On the larval development of Eubranchipus grubii (Crustacea ...

Zoomorphology (2004) 123:107–123DOI 10.1007/s00435-003-0093-0ORIGINAL ARTICLEOle Sten Møller · Jørgen Olesen · Jens Thorvald HøegOn the larval development of Eubranchipus grubii(Crustacea, Branchiopoda, Anostraca), with noteson the basal phylogeny of the BranchiopodaReceived: 19 March 2003 / Accepted: 15 October 2003 / Published online: 11 December 2003 Springer-Verlag 2003Abstract Selected larval stages of Eubranchipus grubii(Anostraca) from Danish temporary waters are examinedby scanning electron microscopy in a phylogeneticcontext. The study focuses on limb development andbody segmentation. It is shown that the large, proximalendite of the trunk limbs in the adult Anostraca is actuallya fusion product of two smaller endites which make theirappearance in the early larval development. This gives atotal of six endites along the inner margin of the trunklimbs. An unsegmented endopod follows more distally. Asmall additional, seventh endite makes a short appearancein late larvae, but has disappeared in the adults. Thenaupliar feeding apparatus is of the same type as found inother branchiopods, and has previously been suggested asan autapomorphy for the Branchiopoda. The similaritiesbetween the naupliar feeding apparatus of E. grubii andother branchiopods include the presence of a longprotopod with a characteristic morphology of the coxaland basipodal masticatory spines/setae, and a threesegmentedmandibular palp (basipod and two endopodsegments) with a largely similar setation in all taxa. Themode of trunk limb development is also the same as seenin most other recent branchiopods. The phylogeneticsignificance for the basal phylogeny of the Branchiopodaof these and other morphological features is discussed inrelation to the phylogenetic position of two branchiopodfossils, Lepidocaris rhyniensis and Rehbachiella kinnekullensis.While R. kinnekullensis has previously beensuggested to be a stem lineage branchiopod, the positionof L. rhyniensis is more uncertain. Three differentpossible phylogenetic positions of L. rhyniensis areO. S. Møller ()) · J. T. HøegDepartment of Zoomorphology, Zoological Institute,University of Copenhagen,Universitetsparken 15, 2100 Copenhagen OE, Denmarke-mail: osmoller@zi.ku.dkFax: +45-35321200J. OlesenZoological Museum,University of Copenhagen,Universitetsparken 15, 2100 Copenhagen OE, Denmarkdiscussed: (a) L. rhyniensis as a stem lineage anostracan,(b) L. rhyniensis as a stem lineage branchiopod or (c) L.rhyniensis as a stem lineage phyllopod. It seems mostplausible to consider L. rhyniensis a stem lineageanostracan.Keywords Ontogeny · Nauplius · Limbs · SEM ·PhylogenyIntroductionThere is still some uncertainty about the basal phylogenyof the Branchiopoda. In recent years a consensus hasappeared that the Anostraca are the sister group to amonophyletic Phyllopoda sensu Preuss (1951) (comprisingthe Notostraca, Spinicaudata, Laevicaudata, Cyclestheridaand Cladocera) (see, for example, Walossek 1993,1998; Spears and Abele 2000; Braband et al. 2002;Olesen 2003), but the phylogenetic positions of twominute branchiopod fossils, Rehbachiella kinnekullensisMüller, 1983 (“Orsten”, Upper Cambrian) and Lepidocarisrhyniensis Scourfield, 1926 (Devonian), are stillbeing debated. Due to favourable preservation conditions,both these fossils are remarkably well-preserved, andlargely all aspects of their external morphology areknown, including the morphology of several larval stages(see Scourfield 1926, 1940; Walossek 1993). Both fossilsare preserved in three dimensions, which is very unusual,and one of the fossils (R. kinnekullensis) has even beenstudied by scanning electron microscopy, and this to suchan extent that our knowledge of many recent crustaceanslags behind (including the Anostraca). Various phylogeneticpositions of R. kinnekullensis and L. rhyniensis havebeen suggested, but in all cases the recent Anostraca playan important role, which is due to the general similarity ofthese three taxa. Already Scourfield (1926) discussed theanostracan affinity of L. rhyniensis, but Walossek (1993)was the first to formally suggest the recent Anostraca andL. rhyniensis as sister groups. Also R. kinnekullensis wasviewed by Walossek (1993) as an early offshoot of the

108anostracan line. Olesen (1999) questioned the anostracanrelationship of R. kinnekullensis and suggested a positionone step down as a stem lineage branchiopod. Schram andKoenemann (2001) had the same view and placed L.rhyniensis in a similar position. Olesen (2003) followedup on the discussion and provided some characters insupport of the position of R. kinnekullensis as a stemgroup branchiopod, but left the position of L. rhyniensisunresolved.Due to the pivotal role of the Anostraca in theunderstanding of the phylogenetic position of R. kinnekullensisand L. rhyniensis, along with the lack ofimportant morphological information for the Anostraca, itis of interest to examine aspects of the external morphologyof an anostracan species in more detail to facilitatecomparison to the two fossils. To address questions oflimb homologies and body segmentation in a comparativecontext, we chose to study the external morphology ofthree different stages of Eubranchipus grubii (Dybowski,1860). This species has been chosen for conveniencesince it is the most common species in Denmark(Damgaard and Olesen 1998), and since it provides analternative to the often-studied species of Artemia. Thespecies has previously been briefly dealt with byOehmichen (1921) in light microscopy. As summarisedby Walossek (1993), the Anostraca have the longest andmost gradual (anamorphic) development among recentCrustacea (see also Heath 1924; Benesch 1969; Bernice1972; Fryer 1983; Schrehardt 1987), and we have chosenlarvae of E. grubii that represent the early, intermediateand late development. Other important studies on thelarval development of various anostracan species includethose of Claus (1873, 1886), Cannon (1926), Anderson(1967), Barlow and Sleigh (1980) and Jurasz et al. (1983).Comparative studies of ontogeny have previously providedclear information useful for phylogenetic considerationswithin the Branchiopoda (Fryer 1988; Walossek1993; Olesen 2003).The end of the discussion will focus on the phylogeneticposition of L. rhyniensis since this is an importantaspect of branchiopod phylogeny that remains to beclarified. As a basis for the discussion, we assume that:(1) R. kinnekullensis is not a stem lineage anostracan, butrather a stem lineage branchiopod, (2) within the Branchiopoda,all branchiopods, except the Anostraca, can begrouped in the Phyllopoda and (3) L. rhyniensis forms amonophylum with the recent Branchiopoda based onsimilarities in the nauplius feeding apparatus and themorphology of the adult trunk limbs, the details of whichwere summarised by Olesen (2003). Accepting the listedassumptions for the basal branchiopod phylogeny, threepossibilities exist for the phylogenetic position of L.rhyniensis: (a) a stem lineage anostracan, (b) a stemlineage branchiopod or (c) a stem lineage phyllopod.Hence, the purpose of this paper is twofold:1. To provide new information on the external morphologyof a species of the Anostraca, with special focuson limb development and limb homologies2. To use the new information and the available literatureto evaluate a debated aspect of basal branchiopodphylogeny: the phylogenetic position of the Devonianbranchiopod fossil L. rhyniensisMaterials and methodsLarvae of E. grubii were collected in the early spring of 2001(January to March) in one of the classic Danish localities for “largebranchiopods” close to “Trepilelågen” in the deer park, (JaegersborgDyrehave), north of Copenhagen (more information about thelocality in Damgaard and Olesen 1998 and Mossin 1986). Thematerial was fixed in a 1% osmium tetroxide solution overnight atroom temperature. Further preparation was by dehydration throughacetone and critical-point drying using CO 2 . The specimens weresputter coated with a mixture of palladium and platinum. The SEMsused were a Jeol JSM-840 and a Jeol JSM 6335-F, placed at theZoological Museum, University of Copenhagen.ResultsIn the following description, we include larvae in thefollowing three categories largely representing the wholelarval sequence: (1) stage without well-developed limbbuds, (2) stage with five to seven well-developed limbbuds and (3) stage with 12–13 well-developed limb buds.Stage without well-developed limb budsThe earliest stage observed is a nauplius larva ofapproximately 670 mm in length (without antennules).The three naupliar appendages, antennules, antennae andmandibles, are developed and partly functional, whiletrunk limbs are only weakly demarcated (Fig. 1).The labrum is large, inflated and evenly roundeddistally (U-shaped); it overhangs the median tips of themandibular coxae and lacks setation (Fig. 1A–C). Theantennules are inserted anteriorly and somewhat laterally,separated by a space corresponding to the width of thelabrum (Fig. 1A, C). They are long (nearly same length astrunk), slender, unsegmented and bear three setae at thetips.The antennae are inserted more laterally at the bodythan are the antennules (Fig. 1A, B). They are large(almost the same length as the whole animal) andbiramous. The protopod is only weakly divided into acoxal and basipodal part. The distal portion of theprotopod, the prospective basipod, is about twice thelength of the proximal portion, the prospective coxa. Thecoxal portion of the antenna has a large spine orientedtowards the mouth (“proximal masticatory spine”, sensuFryer 1983); here we intentionally use the neutral term“spine”, leaving it open whether this structure, or parts ofit, should be a seta. The basipod also bears another spinedirected somewhat against the midline of the animal(“distal masticatory spine”, sensu Fryer 1983, in thefollowing referred to as the “basipodal masticatoryspine”). The endopod is only about the length of the

109Fig. 1A–D Eubranchipus grubii, stage without well-developedlimb buds. A Ventral view. B Lateral view. C View from ventral/posterior. D Ventral view on trunk region. a1 Antennule, a2antenna, en endopod, ex exopod, la labrum, md mandible, tl1 trunklimb 1, tl12 trunk limb 12basipod, which corresponds to one quarter the length ofthe exopod; the segmentation is indistinct and it has threelong setae distally. The exopod has about the length of theprotopod, is multiannulated and has 20 long, natatorysetae on the posterior/median face and two setae distally.The mandibles are inserted laterally on the body(Fig. 1A, C). They each consist of an enlarged coxa with athree-segmented “palp” (basipod and two presumedendopodal segments distally). The coxal processes areonly weakly developed and seem unable to meet mediallywhere they are covered ventrally by the fleshy U-shapedlabrum. The median edge of the coxal process has a smallseta (termed “gnathobasic seta” by Fryer 1983). The palphas seven setae: two setae on the basipod, two on the firstendopodal segment and three setae terminally on thedistalmost second segment.Neither maxillules nor maxillae can be clearly seen inthis stage. The head region of the larva is generally

110“narrower” (i.e. the circumference is smaller) than thethorax with the maxillary region as a cone-shapedtransition zone.The trunk is almost twice as long as the head region.No clearly recognisable trunk limbs are yet present, but12–14 weakly demarcated pairs of buds are presentventrally, posteriorly lying progressively closer to eachother (Fig. 1C). A pair of minute precursors of the furcalrami, with an anal opening in between, completes thelarva posteriorly (Fig. 1D).Stage with five to seven developed limb budsThe length of this stage is about 750 mm (withoutantennules). Apart from an increase in size, the mostsignificant differences between this and the previouslydescribed stage are that the buds of the maxillae andmaxillules have appeared (Fig. 2A, E) and that the trunklimb buds have become differentiated into various limbportions (Fig. 2A).Frontally a pair of separate buds represents theprimordial compound eyes (Fig. 3B). Slightly moreventrally, closer together, a pair of small depressions isseen that probably represent the “frontal organs”(Fig. 3B). Dorsally a large dorsal organ covers the regioncorresponding to the antennae and the mandibles(Figs. 3A–F, 4A).Behind the naupliar portion of the head is a short,segment-like section the same size as posterior trunksegments (Fig. 4A), corresponding to the maxillules andmaxillae, but without external demarcation into twoseparate segments. There are 14–15 postmaxillary segments,decreasing gradually in width posteriorly(Fig. 4A, D). The labrum is large, fleshy and U-shaped(Fig. 2A, C).The naupliar appendages (antennules, antennae andmandibles) are very much like those of the previouslydescribed stage. In this stage they have been examinedsomewhat closer, and since the setae are more developed,more details will be presented here. The antennules arenow long and slender, with three distal setae (Figs. 2A,3A). The antennal protopods are more clearly divided intoa coxa and a basipod (Fig. 2A). The coxa has a longenditic process, the “coxal masticatory spine”, directedtowards the mouth region (Fig. 2A). This process is armedwith setules distally which themselves bear small secondordersetules (Fig. 2B). The basipod is about twice as longas wide and is superficially divided into four portions(Fig. 2A). Distally the basipod carries a long setaequipped with long setules also directed towards themouth region (Fig. 2A). The endopod is short and itssegmentation is somewhat unclear but is probablyrevealed by several transverse rows of minute spines thatindicate a total of four portions (probable segment bordersmarked by arrows on Fig. 4C). The exopod is unchangedfrom the previously described stage and it has 20 setaealong the posteromedian margin, one for each annulation.The mandibular coxa has become larger compared tothe previously described stage (Figs. 2A, 3F, 4A) and thegnathal edges of the coxal processes are now able to meeteach other. The gnathobasic setae are also larger andalmost hidden by the labrum. The mandibular palp hasmore distinct borders between the three segments (basipodand two endopod segments; Fig. 4D, E). The setation of themandibular palp is the same as earlier—basipod with twosetae, the first endopod segment with two setae, and thesecond endopod segment with three setae—but the setaeare longer. The two setae on the basipod and endopodsegment 1, respectively, have a number of unevenlydistributed long setules, arranged pairwise along the seta towhich they are attached in a “V”-like arrangement(Fig. 4D). These setules point towards the mouth region.The three setae on the second endopod segment each havea row of short, closely placed setules along them.A pair of primordial paragnaths, densely covered withsetae, is visible just below and slightly posterior to thelabrum (Fig. 2E). Between the primordial paragnaths is aparagnath channel. Both the maxillules and the maxillaeare now visible and armed with fine setae on their ventralface (Fig. 2E). The maxillulary and maxillary limb budsare attached ventrally at the short, segment-like sectionbetween the naupliar portion of the head and the firsttrunk segment (Figs. 2E, 4A, 5D). The maxillules areelongate limb buds placed laterally, immediately behindthe paragnaths and oriented towards the ventral midline,placing them on the “outside” of the maxillae(Fig. 2A, E). The maxillae are small rounded buds,placed slightly behind the elongate maxillules, and muchcloser together than these (Figs. 2A, E, 5D).The trunk has two rows of limb buds placed ventrolaterally(Figs. 2A, 4A, B, 5); no limb has yet attained theadult, vertical (“upright”) orientation. A gap separates thetwo rows of limb buds in the ventral midline of the trunk(Figs. 2A, 5D). On each side seven to eight limb buds aredeveloped with various limb portions demarcated to avarying degree (Figs. 2A, 3F, 4A, B, 5). More posteriorlythere are seven to eight additional limb buds or trunksegments, with no portions indicated. The anterior limbbuds are the largest and most developed ones. Each limbbud curves or bends slightly posteriorly and overlaps itsmore posterior neighbour, thus reflecting the situation inthe adult (Figs. 3F, 4A, B). The complete and mostdeveloped limb buds have as many as 11 lobes of varyingsizes, to which we henceforward apply the terminologyused for adult trunk limbs. Ventrally (median in adult)each limb bud is divided into seven portions: threemedian (proximal) large ones, called endites in the adult,three smaller lobes more laterally, which are also calledendites in the adult, and one large lobe most laterally,called an endopod in the adult. Lateral to the endopodfollows first a large lobe, the exopod, and thereafter threeadditional lobes that will develop into the adult epipods.The exopod has a distinct setation consisting of two tofour setae on the four most anterior limbs. Small setae arepresent on the three proximal ventral lobes (endites) ontrunk limbs 1 and 2.

111Fig. 2A–E Eubranchipus grubii, stage with five to seven welldevelopedtrunk limbs. A Ventral view. B Tip of antenna coxalmasticatory spine. C Distal margin of labrum. D Left antenna. EClose-up of area behind labrum showing paragnaths (pgn),maxillules (mx1) and maxillae (mx2). a1 Antennule, a2 antenna,bas basis, cox coxa, en endopod, ex exopod, tl1 trunk limb 1

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113Fig. 4A–E Eubranchipus grubii, stage with five to seven welldevelopedtrunk limbs. A Lateral view. B Lateral view of trunklimb buds. C Lateral view of left side antennal endopod (a2 en).Arrows point at possible rudiments of segmentation as indicated byrows of short spines. D Median side of left side mandibular palp. EAnterolateral view of right side mandible. a2 Antenna, bas basis, cecompound eye, cox coxa, do dorsal organ, en endopod, en1, 2endopodal segments 1, 2, ep epipod, ex exopod, md cox mandibularcoxa, mx1 maxillule, tl1–9 trunk limbs 1–9Dorsally each postmaxillary segment carries tworelatively long setae placed some distance apart(Figs. 3E, F, 4A). In contrast, there is no such pair ofsetae on the maxillulary/maxillary segment-like section.In total, 11 postmaxillary segments have a pair of setae.These setae are placed in the same position on mostsegments, but on segment 9, they are placed significantlyFig. 3A–F Eubranchipus grubii, stage with five to seven welldevelopedtrunk limbs. A Dorsal view. B Frontal view showingprimordial compound eyes (ce) and dorsal organ (do). C Close-upof dorsal organ. D Pair of caudal protrusions each with a seta. EDorsal side of trunk. Arrows point to a pair of setae placed closertogether than the remaining dorsal pairs of setae. F Lateral view. a1Antennule, a2 antenna, md mandible, md cox mandibular coxa, tl1trunk limb 1closer together (Fig. 3E arrows). The telson region tapersinto two conical protrusions each carrying a seta, denselyarmed with setules along the distal half (Fig. 3D, E).Stage with 12–13 well-developed limb budsThis stage is about 1.2 mm long and, in most respects, isquite different from the previously described stages.Among the most significant changes are the tips of thecoxal masticatory spines of the antennae have becomebifid and the anterior trunk limbs have attained an“upright”, adult orientation.The frontally placed primordial buds of the compoundeyes have become larger and the separate facets arevisible through the cuticle (Fig. 6B). Between the eyes is

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a pair of small elevations probably being frontal organs(Fig. 6B).The antennules, with three distal setae, are relativelylong and slender (Fig. 6A). The antennae are rathersimilar to those in the previously described stage, but theyare relatively smaller and, most significantly, the tips ofthe coxal masticatory spines have become bifid(Fig. 7A, D, H). Both branches of the bifid tip arecovered with robust setules, arranged semiregularly inrows along each spine. The setules themselves also bearsome second-order setules. The posterior branch of thebifid tip has an annulation close to its basis (Fig. 7E).As earlier, the basipodal masticatory spine has thesame arrangement of mostly pairwise attached, longsetules, directed towards the mouth region and themselvesbearing small, second-order setules (Fig. 7A, F). Along itsposterior margin, the basipodal masticatory spine carries arow of short, densely placed setules (Fig. 7F). Distally,the endopod carries four rather long setae (Fig. 7C) incontrast to the previously described stage, where one wasrather short. The endopod is apparently composed of fourportions as indicated by rows of spines and articulationlikeconstrictions (Fig. 7C arrows). The exopod isunchanged and carries 19–20 long setae (Figs. 6A, 7A).The coxae of the mandibles have increased in size. Theprimordial paragnaths with a paragnath channel in betweenare found behind the mandibles (Fig. 7B). The paragnathsare covered by a dense layer of setae. Placed immediatelybehind the paragnaths, the elongate buds of the maxillulesare also covered by fine setae. The maxillules have becomemore prominent than in the previously described stage(Fig. 7B) and, pointing more or less medially, they have afew setae directed towards the mouth region. The smallerand circular buds of the maxillae are placed closer togetherthan the maxillules, slightly posterior to these, and are alsocovered with small setae.The region of the trunk limbs has developed significantly,carrying 13 pairs of limb buds, which is the fullnumber achieved in adults, including the modified limbsassociated with the genital segments (Fig. 6A, F). Trunklimb 1 is almost fully developed, but the degree ofdevelopment decreases posteriorly, and trunk limbs 9–11are elongate limb buds with only incipient subdivisions(Fig. 6A, F). Buds of limb pairs 11 and 12 on the futuregenital segments show no clear division into substructures(Fig. 6F). The anterior six to seven pairs of trunk limbshave enlarged very much and attained a more verticalorientation; these limbs have a medial row of six to sevenenditic lobes (Fig. 6F, G). In the three anterior trunk limbsthe two proximal enditic lobes have begun to fuse (as isFig. 5A–D Eubranchipus grubii, stage with five to seven welldevelopedtrunk limbs. A Ventral view of buds of trunk limbs 1–6,right side. B Lateral view of buds of trunk limbs 2–4, right side,showing future distal parts. C Lateral view of buds on right side oftrunk limbs 4–11. D Ventral view of approximately 12 limb buds indifferent degrees of development. e1/pe Endite 1/proximal endite,e2–6 endites 2–6, en endopod, ep epipod, ex exopod, mx2 maxilla,tl1–7 trunk limbs 1–7the adult condition; Fig. 6F). At least six endites arepresent, but possible rudiments of a seventh endite can berecognised in trunk limbs 4–6 between the sixth enditeand the endopod (Fig. 6F, G). The exopod has attained aconsiderable size in the anterior four to six trunk limbs(Fig. 6A, D, F). Laterally, the epipod and the twoadditional more proximal exites (or epipods) haveenlarged significantly (Fig. 6D, E). The distalmost epipodis the largest, and it appears to be incipiently articulated tothe stem of the limb. Setation is most developed in theanterior trunk limbs (Fig. 6A, F, G). The proximal enditesof the trunk limbs have a row of curved, well-developedsetae at the posterior edges of the endites. The remainingfive endites also have setae along the posterior edge.Slightly more anteriorly, on endites 1–4, one or two shortspines are found (Fig. 6F, G). Setae are present on theexopods of trunk limbs 1–9. Setation is only present onthe endopods and the most proximal endites on trunklimbs 1–7. The telsonic region tapers into two conicalprotrusions that are more elongate than in the previouslydescribed stage (Fig. 6A); each carries a long seta armedwith setules.DiscussionThis study deals with three different larval instars of theanostracan E. grubii chosen specifically to cover most ofthe variation throughout the developmental sequence ofthe species. Based on what is known, the larvalmorphology of various anostracan species appears to bevery similar, which leads us to assume that E. grubii, inthe context of comparative larval morphology of theBranchiopoda, is a typical representative of the Anostraca.First, selected structures of body segments and limbsof E. grubii will be summarised and compared to similarstructures in relevant branchiopods, especially those oftwo early branchiopod fossils (R. kinnekullensis and L.rhynieniens). Then some of this information and otherinformation from the literature will be used in a discussionof the basal phylogeny of the Branchiopoda wherethe Anostraca play a central role.Body segmentation, dorsal setation of trunkand other details115In the stage with five to seven limb buds there is a short,segment-like body section between the mandibular regionand the segment of the first trunk limbs. This sectioncorresponds ventrally to the maxillulary and maxillarylimb buds. Although two limb pairs (maxillules andmaxillae) belong to this section there is no clear externalindication of its division into two separate segments.Probably this segment-like section is primarily maxillularysince the maxillules in size dominate over themaxillae (Figs. 4A, 5D). It appears likely that the section/segment in question is, at least in part, a fusion product of

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the maxillary and maxillulary segments. The presence ofsuch a “free” body portion immediately behind themandibular region (not fused with naupliar portion toform a continuous head) prevails in the adults. Also adultsof the Devonian fossil L. rhyniensis have such a freeportion behind the mandibles. Since this situation isunique within the Branchiopoda, and apparently differentfrom the condition in other Crustacea, it is suggestedbelow as a possible synapomorphy of L. rhyniensis andthe Anostraca.In E. grubii each trunk segment carries a pair ofrelatively long setae dorsally. They are placed in a quiteregular pattern, in which the distance between setae of apair decreases posteriorly. In all members of the Phyllopodasensu Preuss (1951), even in the aberrant Leptodorakindtii (Focke, 1844) (see Olesen et al. 2003), a dorsalpair of setae is present posteriorly on the trunk (homologiesfirst discussed by Martin and Cash-Clark 1995). In theCladocera they are traditionally termed postabdominalsetae and in the “Conchostraca” sometimes telson filaments.Because of the presumed close relationshipbetween all extant Branchiopoda, it is tempting to considerhomology between the single pair present in the Phyllopodaand one of the setae pairs in the Anostraca (seeMartin and Cash-Clark 1995 on the same subject).However, such a homology can only be established withmajor difficulties since the position of the setae is quitedifferent. Embryological studies of Cyclestheria hislopi(Baird, 1859), (Olesen 1999) and L. kindtii (Cladocera)(Olesen et al. 2003), and thereby most certainly in otherPhyllopoda, have shown that the single dorsal pair of setaeis telsonal, while none of the dorsal setae pairs present inE. grubii can be found that far posteriorly.The latest larval stage examined has 13 pairs of trunklimb buds (Fig. 6A, F), while adults only have 11 truetrunk limbs. We interpret the two posterior buds (Fig. 6F)as either the anlagen to the penis in the male or the ovisacin the female, which indicate a limb-origin for thesestructures.Comparison of larvae of E. grubii with larvaeof two branchiopod fossilsNaupliar feeding apparatus of Eubranchipus, Lepidocarisand RehbachiellaThe naupliar feeding systems of these three taxa are rathersimilar, but those of E. grubii and L. rhyniensis sharesome specific similarities not found in R. kinnekullensis(if not otherwise specified, the information on L.Fig. 6A–G Eubranchipus grubii, stage with 12–13 well-developedtrunk limbs. A Ventral view. B Frontal view showing compoundeyes (ce). C Distal margin of labrum. D Ventral view of trunklimbs. E Close-up of exites of left side trunk limbs 1–3. F Medianview of left side trunk limbs. G Median view of left side trunklimbs 5–8. a1 Antennule, a2 antenna, e1/pe endite 1/proximalendite, e2–7 endites 2–7, en endopod, ep epipod, ex exopod, lalabrum, md mandible, tl1–13 trunk limbs 1–13117rhyniensis and R. kinnekullensis is from Scourfield1926, 1940 and Walossek 1993). These similaritiesinclude a long antennal protopod, a similar arrangementof the coxal and basipodal masticatory setae of theantennae, as well as the segmentation and setation of themandibular palp. Since other large branchiopods (Notostraca,Spinicaudata and Laevicaudata) for the most parthave a similar naupliar feeding apparatus, not found inother Crustacea, Olesen (2003) treated it as a synapomorphyfor the Branchiopoda (or Branchiopoda s. str.),excluding R. kinnekullensis.In the antennae, the specific similarities of the coxalmasticatory spines of E. grubii (Fig. 7D) and L. rhyniensisinclude that they, in certain parts of the development, arepincer-like, divided into two branches of equal length andcovered with setules arranged more or less like the hairson a bottle-brush. This pattern is also present in theSpinicaudata and Laevicaudata (Olesen 2003; Olesen andGrygier 2003). It should be noted, however, that R.kinnekullensis also has long median coxal spines of theantennae in early larvae (see, for example, Walossek 1993plate 2:6), but never with the morphology describedabove. The most import difference is that the antennalcoxae have more than one spine in R. kinnekullensis,while only one is present in the remaining Branchiopoda.Another difference concerns the branched tip of the coxalspine which, in contrast to L. rhyniensis and the recentBranchiopoda, has not been documented for any stage ofR. kinnekullensis, but it is difficult to exclude that thiscould be explained by the fact that setae and spines tendto be broken off in R. kinnekullensis (personal communicationD. Waloszek).The specific similarities of the larval mandible in thefree-living larvae of all large branchiopods (including L.rhyniensis and E. grubii) concern the uniramous conditionof the palp and the identical segmentation pattern(basipod and two endopod segments) (Olesen 2003). Itfurther supports a segmental homology of the mandiblesbetween the groups that the setation pattern is the same inanostracans, notostracans and L. rhyniensis and largelythe same in the spinicaudatans and laevicaudatans (seeOlesen 2003 for more details). R. kinnekullensis isdifferent in having more segments in the endopod andmore coxal and basipodal masticatory spines (seeWalossek 1993). Furthermore, the mandibular palp in R.kinnekullensis is biramous and consists of a basipod, anendopod and an exopod, in accordance with the situationin the larvae of the Cephalocarida and in many “maxillopodan”taxa (see, for example, Sanders 1963; Moyse1984, 1987; Olesen 2001). This makes the uniramous palpa putative synapomorphy of the Branchiopoda excludingR. kinnekullensis.Development of trunk limbs in Eubranchipus, Lepidocarisand Rehbachiella and some other branchiopodsDespite the general similarity between adult limbs of E.grubii and those of the Devonian fossil L. rhyniensis,

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including a leaf-like appearance with a largely identicalarrangement of median and lateral lobes (endites andexopod/epipods; see, for example, Sars 1896; Scourfield1926), the mode of limb development is different. Themain difference is that while the long axis of the limb(proximal/distal axis in the adult) is displaced laterally inthe larvae of E. grubii, L. rhyniensis has the adultdorsoventral orientation of this axis from the onset of thelarval development (Scourfield 1940). In this respect E.grubii is similar to other large branchiopods, as summarisedby Olesen (2003), while L. rhyniensis is similarto R. kinnekullensis and to other crustaceans.The serial displacement of the degree of developmentof the trunk limbs in E. grubii allows the ontogeny of thevarious limb parts to be followed. The trunk limbs firstappear as elongate buds placed ventrolaterally on thebody, following the curvature of the trunk, withoutexternal subdivision. This division appears first as a cleftpractically in the middle of the limb bud that divides itinto a ventral and laterodorsal part between the futureendopod and exopod (Møller et al. 2003 describe a similarcondition in the development of the Notostraca). The nextsubdivision appears further laterally between the exopodand the future epipod. Approximately at the same time thefirst indication of the three to four most proximal enditesappears as well as the primordia of two more laterallyplaced epipods. The differentiation of the (future) medianedge (directed ventrally in the larvae) takes placesgradually from proximal towards distal. The proximalendites appear first and the distal endites and theunsegmented endopod appear last.In general terms, the development of the trunk limbs inE. grubii is very similar to that of two other branchiopodsviz., C. hislopi and Eulimnadia braueriana Ishikawa,1895 which have been studied in some detail by Olesen(1999) and Olesen and Grygier (2003). In those works,the similarities in development have been discussedbriefly as a possible synapomorphy of the branchiopods.Some important similarities are: the developing limb budsare placed as two rows lateroventrally on the trunk withthe future median edge of the limbs facing ventrally, andthe limbs in all mentioned species differentiate firstdistally (directed laterally in larvae) and the median edge(directed ventrally in larvae) differentiates into enditiclobes from proximal towards distal. Significant differencesbetween the species include that the developingtrunk limb buds of E. grubii are divided into 11 portions(six endites, an endopod, an exopod, three “epipods”),while only eight are present in the two “conchostracans”.Homologies of the endites and endopodof anostracans and other branchiopods119In early trunk limbs of E. grubii at least six endites and anunsegmented endopod are found along the medial edge(ventral in larvae). The three proximal, relatively largeenditic lobes are followed by three smaller ones. Largerlarvae occasionally carry a rudiment of a seventh enditeimmediately proximal to the endopod on some of thelimbs but it disappears before adulthood (indicated by e7?on Fig. 6F).Our observations agree with those of Fryer (1983:Fig. 36) that the most proximal, large enditic lobe in theadult trunk limbs of anostracans is the product of a fusionbetween the larval enditic lobes 1 and 2 (Fig. 8). Thepresence of six enditic lobes means that there is noequivalence in the number of endites between anostracantrunk limbs of and those of notostracans and “conchostracans”where only five enditic lobes are present (seeMøller et al. 2003 for Notostraca). The large proximalendite in the trunk limbs of the Anostraca probablycorresponds to endites 1 and 2 in the Notostraca and the“Conchostraca”. With respect to the number of endites,anostracans are more similar to L. rhyniensis, where theanterior trunk limbs also have six enditic lobes (Scourfield1926). Interestingly, previous authors have beenuncertain on the status of the large, proximal endite of thetrunk limbs of the anostracans. Most workers mark thelarge proximal endite as endite 1. Based on work withPolyartemia forcipata Fischer, 1851, Ekman (1902)suggested that this endite is formed by a fusion of twoendites. Borradaile (1926) left the question unresolved,while Baqai (1963) explicitly stated (wrongly) that themost proximal, large endite does not consist of twoendites. Fryer (1983), on the other hand, correctly labelledthe proximal large endite in adults of Branchinecta ferox(Milne-Edwards, 1840) as being composed of twoendites, but did not comment much on the issue.Walossek (1993) also favoured this interpretation.Fig. 7A–J Eubranchipus grubii, stage with 12–13 well-developedtrunk limbs. A Left side antenna (a2) and mandible (md). B Atriumoris: paragnaths (pgn), maxillules (mx1) and maxillae (mx2). CAntennae endopod of left side. D Bifurcated antennal coxalmasticatory spine of left side. E Detail of D showing a distinctannulus of the posterior branch. F Close-up of the basipodalmasticatory spine/seta of the antenna, showing its characteristicsetulation. G Mandible of right side. H Close-up of distal branchesof coxal masticatory spine of the antenna. I Close-up of mandibleendopod segment 1, showing its armature of two setae. J Close-upof distal setae of mandible endopod segment 2. a2 en Antennalendopod, a2 ex antennal exopod, ba, bas basis, bas msp basipodalmasticatory spine, cox coxa, cox msp coxal masticatory spine,en1, 2 endopodal segments 1, 2, la labrum, md cox mandibularcoxa, tl1 trunk limb 1Phylogenetic evaluationIn recent years a growing body of evidence has indicatedthat the Branchiopoda is monophyletic based on informationfrom different sources, such as larval morphology,sperm morphology and molecular data (Sanders1963; Wingstrand 1978; Walossek 1993; Spears andAbele 2000; Olesen 2003). Various authors support theAnostraca as the sister group of the remaining Branchiopoda,and Walossek (1993) and Braband et al.(2002) suggested that the latter should be grouped underthe name Phyllopoda sensu Preuss (1951). This was also

120Fig. 8A–C Eubranchipus grubii. A Ventral view of subadult. B Trunk limb of adult. C Close-up of B showing endites along the medianmargin. e1/pe Endite 1/proximal endite, e2–6 endites 2–6, en endopod, ep epipod, ex exopodfavoured in the comprehensive account of crustaceantaxonomy provided by Martin and Davis (2001). Hence,the basal split in branchiopod phylogeny now seems wellunderstood, but due to the generally primitive appearanceof the Devonian L. rhyniensis and the UpperCambrian R. kinnekullensis, these remarkably wellpreservedcrustaceans are obviously important for ourunderstanding of early branchiopod evolution. It is

121Fig. 9 Basal phylogeny of theBranchiopoda. The DevonianLepidocaris rhyniensis is placedas a stem lineage anostracan(position A), but two otherpossible positions are also indicated(B and C)therefore crucial to evaluate the phylogenetic position ofthese fossils.Olesen (1999), Schram and Koenemann (2001) andOlesen (2003) suggested that at least R. kinnekullensis is astem lineage branchiopod rather than a stem lineageanostracan. In other words, these authors suggested that R.kinnekullensis is not closer related to the Anostraca than itis to other branchiopods. The Branchiopoda together withits stem group were treated as Pan-Branchiopoda byOlesen (2003). What remains to be established is thephylogenetic position of L. rhyniensis which, as notedpreviously, has been suggested to be a fossil anostracan(Scourfield 1926; Walossek 1993).Below is listed a number of putative synapomorphiesof L. rhyniensis and the Anostraca which support that L.rhyniensis is a stem lineage anostracan (see Fig. 9). Thisis the preferred phylogenetic position of L. rhyniensis inthis study, but other possible positions of L. rhyniensis arediscussed more briefly below.– Ventral brood pouch formed from trunk segments 12and 13 (if Scourfield’s 1926 original counting isfollowed)– Median edge of trunk limbs with six endites (onlyanterior limbs in L. rhyniensis)– Carapace lacking– Thirteen limb-bearing segments (including the genitalsegments)– Maxillular/maxillar segment not fused to cephalon(this character may not be independent from carapacelacking)– Maxillar limb buds in larvae placed close togetherScourfield (1926) also listed some “points of agreement”between L. rhyniensis and the Anostraca, but onlytwo of these (“elongated form and general appearance”and “absence of dorsal shield or bivalve shell”) are noteasily dismissible as symplesiomorphies also present inmany other branchiopods (like structure of mandibularpalp and foliaceous structure of trunk limbs).A second possibility is L. rhyniensis as a stem lineagebranchiopod (position B in Fig. 9; suggested by Schramand Koenemann 2001), a hypothesis which can be basedon the following putative synapomorphies for the recentBranchiopoda (excluding L. rhyniensis): (1) early limbbuds have future median edge directed ventrally, themedian limb portions (endites and endopod) becomedifferentiated before the limbs attain a dorsoventralorientation, (2) maxillule reduced in size and (3) antennulesunsegmented in adults (with few exceptions whichthen must be considered secondary).A third possibility would be L. rhyniensis being a stemlineage phyllopod (position C in Fig. 9). At least there is asimilarity in the presence of only five to seven setae (incontrast to many more) on the antennal exopod of thelarvae.However, due to the limited support for L. rhyniensisas a stem lineage phyllopod, we believe that only the twofirst-mentioned possible phylogenetic positions of L.rhyniensis are worth considering: (a) L. rhyniensis as astem lineage anostracan or (b) L. rhyniensis as a stemlineage branchiopod. An anostracan stem lineage positionof L. rhyniensis would mean that the laterally displaceddorsoventral axis of the trunk limbs (enditic portionsfacing ventrally) in the Anostraca and the Phyllopodawould have appeared independently in these two groups(or it is an evolutionary reversal of the taxon L.

122rhyniensis), as would the reduced maxillules and the othercharacters listed above. If L. rhyniensis on the other hand,is considered to be a branchiopod stem lineage, then allthe characters listed in support for an anostracan stemlineage position (brood pouch, six median endites, samenumber of body somites, etc.) would be either convergentor symplesiomorphies. We conclude, as did Walossek(1993), that an anostracan stem lineage position of L.rhyniensis is the most likely.Acknowledgements We thank Jesper T. Stenderup (Copenhagen)for assistance when collecting and sorting the material for thisstudy. Two anonymous reviewers and the editor of Zoomorphology,Prof. Dr. Thomas Bartolomaeus (Berlin, Germany), arethanked for providing many useful suggestions for improvementof the paper. 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