open I16S, '<', $in16S;
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You don't test that open succeeded so this code has the same problem as the OP's code.

my %r_genus16S;
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You don't use this variable anywhere so why are you creating it?

my ($genus16s) = <I16S>;
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That reads the entire file and assigns the first line to $genus16s.

while ( defined( ${'genus16S'} = readline(*I16S) ) ) {
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Which means that this loop will never execute because I16S is already at EOF at this point.    And WTF is up with ${'genus16S'}?