Int egrat ive and Compa rative Bio lo gy Int egrat ive and Compa rative Bio lo gy , volume 0, pp. 1–8 htt p s://doi .or g/10.1093/icb/icae069 Soc ie ty fo r Inte g rati ve and Compa rative Bio lo gy INVITED PAPER Intro gre ssion of the Gamete Recognition Molecule, Bindin, in the Sea Urchin Diadema H. A. Lessios1 Smit hson ian Tropi cal Research Instit ut e, Box 0843-03092, B alb oa, Pan am a 1 E-mai l: LessiosH@si.e du Synopsis Hybr idization is impor tant in evolu t ion , because it is a necess ary (t ho ugh no t su fficient) st ep in the int rog ression of potent ia l ly adapt ive variat ion betwe en spe cies. Bindin is a gamete re cog nit ion protein in echinoids and a steroid s, capable of blocking cross-fert i lizat ion betwe en spe cies to varying deg re es. Four spe cies of the sea urchin genus Dia de ma are bro ad ly symp at ric in the In do-Pacific: D. pau cispin um , D. sav igny i , D. cla rki , and D. seto su m. D at a from three pub lish e d studies, one of ident ificat ion of hybr ids t hrough a l lozyme s, one of the phyloge og raphy of mito ch o nd rial DNA, and one of the phy logeny of bi ndin, were com bi ned to ass ess the deg re e of bi ndin int rog ression betwe en these four spe cies. I ana lyze d se quenc es of the ATPase 8 and ATPase 6 mito ch o nd rial gene s and of bi ndin, s ampled t hrou ghou t the species rang e s, w ith an isol at ion–mig rat ion algor it hm, IMa 3. IMa 3 uses a co a lescent ap proach to pr odu ce Bayesian estimates of effe ct ive pop u lat ion sizes and gene flow b etween p op u lation s. The result s sh o wed that bindin alleles coalesce compl etely within the species bounds of D. clarki and of D. seto su m. The sist er sp ecies D. pa uc ispin um and D. sav igny i , howe ve r, we re est imate d a s h aving exc hang e d a bindin a l lele at an ave rage of eve ry one to two -and-a-half ge nerations sinc e the y spe ciate d from e ach ot her. As t he a l loz yme study dete cte d nine hybrids between three of these species in Okinawa (most of them bet ween D. s eto su m and D. savig nyi ) in a single sample, hybrids between these species are pr odu ced, but bi nd in does no t int rog ress. Therefor e, bi ndin must not be efficient in blocking heterospe cific fert i lizat ion s. Com plete , or almost complete , re pro ductiv e isol ation bet ween species of Dia de ma must resu lt from low hybrid fitness. I c I A s 2 t ( c A r P ( W H i t r a g a o r a m t t 1 f e t e s n g A P G Downloaded from https://academic.oup.com/icb/advance-article/doi/10.1093/icb/icae069/7693114 by Smithsonian Libraries and Archives user on 03 July 2024ntroduction nt rog ression, the inc orporation of genes from one pecies into the genom e of anot her t hrough hybr idiza- ion , is impor tant in evolu t ion . Hybr idization between losel y rel ate d spe cies h a s the potent ia l of t ran sfe r- ing adaptive variation from on e species to ano the r A nderson 1949 ; A rnold 1997 ; Abbott et al. 2013 ; e drick 2013 ; E delman an d Ma l let 2021 ), g ives rise o ne w ho mo ploid or pol y pl oid spe cies ( Ma l let 2007 ), n d reinfo r ces repr odu ct ive b ar r iers t hroug h selec tion g ainst low-fitness offsp ring ( Howard 1993 ). It can also esult in the fusion or ext inct ion of species ( Rhymer nd Simberl off 1996 ; Wolf et al. 2001 ). Onc e tho ught o be rare in marine anim al s (e.g., Hubbs 1955 ; Arnold 997 ), hybr idization was subsequently found in s e v- ral marine gro ups (re vie ws in Gardner 1997 ; Willis t al. 2006 ; Arnold an d Fogar ty 2009 ). Among echi- oi ds, hybr ids we re ge net ica l ly ident ifie d in Strongylo-d vance Ac cess pub lication June 13, 2024 ub lish e d by Oxford University Press on behalf of the Society for Int egrat ive overnment emp loyee(s) and is in the public domain in the US. entr otu s ( Addison and Hart 2005 ; Harper et al. 2007 ; ddison and Pogson 2009 ; G la sen app and Pogson 023 ), Ech inot hrix ( Cop p ard et a l. 2021 ), Pseud o bo le tia Zigler et al. 2012 ), Lytec hi nu s ( Zigler and Lessios 2004 ), rba cia ( Lessios et al. 2012 ), Echino me t ra ( G e yer and alu mbi 2005 ), and Diad ema ( Lessios and Pearse 1996 ) . it h t he no tab le exc eption of Strongylo c entrotus , the mpor tance of int rog ression in echinoid evolution h a s emaine d une xplor ed. Most of the rep orts ab ou t ot her e nera cons ist of the ident ificat ion of a few hybrids in the rwise mo no phylet ic spe cies. How closel y rel ated sy mp at ric species of sea urchins ai ntai n their genetic integrity remains an open ques- ion, as there are few potent ia l b ar r iers to hete rospecific ert i lizat ion s in or gani sms th at sh e d the ir game te s into he water column ( Lessios 2007 ). Non-ove rlapping p awning t ime s are one possib le bar r ier in prezy- ot ic isolat ion, as is re cog nit ion betwe en conspe cificand Comp arat ive Bi ology 2024. This work is written by (a) US 2 H. A. Lessios Downloaded from https://academic.oup.com/icb/advance-article/doi/10.1093/icb/icae069/7693114 by Smithsonian Libraries and Archives user on 03 July 2024game tes. One of the prot eins pot ent ia l ly imp ortant in gamete re cog nit ion is bind in. Bind in is a gamete recog- nit ion pr otein (GRP) that mediat es int eract ion betwe en egg and sperm. It is expressed on the acrosome of sea urchin sperm, and it is re cog nize d by two recepto rs of t he eg g, EBR1 and a 350-kDa protein ( Vacquier 2012 ). Comp at ibi lit y of bi ndin wit h it s receptor s permit s adhesion wit h t he eg g vitel line me mbrane , fusion, and fert i lizat ion ( Vacquier and Moy 1977 ; Ul rich et al. 1998 ; Vacquier 2012 ). Bindin ha s been sh o wn experime n- ta l ly to be capable of block ing hete rospe cific mat ing in sea urchins ( Metz et al. 1994 ). Its DNA sequence dive rg e nce betwe en spe cies is inversely correl ated w ith comp at ibi lit y bet ween heterospecific gametes ( Zigler et al. 2005 ). It, thus, has the potent ia l of being a “spe ciat ion gene ,” as sub stit ut ion s that alt er it s affinit y to t he eg g recepto rs could re pro ductiv ely isol ate pop u lations ( Lessios 2011 ). GRPs genera l ly evol v e at a fast rate under strong positiv e (div ersifying) sele ct ion ( Swanson and Vacquier 2002 ; Turne r and Hoek stra 2008 ; Vacquier and Swanson 2011 ). Sea urchin bindin does not evol v e as rapidly a s th at of som e othe r GRPs ( Lessios and Zigler 2012 ), but it s rate of dive rg e nce betwe en cong e neric species i s u sua l ly hig her w he n the ir ge og raphic ranges over- lap ( Palu mbi and Lessios 2005 ). Posit ive sele ct ion on bindin is present in three sea urchin genera, Echino me - tra ( Metz and Palu mbi 1996 ; McCartney and Lessios 2004 ), Strongylo c entrotus ( Biermann 1998 ), and Para- centr otu s ( Calderon et al. 2009 ), but absent in five, Arba cia ( Metz et al. 1998 ; Lessios et al. 2012 ), Trip- neust e s ( Zigler and Lessios 2003 ), Lytechi nu s ( Zigler and Lessios 2004 ), Pseud o bo le tia ( Zigler et al. 2012 ), He- lio c id a ris ( Hart et al. 2012 , but see Zigler et al. 2003 ), and Dia de ma (except for one branc h) ( G e yer et al. 2020 ). Even in genera in which no posit ive sele ct ion h a s been found , bind in a l lel es gene ra l ly cl uste r into mo no - phyletic clades. The exception is bindin of Dia de ma . Species of Dia de ma are abund ant in tropic al seas around the globe. Four of the eight describ ed sp ecies of the genus are symp at ric over a large part of their rang e in the Indo-Pacific ( Fig. 1 ). Diad ema sav igny i rang e s from the cent ra l Pacific to t he e a st coas t of Africa, D. seto su m is spre ad fro m Ton ga to Afr ica ( Pe arse 1998 ), D. pa uc ispin um was orig ina l ly thought to be endemic to the cent ra l Pacific ( Mortensen 1940 ; Clark 1954 ), but re- cent evidence h a s sh o wn it to be much mor e widespr ead ( Lessios et al. 2001 ), and D. clarki is only known from the isl and s of Japan, the Marsh a l l Isl and s, and Ind one - sia ( Chow et al. 2016 ; Moore et al. 2019 ). All species are mo rpho log ica l ly very simi lar ( Lessios an d Pear se 1996 ), which h a s led to misident ificat ion s and erron eous re- por ts of their pr e sence in various re gion s ( Mor tensen 1940 ; Pearse 1998 ). John Pearse and I, with the help of Bailey Kessing, studied the mito ch ondria l phyloge og raphy of Dia de ma ( Lessios et al. 2001 ). We found D. seto su m to consist of two deepl y div ide d clades, a lmost certain ly differ- ent species, D. seto su m - a, spre ad widely from the West Pacific to v irtu a l ly the ent ire Indian O ce an, and D. seto su m -b, only occur r ing around t he Arabian Penin- sul a, where D. s av igny i is absent and D. paucispin um is rare. These two clades were an ou tgrou p to those of a l l other spe cies in the genus. Mito ch o nd rial DNA (mt- DNA) of D. pa uc ispin um , far fro m being re st ricte d to the cent ra l Pacific a s it wa s onc e tho u ght, was fou nd in the entire Ind o-Pacific ( Fig. 1 ). Dia de ma seto su m was est imate d as having sep arate d from a l l other spe cies of Dia de ma 6.7–13.9 mi l lion ye ars ago (Mya), D. clark i from the clade leading to D. pa uc ispin um and D. savi- gnyi 4–8.5 Mya, while the two sister species split from e ach ot he r 1.02–1.86 Mya. The study of mt-DNA con - fir med t he presence of D. paucispin um outside the cen- t ra l Pacific, as reporte d from semi-diag nost ic a l lozymes in Okinawa by Lessios and Pearse (1996) . We als o dis - covered a distinct clade of mt-DNA in Kyushu , Honshu , and Ma juro, Ma rsha l l Islands—more simi lar to D. sav- ig nyi t han to D. setos um—and su ggest ed th at it wa s a dist inct sp ecies, D. clarki , orig ina l ly describe d by Ikeda (1939) but sy nony mized w ith D. s eto su m by Mortensen (1940) . C how et a l. (2014 , 2016 ) subse quently provide d mo rpho log ica l and addit ion al mt-DNA evidence that thi s clade was , inde e d, the spe cies orig ina l ly describe d by Ikeda. Thu s, the di stribut ion s of four species of Di- ad ema overlap in the West Pacific. The question of hybr idization in the Ind o-Pacific species of Dia de ma was first addressed by Lessios and Pearse (1996) at a time when D. clarki was st i l l ass u med to be a sy nony m of D. s eto su m , and that D. pa uc ispin um was thought to be rest ricte d to the cent ra l Pacific. John Pearse col le cte d Dia de ma in Ok inawa and classified the specime ns according to the ir mo rpho logy as D. se- to su m , D. savig nyi , or as on e of three types of potent ia l hybr ids. To deter mine a l leles diag nost ic of each species, col le ct ion s of D. seto su m were al so m ade at Fant ome Isl and at the Great Barrier Reef in Australia, where it occurs alone, an d of D. paucisp in um at Hawaii, where it is the only species of Dia de ma . A llozy mes, di ag nost ic or semi-diag nost ic for each species, indicate d that nine of the morph ol og ica l ly interme diate spe cimens in Okinawa were hybrids between D. seto su m and D. sav igny i , or between on e (or both) of the se species and D. pa uc ispin um . The a l lozy me d at a est ablished t hat hybr idization between three species was taking place. The y could no t, ho we ve r, provide an estimate of the frequency of these hybrids in the pop u lat ion be cause sampling was del ib eratel y biased in favor of hybrid inclu sion by looking for indiv idu al s th at could not be Introgression of bindin in Diadema 3 Fig. 1 Localities where each of the species of Diadema discussed in the text is known to occur, and a re pre sentation of the species relationships as determined by their mitochondrial DNA (after Lessios et al. 2001 with additions from Chow et al. 2016 and Moore et al. 2019 ). i d P s c i e t c o M v M w f b b n L h D i s o t t t i t I s a s a t A i h t w b C h a c t ( c P t g b c a t t K p t t o L a Downloaded from https://academic.oup.com/icb/advance-article/doi/10.1093/icb/icae069/7693114 by Smithsonian Libraries and Archives user on 03 July 2024dent ifie d to spe cies by the ir mo rpho logy ( Lessios and earse 1996 ). An estimate of historical intro gre ssion ould, thus, no t be obtaine d, except through weak vidence from lin kage dise qui libr iu m and dev i a- ion s from Hardy–Weinb erg equ ilibr iu m. The mit o- ho nd rial phy logeny of Lessios et a l. (2001) , b ase d n ly on m atern a l ly in herite d loci, cou ld not pro- ide a definitive answer as to whe the r ind iv idu als ith D. pa uc ispin um haplotypes were pure mem- ers of this species, or whe the r the ir mt-DNA ow existed in a D. sav igny i nuclear b ackg round, aving spread outside the cent ra l Pacific through nt rog ression. Sequencing of bi ndin by G e yer et al. (2020 ) found hat a l leles of D. seto su m were on a separate clade than hose for me d by a l leles of a l l other spe cies of Diad ema ; he s ame was true for alleles of D. cla rki . Allel es of D. av igny i and D. pa uc ispin um , howe ve r, we re often in the ame cl ade. Four bindin firs t exon sequences of these wo species were ident ica l. The sh ared pol y morph isms n thi s genu s rai se the question of whe the r the y are he result of incom plete sor ting (i. e. , whe the r the y have e en in herite d from their com mon ancestor but did not ave enough time to dive rg e ), or int rog ression that oc- ur red af ter spe ciat ion. D at a from mito ch o nd rial ( Lessios et al. 2001 ) and nu- lear ( G e yer et al. 2020 ) DNA provide an op p or tunit y o estimate the deg re e of histor ica l genet ic exchange of indin between symp at ric species of Dia de ma and thus ss ess t he import ance of int rog ression in their evolu- ion. The deg re e of int rog ression of bi ndin al so h a s im- lications re gard ing its efficacy in re pro ductiv e isol a- ion. Here, I use the data of Lessios and Pearse (1996) , essios et al. (2001) , and G e yer et al. (2020 ) to ad-ress this qu estion, qu ant ify rat e of bi ndin int rog res- ion , and evalu at e the nat ure of re pro ductiv e isol ation n this genus. aterials and methods ito ch o nd rial DN A var iation in Dia de ma wa s a ss ess ed rom sequences of the ATPase8 and ATPase6 gene s (642 p, he reinafter me ntione d as “ATPase86”) reported by essios et al. (2001) . Sequences of eight indiv idu als of . sav igny i and one of D. cl arki from Ma juro, the Ma r- ha l l Islands, were adde d. Lessios et a l. (2001) had found nly hapl otypes of D. clarki (listed as Dia de ma sp.) at hi s i sl and , but the mo re re cently se quence d haplotypes ndicat ed that D. sav igny i i s al s o pres ent . The samp led ndo-Pacific species were D. paucispin um ( n = 79, both and b clades), D. clarki ( n = 21), D. sav igny i ( n = 107), nd D. seto su m- a ( n = 87). Dia de ma seto su m -b fro m the rab ian Penins ula was not included in the ana lysi s of ybr idization , because it was not found in t he s am e ar ea ith any other species of Diad ema . Sampl es came from lip perton , Easter Isl and , Pi tcai r n, Moore a, Kir it imat i, n d Hawai i in the cent ra l Pacific; Samo a, Rarotonga, he Cook Islands, Fiji, New Ca le donia, Fantome Isl and Ea st Au st ra li a), Papu a New Gu inea, Majuro, Gu am, the hilip pi nes, Tai wan , Okin awa, Kyu sh u, Honsh u, Ish i- a ki (Jap an), Hon g Kon g , and Sing apore in the West Pa- ific; the Lam ar ck an d Wh it e Isl and s, Ninga loo, Gera ld- on (West Aust ra li a), R éunion, Sout h Afr ica, Zan zibar , enya, an d Oman in the Ind ian O ce an. D et ai le d loca li- ies, sample sizes per loc alit y in each species, and meth- ds of DNA ext ract ion, amp lificat ion, and se quencing re listed in Lessios et al. (2001) , as is the list of many 4 H. A. Lessios Table 1 Highest probability density points of introgression events between Indo-Pacific species of Diadema 2NM (95% HPD) 2NM (95% HPD) sp1 sp2 sp1 → sp2 sp2 → sp1 D. setosum D. clarki 0.002 (0.000–0.256) 0.002 (0.000–0.486) D. setosum D. savignyi 0.003(0.000–0.597) 0.003(0.000–0.567) D. setosum D. paucispinum 0.003 (0.000–0.571) 0.018 (0.000–0.542) D. clarki D. savignyi 0.002(0.000–0.666) 0.012(0.000–0.342) D. clarki D. paucispinum 0.002 (0.000–0.339) 0.007 (0.000–0.021) D. savignyi D. paucispinum 1.069 (0.046–3.456) 0.412 (0.000–2.791) Range of 95% Highest Probability Densities (HPDs) are shown in paren- theses. Downloaded from https://academic.oup.com/icb/advance-article/doi/10.1093/icb/icae069/7693114 by Smithsonian Libraries and Archives user on 03 July 2024people to whom J. Pearse and I were indebted for col- le ct ions. Sequences of the first exon of mature bindin were from a subsample of indiv idu al s u sed in Lessios et al. (2001) : D. pa uc ispin um ( n = 16), D. sav igny i ( n = 8), D. clarki ( n = 9), and D. seto su m -a ( n = 8) ( G e yer et al. 2020 ). ATPas e86 s e quences provide d spe cies ident ifica- t ions, be cause mo rpho logy in Dia de ma can be mislead- ing. It i s, thu s, a ss u med that mito ch o nd rial DNA del in- eates species of Dia de ma . To determine whe the r ind i- v idu als were hetero zygotes in bi ndin, up to five clones were obtained fro m each. Sequence differe nces between clon es from t he s ame indiv idu al were ass u med to be in- dicative of a hete roz ygote if they differed at more than on e sit e , or if they differed at on ly on e sit e , but this differ- ence was consistent in more than one cl one. The bindin data were fro m Easter Isl and , the Cook Isl and s, Majuro, Okin awa, Kyus hu, Ishigaki, the Philip pi nes, Papua New Guine a, Zanzib ar, Réunion, and South Africa. Gene flow between species of Dia de ma sinc e the ir sep arat ion from e ach ot her wa s estim ated with the isolat ion–mig rat ion pr ogram IMa3 ( Hey and Niel sen 2004 ; Hey et al. 2018 ), incl uding the two mito ch o nd rial genes as a single (haploid) locus, and the bind in se- quences as a dip loi d locus. The program uses gene ge- nea log ies to pr odu ce Bayesian est imates, b ase d on co- a lescence, of effe ct ive pop u lat ion size (2 N eμ, where μ is t he mut ation rate) of ancest ra l and dau ght er pop- u lat ions, the t ime since their init ia l sep arat ion ( T μ), and the rate of gene flow in each dire ct ion (m/ μ). The mito ch o nd rial phy logeny of Lessios et al. (2001) pr o- vided a fixed species tree ( Fig. 1 ). I estimated gene ex- chang e and effe ct ive pop u lat ion sizes in six p airwise com parison s betwe en the four species. A n alyses were imp lement ed wit h t he Hasegawa et al. (1985) (HKY) mo del for both loci. Pr eliminar y runs we re per for med to op t imize the bou nd s of the pr ior s, the n the y were run in 80 chains with ge omet r ic he ating for 107 steps, sampling 105 ge nea log ies every 102 ste ps afte r a burn- ing of 4 × 106 steps. The IMa3 runs were done in the CIPRES porta l ( Mi l ler et a l. 2010 ). Conve rg e nce was de- termined by comparing estimates resulting from three runs with different random se e ds but with the same pri- ors. As the results of the three runs were very simi- lar, est imate d p aramete rs of only one are shown here. Be cause est imates pr odu ce d by IMa3 are a funct ion of mut ation rate , to ca lcu late demog raphica l ly sca le d estim ates, I us ed ATPa se86 dive rg e nce of 4.24% be- tween D. antil l a rum and D. mexic anum , ass u med to have be en sep arate d by t he Ist hmus of Pana ma com- pletion ∼3 Mya ( Lessios et al. 2001 ). There is no re- com bi nation (that con founds co a lescenc e) in the first exon of bi ndin. G e yer et al. (2020) us ed four me tho ds of est imat ing rat ios of rate of amino acid replaceme ntand silent su bst it ut ion s to ass ess sele ct ion on the mature bind in mo le cu le. They provide d no evidenc e of posit ive sele ct ion in eit her t he first or the second exon of bi ndin, except for the branc h leading to D. clarki ( G e yer et al. 2020 ). Results Est imate d effe ct ive pop u lat ion sizes and rate of gene flow between species of Dia de ma are sh o w n in Fig . 2 . Hybr idization bet ween D. s eto su m or D. cl a rki and t he ot her two species was essent ia l ly nu l l; the lowest limit s of the rang e of 95% highest pr obabi lit y densi- t ies (HPD) of a l l mig rat ions in these com parison s we re invariabl y zero ( Table 1 ). Int rog ression in both direc- tions betwe en the sist er sp ecies D. pa uc ispin um and D. sav igny i , on the othe r hand , was mo re than a hund red t imes g re ater t han t hat of any ot her compar ison. From D. pa uc ispin um , an ave rage of 0.4 bi nd in geno types were tran sfe r red into D. savig nyi eve ry ye ar (or eve ry ge nera- tion, as Dia de ma pro bably re aches sexual maturity in 1 ye ar). From D. savig nyi , 1.1 a l leles per ye ar we re trans - ferred into D. pa uc ispin um . Thus, the sp ecies b ound- ary between these two recip r oca l ly mo no phyletic mito- cho nd ri al sister cl ades h a s be en crosse d by bindin ev- ery 1–2.5 years since spe ciat ion , by hybr ids fit enough to back c ross an d tran sfe r their a l leles. The rates of in- t rog ression betwe en D. pa uc isp inum an d D. sav igny i r ival t hos e s een in ston y cor a ls, a g roup in which hybr idization is com mon ( Willis et al. 2006 ), and in w hich retic ulate evolu t ion is thought to be widespread ( Veron 1995 ). 2 N em values of 0.4–2 prop agu les per ge neration we re est imate d by the isolat ion–mig rat ion mo del as tran sfe rred between the scleractinians Acro- pora cythere a and A. hyacinthus ( Ladner and Palu mbi 2012 ), and 0.29 between Por ite s evermann i and P. lo- ba ta ( Hel lb erg et al. 2016 ). The same mo del provided 2 N em estimates of 0.13–1.05 between sister species of snap ping shrimp of the genu s Alp h a e us ( Hurt et al. 2013 ). Introgression of bindin in Diadema 5 Fig. 2 Estimates of effective population sizes and bindin introgression rates between Indo-Pacific species of Diadema , superimposed on the mitochondrial phylogeny of Lessios et al. (2001) . Width of rectangles represents the Highest Probability Density (HiPt) of effective population size ( N e), lines re pre sent the 95% HPD intervals. Labels on arr ow s indicate the HiPt of the number of introgression events (2 N em ) per generation between the species in the direction indicated by arrows (forward in time). 95% HPD ranges are shown in Table 1 . Estimates of introgression events are shown between D. setosum and all other species (panel A), D. clarki and D. savignyi or D. paucispinum (B), and D. savignyi and D. paucispinum (C). r ( t i t ( s ( f v t D v e D o T t h e l w i i t M 2 ( v Downloaded from https://academic.oup.com/icb/advance-article/doi/10.1093/icb/icae069/7693114 by Smithsonian Libraries and Archives user on 03 July 2024Effe ct ive pop u lat ion sizes of D. pa uc ispin um 4.35 × 108 indiv idu als) and D. 8 sav igny i (5.5 × 10 ndiv idu als) were much larger than that of D. cl arki 9.6 7 × 10 indiv idu a ls), whi le that of D. seto su m 2.1 × 108 indiv idu al s) wa s int ermediat e. The rel ative alu es of D. pa uc ispin um and D. sav igny i versus th at of . seto su m may well reflect the expected larg e r ge netic ariation in hybr idizing species, but the much sma l ler st imate d pop u lat ion in D. clarki could be an artifact f sma l ler sam ple sizes an d of sel e ct ion on bi ndin in hi s species, a s co a lescence ass u me s sel e ct iv ely neut ra l volu t ion . Neverthe l ess, pop u lat ion sizes correlate it h t he kno wn rang e s of the species. Dia de ma cla rki s only known from Japan ( Chow et al. 2016 ), the arsha l l Islands ( Lessios et al. 2001 ), and Ind one sia Moore et al. 2019 ), whereas the other three speciesang e ove r the entire Ind o-Pacific. Repor ts of the dis- ribut ion of newly re discovere d spe cies are expe cte d o underestimate their rang e , bu t ou t of 29 location s ampled by Lessios et al. (2001) in the Indo-Pacific or ATPase86, D. cl ark i haplotypes we re only found in wo. iscussion he discovery of hybridization in various animal taxa a s led to the proposal that species bar r iers are se- e ct iv ely por ous, wit h som e gene s int rog ressing read- ly betwe en geno me s, while othe rs ar e bar re d fro m en- ering by natural s election (re vie ws in Abbott et al. 013 ; Edelman an d Ma l let 2021 ). This view, in turn, fa- ors ca l ls for the abandon ment of the bi olog ica l spe cies 6 H. A. Lessios Downloaded from https://academic.oup.com/icb/advance-article/doi/10.1093/icb/icae069/7693114 by Smithsonian Libraries and Archives user on 03 July 2024concept ( Ma l let 2008 ), a s thi s conc ept define s species by their re pro ductiv e isol ation ( May r 1963 ). The docu- ment at ion pr e sented here , that bindin int rog ression oc- curs between siste r species of Dia de ma but no t between more dist ant ly relate d spe cies, does not directly address the rate of a l lele t ran sfe r in othe r loci. Bind in, ho wever, is not a ran dom nuclear locus, but a GRP, potent ia l ly capabl e of bl ocking cross-fert i lizat ion betwe en spe cies. Differences betwe en a l leles in a locu s th at aff ect the ef- ficiency of cross-fert i lizat ion dire ctly aff ect the por osit y of the entire genome. My an alysi s necess ar ily ass u mes that mt -DNA distin- guish e s b etween sp ecies of Dia de ma , and that bind in, rat her t han mt-DN A, is being exc hang e d betwe en D. pa uc ispin um and D. sav igny i . Thi s a ss u mption, made necessary by the unrel iabi lit y of mor ph ol og ica l spe cies ident ificat ions, i s al so ba sed on the mo no ph yly of mt- DNA ( Lessios et al. 2001 ) between the species and the paraph yly of bi ndin ( G e yer et al. 2020 ). Under thi s a s- sumption, the sh aring of bi ndin a l leles betwe en D. pau- cispin um and D. sav igny i is not the result of incom- plete lineage shor ting, but rather of hybr idization that h a s ha ppene d sinc e the s e species were s ep arate d from e ach ot he r. Bind ins of D. cl a rki and D. seto su m co a lesce wit hin e ach of t hese species. The a l lozyme study of Lessios and Pearse (1996) ident ifie d one ind iv idu al tha t wa s an F 2 (or later gener- ation ) hybr id bet ween D. s av igny i and D. pa uc ispin um . It al so di scove red seve n hybrids betwe en D. sav igny i and D. seto su m , and one hybr id car rying a l leles from a l l thre e spe cies. Thus , hybrid s of D. sav igny i with D. seto- sum can be found on the reef at the same time, but, as the present an alysi s sh o ws, bind in int rog ression betwe en the m does no t occur. Sampling by Cho w et al. (2014 , 2016) h a s fai le d to locate D. cla rki at Ok inawa. Altho ugh their exact frequency in the pop u lat ion cannot be calcu- late d, nine natural hybrids in a single generation on two Okinawan reefs, each searched once, indicate that het- erospe cific fert i lizat ions are far fro m rare . That eight of t hese hybr ids we re F 2 or later ge neration off spr ing indi- cat es that hybr ids re ach sexual matur ity an d backc ross or mate wit h e ach ot her. Yet, t he IM an alysi s indicates that bindin int rog ression is pr esent on ly betwe en D. sav- ignyi and D. paucispin um , and that even between these species bindin allele tran sfe r h a s occurre d on ly once in a generation in on e dire ct ion and on ly eve ry 2.5 ye ars in t he ot her. Frequent hybr ids in one gene ration but low int rog ression since spe ciat ion sug gest t hat pre zygot ic isol ation in Diad ema is weak. Uehara et al. (1990) re- ported that gametes of D. sav igny i and D. seto su m are cap able of fert i lizing each other in the labor ator y at un- spe cifie d sperm concent rat ions. In addit ion to bi ndin, a potent ia l pre zygot ic b ar r ier to hybr idization bet ween D. s av igny i and D. seto su m isspawning that peaks at different ph a ses of the moon ( Cop p ar d an d Cam pbell 2005 ). It might be thought that the 15-day face shift in spawning h a s evol v ed by rein- for cement, but a similar difference exists between D. antil l a rum and D. mexic anum , sep arate d by the Cen- t ra l Amer ican Ist hmu s ( Lessios 1984 ). Int raspe cific syn- chron ization of spawning in D. seto su m and D. savig nyi is not tight ( Pearse 1968 ). That hybrids are pr odu ced in- dicat es that game tes of the two species are in the wa- ter column at the same time , and that ne ithe r bind in no r async hrono us spawning cyc les are ve ry efficient in block ing inte rs p ecific cross es. Intro gre ssed bindin al- lel es, ho we ve r, survive ove r some ge nerations , but in deepl y s eparated species, the y are e vent ually eliminat ed from the gene p o ol. Separate species of symp at ric Di- ad ema are, thus, mai ntai ne d as ind epend ent gene tic en- t it ies by post-zygot ic isolat ion resu lt ing from low hybr id fitness. Lower hybr id fit ness cou ld be cause d by a l leles that are not fu l ly comp at ib le whe n int roduce d into the geno me of a sist er sp ecies. A genomic scan of the species of Dia de ma would he l p ident ify such a l lel es and , thus, begin to sh e d light into the causes of post-zygotic isola- t ion betwe en spe cies of sea urchins. That a gre at de al is known about the function of genes in sea urchin devel- opm ent ( Davidson et al. 2002 ) su ggest s t hat t his type of invest igat ion may be f r uit fu l. One might expect that mar ine t axa wit h exter nal fer- t i lizat ion wou ld be mor e pr on e to int rog ression than t axa wit h inter na l fert i lizat ion. A nim al s with beh avioral and ph ysiol og ica l spe cies re cog nit ion sh o uld b e b et- te r insulate d ag ainst inte rs pe cific mat ing and less pr on e to sh o w the effe cts of int rog ression in their evolution- ary histor ies. Yet, t here is a host of documented cases of int rog ression in arthrop o ds, am phibian s, birds, or ma mm al s, but a paucity of examples of inte rs pe cific a l- lele tran sfe r in spong e s, an thozoan s (exc ept cor als), bi - va lves, or e chinoderms. Th at ma ny ca ses of int rog res- sion have been discovered in groups such as arthrop o ds may be simpl y the result of the pr obabi lit y that l arg e , di- verse groups are more likely to illustrate any given phe- no me no n. The trend also und oub te d ly refle cts the in- tensit y of effor t devote d to evolu t ion ary studies in each group. As the study of int rog ression h a s imp licat ions re- garding the por osit y of species genom es, the ut i lit y of species conc epts, and ultimatel y the manne r in which variat ion in nat ure is generat ed an d mai ntai ned, its oc- currence in taxa with external fertilization deserves at- tention. Acknowledgments As t he cit ations in this paper make cle ar, t hi s study wa s made possible by John Pears e, an idea l ly amicabl e, hard- wo rking , and knowledge ab l e col labor ator . I al so th ank Introgression of bindin in Diadema 7 t e a E s G C T G G D S i G A A H A P M H R H A H A H A H A H A A H B C H H C I C L L C C L C L L D sea urchin Dia de ma . Evolu t ion 55:955–75. Downloaded from https://academic.oup.com/icb/advance-article/doi/10.1093/icb/icae069/7693114 by Smithsonian Libraries and Archives user on 03 July 2024he ho st of people (list ed in Less ios et al. 2001 ) who gen- rousl y contrib ute d spe cime ns, and Alexand ra Hi l ler nd Laura G e yer for com ments on the m anu script. Thi s tudy is de dicate d to the memory of John S. 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