Hybridizing toads with different sex chromosomes

A new study pub­lished in Evol­u­tion Let­ters shows that spe­cies with dif­fer­ent sex chro­mo­somes can inter­breed. Lead author Prof Chris­tophe Dufresnes tells us more.

How do organ­isms become male or female? It var­ies between ver­teb­rates. Humans, like all mam­mals, do it with a Y chro­mo­some – a chro­mo­some that car­ries a “male determ­in­ing” gene that is there­fore only ever found in males. When it is the male that car­ries this “sex-lim­ited” chro­mo­some, it is termed an XY sys­tem: males are XY and gen­er­ate daugh­ters and sons by trans­mit­ting the X or the Y, respect­ively, while females are always XX. Anoth­er sys­tem exists, how­ever, in which a gene determ­ines female­ness. The chro­mo­some hold­ing that gene is called the W. In these “ZW” sys­tems, the females carry Z and W, and trans­mit either one to the next gen­er­a­tion to make ZW females or ZZ males.

Mam­mals and birds are known to have extremely old sex determ­in­ing sys­tems (hun­dreds of mil­lion years old). But the situ­ation in amphi­bi­ans (frogs, toads, sala­man­ders) is much more dynam­ic. Closely related lin­eages (some less than 2 mil­lion years diverged) can pos­sess dif­fer­ent sex chro­mo­some sys­tems – either XY or ZW, and not neces­sar­ily involving the same chro­mo­some pairs. Some frogs even use sev­er­al chro­mo­some pairs at the same time. In fact, sex-determ­in­a­tion mech­an­isms have switched so many times that it is hard to keep track of their evol­u­tion, and since only a few muta­tions may suf­fice to trig­ger male vs female devel­op­ment, this makes sex chro­mo­somes chal­len­ging to study. Need­less to say, the amphi­bi­an tree of life is a mosa­ic of sex determ­in­ing sys­tems, and this means that these sys­tems can some­times col­lide. This is what happened between two wide­spread European amphi­bi­ans, the com­mon toad Bufo bufoand its sis­ter spe­cies the spiny toad Bufo spinosus.

This spiny toad (Bufo spinosus), like us humans, determ­ines sex with XY chro­mo­somes, but its closest rel­at­ive (Bufo bufo) uses a dif­fer­ent sys­tem (ZW). Des­pite this, they can still hybrid­ize, but most for­eign genes do not make it very far.

Based on appear­ance alone you would be for­giv­en for think­ing that B. bufo and B. spinosus were the same spe­cies. In fact, they were not offi­cially recog­nized as dif­fer­ent spe­cies until 2012. After the last ice age, they expan­ded from sep­ar­ate refu­gia in south­ern (B. spinosus) and east­ern Europe (B. bufo), and now meet all over France at so called “sec­ond­ary con­tact zones”. These sites are gold­mines for sci­entif­ic stud­ies, as they act as nat­ur­al exper­i­ments to learn what hap­pens when diver­ging lin­eages hybrid­ize. Do they merge back into one spe­cies, or have they become so dif­fer­ent that their gen­omes have accu­mu­lated incompatibilities?

It was as part of a glob­al effort to address these fun­da­ment­al ques­tions using next-gen­er­a­tion sequen­cing in Euras­i­an amphi­bi­ans that I ini­tially focused on the B. bufo / spinosus con­tact zone with Dr. Pierre-André Crochet (CNRS), and we star­ted col­lect­ing samples with loc­al nat­ur­al­ist soci­et­ies. But this con­tact zone came with a sur­prise. Our pre­lim­in­ary ana­lyses revealed XY genet­ic mark­ers in some south­ern samples, assigned to B. spinosus, while toads from the genus Bufo had been believed to be ZW since the 1940s! This is exactly the sort of gen­ome incom­pat­ib­il­ity that would hamper hybrid­iz­a­tion and facil­it­ate the diver­gence of new spe­cies. So, this res­ult war­ran­ted more exploration! 

We sub­sequently sampled dozens more samples includ­ing adults and a few fam­il­ies (par­ents and tad­poles) from each spe­cies, in col­lab­or­a­tion with Dr. Daniel Jef­fries (Uni­ver­sity of Lausanne), who spe­cific­ally developed a new stat­ist­ic­al meth­od to ana­lyze sex chro­mo­somes using our large gen­om­ic data­sets. These efforts cor­rob­or­ated that B. bufoand B. spinosus had dis­tinct ZW and XY sex chro­mo­somes, a strik­ing example of how labile sex-determ­in­a­tion can be in amphi­bi­ans. On this top­ic, we pub­lished anoth­er study detail­ing switches between XY to ZW sys­tems in Euras­i­an tree frogs, togeth­er with col­leagues from River­side, Har­vard and Lausanne.

Our sampling tran­sect along the hybrid zone between Bufo bufo (blue) and Bufo spinosus (red) in south­ern France. On the map, notice how the trans­ition switches from B. bufo to B. spinosus with­in a few short kilo­met­ers (blue-red contact).

The left graph details the trans­ition for hun­dreds of genet­ic mark­ers sep­ar­ately. Notice how much smooth­er the trans­ition is on the B. spinosus side for a few genes, as B. bufo vari­ants reached far south (e. g. the dash line). This is poten­tially a sign that B. bufo was replaced in the south, but some of its genes still persist.

We knew by then that these closely related spe­cies were in close con­tact with each oth­er, but had dif­fer­ent sex determ­in­ing sys­tems, one XY and one ZW. So the big ques­tion was, what hap­pens when they attempt to repro­duce with each oth­er? Using thou­sands of genet­ic mark­ers, we con­firmed gene exchanges between the spe­cies, indic­at­ive of hybrid­iz­a­tion. This is already an import­ant res­ult – they are able to repro­duce. How­ever, the gen­omes showed very restric­ted pat­terns of mix­ing, with no hybrids found more than few kilo­met­ers out­side the con­tact zone. This sug­gests that hybrids prob­ably suf­fer from reduced sur­viv­al and/or fertility.

Des­pite this, faint genet­ic traces of B. bufo were detec­ted as far south as the Medi­ter­ranean coast, with­in the B. spinosus pop­u­la­tions. These res­ults appear to be con­flict­ing at first glance, but by mod­el­ling eco­lo­gic­al pref­er­ences, a col­lab­or­a­tion with Dr. Spar­tak Litvin­chuk (Rus­si­an Academy of Sci­ences), we think that this may be because the south­ern spe­cies B. spinosus, which seems bet­ter adap­ted to Medi­ter­ranean con­di­tions, has been shift­ing north­wards for hun­dreds of years, push­ing B. bufo out of Provence – a phe­nomen­on per­haps intens­i­fied by glob­al warming.

We can­not firmly draw a caus­al link between the restric­ted admix­ture and the dif­fer­ing sex chro­mo­some sys­tems at this stage. Sex chro­mo­somes are often involved in the prob­lems exper­i­enced by hybrids, at least in mam­mals and birds, but this not neces­sar­ily true of amphi­bi­ans, where sex chro­mo­somes are so dynam­ic that they do not accu­mu­late as many defects or sex spe­cif­ic func­tions. So, as always, there is more work to be done. Next on the list is to exam­ine the sex and con­di­tion of hybrids from con­trolled crosses between the two spe­cies and to see if some of the inher­ited com­bin­a­tions of sex chro­mo­somes (e. g. ZW–XY, ZZ–XX) are more viable than the others.

This strange toad case raises aware­ness about the poten­tial role of sex-determ­in­a­tion mech­an­isms on the form­a­tion and main­ten­ance of nov­el spe­cies. This per­spect­ive will be fas­cin­at­ing to explore in addi­tion­al amphi­bi­an groups that sim­il­arly fea­ture evol­u­tion­ary lab­il­ity in sex determination.

Chris­tophe Dufresnes is Pro­fess­or at the Col­lege of Bio­logy and the Envir­on­ment, Nanjing Forestry Uni­ver­sity. The ori­gin­al art­icle is freely avail­able to read and down­load from Evol­u­tion Letters.