Anchored hybrid enrichment, Pleurocerid evolution, and the obovata confirmation

Review: Whelan, N. V., Johnson, P. D., Garner, J. T., Garrison, N. L., & Strong, E. E. (2022). Prodigious polyphyly in Pleuroceridae (Gastropoda: Cerithioidea). Bulletin of the Society of Systematic Biologists, 1(2). https://doi.org/10.18061/bssb.v1i2.8419

Although I myself have never harbored any aspiration to reconstruct a phylogenetic tree [1], I can understand the intellectual appeal of the fancy new whole-genome technique called “Anchored Hybrid Enrichment” (AHE) to the not-insubstantial fraction of my professional colleagues who feel differently.  AHE reminds me of an obsolete technique called single-copy DNA hybridization, which I greatly admired in the 1980s, where pioneering researchers like Sibley & Ahlquist [3] extracted the entire genomic DNA of bird #1 and bird #2, chopped it, melted it to single strands, then cooled it to the temperature that repetitive DNA would reanneal and stick in a hydroxyapatite column, eluting just the single-copy DNA out the bottom.  They would then mark the single-copy DNA of bird #1 with P-32, mix it with a large excess of single-copy DNA of bird #2, melt the mixture again, and gradually elute the mixture back through the hydroxyapatite, cooling slowly, to estimate the similarity of bird #1 and bird #2 across all their single-copy genes together.

The terrible flaw in the technique of single-copy DNA hybridization, as practiced in the 1980s, was that it yielded a matrix of overall genetic similarities across pairs in a set of study individuals, committing the mortal sin of being phenetic.  The Sibley & Ahlquist hypothesis for the evolution of birds disappeared into the footnotes many years ago [4].  If only there were a technique to do exactly the same thing with a bazillion single-character nucleotide differences, that could be sanctioned as cladistic, am I right?

Anchored Hybrid Enrichment [6]

In recent years, the coupling of mind-numbing computer power with high-throughput DNA sequencing technology has spawned a variety of whole-genome phylogenetic techniques (“phylogenomics”) that harken back to the days of Sibley & Ahlquist.  As the name implies, anchored hybrid enrichment [5] depends on the development of “anchors,” DNA probes to target and enrich highly conserved regions within the whole genomes of a set of organisms under study.  The idea is that the regions flanking such probe areas – not the probe areas themselves – are likely to be less conserved, and hence have some utility in phylogenetic construction.

So, in June of 2022 a research group of our own colleagues, headed by Nathan Whelan of Auburn University, published the first application of AHE technology to the reconstruction of a molluscan phylogeny, as far as I know [7].  And it was to the North American Pleuroceridae that their attention was drawn.

Bless their hearts, all five members of the team.  I mean that sincerely.  A lot of what I am getting ready to write over the next couple essays will be critical.  Such is science.  But I do not mean to minimize their innovative spirit, their commendable effort, or their motives, which were the highest.  We really do need a lot more research like this, and a lot more researchers like Nathan Whelan and his colleagues to do it.  Better understanding of the basic evolutionary biology of their living, breathing study animals, before chopping them into massively-parallel chum and high-through-putting them off the stern of a next-generation sauceboat, is all that is wanted.  That, plus some nod toward calibration, for a change.  Oh, and a passing familiarity with the recently published scientific literature on the subject matter would have been really helpful, too.

OK, first.  Regarding the generation of the AHE probes (or “baits”).  Rather than extracting total genomic DNA, the Whelan team extracted mRNA from five pleurocerid species “chosen to maximize phylogenetic diversity,” which we will call “Probe Species A – E.”  Those five mRNA samples were mailed off to Rockville, Maryland, for the generation of transcriptomes, that subset of the genome actually doing something.  I like that.  That’s a good way to factor out junk DNA before stepping up to the starting block.  Ultimately, our colleagues identified 742 baits “present in at least 4 of 5 pleurocerid transcriptomes and larger than 120 bp in length.”

Figure 4 of Whelan et al. [7]

These 742 baits were trolled through a school of 192 sharks – and by sharks, I mean the whole genomes of individual pleurocerid snails – representing 92 putative species, with two rays – and by rays, I mean the whole genomes of two individual Juga plicifera from the west coast – to serve as an outgroup.  And I do want to commend the Whelan group for the care and completeness they took in documenting their study sample.  The shells from each of those 194 individual snails were numbered, photographed, and deposited in the USNM.  And complete locality data for all 194 snails were made available in tabular form from the journal website, as well as a big folder of 194 jpeg images.  We who sail in your chum salute you, colleagues.

One could only wish that the actual fishing trip hadn’t turned so fraught with peril.  Total genomic DNA was extracted from those 194 individuals and sent off to Gainesville, Florida, for AHE library prep and sequencing.  Three different datasets were generated by this process, under three different assumptions (masked, probe, and full), and trees generated using two different algorithms (ML and ASTRAL).  The ASTRAL method also involved two assumptions (TaxMap yes or no), yielding N = 9 gigantic, eye-assaulting, headache-inducing phylogenomic trees.  Eight of these trees were consigned to the Supplementary Material.  And the tree that Whelan and colleagues liked best, probe + ASTRAL + TaxMap, was published in the journal article, in circular format, as though we evolutionary biologists were birds looking down upon a tree of sharks, which are actually snails, which the most spectacular mixture of metaphor I have muddled in recent memory.

But when I first laid eyes on Nathan Whelan’s favorite phylogenomic tree for the gastropod family we both so obviously love, I had to smile.  I honestly cannot remember when the result of an evolutionary study gave me more pleasure.

As (I imagine) my loyal readership will remember, in 2014 I published a paper in Zoological Studies [8] extending my research on cryptic phenotypic plasticity to the widespread set of pleurocerid populations variously identified using the specific nomina livescens, semicarinata, and obovata, variously allocated to the genera Pleurocera or Goniobasis or Elimia or Lithasia.  I showed that all of these populations, including those traditionally identified as Lithasia obovata and Elimia semicarinata, are conspecific, their evolutionary relationship obscured by extreme phenotypic plasticity of shell.  See my blog post of [11July14] for a complete review.

The power of a scientific hypothesis is its ability to predict, to yield new information, to give something back that was not put into it.  That Nathan Whelan and his team successfully recovered the close evolutionary relationship between N = 2 snails they identified as “Elimia semicarinata” from the Middle Fork Vermillion River in eastern Illinois and N = 2 snails they identified as “Lithasia obovata” from the Ohio River between Indiana and Kentucky is a demonstration that this new AHE technique can have great power.

Yes, the assumptions that brought us the Whelan et al. phylogenomic tree are as manifold and diverse as shells on the beach, and yes, the opportunities for experimental error were as countably-infinite as the nucleotides.  And yes, the human error introduced into the Whelan study most certainly did reach embarrassing levels at times, as we shall see.

But hiding in the abstract splatter of drab shells and gaily-colored tangle of branches above – yellow Elimia, blue Pleurocera, green Lithasia and purple Leptoxis – is genuinely important insight regarding the evolution of the North American pleurocerid snails.  There is sweet music to be heard in that noise, if one brings an understanding of the biology of those wonderful organisms to the concert.  Which we will do, next time.

 

Notes:

 

[1] By way of full disclosure, my colleagues and I did reconstruct a species tree for the Physidae in 2011 [2], which we compared to a gene tree previously published.  But please note that every other treelike diagram I have ever published in my entire career has been a nearest-neighbor or cluster analysis, intended to represent genetic similarities only.  Which I have always oriented sideways, to look as little like a tree as possible.

 

[2] Dillon, R. T., A. R. Wethington, and C. Lydeard (2011) The evolution of reproductive isolation in a simultaneous hermaphrodite, the freshwater snail Physa.  BMC Evolutionary Biology 11:144. [pdf]

 

[3] Sibley, C.G. & J.E. Ahlquist (1990) Phylogeny and Classification of birds.  Yale University Press, New Haven.

 

[4] Prum RO, Berv JS, Dornburg A, Field DJ, Townsend JP, Lemmon EM, and Lemmon AR (2015). A comprehensive phylogeny of birds (Aves) using targeted next-generation DNA sequencing. Nature 526(7574):569-73. doi: 10.1038/nature15697.

 

[5] For more about Anchored Hybrid Enrichment, see:

• Lemmon, AR, SA Emme, and EM Lemmon (2012) Anchored hybrid enrichment for massively high-throughput phylogenomics.  Systematic Biology 61: 727 – 744.
• Lemmon, E.M., and A.R. Lemmon (2013) High-throughput genomic data in systematics and phylogenetics.  Annual Review of Ecology and Systematics 44: 99 – 121.
• Jones, M.R. and J.M. Good (2016) Targeted capture in evolutionary and ecological genomics.  Molecular Ecology 25: 185 – 202.

[6] This graphical summary is heavily modified from an original illustration by Bas Blankevoort in

Gravendeel, B., D. Bogarin, A. Dirks-Mulder, R. Kusuma Wati, and D. Pramanik (2018) The orchid genomic toolkit.  Cah. Soc. Fr. Orch. 9.

 

[7] Whelan, N. V., Johnson, P. D., Garner, J. T., Garrison, N. L., & Strong, E. E. (2022). Prodigious polyphyly in Pleuroceridae (Gastropoda: Cerithioidea). Bulletin of the Society of Systematic Biologists, 1(2). https://doi.org/10.18061/bssb.v1i2.8419

 

[8] Dillon, R. T., Jr.  (2014) Cryptic phenotypic plasticity in populations of the North American freshwater gastropod, Pleurocera semicarinata.  Zoological Studies 53:31. [pdf]. For a review, see:

• Elimia livescens and Lithasia obovata are Pleurocera semicarinata [11July14]

The birth, death, and resurrection of Melania acutocarinata

It was from the pen of our old buddy Isaac Lea [5Nov19], early on a frosty morning in 1841, that Melania acuto-carinata was born, in brief Latinate form, number 46 in a litter of 57 pleurocerid puppies [1].  Among its more famous siblings were #2 castanea [12Nov24], #7 ebenum [3Oct19], #10 clavaeformis [20Feb07, 12Oct09], #25 virgata [9May23], and #37 edgariana [5June20].  Lea followed with more complete English descriptions and figures of the entire litter in 1843 [2].

The type locality of Melania acuto-carinata was vague, “Tenn., Dr Currey.”  We first met Dr. Richard Owen Currey (1816 – 1865), impeccably credentialed with Presbyterian heritage and doctorate from the University of Pennsylvania, back in [3Oct19].  Currey was born and raised in Nashville but moved to Knoxville in 1846 to become “the first person with an earned doctorate to teach science” at East Tennessee University, later the University of Tennessee [3].

Melania acuto-carinata [2]

Lea’s description of the acuto-carinata shell was almost as vague as its type locality: “It seems to be distinct in its large carina which extends over all the whorls, but it is scarcely distinct on the last.”  Nevertheless, the nomen was launched into malacological stardom in 1858 by the English naturalists Henry and Arthur Adams, in their justifiably maligned “Genera of Recent Mollusca” [4].

It seems highly unlikely to me that either of the Adams brothers ever laid eyes on a North American pleurocerid snail in their entire, justifiably-maligned lives.  Tryon [5] reprinted their classification verbatim in 1873, “in order that the insufficiency of their genera may become more apparent from the incongruous assemblage of shells of which they have composed them.” 

 

Among the 16 genera Henry and Arthur recognized in the Melanidae was Io (of Lea) “remarkable for the peculiar elongation of the axis anteriorly, and for the spinose nature of the last whorl.”  Fine.  Then, as a subgenus of Io (good grief!) the Adams brothers proposed, “Elimia” for pleurocerids bearing shells fusiformly ovate, “whorls reticulate or nodulose, carinate in the middle.”  And under Elimia they listed 16 apparently random pleurocerid specific nomina in alphabetical order, the first of which was “acuticarinata, Lea,” without the dash, misspelled, bearing a shell that is neither reticulate nor nodulose.

 

Tryon dismissed the Adams classification from further consideration and assigned acutocarinata to the genus Goniobasis, newly proposed by his mentor Isaac Lea in 1862 [6].  And pretty much the entire malacological community fell in line with Tryon and Lea, most notably Calvin Goodrich [23Jan07], toward whom our attention now turns.

 

In a 1939 paper that should be better known than it is, Goodrich [7] used Goniobasis acutocarinata as an example of “depauperization,” by which he meant “the outward manifestation of disease, accident or malnutrition or a reaction to inimical environment.”  He wrote: 

“In Goniobasis, very loose coiling appears to be a sign of depauperization.  Goniobasis acutocarinata, although described as a species, has not been found in pure colonies. Lea, its author, had only one specimen.  The shell occurs as a rare variant among G. clavaeformis in springs and spring branches of East Tennessee.” 

For that reason, in his influential review of 1940, Goodrich [8] synonymized Melania acutocarinata Lea 1841 under Goniobasis clavaeformis (Lea 1841).  And that should have been the end of it.  Pleurocerid nomina that did not survive Calvin Goodrich’s 1930 – 1950 piecemeal crusade to monograph the family were not, as a rule, listed or figured in the Burch Bible [9].  They have died, been buried, and forgotten, RIP.

 

P. clavaeformis from Burch [9]

And that is indeed what happened to 12 of the 13 specific nomina [10] synonymized under clavaeformis by Goodrich in 1940.  They disappeared, never to be seen again.  But not acutocarinata.

Although generally following Goodrich quite closely, in the 1980s Jack Burch [9] gave Goodrich’s system one great big kick and a bunch of little tiny anonymous tweaks [11].  Out of a sense obligation to the letter of the ICZN law [12], Burch elected to resurrect the Adams brothers’ obscure Elimia in place of the widely used Goniobasis, confounding the work of Lea, Tryon, Goodrich and everybody else over the previous hundred years.  Following Henry Pilsbry [26Jan21] and Pilsbry’s local collaborator Samuel Rhoads [13, 14], Burch selected acutocarinata as the type of the Adams polyglot genus, by virtue of its first place in the alphabetized list of 16 odd lot species included.

 

And among his tweaks, Burch saved Isaac Lea’s acutocarinata.  To be precise, what he did was exhume two acutocarinata specimens from the UMMZ collection (Figs 404 and 405 above) and figure them alongside a singleton specimen of typical clavaeformis (Fig 403), labeling both of the former “E. acutocarinata = ? E. clavaeformis.”  Why did Burch dump 12 of Goodrich’s clavaeformis synonyms and double-figure the thirteenth?  I don’t know.  If it was a coincidence that acutocarinata was the type of his resurrected Elimia, it was certainly a convenient one.  But for whatever reason, Isaac Lea’s 1841 acutocarinata was spared into a kind of taxonomic purgatory, a place of shadows, neither alive nor dead.

 

In retrospect, I myself should have rescued the nomen from the netherworld for use at the subspecies level in my 2011 paper on Goodrichian Taxon Shift [15], later renamed cryptic phenotypic plasticity (CPP) [16].  Longtime readers will remember that I selected three sites in East Tennessee (and one in North Georgia) inhabited upstream by what I was calling, at that time, Goniobasis acutocarinata, in the mid-reaches by what I was calling, at that time, Goniobasis clavaeformis, and downstream by what I was calling, at that time, Pleurocera unciale.  Those sites included the famous [17] Indian Creek tributary of the Powell on the VA/TN border (IC, map way down below) and the even more famous [19] Pistol Creek of Maryville (PC), as well as the (rather obscure) Lick Creek tributary (LC) of the Hiwassee.

 

My allozyme analysis showed that each acutocarinata population was more genetically similar to its downstream clavaeformis population than to any other acutocarinata, and that each unciale population was genetically more similar to its upstream clavaeformis population than to any other unciale.  On that basis I folded both acutocarinata (no surprise) and unciale (big surprise) under clavaeformis, and both Goniobasis and Elimia under Pleurocera.

 

P. clavaeformis in Indian Creek, Va - Tn

I elected, however, to save uncialis/unciale at the subspecific level, as Pleurocera clavaeformis unciale (Hald 1841), both because of its utility to describe a familiar shell form, and its indexing function to the older scientific literature.  Note that subspecific designation does not carry with it any assumption regarding the heritability of the distinguishing characteristics, much less genetic relationships [20].  It’s just useful.

By the same reasoning, I should also have elevated acutocarinata back up from taxonomic purgatory to the subspecific level, as Pleurocera clavaeformis acutocarinata (Lea 1841).  That should have happened back in 2011.  In my own defense, all I can offer is that unciale was recognized at the species level by Goodrich in 1940, and acutocarinata was not, and resurrecting old pleurocerid names from the nether regions is not the direction anybody wants to go.  But I’ve done it subsequently [21].  The indexing utility of Lea’s nomen acutocarinata is just as significant as that of Haldeman’s unciale/uncialis, probably greater, and the shell morphology just as distinctive.

 

Then let it be so, better late than never.  This month I have added a new Pleurocera clavaeformis acutocarinata page to the FWGNA website, and a new photo to the FWGTN gallery, and a new entry in the FWGTN dichotomous key – the second time I have found myself doing this in recent memory [22].  The number of freshwater gastropod species now covered by the FWGNA web resource is 145 + 1 = 146. 

 

And I have dug back through my old collections and re-identified 22 populations of P. clavaeformis from the typical subspecies to acutocarinata.  Almost all of these inhabit small streams south of Knoxville, primarily direct tributaries of the main river or of the Hiwassee.  The Indian Creek population, way up on the Virginia/Tennessee Line, appears to be an outlier.

 

In all my earlier writings on Goodrichian Taxon Shift and CPP, I have tended to favor explanations that are not heritably genetic.  Although I have always left room for natural selection, most of the column inches in the discussion sections of the papers I have published on this subject [23] focus on the (many and striking) laboratory demonstrations of ecophenotypic plasticity in freshwater gastropod shell morphology.

 

Reidentified acutocarinata in blue

Quite recently a third possible explanation has dawned on me, in the specific case of the apparent transition between P. clavaeformis populations bearing shells of the typical morphology, and those bearing shells of the acutocarinata morphology.  Hybridization [24].

Populations of P. clavaeformis reach maximum abundance in streams of moderate size – marginally wadeable, where you’d bait up for redeye bass and bream.  Populations certainly extend upstream into colder waters suitable for trout, however, where in East Tennessee they bump into big populations of P. troostiana [25].

 

It is conceivable to me that Pleurocera clavaeformis acutocarinata may be a clavaeformis x troostiana hybrid.  I don’t have any genetic evidence of that [26].  This is just a speculation, based on the acutocarinata shell, which appears morphologically intermediate between the two species, both in its carination and in its overall length-to-width, body-whorl-to-apex slenderness.

 

But to conclude, I must emphasize.  Whether the shell morphological distinction between the (now three) subspecies of P. clavaeformis results from adaptation, hybridization, or ecophenotypic plasticity is irrelevant to their subspecific identity.  Here we resurrect Lea’s 1841 nomen acutocarinata because it is a useful descriptor of shell form, and because it has appeared frequently in the published literature.  And will appear again.  Stay tuned.

Notes:

[1] Lea, Isaac (1841) Continuation of Mr. Lea's paper on New Fresh Water and Land Shells.  Proceedings of the American Philosophical Society 2: 11 – 15.

 

[2] Lea, Isaac (1843) Description of New Fresh Water and Land Shells.  Transactions of the American Philosophical Society (New Series) 8: 163 – 250.

 

[3] Quoting the Richard Owen Currey entry in the University of Tennessee “Volopedia” archives: “In 1861, Currey entered Confederate service as a chaplain-surgeon. In 1865, he started caring for Union prisoners in North Carolina. He died while working in a disease-infested hospital on February 17, 1865.” He must have been a wonderful man.  It would have been an honor to shake his hand.

 

[4] Adams, Henry and Arthur (1854 - 1858) The Genera of Recent Mollusca Arranged According to Their Organization. In Three Volumes.  London: J. Van Voorst.  The Melanidae is covered in Volume 1, pp 293 – 311.

 

[5] Tryon, G. W. (1873)  Land and Freshwater shells of North America Part IV, Strepomatidae.  Smithsonian Miscellaneous Collections 253: 1 - 435.

 

[6] Lea, Isaac (1862) Description of a new genus (Goniobasis) of the Family Melanidae and eighty-two new species. Proceedings of the Academy of Natural Science of Philadelphia 19: 262 – 272.

 

[7] Goodrich, Calvin (1939) Aspects of depauperization.  The Nautilus 52: 124 – 128.

 

[8] Goodrich, C. (1940) The Pleuroceridae of the Ohio River drainage system.  Occasional Papers of the Museum of Zoology, University of Michigan 417: 1-21.

 

[9] This is a difficult work to cite.  J. B. Burch's North American Freshwater Snails was published in three different ways.  It was initially commissioned as an identification manual by the US EPA and published by the agency in 1982.  It was also serially published in the journal Walkerana (1980, 1982, 1988) and finally as stand-alone volume in 1989 (Malacological Publications, Hamburg, MI).

 

[10] Which I will not list, not even in the footnote of an obscure blog post.

 

[11] For the record, here is the complete text of Burch’s Supplemental Note #23: “Elimia H. and A. Adams (type species Melania acutocarinata Lea 1841 = Melania clavaeformis Lea 1841) is used in place of its better-known synonym Goniobasis Lea 1862 (type species Goniobasis osculata Lea 1862).  The classification of the genus Elimia presented here, and the distribution of the various recognized species and subspecies, is that of Goodrich (1930a, 1936, 1939d, e, 1940d, 1941a, b, c, 1942b, 1944d, 1945, 1950).  No attempt has been made to assess the taxonomic validity of the species and subspecies.”

 

[12] Burch, J. B. 2001. On the genus name Goniobasis (Elimia - Gastropoda: Pleuroceridae) and other recent nomenclatural inconsistencies. Walkerana 12:97-105.

 

[13] Pilsbry, H. and S. Rhoads. 1896. Contributions to the Zoology of Tennessee, Number 4, Mollusca. Proc. Acad. Nat. Sci. Phila. 1896:487-506.

 

[14] Note, however, that Pilsbry never used “Elimia” himself.  His imperial majesty continued to prefer Goniobasis in every paper he ever wrote or edited for The Nautilus.  And all the labels in the ANSP collection read “Goniobasis” to this day.  For example:

• Pilsbry, H. 1916. Goniobasis in western Pennsylvania. Nautilus 30:4-5.

[15] Dillon, R. T. (2011) Robust shell phenotype is a local response to stream size in the genus Pleurocera (Rafinesque 1818). Malacologia 53: 265-277. [pdf]

 

[16] For an entry into the rather extensive literature of CPP in North American pleurocerid snails, see:

• Goodrichian taxon shift [20Feb07]
• Mobile Basin III: Pleurocera puzzles [12Oct09]
• Pleurocera acuta is Pleurocera canaliculata [3June13]
• Pleurocera canaliculata and the process of scientific discovery [18June13]
• Elimia livescens and Lithasia obovata are Pleurocera semicarinata [11July14]

[17] I was wading in waters of Indian Creek, just like John, when the scales fell from my eyes.  John Robinson and I published a gray-literature report on those findings to the VDGIF in 2007 [18], and I coined the term “Goodrichian Taxon Shift” on this blog.  See:

• Goodrichian Taxon Shift [20Feb07]

[18] Dillon, R. T. & J. D. Robinson (2007b) The Goniobasis ("Elimia") of southwest Virginia, II. Shell morphological variation in Goniobasis clavaeformis. Report to the Virginia Division of Game and Inland Fisheries, contract 2006-9308. 12 pp. [pdf]

 

[19] It was at Pistol Creek that I ultimately discovered a forgotten pleurocerid species cryptic under P. simplex.  See:

• The cryptic Pleurocera of Maryville [13Sept16]
• The fat simplex of Maryville matches type [14Oct16]
• One Goodrich missed: The skinny simplex of Maryville is Pleurocera gabbiana [14Nov16]

[20] Subspecies are populations of the same species in different geographic locations, with one or more distinguishing traits.  For an elaboration, see:

• What is a subspecies? [4Feb14]
• What subspecies are not [5Mar14]

[21] I resurrected Isaac Lea’s (1862) lyonii, previously subsumed by Goodrich (1940) under laqueata, as a subspecies of P. troostiana in:

• The return of Captain Lyon [6July20]

[22]  I added a new Pleurocera laqueata castanea page to the FWGNA website last fall, with corresponding entries in the FWGTN species gallery and dichotomous key.  See:

• Reticulate evolution in the North American Pleuroceridae [12Nov24]

[23] Published papers on CPP:

• Dillon, R. T. (2011) Robust shell phenotype is a local response to stream size in the genus Pleurocera (Rafinesque 1818). Malacologia 53: 265-277. [pdf]
• Dillon, R. T., S. J. Jacquemin & M. Pyron (2013) Cryptic phenotypic plasticity in populations of the freshwater prosobranch snail, Pleurocera canaliculata.  Hydrobiologia 709: 117-127.  [pdf]
• Dillon, R. T. (2014) Cryptic phenotypic plasticity in populations of the North American freshwater gastropod, Pleurocera semicarinata.  Zoological Studies 53:31. [pdf]

[24] For more on hybridization in pleurocerids, see:

• Widespread hybridization between Pleurocera laqueata and P. troostiana in streams of the Tennessee/Cumberland [15Oct24]
• Reticulate evolution in the North American Pleuroceridae [12Nov24]

[25] And simplex. And gabbiana.  But I don’t think that is relevant to my argument here.

 

[26] Just the opposite, actually.  In our VDGIF allozyme study [27], John Robinson and I did run gels on N = 31 G. clavaeformis (C1) and N = 30 G. “arachnoidea” (A1 = troostiana) from Indian Creek, finding a little bit of evidence of mixing at some loci, but fixed differences at three others.

 

[27] Dillon, R. T. & J. D. Robinson (2007a) The Goniobasis ("Elimia") of southwest Virginia, I. Population genetic survey. Report to the Virginia Division of Game and Inland Fisheries, contract 2006-9308. 25 pp. [pdf]

Water hardness, stream size, and A.E. Boycott: A New River reminiscence

Just across the Blue Ridge, where the high meadows lay,

And the galax spreads through the new mown hay,

There's a rusty iron bridge, 'cross a shady ravine

Where the hard road ends and turns to clay [1].

The New River is the second-oldest river in the world, just a bit younger than the Nile.  I have heard that assertion stated so often and so forcefully that it must be so.  Born on the slopes of North Carolina’s Grandfather Mountain (G, down below), the New River first flows rather improbably to the northeast, through high meadows parallel to the Blue Ridge, into the Commonwealth of Virginia.  Then, quite astoundingly, the river shifts its course northwest, orthogonal to the Appalachian Mountains, near the little city of Blacksburg (B), and cuts a deep notch through the Allegheny Plateau, diagonally across West Virginia to The Ohio.  The earliest explorers of the American interior named its lower half the Kanawha River, never imagining that the New River and the Kanawha River might connect through those hundreds of miles of rugged terrain.

New River at Grandview

My mother was born and raised in the little town of Floyd, Virginia, perched on the New River plateau, looking down over the Blue Ridge (M).  And my father was born and raised in Rock Castle Gorge, deep in the Blue Ridge down below (F).  Many were the sparkling summers I spent rocking on my grandmother’s porch, many were the spring times I fished the chilly creeks for trout, many the falls I hunted the dark forests for squirrels.

 

With a suitcase in his hand

There the lonesome boy stands

Gazing at the river sliding by beneath his feet,

But the dark water springs from the black rocks and flows

Out of sight where the twisted laurel grows.

 

So, I matriculated at Virginia Tech, the University of Blacksburg, in the fall of 1973.  And I have shared with this readership quite a few anecdotes about my education in those hallowed halls [2], especially highlighting the impact of my mentor, Dr. E. F. (Fred) Benfield.  Longtime readers might remember that, even as I was finding a seat in my freshman biology class, plans were well advanced to construct a double-dam pump-storage facility on the upper New River at the Virginia / North Carolina border, which would have sunk 100 miles of the world’s second-oldest river into inky blackness and inundated thousands of square miles of lovely farmland.  And the Virginia Tech Center for Environmental Studies had been contracted to prepare the Environmental Impact Statement [3].

 

And so it was that I, a mere sophomore of age 19, found myself checking out boots, buckets and nets from the storeroom of the Virginia Tech Center for Environmental Studies, pulling the keys to a pickup truck from the pegboard, and driving off into the high meadows of the upper New River for my first systematic survey of a freshwater molluscan fauna [4].  And in my undergraduate research thesis, defended in May of 1977, I reported a modest 6 species of unionid mussels, 4 species of pisiidid clams, 4 species of prosobranch snails, and 6 species of pulmonate snails from 87 sites sampled across the drainage in ten counties of southern Virginia and northwestern North Carolina [5].

 

It materialized that those 20 species of freshwater mollusks were not all evenly distributed across my ten-county study area.  Even to my young and untrained eye, patterns manifest themselves.  And the most striking pattern was closely correlated with the underlying geology of the upper New River basin.  Which sent me to the library, looking for clues.

The geology of the New River Valley of Virginia

I do not remember the day or the hour I first discovered the wonderful 1936 work of A. E. Boycott [6], but I do remember the impact.  Boycott’s 70-page review was a thing of beauty; so complete, so rigorous, so bubbling over with cheerful facts about the biology of the creatures we both obviously loved that 20 years later, I patterned an entire book after it [7].  And in retrospect, may have produced but a pale imitation.

 

So Boycott, after reviewing the general biology and local habitats of the 62 species of freshwater mollusks inhabiting the Island of Great Britain, turned to “The relevant qualities of the habitats.”  Item (b) in his list of eight relevant qualities was “lime,” and item (c)  was “reaction,” by which he meant pH, which (he hastened to point out) was essentially the same thing as lime [8].

 

Boycott went on to classify (and map) the 62 British species as 25 “soft-water” species (“all those which we can find in soft water without surprise”) and 30 species “calciphile or calcicole in the sense that they are habitually found in hard water [9].”  He could not identify any British species as “calcifuge,” i.e., restricted to soft water.  Thus, overall molluscan diversity increased in the hard waters, as the 30 calciphiles were added to the 25 softwater species in the richer environments.

 

Well, that explains a lot right here in the good old USA, I thought to myself.  From its North Carolina origin through the first (roughly) hundred miles of its journey, the New River runs through ancient metamorphic rocks such as gneiss and schist, remaining quite soft.  But about halfway to its hard left turn at Blacksburg, the New enters a region of limestone and dolomite, and the water hardens up.  The distribution of most of the unionids and a couple of the gastropods (Pleurocera shenandoa, Physa gyrina) seemed to be restricted below that invisible barrier, as though they were, in Boycott’s terminology, “calciphile.”

 

But there was a second obvious factor in the distribution of the upper New River mollusk fauna, and Boycott had that one covered as well.  Item (d) of Boycott’s eight “relevant qualities” was “Size and Volume.  The larger units of water are liable to contain the more Mollusca.”  And indeed, most of the unionid mussel species were confined to the main New River, apparently unable to inhabit the smaller tributaries.  That also seemed true for Campeloma decisum.

Dillon & Benfield [16]

That’s pretty much where I dropped the shovel for my 1977 undergraduate thesis and sat down in front of the typewriter.  Each of my four subheadings under Results and Discussion – Unionidae, Sphaeriidae, Prosobranch Gastropods, and Pulmonate Gastropods – had a section entitled “Effect of Hardness” and a section entitled “Effect of Stream Size.”  All four subheadings also had a section entitled “Effect of perturbation” or “Effect of (artificial) enrichment.”  This was the 1970s, after all, I had to get pollution in there somehow.  I defended in May, got married in June, and moved to Philadelphia in July.

 

Ah, but.  Hidden deep inside my thesis was the germ of an idea.  Under the “Unionidae” subheading was also a brief section entitled “Interaction of factors.”  And there I speculated, at the age of 21, “Perhaps hardness and stream size interact in some manner so that a large stream can support Elliptio dilatata even though its hardness may be low, and a small stream can support mussels if it has high hardness.”  That hardness x stream size interaction was also obvious in the pulmonate snails.  About the origin of the phenomenon, at my young age, I would not hazard a guess.

 

Past the coal-tipple towns in the cold December rain

Into Charleston runs the New River train,

Where the hillsides are brown, and the broad valleys stained

By a hundred thousand lives of work and pain.

 

Ecology at The University of Pennsylvania and ecology at Virginia Tech are as different as Philadelphia and Blacksburg.  At Penn, I found the intellectual focus entirely upon the interactions among organisms, not the interactions between organisms and their environment.  Density dependence was the key, density-independence a quaint anachronism.  I remember vividly the argument made by the chairman of my graduate committee, Bob Ricklefs.  “A population without density-dependent control will either go extinct, or cover the world ass-deep.”  Since freshwater gastropods exist [10], and we are not ass-deep in them, they must be under density-dependent control.  Food availability or predation might certainly qualify as potential controls for their distribution, perhaps, certainly not the availability of calcium to build their shells.

 

Robert MacArthur’s theory of island biogeography was also still very much en vogue at Penn in the late 1970s, with its focus on island size.  And somewhere during my first year of graduate training, it dawned on me that both the size of a stream and the hardness of its water might affect its productivity.  And that the quantity of food might be controlling freshwater mollusk distributions in the upper New River, neither the calcium nor the stream size directly.

 

And so, at age 22, I hazarded a guess.  In July of 1978 I drove back down I-95/I-81 south from Philadelphia into those high meadows of the Upper New River where I had spent the summers of my youth, with beakers, cylinders, burettes, bottles of indicators and a 0.02 N sulfuric acid titrant carefully stowed behind the hatch of my 1973 Pinto.  I revisited and re-sampled every one of those 87 sites I had collected in the years previous, this time taking a streamside measurement of alkalinity, more reliable than pH, much easier to measure than calcium or overall hardness.  And this time I estimated a rank abundance for each of the five New River pulmonate snails (excluding limpets, which were omnipresent): Physa acuta, Physa gyrina, Lymnaea humilis, Lymnaea columella, and Helisoma anceps [11].

Dillon & Benfield [16]

I found pulmonate snails at 26 of the 87 sites, marked letters A – Z in the map above.  Confirming and deepening my undergraduate results, pulmonates were common and widespread in the main New River and most of its tributaries downstream from where the river entered the limestone/dolomite zone.  Above that zone, however, pulmonates were generally found only in the main river itself.

 

I summed the rank abundances of the five species into an overall measure of pulmonate abundance for each site.  And I calculated the Kendall rank correlation between pulmonate abundance, alkalinity, stream drainage area and (here’s the key!) the alkalinity x drainage area interaction.  The correlation with interaction (0.35**) was greater than either alkalinity or drainage area alone.

 

OK, one last thing.  Notice in the table above that alkalinity and drainage area were negatively correlated.  I guess that’s not too surprising – a little creek running through a limestone valley can get much harder than a big river, buffered as it is by its large catchment.  This gave me the idea of a nonparametric partial correlation coefficient, analogous to (parametric) partial correlation – a correlation between two variables holding a third variable constant.

Dillon & Benfield [16]

So, I invented nonparametric partial correlation [14].  The table above shows that the Kendall rank correlation between abundance and interaction remains high, even if alkalinity is partialled out (0.31) or if drainage area is partialled out (0.27).  The primary phenomenon is the interaction – the increased productivity that both water hardness and stream size promote – not the calcium nor the stream size directly.

 

When the paper by Dillon & Benfield [16] finally reached publication in 1982, I honestly thought I would become famous.  Brilliant young scientist invents novel statistical technique to answer a fundamental question of freshwater biology!  Alas, no.

 

In a tar-paper shack out of town across the track

Stands an old used-up man trying to call something back

But his old memories fade like the city in the haze

And his days have flowed together like the rain

 

And the dark water springs from the black rocks and flows

Out of sight where the twisted laurel grows

 

Notes:

 

[1] The lovely and haunting lyrics interleaved with this month’s essay come from a song entitled “Twisted Laurel” by The Red Clay Ramblers, one of the greatest bands of the postmodern era.  Some good music is still being made today, but for mysterious reasons has fallen out of fashion with the popular multitude.  Exactly the same could be said for science.

 

[2] A small sample of previous essays in which I have reminisced about my undergraduate experiences at Virginia Tech and traced their subsequent influence on my scientific career:

• To Identify a Physa, 1975 [6May14]
• Pleurocera shenandoa n.sp. [11Mar19]
• Interpopulation gene flow: King Arthur’s lesson [7Sept21]
• Growing up with periwinkles [6Apr23]

[3] For more about APCO’s Blue Ridge Project and its ultimate fate, see:

• Woodard, R. S., Jr. (2006) The Appalachian Power Company along the New River: The defeat of the Blue Ridge Project in historical perspective.  M.A. Thesis, Virginia Tech, Blacksburg.  139 pp. [pdf]

 [4] We never change.  Fifty years later, I am still doing exactly the same thing.

 

[5] Dillon, R.T., Jr (1977) Factors in the distributional ecology of upper New River mollusks (Va/NC).  Undergraduate research thesis, Virginia Tech. [pdf]

 

[6] Boycott, A.E. (1936) The habitats of freshwater molluscs in Britain.  Journal of Animal Ecology 5: 118 – 186.

 

[7] Dillon, R.T., Jr. (2000) The Ecology of Freshwater Molluscs. Cambridge University Press.  509 pp.

 

[8] Boycott verbatim: “Broadly speaking, for the natural waters of this country the reactions run parallel with the quantities of calcium.”

 

[9] Boycott did not categorize 7 of the 62 British species because he felt that his data were insufficient.

 

[10] OK, I realize that I have touched a controversial point.  The vast majority of my colleagues today, including (quite likely) most of the handful of you who will ever read this footnote, believe, as an article of faith, that freshwater gastropod populations are indeed going extinct.  Possibly that every living creature on this earth, except cockroaches, mosquitos, thee and me, is going extinct?  And I will grant you all that the vast majority of all species that have ever lived have, indeed, gone extinct.  That is evolution.  That is not a crisis; that is not even bad.  That is normal.

 

[11] Back in 1978, I was still using George Te’s [12] identifications for the Physa, so Physa acuta = “hendersoni” and Physa gyrina = “pomilia.”  I identified the Lymnaea humilis as “Fossaria obrussa” [13] and used “Pseudosuccinea” as the genus for columella.  Science advances.

 

[12] For the complete story, see:

• To identify a Physa, 1971 [8Apr14]
• To identify a Physa, 1975 [6May14]
• To identify a Physa, 1978 [12June14]
• To identify a Physa, 1989 [3Oct18]
• To identify a Physa, 2000 [6Dec18]

[13] For the complete story, see:

• The American Galba and The French Connection [7June21]
• The American Galba: Sex, Wrecks, and Multiplex [22June21]
• Exactly 3ish American Galba [6July21]

[14] I was unable, however, to offer any statistical inference on my newly-invented Kendall partial rank correlation coefficients.  I was experimenting with Monte Carlo techniques at the time [15], and (in retrospect) should have done so.   Then I would have become famous.  Surely.

 

[15] Dillon, R.T., Jr. (1981) Patterns in the morphology and distribution of gastropods in Oneida Lake, New York, detected using computer-generated null hypotheses. American Naturalist 118: 83-101.  [pdf]

 

[16] Dillon, R.T. and E. F. Benfield (1982) Distribution of pulmonate snails in the New River of Virginia and North Carolina, U.S.A.: Interaction between alkalinity and stream drainage area. Freshwater Biology 12: 179-186. [pdf]