Contributions to Zoology, 71 (4) 123-129 (2002)

SPB AcademicPublishing bv. The Hague

A note on the systematic position of the (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, , 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 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 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 apply these to- wards ones has often of an of the of this had the undesirable effect con- analysis relationship group

with other malacostracan . cealing that neither view, morphological or molecu-

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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

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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 : 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 et al. (Lavane 1984; Rodriguez et al. 1990) was Superorder Syncarida

Order used. Confidence limits for the esti- : 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 a significantly better or Allanaspides hickmani worse than interpretation of the data an alternative (AF 133681) tree, we used the Shimodaira-Hasegawa Paranaspides lacustris test (Shimo- daira & (AFI33682)(AF133682) Hasegawa, 1999), as implemented in PAUP. Order Bathynellacea: Iberobathynella magna

(AF503569, AF503570) Superorder Results Order :Isopoda: Asellus aquations (AJ388077)

TyphlocirolanaTyphlocirolana moraguesi The of (AF356849) alignment the sequences obtained in GenBank Order : Cumopsis fageifagei (AJ388111) with the new together bathynellacean sequences Order : Tanaidacea: Apseudes latreilleilalreillei (AJ388110) (GenBank accession numbers: AF503569 and Superorder AF503570) provided a matrix of 546 characters, Order Two main variable InfraorderInfraorder Palinura: including gaps. areas were found

between the nucleotides caveorum (AF 192869) 237-294 and 340-377. The Scyllarides Scyllarides nodifer (U96088) transition/transversion ratio was 0.8, showing in the lufraorderInfraorder Caridea transversions a saturation tendency for the biggest Macrobrachium atactum divergence values (data not shown). (AF374468) The NJ tree (Fig. 1) showed Speleonectes as the Palaemon serenus (AF439519)

most basal SynalpheusSynatpheus cf. bousjleldibousfieldi ‘A’ species, considering Branchinecta and

(AF230260) Hutchinsoniella as The an out-group. next group ParatypParatya australiensis to appear was the subclass Hoplocarida (class Ma- (AF439523) lacostraca) with and Gonodactylus on the Infraorder Astacida Squilla hand and one the rest of the in-group, subclass (AF235983)

Infraorder Brachyura Eumalacostraca (Class Malacostraca) on the other.

Cancer irroratus The irroratus (AJ130812) analysed specimens of Iberobathynella appeared Infraorder Anomura in a basal just position of this in-group. The rest of Aegla affinis Aegla affinis (AY50036) the species are grouped following their Superorder:

Eucarida and sister Syncarida as groups, and Pera-

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Fig.I. NJ tree based onpartial mt 16S rDNA. Numbers above branches represent the bootstrap values obtained for 5000 replications

corresponding to maximun parsimony and numbers below branches indicate those corresponding to neighbor-joining.

caricia species completing the cluster. The boot- member of the only lineage that has escaped the

strap values mainly supported terminal branches most important evolutionary trend in Crustacea; the

(Fig. 1) where the different taxa of a determinate cephalization of the first thoracic segments. Serban

Superorder are related. justified the necessity of creating a superorder for

the Bathynellacea because of this special and sin-

gular “bathynelloid” structural “Bauplan”. As a

Serban dismissed the idea Discussion consequence, of consid-

the ering Bathynellacea as a degenerate member

On the phylogenetic relationships ofBathynellacea of the Syncarida or a structurally simplified syncarid.

The corresponding question of whether or not the

The different views on phylogenetic relationships Bathynellacea are primitive Malacostraca or sim-

out descendants of this unsolved among crustacean specialists are clearly set by I plified group was an

in 1972. Serban (1972), where he says that the caparace, question

in the is well Schminke the so-called which is lacking Syncarida, very Later, (1981) proposed which made the of the developed in the Eucarida, and is present in inter- zoea theory, origin Bathy- of mediate stages of development in the Hoplocarida nellacea once again a matter discussion. Schram

and Peracarida. Consequently, Serban defined Ba- (1981) approached the problem from the point of

malacostrace view that the “caridoid” structural basis is the thynellacea as: “... un a structure para- primi-

la tive for all Malacostraca and this basis doxale, une forme archai’que evadee de classique pattern on

evolution caridoide et stylisee de maniere sing- suggested some relationships, later modified by

uliere.” (pp. 114)'. The Bathynellacea would be one Hessler (1983). They considered that the differences between Bathynellacea and Anaspidacea could be

due to the fact that the former is to inter- 1 “...a malacostraca with a paradoxical structure, an archaic adapted

form beyond the classic caridoid evolution and stylised in a stitial life while the Anaspidacea is epigean. How-

singular way” this view has been into doubt ever, put since the

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discovery of new syncarids adapted to interstitial

life, the family Stygocaridae in the Order Ana-

spidacea. However, Schram & Hof (1998) in fact,

under some conditions, found Bathynellacea sort-

from It ing separately Anaspidacea. might appear

that, the peculiar body plan of the Bathynellacea,

very different from the typical Syncarida, cannot be explained easily as an ecological specialization.

is clear The Stygocaridae a example of truly primi- tive syncaridans which, after conquering the subter- ranean environment, have maintained theirpeculiar

morphology.

As pointed out by Serban (1972), Bathynellacea has a “bathynelloide” structure (p. 114) that is, a

mixture of archaic simple carioid Malacostraca

characters and a set of specific characters that dif-

ferentiate them from Malacostraca and Syncarida. Fig. 2. Crustacean phytogeny inferred by In this it is morphology. Slightly sense, interesting to quote Brooks (1962) modified from Lange & Schram, 1999. (pp.240): “The Bathynellacea with their eight free thoracic somites and furcal lobes definitely have ancient heritage. They are descendants of is transformed into probably a proper ejaculatory organ,

the archaic ancestors of all the We could this Syncarida”. transformation being unique within the crus- add that share with true the follow- they Syncarida tacean groups. ing characters: Additionally, the development of Bathynellacea is

• absence of (all other malacostraceans the caparace completely different from syncarids as Serban have sometimes one, although ses nombreuses reduced); says (1972) (p. 122): “...par par-

• of 6 abdominal presence segments (number ticularites, fontogenie de Bathynella s’inscrit parmi reduced in other malacostraceans); les principales caracteristiques qui eloignent les

• first thoracic segment always free (in all other Bathynellacea du super-ordre des Syncarida”. The

malacostraceans it is welded the form to cephalic tag- new that comes out of the egg has fewer

and ma), thoracopods (1 to 4) than the adult form, but in

• with thoracopods always or the is epipodites (none syncarids young identical with the reduced in the other malacostraceans). adult (see too Schmincke, 1981).

However, are different bathynelids from syncarids The most recent works on the phylogeny of Crus- in the following: tacea based on morphological characters (Lange &

• the protopod of the retain its ar- & thoracopods Schram, 1999; Schram Hof, 1998) suggest that chaic shape; Bathynellacea are an ancestor of the Malacostraca,

• the has less than endopod always 5 segments including the Anaspidacea. However in their clas- (5 is the typical number in the carioid facies) sification Bathynellacea is an order of Syncarida

and the 4 it is exopod (1 to segments) never within the subclass Eumalacostraca. When fossil

reduced to in a flagelum (which is typical syn- forms are included in the analysis, the Syncarida carids); (Bathynellacea, Anaspidacea and Palaeocaridacea)

• the vast majority of lack remains a bathynelid species paraphyletic group and ancestor of the

and that do 1 pleopods those have them, have Malacostraca (Schram & Hof, 1998; Wills, 1997).

or 2 at most and reduced The always (sometimes to Hoplocarida appears within the Malacostraca,

a simple seta); related sometimes to the Eucarida (Wills, 1997),

• lack on the - and bathynelids statocysts antenna I; at others to the Peracarida or the Syncarida lack • the males a and the 8 (Schram & petasma thoracopod Hof, 1998) (Fig. 2). Taking into ac-

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count that the hoplocarids show several apomorphic References

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