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I think Twigs is agender so you can use the pronouns she/her or him/his but they prefer if you use they/them pronouns with them

Timber certainly has a penis and is capable of ejaculation. It's not ambiguous. 

Warning: explicit language (and spoilers I guess?)

When you have sex with Timber in your tent in the human route, you ask him to "whip it out for you," and you think to yourself, "you're about to give him the best blowjob ever." Timber eventually says you "just can't go on livin' without slurpin' some wolf dick." You even get the choice to spit or swallow.

I tried to use a spoiler for this section but it did not work D:

That's Over

The only point of ambiguity is that we can't say for certain that Timber is cis. He could be intersex, as some intersex folks do have penises, and may be able to ejaculate in some cases, but, whether or not an intersex person identifies as cis or trans is a personal choice that has a ton of factors. But given that Nami isn't really shy about explicit representation and the contrast with how Tofu/Plum/Twigs have no mention of sexually dimorphic characteristics, he's probably cis.