Thread by @BiolBulletin

It's #ReadAlong Day!

@wareslab will #TakeOver our account at 12 pm ET. Follow this thread for live tweets as we #ReadAlong Hines 1978 (journals.uchicago.edu/doi/pdf/10.230…)

Drop your questions and comments in this thread.

Happy Reading! 🧵(1/n)

@wareslab: Hello! For those of you who don’t know me - I study how diversity is distributed, often using molecular markers to define that diversity and work towards knowing the function and mechanism that makes patterns of it. (2/n) #TakeOver #ReadAlong

It’s wonderful! I think of the globe and patterns of movement across it, in so many ways, and I get paid to do that. (3/n)

One of my first publications - with the cover illustration! - was Biol Bull 2000. I had no *real* understanding of what I was doing then, but what I found jived with the work of some great paleobiologists before me &, one of several reasons I’m still in love with sea stars (4/n)

I’ve often gained from @BiolBulletin, as it is the journal that best replicates my experience with and love for the people who end up at places like Friday Harbor, Bodega, Shoals, Whitney, LUMCON - how do the organisms we study help us learn about this world and vice-versa? (5/n)

I still love the fact that I was invited to be an AE of the Bulletin *in the same email* that our current EiC "desk rejected" one of my papers on the pop gen of an intriguing barnacle and patterns along the Chilean coast! (6/n)

I’ll leave that story for another time, this isn’t about me, it’s about the journal. And honestly to work for a journal that has been publishing for well over a century about the stuff I love, well, it’s an honor! (7/n)

And I’m very happy with what Ken and the rest of the AEs actively do to try and boost the journal and its audience. So, when I was asked by our social media liaison, Mihika (@crustyolfactory) to try this virtual #ReadAlong, I loved the idea. (8/n)

At first I was bewildered by which of so many great papers to choose. (9/n)

But just taking a quick look through my refs, it was actually pretty easy. It’s a barnacle paper that incorporates wisdom about local climate effects, written well before we really learned to fear what we are doing to this planet in terms of climate change. (10/n)

Anson Hines 1978! First read this because I was learning how to use population genetic data to explore movement and isolation in a couple of ways, early in my dissertation. (11/n)

Understanding barnacles in particular for oceanic movement and isolation has been a regular pattern in my work. I’m not sure I understood much other than “that’s when they tend to reproduce” when I first read it (you’ll see what I mean). (12/n)

My understanding of movement in marine organisms at the time was about as sophisticated as gleaning figures from paleo & physical oceanography papers of coastal environments that happened to have arrows drawn on them. I hope my colleague Jamie Pringle has fixed me by now. (13/n)

Hines 1978 reappeared in my life as a postdoc with Rick Grosberg. I was paid to study diversity and patterns in Balanus glandula (a recurring theme in my life) and began to get a closer sense of how upwelling, currents, phenology, and other life history worked together. (14/n)

I also learned more about experimental manipulation and observation in the Grosberg lab, for which I’m incredibly grateful. (15/n)

My grad training was all about computational inference, though I had plenty of field experience, and the middle ground is where I try to find myself more and more these days - understand the mechanism, and the organismal response, repeat! (16/n)

Then in 2016 I taught a course on Molecular Ecology at @MarineBiol_FHL with Morgan Kelly (@MorganWKelly). (17/n)

One thing I thought was a valuable experience for the students was to see if the climate velocity work of eg. @JennSunday et al. could tell us about Balanus shifting its notable cline (@ErikSotka), and data and DNA existed from the past as well as now. (18/n)

From that field course & subsequent work with a brilliant undergrad Katie Skoczen, (doi.org/10.1086/703516), we learned a lot about how it is hard to see frequency shifts when sample sizes are small & sampling is haphazard. But, that cline in Balanus hadn’t really shifted.(19/n)

Katie did another great project as an undergrad (doi.org/10.1093/jcbiol…), but this above observation really took off when we read (re-read, for me) Hines 1978. (20/n)

That temperature-based manipulation really spoke to me given all that we’ve seen with climate shifts, and the fact it shifted reproductive potential the most in Balanus kind of tugged at my broke-ass professor mode and we added data. (21/n)

From this we learned a lot, both about the possibility of temperature-based fitness traits holding that cline, but also what that can help us know about spatial genetic clines that fall somewhere between biogeographic boundaries and hybrid zones (22/n)

That paper is about to come out in @JBiogeography. (23/n)

And yet I’ve not met Anson “Tuck” Hines (serc.si.edu/staff/anson-hi…)! Hope I do at some point, because he has played large in my own path as a biologist, repeatedly. And I should say, this paper in particular. So, lets read up! I hope you don’t mind me thinking out loud… (24/n)

So, I’m honored @crustyolfactory asked me to do this - and she also suggested I say something about how I tend to read papers. Obviously, this has changed a lot since I first read this in the Science library when at @DukeU Zoology! (25/n)

I think especially as busy as I think I am these days, many papers I admit to first ‘gleaning’ for key info first... sometimes they then don’t get read, or sometimes only the gleaned info really gets incorporated to what I’m doing. (26/n)

But a consistent thread in my learning and progress is repetition. Learning your inverts or fish? Repeated exposure, turning them over. Learning concepts? Same. When I teach I encourage students to read a paper once, then a few days later read again. (27/n)

As it turns out Hines 1978 @biolbulletin 154: 262-281 is one that I have read repeatedly, because it keeps showing up as something really key to what I’m trying to understand whether my first research on barnacles (@UCSB_OakleyLab @_hickerson_ saw that get off the ground!) (28/n)

Or when they returned to my research life as a Postdoc, or the fact they have been a mainstay in my research on spatial patterns in biodiversity since I started at @universityofga. (29/n)

Anyway, today I’m going to move through pretty linearly but most of you know I think out loud a bit so will take a few digressions as I link things in this paper to the science it has influenced. (30/n)

So. In this paper Tuck Hines points out the deep history of barnacle research in Europe, much of it cited here but also Southward and others were so instrumental. (31/n)

His goal was to add such data on reproductive cycles from the Pacific North American coast, making standardized comparisons. (32/n)

First is Chthamalus fissus - beautiful little #barnacle from a nearly globally-distributed genus. Should note, work Amanda Castañeda did with me shows that where Hines did this work, some of what he studied was likely C. dalli DOI:
dx.doi.org/10.1017/S00253… (33/n)

Second is Balanus glandula, my long-term on-again-off-again partner (here a photo that includes both Balanus and Chthamalus in hand but from up around @MarineBiol_FHL

📸 Rock with some Chthamalus dalli, some Balanus glandula, and (not in Hines) some Semibalanus (34/n)

Finally, Tetraclita squamosa rubescens (these days just T. rubescens, marinespecies.org/aphia.php?p=ta…) - so, 3 different families. (35/n)

Some background on the species comes next, based on key work on competition and only scarce information on reproductive cycles of ‘west-coast’ barnacles (being US-centric here), noting “the relationship of reproduction with temperature is complex.” (36/n)

Now here is what is cool about Hines 1978 - because of that temp sensitivity, Tuck Hines chose a site at Morro Bay CA (35°22.5’N) where a fossil-fuel power plant discharges cooling water, forming a plume about 5°C warmer. So in situ experiment of temperature was possible! (37/n)

Hines made ~monthly trips for this. A little #barnacle #lifehistory: (1) sessile (2) copulatory (3) female broods offspring through the first few molts before releasing them to feed as free-swimming plankton. (38/n)

So, observing reproduction means popping them off the rock and looking for mature ovaries, eggs, or early-stage one-eyed larvae. Oh! These are all hermaphroditic as well - so anybody can be a mother. (39/n)

I hope you all see I’m in the Methods now. A blissfully short and to-the-point intro, seems like contemporary papers have a whole dang thesis about why on earth we would do something as cool as learning life history traits from organisms. (40/n)

(Side note: at an @NSF workshop on #ClimateChange and evolutionary responses in the ocean a decade ago, one of the main things that all us biologists called for needing more of, so we can understand trajectory better? Life history data and reaction norms. Cheap stuff!) (41/n)

Hines also points out - interesting - not just 3 families of barnacles, but 3 very different size classes (chthamalus < balanus < tetraclita in size) that can all be found at roughly same intertidal height so they have same temperature exposures. (42/n)

So, Hines did as above: select barnacles haphazardly, examine for brooded embryos, ripeness of ovary, ripeness of male reproductive system. This is a pretty qualitative approach, but still the typical standard for evaluating ovigery... (43/n)

I’ll admit my own work has focused on the ovarian tissues and eggs/larvae, less so on the detail of the male reproductive system. Hines also measured and weighed each individual, including the egg mass “and, in some cases, the ovary...” *this is not trivial field work!* (44/n)

Eggs counted using a Coulter counter (en.wikipedia.org/wiki/Coulter_c…) and some of them measured as well. Some of this work was done in lab using small rocks with barnacles - it is really not possible to remove an acorn barnacle to keep it alive for studies typically. (45/n)

In the lab work, Hines fed them with Artemia (brine shrimp) and phytoplankton, maintaining temperatures that matched the winter ambient (11.5°C) and power station outflow temps (20°C) - Figure 1 shows how large the temperature shift is and how it changes seasonally (46/n)

It isn’t clear from the paper so far how far away from the outflow is considered ‘ambient’ and how this may vary daily or seasonally. (47/n)

Later on I may point out that this location would be interesting for an evaluation of genomic diversity that is a part of my ongoing work (stay tuned) - a great student project! (48/n)

The lab work, by the way, is to take egg masses and hold them in-lab to evaluate survival and brooding time variation. This is another cool part about barnacles - once you open one up you can immediately see the mass, just waiting their turn, avoiding predation. (49/n)

I have recently been handling a species from Pitcairn Island collected in the 1990s (from @FloridaMuseum) and keep notes on which were ovigerous at the time. (50/n)

https://twitter.com/wareslab/status/1397591187259727875?s=21

Believe it or not we are through Intro and Methods now - straightforward, right? (51/n)

But as I said before, pretty darned important if we want to understand how reproductive cycles interact with temperature, ocean currents (int-res.com/articles/meps_…), distribution of diversity, and more. (52/n)

So... Figure 2 (Results) is probably what I focused on the most 25 years ago. Brooding frequencies for outfall and control populations of all 3 species. Two full years! (53/n)

This study of #phenology in brooding in these barnacles tells us about seasonality in resources for sure, and the cyclic nature of Chthamalus and Balanus is most notable (less so in Tetraclita). (54/n)

This is key for understanding how/when mature larvae (cyprids) will recruit to intertidal ecosystems, and how it will vary spatially (eg doi.org/10.2307/2680048) - that spatial variation is dependent on timing of release relative to upwelling, current reversals... (55/n)

... larval behavior (eg. vertical migration, larval duration), etc. So the timing of major and secondary brood cycles sets the distribution and density of these species and even the genomic diversity within these species (doi.org/10.3354/meps33…). (56/n)

A key part that you don’t necessarily get from the figure: “from June to Sept, 50-75% of (Chthamalus) were brooding, but low levels of about 10% brooding often occurred during the 'off' season." Brood timing similar between warmer, ambient water. (57/n)

Balanus, similarly - most brooding from Jan-May, but also some brood in fall - key for issues of diversity distribution in the Pringle papers noted earlier. But then here is what got me in my most recent (before today) reading of Hines 1978... (58/n)

(I should note, everybody, that if you have a question for me - if you respond to one of these please also tag @wareslab and then I’ll be notified. I’m taking a breather here to make that last tweet a cliff-hanger). (59/n)

... in Balanus, “samples from the warm-water outfall consistently had a lower percentage of brooding...delayed one or more months” (delay not in all years). (60/n)

In past work as PhD student, I just needed to know THAT they have cyclic reproduction and what it meant to be planktotrophic. As a postdoc, that the cycles of planktonic larvae meant something for distribution and abundance of diversity. (61/n)

Most recent work w/ Katie Skoczen in 2019 (doi.org/10.1086/703516) @biolbulletin, we keyed on this fact and that there is an enormous genomic cline in the middle of the distribution of Balanus (see doi.org/10.1111/j.1365… by @ErikSotka et al). (62/n)

The two mitochondrial lineages found in Balanus may have functional ecological significance, much like other recent work in e.g. Carcinus (doi.org/10.1242/jeb.20…) by Coyle, @CTepolt, Dave Carlon, et al - and Morro Bay is in an important (steep) part of that genomic cline. (63/n)

Anyway, Tetraclita has a cyclic brooding pattern - but not so much when it is in the outflow temperature, which makes you wonder if further work should evaluate how it is responding to climate change... (64/n)

... and well look at that: doi.org/10.1111/j.1365… by @MNDlab - this is a species that has expanded up the coast in recent decades, responding to #ClimateVelocity (eg. @JennSunday @pinskylab and others, wonderful work!) (65/n)

Could warmer waters mean distinct times of ovigery/brooding, and responding to currents and upwelling differently? Sounds like a fun follow-up for a student on the California coast! (66/n)

Hines notes size doesn’t affect brooding activity much once they are reproductively mature, that's useful for further study as a factor that you don’t have to worry about as much perhaps. (67/n)

In the next section on lab experiments, Tuck says “for Chthamalus... temperature is not an important factor regulating brooding.... in Balanus...temperature is important” -- look at that drop off in Table 1 in lab experiment!! (68/n)

Remember there are more or less still two ‘types’ or lineages of Balanus glandula here, though the vast majority are the ‘southern’ type that we assume to be somehow more adapted to warmer waters (here from Wares & Skoczen: doi.org/10.1086/703516). (69/n)

Still, that was the clue that Katie and I followed up on, with many thanks to @PiscoScience and colleagues for quick seasonal collections of Balanus glandula throughout the cline region in 2018-2019, so we could assess ovigery relative to mitotype! (70/n)

Again the contrast comes here that Balanus (generally the southern type) needs cooler water for normal reproduction; Tetraclita seems to need warmer waters and it shifts the timing of ovigery and thus release. (71/n)

And the lab work also showed the importance of food availability for brood timing in Chthamalus - so as you might expect, each barnacle has its own cues for reproduction. (72/n)

THIS IS WHY BIOLOGY IS SO DANGED HARD and why we need more life history data, more reaction norms and temperature performance work, etc. (73/n)

A lot of good stuff on brood weight x body size, operculum as a cue to body weight, but generally a lot of noise in these data because body weights fluctuate *erratically* through the year. (74/n)

“The body weights (of the barnacles) are not large compared to brood weight in all species, and it is hard to see how any sizeable quantity of nutrients could be stored there”... (75/n)

Classic trade-offs in reproductive life history, these barnacles are doing their best - but the planktonic phase is clearly necessary to have so many offspring. (76/n)

The male reproductive cycle - as I noted, I have not focused on this as much in the past. Here, there is probably a snarky comment to be made about male reproductive potential being high year-round in Chthamalus, more cyclic in Balanus. Remember Chthamalus is VERY small. (77/n)

So, ability to fertilize early in life and continually may be a good reproductive strategy. I’ll take the time to plug @ewers_saucedo here (doi.org/10.1111/mec.13…), and how some barnacle systems let us separate size and proximity as indicators of fitness! (78/n)

In any case, male repro capacity is pretty much off/on in Balanus, and again quite shifted by warm temperatures in Tetraclita. (79/n)

So which reproductive function is affected by heat, drives? Is it fortunate that both cycles shift with temperature? Another good question to follow up on... (80/n)

(Note that Hines picks up on this cool thing w/ Tetraclita for sure - temperature cycles would effectively reproductively isolate individuals that experienced such differences in their environment!) (81/n)

Ovarian ripeness itself, by the way, does not seem to vary by temp in the two smaller barnacles (Chthamalus and Balanus). (82/n)

Remember that all these factors happen in the life cycle of a hermaphrodite, the female reproductive tract gets cycled up (endocrine), male reproductive (endocrine), fertilization requires both and temp dependency? (83/n)

I’m just going to highlight again that measurement of these components - ovary weights, brood weights, male reproductive tissues (incl measurement of penis in some studies), opercular length, etc. is hard, tedious, microscopic, steady-hand kind of work.😓 (84/n)

Here is @ewers_saucedo again, doing such work in between field outings at Sapelo Island for her Chelonibia project along w/ the field work itself including my kid, @KBockrath, and a friendly Limulus. (85/n)

These results are long, bear with me folks - all cool stuff. One of the keys that comes out of studying these animals is then recognizing how many times they brood annually. (86/n)

Hines shows a brood can hatch (go from fertilization to release) in less than 2 weeks, shorter when water is warmer (Chthamalus), longer (about 3-4 weeks) in Balanus, still longer in Tetraclita. (87/n)

So, huge fecundity potential differences among these genera with maybe 5-fold as many broods in the tiny Chthamalus relative to Tetraclita! Of course, other factors probably influence delays between broods (88/n)

Intriguingly, Hines also evaluated this problem. Nutrient storage appears to be maintained as just enough per-brood in Chthamalus and Tetraclita, but with longer-term storage in Balanus glandula. (89/n)

And as a final wow! from the Results in Hines (1978): (90/n)

This is why when you jump in the ocean you will often be literally bombarded by plankton, and a lot of those beautiful microscopic creatures will be barnacle nauplii! (for more on this please follow @PlanktonPundit). (91/n)

Any questions at this point? Can you ‘like’ if you have actually been reading along? Have you followed @biolbulletin? I’m going to go get some iced tea and a snack and then wrap up the discussion, I appreciate folks listening in on all of this! (92/n)

The Discussion notes that barnacles often follow a small number of basic patterns in reproduction - based on what was known at the time. Some very regular and predictable, some more contingent on season. Hines is giving the context from what has been done. (93/n)

But what was really cool here is doing that hard work, doing it in 3 species at once, doing it with a temperature treatment vs. control, and having lab as well as field observations! (94/n)

The temperature dependencies of Balanus and Tetraclita as I noted above point to further questions to be addressed - would any of the ‘northern’ type Balanus @ErikSotka et al. '04 or Wares & Skoczen 2019 be found in the Morro Bay outfall warm water? Probably not. (95/n)

Would the seasonality of brooding be distinct south of Point Conception where the water is warmer, and the timing of release is not so critical to maintain/retain larvae nearshore because of the large-scale eddy in that region? (96/n)

Has climate influenced Tetraclita in the way predicted in this paper, and is that part of the key to how it has moved up the coast so rapidly? DOI: doi.org/10.3354/meps12… (97/n)

These aren’t the questions posed in Hines 1978 discussion, but he is mulling over what these effects mean in a broad and useful sense - the distribution of fitness over seasonal brooding, and beyond. (98/n)

Much of the rest of the discussion is comparing these 3 species to the other recognized patterns out there, what does it mean given their latitudinal distributions (super important now as we think how many species have cryptic lineages within). (99/n)

How barnacles - and all intertidal species - maintain their distribution, and how the genomic diversity within these species is distributed in that range, depend on fecundity, phenology, climate, and interactions with other taxa. (100/n)

The technical details of how we think about e.g. putting ‘virtual’ larvae in a physical model (here, again, @ewers_saucedo doi.org/10.1002/ece3.2…) depends *greatly* on this knowledge.(101/n)

Genomic diversity itself, the efficacy of selection, and the distribution of variants within a range also depends on these life history traits and how offspring are distributed following each reproductive event. doi.org/10.1186/1471-2… (102/n)

There are a lot of assumptions built into trying to figure out the overall reproductive output of barnacles, or any small aquatic organism, but those data are necessary for us to understand and predict how these organisms respond to #ClimateChange NOW. (103/n)

What I love about reading and re-reading Hines (1978) is that this paper is so relevant to our concern and focus today on species response to climate, to habitat change, and more. (104/n)

My former boss and colleague, Rick Grosberg said this when I noted I’d be doing this #ReadAlong for @biolbulletin: “...a great paper...extraordinary attention to important details, its scope, both geographic and taxonomic...”(105/n)

“...importance as a reference baseline for how climate change etc. is affecting patterns of reproduction...different species of barnacles at the same time and place are affected very differently..." (106/n)

"...a deep understanding of reproductive biology, life-history evolution, and ecology.” -Grosberg. (107/n)

And so - that’s that! My “tweet hurricane” as @ErikSotka put it earlier! A great paper that I have relied on repeatedly over the past 25 years, and a fun chance to explain why it reaches out so broadly to me and my colleagues. (108/n)

Many thanks to @crustyolfactory for this idea and supporting my sketchy typing and thought formation, making it sensible in such a crazy Twitter-format report. and with that, another snack and happy to take questions here or at @wareslab! (fin)

📸 Credit: Wim Van Egmond

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