order used. Confidence limits for the esti- Anaspidacea: Anaspides tasmaniae (AF133691) analyses were mated Anaspides spinulae (AF 133679) by bootstrapping (5000 repetitions) (Felsen- Allanaspides helonomus stein, To determine 1985). whether a particular tree (AF133680) topology corresponded to