HATCH ING MECH A NISMS AND DEATH OF EUPHAUSIID EMBRYOS DURING HATCHING PRO CESS: EVIDENCES FOR EVOLUTIONARY REVERSAL OF THE FREE-LIVING NAUPLIUS?

Transcription

1 CICIMAR Oceá ni des, 21(1,2):63-79 (2006) HATCH ING MECH A NISMS AND DEATH OF EUPHAUSIID EMBRYOS DURING HATCHING PRO CESS: EVIDENCES FOR EVOLUTIONARY REVERSAL OF THE FREE-LIVING NAUPLIUS? Jai me Gó mez-gu tié rrez Departamento de Plancton y Ecología Ma rina, Centro Interdisciplinario de Ciencias Ma ri nas, IPN, A.P. 592, C.P , La Paz, Baja Cal i for nia Sur, México. jagomezg@ipn.mx ABSTRACT. This es say at tempts to sum ma ri ze and hypot he ti cally re cons truct the ori gi nal con di tion for the re pro duc ti ve stra tegy and hat ching me cha nisms of the fa mily Eup hau sii dae (Order Eup hau sia cea). Com pa - ri son is made of the hat ching me cha nisms and hat ching suc cess ra tes among five broad cast-spaw ning (Eup hau sia pa ci fi ca, Eup hau sia exi mia, Eup hau sia dis tin guen da, Thysa noes sa spi ni fe ra, and Thysa noes sa ins pi na ta) and two sac-spaw ning eup hau siid spe cies (Ne ma tos ce lis dif fi ci lis and Nyctip ha nes sim plex) co - llec ted from the Ore gon coast, Bahía Mag da le na (west coast of Baja Ca li for nia pe nin su la), and Gulf of Ca li - for nia. The se along with the dis co very of a no vel sour ce of em bryo mor ta lity du ring hat ching for broad cast-spaw ning spe cies, and re cently pu blis hed ge ne tic and phylo ge ne tic in for ma tion of the eup hau - siids, sup port the hypot he sis that hat ching as a free-li ving nau plius is a re ver sed cha rac ter wit hin the Order Eup hau sia cea in com pa ri son with spe cies be lon ging to ot her or ders in the Class Crus ta cea. The hat ching of em bryos at nau plius sta ge, with dis tinct hat ching me cha nisms, ap pears re pea tedly and in ter mit tently in the Eup hau sii dae fa mily phylo geny both in eup hau siids with broad cast and sac-spaw ning re pro duc ti ve stra tegy. This may re pre sent a con di tion re-emer ging well back in crus ta cean phylo geny, even though it is not ne ces - sa rily pri mi ti ve among the Order Eup hau sia cea as a who le. Key words: Euphausiids, hatch ing mech a nisms, hatch ing suc cess, re pro duc tive estrat egy, phy log eny. Mecanismos de eclosión y muerte de embriones de eufáusidos du rante el proceso de eclosión Son éstas evidencias que expliquen una reversión evolutiva de nauplios de vida li bre? RESUMEN. En este en sa yo se in ten ta re su mir y re cons truir la con di ción hi po té ti ca ori gi nal de las es tra te - gias de re pro duc ción y me ca nis mos de eclo sión de la Fa mi lia Eup hau sii dae (Orden Eup hau sia cea). Se hace la com pa ra ción de los me ca nis mos y ta sas de éxi to de eclo sión de cin co es pe cies de so va do ras ex ter - nas (Eup hau sia pa ci fi ca, Eup hau sia exi mia, Eup hau sia dis tin guen da, Thysa noes sa spi ni fe ra y Thysa noes - sa ins pi na ta) y dos es pe cies de so va do ras en saco oví ge ro (Ne ma tos ce lis dif fi ci lis y Nyctip ha nes sim plex) de la cos ta de Ore gon, Bahía Mag da le na en la cos ta oes te de la pe nín su la de Baja Ca li for nia, y Gol fo de Ca - li for nia. Esto, jun to con el des cu bri mien to de mor ta li dad aso cia da al pro ce so de eclo sión en es pe cies con de so ve ex ter no y la re cien te in for ma ción pu bli ca da so bre ge né ti ca y fi lo ge né ti ca de los eu fáu si dos, apo yan la hi pó te sis de que la eclo sión de nau plios de vida li bre es una ca rac te rís ti ca re ver ti da den tro del Orden Eup - hau sia cea, en com pa ra ción con es pe cies de otros ór de nes den tro de la cla se Crus tá cea. La eclo sión de los em brio nes en el es ta dío nau plio, con dis tin tos me ca nis mos de eclo sión, apa re ce in ter mi ten te men te a lo lar - go de la fi lo ge nia de las es pe cies de la Fa mi lia Eup hau sii dae, tan to en es pe cies de so va do ras ex ter nas como en de so va do ras en saco oví ge ro. Esta eclo sión en es ta dio nau plio apa ren te men te re pre sen ta una con di ción re-emer gen te de an te pa sa dos en la fi lo ge nia de crus tá ceos, y por lo tan to, no es ne ce sa ria men te una con di ción pri mi ti va en tre las es pe cies de la Orden Eup hau sia cea. Palabras clave: Eufáusidos, mecanismos de eclosión, éxito de eclosión, estrategia reproductiva, filogenia. Gó mez-gu tié rrez, J Hatching mechanisms and death of euphausiid embryos during hatching process: evidences for evolutionary reversal of the free-living nauplius?. CICIMAR Oceánides, 21(1,2): Fe cha de re cep ción: 10 de fe bre ro, 2006 Fe cha de acep ta ción: 13 de agos to, 2007

2 64 GÓ MEZ-GU TIÉ RREZ INTRODUCTION The crus ta cean nau plius lar va, cha rac te - ri zed by a re mar kably con ser ved morp ho logy, has be co me the fo cus of stu dies on li fe-his tory evo lu tion that in ves ti ga te the in ter play of ani - mal struc tu re, func tion, eco logy and evo lu tio - nary his tory (Wi lliams, 1994; Dahms, 2000). Se ve ral stu dies claim that the ort ho nau plius is a pri mi ti ve (ple sio morp hic) cha rac ter wit hin the Subphylum Crus ta cea (Lau ter bach, 1986; Dahms, 2000). Thus, any lar val de ve lop ment wit hout a free-li ving nau plius has to be a de ri - ved (apo morp hic) con di tion. In the Class Ma la - cos tra ca, only in the Su bor der Den dro bran - chia ta (De ca po da) and se ven ge ne ra of the Order Eup hau sia cea (Eup hau sia, Me gany - ctip ha nes, Ne ma tos ce lis, Nyctip ha nes, Stylo - chei ron, Thysa noes sa and Thysa no po da; ~91% of all known 86 va lid spe cies) is cu - rrently known to hatch as a nau plius, alt hough se ve ral sac-spaw ning eup hau siid spe cies usually hatch in ol der pseu do me ta nau plius (PMN) or me ta nau plius (MN) lar val sta ges (Gor don, 1955; Brin ton, 1966; Po no ma re va, 1969; Zilch, 1978; Gó mez-gu tié rrez, 2003a,b; Gó mez-gu tié rrez & Ro bin son, 2005). Other ma la cos tra cans, li ke Sto ma to po da, Bath yne - lla cea, Anphio ni da cea, Ca ri dea, Ste no poi dea and Rep tan tia, hatch as a zoea or zoea-li ke lar va; with the ex cep tion of the spe cies with di - rect de ve lop ment (Rich ter & Scholtz, 2001). De ve lop ment with a free-swim ming nau - plius was one of the main ar gu ments why the Order De ca po da was tra di tio nally con si de red the nea rest re la ti ve of eup hau siids, both in clu - ded in the Su pe ror der Eu ca ri da. Gor don (1955), Brin ton (1965), Maas & Wa los zek (2001) and Ca sa no va et al. (2002) al so ar gued that the sha red de ri ved cha rac ters (syna po - morp hies) bet ween De ca po da and Eup hau - sia cea are: 1) the ca ra pa ce at ta ched to 7, not 8, tho ra co me res and 2) that in adult sta ge their tho ra cic seg ments are open. Alwes & Scholtz (2004) con clu ded that the early clea va ge and gas tru la tion pat tern of the eup hau siid Me - gany ctip ha nes nor ve gi ca and Den dro bran - chia ta are may be ho mo lo gous fea tu res in di ca - ting a clo sely phylo ge ne tic re la tions hip bet - ween both groups. In the ot her hand, a re cent mo le cu lar study shows that the sub or der Mysi da is the clo sest ta xon of Eup hau sia cea. The Synca ri - dae are anot her clo sely-re la ted, but mo re ba - sal group and De ca po da are mo re dis tantly re - la ted to eup hau siids (Jar man, 2001; Jar man et al., 2000a). The hypot he sis that Eup hau sia cea and Mysi da cea are clo sely re la ted is not new and has been pro po sed and dis cus sed in the past. The ge nus Thysa no po da (the first eup - hau siid spe cies for mally des cri bed was Thysa no po da tri cus pi da ta Mil ne-edwards, 1837) was ori gi nally pla ced by Mil ne-edwards in his tri be of Mysiens and la ter ot her aut hors in la te ni ne teen and early twenty cen tu ries re - tai ned the as so cia tion of the se two ta xa in the group Schi zo po da (Cal man, 1909). All mysids are sac-spaw ners for ming an ovi ge - rous sac ca lled oos teo gi te. The nau ploid e ye - less lar vae of mysids ha ve the sa me ap pen - da ges as the clas sic ort ho nau plius, but lac king the nau plius eye and swim ming se tae (Wort - ham-neal & Pri ce, 2002). Sac-spaw ning stra - tegy has evol ved in four eup hau siid ge ne ra (Pseu deup hau sia, Nyctip ha nes, Ne ma tos ce - lis and Stylo chei ron) in clu ding 25 out 86 eup - hau siid spe cies (Brin ton et al., 2000). Se ve ral spe cies ha ve dis tinct hat ching me cha nisms hat ching as nau plii, PMN or MN. This may sug gest that sac-spaw ning re pro duc ti ve stra - tegy in eup hau siids may not be con si de red strictly and ex clu si vely as an apo morp hic fea - tu re as men tio ned in pre vious stu dies (Mauch - li ne & Fis her, 1969; Ca sa no va, 1984, 2004). Re cent ar gu ments ba sed on com pa ra ti ve morp ho logy and em bryo logy sug gest that the free-li ving nau plius of Eup hau sia cea and Den - dro bran chia ta has ari sen se con da rily from an an ces tor that de ve lo ped through an an ces tral em bryo nic egg nau plius with hat ching at a la - ter lar val sta ge (Shii no, 1958; Strath mann & Eer nis se, 1994; Scholtz, 2000). The egg-nau - plius is de fi ned as an em bryo in which seg - ments and limbs form gra dually with the pre co - cious de ve lop ment of the nau pliar re gion (Scholtz, 2000). The Ma la cos tra can egg-nau - plius shows a dis tinct morp ho logy, i.e. uni ra - mous man di ble, rich yolk and un-dif fe ren tia ted dor sal si de due to the yolk con tent. In con trast, free-li ving nau plii ha ve bi ra mous man di ble, func tio nal limb mus cles and litt le yolk. The

3 HATCH ING MECH A NISMS IN EUPHAUSIIDS 65 nau plius of Nematoscelis difficilis and three broad cast spaw ning eup hau siid spe cies ha ve bi ra mous man di ble (Gó mez-gu tié rrez, 2003b), but Nyctip ha nes sim plex nau plius has uni ra mous man di ble (Bo den, 1951). Thus, whet her the Order Eup hau sia cea has an egg-nau plius or whet her hat ching as a nau - plius sta ge is a pri mi ti ve (ple sio morp hic) or de - ri ved (apo morp hic) cha rac te ris tic are cu rrently in de ba te and so me scien tists even be lie ve it is an un sol va ble pro blem to de fi ne: which is the most pri mi ti ve re pro duc ti ve stra tegy wit hin the Order Eup hau sia cea, broad cast or sac-spaw - ning stra tegy? A com pa ra ti ve study of the em - bryo nic sta ge at hat ching and hat ching me - cha nisms would be of in te rest in re gard to the pos si ble evo lu tio nary re ver sal of eup hau siid de ve lop ment. Ho we ver, three ex ten si ve re - views of hat ching pro cess of in ver te bra te eggs re por ted no ade qua te des crip tion of hat ching for the Order Eup hau sia cea (Da vis, 1968, 1981; Ander son, 1982) showing a gap in know led ge on egg re lea se and brood ca re in eup hau siids that ac cor ding to Maas & Wa los - zek (2001) may be an im por tant cha rac ter in the in ter pre ta tion of the syste ma tic re la tions - hips wit hin the Order Eup hau sia cea. Until re - cently it was as su med or ge ne rally known wit - hin ex perts stud ying eup hau siid bio logy and eco logy, that all broad cast-spaw ning eup hau - siid spe cies hatch as a nau plius, whi le sac-spaw ning hatch ex clu si vely in ol der sta - ges (Bo den, 1951; Mauch li ne & Fis her, 1969; Go pa la krish nan, 1973; La va nie gos, 1992). Ho we ver, such dis tinc tions are not so clear sin ce broad cast spaw ning spe cies may hatch as PMN or MN (Gó mez-gu tié rrez, 2002) and sac spaw ning spe cies may hatch as nau plius (Gó mez-gu tié rrez, 2003a,b; Gó mez-gu tié - rrez & Ro bin son 2005). Spe cies wit hin each eup hau siid ge nus has ex clu si vely broad cast or sac-spaw ning spaw ning re pro duc ti ve stra - tegy. Ho we ver, the hat ching me cha nisms are highly va ria ble among ge ne ra and among spe - cies of the sa me ge ne ra, sup po sedly to en - han ce lar val sur vi val. Thus, hat ching me cha - nism may be a cha rac te ris tic that hypot he ti - cally could be trac ked throug hout eup hau siid s phylo geny de ri ved from DNA analy sis. The goal of this es say was to sum ma ri ze all pre vious know led ge on eup hau siid hat - ching me cha nisms and hat ching suc cess of fi - ve broad cast and two sac-spaw ning eup hau - siid spe cies from the Nort heas tern Pa ci fic (Gó - mez-gu tié rrez, 2003a,b; Gó mez-gu tié rrez & Ro bin son, 2005) in the con text of pre vious hypot he ti cal phylo ge ne tic trees of eup hau siids de duc ted from morp ho lo gi cal, re pro duc ti ve stra te gies (Ca sa no va, 1984; Maas & Wa los - zek, 2001) and ge ne tic in for ma tion (Jar man, 2001; Jar man et al. 2000a,b) to in ter pret, in an evo lu tio nary pers pec ti ve, whet her the egg-nau plius could re pre sent an ces tral in for - ma tion that has been con ser ved, a li kely con - di tion from which eup hau siids di ver ged by re - tur ning to hat ching of free-swim ming nau plii (Sud haus & Reh feld, 1992). MA TE RI ALS AND METH ODS Euphausiid collection. Euphausiids eggs of Euphausia pacifica, Thysanoessa spinifera, Thysanoessa inspinata and Nematoscelis difficilis were ob tained from gravid fe males col lected in the field at night us ing a 1 m di am - e ter net with black 333 µm mesh and close cod end (0.75 m long, 0.25 m di am e ter) dur ing 15 ocean o graphic cruises ( ) car ried out be tween March and Sep tem ber along the Or e gon coast, USA (Gómez-Gutiérrez, 2003a). Fe males of Nyctiphanes sim plex, Euphausia eximia and Euphausia distinguenda were col lected with a sim i lar net in May, July and De cem ber, 2004 over the con ti nen tal shelf and the mouth of Bahía Magdalena, México (24 N, 112 N) and along the Gulf of Cal i for nia (No vem ber 2005 and July 2007). In all cases, the 10 m to 40 m depth stra tum was sam pled at drift ing tow ing speed to avoid dam age to the live euphausiids. The catches were di luted into 40 L cool ers filled with sur face sea wa ter from the sta tion sam pled. Euphausiid incubation. Du ring all the ocea no grap hic crui ses the in cu ba tions we re set up at sea wit hin a few mi nu tes af ter co llec - tion in high pre ci sion in cu ba tor (Fis her) aboard the R/V We co ma (Ore gon Sta te Uni ver sity), R/V New Ho ri zon (Scripps Insti tu tion of Ocea - no graphy) and in Méxi co in a cold room aboard the R/V El Pu ma (Uni ver si dad Na cio -

4 66 GÓ MEZ-GU TIÉ RREZ nal Au tó no ma de Mé xi co). We gently re mo ved gra vid fe ma le eup hau siids from the di lu ted plank ton sam ple with a plas tic spoon and pla - ced each fe ma le in a 1 L bott le fi lled with fil te - red (200 µm) sur fa ce sea wa ter. From 2 to 50 gra vid fe ma les we re in cu ba ted per sta tion ac - cor ding to avai la bi lity. Se ve ral fe ma les we re in cu ba ted and mo ni to red fre quently (< 4 hours) un til they spaw ned, and the em bryos we re coun ted and their em bryo nic de ve lop - ment ob ser ved every ~10 mi nu tes be fo re gas - tru la sta ge and every 1 h to 4 h for ol der em - bryos un til hat ching ti me at cons tant tem pe ra - tu re 10.5 C ± 0.5 C for Ore gon spe ci mens and 16 C ± 1 C for Ba ja Ca li for nia and Gulf of Ca li for nia spe ci mens. Hat ching suc cess (HS) and field hat - ching suc cess (FHS). In broad cast spaw ning spe cies HS was mea su red as the per cen ta ge of eggs hat ched up to the mo ment all the un - hat ched eggs be gan to spoil and the hat ching me cha nism was mo ni to red using a di gi tal ca - me ra (Olympus Ca me dia 3040, 3.3 x 10 6 pi - xels re so lu tion). The HS was com pa red with that of eggs co llec ted from the field > 48 hours and in cu ba ted in the la bo ra tory at 10.5 C ± 0.5 C (E. pacifica), he re de fi ned as field hat ching suc cess (FHS). In sac-spaw ning eup hau siids hat ching suc cess was ob ser ved du ring se ve - ral days ob ser ving the em bryos in si de the ovi - ge rous sac un til the fe ma les com ple tely re lea - se their em bryos of the ovi ge rous sac. At the end of an ex pe ri ment, both fe ma les and em bryos of the in cu ba tions we re pou red through a sie ve (120 ìm) and pre ser ved with 5% for ma lin buf fe red with so dium bo ra te for Ore gon sam ples and 96% et ha nol for sam ples co llec ted in Mé xi co. Pre ser ved fe ma les we re mea su red from the pos te rior part of the eye to the tip of the tel son (to tal length, mm). Brood si ze was the num ber of eggs pro du ced per fe - ma le per spaw ning event (eggs brood -1 ) du ring the 48 h of in cu ba tion. Interpretation of the hatching mecha - nisms and death of em bryos du ring hat - ching from a phylogenetic perspective. The hat ching me cha nisms and ave ra ge hat ching suc cess we re com pa red by a par si mo nious in - duc ti ve pro cess with the phylo ge ne tic trees pro po sed in se ve ral pre vious pu bli ca tions. I used 13 morp ho lo gi cal cha rac ters and re pro - duc ti ve stra tegy (Ca sa no va, 1984), cla dis tic analy sis of adult fea tu res and on to ge ne tic cha - rac ters (Mass & Wa los zek, 2001) and a phylo - ge ne tic tree ba sed on the ave ra ge di ver gen ce ti me using slowly evol ving nu clear DNA se - quen ces (par tial 28S rdna) (Jar man, 2001) to at tempt to re cons truct a con cep tual mo del of the re la ti ve chro no lo gi cal ap pea ran ce of broad cast spaw ning and sac-spaw ning re pro - duc ti ve stra tegy in the fa mily Eup hau sii dae. I al so at temp ted to in ves ti ga te whet her free nau plius lar vae re pre sent a ple sio morp hic fea - tu re wit hin the Eup hau sii dae fa mily as pre - vious stu dies ha ve sug ges ted (Lau ter bach, 1986; Dahms, 2000). The Jar man s (2001) ge - ne tic phylo ge ne tic tree was ori gi nally shown un roo ted, but an in ter pre ta tion was ma de to re cons truct the phylo ge ne tic tree for the ge ne - ra of the fa mily Eup hau sii dae from the ave ra ge di ver ged ti mes for clo se ge ne ra es ti ma ted by slow evol ving nu clear DNA se quen ces from Jar man (2001). To recons truct the Eup hau sii - dae phylo ge ne tic tree, di ver gen ce ti mes we re ob tai ned mostly from the empty-cir cles no des shown in Jar man s Fi gu re 4 and clus ter of ge - ne ra we re ma de from the clo sest bran ches in the un roo ted tree from Jar man s Fi gu re 3 ori gi - nally ma de with clus ter analy sis. RESULTS Hatching mechanism and hatching suc cess of broad cast-spaw ning spe cies The emer gen ce of a lar va from its egg is a cri ti cal pe riod in the li fe cycle in crus ta ceans and for broad cast-spaw ning eup hau siid em - bryos it may in vol ve a re la ti vely low ave ra ge hat ching suc cess, sho wing lar ge fe ma le-to-fe - ma le va ria bi lity ran ging from ~0% to 100%. Embryos of broad cast spaw ning spe cies can die du ring hat ching pro cess. When the em - bryos are ready to hatch (twit ching sta ge), the nau plius pus hes against the cho rion with the pos te rior part of the ab do men pro du cing a pro - tu be ran ce. The pres su re breaks the cho rion and the nau plius pus hes it self back ward with all its ap pen da ges to sli de out from the cho - rion. But so me ti mes the ho le whe re the nau - plius ought to pass through is so small that the

5 HATCH ING MECH A NISMS IN EUPHAUSIIDS 67 lar va rup tu res. The nau pliar body is mainly fi - lled with yolk-con tai ning en do derm cells be - cau se the gut is not for med yet and much yolk is re lea sed when the lar va rup tu res du ring hat - ching. This newly dis co ve red sour ce of mor ta - lity was fre quently ob ser ved du ring in cu ba - tions un der la bo ra tory con di tions for eggs of E. pa ci fi ca (Fig. 1a-d), T. ins pi na ta (Fig. 1e-h) and T. spi ni fe ra in Ore gon. Rup tu re of the lar - va de pends on the si ze of the cho rion ho le. If an ani mal can pass most of its body through the small aper tu re, the nau plius shows a cha - rac te ris tic fi gu re-eight sha pe with a na rrow waist. The brea king point can be in the an te - rior (Fig. 1 a-d) or pos te rior part of the em bryo body (Fig. 1 e-h). Eup hau sia pa ci fi ca em bryos, from fe ma les spaw ned un der la bo ra tory con - di tions, had low hat ching suc cess (high mor ta - lity) due to fai lu re to break a lar ge enough ho le in the cho rion du ring hat ching; ra tes ran ged bet ween 0.5% and 5% (n=9 broods). In con - trast, T. ins pi na ta (1% to 98%, n=3 broods) and T. spi ni fe ra (0.2% and 25%, n=5 broods) sho wed so me ti mes re la ti vely hig her hat ching mor ta lity ra tes (Ta ble 1). The fact that so me of the eggs from the sa me brood died du ring hat - ching whi le ot hers hat ched nor mally sug gests dif fe rent sur vi val pro ba bi li ties for si bling em - bryos, ba sed on their in di vi dual abi lity to rup tu - re the cho rion. It is un cer tain whet her la bo ra - tory con di tions pro mo te the death du ring hat - ching. Ho we ver, em bryos that died du ring hat - ching pro cess, in di ca ted by tis sue frag ments at ta ched to the cho rion, we re oc ca sio nally ob - ser ved from pre ser ved zoo plank ton sam ples co llec ted in the field, in di ca ting that mor ta lity du ring hat ching pro cess al so hap pens in the sea, alt hough in very low pro por tion (<0.1%) a b c d e f g h Figure 1. Se quen ce of the death of the em bryos du ring hat ching of Eup hau sia pa ci fi ca (a-d) and Thysa noes sa ins pi na ta (e-h). (a) Twit ching nau plius (528 µm dia me ter) trying to hatch back ward and the aper tu re of the cho rion is not big enough to pass through it. (b-d) The pres su re on the em bryo is high and the nau plius mem bra ne breaks in the an te rior part of the nau plius body. This se quen ce of pic tu res took <5 se conds. (e) Egg in early limb-bud sta ge (367 µm) ~18 h af - ter spaw ning. (f) The egg is in la te limb-bud sta ge 25 h af ter spaw ning. (g) The twit ching nau plius is hat ching back ward and the aper tu re of the cho rion is al so too small to pass through it. (h) The brea king point he re is the pos te rior part of the body and most of the nau plius body re mains in si de the cho rion. The sca le bars are 100 µm. Figura 1. Se cuen cia de la muer te de em brio nes du ran te la eclo sión de Eup hau sia pa ci fi ca (a-d) y Thysa noes sa ins pi na - ta (e-h). (a) Nau plio den tro del co rión (528 µm diá me tro) in ten tan do eclo sio nar ha cia atrás en el cual la aber tu ra del co - rion no es su fi cien te men te gran de pa ra que el em brión pa se por el. (b-d) La pre sión del em brión es al ta y la mem bra na del em brión se rom pe de la par te pos te rior del nau plio. Esta se cuen cia de fo tos du ró <5 se gun dos. (e) Embrión en es ta - dio de apén di ces en de sa rro llo (367 µm) ~18 h des pués del de so ve. (f) Embrión en es ta dio de apén di ces ya de sa rro lla - dos 25 h des pués del de so ve. (g) Nau plio den tro del co rión es ta eclo sio nan do ha cia atrás pe ro la aber tu ra tam bién es de ma sia do pe que ña pa ra pa sar a tra vés de ella. (h) El pun to de rom pi mien to del em brión es en la por ción pos te rior del cuer po y la ma yor par te del nau plio se que da den tro del co rión. La es ca la mi de 100 µm.

6 68 GÓ MEZ-GU TIÉ RREZ Ta ble 1. Brood si ze (egg fem -1 ) and num ber and per cen ta ge of dead em bryos du ring hat ching (in ver se of hat ching suc - cess) from fe ma les that spaw ned un der la bo ra tory con di tions from Ore gon (Gó mez-gu tié rrez, 2002, 2003a,b) and west coast of Ba ja Ca li for nia pe nin su la and Gulf of Ca li for nia (Gó mez-gu tié rrez & Ro bin son, 2005) and this study. Num bers shown bet ween pa rent he ses are ave ra ges. Ta bla 1. Ta ma ño de pues ta (em brio nes hem -1 ) y nú me ro y por cen ta je de em brio nes muer tos du ran te la eclo sión (in ver - so de éxi to de eclo sión) de hem bras que de so va ron ba jo con di cio nes de la bo ra to rio en Ore gon (Gó mez-gu tié rrez, 2002, 2003a,b) y cos ta oes te de la pe nín su la de Ba ja Ca li for nia y Gol fo de Ca li for nia (Gó mez-gu tié rrez & Ro bin son, 2005) y es te es tu dio. Los nú me ros en tre pa rén te sis son pro me dios. Eup hau siid spe cies Da te La ti tu de, Lon gi tu de Brood si ze Broad cast spaw ning spe cies per fe ma le No. of eggs dead du ring hat ching Per cen ta ge Eup hau sia pa ci fi ca (n=9) Jul 18, N, W Aug 5, N, W Aug 5, N, W Sep 10, N, W Apr 10, N, W Jun 12, N, W Jun 12, N, W Jul 23, N, W Jul 23, N, W Thysa noes sa spi ni fe ra (n=5) Jun 12, N, W Aug 6, N, W Aug 6, N, W Aug 6, N, W Aug 6, N, W Thysa noes sa ins pi na ta (n=3) Jun 13, N, W Jun 13, N, W Jun 13, N, W Eup hau sia exi mia (n=1) Dec 15, N, W Eup hau sia dis tin guen da (n=1) Jul 26, N, W Sac spaw ning spe cies Ne ma tos ce lis dif fi ci lis (n=7) Aug 3-17, N, W Nyctip ha nes sim plex (n=55) Mar, Jul, Dec, N, W Nyctip ha nes sim plex (n=3) Nov, 2005; Jan, N, W (%) < (27.9) (46) 100 pro bably due to the dif fi culty to co llect and pre - ser ve em bryos exactly du ring the hat ching pro cess. E. exi mia in Bahía Mag da le na and Gulf of Ca li for nia was fre quently ob ser ved hat - ching back ward, but was al so ob ser ved hat - ching for ward, most ti mes thought it was not pos si ble to mea su re brood si ze and hat ching suc cess. The only gra vid fe ma le spe ci men of Eup hau sia dis tin guen da that spaw ned in the la bo ra tory hat ched as nau plius using back - ward hat ching me cha nism (Ta ble 2). Quan ti fi ca tion of the mor ta lity ra tes, via field hat ching suc cess (FHS), was es ti ma ted se ve ral ti mes at mo nos pe ci fic spaw ning events of E. pacifica with high den si ties of eggs in early limb-bud (elb) and la te limb-bud (llb) sta ges (Fig. 1) (pro bably ~20 h - 25 h

7 HATCH ING MECH A NISMS IN EUPHAUSIIDS 69 Ta ble 2. Expe ri ments of field hat ching suc cess (FHS) of E. pa ci fi ca em bryos co llec ted in Ore gon (44 65 N, W, July 2002, and N, W, Au gust 2002) and in cu ba ted un der la bo ra tory con di tions. Most eggs we re co llec ted and in cu ba ted in early (elb) and la te limb-bud sta ge (llb). Ta bla 2. Expe ri men tos de ta sas de eclo sión de cam po de em brio nes de E. pa ci fi ca re co lec ta dos en Ore gon (44 65 N, O, Ju lio 2002, y N, O, Agos to 2002) e in cu ba dos ba jo con di cio nes de la bo ra to rio. La ma yor par - te de los hue vos fue ron re co lec ta dos e in cu ba dos en es ta dio tem pra no de apén di ces (elb) y es ta dio tar dío de apén di - ces (llb). Date Ini tial num ber of em bryos (N) Abun dan ce per egg and nau plius af ter 48 h of la bo ra tory in cu ba tion elb-tw sta ges Embryos hat ching Embryos dead du ring hat ching Hat ched nau plius FHS (%) July, Total % 2.14% 1.64% 55.56% 53.8 August, Total % 0.8% 1.5% 76.9% 76.7 af ter spaw ning) co llec ted near sho re du ring July 2002 (44 65 N and W, eggs m -3 ) and Au gust 2002 (44 65 N and W, eggs m -3 ). At each ex pe ri - ment 10 groups with mo re than 40 eggs per group we re in cu ba ted > 48 h (Ta ble 2). Eggs spaw ned at the sea and in cu ba ted in the la bo - ra tory hat ched by the mo re fre quent back ward hat ching me cha nism to yield healthy nau plii (23.5% and 89.0 % hat ching suc cess) with an ave ra ge mor ta lity of em bryos du ring hat ching of 1.5% at both ex pe ri ments (Ta ble 2). Hatching mechanism and hatching suc cess of sac-spawn ing spe cies The death of eggs du ring hat ching was ne ver ob ser ved for eggs of the sac-spaw ning N. difficilis when the em bryo used the push-off me cha nism as PMN or MN (n=11 broods),

8 70 GÓ MEZ-GU TIÉ RREZ which ap pa rently use a mo re ef fi cient hat ching me cha nism (Gó mez-gu tié rrez un publ. da ta), but death du ring hat ching was ob ser ved when the em bryo hat ched ear lier as N2 (Gó mez-gu - tié rrez, 2003). I in ter pret the death of the nau - plius 1 du ring hat ching in broad cast-spaw ning spe cies as an in he rent di sad van ta ge as so cia - ted with the back ward hat ching sha red with the less com mon ac ce le ra ted hat ching in sac-spaw ning spe cies. In sac-spaw ning spe - cies when the fe ma le dies for any ot her cau se than pre da tion du ring early em bryo nic de ve - lop ment the who le brood si ze can not sur vi ve. Ho we ver, when a fe ma le dies (ex clu ding pre - da tion) du ring la te em bryo nic de ve lop ment clo se to hat ching ti me, em bryos may sur vi ve wit hout the oxy ge na tion pro vi ded by the fe ma - le when it fre quently mo ves up and down its ovi ge rous sac with the se venth pair of tho ra cic legs. A sum mary and des crip tion of all hat - ching me cha nisms is shown in Ta ble 3. DISCUSSION Cu rrently fi ve dis tinct hat ching me cha - nisms are known in eup hau siid em bryos, ob - ser ved so far in ten eup hau siid spe cies around the world (Ta ble 3). Dif fe rent spe cies sha re the sa me hat ching me cha nisms in de pen - dently of their dis tri bu tion ran ge (i.e. tem pe ra te eup hau siids Eup hau sia pa ci fi ca ver sus tro pi - cal eup hau siids Eup hau sia exi mia and Eup - hau sia dis tin guen da) and the sa me spe cies co llec ted in two or mo re dis tinct re gions seem to ha ve si mi lar hat ching me cha nisms (Ne ma - toscelis difficilis). This in di ca tes the hat ching me cha nism is an in he rent fea tu re of the spe - cies rat her than a re gio nal in du ced beha vior. Gó mez-gu tié rrez (2002) re por ted three dif fe - rent hat ching me cha nisms: back ward, for ward and flip ping for three broad cast spaw ning spe - cies, E. pa ci fi ca, T. ins pi na ta and T. spinifera. Back ward hat ching as nau plius 1 (N1) was the most com mon hat ching me cha nism of broad - cast spaw ning spe cies and was as so cia ted with re la ti vely hig her brood si ze and HS. The pre sent study shows that the tro pi cal eup hau - siids E. exi mia and E. dis tin guen da al so hatch pre fe ren tially as nau plius, but it al so has been ob ser ved hat ching as me ta nau plius (Ta ble 3). Broad cast eup hau siid em bryos in fre quently hatch in la ter de ve lop men tal sta ges using two unu sual hat ching me cha nisms as so cia ted with re la ti vely low HS: for ward and flip ping hat - ching me cha nisms (Gó mez-gu tié rrez, 2002). The push-off hat ching me cha nism of the sac-spaw ning spe cies N. difficilis, has been so far ob ser ved ex clu si vely in this spe cies (Gó - mez-gu tié rrez, 2003a,b) (Ta ble 3). Alter na ti - vely, the N. difficilis em bryo can hatch pre ma - tu rely as N2 using the for ward hat ching me - cha nism ob ser ved in broad cast spaw ning spe - cies. Nyctip ha nes sim plex em bryos hatch as nau plius with the ex pan sion hat ching me cha - nism (so far ob ser ved only in this spe cies), they furt her de ve lop in to pseu do me ta nau plii and me ta nau plii in si de the ovi ge rous sac. The nau plius breaks the thin and fra gi le cho rion by in crea sing the vo lu me of the body be cau se the em bryos ha ve a qui te small pe ri vi te lli ne spa ce. Nyctip ha nes sim plex lar vae es ca pe from the ovi ge rous sac la te in the MN sta ge, usually 5 days af ter spaw ning, just a few hours be fo re mol ting in to caly pto pis 1. This de la yed re lea se ex tends pro tec tion by the fe ma le, li kely de - crea sing the risk of pre da tion or early can ni ba - lism (Gó mez-gu tié rrez & Ro bin son, 2005). The se fi ve dif fe rent hat ching me cha nisms ob - ser ved in eup hau siids may ha ve an evo lu tio - nary sig ni fi can ce as so cia ted with their po pu la - tion in crea se of fit ness and lar val re cruit ment ra tes, par tially ex plai ning why in tem pe ra te and po lar en vi ron ments nu me ri cally do mi nant eup hau siid spe cies ha ve broad cast spaw ning re pro duc ti ve stra tegy, whi le in tro pi cal and sub tro pi cal en vi ron ments nu me ri cally do mi - nant eup hau siid spe cies fre quently ha ve sac-spaw ning re pro duc ti ve stra tegy. The only pos si ble way to know which of the se hat ching stra te gies ap pea red first in the evo lu tion of the eup hau siids is to in fer it from the com pa ri son of such hat ching me cha nisms ob ser ved among spe cies and ge ne ra with the phylo ge - ne tic trees pro po sed in pre vious stu dies using morp ho lo gi cal and re pro duc ti ve stra te gies (Ca sa no va, 1984; Maas & Wa los zek, 2001), ba sed on par tial 28S rdna se quen ces (Jar - man, 2001), or COI (Za ne & Pa tar ne llo, 2000; Buc klin et al., 2007). A com pa ri son of hat ching me cha nisms bet ween broad cast (se ven spe cies) and sac-spaw ning (three spe cies) re pro duc ti ve stra te gies (Ta ble 3) and the ob ser va tion re la ti -

9 HATCH ING MECH A NISMS IN EUPHAUSIIDS 71 Ta ble 3. Hat ching me cha nisms of the broad cast-spaw ning (B) or the sac-spaw ning (S) eup hau siid spe cies ob ser ved un - der la bo ra tory con di tions from ma tu re fe ma les around the world. The eup hau siid spe cies in which the hat ching me cha - nism has been in fe rred from a brief des crip tion of this pro cess, dra wings and/or pho to graphs, are in di ca ted with as te risks (Mo di fied from Gó mez-gu tié rrez & Ro bis non, 2005). Ta bla 3. Me ca nis mos de eclo sión de es pe cies de eu fáu si dos de so va do ras ex ter nas (B) o de so va do ras en sa co oví ge ro (S) ob ser va dos ba jo con di cio nes de la bo ra to rio de hem bras grá vi das re co lec ta das en el cam po en el mun do. Las es pe - cies de eu fáu si dos en las cua les han si do in fe ri dos los me ca nis mos de eclo sión a par tir de una bre ve des crip ción, fo to - gra fías o dia gra mas son in di ca das con as te ris co (Mo di fi ca do de Gó mez-gu tié rrez & Ro bin son, 2005.) Hat ching me cha nism Eup hau siid spe cies Re gion Re fe ren ces Back ward: The nau plius 1 (N1) pus hes against the cho rion with the pos te rior part of the ab do - men pro du cing a pro tu be ran ce. The pres su re breaks the cho - rion, and the N1 pus hes it self back ward with the first and se - cond an ten nae and man di ble to sli de out from the chorion. Eup hau sia su per ba (B)* Eup hau sia pa ci fi ca (B) Eup hau sia exi mia (B) Eup hau sia dis tin guen da (B) Thysa noes sa spi ni fe ra (B) Thysa noes sa ins pi na ta (B) Ne ma tos ce lis dif fi ci lis (S) Antarc tic Sea Ore gon, USA Bahía Mag da le na and Gulf of Ca li for nia Bahía Mag da le na and Gulf of Ca li for nia Ore gon, USA Ore gon, USA Ore gon, USA and Gulf of Ca li for nia Ross & Que tin (1982)*; Geor ge (1984)*; Gó mez-gu tié - rrez (2002, 2003a,b); This study For ward: The nau plius 2 (N2) and me ta nau plius (MN) break the cho rion with the first and se - cond an ten nae, hat ching forward Thysa noes sa iner mis (B)* Stylo chei ron ca ri na tum (S)* Eup hau sia pa ci fi ca (B) Thysa noes sa spi ni fe ra (B) Thysa noes sa ins pi na ta (B) Eup hau sia exi mia (B) Ba rent Sea Indian Ocean Ore gon, USA Ore gon, USA Ore gon, USA Bahía Mag da le na and Gulf of Ca li for nia Ze lik man (1961)*; Po - no ma re va (1969)*; Gó mez-gu tié rrez (2002); Gó mez-gu tié - rrez & Ro bin son, 2005; This study Flip ping: The caly pto pis (C1) slit the cho rion using their tel son spi nes ex ten ding and flip ping the ab do men outside the egg Eup hau sia pa ci fi ca (B) Ore gon, USA Gó mez-gu tié rrez (2002) Push-off: The pseu do me ta nau - plius (PMN) or MN em bryos ex - tend and con tract their first and se cond an ten nae in a swim ming mo ve ment, brea king the cho rion in al most equal hal ves joi ned by one small sec tion in the an te rior part of the cho rion. The PMN or MN hatch and es ca pes from the ovi ge rous sac almost simultaneously Ne ma tos ce lis dif fi ci lis (S) Ore gon, USA and Gulf of Ca li for nia Gó mez-gu tié rrez (2003a,b) ; This study Expan sion: The nau plius hatch brea king the thin cho rion with the body growth, but is the MN sta ge that es ca pes from the ovi - ge rous sac about 2 days af ter hatching Nyctip ha nes sim plex (S) Bahía Mag da le na and Gulf of Ca li for nia Gó mez-gu tié rrez & Ro bin son, (2005); This study

10 72 GÓ MEZ-GU TIÉ RREZ vely high mor ta lity ra tes du ring hat ching pro - cess in broad cast spaw ning eup hau siids com - pa red with Jar man s (2001) phylo ge ne tic tree sup ports the hypot he sis that hat ching at the N1 (ort ho nau pliar) sta ge is a de ri ved (apo - morp hic) cha rac ter in the Order Eup hau sia - cea. Thus, the abi lity of broad cast-spaw ning spe cies to hatch as N2, MN and C1 re pre sents an in ter me dia te fea tu re bet ween broad cast and sac-spaw ning re pro duc ti ve stra te gies, in ot her words an evol ving ata vism of an an ces - tral egg-nau plius con di tion (Fig. 2a). Thus, the early hat ching of the N2 (for ward hat ching) ob - ser ved in the sac-spaw ning spe cies N. difficilis or the N. sim plex eclo sion as nau pli with the ex pan sion hat ching me cha nisms may be dis - tinct steps to ward an ear lier hat ching sche du le typi cal of broad cast spaw ning spe cies. The ge ne ral pic tu re emer ging from the com pa ri son of hat ching me cha nisms and hat ching suc - cess of both eup hau siid re pro duc ti ve stra te - gies ap pears to sup port the hypot he sis that a free-li ving nau plius lar va is a re ver sal fea tu re for the Order Eup hau sia cea and that the push-off hat ching me cha nism in sac-spaw ning eup hau siids re pre sents a pri mi ti ve (ple sio - morp hic) hat ching me cha nism mo de si mi lar to mysids (Fig. 2a). It is in te res ting that in the eup hau siid phylo geny spe cies with sac-spaw - ning re pro duc ti ve stra tegy ap pa rently evol ved in de pen dently among ge ne ra sin ce se ve ral me cha nisms of hat ching ha ve been ob ser ved in the sac-spaw ning spe cies Stylo chei ron ca ri - na tum (nau plii, for ward), N. difficilis (nau plii, back ward, PMN or MN, push off) and N. sim - plex (nau plii, ex pan sion) (Ta ble 3) which ap - pears re pea tedly and in ter mit tently in the Eup - hau sii dae phylo geny, both in eup hau siids with broad cast and sac-spaw ning re pro duc ti ve stra tegy. This agrees with Strath man & Eer - nis se (1994) in that the most par si mo nious in - ter pre ta tion is ata vism ( re su rrec tion ) of a free nau plius from an egg-nau plius at least on ce for Eup hau sia cea and Den dro bran chia ta. Be - cau se this in ter pre ta tion agrees with one of the most ro bust ge ne tic phylo ge ne tic trees cu - rrently known (Jar man, 2001) (Fig. 2c), this may be the most par si mo nious ex pla na tion for evo lu tion of the eup hau siid re pro duc ti ve stra - te gies. Ho we ver, this pers pec ti ve of Eup hau - sii dae evo lu tion is qui te un con ven tio nal sin ce in the twenty cen tury most scien tists pro po sed that broad cast spaw ning re pro duc ti ve stra tegy is a ple sio morp hic re pro duc ti ve stra tegy and sac-spaw ning a apo morp hic re pro duc ti ve in the eup hau siid phylo geny ba sed on so me con - vin cing evi den ce to sup port this view. The Order Eup hau sia cea com pri ses two fa mi lies: the mo noph yle tic Bent heup hau sii dae (Bent heup hau sia am blyops, G.O. Sars 1885) and the Eup hau sii dae with 85 va lid spe cies (Brin ton et al., 2000). The first pro po sed dia - gram of phylo ge ne tic re la tions hips of the Order Eup hau sia cea was ba sed on the exa mi - na tion of 13 ge ne ric cha rac ters dea ling with morp ho logy and re pro duc tion (Fig. 2b) (Ca sa - no va, 1984). In this hypot he ti cal tree the mo - nos pe ci fic ge nus Bent heup hau sia seems to be the most ar chaic (Ca sa no va, 2004), ha ving the en do pod of the first ma le pleo pod un mo di - fied as a pe tas ma, no pho top ho res, three seg - men ted ma xi lla and three en do po dal po do me - res in the ma xi llu la (Brin ton, 1967; Maas & Wa los zek, 2001). Bent heup hau sia am blyops is pre su mably a broad cast-spaw ning spe cies be cau se no ovi ge rous fe ma le has ever been co llec ted du ring ex ten si ve deep sam pling co - llec tions around the world, but waits for ex pe ri - men tal co rro bo ra tion (Ca sa no va, 1984; Brin - ton et al., 2000). This is a con vin cing ar gu ment that eup hau siids first evol ved from a crus ta - cean with broad cast spaw ning stra tegy, but ge ne tic analy sis so far does not sup port this view. The Order Eup hau sia cea lack a fos sil re - cord as adult and em bryos, thus ex tinct eup - hau siid spe cies are ab sent, for exam ple du ring the Cre ta ceous ex tinc tion 65 Mya and the re fo - re not in clu ded in any of the cu rrent eup hau siid phylo ge ne tic trees (Jar man, 2001). Thus, it is not pos si ble to know if an an ces tral eup hau siid with sac-spaw ning re pro duc ti ve stra tegy is cu - rrently ex tinct or evol ved in so me of the cu rrent spe cies with dis tinct re pro duc ti ve and hat - ching me cha nism stra tegy. Ca sa no va (1984) clai med that from the Thysa no po da, which re - pre sents the first really eup hau sia cean step, four ra dia tions can be dis tin guis hed. This ra - dia tion may be ex plai ned if sympa tric spe cia - tion of eup hau siids may ha ve oc cu rred si mul - ta neously (in geo lo gi cal ti me sca les) in se ve ral re gions around the world with chan ges of the ocean tem pe ra tu re mo dif ying the dis tri bu tion of in di vi dual spe cies ac cor ding to their physio -

11 HATCH ING MECH A NISMS IN EUPHAUSIIDS 73 Fi gu re 2. (a) Con cep tual mo del of evo lu tion of hat ching me cha nism of broad cast spaw ning and sac-spaw ning stra te gies in the fa mily Eup hau sii dae. La te hat ching me cha nism (for ward and flip ping) in broad cast spaw ning spe cies is in ter pre ted he re as an ata vis tism and early hat ching sche du le (for ward and ex pan sion hat ching me cha nism) in sac-spaw ning spe - cies as an in ter me dia te hat ching me cha nism bet ween both re pro duc ti ve stra te gies. (b) Phylo ge ne tic re la tions hips and evolutionary cha rac ters of the Order Eup hau sia cea ba sed on 13 ge ne ric cha rac ters dea ling with morp ho logy and re pro - duc tion (Ca sa no va, 1984; re drawn from his Fi gu re 3). (c) Dia gram of the phylo ge ne tic tree for the ge ne ra of the fa mily Eup hau sii dae sho wing ave ra ge di ver ged ti mes for clo se ge ne ra es ti ma ted by slow evol ving nu clear DNA se quen ces (mo di fied and in ter pre ted from Jar man, 2001, from his Fi gu re 4). (d) Re-drawn and simplified of the Order Euphausiacea phylogenetic tree where the subfamily Euphausiinae was proposed (Maas & Waloszek, 2001). Fi gu ra 2. (a) Mo de lo con cep tual de la evo lu ción de los me ca nis mos de evo lu ción de las es tra te gias de re pro duc ción de de so ve ex ter no o en sa co oví ge ro en la fa mi lia Eup hau sii dae. Los me ca nis mos de eclo sión tar díos (ha cia ade lan te y por mo vi mien tos del ab do men) en es pe cies de so va do ras ex ter nas se in ter pre ta co mo un ata vis mo y la eclo sión tem pra na (me ca nis mos ha cia de lan te y de ex pan sión) en es pe cies con de so ve en sa co oví ge ro co mo un me ca nis mo de eclo sión in ter me dio en tre am bas es tra te gias de re pro duc ción. (b) Re la cio nes fi lo gené ti cas y ca rac te res evo lu ti vos del Orden Eup hau sia cea fun da men ta do en 13 ca rac te rís ti cas gené ri cas con cer nien tes a la mor fo lo gía y re pro duc ción (Ca sa no va, 1984; re di bu ja do de su Fi gu ra 3). (c) Dia gra ma del ár bol fi lo genéti co de los gé ne ros de la fa mi lia Eup hau sii dae mos tran - do los tiem pos de di ver gen cia pro me dio pa ra gé ne ros cer ca nos, es ti ma dos de se cuen cias de re du ci da ta sa de mu ta ción de ADN nu clear (mo di fi ca do e in ter pre ta do de Jar man, 2001, Fi gu ra 4). (d) Árbol fi lo gené ti co re di bu ja do y sim pli fi ca do del Orden Eup hau sia cea, don de fue pro pues ta la sub fa mi lia Eup hau sii nae (Maas & Waloszek, 2001).

12 74 GÓ MEZ-GU TIÉ RREZ lo gi cal to le ran ces (Brin ton, 1962; John son & Brin ton, 1963). Ca sa no va (1984) sug ges ted that the broad cast-spaw ning is a pri mi ti ve re - pro duc ti ve stra tegy in the Order Eup hau sia - cea. Accor ding to Ca sa no va (1984, 2004) the evo lu ti ve trend in eup hau siids is the re duc tion of pairs of tho ra co pods in the two sex gen ders, the elon ga tion of the se cond and third pair of pe rio pods and the ac qui si tion of bi lo bed eyes and ovi ge rous sac. Accor ding to Brin ton (1965) and Maas & Wa los zek (2001) who ha - ve a si mi lar pers pec ti ve as Ca sa no va, the sac-spaw ning ge ne ra Ne ma tos ce lis and Stylo chei ron sha re ad van ced morp ho lo gi cal cha rac te ris tics li ke elon ga tion of one or two pairs of tho ra co pods, a bi lo bed eye and neo te - nic ex ter nal morp ho logy as adults, in di ca ting that the se are apo morp hic cha rac te ris tics in com pa ri son with spe cies of ot her ge ne ra wit - hin the Order Eup hau sia cea. Ho we ver, the mo le cu lar phylo geny for all the eup hau siid ge - ne ra of the fa mily Eup hau sii dae, shows a dif fe - rent pers pec ti ve (Jar man, 2001). Jar man s un - roo ted tree ba sed on par tial 28S rdna se - quen ces, which sta tes that no eup hau siid is ta - ken to be mo re an cient than any ot her, is dif fe - rent from Ca sa no va s phylo ge ne tic tree, ex - cep ting the clo se re la tions hip bet ween the broad cast-spaw ning ge ne ra Thysa noes sa and Me gany ctip ha nes and that Tes sa ra bra - chion di ver ges from any ot her eup hau siid ge - ne ra clus te red in ot her phylo ge ne tic groups (Fig. 2c). The main dif fe ren ces we re that Pseu deup hau sia (a sac-spaw ner) was con si - de red a pri mi ti ve eup hau siid ge nus which pos - sibly di ver ged about 100 Mya (Cre ta ceous) from the com mon an ces tor of the fa mily Eup - hau sii dae (~130 Mya) and that Nyctip ha nes (sac-spaw ner) and Eup hau sia (broad cast spaw ner) we re clo sely re la ted (he re is shown that both hatch pri ma rily as nau plii), even though they ha ve dif fe rent ex ter nal morp ho - logy and re pro duc ti ve stra tegy. Anot her no vel as pect of Jar man s pers pec ti ve is that Pseu - deup hau sia la ti frons (sac-spaw ner) and Ne - ma to bra chion boo pis (broad cast spaw ner) are clo sest re la ti ves, whi le Ca sa no va con si de red Pseu deup hau sia clo sely re la ted with Nyctip - ha nes (both sac-spaw ning ge ne ra). Jar man et al. (2000a) sup por ted the idea that if the sac and broad cast spaw ning ge ne ra are dis tri bu - ted throug hout the Order Eup hau sia cea, it is mo re li kely that sac-spaw ning is pri mi ti ve and has been lost se ve ral ti mes, ex plai ning why eup hau siids are most clo sely re la ted to Mysids. Gó mez-gu tié rrez (2003) sug ges ted, from the hat ching des crip tion of Stylo chei ron ca ri na tum (Po no ma re va, 1969), that this spe - cies hat ches as N2 using for ward hat ching mechanism like sometimes N. difficilis, Ne ma - tos ce lis and Stylo chei ron do, which seem to be re cent ge ne ra that di ver ged about 23 Mya (Jar man, 2001). This sug gests that the hat - ching me cha nism of both ge ne ra might not be ne ces sa rily sha red with ot her sac-spaw ning spe cies li ke Pseu deup hau sia (so far not ob - ser ved), or Nyctip ha nes. Ho we ver, it is re qui - red to ob ser ve the hat ching me cha nism of all the sac spaw ning ge ne ra and spe cies in fu tu re re search. Accor ding with Jar man s phylo ge ne - tic tree ap pa rently broad cast spaw ning is an ad van ced re pro duc ti ve stra tegy be cau se nau - plii can swim and es ca pe mo re ef fi ciently than when hat ching in ol der de ve lop men tal sta ges. No eup hau siid spe cies ha ve di rect em bryo nic de ve lop ment, con si de red an apo morp hic fea - tu re in ot her crus ta cean groups (Rich ter & Scholtz, 2001). Ho we ver, be cau se this study in clu ded only three sac-spaw ning spe cies it can not be ex tra po la ted to ot her spe cies or ge - ne ra around the world. Such ob ser va tions must be do ne in fu tu re stu dies. Re la ti vely less com prehen si ve ge ne tic stu dies (fe wer ge ne ra and spe cies) sho wed a re la ti vely clo se match to Ca sa no va s morp ho lo gi cal and mo le cu lar phylo ge nies (mi to chon drial lar ge-sub u nit ri bo - so mal par tial 28S rdna and cytoch ro me oxi - da se sub u nit I, COI DNA se quen ces) (Jar man et al., 2000b; Za ne & Pa tar ne llo, 2000; Buc kin et al., 2007). This ma kes evi dent the dif fi culty to com pa re and reach phylo ge ne tic con clu - sions from in for ma tion with un re pre sen ted spe cies. Anot her re cent eup hau siid cla dis tic phylo ge ne tic study ba sed so lely on morp ho - logy and de ve lop ment of eup hau siid ap pen da - ges emp ha si zed the pro blem of egg re lea se (Fig. 2d) (Maas & Wa los zek, 2001). Accor ding to tho se aut hors, as su ming that free re lea se of eggs is a pri mi ti ve fea tu re, their phylo ge ne tic tree im plied that brood ca re could ha ve de ve - lo ped four ti mes in de pen dently wit hin the Eup - hau sia cea. This pers pec ti ve aro se be cau se

13 HATCHING MECHANISMS IN EUPHAUSIIDS 75 the me cha nisms of hol ding the eggs and the num ber of limbs in vol ved dif fer among ta xa (ge ne ra). Alter na ti vely, Maas & Wa los zek (2001) sug ges ted that if egg ca rria ge is the pri - mi ti ve sta ge wit hin Eup hau sia cea, then loss of this ha bit should ha ve oc cu rred at least six ti - mes in de pen dently wit hin Eup hau sia cea. Ho - we ver, they we re in con clu si ve about what was mo re li kely to ha ve oc cu rred throug hout the evo lu tion of the Order Eup hau sia cea. Also the re is a com ple te lack of in for ma tion on hat - ching mo de and con fir ma tion of re pro duc ti ve strategy (broad cast or sac-spaw ning) for three eup hau siid ge ne ra Bent heup hau sia, Ne ma to - bra chion and Tes sa ra bra chion (Gó mez-gu tié - rrez, 2002). Maas & Wa los zek (2001) pro po - sed a phylo ge ne tic tree with a new sub fa mily (Eup hau sii nae), ex clu ding the ge ne ra Thysa - no po da and Ne ma to bra chion, in clu ding Me - gany ctip ha nes nor ve gi ca and two ot her sub - groups na med Eup hau sii ni and Ne ma tos ce li - ni. Pseu deup hau sia and Eup hau sia in ter pre - ted as sis ter ge ne ra (Eup hau si nii) and the Ne - ma tos ce li ni which in clu ded the ge nus Nyctip - ha nes clo sely re la ted with anot her sub group na med Ne ma tos ce li na whe re they in clu ded Ne ma tos ce lis, Thysa noes sa, Tes sa ra bra - chion and Stylo chei ron pro vided mo re phylo - ge ne tic re la tions hips than ot her pre viously pro po sed phylo ge ne tic trees (Fig. 2d). Ho we - ver, they did not quo te Ca sa no va and Jar - man s pu bli ca tions and the re fo re pro bably they we re una wa re of such stu dies sho wing re la ti vely un sup por ted clus te ring of spe cies. Re cently Buc klin et al. (2007) ma de a ge ne tic com pa ri son of ~650 bp re gion of mi to chon drial cyto cro me oxi da se I (mtcoi) among 40 eup - hau siid spe cies (10 ge ne ra) around the world with the groups of eup hau siids de ri ved using morp ho lo gi cal fea tu res (Brin ton et al., 2000). He sho wed a clo se in ter spe cies match sug - ges ting that this is a use ful met hod to re sol ve re la tions hips among clo sely re la ted spe cies to en su re iden ti fi ca tion, re cog ni tion of cryptic spe cies and eva lua tion of ta xo no mi cally mea - ning ful geo grap hic va ria tion. Ho we ver, be cau - se they did not make com pa ri sons among ge - ne ra they did not pro vi de an in sight in to evo lu - tio nary re la tions hips of eup hau siids. Although the pre sent study pro po ses that hat ching in a nau plius sta ge in the Order Eup - hau sia cea is a de ri ved (apo morp hic) cha rac te - ris tic in their evo lu tio nary phylo geny (Fig. 2a), the re is a cost to early hat ching, im po sed by mor ta lity and cer tainly high mor ta lity by pre da - tion in free-swim ming lar vae that must be com - pen sa ted with high fe cun dity ra tes typi cal of broad cast spaw ning spe cies (Ni col, 1995; Gó - mez-gu tié rrez et al., 2006). Most eup hau siids are highly gre ga rius ani mals for ming den se swarms that at tract mul ti ple pre da tors, if spaw - ning oc curs. At least mor ta lity due pre da tors on fe ma les af ter spaw ning is in de pen dent from the eggs, si tua tion that does not hap pen with sac-spaw ning spe cies. In broad cast spaw ning spe cies, mor ta lity du ring hat ching pro cess seems to be re la ti vely small (<2%) in na tu re cou pled with the abi lity to avoid plank to - nic pre da tors as soon as the nau plius hatch (pro bably al most ne gli gi ble in a po pu la tion pers pec ti ve), or to avoid dee per la yers de void of food when the eggs are sin king af ter spaw - ning. Be cau se both re pro duc ti ve stra te gies allow sur vi val of the spe cies, re flec ting the dif - fe rent eco lo gi cal stra te gies, hat ching mo de per haps does not re flect an evo lu tio nary ad - van ta ge for most re cent ge ne ra ex plai ning why ap pear irre gu larly wit hin the Eup hau sia - cea phylo geny. Even if hat ching as nau plius is ple sio morp hic wit hin Eup hau sia cea, it might be an apo morphy for Eup hau sia cea in re la tion to ot her Ma la cos tra ceans (Sud haus & Reh - feld, 1992). This study, li ke all of the stu dies pu blis hed so far on morp ho logy, de ve lop ment and ge ne tic phylo ge ne tic trees, shows par tial in for ma tion of eup hau siid bio logy and phylo - geny. Thus, the in ter pre ta tion pro vi ded in this work must be ta ken with cau tion. The ob ser va - tions of hat ching me cha nisms and hat ching suc cess re por ted and sum ma ri zed he re (Ta - ble 3) com pa red chro no lo gi cally with the phylo geny of eup hau siids de duc ted from DNA analy sis, sup port the evo lu tio nary trend that the free-nau plius has re ver ted se ve ral ti mes wit hin the fa mily Eup hau sii dae and sac-spaw - ning may be ne ces sa rily con si de red ex clu si - vely as an apo morp hic con di tion sin ce se ve ral dis tinc ti ve hat ching me cha nisms oc cur in Ne - ma tos ce lis, Stylo chei ron and Nyctip ha nes per haps re flec ting dif fe rent ti me adap ta tions throug hout the eup hau siid s phylo geny. Evi - dently the re pro duc ti ve and hat ching me cha - nisms of at least one or two spe cies of each