It makes a difference because with the added quotes, you're splitting on a literal string rather than a regular expression.

If you want to split along tab characters, then the following are equivalent:

    @data{@speciesList} = split /\t/, $_, scalar @speciesList;

    @data{@speciesList} = split "\t", $_, scalar @speciesList;
[download]

But the following is not:

    @data{@speciesList} = split "/\t/", $_, scalar @speciesList;
[download]

as that splits on a slash followed by a tab followed by another slash, rather than just a slash. Here's a quick demonstration that may be instructive:

#!/usr/bin/perl

use strict;
use warnings;
use feature qw/say/;
use English;

$LIST_SEPARATOR = ",";

while(<DATA>) {
    chomp;

    my @a = split /\t/;
    my @b = split "\t";
    my @c = split "/\t/";

    say "@a";
    say "@b";
    say "@c";
}

__DATA__
foo     bar
foo/    /bar
[download]

This outputs:

$ perl 1119187.pl
foo,bar
foo,bar
foo     bar
foo/,/bar
foo/,/bar
foo,bar
$
[download]

EDIT: posting the above script converted tab characters to spaces in the __DATA__ section, so you'll have to change those back before running the script to get the correct output.