I I BULLETIN DE L'INSTITUT ROYAL DES SCIENCES NATURELLES DE BELGIQUE, BIOLOGIE, 66: 73-92, 1996 BULLETIN VAN HET KONINKLIJK BELGISCH INSTITUUT VOOR NATUURWETENSCHAPPEN, B10LOGIE, 66: 73-92, 1996

Redescription and morphological variability of Darwinula stevensoni (BRADY & ROBERTSON, 1870) (Crustacea, Ostracoda)

by Giampaolo ROSSETTI & Koen MARTENS

Abstract Introduction

The species Darwinula stevensoni is extensively redescribed. Mor­ The Darwinuloidea are, together with the Bdelloidea, one phological variability of both valves and soft parts is assessed of the two prominent examples of so-called ancient asex­ in several geographically and climatically distant populations uals (BUTLIN & GRIFFITHS, 1993, JUDSON & NORMARK, and is found to be minimal or non-existant. Only size 1996), organisms which have persisted over geologically significantly varies between populations and this can be long periods of time without sexual reproduction. Accord­ attributed to the differences in ambient temperatures during the larval development. Number and shape of muscle scars also vary, ing to ruling theory, such lineages are doomed to early but this both within and between populations. Valve shape and extinction for several reasons: co-evolving parasites ('Red chaetotaxy of limbs are remarkably constant. One female from Queen' hypothesis of HAMILTON (1980), not VANVALEN, an Italian population has aberrant Mx2-palps, but this specimen 1973), Muller's ratchet and mutation-load-reduction­ is considered a teratological case. theory (MAYNARD SMITH, 1986, JUDSON & NORMARK, Earlier records of males of D. stevensoni and the taxonomic posi­ foe. cit.). Nevertheless, indeed appear to tion of the infraorder Darwinulocopina within the suborder have survived without sexual reproduction at least since Podocopina are briefly rediscussed. A hypothesis on the the end of the (c. 70 Ma), or the (c. biological strategy of darwinulids is tested using data on 120 Ma) (SOHN, 1988), but as quotations of some morphological variability and taxonomic diversity. Mesozoic males are doubtful, possibly since the Key-words: reproduction, diversity, evolution, . (250 Ma) (SCHOEN eta!., 1996). It should be noted that 'asexuality' has always equalled to 'absence of males', both Resume in extant populations and in fossil assemblages. A European research network, consisting of six labora­ L'espece Darwinula stevensoni est redecrite en detail. La tories in five countries, has investigated the evolutionary variabilite morphologique, aussi bien des valves que des par­ ecology of reproductive modes of the three extant lineages ties molles, est evaluee dans plusieurs populations geographi­ of non-marine with a variety of techniques: quement et climatiquement eloignees les unes des autres, et s'est morphological and morphometrical analyses, clonal averee negligeable ou inexistante. Seule Ia taille varie de far;on autecology, starch-gel electrophoresis of allozymes and significative d'une population a !'autre, ce qui peut etre attribue amplification and automated sequencing of both a Ia difference de temperature de !'environment au cours du mitochondrial and nuclear DNA. Three species, one of developpement larvaire. Le nombre et Ia forme des attaches musculaires sont egalement variables, aussi bien, cependant, each lineage, were investigated and Darwinula stevensoni entre differentes populations qu'a l'interieur d'une meme popula­ (BRADY & ROBERTSON, 1870) was the obvious represen­ tion. La forme des valves et Ia chetotaxie des pattes sont remar­ tative of the Darwinuloidea. The present paper forms part quablement stables. Une femelle d'une population italienne of this concerted research effort and reports on mor­ presente d'aberrants palpes Mx2, mais c'est un cas teratologique. phological variability of both valves and soft parts in Les mentions precedentes de males D. stevensoni et Ia position several geographically isolated populations from Europe taxonomique des Darwinulocopina au sein des Podocopina sont and Africa; the clonal and genetic variability of the same reconsiderees. Une hypothese sur Ia strategie biologique des populations will be reported on elsewhere. Darwinula darwinulides est verifiee a I'aide de donnees sur Ia variabilite stevensoni is extensively redescribed. Earlier redescriptions morphologique et Ia diversite taxonomique. Mots-clefs: reproduction, diversite, evolution, taxonomie. of this species (PINTO & SANGUINETTI, 1958, SOHN, 1987) give an incomplete picture of the chaetotaxy and were thus unsatisfactory for our purposes, which included detailed morphological comparisons within and between populations. The position of the Darwinulocopina within the is briefly discussed. A hypothesis on the biological strategy of Darwinula is outlined and is tested using the present morphological evidence. 74 G. ROSSETTI & K. MARTENS

Materials and methods Hollandersgatkreek, NW Belgium Coordinates: N 51°15'47" E 03°31 '58 " The morphology of both soft parts and valves of Date: 18.5.1995 specimens of four populations (Finland, Belgium, Italy Coli.: K. Martens & M.E. Montenegro and South Africa) was investigated with both Scanning Habitat: slightly saline lake, sandy bottom, col­ Electron Microscopy (SEM) and light microscopy. lected at c l-1.5m deep, along edge of littoral Typha-stand. Illustrations of valves and limbs of representative pH= 8.8 specimens are here reproduced. Specimens from the EC = 3.44 mS/cm (S= 2.0) following localities were investigated (Fig. I): water temperature = 12.8 oc

Canale Corniano, Italy Lake Paajarvi, Lammi Biological Station, Finland. Coordinates: N 45°1 0'06" E 10°44'44" Coordinates: N 6! 0 03 '02" E 25°02'32" Date : 15.4.1996 Date: 14.11.1995 Coli.: G. Gentile Coli. : K. Martens & K. Salonen Habitat: Material collected in littoral at 1m depth in the Habitat: medium-sized lake, material collected in littoral final stretch of a polluted canal (Canale Corniano) , flow­ at 1-2m deep, on soft mud. No ice at time of collecting. ing into to Lake Superiore of Mantova, a large wetland Station: near field station, stations 'c' and 'e' of RANTA situated along the low course of the Mincio River (a (1979) tributary of the Po River). pH= 7.0 pH= 7.5 EC = 85.8 f.J.S /cm EC = 490 f.l.S/cm water temperature = 4.0 °C. water temperature = 14.3 °C

Fig. I.- Location of sampling sites (1: Pii ; 2: Ho ; 3: Os ; 4 : Mo; 5: IM ; 6: CC; 7: Si). I I Morphology of D. stevensoni 75

Lake Sibaya, KwaZulu-Natal, R . South Africa recorded. Size differences among populations were Coordinates: S 27°25'13" E 32°41 '53" analyzed by one-way ANOVA test (SOKAL & ROHLF, Coli.: K. Martens, M. Hamer & M. Coke. 1981 ). Date: 16.10.94 Habitat: oligotrophic lake, situated very near to the ocean, Nomenclature proposed by D ANIELOPOL (1968, 1970) is with sediments generally consisting of clean white sand. used in the description of the chaetotaxy of Antennula Age is presumed Holocene (max 0.0 I Ma). Station: and Antenna and the morphology of the mandibular southernmost arm of lake, at pumping station, opposite masticatory process (Fig. 2). water level gauge jetty. At edge of emergent macrophyte (Typha, Phragmites) stands, amongst submerged macrophytes on very muddy and organically rich sedi­ Abbreviations used in text and figures ment. Max depth of collecting: c. 1 m. pH= 8.6 Soft parts EC = 684 11Sicm A1 antennula water temperature = 22.0 oc A2 antenna Ill incisor on Md coxa Specimens of three other Austrian and Italian populations LaMo lamina molaris are also illustrated (Fig. I) : Md mandibula Mx1 maxilla - Ossiacher See, Austria (N 46°40' E 13°57') (Lab Mx2 maxillula culture). Pg guidance seta ('poi] de guidance') - Lake Montorfano, Italy (N 45°46'57" E 09°08'15"). on Md coxa Sampling date: 25.4.1996. Coli.: V. Rossi & G. Gentile. PI 'poi] laciniforme' on Md coxa - Lake Inferiore of Mantova, Italy (N 45°09'08" RMo molar region on Md coxa E 10°48'21 "). T(l-2) thoracic limbs Sampling date: 11.4.1996. Coli.: G. Rossetti & G. Gentile. Abd abdomen

Line drawings of limbs are all made with camera Iucida. Valves Valves are prepared for SEM by washing iil absolute Cp carapace EtOH and simply drying in air. Error on SEM magnifica­ ms muscle scars tions is ±5%. In Darwinula stevensoni often all specimens RV right valve from a preserved recent sample have tightly closed valves, LV left valve which makes dissection without damage to at least one dv dorsal view of the valves very difficult. We developed the following vv ventral view simple technique: glue a piece of double-sided sticky tape llv left lateral view in a small Petri dish and attach the carapace to it. Use IV internal view a very fine brush (no. 000) bearing a small piece of the same tape to push against the edge of the overlapping Sampling sites valve (the R V in D. stevenson f); after a while the valves Pa Lake Paajarvi (Finland) will open slightly. Then open carapace completely with Ho Hollandersgatkreek (Belgium) either brush or fine needle. Add 100% EtOH to the Os Ossiacher See (Austria) mounted carapace in the Petri dish. The valves will come Mo Lake Montorfano (Italy) loose from the tape. Soft parts are then dry and presumed IM Lake Inferiore Mantova (Italy) lost, but can, when needed, be re-soaked again overnight cc Canale Corniano (Italy) in a phosphate rich detergent. Si Lake Sibaya (South Africa)

Limbs are prepared for SEM photography in much the same way, namely by submerging and dissecting the body Figured specimens (all adult females) in absolute EtOH and then drying the limbs in air. For limbs, no glue or sticky tape is used, they are directly stuck OC. l817-l 824: Pa; OC.l825- l837: Ho; OC.l838: Os; on a cover slip glued to the stub, and remain attached OC.l839-l845: Mo; OC.l846-l849: IM; OC.l850-1857 : when dried. This has several advantages: small setae and CC; OC.l858-1863 : Si. All specimens stored in the claws cannot sink into the glue and the background of Collection of the Royal Belgian Institute of the micrograph is more uniformous. Natural Sciences, Brussels.

Right and left valves of ten specimens from Lake P;Hi­ jarvi, Hollandersgatkreek and Canale Corniano popula­ tions were measured to the nearest 4 Jlm under a light microscope. Length and greatest height of each valve were II 76 G. ROSSETTI & K. MARTENS

c

Fig. 2. - Terminology used in description of darwinulid appendages (from DANIELOPOL I 968, 1970). A. AI, endopodite. B. A2 (Ac: aesthetasc clump). C. Md, masticatory process.

Taxonomic description Darwinula stevensoni (BRADY & ROBERTSON, 1870)

Class Ostracoda LATREILLE, 1806 Argilloecia aurea BRADY & ROBERTSON, 1870 (partim) Subclass Podocopa G.W. MOLLER, 1894 Polyche/es stevensoni BRADY & ROBERTSON , 1870 Order Podocopida SARS, 1866 Darwinel/a stevensoni (BRADY & ROBERTSON, 1870) Suborder Podocopina SARS, 1866 BRADY & ROBERTSON, 1872 Infraorder Darwinulocopina SOHN, 1988 Darwinula stevensoni (BRADY & ROBERTSON, 1870) BRADY & ROBERTSON, 1885 Superfamily Darwinuloidea BRADY & NORMAN, 1889 Darwinula improvisa TURNER , 1895 Family Darwinulidae BRADY & NORMAN, 1889 Darwinula aurea (BRADY & ROBERTSON, 1870) G.W. MOLLER, 1912 (partim) Genus Darwinula (BRADY & ROBERTSON in Darwinula variabilis T AGLIASACCHI , 1968 nov. syn. JONES, 1885) Darwinula protracta ROME, 1953 nov. syn. Po/yche/es BRADY & ROBERTSON, 1870 Darwine/la BRADY & ROBERTSON , 1872 '' Morphology of D. stevensoni 77

REDESCRIPTION "incisor", a "molar region" and an internal "lamina molaris". Incisor consisting of an acuminate tooth, par­ Carapace (Figs. 9A-R, lOA-M, llA-H) cigar-shaped. tially fused with a very thin external spine, the former hir­ Valves thin with smooth surface and widely spaced setae, sute and with an additional small spine ("poi! pearly white, with dark spots above the adductor attach­ laciniforme") at the internal edge; molar region formed ment transparent through the valves, more evident in fresh by "lamellar teeth" of different sizes, the outermost larger, specimens than in preserved ones. Valves unequal, RV with bifide apex rather flattened in lateral view ; lamina overlapping LV on all sides except at the hinge. Seen ven­ molaris provided with a basal plate carrying a series of trally, shell ovoid, posterior part rounded, forming a subequal, closely spaced slender setae and, at the inner brood chamber, anterior extremity rather pointed; margin edge, with a thin, curved " guidance seta" (poi! de of the R V sinuous anteriorly and quite straight at the guidance); one subapical seta inserted along the external posterior three fifths; greatest width at about 2/3 of the margin; proximal part of coxa curved and· pointed (not length. In lateral view, shell elongate, greatest height in figured). Basal joint of palp bearing 8 long feathery fan­ the posterior quarter; posterior margin more broadly arranged apical setae and a group of 5-6 subapical setae rounded than the anterior one, the latter more pointed of different size, some with a widened base (not figured). and slightly curved towards the ventral side; ventral Exopodial branchial plate small and delicate, consisting margin almost straight, dorsal margin gently curved. of 8 hirsute rays (not figured). First podomere of Internal surface of valves from smooth to weakly endopodite apically widened, with four terminal setae, sculptured; central ms consisting of9-12 spots arranged three short and one longer, the latter reaching beyond the in a circular rosette. Calcified inner lamellae absent. next segment; second podomere narrow, with two subter­ Hingement adont, with one groove on both valves. minal and two terminal setae, the longer one wider in the Pigmented eye fused, usually well marked, in frontal first half, and four apically hirsute, subequal claws. position. Mxl (Figs. 3E,F, 4E,F, 5E,F, 5E,F, 8K). Masticatory part Al (Figs. 3A, 4A, SA, 6A, 8A,B). Uniramous, short and consisting of three endites with indeterminate number of stout. Protopodite consisting of two segments, the first apical setae (not figured). Palp two-segmented; first seg­ one rather enlarged with two short setae in dorsal posi­ ment rather broad, with two sub-terminal setae, one on tion, the second one bearing a short dorsal seta. Exopodite the external edge and one close to the second endite, and rudimentary, with two short setae and one long seta four distal setae, two curved and plumose, two straight pointing backwards. Endopodite four-segmented; first and hirsute, three of which grouped at the external cor­ podomere with one ventral and one dorso-apical seta, the ner; second segment small, with one thin median seta and latter longer; second podomere with two dorso-apical two larger unequal setae, provided with a double row of bristles, the one nearly twice as long as the other, and one setulae. Branchial plate large, with four long reflexed setae shorter ventro-apical bristle; third podomere with four at the proximal, external margin, followed by a small con­ apical bristles, two ventral and two dorsal, the latter ical seta and then by 23-26 hirsute rays; inner, proximal longer; fourth podomere with two apical bristles and an margin of the respiratory plate hirsute, except along the aesthetasc at the ventral side ; last three segments of insertion of the masticatory process. endopodite with ventro-distal "alpha" setae.

A2 (Figs. 3B, 4B, 5B, 6B, 8C-H). Protopodite two­ Mx2 (Figs. 3G, 4G, 5G, 6G). Protopodite with many segmented, proximal segment with two dorsal setae and distal setae (not figured); two isolated long, unequal setae a sturdy ventral hook, distal segment with one long seta. close to the articulation with the endopodite. Epipodal One-segmented exopodite with three setae, two long apical branchial plate bearing 11 respiratory rays, and set with ones and one lateral conical one, the latter reduced. thin setulae at the free margin. Three-jointed endopodite; Endopodite three-jointed; first segment with a group of first and second segment both with two lateral setae along 4-5 aesthetascs in ventral, proximal position, this segment the anterior edge ; third segment shorter with two une­ with an expanded distal corner corresponding to the inser­ qual subapical setae and a strong terminal claw, the tion of two long setae; dorso-apical margin ending as a latter about three times the length of the last segment. spine; second segment with four distal claws of different lengths, one of which rather small (X), one seta (t) and T1 (Figs. 3H, 4H, 5H, 6H, 8L). Protopodite one­ two aesthetascs (Yl and Y2) on the ventral margin and segmented, with three ventral setae, one short proximal one short subapical seta (Z); third endopodite segment and two long distal ones. Endopodite four-jointed ; first inserted on the second segment in subapical position, with segment long, with two unequal ventro-apical setae and two distal claws and one ventral aesthetasc (Y3). one long seta ("poi! darwinulo!de" - DANIELOPOL 1968) more inward, slightly longer than the next segment; Md (Figs. 3C,D, 4C,D, 5C,D, 6C,D, 8I,J). Consisting of second and third podomere with one apical seta each ; last sclerotized coxa and three-segmented palp, the latter cor­ segment short, with one stout apical claw, flanked by two responding to basis and two-segmented endopodite. subapical claws, the ventral one longer than the dorsal Masticatory process divided into three part: an external one and slightly less than half the length of the distal claw. 78 G. ROSSETTI & K. MARTENS

Fig. 3. - Darwinula stevensoni BRADY & ROBERTSON. Lake Piiiijiirvi, Finland. A. AI (OC.l824). B. A2 (OC.I824). C. Md, endopodite (OC.I825). D. Md, masticatory process (OC.I824). E. Mxl, branchial plate (OC.I824). F. Mxl, palp (OC.l817). G. Mx2 (OC.l824). H. TI (OC.l824). I. T2 (OC.l824). J. Abd (OC.I822). Note that the aberrant position of the A2 exopodite in Fig. 3B is due to a twist during slide preparation. Scale = 49 pm for A-1. Morphology of D. stevensoni 79

Fig. 4. - Darwinula stevensoni BRADY & ROBERTSON. Hollandersgatkreek, Belgium. A. AI (OC. I830). B. A2 (OC.I834). C. Md, endopodite (OC.l829). D. Md, masticatory process (OC.i834). E. Mx l, branchial plate (OC.l832). F. Mx l, palp (OC.i83!). G. Mx2 (OC. I830). H. Tl (OC.l830). I. T2 (OC.I830). J. Abd (OC.l829). Scale = 49 .urn for A-J. II 80 G. ROSSETTI & K. MARTENS

I/

Fig. 5. - Darwinula stevensoni BRADY & ROBERTSON. Canale Corniano, Italy. A. A I (OC. l855). B. A2 (OC.l855). C. Md, endopodite (OC.l856). D. Md, masticatory process (OC.l856). E. Mxl, branchial plate (OC. !857). F. Mx l, palp (OC.l857). G. Mx2 (OC.l856). H. T1 (OC.l853). I. T2 (OC.l854). J. Abd (OC.l856). Scale = 49 pm for A-J. Morphology of D. stevensoni 81

Fig. 6. - Darwinula stevensoni BRADY & ROBERTSON. Lake Sibaya, South Africa. A. Al (OC.l858). B. A2 (OC.l858). C. Md, endopodite (OC.l859). D. Md, masticatory process (OC.l859). E. Mxl, branchial plate (OC.l859). F. Mxl, palp (OC.l859). G. Mx2 (OC.l858). H. T1 (OC.l858). I. T2 (OC.l858). J. Abd (OC.l859). Scale = 49 pm for A-J. '' 82 G. ROSSETTI & K. MARTENS

T2 (Figs. 31, 41, 51, 61). Protopodite one-segmented, with • the clump of aesthetascs on the second podomere of the two ventro-apical setae (not figured). Endopodite four­ A2 are figured in Plate 2, Figs. 7, II , but were not men­ segmented; first three segments bearing one distal seta tioned in the description; each; last segment with a long, curved apical claw, a • the chaetotaxy of the second podomere of the A2 was subapical ventral claw about half the length of the cen­ missing; tral claw and a shorter dorso-apical seta. • the third podomere of endopodite of the A2 has 2 distal claws and one aesthetasc, not "three long and one short Furca absent in adult specimens. claws"; • the basal podomere of the endopodite of the Md does Abdomen (Figs. 3J, 4J, 51, 61, 8M) ending as a conical, not have "one long and one shorter seta", but four apical vermiform process (postabdomen). setae; • the Mx I palp was described as follows: " the first joint Male unknown. of the palp has three long bristles and the end joint of the palp has two". Actually, the first segment bears 6 setae and the second segment 3 setae. MEASUREMENTS • we prefer to follow the nomenclature of DANIELOPOL ( 1968) and consider a four-jointed endopodite, not See Tables I and 2. exopodite, for Tl and T2. • certain other details are different (for example number of branchial rays), but these are difficult to interpret. They might follow either from difficulties during observation Table 1 or might constitute " normal" variability (see below). Results of one-way ANOVA's on length (L, in ,urn) and height (H, in ,urn) of R V and LV in Darwinula stevensoni. N = 30 for each test (I 0 per population); Pa = mean value for specimens from Pa; Ho =mean value for specimens MORPHOLOGICAL VARIABILITY OF D. stevensoni from Ho, CC= mean value for specimens from CC The most marked differences between the populations p Valve L/H Pa Ho cc F(2,27) Sign. presently investigated are in carapace size (Tabs. 1,2; Figs. RV L 734 752 706 24.987 0.000 *** 9, 10); one-way ANOVA's performed on length and RV H 303 312 298 9.137 0.001 *** height measurements of 3 populations (Pa, Ho, CC) are all highly significant. Differences in food and salinity, but LV L 718 730 691 15.950 0.000 *** especially in temperature, during the larval development LV L 290 288 278 7.867 0.002 *** can cause significant differences in adult size (MARTENS eta!., 1985) and we thus attribute size differences to these epigenetically active environmental factors. There are also Table 2 occasional small differences in carapace shape (for exam­ Measurements of valves (length = L, in ,urn and ple slightly different LIH ratio, curvature of postero­ height = H, in ,urn) of D. stevensoni, pooled values of ventral corner etc), but, although difficult to quantify, three populations (Pa, Ho, CC); N = 30 these appear to be similar within and between popula­ tions and in any case are neglectable. Valve L/H mm1mum maximum mean± S.D. Although conserving a general pattern in the shape of the · muscle scars (ms) in Darwinula stevensoni, the number of RV L 686 771 730 ± 24 spots varies amongst individuals of the same population, RV H 288 322 304 ± 10 is not related to development stage and may even be dif­ ferent in the two valves of the same specimen. Therefore, LV L 669 746 713 ± 23 ms seem to have low diagnostic importance at the LV H 263 305 286 ± 9 taxonomic levels lower than superfamily (PINTO & KOTZIAN , 1961 ; SOHN , 1976, 1987), although WAKEFIELD (1994, in press) used ms patterns for the identification of species and growth stages in fossil Dar­ REMARKS winula. In the populations of D. stevensoni analyzed in the present study, number ofms varies between 9 and 12, The above redescription amends and corrects the one by and this is in accordance with the range commonly SOHN (1987) in the following details : reported for this species (Table 3) ; in most specimens the • "alpha" setae on third and fourth podomeres of the A I number of ms in the R V is larger than in the LV (Fig. II). endopodite and aesthetasc on the fourth segment of AI Differences amongst populations were further observed endopodite were not mentioned. in size of appendages, although morphometric analyses • there is a third setae on the A2 exopodite ; were not carried out. In any case, no evident variations I I Morphology of D. stevensoni 83

. ' r fi r ~ d3 d2 I!l' A ~ B Fig. 7. - Darwinula stevensoni BRADY & ROBERTSON. Lake Montorfano, Italy. A,B. Mx2 (OC.l841): aberrant form. C. Mx2 (OC. l842) : normal form. Scale = 49 Jim for A-C.

were noticed in size ratios between podomeres or in length the limit of observation with light microscopy, and are ratios in the chaetotaxy. thus not discussed here. Number of aesthetascs in the clump on the second Fifteen gravid females of D. stevensoni from Lake Mon­ podomere of the A2 varied slightly: the most common torfano were dissected. One of these presented anomalous pattern is a group of 4 aesthetascs, but 5 were occasionally Mx2, with asymmetrical limbs (Figs. 7 A, B), both with observed (Figs. 8F, G). only one lateral seta on the second endopodial segment Number of setae along the distal margin of the Mx2 bran­ (tl and t2, respectively) and with d2 on the third chial plate varies between 24 and 27 and a similar varia­ endopodial segment claw-like and of the same length of tion (20-26) was also reported by SOHN (1987). As these d3; d 1 was furthermore absent in one of the limbs structures are small and weakly sclerified, it is difficult (Fig. 7B, arrow). As all other appendages, as well as to ascertain if this represents a bias in observed variation carapace morphology (Fig. 1OM), were typical of or if it reflects genuine variability. No differences were D. stevensoni, this specimen is here considered an isolated found in the number of respiratory rays of Md and Mx2 teratology rather than a stable (clonal) morphology. A branchial plates, which showed a constant number of further argument for this is the observed asymmetry in 8 and 11, respectively. its Mx2, a feature not common in females, which indicates Small details (such as "sclerotization", presence ofsetulae, that this female had a deficient development. barbulate setae, spines, hirsute parts, etc.) are mostly at

Table 3 Carapace length (L, in mm) and number of muscle scars (N) in Darwinula stevensoni (n.r. : not reported)

L N BRADY & NORMAN (1889) 0.80 10-12 TURNER (1895) (D. improvisa) 0.68-0.70 n.r. SARS (1928) 0.72 9 BRONSHTEIN (1947) 0.75 9 ROME (1953) (D. protracta) 0.78 11 PINTO & SANGUINETTI (1958) 0.75 9-10 TAGLIASACCHI-MASALA (1968) (D. variabilis) 0.65 7-11 SOHN (1976) 0.58-0.70 9-12 RANT A (1979) 0.65-0.68 n.r. '' 84 G. ROSSETII & K. MARTENS

Fig. 8. - Darwinula stevensoni BRADY & ROBERTSON. A,D,E,F ,H,J-M : Ho; B,G: Si ; C,I: Pii. A. AI (OC.I836). B. AI , detail poils «alpha» (preparation not kept). C. A2, detail aesthetascs Yl-3 (preparation not kept). D. A2, detail exopodite (preparation not kept). E. A2, detail aesthetasc (OC.I835). F. A2, aeshetasc clump (prepara­ tion not kept). G. A2, aesthetasc clump (preparation not kept). H . A2, detail hook (OC.I836). I. Md, distal segment ofpalp (preparation not kept). J. Md, masticatory process (preparation not kept). K. M x I, palp (OC.I837). L. Tl, detail (OC.I837). M. Abd (OC.I837). Scale = 114 ,urn for A ; 26 ,urn for B and L; 23 ,urn for C ; 12 ,urn forD ; 9 ,urn for E,F; II ,urn for G ; 21 ,urn for H and M; 24 ,urn for I; 16 ,urn for J; 42 ,urn forK. I I

Morphology of D. stevensoni 85

Fig. 9.- Darwinula stevensoni BRADY & ROBERTSON. A-C : Pii; D-F: Ho; G-I : Mo ; J-L:IM; M-0 : CC; Q-R : Si . A. Cp, dv (OC.l820). B. Cp, vv (OC.l819). C. Cp, llv (OC.l821). D. Cp, dv (OC.l825). E. Cp, vv (OC.l826). F. Cp, llv (OC.l827). G . Cp, dv (OC.l845). H. Cp, vv (OC.l844). I. Cp, llv (OC.i843). J. Cp, dv (OC.l846). K. Cp, vv (OC.l847). L. Cp, llv (OC.l848). M. Cp, dv (OC.l851). N . Cp, vv (OC.i850). 0 . Cp, llv (OC.l852). P . Cp, dv (OC.l863). Q. Cp, vv (OC.l861). R. Cp, llv (OC.l862). Scale= 417 ,urn for A-R. II 86 G. ROSSETII & K. MARTENS

Fig. 10. - Darwinula stevensoni BRADY & ROBERTSON. A,B: Pii ; C,D : Ho ; E,F,M: Mo ; G,H: IM ; l,J : CC; K,L : Si. A. LV , iv (OC. I818). B. RV, iv (OC.l818). C. LV, iv (OC.l828). D. RV, iv (OC.I828). E. LV, iv (OC.l840). F. RV, iv (OC.I840). G. LV, iv (OC.l849). H. RV, iv (OC.I849). I. LV, iv (OC.I853). J. RV, iv (OC.I853). K. LV, iv (OC.l860). L. RV, iv (OC.I860). M. LV, iv (OC.l841). Scale = 417 ,urn for A-M. I I Morphology of D. stevensoni 87

Fig. II. - Darwinula stevensoni BRADY & ROBERTSON. All specimens adult females. A,B: Ho ; C,D: Os ; E,F: Mo ; G,H : CC. A. RV, ms (OC.l833). B. LV, ms (OC.l833). C. RV, ms (OC.l838). D. LV, ms (OC.l838). E. RV, ms (OC.l839). F. LV, ms (OC.l839). G. RV, ms (OC.l853). H. LV, ms (OC.l853). Scale = 73 11m for A-H.

Discussion ventro-distal seta in the first endopodial segment of T2 as typical of D. variabilis; however both in the descrip­ NEW SYNONYMIES tion and in the figures (Plate III, fig. L, p. 626) the presence of this seta is ascertained. (4) The Darwinula variabilis is very similar to D. stevensoni in its shape of the abdominal process (postabdomen) in D. all round morphology. TAGLIASACCHI (1968, variabilis (Plate III, Fig. M, p. 626) does not differ from p. 627) listed the following features of Darwinula variabilis, that of D. stevensoni. a species collected from hyperthermal waters of Sardinia, Unfortunately, the type material of D. variabilis, stored as being different from D. stevensoni: number and posi­ at the University of Cagliari, was completely lost in a fire tion of muscle scars ; setae on the basal segment of the (T AGLIASACCHI, pers. com. 1996), so that the types can­ mandibular palp as well as setae of the branchial plates not be reinvestigated. Moreover, recent sampling in the of Md, Mx 1 and Mx2 not plumose ; absence of ventral type locality did not confirm the occurrence of Darwinula seta on the first endopodial segment of T2 ; shape of sp. (DE MIRANDA RESTIVO eta!., 1995), which makes the abdominal process. investigation of topotype material inpossible. We propose to sink D. variabilis into the synonymy of ROME (1953) described Darwinula protracta from a cave D. stevensoni, because of the following reasons: (1) as is on Mont Hoyo, lrumu, Zaire, and indicated greater size reported below, the number of muscle scars in Darwinula (length = 0. 78 mm, greatest height = 0.31 mm), greater is not a valid taxonomic criterium, and moreover, the number of muscle scars (11), different chaetotaxy of A2 muscle scars figured for D. variabilis (T AGLIASACCHI , lac. (summarily reported but not figured) and of the man­ cit., Plate IV, p. 628) fall into the range of variability dibular palp as major differences with D. stevensoni. observed for D. stevensoni. (2) In many cases, detection However, size and the number of muscle scars fit within of feathery setae in Darwinula can be difficult with a light the normal ranges of variability reported for D. steven­ microscope and again, this can therefore not be regarded soni (see below). The incomplete description of the A2 as a valid specific character. (3) In the discussion, in D. protracta makes a careful comparison of the T AGLI ASACCHI (1968 : 627) cited the absence of one chaetotaxy of this limb in both species impossible. Con- 88 G. ROSSETII & K. MARTENS I I

tatrary to ROME's claim, the two distal segments of the aesthetascs Ac1-4 is smooth, in aesthetascs Y1-3 this sur­ mandibular palp of D. protracta (Plate II, Fig. 20, p. 40) face is pitted, as is also the case in the single aesthetascs are almost identical to those of D. stevensoni. ROME (lac. Y of most Cypridoidea (DANlELOPOL, 1973, 1978). Com­ cit.) also doubted the presence of an eye in D. protracta, parative investigations of these morphologies in the five but he dissected only one specimen and even the lineages of the Podocopina are required to assess the interstitial D. boteai DANlELOPOL, 1970 has a pigmented homology of these structures, and hence of its validity as eye. Because of the apparent absence of structural dif­ a taxonomic feature. Meanwhile, the presence of this ferences between both nominal species, we propose to con­ clump of 4-5 aesthetascs in Danvinula stevensoni confirms sider D. protracta a synonym of D. stevensoni. the affinity of the Darwinulocopina to the Sigillioidea (McKENZIE, 1982), but does not allow both taxa to be grouped into a separate lineage. POSITION OF THE INFRAORDER DARWINULOCOPINA WITHIN THE SUBORDER PODOCOPINA PAST AND PRESENT DIVERSITY The Darwinulocopina were described as a suborder by SOHN (1988), but lowered to the rank of infraorder by The history of scientific discoveries in the Dar­ MARTENS (1992), one of five within the suborder winulocopina, and more specifically in the Darwinulidae, Podocopina. As is usually the case at such a high has for a long time been rather confused, but recently taxonomical level, it is very difficult to assess affinities some order has been restored by SOHN (1987, 1988). The between these lineages and few characters are available. early nomenclatorial confusion around the type species For example, a spermpump outside of the hemipenis of the genus Darwinula (and thus of the entire group) is (Zenker organ) is present in Cypridocopina and in the briefly summarized above, but was extensively discussed Sigillioidea (this group still requires the formal erection by SOHN (1987: 150-152), who has also designated a of an infraorder- MARTENS, 1992), while the spermpump lectotype from the collections in the Hancock Museum is incorporated within the hemipenis in Cytherocopina (Newcastle-upon-Tyne), thus making Somerton Broad and in Bairdiocopina. Because no males of Darwinulo­ (Norfolk, England) the type locality. The type series on copina have ever been conveniently described, the posi­ which the original description was based, indeed consisted tion of this lineage remains unclear. of material from several localities, so that an unequivocal The presence of a clump of aesthetascs on the first designation was needed. endopodial segment of the A2 in Danvinula stevensoni Within the Darwinulocopina, only one superfamily, the (Figs. 2, 3B, 4B, 5B, 6B, 8F,G) might be useful in this Darwinuloidea BRADY & NORMAN, 1889, is recognized. regard. These structures are considered homologous with SOHN ( 1987) listed 6 families in this group, but later the group of six to eight aesthetascs in Saipanetta (1988) synonymized Microdarwinulidae KASHEVAROVA (Sigillioidea) and with the group of six in Macrocypris & NEUSTRUEVA, 1982 with the Darwinulidae BRADY & (Macrocypridoidea, Cypridocopina), but also with the NORMAN, 1889; he then also explicitely cited Sucho­ single aesthetasc Y in Pontocyprididae and Cypridoidea nellininae KUKHTINOV, 1985 (written with subfamilial (both Cypridocopina) (MADDOCKS, 1976, 1990). There name ending) as a synonym of Darwinulidae. Of the five are up to three setae-like structures (DANlELOPOL, 1972), families thus left, one is unnamed (containing the marine possibly aesthetascs, in that position in a number of Middle genus Predarwinula JIANG, 1978), one Bairdiocopina; there are no such aesthetascs in the did not survive the Permian- extinction (Dar­ Cytherocopina. winuloididae MOLOSTOVSKAJA, 1980) and two were These observations provoke two remarks. First, if all of greatly reduced by the same extinction and fully disap­ these structures are indeed homologous, then a convergent peared at the end of the Triassic (Suchonellidae reduction in number of aesthetascs has occurred in at least MISCHINA, 1972 and Panxianidae WANG, 1980). The 3 of the 5 lineages, namely in Bairdiocopina and Cypri­ Darwinulidae are thus the only survivors of the lineage docopina (partly) and in Cytherocopina (whole lineage?). since the Jurassic. SOHN (1988) recognized three genera As taxa with several aesthetascs in that position are pre­ in this group: Danvinula s.s. and Microdarwinula sent at least in Bairdiocopina and Cypridocopina, these DANIELOPOL, 1968 with Recent representatives and the observations can thus not be used to lodge Sigillioidea late Triassic Paradarwinula KOZUR, 1970. Two fossil and Darwinulocopina in a separate group and the genera, Neudarwinula MISCHINA, 1972 (Late Permian) 5-partite division of the Podocopina remains unchallenged and Dictyodarwinula JIANG, 1983 (upper Triassic) are sur­ by these observations. pisingly maintained as subgenera of Darwinula only. However, it can also be questioned if the clump of We can thus conclude that the largest supra-generic diver­ aesthetascs are indeed fully homologous with the single sity occurred in the Palaeozoic, while the post-Triassic large aesthetasc Y in, for example, Cypridoidea fauna in this lineage is represented by one family and two (DANIELOPOL, 1972). Comparisons between the mor­ genera only. According to SOHN (1987), more than 250 phology of the clump-aesthetascs (Ac1-4) and of the other species, of to Holocene age, have been aesthetascs on the A2 in D. stevensoni (see above) revealed referred to Darwinula s.s., although several have mean­ potentially important differences: the distal part of while been lodged in other genera and families. KEMPF Morphology of D. stevensoni 89

(1980), however, already listed not less than 350 different teratological specimen. When this is compared to dif­ species and subspecies at some stage referred to the genus ferences in shape of valves in for example Eucypris virens Darwinula and another I80 species in the update of his (TETART I982; BALTANAS et al. in prep.) or in differences list (KEMPF, I995). SOHN (foe. cit.) furthermore listed 25 in natatory setae on A2 in species of Herpetocypris (GON­ described and one undescribed Recent species and ZALEZ et al., in prep), the morphological uniformity in subspecies in this genus (D. dicastrii LOFFLER, I966 was D. stevensoni is truly astonishing. not mentioned). Twenty-two described Recent species are How, then, should one interpret the teratologic.al female retained here worldwide, while evidence for at least from Lake Montorfano, which has aberrant Mx2 palps another 7 extant taxa exists. There is only one extant only, and no special morphology in any other of the soft species in the genus Microdarwinula. It is thus clear that parts or valves? We here deem these aberrations to be epi­ the relatively extensive fossil record, more than 500 genetic, rather than genetically induced, for example a as (sub-) species, most of which are furthermore pre-Jurassic, a consequence of problems experienced during one of the is in sharp contrast with the paucity of post-Jurassic and 9 moults in this particular specimen. This hypothesis can extant specific diversity. It is true that Recent species of be falsified by checking long series of specimens from this Darwinula occur in a variety of habitats (high mountain population. lakes, rivers, bogs, springs, interstitial aquifers, etc.) (SOHN, 1987, DANIELOPOL, I980), but only one species, Darwinula stevensoni can truly be termed successful, as THE ELUSIVE MALES it has a global distribution, occurs in a variety of habitats and is seldom rare. Other species are rare to very rare and BUTLIN & GRIFFITHS (1993) and JUDSON & NORMARK generally occur in marginal habitats in low densities. (I996) consider Darwinula as an ancient asexual, i.e. as SOHN's (I987) statement that the 'darwinulid group' is one of the few lineages which has persisted without sexual highly successful, thus largely refers to pre-Jurassic situa­ reproduction for a considerable time, in spite of assumed tions and is disputable with regard to extant diversity. theoretical impossibilities (see introduction). According Looking at the diversity of non-marine ostracods as a to BUTLIN & GRIFFITH (foe. cit.), the Darwinuloidea whole, the Darwinulocopina, being one of three Recent have been asexual for at least 70 Ma and this is a conser­ non-marine infraorders, comprise only I% of the extant vative estimate, as only few putative males have been cited specific diversity in Europe (LOFFLER & DANIELOPOL, since the Permian (c. 250 Ma) (SCHOEN et al. I996). From I978) and about 2% in both Africa and South America recent fauna's, putative males of D. stevensoni have been (MARTENS, in press). In spite of the fact that the Dar­ reported twice. In their original description of winulocopina have managed to persist since the Car­ D. stevensoni, BRADY & ROBERTSON (I870) (see also boniferous (c. 360 Ma), they constitute to date only a BRADY & NORMAN, 1889) reported and even illustrated marginal fraction of the extant non-marine ostracod hemipenes and carapace of a male of this species. fauna. It is important to note, however, that this decrease TURNER (I895) reported a male of D. improvisa (a in diversity of the Darwinulidae is not only relative, and synonym of D. stevensoni) from North America, but only due to the explosive radiation of the Cypridocopina in illustrated the AI and T2 of this presumed male. Since non-marine habitats since the lower Jurassic (WHATLEY, then, researchers have intensively looked for males in this 1988, I990a, b), but also represents a genuine (absolute) lineage (for example RANTA, 1979 and MCGREGOR, decrease in specific and supra-specific diversity. I969) but without any success. The record of TURNER (foe. cit.) cannot be validated on the evidence provided. The illustrations of BRADY & MORPHOLOGICAL UNIFORMITY ROBERTSON (1870) and BRADY & NORMAN (1889), however, constitute more solid evidence. The illustration From the results presented above and the foregoing of the carapace in dorsal view indeed shows that it is more discussion, it appears that this lineage not only presents laterally compressed, which could be related to the low supra-specific and specific (extant) diversity, but also absence of a brooding chamber in this specimen. The neglectable morphological variability in at least one of drawing of the hernipenis is difficult to interpret, although its species. The distinction between species in Darwinula MADDOCKS (I973) compared it to the morphology of the relies on combinations of small structural differences only hemipenes in Saipanetta. It seems that at least the male (number and shape of internal teeth in LV, presence and found by BRADY & ROBERTSON (1870) cannot be shape of ventral keel in R V) and that overall morphology disgarded. How, then, should this be interpreted? in the genus is very uniformous. Within one species then, Asexual and sexual reproduction are two extremes, whith Darwinula stevensoni in this case, there are almost no mor­ several possible intermediates. Single gender reproduction phological differences between geographically and can also be automictic, i.e. with internal meiosis, in stead ecologically significantly different populations: the of apomictic (without meiosis). Cyclic reproduction, like latitudinal gradient and the ecological differences in Cladocera, can occur, while both reproductive modes (temperature, salinity) are only reflected in differences in can exist in different populations of the same species, or size, almost not in shape of carapace, while all soft parts can even occur sympatrically (CHAPLIN, I993). In all of are absolutely identical, with the exception of one these cases, instances of sexual reproduction or at least II 90 G. ROSSETII & K. MARTENS of meiosis are incorporated into the general reproductive research program, some of the ideas developed herein strategy. originated from discussions with other team members · A final possibility is the so-called single-male (or rare their contribution is here strongly acknowledged. ' male) occurrence, repeatedly reported for Limnocythere inopinata (GEIGER, unpubl.), where single male individuals occur amongst thousands of parthenogenetic females. Although seemingly exciting at first, the impor­ tance of such males must not be over-estimated. Such References single-male populations cannot be considered bisexual as the functionality of these males is doubtful and thei; BARCLAY, M .H., 1968. Additions to the freshwater ostracod impact on a population and meta-population level is at fauna of New Zealand. New Zealand Journal of Marine and best marginal. More likely, however, such males are non­ Freshwater Research, 2: 67-80. functional relicts or attavisms, only useful from a BRADY, G .S. & NORMAN, A.M ., 1889. A monograph of the morphological-taxonomical point of view, and of little marine and freshwater Ostracoda of the North Atlantic and of importance for the reproductive strategies. Further discus­ North-Western Europe. Section I: Podocopa. Scientific Tran­ sactions of the Royal Dublin Society, series 2, 4 (2) : 63-270. sions on rare-male occurrences in non-marine ostracods will be presented elsewhere, but in conclusion we here con­ BRADY, G .S. & ROBERTSON , D., 1870. The Ostracoda and Foraminifera of Tidal Rivers. Annals and Magazine of Natural sider that the reports on the male(s) of Darwinula steven­ History, ser. 4, 6: 1-33, 307-309. soni, if at all valid, deal with such rare-male occurrences with no conceivable impact on reproductive strategy i~ BRONSHTEIN, Z.S., 1947. Ostracoda Presnych Vod. Fauna this lineage. SSSR, Rakoobraznye, Tom II, Vyp. I. English translation 1988: Fresh-water Ostracoda of the USSR, , Volume II, Number I, Amerind Publishing, New Delhi, 470 pp. BUTLIN, R.K. & GRIFFITHS, H.l. 1993. Ageing without sex? CONCLUSION : EVOLUTIONARY STRATEGY Nature, 364: 680. Taking into account the combination of low post-Jurassic CHAPLIN , J.A., 1993. The local displacement of a sexually reproducing ostracod by a conspecific parthenogen. Heredity, diversity in the Darwinulocopina at all taxonomic levels 71 : 259-268. low inter- and intra-specific morphological variability and the fact that they are ancient asexuals, the Darwinula­ CHAPMAN, A.N., 1963. A review of the Freshwater Ostracods of New Zealand. Hydrobiologia , 22 (1-2): 1-40. paradox is not concerned with reasons behind the low diversity in this group, but with the fact that it managed DADAY, J. , 1902. Mikroskopische Siisswasserthiere aus Patago­ to persist at all for such a long time. The Darwinulocopina nien. Ternu!szetrajzi Fiizetek, 25 : 201-310. are therefore of interest, not so much from an ecological DANIELOPOL, D.L. , 1968. Microdarwinula n.g. et quelques point of view, but for their aberrant evolutionary strategy. remarques sur Ia repartition de Ia famille Darwinulidae Br. et All of the above, combined with the wide ecological Norm. (Crustacea, Ostracoda). Annales de Limnologie, 4 (2): 153-174. tolerance of for example Darwinula stevensoni (GRIFFITH & BUTLIN, 1994), seems to indicate that at least this DANIELOPOL, D.L., 1970. Une nouvelle espece du genre Dar­ species has developed a conservative, multi-purpose winula des eaux souterraines de Roumanie et quelques remar­ ques sur Ia morphologie des Darwinulidae (Ostracoda-Podo­ phenotype. The genetic bases of this hypothesis will be copida). Travaux de I'Institut de Speologie "Emile Racovitza", discussed elsewhere. 9 : 135-149. . DANIELOPOL, D .L., 1972. Supplementary data on the mor­ phology of Neonesidea and remarks on the systematic position Acknowledgements of the family Bairdiidae (Ostracoda: Podocopida). Proceedings of the Biological Society of Washington, 85(2): 39-48. Dr D .L. DANIELOPOL (Mondsee) and Dr K. WOUTERS DANIELOPOL, D .L., 1973. Sur Ia morphologie des aesthetascs (Brussels) read the manuscript and suggested important chez quelques Ostracodes hypoges de Ia sous-famille des Can­ improvements. The authors also thank Dr R. MADDOCKS doninae (Cyprididae, Podocopida). Annates de Speleologie, 28 (Houston) for useful information with regard to (2) : 233-245. A2-aesthetascs. J. CILLIS and C. BEHEN (Brussels, DANl ELOPOL, D .L. , 1978. Ober Herkunft und Morphologie der Belgium) offered technical assistance with the SEM images S~sswasser-hypogiiische Candoninae (Crustacea, Ostracoda). and with the line drawings respectively. The following S1tzungsber. Osterreichisches Akademie fiir Wissenschaften, people kindly assisted with the collection of material : mathematische und naturwissenschaftliche Klasse 187( 1-5) : M . HAMER (Pietermaritzburg, South Africa), J. POKKI 162 pp. (Tvarminne, Finland), J. SARVALA (Turku, Finland), DANIELOPOL, D .L., 1980. An essay to assess the age of the K. SALONEN (Lammi, Finland), V. ROSSI (Parma, Italy), freshwater interstitial ostracods of Europe. Bijdragen tot de G . GENTILE (Parma, Italy). We gratefully acknowledge Dierkunde, 50 (2): 243-291. support from the E.U. Human Capital and Mobility DELACHAUX , T., 1928. Faune invertebree d'eau douce des Program (contract ERBCHRXCT/93/0253). As this study hauts plateaux du Perou. Bulletin de Ia Societe neuchateloise des forms part of a larger, multidisciplinary and interactive sciences naturelles ( nouv. ser.), I : 45-77. ,, Morphology of D. stevensoni 9!

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