So...if I cancel rental car, how to best change itinerary for May-Tuscany/Puglia-

I think I suffer from Attention Deficit Disorder. Sorry but I could read only half of your post.

You plan A works although you don’t say where you intend to spend nights. My preference is always to minimize the number of bases so that I can maximize the number of nights at each location and use that base for day trips to locations within one hour drive. Changing hotels is time consuming (unpacking, move, check in, unpack, repack, checkout etc.).

Many of the locations you listed are close to one another so you should find a central hub in each area and visits the towns within one hour radius from that hub.

Regarding the fuel shortage, stop being negative. You are going in May. There will be no total shortage of gasoline in the next month. Italy has an enormous refining capacity and although it imports crude oil (mostly from North Africa, Libya in particular, our former colony) it actually exports refined fuels. The problem is only jet fuel, which Italy imports for 40% of its needs (only 60% is refined in Italy). Gasoline will be available and so far there are only some delays in the deliveries which are causing some stations to run out before the next delivery. Just make sure you refuel often, and not let your tank fall too low so that you don’t get stranded somewhere. Obviously prices might go up. So far they haven’t because the Italian government has temporarily cut the gas tax by 25 Eurocents per liter, but that is a temporary measure. Your only risk is that your plane may be stuck in Italy because of lack of jet fuel and that you may have to ask for asylum in Tuscany or Puglia. If it comes to that I can suggest some long term accommodations in either region, and the cost of living in Italy is about half the one here, so you might not mind to be stuck there for a while. Stop worrying also about the possibility of exceeding the 90 day visa stay. Prime Minister Giorgia Meloni has come to your rescue. In case of overstay there will be no penalties if you exceed your time in the Schengen area, and the airlines will need to provide food and shelter for the additional time in Italy. See. There is a reason why both Joe Biden and Donal Trump like her so much. It’s not just because of the blond hair and blue eyes.

Interesting- I guess I do have concerns of being stuck in Italy but I actually had never considered going over the 90 days, thanks for that thought. I have a disabled adult child that I need to get back to take care of and my husband has to get back to work, he cannot do his job remotely. Yes my post is a detailed, I should have said 2 nights florence, 3 nights siena, 3 nights orvieto, 2 nights matera, 4 nights locorotondo, 3 nights otranto. The only thought I came up with was to remove matera and or otranto and add to locorotondo, but the place booked already. Our travel is a respite we need and they are very planned in hopes to not have to think much while on vacation if we don't want to. We considered the train while I was planning, your posts actually encouraged us to get the car, I have your instructions written down to avoid the ZTL for leaving Florence from Borgo Ognissanti to get to S222. I certainly have no idea what the future will hold.

Norhtwestern - Although not the crux of your post, I just wanted to add that Roberto's instructions to avoid the ZTL for leaving Florence from Borgo Ognissanti to get to S222 were excellent. I followed them a couple of years ago and made for an easy departure from downtown Florence. Best of luck with your trip.

You have 17 nights on the ground, but a lot of towns in your bucket list. You definitely need a car to squeeze them all in your time available, as the public transport would not be as efficient. Just remember that 2 nights is really just one day in a place (plus a few hours on the day of arrival), 3 nights is really just 2 full days (plus a few hours on the day of arrival) and so on.

Matera can also be visited on a day trip from Locorotondo (about 1h20m drive), although many prefer to spend at least one night as the Sassi area is really beautiful at night. So it's up to you.
When I'm in the area I usually stay at the resort below, it's actually just outside Alberobello (10 min drive from Locorotondo). Since I always travel to Puglia in summer, a pool is a must for me.
https://maps.app.goo.gl/EPN1T8e2j1SVFYpV6

3 nights in Otranto give you 2 full days. May is a bit early for a beach vacation, but Otranto is beautiful (a fortified town) and you can certainly enjoy it on the day of arrival and the evenings. There is a nice cathedral (Duomo) and the Aragonese castle that are worth a visit. The two full days you have available can be utilized for day trips. One certainly to Lecce (40 min drive from Otranto), and one possibly to Gallipoli (less than one hour away). Basing in Lecce instead of Otranto, is another possibility if you prefer a larger city, but in your shoes I'd rather stay in Otranto. Below is where I stayed in Otranto, it's convenient for a beach, but the historical center is also only 10-15 min walk away.
https://maps.app.goo.gl/cxmCXsyU2sLfiKLi6

Regarding the rental car pick up in Florence, although many offices are still on Borgo Ognissanti area (Avis-Budget, Europcar), however Hertz has moved to Via Alamanni, a street next to the SMN station, while Europcar and Avis-Budget no longer use the Europa garage on Borgo Ognissanti, therefore, unless they bring the car to you on Borgo Ognissanti, your car pick up may actually be at the underground garage on Piazza della Stazione (under the station basically), where Sixt also is.

If the pick up is at the underground garage at the station below is the directions out of the city center. It is actually to a gas station across the river. Once you are at the gas station, reset your next directions from there to the SR222
DIRECTIONS OUT OF THE SMN STATION GARAGE TO TAMOIL GAS STATION: https://maps.app.goo.gl/NMDCpyKcJRgURryQ7
DIRECTIONS FROM TAMOIL GAS STATION ABOVE TO RADISSON BLU HOTEL (Start of SR222): https://maps.app.goo.gl/sh9i8NakiZGEgVQo9
Once at the Radisson Blu hotel, follow directions to Siena (SR222). If you set your navigator to Siena, the navigator will put you back on the freeway because the freeway to Siena is faster than the SR222, therefore, set your navigator to GREVE IN CHIANTI, which is on the SR222, maybe 30 min drive after the Radisson Blu. Once in GREVE then you can set the navigator to Siena and be assured that the navigator will not send you to the freeway Florence-Siena (which runs parallel to the SR222, west of it, but far enough from Greve)

If you rent from Hertz on Via Alamanni, these are the directions to the same Tamoil gas station indicated above:
DIRECTIONS FROM HERTZ TO TAMOIL GAS STATION: https://maps.app.goo.gl/xN2gosYhDWZqqDEM9
Be aware of the bus lane (right lane) on via della Scala. Once you turn on via della Scala (one way street) move to the left lane otherwise if you stay on the right lane (bus lane) the camera takes a photo and you get a ticket later.
Then from the Tamoil Gas station you can follow the rest of the directions to the SR222 (Radisson Blue hotel) above.

Warning: between the Tamoil gas station and the freeway entrance at Firenze Scandicci toll booth, there is a speed camera on Viale Etruria which famously collects more than 1000 fines a day. The cameras are on the pole in the median pictured in the image below:
https://maps.app.goo.gl/uhEEVw9YCArDojyY8

Thank you Roberto as I do have a Europcar reservation through autoeurope- need to change all my directions and see if I can follow it. The pick up address on the voucher/reservation shows the borgo ognissanti address, I had no idea I would need to go somewhere else to pick up the car after I get the paperwork, my biggest uneasy about this trip was this section of driving and the google maps photos are "helpful" but intimidating. I guess we will be walking all over with our luggage that morning and praying we get out of town successfully. I am thankful for the new set of directions- will show the driver when he gets home from work, I am not comfortable- but I am not driving- just trying to make sure the phone is set to the right destination,

You’ll be fine no matter where you start from (Europcar office on Borgo Ognissanti or garage under station). The last time I used Europcar from Borgo Ognissanti was maybe 10 years ago. Since then, when I’ve picked up the car in Florence, I’ve done so directly at the Florence airport upon landing. I can tell you that that time at Europcar on Borgo Ognissanti was a nightmare. It was a Saturday in June and there were dozens of Americans in line picking up their rental cars. It took me 2 to 3 hours to finally get mine. After that time I swore to never pick up the car again downtown in summer when there are millions of North Americans in town. At that time they brought us the cars just around the corner on Piazza Ognissanti, in front of the French Consulate. However in more recent years that Piazza is no longer accessible to cars, as it has been made into a large pedestrian only plaza, and I doubt they can drive that huge amount of cars in front of the Excelsior Hotel across the street from the French Consulate. Maybe more recent renters from Europcar can chime in, but I think I read in this forum of someone who rented from that office, and after doing the paperwork was sent to the station, luggage and all, to pick up the car at the garage there. So be ready for that possibility.

OP- Do you ever plan on sleeping?