So that everyone is “on the same page,” let’s consider the classical example of horse-donkey breeding (i.e. two different species). Usually, the male donkey (jack) mates with a female horse (mare) to produce a mule (they can be either sex). Less commonly, a male horse (stallion) mates with a female donkey (jenny) to produce a hinny (again, they can be either sex). Whereas humans have 23 chromosomal pairs — horses have 32 chromosomal pairs, and donkeys have 31. During fertilization of an egg by a sperm (meiosis), therefore, the extra chromosome cannot pair up. During divergence of horse and donkey, not only has the donkey lost one chromosome, but chromosomal rearrangements and inversions have occurred over evolutionary time.
For the mule or hinny, each cell ends up with 63 chromosomes; because of these dissimilarities, the end result is usually (but not always) sterile offspring. There are no recorded cases of fertile mule stallions. There have been a few dozen cases of mule mares giving birth after mating with a horse or donkey. 😊 According to a British Broadcasting Company (BBC) report, “Only 60 cases of mules giving birth were recorded from 1527 to 2002 (spanning nearly 500 years). In recent times, mules produced a filly in China in 2001, and colts produced mules in Morocco (2002) and Colorado (2007).” According to the American Donkey and Mule Society, however, only one hinny mare has ever been known to give birth (in China in 1981). On the other hand, male mules breeding with hinnies apparently cannot produce offspring. ☹
To answer your question: successful reproduction becomes increasingly difficult, as more chromosomal rearrangements and inversions have occurred over evolutionary time. Having an extra (unpaired) chromosome makes things even more difficult — but not impossible — in this stochastic world. There needs to be a better definition (but perhaps that is impossible?) for “what represents a ‘species’ vs what represents a ‘subline’ capable of generating offspring.” This is the problem with Mother Nature; we always seem to see GRADIENTS. I do not think that Fundulus grandis and Fundulus heteroclitus should be defined as “distinct species.”