Editor’s Note – This essay was subsequently published as:
Dillon, R.T., Jr. (2023b) Intrapopulation gene flow, the Leptoxis of the Cahaba, and the striking of matches. Pp 133 – 146 in The
Freshwater Gastropods of North America Volume 6, Yankees at The Gap, and Other
Essays. FWGNA Project, Charleston, SC.
Last month we reviewed a research project I conducted in the
late 1970s on isolation-by-distance and barriers to dispersal in a population
of Pleurocera proxima inhabiting a small stream in NW North Carolina [1]. The markers I used were allozyme variants –
bands of protein migrating at different speeds in starch gels. Even back in the 1970s, there was already a
lot of hand-wringing about the genetic variation being missed by such a gross
and clumsy technique.
I was missing, of course, all of the silent variation –
variation due to the redundancy of the genetic code. And of the non-redundant variation, mutations
that yield variation in the amino-acid sequence, I was only catching a tiny
fraction – that subset changing the charge on the protein. And the technique I was using worked only on
loci that encode enzymes, the evolution of which must certainly be constrained
by selection, right? What about junk
DNA? What about everything else?
A lot of my colleagues worried about these problems in the
1970s. And I can see why, if one
invested in all that cumbersome equipment for protein electrophoresis, and all
those expensive reagents to demonstrate allozyme bands, and conducted one’s
field work, and ran one’s gels, and found no variation, and scraped one’s gels
into one’s trash can, and washed one’s giant sink-full of dishes, one might
grumble.
 |
| Allozyme variation in Naked Creek |
But the grossness and clumsiness of allozyme electrophoresis
never bothered me, through my entire career, even into the 21st century [5]
because I did find allozyme variation.
With hard work, patience, and good technique, I found a lot of very
useful genetic markers. I realized that
I was “binning” a bazillion silent variants together when I scored a snail
homozygous for Odh106 back in 1979. But
I could distinguish those bazillion variants together from the bazillion silent
variants I had binned together in snails I scored as homozygous Odh109F. And those two big bins were inherited in
Mendelian fashion. Fine.
Well, technology marches on.
The first fully automated sequencing DNA machine came onto the market in
1987, even as Kerry Mullis’ patent for PCR amplification was approved, and by
the 1990s everybody was sequencing DNA.
Massively-parallel (“Next-Generation”) DNA sequencing machines were
introduced for commercial use around 2005, enabling rapid and cheap sequencing
of gigabases of DNA. And in 2008 a
method was first proposed to identify single-nucleotide polymorphisms in random
lengths of DNA amplified from population samples, “Restriction Associated DNA
sequencing,” or RADseq for short.
The DNA from each individual (let’s say, for example, each
snail) is isolated and cut into a bazillion little pieces with a carefully-chosen
restriction enzyme or set of enzymes.
The millions of pieces in this mess that are (usually) a couple hundred
base pairs long [6] are electrophoretically separated from the zillions of
littler pieces and bigger hunks and ligated to adapters that facilitate their
amplification and uniquely identify each individual snail. These millions of pieces are called
“reads.” Feed all those reads through
the front door of your local Next-Generation sequencing factory.
As mind-boggling as the previous paragraph most certainly
is, I will now ask you to imagine repeating that process for (let’s say) 20
snails from (let’s say) 8 populations.
So 160 times. And as
mind-boggling as the preparation and the sequencing of all those 160 million
reads most certainly is, I will now ask you to imagine that all of those reads
can be “quality-filtered” to remove the crappy ones, screened by their utility
as markers across the entire 160-individual population, and analytically
matched to each other using some gargantuan gear-grinding smoke-belching
computer program. If you’re interested
in the technical details, see the references at footnote [7] below.
What comes back out of the sequencing factory is, in our
example, a comparison of 8 sets of 20 snails amplified at some huge number of
random, anonymous (“restriction-site associated”) reads of DNA. If there is any polymorphism, even for one
single synonymous nucleotide, the researcher will know it. The data are typically reported in
tightly-packed bar graph form, standardized by the number of matching reads,
coded by some color, let’s say orange to start.
If snail #1 and snail #2 are genetically identical, as far as can be
determined by this mind-boggling technique, the bar graphs depicting their
genomes will match in orange along 100% of their height. If snail #1 and snail #2 differ by (let’s
say) 40%, a second color is selected, let’s say blue, and the bar graph
depicting snail #2 is 60/40 orange/blue.
So in 2019 our colleague Nathan Whelan, together with six coworkers,
published the first RADseq study of a population of pleurocerid snails
[8]. His results nicely augment and
compliment the results I myself obtained using allozyme variants in the 1970s,
and as such make an important contribution to our understanding freshwater
gastropod evolution generally.
 |
| Whelan et al. Figure 2 [8] |
Nathan collected samples of 20 Leptoxis from each of 8 sites
in the Cahaba River drainage of central Alabama, stretching approximately 45 km
(30 miles) from the Helena suburbs at County Road 52 (“CR52”) downstream to
Centreville. Four of his eight samples
came from the main river, and four from the tributaries, as shown in his Figure
2 reproduced above. This is a
marvelously data-rich figure, from which those of us interested in pleurocerid
evolution can learn at least four important lessons. Let’s unpack each lesson, one at a time.
First, all eight of Nathan’s samples (let’s call them
“subpopulations”) were genetically unique – some entirely unique, others just
mostly unique. I am not a fan of
Nathan’s palate, but what he is trying to convey with his four shades of blue
and four shades of orange is eight unique genomes associated with eight
distinct Leptoxis subpopulations. And
the shade of orange 100% covering the Shades Creek box is completely different
from the shade of orange 100% covering the Schultz Creek box. And both of those oranges are completely
different from the shade of blue 100% covering the CR52 box and the shade of
blue 100% covering the Bulldog Bend box.
Those four subpopulations seem to be entirely unique, as far as Nathan’s
data extend. The other four subpopulations
– Schultz Creek, Marvel Slab, Sixmile, and Centreville, are mostly unique, with
other shades of color more or less impinging around the edges of their
mostly-uniquely-colored box interiors.
Apparently, gene flow among most of Nathan’s subpopulations
of Leptoxis is zero, at least on the time scale of single-nucleotide
mutations. That is the most surprising
result of the 2019 study conducted by Whelan and colleagues. All eight of their subpopulations are
connected by water, with stepping-stone distances generally in the 10 – 30 km
range. The research we reviewed in
September would have led us to expect slow but measurable active migration
upstream, with rapid and episodic transport downstream [9]. My previous research on Pleurocera [1]
suggested an average gene flow of 6.5 migrants among sites sampled 1 kilometer
apart which, one would assume, ought to extend 10 km to some value greater than
0.0. No?
The second important lesson conveyed by Nathan’s Figure 2 is
that what little dribble of gene flow does occur among a few of his
subpopulations seems to occur 100% downstream.
The four completely-unique subpopulations (Shades Creek, Schultz Creek,
CR52, and Bulldog Bend) are from the four most upstream sites. All four of Nathan’s downstream boxes show at
least a little bit of upstream color.
One’s eye is especially drawn to the box at Nathan’s downstream-most
subpopulation, Centreville, which is primarily painted dark blue, but
demonstrates significant bars of NWR yellow, Schultz Creek dark orange, Shades
Creek orange, Marvel Slab blue, and Bulldog blue.
More than any other pleurocerid, Leptoxis populations are
strongly associated with rock substrate and rapid water flow. Individuals of the genus Pleurocera, by
contrast, are at least occasionally observed grazing across softer
substrates. This includes P. proxima,
which although apparently adapted for small, trout-stream-sized creeks tumbling
through the southern Appalachians, is not uncommonly spotted crawling on sand
and firmer mud. See the last photo I
published in last month’s post [12Oct21].
But I can never, in my 60 years of field experience, ever
remember collecting an individual Leptoxis on anything but rock. So, Nathan’s eight Leptoxis subpopulations
were collected from eight riffle areas that must (inevitably) have been
separated by pool areas, with extensive bottoms of mud substrate. Leptoxis can wash downstream through such
pools but (apparently) cannot effectively crawl upstream through them.
Then how did Leptoxis get upstream in the first place? The two answers to that question are great
age + dirty birds. While the lower
regions of the Mobile Basin were yet covered by the Cretaceous embayment, the
upper Mobile Basin had long been flowing free from the mountains of what is now
North Georgia. In 2009 I offered several
lines of evidence suggesting that the pleurocerid populations of this region
are “The Snails The Dinosaurs Saw,” living fossils of great antiquity [10]. I subsequently penned a series of essays
showing that aerial dispersal among such populations is not as unlikely as one
might think [11].
 |
| Whelan et al. Fig 1, modified [8] |
And in 2016, Nathan together with our colleague Ellen Strong
published a paper documenting extensive mitochondrial superheterogeneity among
these same Cahaba River populations of Leptoxis for which he and his co-workers
now report RADseq data [12]. Nathan’s
2016 results strongly imply very long periods of isolation, punctuated by very
rare introductions of genomes from very great distances away. Now in light of Nathan’s 2019 research
findings, his 2016 paper makes more sense.
The third important lesson to be taken from Nathan’s RADseq
study is that divergence among Leptoxis subpopulations of the Cahaba is
phenotypic, as well as genotypic. The
shells born by most of Nathan’s eight samples were almost entirely smooth, as
shown in (B) of his Figure 1, modified above.
But the subpopulation inhabiting the Little Cahaba River below Sixmile
Creek bears lightly tuberculate shells, typically with carination, as shown in
(C).
In the figure below I have reproduced my diagram of Naked
Creek from last month’s post and inset a slice of topographic map showing the
Little Cahaba River between Bulldog Bend and Sixmile Creek. These two maps are depicted at the same
scale, see the Naked Creek scale bar at upper left. The water distance from Bulldog to Sixmile is
a serpentine 7.85 km (flowing from right to left), comparable to the distance
between Naked Creek Site 7 and Naked Creek Site 8.
The Bulldog Bend box is 100% blue, demonstrating no gene
flow from any other subpopulation sampled.
The Sixmile box is almost entirely orange but shows a bunch of little
Bulldog-blue nibbles at the bottom, plus one big dramatic blue cut. That singleton snail, the one individual
whose genotype seems [13] to match Bulldog more than Sixmile, also bore a
smooth shell (B) like the Bulldog subpopulation, not a tuberculate/carinate
shell (C) like the other 19 in Nathan’s Sixmile sample.
Clearly this phenomenon is attributable to washdown gene
flow from Bulldog to Sixmile. The
Smooth-Shelled Singleton in Nathan’s Sixmile Sample (Let’s call him “5S.”) did
not demonstrate a 100% Bulldog genome, however, but rather only about 50% [13]
matching Bulldog. The implication is
that Snail 5S is not a first-generation washdown, but a second generation washdown,
born at Sixmile but retaining the shell morphology along with half the genome
of a Bulldog parent. This is indirect
but nevertheless compelling evidence for the heritability of
tuberculate/carinate shell morphology in pleurocerid snails. But wait, there’s more.
The final lesson from Nathan’s RADseq study, and the most
important lesson, is this. Although
these eight subpopulations have diverged both genetically and morphologically,
they have not speciated. The isolation
between them is physical, not reproductive.
When snails wash down, albeit rarely, they are apparently able
interbreed freely with the snails in the riffles downstream. The Cahaba River at Centreville is not
populated by an admixture of five different Leptoxis species. All 20 of the snails Nathan collected at
Centreville belonged to the same biological species as the seven subpopulations
Nathan sampled upstream.
Nathan identified all eight of his subpopulations as
“Leptoxis ampla.” OK, that’s a good
start. We all agree on the conspecific
status all the Leptoxis subpopulations of the Cahaba. Now let us see if we can apply the lessons we
have learned in the Cahaba to the greater Mobile Basin beyond.
I have reviewed the tangled taxonomic history of the Mobile
Basin Leptoxis fauna on several occasions in the tangled epistemological
history of the FWGNA blog [15]. But not
recently. So to refresh the collective
memory.
Timothy Abbot Conrad got the ball rolling back in 1834,
describing four species, two from the Alabama/Coosa and two from the Black
Warrior. Isaac Lea [16] added seven, J.
G. Anthony added three, H. H. Smith added eleven, and Calvin Goodrich [17] one,
so that by 1922, Goodrich tallied 26 nominal species of Leptoxis in drainages of
the Mobile Basin [18]. I reproduced
Goodrich’s figure of all 26 in my essay of [15Sept09] and have re-reproduced it
below.
Most of these nominal species were nominally-extincted by
extensive damming and impoundment conducted throughout the Mobile Basin,
starting in 1912, accelerating in the 1920s and 1930s, and continuing into the
1960s. By the 1990s, Goodrich’s Leptoxis
list had been reduced to four nominal species, each inhabiting small fragments
of its former range: L. picta (Conrad 1834) in the main Alabama River, L. ampla
(Anthony 1855) in the Cahaba, L. taeniata (Conrad 1834) in the lower reaches of
three creeks in the Coosa drainage, and L. plicata (Conrad 1834) in the Black
Warrior.
 |
| Goodrich [18] |
How many of these might be biologically valid? In 1998 I published a paper with Chuck
Lydeard
[19], reporting that the allozyme divergence among
L. picta,
L. ampla,
and
L. taeniata was no greater than the allozyme divergence among a set of
conspecific
Leptoxis praerosa controls sampled from equally-distant quarters of
Tennessee
[20]. We suggested that
ampla
and
taeniata be synonymized under
picta (Conrad 1834).
Modern fashion has trended in the other direction,
however. Even as our 1998 paper was in
review, a pleurocerid population identified as “Leptoxis downei” was discovered
in the Oostanaula River of Georgia, a nomen subsequently dropped in favor of L.
foremani. And in 2011 a population identified
as Leptoxis compacta was discovered in the Cahaba River at the Shades Creek
confluence, sympatric with snails Nathan identifies as L. ampla [21]. Today the list of nominal Leptoxis species
inhabiting the Mobil Basin has rebounded to six [22].
Now research results have crossed our desk demonstrating that the
Leptoxis population of the Cahaba River is strikingly fragmented into
subpopulations, that these subpopulations have diverged both genetically and
morphologically, and that they have not speciated. Another 50 km downstream will bring us to the
main Alabama River, and 100 km back up the Alabama/Coosa will bring us to the
mouth of Buxahatchee Creek. Does this
new evidence support the assignment of three different specific nomina to
“Leptoxis ampla” in the Cahaba, “L. picta” in the main river, and “L. taeniata”
in Coosa tributaries such as Buxahatchee Ck?
Forty years ago, while I was yet a doe-eyed graduate
student, it was clear to me that the key to understanding speciation was to
understand population divergence, and the key to understanding population divergence
was to understand intrapopulation gene flow.
I did not have any big grants, and I did not have any fancy tools, and I
did not have legions of collaborators.
But I did have, even at that tender age, quite a few years of field
observation on the biology of pleurocerid populations in rivers of the American
southeast, and an openness to learn more, and that took me a long way.
Now I am delighted to discover colleagues in Alabama
bringing sophisticated tools to bear on questions I myself pondered in my
youth. Can my colleagues extend their
newfound understanding of intrapopulation gene flow forward through population
divergence and generalize to the species level?
Can they bring dawn to the darkness that has enveloped the pleurocerid
fauna of the Mobile Basin for 200 years?
That remains to be seen. But
Nathan Whelan and his colleagues have struck the first match.
Notes
[1] My research project on gene flow in the Naked Creek
population of Pleurocera proxima was ultimately published in three different
ways. The 1979 results, in their
entirety, were published as Chapter 2 of my 1982 dissertation [2]. The barrier-to-dispersal portion of that 1979
study was combined with data from 1980 and 1985 and published in 1988 [3]. The isolation-by-distance portion of my 1979
study was published just last year in Ellipsaria [4]. For an overview of the entire research program, see
last month’s post:
- Intrapopulation gene flow: The polymorphic Pleurocera of
Naked Creek [12Oct21]
[2] Dillon, R.T. Jr (1982)
The correlates of divergence in isolated populations of the freshwater
snail, Goniobasis proxima. Ph.D.
Dissertation, University of Pennsylvania. 182 pp. Dissertation Abstracts 43: 615B
[3] Dillon, R.T., Jr. (1988) The influence of minor human
disturbance on biochemical variation in a population of freshwater snails.
Biological Conservation 43: 137-144.
[PDF]
[4] Dillon, R. T. (2020) Fine scale genetic variation in a
population of freshwater snails. Ellipsaria 22(1): 24 - 25. [PDF]
[5] I was still frantically running allozyme gels when I was
kicked out of my lab at the College of Charleston in the spring of 2016. And still getting interesting results,
too! See:
- The best estimate of the effective size of a gastropod
population, of any sort, ever [14Jan19]
[6] Actually, Nathan and his colleagues used a clever
modification called 2b-RADseq, involving a special restriction enzyme called
ALF1, that cuts DNA into fragments exactly 36 bp long.
[7] A few of the better references on RADseq:
- Baird N, Etter P, Atwood T, et al. (2008) Rapid SNP
Discovery and Genetic Mapping Using Sequenced RAD Markers. PLoS ONE 3:e3376.
- Davey, JW & M.L Blaxter (2010) RADSeq: next-generation
population genetics. Briefings in
Functional Genomics 9: 416-423. doi: 10.1093/bfgp/elq031
- Rubin, B.E.R., R.H. Ree, and C.S. Moreau (2012) Inferring phylogenies from RAD sequence
data. Plos One 7(4): e33394.
- Wang, S, E. Meyer, J.K. McKay, and M. Matz (2012) 2b-RAD: A simple and flexible method for
genome-wide genotyping. Nature Methods
9: 808 – 810.
[8] Whelan, N.V., M.P. Galaska, B.N. Sipley, J.M. Weber,
P.D. Johnson, K.M. Halanych, and B.S. Helms (2019) Riverscape genetic variation, migration
patterns, and morphological variation of the threatened Round Rocksnail,
Leptoxis ampla. Molecular Ecology 28:
1593 – 1610.
[9] For a review of previous research on this important
topic, see:
- Intrapopulation gene flow: King Arthur’s lesson [7Sept21]
[10] Dillon, R.T., Jr. and J.D. Robinson (2009) The snails the dinosaurs saw: are the
pleurocerid populations of the Older Appalachians a relict of the Paleozoic
Era? Journal of the North American
Benthological Society 28: 1 – 11 [PDF].
For more, see:
- The snails the dinosaurs saw [16Mar09]
[11] My four-part
series on aerial dispersal:
- Freshwater gastropods take to the air, 1991 [15Dec16]
- A previously missed symbiosis? [11Jan17]
- Accelerating the snail’s pace 2012 [24Apr17]
- Freshwater snails and Passerine Birds [26May17]
[12] Whelan, N.V.
& E. E. Strong (2016) Morphology,
molecules and taxonomy: extreme incongruence in pleurocerids (Gastropoda,
Cerithiodea, Pleuroceridae). Zoologica Scripta 45: 62 – 87. For an independent
analysis of these fascinating results, see:
- Mitochondrial superheterogeneity: What we know [15Mar16]
- Mitochondrial superheterogeneity: What it means [6Apr16]
- Mitochondrial superheterogeneity and speciation [3May16]
[13] The singleton blue streak in the orange Sixmile box
seems to slice much more than halfway through.
Possibly 75 – 80%? I feel sure
that’s just slop [14]. If a snail
collected at Sixmile doesn’t bear 100% of the Bulldog genome, it must bear 50%
or less.
[14] The only other explanation would be that Snail 5S has
one parent and one grandparent washed down from Bulldog. In other words, the mother of 5S washed down
from Bulldog and was inseminated by a father with one parent born at Bulldog,
yielding Snail 5S, with 75% Bulldog genome.
That scenario seems quite unlikely. The proportion of first-generation
Bulldog-washdowns at Sixmile seems to be less than 1/20 = 0.05, and the
proportion of second-generation washdowns approximately 1/20 = 0.05, so the
likelihood of a first-generation x second-generation mating must be less than
0.05^2 = 0.0025.
The implication of the highly-unlikely scenario outlined
above would be that the father of Snail 5S actively sought the mother of 5S. In other words, there is some sort of
reproductive isolation between the Bulldog and Sixmile Leptoxis
populations. The two populations have
speciated.
Might a unique species of Leptoxis have evolved on the
Sixmile rapids in the middle of the Cahaba? Nah.
Just look downstream at Centreville.
I count six snails bearing hunks of Sixmile genome together with native
Centreville genome, upstream Schultz Creek genome, and everything else. So I agree with Nathan on this one. The apparent excess in the length of that
skinny blue cut in the orange Sixmile box is almost certainly just slop.
[15] For additional background on the taxonomy of Leptoxis
populations in the Mobile Basin:
- Mobile Basin I: Two pleurocerids proposed for listing
[24Aug09]
- Mobile Basin II: Leptoxis lessons [15Sept09]
[16] For the record:
- Isaac Lea Drives Me Nuts [5Nov19]
[17] Actually, if you’re digging around in these footnotes
for more homework, I would recommend reading my 2007 biographical sketch of
Calvin Goodrich before reading the two Mobile Basin essays I posted in
2009. Start here:
- The Legacy of Calvin Goodrich [23Jan07]
[18] Goodrich, C. (1922) The Anculosae of the Alabama River
Drainage. University of Michigan Museum
of Zoology Miscellaneous Publication 7: 1 – 57.
[19] Dillon, R.T., and C. Lydeard (1998) Divergence among
Mobile Basin populations of the pleurocerid snail genus, Leptoxis, estimated by
allozyme electrophoresis. Malacologia.
39: 111-119. [PDF]
[20] Leptoxis plicata populations of the Black Warrior
appear to be genetically distinct.
[21] Whelan, N.V. P.D. Johnson and P.M. Harris (2012) Rediscovery of Leptoxis compacta (Anthony
1854) (Gastropoda: Cerithioidea: Pleuroceridae)
PlosOne 7(8) e42499 [html]
[22] Shelton-Nix, E. (2017)
Alabama Wildlife, Volume 5.
University of Alabama Press, Tuscaloosa. 355 pp.
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